2012 - Oxygen Club of California

oxyclubcalifornia.org

2012 - Oxygen Club of California

OXYGEN CLUB

OF CALIFORNIA

UNIVERSITY OF TURIN

2012

OXIDANTS AND ANTIOXIDANTS IN BIOLOGY

Cell Signaling and Nutrient-Gene Interactions

BOOK OF ABSTRACTS

20-23 JUNE 2012

ALBA, ITALY


ORGANIZERS

Giuseppe Poli

DEPARTMENT OF CLINICAL AND BIOLOGICAL SCIENCES, UNIVERSITY OF TURIN

SAN LUIGI GONZAGA HOSPITAL, ORBASSANO, TURIN, ITALY

Maret G. Traber

LINUS PAULING INSTITUTE, OREGON STATE UNIVERSITY, CORVALLIS, OREGON, USA

SCIENTIFIC ADVISORY COMMITTEE

Enrique Cadenas

DEPARTMENT OF PHARMACOLOGY AND PHARMACEUTICAL SCIENCES, SCHOOL OF PHARMACY,

UNIVERSITY OF SOUTHERN CALIFORNIA, LOS ANGELES, CALIFORNIA, USA

César G. Fraga

PHYSICAL CHEMISTRY–INSTITUTE FOR BIOCHEMISTRY AND MOLECULAR MEDICINE,

SCHOOL OF PHARMACY AND BIOCHEMISTRY, UNIVERSITY OF BUENOS AIRES-CONICET,

BUENOS AIRES, ARGENTINA

Klaus Kraemer

SIGHT AND LIFE, DSM NUTRITIONAL PRODUCTS LTD., BASEL, SWITZERLAND

Lester Packer

DEPARTMENT OF PHARMACOLOGY AND PHARMACEUTICAL SCIENCES, SCHOOL OF PHARMACY,

UNIVERSITY OF SOUTHERN CALIFORNIA, LOS ANGELES, CALIFORNIA, USA

Helmut Sies

INSTITUTE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY I, FACULTY OF MEDICINE,

HEINRICH-HEINE-UNIVERSITY DÜSSELDORF, DÜSSELDORF, GERMANY


CONTENTS

KEYNOTE LECTURE ................................................................................. 5

SESSION I

NRF2-DRIVEN REGULATION OF ANTIOXIDANT DEFENSES ............................ 7

SESSION II

NUTRIENT-GENE INTERACTIONS AND EPIGENETICS ..................................... 15

SESSION III

NOVEL ROLES OF MICRONUTRIENTS ........................................................ 21

SESSION IV

LIPID OXIDATION AND SIGNALING ............................................................. 31

SESSION V

EPIGENETICS, METABOLISM, AND AGING ................................................... 41

POSTERS................................................................................................ 57

AUTHOR INDEX .................................................................................... 175

SPONSORS ............................................................................................ 183

3


5

KEYNOTE LECTURE


Metabolic reprogramming, caloric restriction and aging

ROZALYN M. ANDERSON 1 , JAMIE BARGER 2 , TOMAS PROLLA 3 ,

RICKI J. COLMAN 4 , AND RICHARD WEINDRUCH 1

1 Department of Medicine / VA Geriatric Research, Education and Clinical

Center; University of Wisconsin-Madison, WI 53705; 2 LifeGen Technologies,

LLC, Madison, WI 53719; 3 Department of Genetics and Medical Genetics,

University of Wisconsin-Madison, WI 53706; 4 Wisconsin National Primate

Research Center, University of Wisconsin-Madison, Wisconsin 53715

The number of older persons in many nations is rapidly growing

and should continue to do so. This demographic reality supports the

urgent need for interventions that can extend the span of good health.

Caloric restriction (CR) is one such candidate intervention because

health span extension has been demonstrated in rodents and rhesus

monkeys. Importantly, because it is unlikely that millions of people

could adhere to long-term CR, there is great interest in identifying

nutrients or drugs that can mimic the effects of CR (so-called “CR

mimetics”) in animals/people consuming normal calorie intakes. A

mechanistic understanding of CR will facilitate the discovery of CR

mimetics. An inverse linear relationship between calorie intake and

lifespan in mice suggests that regulators of energy metabolism are

important in CR’s actions. Many studies reveal tissue-specific changes

in energy metabolism with CR and suggest that metabolic reprogramming

plays a critical role in its mechanism of aging retardation. I will

discuss data germane to these points derived from my 37 years of

studying CR, in collaboration with many gifted colleagues, in mice

and monkeys. Lastly, unpublished data based on a transcriptomic

meta-analysis revealing conserved pathways to longevity will be presented.

The rapid growth of knowledge on the retardation of aging

and diseases by CR provides optimism in the search for needed interventions.

6


SESSION I

NRF2-DRIVEN REGULATION OF ANTIOXIDANT DEFENSES

7


Molecular basis of Keap1-Nrf2 system function

MASAYUKI YAMAMOTO 1 , KEIKO TAGUCHI 1 , TAKAFUMI SUZUKI 1 ,

AND HOZUMI MOTOHASHI 1,2

1 Department of Medical Biochemistry and 2 Center for Radioisotope Sciences,

Tohoku University Graduate School/ School of Medicine,

Sendai 980-8575, Japan

Toxic chemicals (often electrophiles) and reactive oxygen species

(ROS) activate expression of detoxifying and antioxidant genes

through antioxidant/electrophile responsive element (ARE/EpRE).

Transcription factor Nrf2 is essential for the coordinated induction of

cellular defense enzymes through ARE. This notion is best demonstrated

in animal models, showing that Nrf2-null mice are sensitive

to a wide variety of electrophiles and ROS. Keap1 is a component

of ubiquitin-E3 ligase and degrades Nrf2 constitutively. Electrophiles

and ROS liberate Nrf2 from the repression by Keap1 and provoke

nuclear accumulation of Nrf2. Keap1 possesses reactive cysteine

residues that act as sensors for xenobiotic and oxidative stresses. We

refer this system to as the Cysteine Code. Mouse and zebrafish models

demonstrate that multiple sensor functions reside within Keap1.

The Hinge and Latch model proposed for the Keap1-Nrf2 system

describes the mechanism of nuclear accumulation of Nrf2 in a Cul3-

Keap1 E3 ubiquitin ligase-dependent manner. We have verified this

model through structure biology, mouse/zebrafish genetics, and human

cancer analyses. In human cancers missense mutations have been

identified in KEAP1 and NRF2 genes. These mutations disrupt the

KEAP1-NRF2 complex and result in constitutive activation of NRF2.

Elevated expression of NRF2 target genes confers advantages on

cancer cells. Transgenic mouse models provide evidence that mutated

form of Keap1 analogous to cancer genotypes lose the ability

to repress Nrf2 in vivo. Thus, the Keap1-Nrf2 system opens a new

avenue to the understanding of the signal transduction and regulatory

processes underlying the stress response and cancer progression.

8


Keap1-Nrf2 signaling: targets for disease prevention

THOMAS W. KENSLER 1,2

1 Department of Pharmacology & Chemical Biology, University of Pittsburgh,

Pittsburgh, PA 15261 and Department of Environmental Health Sciences, 2

Johns Hopkins Bloomberg School of Public Health, Baltimore,

MD 21205, USA

Health reflects the ability of an organism to adapt to stress. Stressesmetabolic,

proteotoxic, mitotic, oxidative and DNA-damage stresses

not only contribute to the etiology of cancer and other chronic degenerative

diseases but are also hallmarks of the cancer phenotype.

Activation of the Kelch-like ECH-associated protein 1 (KEAP1)–

NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response

to environmental and endogenous stresses and serves to render

animals resistant to chemical carcinogenesis, other forms of toxicity,

and inflammation whilst disruption of the pathway typically exacerbates

these outcomes. Protection against these stresses is manifest in

multiple ways: (i) prevention of macromolecular damage through

induction of electrophile detoxication and antioxidative enzymes, as

well as dampening of inflammatory processes, (ii) induction of

macromolecular damage repair/removal systems including the

proteasome, DNA repair and autophagy, and (iii) activation of tissue

repair/regeneration pathways. These cytoprotective effects of Nrf2

reflect responses mediated by direct activation of downstream effector

genes and through cross-talk with other signaling networks contributing

to cellular plasticity including aryl hydrocarbon receptor,

NF-kB, p53 and Notch1. In addition to endogenous signaling molecules

such as reactive oxygen and nitrogen species, growth factors

and oxidized lipids, the Keap1-Nrf2 pathway can also be induced by

thiol-reactive small molecules including dithiolethiones (e.g., oltipraz),

isothiocyanates (e.g., sulforaphane) and triterpenoids (e.g., CDDO-

Im) that demonstrate protective efficacy in preclinical

chemoprevention models and in clinical trials. Thus targeting the

pathway may provide important opportunities for disease prevention.

Supported by NIH grants CA039416, CA094076 and ES006052.

9


Nrf2-mediated redox signaling in endothelial cells:

consequences for cardiovascular disease

GIOVANNI E. MANN

Cardiovascular Division, British Heart Foundation Centre of Research

Excellence, School of Medicine, King’s College London, 150 Stamford Street,

London, SE1 9NH, UK

Fetal exposure to pre-eclampsia (PE) and gestational diabetes

(GDM) in utero is strongly associated with a higher risk of cardiovascular

disease and insulin resistance in later life, and accumulating

evidence suggests this may be a consequence of fetal programming. 1

Notably, offspring of mothers with PE or GDM exhibit elevated blood

pressure and reduced endothelium-dependent reactivity.

We previously reported abnormal NO production, insulin resistance

and reduced cell proliferation in fetal umbilical vein endothelial

cells (HUVEC) from GDM pregnancies, 2,3 and in studies with fetal

cells from PE pregnancies identified abnormalities in NO production

and regulation of [Ca 2+ ] i . 4,5 These phenotypic changes are maintained

during culture, highlighting the involvement of fetal programming

and epigenetic influences. As in PE and GDM, placental oxidative

stress is propagated to the maternal and fetal vasculature via circulating

lipid peroxides and H 2 O 2 , we hypothesised that sustained

oxidative stress in the fetal vasculature impairs endogenous antioxidant

defences.

We have recently established that activation of the redox sensitive

transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) 6

and its downstream target antioxidant enzymes is markedly inhibited

in HUVEC from PE or GDM pregnancies. Both cell types exhibited

marked deficits in glutathione synthesis, increased basal mitochondrial

superoxide production, reduced nuclear translocation of Nrf2,

and diminished adaptive increases in the expression of the Nrf2 target

genes heme oxygenase-1 (HO-1) and/or NAD(P)H quinone oxidoreductase

1 (NQO1) in response to the lipid peroxidation product

4-hydroxynonenal. A proteomic analysis of normal and GDM

HUVEC confirmed the altered GDM phenotype, characterised by

markers of increased oxidative stress, reduced antioxidant protection

and reduced cell proliferation. The altered phenotype of fetal endothe-

10


lial cells derived from PE and GDM pregnancies may well contribute

to the increased risk of developing type 2 diabetes and cardiovascular

disease in childhood and adulthood. 7-9

Supported by BHF

1. Simmons, R. (2005). Trends Endocrinol Metab 16, 390-394.

2. Sobrevia, L., Cesare, P., Yudilevich, D.L., & Mann, G.E. (1995). J Physiol 489,

183-192.

3. Sobrevia, L., Yudilevich, D.L., & Mann, G.E. (1998). J Physiol 506, 219-230.

4. Ishii, T., Itoh, K., E. Ruiz, Leake, D.S., Unoki, H., Yamamoto, M. & Mann, G.E.

(2004). Circ Res 94, 609-616.

5. Steinert, J.R., Wyatt, A.W., Poston, L., Jacob, R. & Mann, G.E. (2002). FASEB

J 16, 721-723.

6. Steinert, J.R., Poston, L., Mann, G.E. & Jacob, R. (2003). FASEB J 17, 307-

309.

7. Chapple, S. et al. (2010). Free Radical Res 44, 1125-1171.

8. Gao, L. & Mann, G.E. (2009). Cardiovasc Res 82, 9-20.

9. Cheng, X., Siow, R.C.M. & Mann, G.E. (2010). Antiox Redox Signaling 14,

469-487.

11


Adaptive survival response mediated by the Nrf2-induced

HO-1 upregulation

YOUNG-JOON SURH

Tumor Microenvironment Global Core Research Center and WCU Department

of Molecular Medicine and Biopharmaceutical Sciences, College of

Pharmacy, Seoul National University, Seoul 151-742, South Korea

Living organisms are constantly subjected to diverse types of stress

both external and internal sources. While excessive stress leads to

necrotic or apoptotic death, moderate amounts of noxious stimuli may

render the cells adaptive or tolerant to ongoing or subsequent insults.

Such adaptive survival response normally accompanies de novo synthesis

of proteins through activation of distinct stress-responsive

signaling. The induction of heme oxygenase-1 (HO-1) gene expression

represents the first line of cellular defence against oxidative/

nitrosative stress and other noxious stimuli. Experimental models of

various diseases including acute inflammation, atherosclerosis,

neurodegenerative disorders and cancer have demonstrated that the

induction of HO-1 can prevent or mitigate the symptoms associated

with these ailments. Nuclear factor E2-related factor-2 (Nrf2) has

been identified as a major transcription factor responsible for regulating

HO-1 gene expression. Our previous studies have demonstrated

that hydrogen peroxide and peroxynitrite induce HO-1 expression

through activation of Nrf2 signaling, which confers the cellular

cytoprotection. Such adaptive response to oxidative or nitrosative

stress was mediated, in part, by 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-

PGJ2). This cyclopentenone prostaglandin activated Nrf2 signaling

and subsequently induced expression of HO-1 and other cytoprotective

proteins. HO-1 induction is also associated with cytoprotection against

inflammatory tissue injuries. Thus, HO-1 induction occurred before

inflammatory damage manifests in colonic mucosa following administration

of dextran sulfate sodium (DSS) to ICR mice. Some

chemopreventive/chemoprotective natural products can induce ARE/

EpRE-driven upregulation of HO-1 expression, thereby fortifying

cellular defence against oxidative, nitrosative and inflammatory insults.

Supported by the Global Core Research Center (GCRC) grant, National

Research Foundation-MEST, Republic of Korea

12


Nrf2 target genes are induced under marginal

selenium-deficiency

REGINA BRIGELIUS-FLOHÉ, MIKE MÜLLER AND ANNA P. KIPP

Department Biochemistry of Micronutrients, German Institute of Human

Nutrition Potsdam-Rehbruecke, D-14558 Nuthetal, Germany

Mice were fed a selenium-adequate (0.15 mg Se/kg) or a moderate

selenium-deficient (0.086 mg/kg) diet to test consequences for

gene expression under a suboptimal selenium supply as it appears to

prevail in Europe. Microarray analyses in the colon of these mice

identified GPx1 and the selenoproteins W, H, and M as highly sensitive

to changes in the selenium state. Most sensitive pathways were

protein biosynthesis, and those of inflammation, Delta-Notch and

Wnt signaling. Activation of the Wnt pathway was deduced from an

increase in the expression of β-catenin, Dvl2, Lef1 and c-Myc accompanied

by a decrease in GSK3β. Nrf2 target genes were manually

selected and their up-regulation under the selenium-poor diet

confirmed by qPCR in the colon and duodenum. Up-regulation of

phase II enzymes (Nqo1, Gsts, Sult1b1 and Ugt1a6) was most significant

in the colon whereas antioxidant enzymes (Hmox1, Prdx1,

Srxn1, and Gclc) increased in both intestinal areas. This might be

taken as compensation for the loss of selenoproteins. mRNA of the

Nrf2-regulated selenoproteins TrxR1 and GPx2 was higher in the

selenium-poor duodenum which would enable their immediate translation

upon selenium refeeding. In contrast mRNA of SelW decreased

further supporting its sensitivity to limited selenium supply. Possible

mechanisms for the obvious activation of Nrf2 under moderate selenium

deficiency are discussed in the context of a decrease in GSK3β

and TrxR1 under the selenium-poor diet which in an adequate status

can turn off the Nrf2 signal.

13


SESSION II

NUTRIENT-GENE INTERACTIONS AND EPIGENETICS

15


Micronutrient genomics - an overview

BEN VAN OMMEN

TNO, Utrechtseweg 48, Zeist, the Netherlands

We ever more understand the molecular and mechanistic intricacies

of micronutrient biological functions and the physiological implications

of suboptimal functioning. This has three major implications

for establishing trace element requirements:

1) by quantifying the biological function, we automatically include

all variability related to micronutrient ADME, mode of action,

and all genetic/environmental variation on these processes;

2) if multiple micronutrient influence the biological function (and

this is often the case), we can assess the requirements each of these

by studying the biological process and its biomarkers;

3) if a micronutrient is affecting multiple biological processes, we

can define requirements for each of these processes by quantifying

these.

A number of initiatives are working along these lines and will be

illustrated. Based on these assumptions, the combination of modern

analytical technologies, genomics knowledge and network biology

can be used to derive personalized requirements of multiple micronutrient.

More importantly, this approach may be needed to tackle

the real issues in micronutrient: strengthening robustness of challenged

populations.

16


Functional genetic polymorphisms in genes of choline

metabolism: an unexpected link to energy metabolism

STEVEN H. ZEISEL

University of North Carolina at Chapel Hill, Nutrition Research Institute

It is not widely appreciated that there is significant crosstalk

between choline/1-carbon metabolism and energy metabolism. There

are several common SNPs in genes of choline and folate metabolism

that cause metabolic inefficiencies that affect crosstalk between

choline metabolism and energy homeostasis. A number of observations

in mice with deleted genes of choline metabolism suggest that

such crosstalk occurs. Bhmt -/- mice gained less body weight had reduced

adiposity, increased fatty acid oxidation and increased FGF21

signaling. Pemt -/- mice fed a high fat diet had increased energy expenditure

and did not gain weight and remained insulin sensitive;

these differences disappeared when Pemt -/- mice were supplemented

with choline. BMI is significantly lower in women with 2 copies of

the minor allele of the PEMT SNP rs12325817 when compared to

the women without the SNP. Chdh -/- mice have abnormal mitochondrial

structure and function. Mice fed a methionine and choline deficient

diet were hypermetabolic, lost weight, and had better insulin

sensitivity and glucose tolerance. Betaine administration enhanced

insulin sensitivity in diet-induced-obese mice and increased insulin

signaling pathways in isolated adipocytes. Both BHMT and choline

dehydrogenase (CHDH) activities are decreased by insulin and increased

by diabetes in rats. Thus, many different lines of investigation

suggest that there is a physiological link between of choline/1carbon

metabolism and energy metabolism, insulin sensitivity and

body fatness.

This work was supported by grants from the NIH (DK55865 and DK36530)

1 To whom correspondence should be addressed: 500 Laureate Way, Kannapolis,

NC 28081.Phone: 704-250-5003;Fax: 704-250-5001; E-mail:

steven_zeisel@unc.edu.

17


The redox basis of epigenetic control

FREDERICK E. DOMANN

Free Radical & Radiation Research Program, Department of Radiation

Oncology, Carver College of Medicine, and Holden Comprehensive Cancer

Center,University of Iowa, Iowa City, IA 52242

Epigenetic modifications represent mechanisms by which cells

may effectively translate multiple signaling inputs into phenotypic

outputs. Recent research is revealing that redox metabolism is an

increasingly important determinant of epigenetic control that may have

significant ramifications in human health and disease. Numerous

characterized epigenetic marks, including histone methylation,

acetylation, and ADP-ribosylation, as well as DNA methylation, have

direct linkages to central metabolism through critical redox intermediates

such as NAD+, S-adenosyl methionine, 2-oxoglutarate, ascorbate,

FeII, and molecular oxygen. Fluctuations in these intermediates

caused by both normal and pathologic stimuli may thus have

direct effects on epigenetic signaling that lead to measurable changes

in gene expression through alterations to DNA methylation and/or

histone modifications. This lecture will present a survey of the metabolism-sensitive

epigenetic enzymes and the metabolic redox processes

that participate in their regulation. In addition, we will provide

a series of clinically relevant illustrations of the communication between

metabolism and epigenetics in the pathogenesis of various

human disease states including cancer, diabetes, and

neurodegenerative disease. We anticipate that the regulatory mechanisms

described here will play increasingly large roles in our understanding

of human health and disease as metabolic epigenetics research

progresses.

Supported by NIH R01 CA115438

18


MTHFR genotype and riboflavin: a novel gene-nutrient

interaction affecting blood pressure

HELENE MCNULTY, J.J. STRAIN, AND MARY WARD

The Northern Ireland Centre for Food and Health (NICHE), School of

Biomedical Sciences, University of Ulster, BT52 1SA Northern Ireland

Hypertension is estimated to carry an almost 3-fold increased

risk of developing cardiovascular disease (CVD), while treating hypertension

significantly reduces cardiovascular events, and stroke in

particular. One genetic variant under recent investigation in relation

to hypertension is the 677C’!T polymorphism in the gene encoding

the folate-metabolizing enzyme methylenetetra-hydrofolate reductase

(MTHFR). The homozygous mutant (TT) genotype has a reported

frequency of 10% worldwide, but can be as high as 32% in

some populations. We have been studying the modulating role of the

B-vitamin, riboflavin, the cofactor (as FAD) for MTHFR. The variant

enzyme is known from molecular studies to become inactive as a

result of having an increased propensity to dissociate from FAD, but

our earlier work suggested that supplementation with low-dose riboflavin

could stabilise its activity in vivo. More recently we found that

CVD patients with the MTHFR 677TT genotype (compared to CC

or CT genotypes) had significantly higher blood pressure, and more

importantly, that blood pressure was highly responsive to riboflavin

intervention specifically in patients with the TT genotype. Further

investigation of these patients 4 years later showed that those with

the TT genotype had remained hypertensive despite marked changes

in the number and type of antihypertensive medications being prescribed;

with riboflavin administration however there was once again

a marked blood pressure-lowering response. Thus riboflavin may

offer a targeted strategy for managing hypertension, and potentially

modulating the risk of stroke, specifically in this genetically at-risk

group. Further work is required to elucidate the mechanism through

which this gene-nutrient interaction affects blood pressure.

19


Dietary histone deacetylase inhibitors for cancer prevention

EMILY HO

School of Biological & Population Health Sciences and Linus Pauling

Institute, Oregon State University, Corvallis, OR, 97331 USA

The reversible acetylation of histones is an important mechanism

of gene regulation. During cancer progression, specific modifications

in acetylation patterns on histones are apparent. Targeting the

epigenome, including the use of histone deacetylase (HDAC) inhibitors,

is a novel strategy for cancer chemoprevention. Recently, drugs

classified as HDAC inhibitors, have shown promise in cancer clinical

trials. Based on the similarity of sulforaphane (SFN) metabolites

and other phytochemicals to known pharmacological HDAC inhibitors,

we previously demonstrated that sulforaphane acts as an HDAC

inhibitor in the prostate, causing enhanced histone acetylation, derepression

of P21 and Bax, and induction of cell cycle arrest/apoptosis,

leading to cancer prevention. SFN also causes the down-regulation

of both Class I and II HDAC protein expression. These epigenetic

effects are specific to cancer cells, not normal cells. The ability of

SFN to target aberrant acetylation patterns, in addition to effects on

phase 2 enzymes, may make it an effective chemoprevention agent.

These studies are significant because of the potential to qualify or

change recommendations for high-risk prostate cancer patients and

thereby increase their survival through simple dietary choices incorporating

easily accessible foods into their diets. These mechanistic

pre-clinical studies have provided a strong scientific foundation for

on-going human clinical trials.

20


SESSION III

NOVEL ROLES OF MICRONUTRIENTS

21


Biomedical action of pomegranate ellagitannins

DAVID HEBER, ROBERTO VICINANZA, YANJUN ZHANG, AND ZHAOPING LI

UCLA Center for Human Nutrition, David Geffen School of Medicine at

University of California, Los Angeles CA 90095

Pomegranates have been used medicinally for centuries and are

popularly consumed as juice. Pomegranate juice (PJ) and extract (PE)

contain high molecular weight (ca. 1000 Dalton) hydrolyzable tannins

called ellagitanins (ET). The ET are hydrolyzed in the gastrointestinal

tract and ellagic acid (EA) appears in the circulation between 30 minutes

and 5 hours after consumption of PJ or pomegranate extract

(PE). Through the action of human colonic microflora, EA is converted

into metabolites including urolithin A (UA), which are then

absorbed, transported in the blood, conjugated in the liver, and excreted

in glucuronidated form in the urine between 12 and 56 hours

after PJ consumption. Consumption of PJ was associated with a significant

increase (15 to 54 months) in the PSA doubling time in men

with rising PSA after prostatectomy suggesting a direct action on

prostate cancer cells. The anti-tumor effects of PE were shown to be

due to inhibition of NF-kappaB activation in tumor xenografts. Recent

investigations have demonstrated in vitro synergy of EA and

UA in human prostate cancer cells and defined unique molecular

mechanisms of action. In LAPC-4 human prostate cancer xenografts

in SCID mice, there was also evidence of enhanced inhibition of

tumor growth. These observations suggest the potential of pomegranate

juice and extracts as food ingredients and as dietary supplements

in conditions where inflammation and oxidant stress play an

important role.

Research supported by unrestricted gifts and research grants from POM

Wonderful, Inc. Los Angeles, CA

22


Zinc and redox signaling in the developing brain

SUANGSUDA SUPASAI, JONATHAN NUTTALL, AND PATRICIA I. OTEIZA

Department of Nutrition and Department of Environmental Toxicology,

University of California Davis, Davis CA 95616

The developing brain is highly susceptible to fluctuations in zinc

availability. Deficits of zinc activate the NMDA receptor, leading to

calcium influx, increased oxidant production, alterations in thiol redox

homeostasis, and to a redox-modulation of cell signals. Zinc, a

redox inactive metal, has been long viewed as a component of the

antioxidant network, and growing evidence points to its involvement

in redox-regulated signaling. Zinc can contribute to maintain the cell

redox balance through its capacity to: i) regulate oxidant production

and metal-induced oxidative damage; ii) dynamically associate with

sulfur in cysteines, and be released from these associations by reactive

nitrogen and oxygen species; iii) regulate glutathione (GSH)

metabolism and the overall protein thiol redox status; and iv) to modulate

redox signaling either directly or indirectly. In neuronal cells

and embryonic brain, zinc deficiency impairs Nrf2 activation and

GSH synthesis, and leads to a deregulation of MAPK, NF-κB and

STAT signaling pathways. Given the role of Nrf2 in the upregulation

of antioxidant defenses, zinc deficits increase the susceptibility of

neurons to oxidant stressors. MAPK, NF-κB and STAT play key

roles in the decisions of progenitor cells to proliferate, survive, and

differentiate into neurons or glial cells. Thus, a suboptimal zinc availability

to the fetus, either of nutritional or secondary (maternal exposure

to toxicants, infections, chronic diseases) can result in altered

brain development.

23


Vitamin E in the prevention of cardiovascular disease

- the importance of proper patient selection

ANDREW P. LEVY

Department of Anatomy and Cell Biology, Technion Faculty of Medicine,

Technion-Israel Institute of Technology, Haifa, Israel 31096

Vitamin E is a naturally occurring fat-soluble antioxidant which

has been proposed as a treatment for both primary and secondary

protection against cardiovascular (CV) events. Promising data from

observational epidemiological studies associating higher vitamin E

dietary intake with lower risk of CV events have not been validated

in randomized, controlled clinical trials assessing the effect of vitamin

E on CV outcomes. While the pendulum of medical opinion has

swung to suggest that high dose vitamin E supplements have no place

in the treatment and prevention of CVD, new data is emerging that

allows identification of a specific target population for this treatment,

namely patients with Diabetes Mellitus and the haptoglobin genotype

2-2. This review details the scientific basis and clinical evidence

related to the effect of vitamin E on CV outcomes, and the importance

of proper patient selection in gaining therapeutic benefit from

this intervention.

24


CoQ, adipocytes and metabolism

LOUIS CASTEILLA

STROMALab, UMR 5273, UPS/CNRS/EFS, Inserm U1031 Toulouse, France

Coenzyme Q (CoQ) is an obligatory partner of mitochondrial

biology and the unique lipophilic antioxidant synthesized in humans.

Although increasing data underline the importance of mitochondria

and oxidative stress in adipocyte biology, few link between CoQ

and metabolic disorders has been yet established. We demonstrated

that CoQ synthesis is a feature of adipocyte differentiation. Its content

is specifically and significantly decreased in adipose tissue of

obese mice and humans. Using in vitro model, we showed that the

inhibition of CoQ synthesis in preadipocytes impairs adipocyte differentiation,

lipid storage, and hypertrophy. The full restoration of its

content in adipose tissue of obese mice can be achieved via the coadministration

with PPARg ligand. This improves body weight,

oxidative metabolism, metabolic parameters and fat functions. Furthermore,

this counteracts adverse side effects classically induced by

PPARg ligands alone. Finally, we determined a threshold for CoQ

content in adipose tissue below which individuals are obese. In another

tissue such as liver, High fat diet induces a decrease in CoQ

content that is associated with the inhibition of fatty acid oxidation

and of mitochondrial oxidative-phosphorylation associated increased

mitochondrial ROS production. As a whole, these data demonstrate

that CoQ behaves as a key and new regulator of lipid metabolism

and that the manipulation of its content could open new therapeutic

perspectives.

25


Downstream effects of GSH depletion as inducers of longevityassociated

pathways

MARIA ROSA CIRIOLO

Department of Biology, University of Rome “Tor Vergata” and IRCCS

San Raffaele, Rome, Italy

GSH is the main non-enzymatic antioxidant implicated in maintenance

of redox homeostasis, detoxification and direct scavenging of

oxyradicals. GSH has also a pivotal function in buffering the intracellular

production of nitric oxide (NO), particularly in brain wherein

NO acts as an important neuromodulator and neurotransmitter. Indeed

chemical GSH depletion triggers NO unbalance and

nitroxidative stress, which is the primary cause of death in proliferating

neuroblastoma or embryonic neurons. The importance of GSH

in limiting the detrimental effect of NO is corroborated by the evidence

that in adult brain even atrivial GSH decrease is able to cause

protein nitration. Interestingly, neurodegenerative manifestations are

not observed in vivo owing to the connected induction of an adaptive

antioxidant response by way of a novel NO/PGC-1alpha-dependent

mechanism. Nutrient limitation, a dietary regimen that is generally

linked to longevity, induces a prominent efflux of intracellular GSH

and redox alterationthat is mandatory for activating NO-driven autophagy.

On the basis of these evidence it can be assumed that the decline

of GSH level occurring with normal ageing is a beneficial rather

than a detrimental event and may serve as a molecular signal inducing

a NO-mediated endogenous adaptive response (i.e. antioxidants

expression and/or autophagy) that culminates in increased resistance

to environmental/pathological stress and possibly in the extension of

lifespan.

26


Redox modulation of cell signaling by lipoic acid

and derivatives

DAVID A. CARLSON 1 , DAO YAO HE 2 , EKATERINA KALASHNIKOVA 1 , RINA

GENDELMAN 1, 3 , SARAH J. FISCHER 1 , AND LESTER PACKER 4

1 GeroNova Research Inc, 4677 Meade Street Richmond Ca 94804, 2 C&M

Biolabs, 5221 Central Avenue, Suite 8A, Richmond, CA 94804, 3 UCSF

Cancer Center, 2340 Sutter Street, N361 San Francisco, CA 94115,

4 Pharmacy and Pharmaceutical Sciences, School of Pharmacy, University of

Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089

Lipoic acid (LA) is a chiral, medium chain (C8) fatty acid existing

as two enantiomers (RLA and SLA) and a racemic mixture

(RS-LA). The only form synthesized in biological systems is RLA.

Unsolved problems in LA research include the contributions of stereochemistry

and carboxyl substitution toward the mechanisms of action

of exogenously applied LA. The enantiomers and racemates of

a neutral and a positively charged amide derivative; Lipoamide (LAM)

and LA Plus (LAP) were synthesized and compared directly with

those of LA. Previous studies with the racemates; RS-LAM and RS-

LAP and the enantiomer, RLAP indicated similarities and differences

relative to LA in vitro. SLAM and SLAP have not been previously

evaluated. Widely characterized as “antioxidant” LA and derivatives

may function simultaneously and differentially as oxidative, reductive

and redox modulators of various signal transduction pathways. Additionally,

at sufficient concentrations LA and derivatives may induce

stress responses leading to increased adaptation and cellular

stress resistance. The effects of R, S and racemic forms of 9 test

compounds were studied in multiple signaling pathways in HepG2

cells using a dual-luciferase reporter array. The reporter is a mixture

of an inducible transcription factor responsive construct encoding

firefly luciferase and an internal control plasmid constitutively expressing

Renilla luciferase construct. Each inducible construct monitors

changes in the activity of a select transcription factor serving as a

marker for activation or suppression of a specific signaling pathway.

Preliminary results will be presented of novel, stereoselective and

non-stereoselective redox protein targets and pathways differentially

affected by the test compounds.

27


Vascular oxidative stress and inflammation in atherosclerosis

and aging: ameliorating effects of alpha-lipoic acid

supplementation

BALZ FREI, WEIJIAN ZHANG, LIXIN LI, ANTHONY SMITH, AND

TORY HAGEN

Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA

Increased oxidative stress and inflammation have been proposed

as etiologic factors for atherosclerotic vascular diseases, for which

advanced age is a major risk factor. We have shown that á-lipoic

acid (LA) exerts anti-inflammatory effects by inhibiting TNFá or lipopolysaccharide

(LPS)-induced endothelial cell and monocyte activation

in vitro and LPS-induced acute inflammatory responses in

vivo. We also reported that LA inhibits atherosclerotic lesion development

in apolipoprotein E (apoE)-deficient and apoE/LDL receptordeficient

mice, two well-established animal models of human atherosclerosis.

The anti-atherosclerotic effect of LA was paralleled by reduced

body weight gain, lower serum and VLDL triglyceride levels,

and reduced aortic macrophage accumulation and gene expression

of adhesion molecules and pro-inflammatory cytokines. More recently,

we reported that dietary supplementation with LA caused a decrease

in aortic NADPH oxidase activity in old rats, accompanied by a significant

decrease in mRNA levels of NOX4 and vascular cell adhesion

molecule-1 (VCAM-1). Furthermore, LA supplementation reversed

the age-dependent decrease in aortic SOD activity and the

age-dependent increase in plasma levels of monocyte chemotactic

peptide-1 (MCP-1). Most of the observed effects of LA supplementation

are mediated by the transcription factors, NFkB and Nrf-2,

which regulate expression of inflammatory genes and phase II detoxification

and antioxidant enzymes, respectively. Mechanistic aspects

of this interaction of LA with gene expression will be further

discussed.

This work was supported by grant 0760018Z from the American Heart

Association and NIH Center of Excellence grant P01 AT002034.

28


Redox epigenetic modifications by dietary bioactive

compounds in inflammation

IRFAN RAHMAN

Department of Environmental Medicine, University of Rochester Medical

Center, Rochester, NY, USA.

Chromatin modifications and epigenetic regulation are critical for

regulation of gene expression. Acetylation of histones facilitates gene

expression by allowing the access of transcriptional machinery to

DNA. Deacetylation of histones suppresses gene expression by closing

the transcriptional machinery on promoters. Oxidative stress

causes pro-inflammatory gene transcription, which is associated with

changes in chromatin remodeling. Steroids are potent anti-inflammatory

agents, but their function is lost during oxidative stress leading

to steroid resistance. Cigarette smoke-mediated oxidative stress in

cell culture (macrophages and epithelial cells) and mouse models were

used to test the hypothesis that dietary bioactive compounds reverse

steroid resistance via an effect on histone modifications. Oxidants/

cigarette smoke down-regulated the levels and activities of histone

deacetylase 2 (HDAC2) and sirtuin 1 (SIRT1) by oxidative posttranslational

modifications, and induce gene expression of pro-inflammatory

mediators. Cigarette smoke/oxidant stress caused reduced

glucocorticoid sensitivity by reducing deacetylases via Nrf2-dependent

mechanism. This was associated with the loss in recruitment of

deacetylases onto the promoters of pro-inflammatory genes.

Resveratrol (phytoalexin, a flavanoid found in red wine) was found

to activate SIRT1 and Nrf2, and protected against oxidants/cigarette

smoke-induced pro-inflammatory cytokines release. Similarly,

curcumin (diferuloylmethane, an active component of spice turmeric)

attenuated the reduction in HDAC2, inhibited NF-kB, and reversed

the steroid resistance. Hence, overcoming the steroid resistance by

dietary bioactive compounds via molecular epigenetic chromatin

mechanisms is an important aspect in treatment of chronic inflammatory

diseases where oxidative stress occurs. Current knowledge on

epigenetics of steroid resistance on cytokine genes involved in chronic

inflammation in light of HDAC2/SIRT1-Nrf2, and ways to overcome

steroid resistance by dietary bioactive polyphenols based on chromatin

modifications will be presented.

29


31

SESSION IV

LIPID OXIDATION AND SIGNALING


Physiological and unregulated membrane lipid oxidation

ETSUO NIKI

Health Research Institute, National Institute of Advanced Industrial Science

& Technology, Ikeda, Osaka 563-8577, Japan

Lipids such as unsaturated fatty acids, cholesterol, and their esters

are susceptible to oxidation and oxidized continuously in vivo by

free radical-mediated and radical-free mechanisms. Above all, polyunsaturated

fatty acid esters are readily oxidized by free radicalmediated

chain mechanisms to produce diverse products. Recent

lipidomics technology enables us to analyze lipid oxidation products

from biological samples accurately and quantitatively. Lipid oxidation

products are potentially cytotoxic and modify proteins and DNA

bases. Many studies from our groups and others show that the levels

of lipid oxidation products are in general associated with the levels

of oxidative stress and progress of diseases, although it has not been

demonstrated clearly if the lipid oxidation products play a causal role

or serve as reliable biomarker of diseases. On the other hand, some

lipid oxidation products are produced in vivo on purpose to exert

physiological function. Further, lipid oxidation products such as

phosphatidylcholine (PC) hydroperoxide, 4-hydroxy-2-nonenal,

hydroxyoctadecadieoic acid, lyso PC, and 7-hydroxycholesterol may

induce adaptive response and enhance defense capacity. Such adaptive

response has been observed in experimental animals as well as

cultured cells. However, it should be noted that, unlike ROS/RNS or

enzymatic oxidation products which act as signaling messenger in

physiological settings, it may be difficult for free radical-mediated

lipid oxidation products to function as physiologically important

signaling messenger, since it is difficult to program and regulate the

formation of free radicals and control many competing reaction pathways.

32


Decision-making by macrophages and dendritic cells using

RXR heterodimeric receptors to sense their lipid environment

LASZLO NAGY

University of Debrecen, Medical and Health Science Center

A key issue in the immune system is to generate specific cell types

often with opposing activities. We have explored how intracellular

lipid signaling in immune cells via a set of transcription factors, regulates

cellular differentiation, subtype specification, immune as well

as metabolic homeostasis. Peroxisome proliferator-activated receptor

γ (PPARγ) is a lipid-activated transcription factor regulating lipid

metabolism and inflammatory response in macrophages and dendritic

cells (DCs). These immune cells exposed to distinct inflammatory

milieu show cell type specification as a result of altered gene expression.

We identified a set of mechanisms how inflammatory molecules

modulate PPARγ signaling in distinct subsets of cells. Proinflammatory

molecules inhibited, whereas interleukin-4 (IL-4) stimulated PPARγ

activity in macrophages and DCs. Furthermore, IL-4 signaling augmented

PPARγ activity through an interaction between PPARγ and

Signal Transducer and Activators of Transcription 6 (STAT6) on promoters

of PPARγ target genes, including FABP4. An in silico analysis

predicted activation of PPARγ signaling as well as experimental

data suggested the presence of alternative macrophage activation

specific endogenous PPARγ ligand producing mechanisms. We found

three enzymes, which can potentially generate endogenous PPARγ

ligands in an IL-4 dependent manner. These are MAOA, ENPP2

and ALOX15 producing 5-methoxy-indole acetate, lysophosphatidic

acid (LPA) and 13-hydroxyoctadienoic acid (13-HODE) as well as

15-hydroxyeicosatetraenoic acid (15-HETE), respectively. More recently

we have used genomic approaches to map the localization

and the cistromic interactions of nuclear receptors and histone marks

as well as inflammatory transcription factors. These interactions, underpinning

cell type-specific responses represents a unique way of

controlling nuclear receptor signaling by inflammatory molecules in

immune cells.

33


Lipid oxidation-mediated mechanisms of destabilization

of the atherosclerotic plaque

GABRIELLA LEONARDUZZI AND GIUSEPPE POLI

Department of Clinical and Biological Sciences, University of Turin,

Orbassano, Torino, Italy

An imbalance in the matrix metalloproteinases/tissue inhibitors of

metalloproteinases (MMPs/TIMPs) contributes to atherosclerotic

plaque destabilization and rupture. Among MMPs, MMP-9 has been

consistently implicated in the pathophysiology of plaque rupture.

Oxidative modifications of low density lipoproteins (LDLs), major

carriers of cholesterol in human circulation, provide them with several

pro-atherogenic properties which might contribute to plaque

destabilization. Among the different molecules present in oxidized

LDLs and potentially responsible for its bioactivity, there are cholesterol

oxidation products, termed oxysterols.

To clarify whether oxysterols might play a role in plaque

destabilization, we investigated, in human promonocytic U937 cells,

the effect of an oxysterol mixture of composition similar to that in

advanced human carotid plaques on expression and synthesis of MMP-

9 and its endogenous inhibitors TIMP-1 and TIMP-2. A marked increment

of MMP-9 gene expression and protein levels, but not of its

inhibitors, was observed, as well as of MMP-9 gelatinolytic activity.

Using antioxidants or specific inhibitors or siRNAs, we demonstrated

that the oxysterol mixture induces MMP-9 expression through: i) overproduction

of reactive oxygen species (ROS) likely by NADPH oxidase

and mitochondria, ii) up-regulation of mitogen-activated protein

kinases signaling pathways via protein kinase C, iii) up-regulation

of activator protein-1- and nuclear factor-κB-DNA binding.

Moreover, oxysterols induce the expression of various inflammatory

molecules, such as IL-1β, IL-8 and TNF-α, presumably through

TLR4 involvement. These inflammatory molecules appear to contribute

to plaque vulnerability by their subsequent induction of MMP-

9 expression.

These results suggest that oxysterols, accumulating in advanced

atherosclerotic lesions, significantly contribute to plaque vulnerability

by promoting MMP-9/TIMP-1 and TIMP-2 imbalance.

34


Oxysterols in the pathogenesis of inflammatory-based diseases

LUIGI IULIANO

Vascular Biology and Mass Spectrometry Laboratory, Department of Medico-

Surgical Sciences and Biotechnology, Sapienza University of Rome, Latina,

Italy

Cholesterol, 3β-hydroxycholest-5-ene, is a principal lipid of

mammalian cells and is present in all membrane compartments of

cells. The addition of oxygen to the cholesterol molecule – via either

enzymatic or non-enzymatic mechanisms – results in the formation

of derivatives known commonly as oxysterols. In the absence of

enzymatic catalysis, oxysterols may be formed when cholesterol reacts

with free radical species (i.e. the superoxide/hydrogen peroxide/

hydroxyl radical system) and by non-radical reactive oxygen species

(e.g. singlet oxygen, HOCl or ozone). Oxysterols are known to exert

a multitude of biological effects of potential pathophysiological relevance,

which are dependent on the position of the additional oxygen

function in the cholesterol molecule. These effects are mediated

by direct biophysical effects on membranes and/or stereospecific interactions

with proteins. Oxysterols can be viewed as part of the cellular

machinery that governs the integrity of the cell and function by

acting at the level of signalling, and translational- and post-translational-gene

expression. Key proteins in the control of metabolism

reportedly targeted by oxysterols include liver X receptors (LXRs),

which exert a key role in lipid metabolism, sterol regulating element

binding proteins (SBREPs); oxysterol-binding proteins (OSBP);

estrogen receptor; Nieman Pick disease proteins (NCP1, NCP2);

Hedgehog system; and Epstein-Barr virus induced gene 2 (EBI2).

The pace of discovery in the field of oxysterols is accelerating rapidly

and oxysterols of valuable potential importance, including

3β-hydroxy-5-oxo-5,6-secocholestan-6-al (i.e., 5,6-secosterol) and

its carboxyaldehyde generated in close proximity of activated inflammatory

cells, are emerging as putative effectors and biomarkers of

inflammation-oxidative stress processes.

35


Electrophilic nitro-fatty acids as anti-inflammatory mediators

in the vascular compartment

FRANCISCO J. SCHOPFER

Department of Pharmacology & Chemical Biology, University of Pittsburgh,

Pittsburgh, PA 15261, USA

Nitration of unsaturated fatty acids by oxides of nitrogen yield

electrophilic derivatives that modulate protein function via post-translational

protein modifications. The electrophilicity is conferred by

the conjugated nitroalkene group, making them highly reactive with

thiols and histidines through Michael addition reactions. Nitrated fatty

acids are formed endogenously during ischemia and reperfusion, inflammatory

conditions and gastric acidification from addition reactions

between nitrogen dioxide and conjugated linoleic acid (CLA).

Conjugated linoleic acid is reported to be the primary endogenous

substrate for fatty acid nitration in vitro and in vivo resulting in up to

10 5 greater yields of nitration products when compared to bis-allylic

linoleic acid. In particular, two main isomers are formed by mitochondria,

activated macrophages and gastric acidification that activate

PPARgamma, react with KEAP1 to turn on Nrf2 signaling, activate

heat shock responses and inhibit Nf-kB. Nitroalkene derivatives

of CLA and their metabolites are detected in the plasma and

urine of healthy humans and are increased in tissues undergoing episodes

of ischemia-reperfusion. Dietary CLA and nitrite supplementation

in rodents elevates NO 2 -CLA levels in plasma, urine and tissues

and induces electrophile-regulated HO-1 expression in the colonic

epithelium. These results affirm that metabolic and inflammatory

reactions yield electrophilic signaling mediators that modulate

cellular adaptive responses and lead to tissue protection and resolution

of inflammation.

36


High cholesterol diet-induced impairment of glucose

homeostasis in mice with fatty liver: role of inducible NOS

OREN TIROSH

Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith

Faculty of Agriculture, Food and Environment, The Hebrew University of

Jerusalem, P.O. Box 12, Rehovot 76100, Israel

This study was designed to elucidate the role of inducible nitric

oxide synthase (iNOS) in septic fatty livers with the accentuation on

glucose metabolism. Wild-type (WT) and iNOS-knockout (iNOS-/-

) mice were fed with high-cholesterol diet (HCD) (1%w/w) for 6

weeks. Following diet period, mice were injected with LPS (5 mg/

Kg) to induce endotoxemia. Chronic consumption of HCD led to a

significant increase in hepatic steatosis in WT and iNOS-/- mice.

LPS administration caused marked liver damage which was further

exacerbated in the absence of iNOS. Enhanced liver injury in iNOS-

/- mice was in association with a severe hypoglycemia. In the knockout

group, glycogen contents were significantly higher while Hypoxia-induciable

factor-1 (HIF1) signaling was markedly attenuated

compared to control WT. Conversely, LPS treatment equally suppressed

the expression of key gluconeogenic enzymes. The role of

iNOS and HIF1 in hepatic glucose metabolism was further confirmed

in vitro. Hepatic glucose output was augmented by overexpression

of HIF1 or by using NO-donor in AML-12 hepatocytes. Results also

demonstrated increased oxidative stress and reduced heme oxygenase-1

(HO-1) mRNA in the livers of iNOS-/- mice. Furthermore, the

amounts of plasma tumor necrosis factor-α (TNFα) and intrahepatic

TNFα mRNA were significantly elevated in the absence of iNOS.

Conclusion: these data highlights the essential role of iNOS in the

glycemic response to LPS in fatty livers and further argues for the

beneficial effects of iNOS in these livers.

37


Non-alcoholic fatty liver disease, oxidative stress,

and mitochondria

GAETANO SERVIDDIO

C.U.R.E. Centre for Liver Diseases Research and Treatment, Department of

Medical and Occupational Sciences, University of Foggia, Italy

Non-alcoholic steatohepatitis (NASH) is an increasingly recognized

condition that may progress to end-stage liver disease. NASH

pathogenesis is not well understood: Day and James proposed a double

hit model, where steatosis represents the first hit, making the liver

vulnerable to a second hit, which leads to hepatic inflammation and

fibrosis. Oxidative stress, lipid peroxidation and cytokines are considered

the best candidates for the role of second hit. Several recent

studies have revised this model of pathogenesis assigning to oxidative

stress a central role and limiting steatosis to being epiphenomenon of

the injurious mechanism. Oxidative stress in NASH is closely related

to mitochondrial dysfunction. We have defined that, during

NASH progression, oversupply of free fatty acids induces increase

of mitochondrial H 2 O 2 production that in turn oxidizes mitochondrial

membranes and regulates activity of the Uncoupling Protein 2

(UCP2) and Carnitine Palmitoyl Transferase 1 (CPT1). Mitochondria

play a key role in hepatocytes metabolism, being the site of âoxidation

and oxidative phosphorylation. We and others demonstrated

that mitochondrial abnormalities are closely related to the pathogenesis

of NAFLD, which raises the possibility that NAFLD is a mitochondrial

disease. At present, there is no proven therapy for NASH, and

pharmacotherapy is an area of active research. The introduction of

drugs directly able to reduce oxidative stress, in association with clinical

strategies directed at lowering lipid accumulation, would be important

in the treatment of these disorders. Recent approaches in mitochondrial

drug delivery systems and mitochondria-targeted molecules

would be potentially effective in the targeting of mitochondrial

dysfunction in NASH.

38


Lipid oxidation products: signalling and biomarkers for

inflammatory disease in humans

ANA REIS 1 , NORSYAHIDA MOHD FAUZI 2 , ANDREW R. PITT 1

AND CORINNE M. SPICKETT 1

1 School of Life and Health Sciences, Aston University, Birmingham, B4 7ET,

UK; 2 Strathclyde Institute of Pharmacy and Biomedical Science, University of

Strathclyde, Glasgow, G4 0NR, UK

Phospholipid oxidation occurs in inflammation and generates

a wide range of different products, including intact esterified oxidized

phospholipids, chain shortened phospholipids and non-esterified

alkenal products, as well as chlorinated and nitrated lipids. Biological

effects such as toxicity, cell-cell adhesion, ROS and cytokine

production have been reported for several of these products and suggested

to play important roles in many diseases involving oxidative

stress. We have shown previously that oxidized 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphocholine

oxPAPC, a model of

oxLDL) does not induce classical activation of the pattern recognition

receptors TLR2 and TLR4 leading to NFkB activation, and indeed

can inhibit LPS or bacterial lipoprotein-induced signalling via

these receptors. In contrast, chlorinated lipids do not appear to affect

LPS-induced inflammatory processes, having little effect on cytokine

production in myeloid cells. oxPAPC products such as 1-palmitoyl-

2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), which

contains an aldehyde group, can form adducts with proteins and alter

their biological function. Modern mass spectrometry methods are

facilitating both lipidomic and proteomic analysis of LDL. We have

developed targetted LCMS and LCMS ion scanning methods to facilitate

the analysis of oxidized and chlorinated phospholipids and

detect glycosylation and alkenal adducts on albumin and ApoB-100.

This approach is being applied to study LDL changes in cardiovascular

and related inflammatory diseases. A combination of lipidomics

and proteomics presents a powerful approach to understanding lipid

signalling and identifying biomarkers in inflammatory diseases.

39


Protein oxidation: reducing the toxic effects

TILMAN GRUNE

Department of Nutritional Toxicology, Institute of Nutrition, Friedrich

Schiller University Jena, 07743 Jena, Germany

Proteins which are oxidatively modified are degraded by the 20S

proteasome in an ATP- and ubiquitin-independent pathway. However,

in several studies an enhanced ubiquitination of yet unknown

proteins was reported. By determination of oxidized- and

ubiquitinated proteins we demonstrated that the oxidized proteins are

not preferentially ubiquitinated. Identification of the ubiquitinated

proteins revealed a number of metabolic and regulatory proteins to

be ubiquitinated together with a number of chaperons. This reveals a

close interaction on the function of chaperones and the ubiquitin/

proteasome system.

Interestingly, in the case of strong oxidative stress, if the

proteasomal system is overwhelmed and inhibited an up-regulation

of several heat shock proteins (Hsps), including Hsp27, Hsp70 and

Heme Oxygenase-1 (HO-1) takes place. It was demonstrated that

the induction of classical Hsps, such as Hsp27 and Hsp70, is dependent

on an HDAC6-dependent mechanism. Our data demonstrate

that also HO-1 induction is mediated by HDAC6 via p38MAPK

deacetylation and Nrf-2 activation.

If the proteasomal system is overwhelmed and the chaperones are

not sufficient to maintain the solubility of oxidized proteins, these

proteins aggregate and form lipofuscin. In the current literature, the

lysosomal system is considered to be essential in the intracellular formation

and accumulation of lipofuscin. In contrast to that, our experimental

results demonstrated that both autophagosomes and the

lysosomal system are not mandatory for the formation of lipofuscin.

However, an inhibition of these systems is not leading to a decline in

the lipofuscin formation, but rather in an enhanced toxicity of the

formed protein aggregates.

40


SESSION V

EPIGENETICS, METABOLISM, AND AGING

41


Epigenetic regulation of aging

MARIO F. FRAGA 1,2

1 Department of Immunology and Oncology, National Center for

Biotechnology, CNB-CSIC, Cantoblanco, Madrid E-28049, Spain

2 Cancer Epigenetics Laboratory, Instituto Universitario de Oncología del

Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo,

Oviedo, Spain

Epigenetics of aging is an emerging field that promises exciting

revelations in the near future. Epigenetic mechanisms, including DNA

methylation and histone modification, are determinants of normal

development and can change during aging. Some of the epigenetic

alterations described during aging, as hypermethylation at specific

promoters and decrease of global DNA methylation, are also associated

with tumor development. The epigenetic alterations occurring

during development and aging can be stochastic and depend on genetic

(intrinsic) and environmental (extrinsic) factors. Future challenges

in the field involve the determination of the real role of these

epigenetic alterations in the establishment of aging-associated phenotypes.

42


Epigenetic oxidized redox shift theory of aging (EORS)

in brain

GREGORY J. BREWER 1,2 , M.P. WALKER 2 , K.R. LEVAULT 2 ,

AND DEBOLINA GHOSH 2

1 Departments of Neurology and 2 Medical Microbiology, Immunology and Cell

Biology, Southern Illinois Univ. Sch. Med., Springfield, Illinois,

USA 62794-9626

Our 2010 epigenetic oxidized redox shift theory of aging (EORS)

proposed to revise the ROS theory of aging with an epigenetically

enforced metabolic redox shift that leads to a bioenergetic downward

spiral. Since neuron loss, epigenetic changes, less glutathione

(GSH) and increased ROS are associated with aging and Alzheimer’s

Disease (AD), we wanted determined the relative importance of

each of these to neuron death. In the brains of aging and a AD- model

mice, we found reversible epigenetic histone modifications and Bdnf

expression (Age 2012). We also found an oxidative redox deficit in

aging mice and AD-mouse neurons, as well as lower NADH regenerating

capacity and GSH levels, even before ROS-mediated

macromolecular damage and elevated ROS levels (J. Neurosci. 2012).

Here, our strategy imposed a stress of incremental GSH depletion +/

- incremental neuroprotection. GSH depletion increased neuronal

death of AD-mouse neurons at increasing rates across the age-span,

while non-transgenic neurons were resistant to GSH depletion until

old age. Remarkably, the rate of neuron loss with ROS did not increase

in old age and was the same for both genotypes, which suggests

that cognitive deficits in the AD-model are not caused by ROS.

In the opposite approach of neuroprotection, activation of the redox

sensitive Nrf2-ARE along with the NAD + -precursor, nicotinamide

increased neuron survival, decreased ROS and increased GSH and

NAD(P)H in an additive manner. In summary, the reversible redox

environment of GSH and NAD(P)H is more important to neuron

survival than ROS in aging.

Supported by NIH RO1 AG032431

43


Aging, single-cell methylomes

SILVIA GRAVINA AND JAN VIJG

Department of Genetics Albert Einstein College of Medicine,

1301 Morris Park Ave Bronx, New York 10461

Recent data suggest that the epigenome is highly dynamic and

serves as an interface between the environment and the inherited static

genome. The large volume of epigenomic events and its continuous

need of maintenance, i.e., after DNA repair or replication, suggest a

high chance of errors. The question we wish to address is how unstable

the epigenome really is. Do epimutations accumulate with age

and do they occur in a random fashion, i.e., as ‘epigenomic drift’?

Do they ever reach levels that are high enough to have functional

consequences?

To experimentally determine epigenetic drift we focused on DNA

methylation, a major layer of epigenomic control. To study intraorgan

variation in DNA methylation during aging, it is necessary to

have access to procedures that allow assessing DNA methylation

patterns at the level of single-cells or single-molecules. Such methodology

is currently entirely lacking: to fill this void we have optimized

bisulfite sequencing for single cell analysis.

The procedure developed allows us to analyze DNA methylation

patterns in single cells, within promoter regions of genes or genomewide.

Data on mouse fibroblasts, neuronal nuclei and hepatocytes

will be presented and discussed. We are currently applying the method

to test the hypothesis that random DNA methylation changes accumulate

in the mouse brain during aging, contributing to functional

decline of somatic cells that gives rise to chronic pathology and

aging.

44


Selective autophagy on the cellular energetic balance

ANA MARIA CUERVO

Department of Anatomy and Structural Biology, Albert Einstein College of

Medicine, Bronx, NY

Autophagy mediates the digestion of cytosolic material in

lysosomes. All types of autophagy fulfill two important functions in

mammalian cells, serving both as an alternative source of energy,

when nutrients are scarce, and as an efficient mechanism for the removal

of any intracellular damaged structures. In this talk, I will focus

on the connections between selective autophagy and cellular

metabolism. Prolonged starvation has been classically known as the

best stimuli for chaperone-mediated autophagy (CMA) but the contribution

of this pathway to cellular metabolism has not been studied

in depth. Working with transformed cells in culture, we have found

that blockage of CMA results in a marked decrease in intracellular

ATP content. To our surprise, reduced ATP seems to mainly resulting

from alterations in main intracellular metabolic pathways. We

have found that both glycolysis and beta-oxidation are compromised

upon CMA blockage. I will discuss the molecular components identified

until date to participate in this effect of CMA over these metabolic

pathways. In addition, I will also comment on the reverse situation,

namely the impact that different macromolecules incorporated

with the diet have on CMA. On this respect we have identified a

regulatory effect of nutritional lipids on CMA and have elucidate the

molecular mechanisms behind this effect. The active interplay between

nutrients and CMA could be of particular importance on conditions

as aging and age-related disorders in which CMA activity

has been shown to be compromised.

45


Modulation of aging genes: importance in longevity and ageassociated

frailty

JOSÉ VIÑA, CONSUELO BORRÁS JUAN GAMBINI, KHIRA MOHAMED,

JUAN-ANTONIO AVELLANA, LEOCADIO RODRIGUEZ-MAÑAS,

AND EVA SERNA

Departamento de Fisiología, Facultad de Medicina, Universidad de Valencia;

Servicio de Geriatría. Hospital de la Ribera, Alzira y Red Española de

Envejecimiento y Fragilidad RETICEF

The concept of longevity associated genes received serious support

after a review by Sinclair and Guarente (1). Many genes have

been associated with longevity. In our laboratory, we have studied

some of these. For instance we could attribute the increased longevity

in females to the estrogen- dependent over-expression of Mn-

SOD and Glutathione peroxidase (2). Other genes that we have

shown to be involved in longevity are p53 (3); p16/Arf (4) RAS-Grf

(5) and telomerase (6).

We have recently observed that, in humans, oxidative stress is

associated more with frailty than with age itself. An obvious approach

is to find physiological, nutritional or pharmacological intervention

to promote the activity of longevity associated genes. Exercise is a

paradigmatic intervention to promote such genes (7)

We have recently observed that centenarians, not only reach extreme

longevity but also display remarkable low levels of frailty. Thus

we decided to search for longevity genes that could be over expressed

in centenarians.

We have performed the whole mRNAi-ome and correlated it with

the transcritome in centenarians (100 ± 2), octogenarians (80- 85)

and young persons of the area of Alzira (Valencia, Spain)

The main conclusions are:

1. The Principal Component Analysis (PCA) of the mRNAi-ome of

centenarians is remarkably similar to that of young persons and

different from octogenarians

2. Centenarians up-regulate the expression of mRNAi when compared

with young persons. Octogenarians down regulated it. This

46


shows that transcription in centenarians is much more regulated

than in octogenarians.

3. Comparison of the variations in miRNA-ome and whole

transcriptome of centenarians has allowed us to identity mitochondrial

proteins that are specifically up regulated in centenarians.

References

1 Sinclair DA, Guarente L. Sci Am. 2006 Mar;294(3):48-51, 54-7

2 Borrás C, et al. Free Radic Biol Med. 2003 Mar 1;34(5):546-52

3 Matheu A, et al. Nature. 2007 Jul 19;448(7151):375-9

4 Tomás-Loba A, et al. Cell. 2008 Nov 14;135(4):609-22

5 Matheu A, et al Aging Cell. 2009 Apr;8(2):152-61

6 Borrás C, et al. Aging (Albany NY). 2011 Mar;3(3):262-76

7 Viña J, Gomez-Cabrera MC, Borras C. Br J Nutr. 2007 S36-40

47


Redox regulation of cellular stress response in aging and

neurodegenerative disorders: role of hormesis and vitagenes

VITTORIO CALABRESE 1 , CAROLIN CORNELIUS 1 ,

ANGELA TROVATO SALINARO 1 , AND EDWARD J. CALABRESE 2

1 Clinical Biochemistry, Faculty of Medicine, University of Catania, Italy,

2 Environmental Health Sciences Division, School of Public Health, University

of Massachusetts, Amherst, MA, USA

Protein quality control is a critical feature of intracellular

homeostasis 1 . In addition, modulation of endogenous cellular defense

mechanisms via the stress response signaling represents an innovative

approach to therapeutic intervention in diseases causing chronic

tissue damage, such as neurodegeneration and cancer 1 . Protein thiols

play a key role in redox sensing, and regulation of cellular redox

state is crucial mediator of multiple metabolic, signalling and transcriptional

processes. Under optimal conditions long-term health protection

is accomplished by protein homeostasis, a highly complex

network of molecular interactions that balances protein biosynthesis,

folding, translocation, assembly/disassembly, and clearance 2,3 . Efficient

functioning of maintenance and repair processes is crucial for

both survival and physical quality of life. This is accomplished by a

complex network of the so-called longevity assurance processes,

which are composed of several genes termed vitagenes 4-8 . The term

vitagenes refers to a group of genes which are strictly involved in

preserving cellular homeostasis during stressful conditions. The

vitagene family is actually composed of the heat shock proteins (Hsp)

Hsp32, Hsp70, the thioredoxin system and the sirtuin system 9 . Dietary

antioxidants, such as polyphenols and L-carnitine/acetyl-L-carnitine,

have recently been demonstrated in vitro to be neuroprotective

through the activation of hormetic pathways, including vitagenes 10-

12 . Over the past decade there has been a remarkable increase of interest

in hormesis as a result of more significance being given to low

dose effects and the use of more powerful study designs which have

enabled to identify rational approaches to detect hormetic biphasic

dose responses in the low dose zone. The hormetic dose–response,

challenging long-standing beliefs about the nature of the dose–response

in a lowdose zone, has the potential to affect significantly the

48


design of pre-clinical studies and clinical trials as well as strategies

for optimal patient dosing in the treatment of numerous diseases, including

oxidant disorders. Given the broad cytoprotective properties

of the heat shock response there is now strong interest in discovering

and developing pharmacological agents capable of inducing stress

responses. We have recently focused our research on the role of

acetylcarnitine in the defence mechanisms against cellular stress and

neurodegeneration. In addition, with a redox proteomics approach,

we identified mitochondrial oxidatively modified proteins as a function

of brain aging, specifically in those brain regions, such as cortex

and hippocampus, that are commonly affected by the aging process.

In all brain regions examined, many of the identified proteins were

energy-related, such as pyruvate kinase, ATP synthase, aldolase, creatine

kinase, and a-enolase. These alterations were associated with

increased expression of Hsps, as well as carnosinase and thioredoxin

reductase and with significant changes in both cytosolic and mitochondrial

redox status in all brain regions analyzed. This findings are

relevant to potential pharmacological interventions in healthy medicine

strategy, pointing to maximize cellular stress resistance of the

brain thus providing neuroprotection 9-12 , and will be extended to other

systemic oxidant disorders such as diabetes and cancer.

1. Halliwell B. (2008) Arch Bioch Biophys 476:107.

2. Calabrese V. (2007) Nature Neurosci 8,766.

3. Calabrese V. (2011) Mol Aspects of Med 32:279.

4. Calabrese V. (2011) Chem Biol 18:1355.

5. Calabrese V. (2011) Mol Aspects of Med 32:258.

6. Calabrese V. (2012) BBA 1822:75388.

7. Calabrese V. (2010) Antiox Redox Signal 13:1763.

8. Calabrese V. (2010) Neurochem Res 35:1880.

9. Calabrese E.J. (2012) Biogerontology epub

10. Calabrese E.J. (2010) HET 29:980.

11. Calabrese V. (2012) BBA 1822:729.

12. Mattson M.P. (2010) Neuron 67:900.

49


MitoSENS: allotopic expression of mitochondrial genes using a

co-translational import strategy

MATTHEW S. O’CONNOR

SENS Foundation Research Center, Mountain View, CA 94041

The mitochondrion contains its own genome and encodes 13 proteins

that are essential for electron transport chain function and ATP

synthesis to function properly. Congenital mutations in many of the

mitochondrial genes are the cause of serious disease phenotypes including

diabetes, blindness, dementia, ataxia, epilepsy, and many other

neurological disorders. Somatic mutations also accumulate in the

mitochondria with normal aging. Allotopic expression of mitochondrial

genes in the cell’s nucleus is one approach to rescuing mitochondrial

mutations. In our strategy, we utilize 5’ and 3’ elements to

target the mRNA to the mitochondria; this approach is a variation of

allotopic expression and is hypothesized to result in the co-translational

import of these proteins into the mitochondria. This approach

is hypothesized to overcome the obstacles experienced by many

groups in the import of allotopically expressed proteins into the mitochondria,

such as clogging of the mitochondrial import machinery

by hydrophobic proteins. Thus far, we have stably transfected 5 of

the 13 mitochondrial genes into the nuclear genome of human cell

lines and are characterizing the expression and function of these

exogenously expressed genes. We will discuss current progress and

future plans for replacing and/or making redundant the entire mitochondrial

genome. We will also discuss potential applications of the

MitoSENS approach in treating mitochondrial diseases, as well as

the diseases and pathologies of aging.

50


Ageing - from molecules to hormesis

SURESH RATTAN

Laboratory of Cellular Ageing, Department of Molecular Biology and

Genetics, Aarhus University, Denmark

Ageing can be understood at various levels, from evolutionary

and biological levels to psychological and sociological levels. At the

molecular biological level ageing is characterized by the stochastic

occurrence and progressive accumulation of molecular damage. Failure

of homeodynamics, increased molecular heterogeneity, altered

cellular functioning and reduced stress tolerance are the determinants

of health status, probability of diseases and the duration of survival.

The inefficiency and imperfection of the maintenance and repair systems

underlie the biological basis of ageing. Two major issues yet to

be resolved in biogerontology are determining the physiological relevance

of various types of molecular damage, and establishing the

nature of healthy and unhealthy gene-protein networks. Gene therapy,

stem cells, and modulation through functional foods, nutriceuticals,

cosmeceuticals and other life style alterations are examples of ageing

interventions. A promising healthy-ageing approach is that of hormesis

by strengthening the homeodynamic ability of self-maintenance

through transient and repetitive mild stress-inducing hormetins.

Achieving the goal of extended health-span will depend on elucidating

and exploiting successful and dynamic interactions among biological,

psycho-social and environmental factors.

51


Proteolytic signaling dysfunction in aging

KELVIN J.A. DAVIES

Ethel Percy Andrus Gerontology Center of the Davis School of Gerontology,

and Division of Molecular & Computational Biology, Department of

Biological Sciences of the Dornsife College of Letters, Arts & Sciences,

University of Southern California, Los Angeles, California 90089-0191, USA

Proteins are major targets for oxidative damage, and both the intracellular

and extracellular accumulation of oxidized proteins has

been reported in many ageing and disease models. In young and

healthy individuals, moderately oxidized soluble cell proteins are

selectively and rapidly degraded by the Proteasome in the cell cytoplasm,

nucleus, and endoplasmic reticulum. Severely oxidized, aggregated,

and cross-linked proteins, however, are poor substrates for

degradation and actually inhibit the Proteasome. Some mildly aggregated

and cross-linked proteins are degraded by autophagy, but more

severely aggregated and cross-linked proteins seem to accumulate as

inclusion bodies or lipofuscin inside lysosomes. It is, therefore, vitally

important that cells rapidly and selectively degrade mildly oxidized

proteins, before they undergo more severe oxidation, aggregation,

and cross-linking. Young mammalian cells can readily adapt to

mild oxidative stress such that they become (temporarily) much more

resistant to oxidative damage. Such adaptive responses include the

immediate disassembly of the 26S proteasome (catalyzed by HSP70)

to form free 20S proteasome and 19S regulator complexes, at which

point and ATP/Ubiquitin-dependent proteolysis is temporarily lost.

The additional free 20S Proteasomes are of immediate help in degrading

oxidized proteins. Next, over a 3-20 hour period, new 20S

Proteasomes, Immunoproteasomes, and both 11S (PA28) and PA200

proteasome regulators are synthesized, partially under the control of

the Nrf2 signal transduction pathway. We have now obtained similar

findings in C. elegans worms, and D. melanogaster fruit flies. The

original 26S Proteasomes are re-assembled, and ATP/Ubiquitin-dependent

proteolysis is restored. Recent work suggests that certain

proteasome subunits and regulators should be classified as true stress

proteins. Induction of Proteasome expression provides significantly

greater capacity to remove oxidized proteins, and such adaptive re-

52


sponses contribute to overall stress resistance. In older cells, however,

Proteasome activities decline, and adaptational responses mediated

by Nrf2 become sluggish or even ineffectual. In some cases,

the antagonistic effects of Nrf1 may actually blunt adaptive proteolytic

responses to stress in older cells and organisms. Studies in animals

and humans suggest that declining Proteasome activities and,

perhaps, declining responsiveness to stress, may contribute to the

ageing process, and to various age-associated diseases.

53


Reactive oxygen species in muscle wasting during aging

MALCOLM J. JACKSON, GIORGOS SAKELLARIOU, TIM PEARSON,

APHRODITE VASILAKI, ANNA KAYANI, SIOBHAN SCULLION,

AND ANNE MCARDLE

Institute of Ageing and Chronic Disease, University of Liverpool,

Liverpool L693GA, UK

Chronic loss of skeletal muscle mass and function is a major contributor

to frailty and weakness in the elderly. The major cause of

age-related loss of muscle mass is a decrease in the number of skeletal

muscle fibres associated with atrophy and weakness of the remaining

fibres. A large number of studies have reported a

dysregulation of reactive oxygen species (ROS) homeostasis during

ageing that may potentially lead to increased oxidative damage to

tissues and/or to defective redox signaling. Skeletal muscle from aged

mammals contains increased amounts of oxidative damage, but

whether this is the cause of age-related deficits in muscle or a consequence

of ageing has been the subject of controversy. Studies from

our group have examined the changes in ROS generation that occur

with ageing in man and experimental models and have also utilised a

transgenic approach to modify ROS generation in model organisms.

Data from these experiments support the hypothesis that aberrant ROS

regulation plays a role in the decline in skeletal muscle and in motor

neurons that occur during ageing. The key processes involved do not

appear to involve merely oxidative damage to tissues, but are associated

with defects in important redox-sensitive adaptive responses to

contractile activity.

This research is supported by the Medical Research Council, Biotechnology

and Biological Sciences Research Council and United States National

Institute on Aging.

54


Testosterone, estrogens and inflammation: a clue for the

understanding of frailty in older people?

LEOCADIO RODRIGUEZ-MAÑAS 1,2 , LAURE CARCAILLON 2 , CARMEN

BLANCO 3 , JESUS F. TRESGUERRES 4 , GONZALO GUTIÉRREZ-AVILA 5 ,

AND FRANCISCO JOSE GARCIA-GARCIA 6

1 Department of Geriatrics, Hospital Universitario de Getafe, Madrid, Spain;

2 Fundación para la Investigación Biomédica, Hospital Universitario de

Getafe, Madrid, Spain; 3 Department of Clinical Analysis, Hospital

Universitario de Getafe, Madrid, Spain; 4 Department of Physiology, School of

Medicine, Universidad Complutense de Madrid, Spain; 5 Department of

Epidemiology and Public Health, Health Department of Castilla-La Mancha,

Toledo, Spain; 6 Department of Geriatrics, Hospital Virgen del Valle,

Complejo Hospitalario de Toledo, Toledo, Spain

Age-associated changes in sexual hormones represent one of the

potential mechanisms leading to frailty. We studied the association

between testosterone, estrogen and frailty and assessed sex-differences

in the relationship between hormones and frailty.

We used cross-sectional data from the Toledo Study for Healthy

Aging, a population based cohort study of Spanish elderly. Frailty

was defined according to Fried’s approach. Multivariate odds-ratios

(OR) and 95% confidence intervals (CI) associated with total testosterone

(TT), free testosterone (FT) and estradiol (E2) levels were

estimated using polytomous logistic regression.

In women, there was a U-shaped relationship between FT levels

and frailty (p for FT²=0.03), confined to obese women (p-value for

interaction=0.05). In men, the risk of frailty linearly decreased with

testosterone levels: adjusted OR =2.9 (95%CI, 1.6-5.1) and 1.6

(95%CI, 1.0-2.5), for 1 SD decrease in TT and FT, respectively. No

interaction was found with BMI.

Higher E2 levels were associated with frailty among women

younger than 79 years old but not in the oldest group (p interaction=0.047).

After adjustment, OR of frailty associated with 1 standard

deviation increase of E2 was 1.51 (1.04-2.20), p=0.03. We identified

an interaction between E2 and hs-CRP on the prevalence of

frailty (p-value=0.042). Women having both higher E2 and hs-CRP

had an age-adjusted OR of 4.2 (1.7-10.5), p=0.002, compared with

women with low levels of both E2 and hs-CRP.

There is a relationship between sexual hormones and frailty both

in older men and women. However its relevance and its interactions

with other mechanisms differ by gender.

55


Oxidative stress and the link of pathophysiology of Alzheimer’s

disease with vascular pathology

M. CRISTINA POLIDORI 1 , LUDGER PIENTKA 1 AND PATRIZIA MECOCCI 2

1 Department of Geriatrics, Marienhospital Herne, Ruhr University Bochum,

Germany, 2 Department of Gerontology and Geriatrics, Perugia University

Hospital, Perugia, Italy

Vascular risk factors (VRF) like hypertension, diabetes and atherosclerosis

linked to dementias and Alzheimer’s disease (AD) are known

to cause ischemia- and inflammation-related oxidative stress as well

as subsequent reperfusion-related free radical production. Oxidative

stress is known to occur early in the onset of cognitive impairment,

but the mutual relationships between oxidative stress, vascular

comorbidities and cognitive impairment progression are poorly understood.

The longitudinal evaluation of elderly patients with mild

cognitive impairment and AD with or without vascular comorbidities

(groups MCI plus and AD plus, respectively) shows that AD and

control subjects have significantly higher plasma levels of carotenoids,

tocopherols and retinol than MCI Plus and AD Plus patients. The

results are independent of age, gender, BMI as well as intake of fruits

and vegetables. In a subset analysis of MCI, AD, MCI Plus and AD

Plus subjects, baseline plasma levels of F2 isoprostanes prove to be

in average 20% higher in MCI Plus and AD Plus patients whose

neuropsychological scores worsened after two years than F2

isoprostane plasma levels in MCI and AD patients neuropsychologically

stable at follow up. Vascular comorbidities and risk factors

as well as biomarkers of oxidative damage should be early identified

in the elderly and always evaluated in aged patients with cognitive

impairment to allow primary and secondary lifestyle-based preventive

strategies.

56


57

POSTERS


Natalizmab caused changes in Nrf2 in patient

with multiple sclerosis

EDUARDO AGÜERA 1 , FERNANDO SÁNCHEZ-LÓPEZ 1 , ANA I. GIRALDO 2 ,

ALEYDA PÉREZ-HERRERA 3 , AND ISAAC TÚNEZ 2

1 Servicio de Neurología, Instituto Maimónides de Investigación Biomédica de

Córdoba IMIBIC/Hospital Universitario Reina Sofía, Córdoba, Spain;

2 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina /

IMIBIC/Universidad de Córdoba, Spain, 3 Lipids and Atherosclerosis Research

Unit, Reina Sofía University Hospital, University of Cordoba IMIBIC/Reina

Sofía University Hospital University of Cordoba, Spain and CIBER

Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud

Carlos III, Spain

Multiple sclerosis (MS) is an inflammatory demyelinating disease

disease of central nervous system (CNS). A recent study of our group

has shown that MS presents a peripheral oxidative stress.

Immunomodulatory agents have provided new therapeutic options.

Natalizumab (Tysabri, Biogen Idec, Inc. and Elan Pharmaceuticals,

Inc.; a monoclonal antibody directed against alpha-4-beta-1-integrin)

is the selective adhesion inhibitor approved for treatment of relapsing-remitting-MS

(RR-MS). Thus, the present work evaluates the

effects of natalizumab on Nrf2 concentrations and oxidative damage

levels in patients with RR-MS. Twenty-two patients with RR-MS

treated with natalizumab were included in this work. Patients fulfilled

the revised McDonald criteria and were scheduled to star treatment

with 300 mg natalizumab intravenously once monthly (infusion

every 4 weeks), for 14 months, according to Spanish guidelines.

Carbonylated proteins, 8-hydroxy-2’-deoxyguanosine

(8OHdG) and Nrf2 levels were measured at baseline and following

treatment. Treatment with natalizumab caused a significant effect on

Nrf2 expression and translocation, increasing in nuclei and cytoplasms

in the white blood cells (WBC) of patients with RR-MS. This phenomenon

was associated with a reduction in oxidative damage. This

data suggest that natalizumab the cytoplasm and nucleus levels of

WBC in patients with RR-MS.

58


The role of iNOS in colon tumor viability

GRACIELE ALMEIDA DE OLIVEIRA, TATIANA ALVAREZ RINALDI,

AND HUGO PEQUENO MONTEIRO

Laboratório de Sinalização Celular, Centro de Terapia Celular e Molecular,

Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil

The regulation of cell proliferation and longevity through cell

signaling pathway is important to cellular homeostasis. Aberrations

in this regulation are associated with tumor development. Nitric Oxide

(NO) is a molecule permeable to cell lipid layer working as a

messenger in intercellular communication and intracellular signaling.

In mammalian, NO synthase enzymes (NOS) are responsible by NO

biosynthesis. Three NOS isoform are well known: neuronal (nNOS),

endothelial (eNOS) and inducible NOS (iNOS).

Caloric restriction has been widely employed as a protocol to increases

cell viability and longevity (1) and nutrients decrease without

mal-nutrition were showed to promote eNOS expression (2,3).

The aim of this study is to investigate the regulation of iNOS expression

in metastatic human colon cancer cell line SW 620 maintained

in restricted levels of nutrients. We will also investigate NO production,

cell proliferation, and viability.

We observed that cells cultured in absence or low concentrations

of fetal bovine serum (FBS) exhibit increased iNOS protein expression

and NO production. Cell proliferation decreases as compared to

cells growing in the presence of 10% FBS. Furthermore, these cells

are more viable. To test the importance of iNOS in this process we

silence the expression of iNOS through permanent transfection of

interference RNA. As opposed to parental SW 620 cells, iNOS silenced

cells have no difference in NO generation and viability when

growing in absence or presence of FBS (10%). Altogether, these results

suggest that NO and iNOS play an important role in metastatic

tumor cell culture viability.

1. Barros, M.H., da Cunha, F.M., Oliveira, G.A., Tahara, E.B., Kowaltowski, A.J.

(2010). Mech Ageing Dev. 131:494-502.

2. Cerqueira, F.M. Laurindo, F.R., Kowaltowski, A.J. (2011) PLoS One. 6:18433.

3. Nisoli, E. et al. (2005) Science. 310:314-7.

59


Anthocyanin metabolites improve human adipocyte insulin

sensitivity by activating Nrf2/PPARgamma signalling pathway

GIADA AMBROSINI, MASSIMO D’ARCHIVIO, BEATRICE SCAZZOCCHIO,

ROSARIA VARÌ, CARMELINA FILESI, CARMELA SANTANGELO,

CLAUDIO GIOVANNINI, AND ROBERTA MASELLA

Department of Veterinary Public Health and Food Safety, Istituto Superiore

di Sanità, Roma, Italy

Anthocyanins naturally occurring in vegetables, fruits and beverages

appear to exert beneficial effects in preventing oxidative stressassociated

diseases, such as type 2 diabetes and insulin resistance,

likely because of their anti-oxidant/anti-inflammatory properties. In

particular, anthocyanins have been demonstrated to ameliorate

hyperglycemia and insulin sensitivity. We have recently demonstrated

that the main anthocyanin metabolite, the protocatechuic acid (PCA),

exerts insulin-like activities in human visceral adipocytes by increasing

PPARγ expression and activation, as well as adiponectin secretion.

Aim of the study was to investigate the molecular mechanisms

responsible for such activation. In particular we evaluated the involvement

of the transcription factor Nrf2. Human adipocytes were

isolated from visceral adipose tissue biopsies. In cells treated with

25μM PCA, the nuclear translocation of Nrf2, and adiponectin and

PPARγ activation were evaluated by immunoblotting. To define the

role of Nrf2 in modulating PCA effects, small-interfering RNA

(siRNA) technique was used to inhibit transcription factor activity.

In a set of experiments aimed at defining the specific signalling kinase

involved in Nrf2 activation, the cells were treated with specific

kinase inhibitors before PCA addition. We found an increase in Nrf2

nuclear level (p


LY294002 (PI3K inhibitor) and SP600125 (JNK inhibitor) significantly

attenuated the PCA-induced Nrf2 increase strongly suggesting

the involvement of JNK and PI3K. This study was directed at

investigating the molecular events elicited by PCA in human visceral

adipocytes responsible for the up-regulation of PPARg, which, eventually,

determines the activation of glucose uptake machinery. The

results raise a novel biologic role for Nrf2 that may be an interesting

new target for exploring pharmacologic interventions directed at prevention

and treatment of obesity and related clinical disorders.

61


Protocatechuic acid reverts oxLDL-induced p53-dependent

apoptosis in macrophages by activating JNK/Nrf2 signaling

pathway

GIADA AMBROSINI, ROSARIA VARÌ, CARMELA SANTANGELO,

BEATRICE SCAZZOCCHIO, CARMELINA FILESI, MASSIMO D’ARCHIVIO,

ROBERTA MASELLA, AND CLAUDIO GIOVANNINI

Dept. Veterinary Public Health and Food Safety, Istituto Superiore di Sanità,

Viale Regina Elena 299, 00161, Roma

The atherogenic properties of oxLDL depend on the cytotoxic

effects exerted on the arterial wall cells. Apoptosis has been demonstrated

to be the main mechanism of cell death in atherosclerotic

plaques promoting core expansion and plaque instability. In advanced

lesions most of the apoptotic cells are macrophages. We have recently

demonstrated that oxLDL induce p53-dependent apoptosis

by activating p38MAPK and PKCd signaling pathways in the macrophage

cell line J774.A1. The phenolic protocatechuic acid (PCA),

despite the low concentration in food, deserves great nutritional interest

because it is one of the main metabolites of complex polyphenols

such as anthocyanins present at high concentrations in vegetables

and fruit and whose daily intake has been estimated to be much higher

than that of other polyphenols. Aim of the present study was to define

the protective anti-apoptotic effect of PCA on oxLDL-induced

macrophage death and to understand the fine mechanisms of action.

J774.A1 macrophages treated with 0.1 mg/ml oxLDL were assessed

for: i) apoptosis (annexin V positive cells); ii) oxidative stress (ROS

production and GSH content), iii) active-phosphorilated p38MAPK

and PKCd expressions, iv) p53 activation, and v) its target gene

(i.e.p66shc and BAX proteins) expressions. To define the effects of

PCA, the cells were treated with 0.25 mM PCA for 2 h before oxLDL

exposure. In a set of experiments aimed at defining the PCA induced

signaling pathways we evaluated the involvement of Nrf2, and the

role played by JNK. First of all, we showed that PCA counteracted

the induction of apoptosis mediated by oxLDL. In particular, PCA

inhibited the onset of oxidative stress by preventing GSH depletion,

p38MAPK, PKCd, and p53 activation, and p66shc and BAX overexpressions.

Furthermore, we found that after phenolic treatment the

62


Nrf2 nuclear level increased (p


Sulforaphane counteracts glycative stress in cardiomyocytes

through glyoxalase 1 induction

CRISTINA ANGELONI, DANIELE FABBRI, AND SILVANA HRELIA

Department of Biochemistry “G. Moruzzi”, University of Bologna,

Bologna, Italy

Methylglyoxal is a reactive dicarbonyl precursor of advanced

glycation end products (AGEs). AGEs play an important role in the

development and progression of cardiovascular disease in diabetes.

Serum levels of AGEs in patients with type 2 diabetes with coronary

heart disease (CHD) are increased compared to patients without CHD,

and correlate with CHD severity. Methylglyoxal is effectively detoxified

by the glyoxalase system, that comprises glyoxalase1 and 2.

Overexpression of glyoxalase1 prevents the accumulation of intracellular

methylglyoxal and AGEs in endothelial cells exposed to high

glucose concentrations. Recently it has been demonstrated that glyoxalase1

expression is regulated by the nuclear factor E2-related factor

2 (Nrf2). Sulforaphane is an isothiocianate formed from

glucoraphanin present in Cruciferous vegetables. As SF is a potent

inducer of Nrf2-modulated phase II enzymes, we investigated its protective

effect against methylglyoxal-induced damage in primary cultures

of rat cardiomyocytes. Cell viability was evaluated by MTT

assay, caspase3 activity by a fluorimetric method, caspase3 and glyoxalase1

expression by immunoblotting, glyoxalase1 activity spectrophotometrically

and ROS production by the 2',7'dichlorofluorescein-diacetate

assay. We observed that methylglyoxal

reduced cardiomyocytes viability, induced apoptotic cell death and

increased ROS production. Sulforaphane treatment significantly increased

cell viability, and reduced apoptotic cell death and ROS production

of methylglyoxal-damaged cardiomyocytes. Moreover, for

the first time, we demonstrated that sulforaphane is able to significantly

increase glyoxalase1 protein expression and activity. These

findings suggest that targeting the glyoxalase system might be a promising

therapeutic strategy to counteract cardiovascular diseases of

diabetic patients.

Supported by Fondazione del Monte di Bologna e Ravenna

64


Postconditioning (PostC) cardioprotection by chromogranin A

(CgA)-derived catestatin (CTS) in isolated rat hearts is

mediated by RISK (Reperfusion Injury Salvage Kinases)

pathway and redox-mechanisms

CARMELINA ANGOTTI 1 , CLAUDIA PENNA 1 , TOMMASO ANGELONE 2 ,

MARIA-GIULIA PERRELLI 1 , AND PASQUALE PAGLIARO 1

1 Department of Clinical and Biological Sciences, University of Turin Italy;

2 Department of Pharmaco-biology, University of Calabria, Italy

Background: CST exerts anti-hypertensive, vasodilator and cardiac

inotropic effects acting via nitric-oxide signaling. We tested

whether CST cardioprotection activates RISK pathway and a reactive

oxygen species (ROS) signaling.

Methods: Isolated rat hearts underwent 30-min ischemia and 120min

reperfusion (I/R) without or with CST (75 nM, CST-Post) given

for 20-min in early reperfusion. To evaluate the role of PKC and

mitochondrial K ATP (mitoK ATP ) channels, hearts were co-infused with

CST, and either the PKC inhibitor (chelerythrine) or the mitoK ATP

channel blocker (5-hydroxy-decanoate). We tested the phosphorylation

(PP) of RISK elements (Akt, PKCe and GSK3â) and the levels

of anti-apoptotic marker (Bcl-2). Coronary pressure and left ventricular

pressure (LVP) were monitored. Infarct size (IS) was evaluated

with NBT-staining.

Results: IS was 67±6% of risk area in hearts subjected to I/R

only. CST-Post reduced IS to 37±4%, and improved postischemic

contracture and recovery of developed LVP. CST-Post induced PP

of all tested RISK elements and preserved Bcl-2 levels. The PKC

inhibitor chelerythrine abolished CST protection and allowed a partial

PP of Akt only. Blockade of mitoK ATP channels abolished the IS

limitation and GSK3β-PP, but allowed contracture limitation and

partial PP of Akt and PKCe. Also 2-mercaptopropionylglycine

(MPG) abolished the infarct-sparing effect of CST.

Conclusions: Results suggest that CST-Post cardioprotection

against IS and postischemic contractile dysfunction depends on Akt

and PKCe PP/activation and on PP/inhibition of GSK3β via a redox-sensible

mechanism. We propose a reverberant circuit from Akt

to PKCe and to mitoK ATP channels and again to PKCε and to GSK3β

via the ROS signaling.

65


Glutathione depletion induces antioxidant response via the

p53-mediated transcription of PGC-1a: a novel longevity

pathway?

KATIA AQUILANO, SARA BALDELLI, BEATRICE PAGLIEI,

AND MARIA ROSA CIRIOLO

Dept. Biology, University of Rome “Tor Vergata”, via della Ricerca

Scientifica 1, 00133 Rome, Italy and IRCCS San Raffaele “La Pisana”, Via di

Val Cannuta 247, 00166, Rome, Italy

The transcriptional coactivator peroxisome proliferator-activated

receptor gamma coactivator 1 a (PPARGC1A or PGC-1α) is a powerful

regulator of cell metabolism. By inducing mitochondrial biogenesis

and antioxidant enzymes, PGC-1α assures the balance between

production and scavenging of pro-oxidant molecules, and may

be considered a key factor in counteracting the oxidative stress that

lies behind several human pathologies. However, the regulation of

PGC-1α transcription and the upstream signalling events remain to

be clarified. Here we demonstrate that upon glutathione (GSH) depletion

p53 binds to the unique predicted region on human

PPARGC1A promoter (-1237), increasing PGC-1α expression. p53mediated

PGC-1 induction was decreased by inhibiting nitric oxide

(NO) production or guanylate cyclase, implicating NO/cGMP signalling

in this process. PGC-1α in turn co-activates Nrf2 inducing

manganese superoxide dismutase (SOD2) and g-glutamylcysteine

ligase. Accordingly, in mice we found that GSH deficiency leads to

NO-mediated p53, PGC-1α and SOD2 accumulation. These results

suggest that PGC-1α up-regulation mediated by p53 could represent

an adaptive response to limit oxidative damage elicited by GSH deficiency.

Furthermore the evidence provided indicate that the modulation

of the p53/PGC-1a pathway could become the cornerstone for

new therapeutic approaches to combat ageing and human pathologies

associated with oxidative stress.

66


Induction of oxidative stress, apoptosis and inflammation in

rat brain regions by D-serine

GULIZ ARMAGAN 1 , LUTFIYE KANIT 2 , AND AYFER YALCIN 3

1 Department of Biochemistry, Faculty of Pharmacy, Ege University, 35100

Bornova, Izmir, TURKEY; 2 Center for Brain Research, Ege University, 35100

Bornova, Izmir, TURKEY and Department of Physiology, Faculty of Medicine,

Ege University, 35100 Bornova, Izmir, TURKEY and Department of

Neurosciences, Institute of Health Sciences, Ege University, 35100 Bornova,

Izmir, TURKEY; 3 Department of Biochemistry, Faculty of Pharmacy, Ege

University, 35100 Bornova, Izmir, TURKEY and Department of Neurosciences,

Institute of Health Sciences, Ege University, 35100 Bornova, Izmir, TURKEY

D-serine is synthesized in the brain by serine racemase from Lserine.

It presents at very high levels in the mammalian brain particularly

in the forebrain area, including cerebral cortex, hippocampus,

striatum and limbic forebrain. This amino acid is recognized as one

of the physiological ligands of the N-methyl-D-aspartate (NMDA)

receptor co-agonist site. NMDA receptors play important roles in

brain function, brain development, learning, memory and neurotoxicity.

Altered neurotoxic levels of D-serine in the hippocampus of

Alzheimer disease’s patients indicates that D-serine might participate

in the pathogenesis of Alzheimer disease, excitotoxicity and

neuroinflammation. The present study, focused on the hippocampus,

cortex, and cerebellum, was designed to investigate the changes in

possible mechanisms; oxidative stress (reactive oxygen species (ROS)

production, lipid peroxidation and protein carbonyl content), inflammation

(tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and

apoptosis (Bax, Bcl-2) after D-serine administration. It is found that

D-serine significantly increased oxidative stress, levels of inflammation-

and apoptosis-related molecules in a region specific manner.

Therefore, it can be suggested that the controlling brain D-serine levels

may constitute a novel strategy for neuroprotection against free

radical-induced oxidative damage.

67


Resveratrol attenuates oxidative stress in neuronal

and glial restricted precursor cells

GULIZ ARMAGAN 1 , SIBEL KONYALIOGLU 1 , TANER DAGCI 2 ,

AND AYFER YALCIN 3

1 Department of Biochemistry, Faculty of Pharmacy, Ege University, 35100

Bornova, Izmir, TURKEY; 2 Center for Brain Research, Ege University, 35100

Bornova, Izmir, TURKEY and Department of Physiology, Faculty of Medicine,

Ege University, 35100 Bornova, Izmir, TURKEY; 3 Department of Biochemistry,

Faculty of Pharmacy, Ege University, 35100 Bornova, Izmir, TURKEY and

Department of Neurosciences, Institute of Health Sciences, Ege University,

35100 Bornova, Izmir, TURKEY

Food-derived antioxidants have received growing attention as

chemopreventive agents against oxidative damage. Resveratrol is a

polyphenolic compound with strong antioxidant properties that is

found abundantly in grapes, berries, nuts and red wine. Neural stem

cells that exist in various regions of the CNS throughout the mammalian

lifespan, can be expanded and induced to differentiate into

neurons and glia in vitro and in vivo. ENSC-based approaches have

received much attention due to the potential to develop treatments

for neurodegenerative disorders. In this study, we aimed to investigate

the effects of resveratrol treatment on the levels of nuclear DNA

(nDNA) and mitochondrial DNA (mtDNA) damage, the activities of

antioxidant enzymes [catalase, superoxide dismutase (SOD), glutathione

peroxidase (GPx)], the levels of nitric oxide (NO) and nitric

oxide synthase (NOS) activity in neuronal and glial restricted precursor

cells against H2O2-induced oxidative stress. H2O2 treatment

alone increased catalase and GPx activities but did not change SOD

levels when compared to H2O2+ resveratrol group. NOS activity

and concomitant NO levels were increased in ENSCs following H2O2

treatment. Conversely, resveratrol treatment against H2O2 decreased

NOS activity and NO levels. Resveratrol also attenuated H2O2-induced

n-or mtDNA damage. It is concluded that resveratrol may be a

promising agent protecting neuronal and glial restricted precursor cells

due to its potential to decrease oxidative stress through the increased

activity of antioxidant enzymes, decreased NO production and NOS

activity, and alleviated both nuclear and mtDNA damage.

68


DHA restores oxidative damage in RPE at high glucose levels

EMMA ARNAL, SIV JOHNSEN-SORIANO, AND FRANCISCO JAVIER ROMERO

FOM, Valencia, Spain

Purpose: Diabetic retinopathy is a leading cause of visual loss

and have been related to oxidative stress. The aim of the present

study was to evaluate protective properties of Docosahexaenoic acid

(DHA) at retinal pigment epithelial (RPE) cells exposed to high glucose

levels.

Methods: Human RPE cells (ARPE-19) were cultured 4 days

with normal blood glucose concentration, followed by ten days exposure

to either normal or high D-glucose concentration. DHA was

administered according to the following groups: control group (C),

control+DHA group (CDHA), glucose group (G) and glucose+DHA

group (GDHA). At the end of the experiment cells were washed,

sonicated and lysed and the homogenates cryopreserved until assayed.

Results: Antioxidant Capacity, (AC), and glutathione concentration

(GSH) in G group decreased significantly when compared to C

group; DHA administration normalized these alterations. Moreover,

the total nitrites (TN) and malondialdehyde (MDA) concentrations

were significantly elevated in G group when compared to the C

group, and DHA administration again normalized these alterations

to control values. In addition Pigment Epithelial Derived Factor

(PEDF) immunohistochemical study was performed. PEDF-positive

cell number in GDHA group increased significantly (48.54 ± 12.53

PEDF-positive cells/total cells) when compared to G group (28.71 ±

7.73 PEDF-positive cells/total cells; p< 0,05).

Conclusion: These data further support previous findings that suggest

that DHA might be useful in protecting diabetic retina.

69


Oxidative stress: recipe for age-related hypertension

MOHAMMAD ASGHAR, GAURAV CHUGH, AND MUSTAFA LOKHANDWALA

Heart and Kidney Institute, College of Pharmacy, University of Houston, USA

Introduction: The aging process causes structural and functional

changes in body’s organs, including in the kidney, resulting in different

pathological outcomes, namely hypertension. The hall-mark of

aging process is increase in oxidative stress. We tested the hypothesis

that age-associated oxidative stress is causal to alterations in kidney

dopamine D1 and angiotensin II AT1 receptors functions and

hypertension in aging. Both dopamine (a natriuretic factor), via D1

receptor (D1R), and angiotensin II (an anti-natriuretic factor), via

AT1 receptor (AT1R), by maintaining sodium homeostasis regulate

blood pressure (BP).

Methods: We determined BP and D1R and AT1R functions in

two rat aging models, Fischer Brown Norway (FBN) and Fischer

344 (F344) rats. D1R and AT1R functions were determined by measuring

natriuresis and diuresis in responses to D1R agonist, SKF 38393,

and AT1R antagonist, candesartan. The role of oxidative stress was

determined by treating the rats with antioxidant tempol.

Results: BP was higher only in aged FBN rats, which decreased

with tempol. Systemic and kidney levels of oxidative stress markers

were elevated in both aged FBN and F344 rats, which decreased

with tempol in these rats. Natriuretic and diuretic responses to

SKF38393 were diminished in both aged FBN and F344 rats,

whereas those to candesartan were higher only in aged FBN rats.

Tempol treatment restored natriuretic and diuretic responses to

SKF38393 and candesartan.

Conclusion: Age-associated oxidative stress is causal to decreased

D1R and higher AT1R functions and hypertension in aging. Aberrant

functioning of both these receptors is required for hypertensive

phenotype in aging.

Financial support: NIH/NIA grants (AG039836, AG029904, AG025056)

70


TGRL lipolysis products induce lipid droplet accumulation

and mitochondrial reactive oxygen species production in

endothelial cells

HNIN H. AUNG 1 , MICHAEL W. LAMÉ 2 , JITKA PETRLOVA 3 , TUN NYUNT 4 ,

DENNIS W. WILSON 5 , AND JOHN C. RUTLEDGE 1

1 Department of Internal Medicine, School of Medicine, University of

California, Davis, CA 95616; 2 Department of Molecular Biosciences, School

of Veterinary Medicine, University of California, Davis, CA 95616;

3 Biochemistry & Molecular Medicine School of Medicine University,

University of California, Davis, CA 95616; 4 NSF Center for Biophotonics

Science and Technology, University of California, Davis, Sacramento, CA

95817; 5 Department Pathology, Microbiology and Immunology, School of

Veterinary Medicine, University of California, Davis, CA 95616

Elevation of triglyceride-rich lipoproteins (TGRL) is a known

cardiovascular risk factor. Increased production of reactive oxygen

species in mitochondria, accumulation of mitochondrial DNA damage,

and progressive respiratory chain dysfunction are associated with

atherosclerosis in human investigations and animal models of oxidative

stress. Our previous studies indicate that TGRL lipolysis products

injure human aortic endothelial cells and induce activating transcription

factor 3 (ATF3) through a JNK signaling pathway.

In the present study, to determine the role of TGRL lipolysis products

in the development of mitochondrial dysfunction due to oxidative

stress and contribute to pathogenesis of atherosclerosis by inducing

the expression of multiple pro-inflammatory cytokines. Human Aortic

Endothelial Cells (HAEC) were incubated with media, lipoprotein

lipase (LpL), TGRL, or TGRL lipolysis products.

Our data indicated that TGRL lipolysis products produced

superoxide (O2 -. ) in HAEC and the production of reactive oxygen

species (ROS) is localized to mitochondrial. Mito-TEMPO suppressed

lipolysis product-induced mitochondria ROS production. Additionally,

TGRL lipolysis products induced expression of genes important

in oxidative stress, and endothelial inflammation. We also found

that TGRL lipolysis products induced lipid droplet formation in

HAEC compared to Media, LpL or TGRL alone treatment. These

results support the involvement of TGRL lipolysis products in endothelial

dysfunction by increasing mitochondria-dependent oxidative

stress and induction of pro-inflammatory genes.

71


Effects of 7-ketocholesterol and 27-hydroxycholesterol on

cellular and mitochondrial respiration in HepG2 cells

FRANCESCO BELLANTI, MARIA BLONDA, ANTONINO DAVIDE ROMANO,

GIUSEPPINA IANNELLI, TIZIANA ROLLO, ROSANNA TAMBORRA,

AND GAETANO SERVIDDIO

C.U.R.E. Centre for Liver Diseases Research and Treatment, Department of

Medical and Occupational Sciences, University of Foggia, Italy

Oxidative stress is a common feature of liver diseases. Cholesterol

oxidation products (oxysterols) produced endogenously in the

body, are new and reliable markers of oxidative stress in vivo. Patients

affected by chronic liver diseases present high plasma levels of

oxysterols, raising the question of the origin and biological relevance

of these compounds in the pathophysiology of chronic liver damage.

Even though it is well known that some oxysterols enhance apoptosis

by activating the mitochondrial pathway, there are currently no data

on mitochondrial respiration. Thus, we aimed to investigate the biological

effects of oxysterols on cellular and mitochondrial respiration

in liver-derived cells HepG2. Cells were treated for 24 and 48 hours

with different concentrations (10 -9 to 10 -6 M) of 7-ketocholesterol

(7K), which is metabolized by mitochondria, and 27hydroxycholesterol

(27OH), which is synthesized by mitochondria.

Oxygen consumption was measured by a Clark type electrode, using

specific mitochondrial substrates and inhibitors. Intact and digitonin-permeabilized

HepG2 cells were used for analyses of respiratory

properties of mitochondria. Increasing concentrations of 7K

impaired cellular but not mitochondrial respiration. Very interestingly,

27OH enhanced mitochondrial respiration by Complexes I-III and

II-III, particularly at 10 -9 and 10 -8 M.

These results indicate that 7K and 27OH may have a different

effect on mitochondrial function, but also on cellular oxygen consumption.

Further experiments are needed to define the impact of

oxysterols on different free radicals sources which cause oxidative

stress during chronic liver diseases.

72


Red palm oil (Elaeis guineensis) offers myocardial protection

in a rat cardiac ischaemia/reperfusion model: proposed

mechanism of protection

D.J. BESTER, A.J. ESTERHUYSE, AND E. TRUTER

Department of Biomedical Sciences, Faculty of Health and Wellness, Cape

Peninsula University of Technology, South Africa

Background: Studies in our laboratory have demonstrated that

dietary supplementation with red palm oil (RPO) is able to offer cardiac

protection against ischaemia/reperfusion injury. Functional recovery

in this model has been measured using both Langendorffand

working heart perfusion apparatus. Dietary RPO supplementation

was also shown to significantly reduce myocardial infarct size.

Several mechanisms of protection have been suggested for above

mentioned protection, including Akt phosphorylation and nitric oxide

(NO) signaling. Materials and Methods: Hearts from rats on dietary

RPO supplementation for 5 weeks were perfused. Hearts were

snap frozen for biochemical analysis before and after a period of

global ischaemia according to the perfusion protocol. Coronary effluent

was collected after ischaemia to determine matrix

metalloproteinase (MMP2) activity.

Results: Dietary RPO supplementation proved to decrease MMP2

activity when compared to controls. Akt phosphorylation was increased

with RPO supplementation when compared to sunflower oil

supplemented controls. We also found that dietary RPO supplementation

was able to upregulate glutathione peroxidase 4 (GPX4) transcription.

Conclusion: Dietary RPO supplementation may partially increase

the availability of NO through phopsporylation of Akt. Scavenging

of superoxide by antioxidants present in RPO may also account for

increased bio-availability of NO. This in turn would lead to decreased

formation of peroxynitirite through the combination of NO and

superoxide which may account for the lower MMP2 activity in hearts

of RPO supplemented rats when compared to control hearts. This

decrease in MMP2 may be partially responsible for decreased necrosis

in the hearts of RPO supplemented rats. Afore mentioned phosphorylation

of Akt is also known to lead to phosphorylation of Bcl-

73


2-accociated death promoter (BAD) which inhibits apoptosis. Increased

transcription of GPX4 in hearts from RPO supplemented

rats suggest that the body’s natural antioxidant defenses are also improved

by RPO supplementation to the diet. Our studies indicate

that several mechanisms for cardiac protection may exist by supplementation

of the diet with RPO.

74


Modulation of neuroinflammatory pathway by centella asiatica

MUSALMAH BINTI MAZLAN 1 , NORFAIZATUL SHALIDA OMAR 1 ,

THEN SUE MIAN 2 , AND WAN ZURINAH WAN NGAH 1

1 Medical Faculty and 2 UKM Institute of Molecular Medicine, The National

University of Malaysia

Reduced cognitive and motor functions have been attributed

to neuronal loss as a result of accumulating reactive oxygen species

(ROS) during aging. ROS is thought to induce apoptosis by directly

activating the apoptotic pathway and indirectly through inflammation

pathway via the release of microglial cytokines and eventually

neuronal apoptosis. Several studies have shown the neuroprotective

potentials of antioxidants. Therefore in this study, the effect of Centella

Asiatica (CA) and its active compounds, asiaticosside (AC) and

madecassoside (MD) in preventing neuroinflammation was elucidated.

Microglial inflammation was induced with lipopolysaccharide

(LPS) and cells were incubated with either CA, AC, or MD.

Cell viability were measured using MTS assay while cytokines (TNFα

and IL-1b) were measured using commercial ELISA kits. Gene expressions

were determined using RT-PCR. Expressions of MAPK

pathway proteins were measured by using western blot. Results

showed that CA, AC and MD significantly reduced TNFα and IL-

1β gene expressions and production induced by LPS. CA, AC and

MD were able to inhibit JNK 1/2 proteins but only CA showed reduced

production of ERK1/2. Thus the study shows that CA inhibits

inflammation in neuron cells probably by inhibiting the activation of

ERK 1/2 and JNK ½. This in turn reduced gene expressions and

production of pro-inflammatory cytokines TNFα and IL-1β in microglia

and prevents neurons from undergoing apoptosis. The study

also suggests that other polyphenols in CA may be involve in its anti

inflammatory effect. In conclusion, this study demonstrates the

neuroprotective potential of CA via its modulation of the inflammatory

pathway.

75


Sulforaphane attenuates microglia activation induced by

amyloid-beta peptide oligomers

CECILIA BOLONDI 1 , FABIANA MORRONI 1 , PATRIZIA HRELIA 1 ,

GIORGIO CANTELLI-FORTI 1 , ERMINIA MARIANI 2 , AND ANDREA TAROZZI 1

1 Department of Pharmacology, Alma Mater Studiorum-University of Bologna,

Bologna, 2 Laboratory of Immunorheumatology and tissue regeneration,

Rizzoli Orthopaedic Institute, Bologna

Neuroinflammation has been known to play a critical role in the

pathogenesis of chronic neurodegenerative disease such as Alzheimer’s

disease (AD). Different studies show the presence of a number

of markers of inflammation in the AD brain including elevated inflammatory

cytokines and accumulation of activated microglia in the

damaged regions. More recent studies suggest that neuroinflammation

might be one of the earliest pathological responses to accumulation

of soluble amyloid-beta (Aβ) peptide oligomers, an important causal

factor in AD. Many studies highlighted the efficacy of diet components,

such as polyphenols and isothiocyanates (ITCs) from vegetables

to reduce inflammation processes and/or block neuronal death

occurring in AD. In particular, sulforaphane (SF), a natural dietary

ITC produced by the enzymatic action of myrosinase on

glucopharanin, a glucosinolate contained in cruciferous vegetables

of the genus Brassica, has recently gained attention as a potential

neuroprotective compound with indirect antioxidant properties. Consistent

with this evidence, this study was aimed at evaluating the effects

of SF on Aβ oligomers and lipopolysaccharide (LPS)-induced

microglia activation. In particular, we recorded the ability of SF to

reduce rat microglia activation in terms of iNOS expression, nitric

oxide production and reactive oxygen species formation induced by

both Aβ oligomers and LPS. In parallel, the attenuation of

microglial activation was associated with inhibition of inflammatory

cytokines (TNFα, Il-1β and IL-6) release. Taken together, these results

show that the SF could be a promising diet component capable

of counteracting neuroinflammation in AD.

76


Keap1-dependant and -independant regulation of Nrf2 by

flavonoids

LAURA J. BROWN 1 , DEREK STEWART 2 , AND JOHN D. HAYES 1

1 Medical Research Institute, University of Dundee;

2 The James Hutton Institute, Dundee

Flavonoids are a family of polyphenolic compounds, which have

been linked with the reduction in degenerative diseases associated

with consuming a diet high in fruit and vegetables. Flavonoids are

known for their antioxidant properties and it is probable that Nuclear

factor-erythroid 2-related factor 2 (Nrf2) mediates may of these properties.

Transcription factor Nrf2 regulates a battery of genes containing

an ARE in their promoter region. Its function is controlled at the

protein level by two distinct negative regulators; Keap1 and beta-

TrCP, through binding to the Neh2 and Neh6 domains in Nrf2, respectively.

A wide array of structurally diverse flavonoids have been screened

using an AREc32 luciferase reporter cell line and many were found

to be good induces of Nrf2-driven gene expression.

To further characterize induction of ARE-driven gene expression

by flavonoids, Neh2 and Neh6 Nrf2 domains were tagged with

betaGal-V5. Interestingly, three flavonoids Luteolin, Apigenin and

Chrysin all led to stabilization of the Neh6 domain, while known

Nrf2 inducers Quercetin and Kaempferol only stabilized the Neh2

domain.

The flavone Luteolin stabilized Nrf2 protein and lead to a modest

induction in Nrf2 target gene expression in Keap1-null mouse embryonic

fibroblasts. This induction was dependant on the PI3K

signaling pathway.

Our data shows that flavonoids can stabilize Nrf2 through Keap1–

independent mechanisms, possibly through activation of the PI3K

signaling pathway.

77


Oxidative stress and corticosterone production in epididymal

white adipose tissue (EWAT) of fructose fed rats

ANDREA CARRANZA 1 , CRISTIAN HOCHT 1 , ESTEFANÍA GERÉZ 3 ,

ARIEL POLIZIO 1 , MARCOS MAYER 1 ,CARLOS A. TAIRA 1 , ANA M. PUYÓ 2 ,

CESAR G. FRAGA 3 , AND MONICA GALLEANO 3

1 Pharmacology and 2 Anatomy, School of Pharmacy and Biochemistry,

University of Buenos Aires, Buenos Aires, Argentina;

3 Physical Chemistry-Institute of Molecular Biochemistry and Medicine

(IBIMOL), UBA-CONICET, Buenos Aires, Argentina

Adult male Sprague-Dawley rats received fructose 10% (W/V) in

the tap water (fructose fed group = F) or tap water (control group =

C) for 7 weeks as a model of metabolic syndrome (MS). At the end

of the treatment F animals showed the characteristic features of MS

in rat models: significant increases in plasma triglycerides (+116%),

plasma insulin (+194%), and blood pressure (+19%). Plasma glucose

levels were not affected by the treatment. Additionally, corticosterone

plasma levels were higher in F than in C rats (190±22 vs.

90±12 pg/ml, p


Different response of proteasomal degradation in normal and

tumor cells during heat stress

BETUL CATALGOL 1 , PERINUR BOZAYKUT 1 , ERDI SOZEN 1 , ASLÝ ECE 1 ,

ESRA OZALTIN 1 , NESRIN KARTAL OZER 1 , AND TILMAN GRUNE 2

1 Department of Biochemistry, Medicine Faculty, Marmara University,

Istanbul, Turkey; 2 Department of Nutritional Toxicology, Institute of

Nutrition, Friedrich Schiller University, Jena, Germany

Proteasomal degradation of oxidized proteins is a crucial mechanism

to prevent the accumulation of cellular damage. The removal of

the damage is generally a required process for healthy organisms to

keep the integrity while in cancer cells the situation may be different.

In normal conditions, cancer cells have higher proteasome activity

compared to normal cells. During cancer treatment, cellular damage

by chemotherapy is an expected process to be able to kill the tumor

cells. And the accumulation of this damage accompanied by the decrease

in protein repair and removal systems may increase the efficacy

of the cancer therapy.

Heat shock proteins (Hsp) as molecular chaperones are involved

in the folding, activation and assembly of a variety of proteins. Among

these Hsp40, Hsp70 and Hsp90 are believed to act as a chaperone

system to regulate the proteasomal degradation.

In this study, we tested the role of heat stress response on the

proteasomal degradation of oxidized proteins. We used two different

cell lines to observe the difference in normal and tumor cells. First

the effect of heat stress (42 ºC, 1h) were tested in terms of protein

oxidation tested by protein carbonyl formation and proteasomal degradation.

The results were extremely different in normal fibroblast

cells and hippocampal tumor cells. In the same direction, the expressions

of Hsp40, Hsp70 and Hsp90 were affected in a different manner

in two cell lines, will be discussed in detail.

Supported by TUBITAK COST-CM1001-110S281

79


Cobalt chloride induced hypoxia and lipid peroxidation in

hRPE cells

F. CERVELLATI 1 , C. CERVELLATI 2 , B. PAVAN 1 , C. STICOZZI 1 ,

AND G. VALACCHI 1-3

1 Dipartimento di Biologia ed Evoluzione, Università di Ferrara, Ferrara,

Italy; 2 Dipartimento di Biochimica Clinica, Università di Ferrara, Ferrara,

Italy; 3 Department of Food and Nutrition, Kyung Hee University, Seoul, South

Korea

It has been postulated that diabetic retinopathy might be initiated

by hypoxia during the hours of darkness. The pathogenesis of diabetic

retinopathy has been a long-standing puzzle. Although capillary

function generally is affected in diabetes, the reason why this

situation develops into uncontrolled vasculopathy in the retina is unknown.

Evidence suggests that the retina is exposed to suboptimum

oxygen concentrations before the development of diabetic retinopathy.

It has been postulated that the development of diabetic retinopathy

is triggered by tissue hypoxia due to the exceptionally high oxygen

consumption of rod receptors during dark adaptation, combined

with impaired capillary function. The mechanism that is behind the

development of this pathology is still under investigation although

hypoxia could be one of the triggers in the development of retinopathy.

In the present study we have investigated the effects of the hypoxia-mimetic

cobalt chloride (CoCl 2 ) in the retinal cell hRPE.

After treatment of hRPE cell with 200mM CoCl 2 there was a strong

induction of HIF1a, confirming that the experimental approach was

an acceptable model for in-vitro hypoxia. The cells showed a significantly

reduced viability and an increased number of cell death. These

data, together with the increased expression of VEGF confirmed that

the retina cells respond to the lost of oxygen by increasing cell death

(most likely apoptosis as shows by a dramatic increase in caspase 3

expression) and by trying to increase blood flow through the release

of VEGF.

These effects seem to be related to oxidative stress as they parallel

with an increased level of 8-iso PGF 2 isoprostane and 4HNE protein

adducts. The responses of the cell to the increased oxidative stress is

the induction of NFkB activity and this was confirmed by

80


immunohystochemistry analysis. The activation of the transcription

factor NFkB could be also the reason of VEGF increased levels in

cells exposed to hypoxia. In addition to oxidative stress, the nuclear

translocation of NFkB could also depend of the early induction of

CD36 expression that has been shown to be able to stimulate NFkB

activation.

The present results provide evidence of the possible mechanistic

pathway involved in retynopathy, where the first step is the existence

of and hypoxic event, which leads to increased oxidative stress that

via the activation of NFkB stimulates the production of VEGF. In

parallel, oxidative stress will consequently damage the cells with an

increased mortality via the induction of apoptosis. Of note is that,

using TEM technique, cells exposed to CoCl 2 showed an extended

vacuolation and this could also be a consequence of an autophagic

process induced by HIF1a but it needs to be investigated in the future

experiments.

81


The role of Keap1/Nrf2 pathway in brown adipose tissue

differentiation and function; implications for high-fat dietinduced

obesity in mice

DIONYSIOS CHARTOUMPEKIS, STEPHEN SLOCUM, JOHN SKOKO,

NOBUNAO WAKABAYASHI, AND THOMAS KENSLER

Department of Pharmacology and Chemical Biology,School of Medicine,

University of Pittsburgh, Pittsburgh, PA, USA

Introduction. White (WAT) and brown (BAT) adipose tissue are

two functionally distinct tissues. WAT stores and releases triglycerides

and BAT is specialized in thermogenesis that dissipates energy in the

form of heat after cold exposure or diet. Nrf2 deletion has been shown

to protect from high-fat diet (HFD) induced obesity and to negatively

affect adipocyte differentiation but nothing is known about the

role of Nrf2 in the BAT.

Research methods. Immortalized wild-type (WT) mouse brown

preadipocytes (BAT cells) were treated with the Nrf2 inducers CDDO-

Im or sulforaphane during differentiation. WT and Keap1 flox/flox

mice (whose Keap1 allele is hypomorphic and thus it is a model of

gain of Nrf2 function) were fed a standard or an HFD for 3 months.

Gene expression analysis was performed with qRT-PCR and oilred-O

staining was used for estimation of differentiation.

Results. The pharmacological Nrf2 activation during BAT cells

differentiation had no effects in the accumulation of lipid droplets.

However, the basal and the induced-after isoproterenol treatment (a

general beta-adrenergic agonist) - mRNA levels of Pgc-1α, a known

mediator of thermogenesis in BAT, and of its downstream target Ucp-

1 were found to be approximately 2 times lower in the treated BAT

cells. Consistently, Pgc-1α levels were also lower in the Keap1 flox/

flox mice compared to the WT under standard diet. The BAT Pgc-1a

levels were reduced in WT mice after HFD, as expected, but not in

the Keap1 flox/flox mice which were partially protected from obesity

as it also happens with their Nrf2-/- counterparts.

Conclusions. Nrf2 activation affects the functional but not the

morphological phenotype of BAT cells. Both gain and loss of Nrf2

function at least partially protect mice from HFD-induced obesity.

Pgc-1α is a novel candidate gene that is directly or indirectly regulated

by Nrf2 and may mediate some of the Nrf2 metabolic effects.

82


Evaluation of oxidative status in human serum: comparison of

different methodological approaches

GIULIANA CIGHETTI 1 , CRISTINA NOVEMBRINO 2 , CAROLINE AMAN 1 ,

RACHELE DE GIUSEPPE 2 , FEDERICA DE LISO 2 , AND FABRIZIA BAMONTI 2

1 Dipartimento Scienze Cliniche “Luigi Sacco”, Università degli Studi di

Milano, Milan, Italy; 2 Dipartimento Scienze Mediche, Università degli Studi

di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,

Milan, Italy

Different biomarker assays have been developed for assessing

oxidative stress in human serum. In this retrospective study the analytical

performance of two different methodological approaches was

evaluated in subjects with known increased oxidative stress to measure

serum peroxidation indices: mild (n=35) and heavy (n=61) smokers,

chronic renal failure (n=19) and kidney transplanted patients

(n=59) compared to healthy controls (n=56).

Serum oxidative stress, assessing Reactive Oxygen Metabolite

derivatives (d-ROMs, Diacron International, Italy) levels and Total

Antioxidant Capacity (TAC, Diacron International, Italy) by commercial

spectrophotometric assays, was compared with

malondialdehyde (MDA) concentrations, quantified both in free (F-

MDA) and total (T-MDA) forms, by isotope-dilution gas chromatography-mass

spectrometry (GC-MS) technique (“gold standard”

reference method), generally unsuitable for routine use.

Sensitivity, specificity and cut-off points of T-MDA and F-MDA,

d-ROMs, TAC assays were evaluated by receiver-operator characteristic

(ROC) analyses together with the area under ROC curve

(AUC).

ROC analyses accuracy: the best for T-MDA (AUC: 1; sensitivity

and specificity: 100%), good for d-ROMs (AUC: 0.87; sensitivity

72.8%, specificity: 100%) and F-MDA (AUC: 0.82; sensitivity

74.7%, specificity: 100%), and not as good as the previous for TAC

(AUC: 0.66; sensitivity: 52% and specificity: 92.9%). The increased

peroxidative damage was best proved only by T-MDA levels. The

assessment of d-ROMS concentrations and TAC by reliable assays

is useful for routine clinical purposes; even if less sensitive, it determines

the balance of oxidative status. The comparison between “gold

standard” and routine methods allows biochemists and clinicians to

evaluate data more thorough.

83


Cellular adaptive response to glutathione depletion modulates

endothelial dysfunction triggered by TNF-a: involvement of

Nrf2/ARE pathway

FRANCESCO CIMINO 1 , SIRAJUDHEEN ANWAR 1 , RAFFAELLA CANALI 2 ,

DEBORAH FRATANTONIO 1 , ELISABETTA RICCIARDI 1 , ANTONELLA SAIJA 1 ,

FABIO VIRGILI 2 , AND ANTONIO SPECIALE 2

1 Dep. Farmaco-Biologico, Faculty of Pharmacy, University of Messina,

Messina, Italy; 2 National Research Institute on Food and Nutrition (INRAN),

Rome, Italy

Cells are continously exposed to the deleterious consequences of

reactive oxigen species generated endogenously and exogenously.

Cells have developed multiple stress adaptive response, including

the system regulated by the transcription factor Nrf2 that modulates a

set of cytoprotective genes. Up today there are few data supporting

the atheroprotective and anti-inflammatory activity of Nrf2 pathway

in endothelial cells. Thus to test this hypothesis we have investigated

if some changes in the intracellular redox status, such as those related

to endogenous antioxidants depletion, might be able to alter the response

to the inflammatory cytokine TNF-α in cultured human endothelial

cells through the activation of the Nrf2 pathway.

In this study, we revealed that an oxidative intracellular status by

GSH depletion is able to induce a cellular adaptive response. Interestingly

we demonstrated that, in our experimental conditions, activation

of Nrf2 pathway is able to reduce endothelial dysfunction by

decreasing NF-kB nuclear translocation and adhesion molecules gene

expression in HUVECs. Furthermore, we confirmed that Nrf2 nuclear

translocation activated by GSH depletion is dependent on extracellular

signal-regulated kinases 1/2 phosphorylation.

In conclusion we showed that the coordinate induction of endogenous

cytoprotective proteins through activation of the Nrf2 pathway

might serve as a new therapeutic approach for prevention or

treatment of endothelial dysfunction.

84


Deficiency of Nrf2 expression in target neural tissues of the

Friedreich ataxia mouse model YG8

GINO CORTOPASSI 1 , YUXI SHAN 1 , MARK POOK 2 , ROBERT SCHOENFELD 1 ,

AND SUNIL SAHDEO 1

1 University of California, Davis, California, USA; 2 Brunel University, London,

England

Friedreich’s ataxia is an inherited neurodegenerative disease that

phenocopies AVED, a genetic deficiency of the Vitamin E (tocopherol)

transporter. Friedreich’s patients inherit a deficiency in the mitochondrial

protein frataxin, which appears to function in the biogenesis

of iron-sulfur clusters, prosthetic groups of redox-active proteins.

We have studied the YG8 model of Friedreich ataxia, that is transgenic

for one or two mutant human alleles of frataxin, in a mouse that lacks

its own murine frataxin. These animals have elevated oxidative stress

and behavioral deficits, and demyelination and neurodegeneration.

We carried out microarray on the dorsal root ganglion neural tissue

in the mice, that degenerates first in human patients. Expression of

multiple thiol antioxidants were decreased in DRG, which was confirmed

by QRTPCR. Since many of these antioxidant genes were

targets of the Nrf2 transcription factor, its expression was also

analyzed, and was observed to be deficient in mutant mice at the

RNA and protein level in DRG and cerebellar tissue. DRG cells

were more sensitive to the inhibitor of the Nrf2-regulated thioredoxin

reductase inhibitor auronofin. Frataxin knockdown in Hela cells similarly

decreased Nrf2 and thioredoxin reductase activity, and were

more sensitive to auranofin, and the redox state of peroxiredoxins

was more oxidized. Thus, a frataxin-dependent deficiency in Nrf2

exists in neural tissues of an animal model of Friedreich’s ataxia,

suggesting a neuroinflammatory mechanism of the disease, and suggesting

potential therapeutic routes.

85


Modifications of the redox intracellular environment during

the human immunodeficiency virus type I (HIV-1) infection

associated with the Src kinase activation

M. CURCIO 1 , H. MONTEIRO 2 , A. SARTORI 2 , S. STRUMILO 1 , W. MAIA 1 ,

E. CASTRO 2 , S. ANDRADE 3 , S. LIMÃO 1 , M. SOANE 1 , AND M. JANINI 1

3 Department of Microbiology, Imunology and Parasitology (DMIP)-

Laboratory of Retrovirology 3 Department of Biochemistry and Molecular

Biology - Laboratory of Cell Signaling, Brasil 3 Department of Gynecology

2 Universidade Federal de São Paulo, São Paulo and 1 Charitable Association

of Blood Collection - COLSAN

Human Immunodeficiency Virus (HIV) and other structurally simple

retroviruses developed, as a defense mechanism, numerous replication

strategies to escape from apoptotic mechanism triggered by

host cells. HIV maintains a persistent infection by stimulating intracellular

(host) production of cytokines and of reactive oxygen and

nitrogen species (ROS and RNS). Levels of ROS and RNS and of

their antioxidant counterparts determine the redox environment. Alterations

on the redox environment are sensed by raising levels of

the antioxidant enzyme Cu/Zn and Mn Superoxide dismutase (SOD).

Such alterations are potentially associated with the regulation of HIVreplication

signaling pathways.

In the present study we used isolated human CD4 T lymphocytes

submitted to a protocol of in vitro infection with purified HIV viral

particles. Levels of the antioxidant enzymes, Cu/Zn and Mn-SOD,

Glutathione peroxidase (GPx), Glutathione reductase (GR), and of

the tri-peptide Glutathione (GSH) were measured in infected and

non-infected CD4 T lymphocytes. There was an increase on the activities

of the antioxidant enzymes followed by a decrease on intracellular

GSH levels. In addition we followed the temporal pattern of

activation of Src kinase. Src is a cytoplasmic protein tyrosine kinase

which has been shown to play an important role in HIV-1 replication.

86


Beneficial effects of CELLFOOD® SILICA supplementation

on oxidative status and bone remodelling in osteopenic women

RACHELE DE GIUSEPPE 1 , LUISELLA VIGNA 2 , FEDERICA DE LISO 3 ,

CRISTINA NOVEMBRINO 3 , PAOLA BONARA 4 , AND FABRIZIA BAMONTI 3

1 Fondazione F.lli Confalonieri, Dip. Scienze Mediche, Università degli Studi

di Milano, Fondazione IRCCS, Cà Granda Ospedale Maggiore Policlinico,

via F. Sforza 35, 20122 Milano, Italy; 2 Dip. Medicina Preventiva, UO

Medicina del Lavoro I Fondazione IRCCS, Cà Granda Ospedale Maggiore

Policlinico, via F. Sforza 35, 20122 Milano, Italy; 3 Dip. Scienze Mediche,

Università degli Studi di Milano, Fondazione IRCCS, Cà Granda Ospedale

Maggiore Policlinico, via F. Sforza 35, 20122 Milano, Italy; 4 Dip. Medicina

Interna, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico,

via F. Sforza 35, 20122 Milano, Italy

Osteopenia (bone mineral density lower than normal) is considered

a precursor of osteoporosis According to several studies, patients

with osteoporosis frequently display vascular diseases, including

atherosclerosis, probably due to the presence of oxidative stress.

Thirty one osteopenic women (aged 59±6.4) were enrolled to

evaluate the effects on bone density and oxidative status of

CELLFOOD® SILICA (Nu Science Corporation, CA, USA), antioxidant

compound enriched with silicon dioxide. Osteopenia was

diagnosed by phalangeal ultrasonographic examination (DMB Sonic

Bone profiler, IGEA, Italy; reference values: -3.2


significantly lower (80±30 vs 65±23 U/L, p=0.01), after supplementation.

Our preliminary results suggest beneficial effects of CELLFOOD®

SILICA in osteopenia: silicon dioxide seems to reduce bone mineral

resorption; notably, CELLFOOD® SILICA might be a valuable

coadjuvant in the prevention and treatment of oxidative stress, thanks

to its antioxidant properties.

88


Impairment of inflammatory parameters and oxidative status

in overweight-obese subjects

FEDERICA DE LISO 1 , LUISELLA VIGNA 2 , RACHELE DE GIUSEPPE 1 ,

CRISTINA NOVEMBRINO 1 , PAOLA BONARA 3 , AND FABRIZIA BAMONTI 1

1 Dip. Scienze Mediche, Università degli Studi di Milano, Fondazione IRCCS,

Cà Granda Ospedale Maggiore Policlinico, via F. Sforza 35, 20122 Milano,

Italy; 2 Dip. Medicina Preventiva, UO Medicina del Lavoro I Fondazione

IRCCS, Cà Granda Ospedale Maggiore Policlinico, via F. Sforza 35, 20122

Milano, Italy; 3 Dip. Medicina Interna, Fondazione IRCCS Cà Granda

Ospedale Maggiore Policlinico, via F. Sforza 35, 20122 Milano, Italy

Obesity is associated with increased risk of cardiovascular diseases.

Adipocytokines (i.e. leptin), produced by the endocrine function

of adipose tissue, can contribute to cardiovascular diseases in

overweight and obese people. Oxidative stress, imbalance between

oxidants and antioxidants, is considered a cardiovascular risk factor.

High serum oxidized LDL levels (oxLDL), marker of lipid

peroxidation, a primary cause of atherosclerosis, can contribute to its

progression.

Obese subjects (141: 94 F, 47M; aged 46.6±9.8; BMI 34.5±5.3

Kg/m2), without previous cardiovascular diseases, were enrolled and

compared with controls, well matched for sex and age (BMI 21.6±2.0

Kg/m2), to evaluate the possible role of oxidative stress and

“cytokines profile”.

Oxidative status was measured by evaluating serum Reactive

Oxygen Species levels (ROS) and Total Antioxidant Capacity (TAC)

by spectrophotometric methods (Diacron International, Italy) and

oxLDL by competitive ELISA method (Mercodia, Sweden); soluble

cytokines and adhesion molecules levels by cytofluorimetric

method (Flow Cytometry, Human Obesity 9plex Kit; Bender

MedSystems, Austria)

ROS and oxLDL levels were high in 87% and 93% of obese

subjects, respectively, despite adequate TAC (72%). Females’ ROS

levels were significantly higher than males’ (423.1± 86.1 vs 324.7±

53.7 UCarr, p


sion molecule involved in leukocyte migration to inflamed area) were

significantly higher than controls’ (93.8±89.1 vs 25.3±23 ng/mL,

p=0.002; 505.8±236.7 vs 339.2±119.6 ng/mL, p=0.01, respectively).

Our obese subjects showed oxidative stress accompanied by low

chronic inflammatory status, possibly contributing to increased cardiovascular

risk. Early intervention on obesity must be a primary

aim.

90


The intake of a single blueberry (Vaccinium corymbosum L.)

portion affects blood pressure and peripheral arterial function

in smokers: preliminary data

CRISTIAN DEL BO’ 1 , DANIELA FRACASSETTI 1 , PATRIZIA RISO 1 ,

JONICA CAMPOLO 2 , DOROTHY KLIMIS-ZACAS 3 , AND MARISA PORRINI 1

1 Università degli Studi di Milano, Department of Food, Environmental and

Nutritional Sciences, Milan, Italy; 2 Istituto di Fisiologia Clinica CNR,

Dipartimento Cardiovascolare, Ospedale Niguarda Cà Grande, Milan, Italy;

3 Department of Food Science and Human Nutrition, University of Maine,

Orono, Maine, USA

Cigarette smoking is one of the primary risk factors for the

development of endothelial dysfunction, an early event in atherosclerosis,

due to an increase of oxidative stress and the impairment of

nitric oxide production. Anthocyanin-rich foods, have been suggested

to offer protection against oxidative stress and modulate endothelial

function. This study aims to investigate whether a single portion of

blueberry improves endothelial dysfunction induced by acute cigarette

smoking. Fifteen healthy smokers were recruited for a crossover

study. Three types of conditions were assessed: 1-blueberry treatment

(300g of blueberry) + smoking (1 cigarette); 2-control treatment

(300mL of water with 27g of sugars; the same amount present

in blueberry) + smoking (1 cigarette); 3- smoking only (1 cigarette).

Peripheral arterial function (determined through Endo-PAT2000) and

blood pressure were evaluated. Preliminary results on 8 subjects

showed that acute smoking caused a reduction in peripheral arterial

function (from 2.22 ± 0.3 to 1.62 ± 0.4 RHI, p


Set up of methodological analysis to evalutate antioxidant

capacity in serum and saliva using DPPH test method

ENRICO DORIA, DANIELA BUONOCORE, AND FULVIO MARZATICO

University of Pavia, Department of Biology and Biotechnology, Pavia

Body oxygen consumption, during physical activity, especially

of high intensity, in the muscle fibres engaged in the performance

can increase up to 200 times compared to that consumed at rest. Part

of these oxygen molecules, in mitochondria, can undergo reduction

with an increase in ROS production, the latter can have different

negative influences on many cellular functions with both acute and

chronic mechanisms. Human body has a complex defence system

from oxidizing agents, which acts synergistically promoting ROS

inactivation. These systems may not be sufficient to cover oxidative

stress that can occur in strenuous exercise or during strenuous athletic

seasons as a professional football league. In this work, we explored

the possibility to monitor antioxidant capacity of professional

football players, during 2010/2011 season. We have applied method

developed by Atsumi in 1999, which used 1,1’-diphenyl-2picrylhydrazyl

(DPPH radical) and put it in contact with human samples,

such as saliva and serum. We have tested two different methods

to treat sample serum and we have compared serum vs saliva results,

the latter is considered a diagnostic window on the blood and a valid

non invasive method. Our results show that trend of antioxidant activity

both in serum and saliva is comparable, in a athletes population,

during the examined months showing qualitative efficacy of the

method; regarding to the serum treatment with TCA which was setup

in this work, data revealed higher values of DPPH scavenging

activity than those obtained from standard boiling treatment previously

developed by Atsumi in 1999. The evaluation of antioxidant

capacity of saliva along with other parameters allow a close monitoring

of athletes and, moreover, corrections in nutrition, supplementation

and workout in a quick and timely manner to optimize the performance

and monitor their health by medical staff.

92


The effect of NOS inhibitors on ROS/NO balance and on

antioxidant and detoxication response in young Wistar rats

IMA DOVINOVA 1 , MIROSLAVA MAJZÚNOVÁ 1 , ZUZANA PAKANOVÁ 1,2 ,

SOÒA ÈAÈÁNYIOVÁ 1 , ŠTEFAN ZORAD 3 , AND FRANTIŠEK KRISTEK 1

1 Institute of Normal and Pathological Physiology, Slovak Academy of

Sciences, Bratislava, Slovakia; 2 Faculty of Chemical and Food Technology,

Slovak University of Technology, Bratislava, Slovakia; 3 Institute of

Experimental Endocrinology, Slovak Academy of Sciences, Bratislava,

Slovakia

Numerous experimental studies focus on the effect of NOS inhibitors.

This study contributes data about radical and antioxidant

signaling in NO-deficient models of Wistar rats. The aim was to investigate

the effects of 7-NI and L-NAME on ROS/NO balance in

young Wistar rats and to observe changes in oxidative stress, antioxidant

response, and the involvement of the MDR detoxication pump

in the left ventricle (LV) and brain stem (BS).

Young, 4 weeks old male Wistar rats were divided into 3 groups.

The animals obtained 7-NI (10 mg/kg/day) or L-NAME (50 mg/kg/

day), or (the control group) only tap water ad libitum for 6 weeks.

Basal blood pressure was measured using the plethysmography tailcuff

method. We observed the activation of NAD(P)H ox subunit

p22phox by real-time PCR and measured the superoxide level using

Lucigenin-enhanced chemiluminiscence. Changes in NO levels were

observed via gene expression of NOS isoforms and by measurement

of NOS activities. The antioxidant and detoxication responses were

detected via SOD and MDR1A gene expression and SOD activity

determinations.

Blood pressure was significantly increased only in the L-NAME

group and only L-NAME administration significantly decreased NOS

activity in the LV and BS. In the left ventricle, we did not observe

any changes in p22phox subunit. The superoxide level was unchanged

or slightly decreased in the 7-NI group. No differences in

SOD activities were observed, but MDR1A in the heart increased.

In the brain stem, SOD activities increased in the 7-NI group, while

no change in MDR1A expression was observed in both treatment

groups.

93


The application of NOS inhibitors L-NAME or 7-NI in the left

ventricles of young Wistar rats did not induce ROS increase or antioxidant

response of SOD activity, but it induced MDR1A activation.

In the brain stem, the effect was different leading to an increase

of SOD activity but not of MDR1A expression.

94


Oncolyn induces apoptosis of cancer cells, causes clinical

remission of solid tumors, ameliorates neurodegeneration and

exhibits radiation protection

ARTHUR H.K. DJANG, M.D., PH D., M.P.H. 1 , M. BUD NELSON, PH.D.,

J.D. 1 , ZHOU DIAN-YUAN, M.D. 2 , LAI ZHO-SHENG, M.S. 2 , BAI JIANG

M.D. PH.D2 , M. EDEAS, M.D., PH.D. 3 , MIKHAEL ADAMS, N.D. 5 ,

ALISON F. ADAMS, D. AC. 5 , SUN HUI, M.D. 7 , WANG AI-PING, M.D. 7 ,

ZHANG YANSHU, M.B.,PH.D. 6 , SUN QING-YU, M.D. 9 , GINNETTE

PELLETIER, N.D. 8 , LUCY LAFONTAINE, N.D. 8 , PETER MOSCOW, PH. D.

10 4 10 , DIMITRIOS GALARIS, PH.D. , HEATHER MURLEY, D.V.M. , AND

ZHANG ZHONG-LI, DVM. 11

1 Santé International, Inc., Jamestown, New York, USA, 2 Southern Medical

University, Guangzhou, China, 3 Hospital Antione Beclere, Paris, France,

4 University Ioannina, School of Medicine, Greece, 5 Renascent Integral Health

Centre, Ontario, Canada, 6 School of Public Health, North China Associated

University, Tangshan, China, 7 Tianjin Cancer Center and Medical University,

Tianjin, China, 8 Private Practice, Montreal & Ottawa, Canada, 9 Private

Practice, Dalian, China, 10 Private Practice, Louisville, KY & Ithaca, NY., 11

Tianjin Environmental Protection Institute, Tianjin, China

Oncolyn ® is composed of edible plant extracts with main ingredients

including flavonoids, polyphenols and saponins. It protects skin

from UV damage, respiratory system from silica and asbestos, endothelium

from ox LDL. It has shown anti-cancer function in clinical

application and nude mouse against various cancer cells implants

both for prevention and therapy. Oncolyn has further shown radiation

protection in mice and man verified with comet assay and micronucleus

test.

Oncolyn was demonstrated to protect DNA against H 2 O 2 , silica

and asbestos particles by single cell gel electrophoresis and histopathology.

Oncolyn inhibits telomerase activity and induces apoptosis

of cancer cells. Effective management of malignant mesothelioma

was verified by CT in clinical application.

Oncolyn effectively scavenged free radicals (O 2 Superoxide and

OH Hydroxyl), generated by Co-60 irradiation on thymidine 5monophate

as measured by free radical Resonance Spectroscope. It

further protected mice exposed to Co-60 irradiation from radiation

95


sickness and leucopenia, increase of circulating micronucleus RBC

and platelets reduction.

Concomitantly, Oncolyn also caused amelioration of rheumatoid

and osteoarthritis and produced a feeling of wellness in patients with

various chronic debilitating conditions including COPD, and prevented

the reoccurrence of TIA’s and caused speedy recovery of stroke

patients. Oncolyn, caused remission for canine mammary cancer and

amelioration of canine liver adenocarcinoma.

In aging mice, the activities of ATPase in the brain and nerve

growth factor (NGF), and brain-derived neurotrophic factor (BDNF)

in hippocampus showed significant decrease. Oncolyn administration

reversed this trend of decrease in mice and caused increase of

acetylcholine, dopamine and norepinephrine in the brain of aged mice.

Clinical observation of a senior population taking Oncolyn (since

2005) showed decreased incidence of neurodegenerative diseases

such as Parkinson and Alzheimer. (Sun Q.Y., et al, Aging and

Neurodegeneration Seminar, Dalian, China 7/2010).

Conflict of Interest: None

Oncolyn ® , 3 United States patents, 1 US Provisional patent, 1 Canadian

patent and 1 European patent and 2 international trademarks.

Supported in part by a grant from Santé International, USA

96


Fisetin blocks protein glycation by two complimentary

mechanisms; significance to diabetes

JENNIFER EHREN, DAVID SCHUBERT, AND PAMELA MAHER

Cellular Neurobiology Laboratory, Salk Institute for Biological Studies La

Jolla, CA

The diabetic complication, diabetic nephropathy (DN), oftentimes

leads to kidney failure but has no known therapy. Fisetin, an orally

active natural product, reverses the increased kidney weight seen in

untreated diabetic mice and decreases urinary protein secretion levels.

Advanced glycation endproducts (AGEs), chemically modified

molecules resulting from nonenzymatic sugar and aldehyde addition,

increase in diabetic patients and contribute towards the development

of DN. Fisetin increases cellular glutathione (GSH, the major intracellular

antioxidant) levels under oxidative stress conditions in mouse

kidney cells. Importantly, fisetin also reversed the increase in AGEs

in kidney cells exposed to high glucose conditions as well as in the

kidneys of diabetic mice.

Fisetin decreases glycation in two complimentary ways. In vitro

data indicates that fisetin can directly block glycation. In addition,

fisetin maintains GSH levels in vivo. GSH is an essential co-factor

for glyoxylase 1, the rate-limiting enzyme in the removal of glycating

agents that when added to proteins, increases inflammation and

oxidative stress in high glucose environments (1, 2). Among the transcription

factors that play critical roles in GSH metabolism are Nrf2

and ATF4. We demonstrate that fisetin rapidly increases the levels of

Nrf2 and ATF4 as well as Nrf2- and ATF4-dependent gene transcription.

In addition, fisetin increases the stability of Nrf2 and ATF4.

Using siRNA we found that ATF4, but not Nrf2, is important for the

ability of fisetin to increase GSH levels under basal condition, whereas

both ATF4 and Nrf2 appear to cooperate to increase GSH levels

under conditions of oxidative stress.

97


Role of the Polycomb group gene family in aging

and neurodegeneration

EL HAJJAR JIDA, ABDOUH MOHAMED, AND BERNIER GILBERT

Department of Developmental Biology, Research Center of the Maisonneuve

Rosemont Hospital

Aging and oxidative stress are the prime risk factors for development

of neurodegenerative diseases and dementia. The p16Ink4a/

p19Arf locus mediates cellular senescence and apoptosis via p53

activation and is greatly implicated in the aging process. Polycomb

group proteins (PcG) are chromatin remodeling complexes that instrument

transcriptional silencing in higher eukaryotes. They mainly

repress the expression of the p16Ink4a/p19Arf locus. Mutations in

PcG instigate degeneration in neuronal dividing cells, mainly the

cerebellar precursor cells and the neural stem cells (NSCs), and induce

oxidative damage in post-mitotic neurons via p53 activation.

Our aim is to scrutinize the function of the PcG proteins in these nondividing

cells, their implication in p53 and aging regulation, and ultimately

their effect on neurodegeneration. We observed a robust decline

in the expression of PcG proteins in neurons of aged mice and

humans with dementia compared to their controls. This decrease corroborated

with an up-regulation of 1) senescence biomarkers p16,

p19, and p21; 2) apoptosis markers p53, Noxa and Caspase-3; 3)

neuroinflammation markers GFAP and Il-6; and 4) the oxidative stress

marker 8-Oxoguanine (8-OHG) indicating an elevated oxidative

damage in neurons. Moreover, we delineated that this neurodegenerative

phenotype was relatively p53 dependent.

Our preliminary results demonstrate that PcG proteins are essential

for a normal physiological aging and that loss of expression would

initiate neurodegeneration. We find it particularly compelling to evaluate

if the overexpression of PcG would mitigate the neurodegenerative

phenotype and could promote extended healthy lifespan; if so, then

this is highly significant as it might be possible to treat late-onset

human disorders with a single therapeutic modality.

98


Exhaustive exercise modulates expression of RCAN1-4 more

than other RCAN1 isoforms in rat skeletal muscles

RAMIN EMRANI 1 , AMÉLIE RÉBILLARD 2 , LUZ LEFEUVRE 2 ,

ARLETTE DELAMARCHE 2 , GENNADY ERMAK 3 , KELVIN J.A. DAVIES 3 ,

AND JOSIANE CILLARD 1

Laboratoire « Mouvement, Sport , Santé » EA 1274, Université de Rennes 1 1

et Université de Rennes 2 2 , and Ethel Percy Andrus Gerontology Center of

the Davis School of Gerontology and Division of Molecular and

Computational Biology, Department of Biological Sciences of the Dornsife

College of Letters, Arts & Sciences, University of Southern California 3 , Los

Angeles, California 90089-0191, USA

The Regulator of Calcineurin1 (RCAN1) is an endogenous intracellular

phosphatase that inhibits calcineurin. Calcineurin is a ubiquitously

expressed serine/threonine protein phosphatase under the

control of calcium/calmodulin which has been identified in skeletal

muscles. However, so far, RCAN1 expression has not been studied

under exhaustive exercise.

Experiment: 24 rats in three groups were subjected to the same

physical exercise protocol and then sacrificed either immediately, or

3 hours or 6 hours after exercise. A control group of 8 rats were

sacrificed in the same condition. Three different skeletal muscles were

collected: Soleus, a slow twitch muscle rich in type 1 fibers; Extensor

Digitorum Longus (EDL), a fast twitch muscle rich in type 2

fibers; and Gastrocnemius which contains both types of fibers. Qualitative

and quantitative western blot analyses of RCAN1 were performed.

Results: Three RCAN1 isoforms were each expressed differently

in the three muscles. RCAN 1-1L , RCAN1-1S and RCAN1-4 were

detected in Soleus and EDL, whereas RCAN1-1S was not observed

in Gastrocnemius. Exercise differentially modulated the RCAN1

isoforms. RCAN1-4 increased significantly after exercise until 6

hours in EDL and Gastrocnemius, but no significant changes were

seen in Soleus. RCAN1-1L and RCAN1-1S were not affected by

exercise in any muscle.

99


Conclusion: Exhaustive exercise up-regulated RCAN1-4 expression

in rat EDL and Gastrocnemius. Exhaustive exercise is known

to induce oxidative stress, and RCAN1-4 induction has been shown

to help protect against oxidative stress in other tissues. Therefore, we

propose that RCAN1-4 expression represents an important component

of the physiological adaptation to exercise.

100


A combination of lipoic acid plus coenzyme Q10 induces

PGC1a, a master switch of energy metabolism, improves stress

response and increases cellular glutathione levels in cultured

C2C12 skeletal muscle cells

INSA M.A. ERNST 1 , ANIKA E. WAGNER 1 , MARC BIRRINGER 2 ,

ÖZGÜR SANCAK 3 , LUCA BARELLA 3 , AND GERALD RIMBACH 1

1 Institute of Human Nutrition and Food Science, Christian-Albrechts-

University Kiel, Germany; 2 Department of Nutritional, Food and Consumer

Studies, University of Applied Sciences Fulda, Germany; 3 Bayer Consumer

Care AG, Global Research and Development, Basel, Switzerland

Skeletal muscle function and integrity largely depend on intact

energy metabolism, stress response and antioxidant defense

mechanisms. In this study we tested the effect of a combined

supplementation of α-lipoic acid (LA) plus coenzyme Q10 (Q10)

on peroxisome proliferator-activated receptor γ (PPARγ)-coactivator

α (PGC1α) activity, expression of glutathione-related phase-II

enzymes and glutathione (GSH) levels in cultured C2C12 myotubes.

Supplementation of myotubes with 250 μmol/l LA plus 100 μmol/l

Q10 significantly increased nuclear levels of PGC1α, a master switch

of energy metabolism and mitochondrial biogenesis. The increase of

nuclear PGC1 was accompanied by an increase in PPARγ

transactivation, a downstream target of PGC1α, and an increase in

mitochondrial transcription factor A (TFAM) mRNA centrally

involved in mitochondrial replication and transcription. Furthermore,

supplementation of myotubes with LA plus Q10 resulted in an

increase of genes encoding proteins involved in stress response

(glutathione-S-transferases; GST), GSH synthesis γ-glutamylcysteinesynthetase

(γGCS) and its recycling. In LA plus Q10 treated myotubes

a significant 4-fold increase in GSH was evident. This increase in

GSH was accompanied by increased nuclear Nrf2 protein levels,

partly regulating γGCS and GST gene expression. Present data

suggest that the combined supplementation of skeletal muscle cells

with LA plus Q10 may improve energy homeostasis, stress response

and antioxidant defense mechanisms.

101


Iron preconditioning against ischemia-reperfusion (IR) liver

injury is abolished by N-acetylcysteine

VIRGINIA FERNANDEZ, ROMINA VARGAS, VALENTINA CASTILLO,

NICOLAS CADIZ, GLADYS TAPIA, AND LUIS A. VIDELA

Program of Pharmacology, ICBM, Faculty of Medicine, University of Chile,

Santiago, Chile

Liver ischemia reperfusion (IR) injury is a major clinical drawback

during surgery. Significant hepatoprotection against IR injury,

underlying transient oxidative stress (OS) induction, is achieved by

Fe administration. Abrogation of IR injury is related to abolishment

of the IR-induced liver glutathione depletion and recovery of redoxsensitive

NF-κB signaling pathway, lost during IR. The aim of this

study was to assess the role of transient OS in Fe preconditioning,

using N-acetylcysteine (NAC) before Fe administration. Male

Sprague–Dawley rats, pretreated with 1.0 g of NAC/kg 0.5 h before

each Fe dose (6 doses of 50 mg/kg, ip, every second day for 10 days)

were subjected to partial IR (1 h of vascular clamping and 20 h of

reperfusion) or sham laparotomy (control). At the end of the

reperfusion period, blood and liver samples were taken for analysis

of serum aspartate (AST) and alanine (ALT) aminotransferases,

hepatic histology, and liver glutathione (GSH) content. Fe-induced

transient OS (changes in liver protein carbonyl/total GSH contents,

24-72 h after Fe administration) was abolished by NAC pretreatment.

In concomitance, Fe protection against hepatic IR injury (serum AST

and ALT values and liver histologies) was suppressed by NAC

administration, with abrogation of the normalization in the IR-induced

liver OS (GSH levels), induced by Fe preconditioning. Data presented

suggest that a transient OS of the liver is an important trigger for Fe

preconditioning, evidenced in a warm IR injury model.

Supported by FONDECYT 1110006

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Comparative study of antioxidants and protective effects of

Rutin and 2,4,6-trihydroxiacetophenone from Myrcia

multiflora in hepatotoxicity induced by carbon tetrachloride

in mice

EDUARDO ANTONIO FERREIRA 1 , ELIANA FORTES GRIS 1 , KARINA

BETTEGA FELIPE 2 , JOÃO FRANCISCO GOMES CORREIA 2 , DANILO

WILHELM FILHO 3 , AND ROZANGELA CURI PEDROSA 2

1 University of Brasilia, Campus Ceilândia, Brasília, DF, Brazil; 2 Federal

University of Santa Catarina, Department of Biochemistry; 3 Federal

University of Santa Catarina, Department of Ecology and Zoology

The hepatoprotective and antioxidant effects of Rutin and 2',4',6'trihydroxyacetophenone

(THA), an acetophenone derived from the

plant Myrcia multiflora, were evaluated against carbon tetrachloride

(CCl 4 )-induced liver damage in mice. The mice were orally treated

with saline or THA (6.0 mg/kg bw) or Rutin (6.0 mg/kg bw) or

Silymarin (SIL) (24 mg/kg bw), once a day for 7 days and on the 8th

day were administrated CCl4 (0.5 ml/kg bw, 20% in olive oil, i.p.).

Approximately 24 h after the i.p. administration of CCl 4 , were evaluated

the oxidative stress biomarkers (lipoperoxidation-TBARS, protein

carbonyl, DNA damage and GSH content) and hepatic serum

enzymes (AST and ALT). CCl 4 induced mice liver damage increased

the AST (95%) and ALT (71%) levels in serum as compared with

the control group significantly (p < 0.01), as well as increasing of the

toxicity liver evaluated by TBARS (61%), protein carbonyl (41%),

DNA damage (100%) and GSH content (65%). The assays showed

that pre-treatment with THA or Rutin prevented the elevation of serum

enzymatic activities of AST (23% and 19%, respectively), ALT

(46% and 27%), as well as the increases of hepatic lipid peroxidation

as measured by the levels of TBARS (83% and 54%), protecting

also the protein carbonylation (38% and 34%) and oxidative DNA

damage the hepatocytes (55% and 35%), and increased the levels of

GSH (24% and 18%) significantly (p


Atherosclerosis-related genes are differentially expressed by

methionine-supplemented or methionine-deficient diets in

female mice

ALEXANDRE FERRO AISSA 1 , TARSILA DAYSY URSULA HERMOGENES

GOMES 2 , MARA RIBEIRO DE ALMEIDA 2 , LÍVIA CRISTINA HERNANDES 2 ,

JOANA D’ARC CASTANIA DARIN 2 , MARIA LOURDES PIRES BIANCHI 2 ,

AND LUSÂNIA MARIA GREGGI ANTUNES 2

1 Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo,

Ribeirão Preto, SP, Brazil; 2 Faculdade de Ciências Farmacêuticas de

Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

Methionine is an essential amino acid obtained from the diet which

plays a pivotal role in DNA methylation. Consistent with the essential

role of methionine in the methylation process, diets with inadequate

methionine content result in severe implications for cellular

processes. Furthermore, an imbalance in methionine can elevate the

homocysteine level which is associated with an increased risk of cardiovascular

disease (CVD). Therefore, it may also be of interest to

investigate how methionine intake can induce CVD by identifying

differentially expressed genes. So, adult female Swiss mice were fed

a control diet, a methionine-supplemented diet (MSD) or a methionine-deficient

diet (MDD) over a 10-week period (n = 6/group). The

animals were euthanized at the end of the treatment, and heart tissue

was collected for gene expression profiling analysis. We analyzed

the gene expression changes influenced by the diets using the Mouse

Atherosclerosis RT² Profiler PCR Array, which profiles the expression

of 84 genes related to atherosclerosis. All these genes represent

diverse functional categories, including adhesion molecules,

apoptosis, cell growth and proliferation, extracellular molecules, lipid

transport and metabolism, response to stress, and transcription

regulators. In the animals treated with MSD, we identified 39 differentially

expressed genes, including 31 up-regulated and 8 down-regulated.

In these animals, the groups of genes involved in cell growth

and adhesion molecules had the largest change of expression compared

to the control group. In the animals fed the MDD, 22 genes

were differentially expressed, 4 up-regulated and 18 down-regulated.

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The genes with the most different expression were Apoa1, Fgb, Apob

and Fga, which were down-regulated 171-, 113-, 49- and 31-fold,

respectively. Here, we reported that methionine imbalance in the

diet can induce difference in expression of several genes related

to CVD.

Financial Support: FAPESP.

105


Flavonoid profile and effect of chronic consumption of

Brazilian red wines on in vivo antioxidant activity

ELIANA FORTES GRIS 1,2 , FULVIO MATTIVI 3 , EDUARDO ANTONIO

FERREIRA 1,2 , URSKA VRHOVSEK 3 , DANILO WILLIAM FILHO 1 , R. C.

PEDROSA 1 , AND MARILDE BORDIGNON-LUIZ 1

1 Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346,

Itacorubi, Florianópolis, SC, Brasil; 2 Universidade de Brasília, Faculdade

Ceilândia, QNN 14, Ceilândia, Brasília, DF, Brasil; 3 Edmund Mach

Foundation, IASMA Research and Innovation Centre, Food Quality and

Nutrition Department, Via E. Mach 1, 38010 San Michele all’Adige, TN, Italy

In this study, Vitis vinifera L wines cv. Cabernet Franc, Merlot,

Sangiovese and Syrah, 2006 and 2007 vintages, produced in São

Joaquim, a new wine-producing region in southern Brazil, were evaluated.

As phenolic compounds, and specially flavonoid compounds,

are one of the most important parameters in assessing the quality of

wines and possible responsible for beneficial properties of wines, in

this study, the levels of the main anthocyanins and their respective

derivatives (esters of acetic and p-coumaric acids) and flavonols

(myricetin, quercetin, laricitrin, kaempferol, isorhamnetin and

syringetin) (HPLC-DAD and HPLC-DAD-MS analysis), and also

the in vivo antioxidant activity in mice are reported. The antioxidant

capacity of plasma was assessed through the reduction of ferric iron

(FRAP). Moreover, lipid peroxidation (TBARS), carbonyl protein

(CP) and reduced glutathione (GSH) levels were determined. The

results for the phenolic compounds content of the wine samples were

considered appropriate for quality red wines; the mean total

anthocyanins content was 47.43 mg L -1 , and ranged from 12.94 to

125.60 mg L -1 ; the main flavonols in these wine samples were quercetin

and myricetin and the concentration of total flavonols in the

wine samples ranged from 20.81 to 46.79 mg L -1 . The wine consumption

promoted a significant increase in FRAP, decreases in the

TBARS and CP levels. Moreover, the phenolic content of the wines

was positively correlated with the in vivo antioxidant capacity promoted

by chronic wine consumption. Thus, the data presented herein

provide evidence that red wine ingestion contributes to increasing

the in vivo antioxidant activity and suggest that this effect can be

attributed to the flavonoid composition of the wine.

106


Hypolipidemic activity of brazilian red wines and influence of

tyrosol and resveratrols compounds

ELIANA FORTES GRIS 1,2 , FULVIO MATTIVI 3 , EDUARDO ANTONIO

FERREIRA 1,2, URSKA VRHOVSEK 3 , ROZANGELA CURI PEDROSA 1 , AND

MARILDE T. BORDIGNON-LUIZ 1

1 Federal University of Santa Catarina; Department of Food Science and

Technology, Florianópolis, SC, Brazil; 2 University of Brasilia, Campus

Ceilândia, Brasília, DF, Brazil; 3 Edmund Mach Foundation, IASMA Research

and Innovation Centre, Food Quality and Nutrition Area, San Michele

all’Adige, TN, Italy

In this study Vitis vinifera red wines, cv. Cabernet Franc, Merlot,

Sangiovese and Syrah, 2006 and 2007 vintages, from the São Joaquim

region, a new grape growing region at southern Brazil, were evaluated.

The effect of chronic consumption of these samples on the

hypolipidemic activities was monitored in C57BL6 mice knockout

LDL, treated with hypercholesterolemic diet, through biomonitoring

of lipid levels in animals. Moreover, the contents of tyrosol and

resveratrol monomers were quantified and correlated with the results

of lipids levels in animals. Hypercholesterolemic diet promoted

hyperlipidemia in the animals, and it was characterized by an increase

in total cholesterol levels (hypercholesterolemia), and in

triglycerides (hypertriglyceridemia) (p< 0.05). Moreover, the

hypercholesterolemia in the mice showed a highly atherogenic lipoprotein

profile (p< 0.05). The results revealed high concentrations of

tirosol and resveratrols in the wine samples. Biomonitoring of

hypolipidemic activities in vivo revealed that consumption of these

wines reduced significantly the hypercholesterolemia (32 to 40%)

and hypertriglyceridemia (37 to 51%) promoted by hypercholesterolemic

diet; in addition, decreased the atherogenic index (42 to 59%)

and increased significantly the values of cholesterol HDL (22 to 26%)

(p< 0.05). The lipid levels showed strong correlations with the tyrosol

and resveratrols contents, particularly with total resveratrols and cis

isomers, suggesting the possible involvement of these compounds,

or their metabolites, in the mechanism of action. These findings confirm

the important biological activity of these compounds when

present in high concentrations, as in the case of the wines analyzed

in this study.

107


Immunomodulatory potential of wild blueberries (Vaccinium

angustifolium) in human intestinal epithelial Caco-2 cells:

preliminary data

DANIELA FRACASSETTI 1 , CRISTIAN DEL BO’ 1 , SIMONE GUGLIELMETTI 1 ,

PATRIZIA RISO 1 , VALENTINA TAVERNITI 1 , DOROTHY KLIMIS-ZACAS 2 ,

AND MARISA PORRINI 1

1 Università degli Studi di Milano, Department of Food, Environmental and

Nutritional Sciences, Milan, Italy; 2 Department of Food Science and Human

Nutrition, University of Maine, Orono, Maine, USA

Wild blueberries (WB) are a rich source of polyphenols, particularly

anthocyanins, which may be beneficial for human health because

of their antioxidant, antiinflammatory and immunomodulatory

potential. This study aims to investigate the immunomodulatory potential

of three WB fractions (anthocyanin, phenolic and soluble fraction)

in an inflammatory state model, based on human intestinal Caco-

2 cells. A reporter gene system was prepared by transfecting Caco-2

cells with the pNiFty-SEAP reporter- construct containing an engineered

ELAM promoter with 5 NF-kB binding sites and an insect

luciferase reporter gene. The transfected Caco-2 cells were stimulated

with interleukin (IL)-1β to mimic an inflammatory state and

then supplemented with 5 mg/L, 25 mg/L, 50 mg/L and 100 mg/L of

the anthocyanin, phenolic and soluble fractions. Immunomodulatory

activity for each fraction was monitored in real time by quantification

of bioluminescence with a luminometer. The addition of

anthocyanins at the concentration of 50 and 100 mg/L reduced the

bioluminescence, and therefore NF-κB activation, up to 70% and

85% (p


Antihypertensive effects of epicatechin

CESAR FRAGA, CORINA LITTERIO, AND MONICA GALLEANO

University of Buenos Aires

Flavanols and their oligomers (procyanidins) are naturally occurring

plant compounds, which biological effects could explain some

of the cardiovascular benefits linked to the consumption of fruit and

vegetables. Dietary intervention studies in humans and animals indicate

that flavanol-rich foods, i.e. wine, tea, and chocolate may exert

blood pressure (BP) lowering effects in humans and rats. Recent reports

have shown that such BP decrease could be dependent on NO

production, although the mechanisms and the fruit and vegetable

components responsible of such associations remain unclear. Hypertension

was induced in male Wistar rats by treatment with the NOsynthase

inhibitor L-NAME (360 mg/l drinking water, about 10 mg/

rat/day). The addition of (-)-epicatechin (EPI) in the diet (0.2-4.0

mg/g diet) significantly modulated the L-NAME-dependent increase

in BP. EPI had an antihypertensive effect that was observed when

EPI was administered simultaneously with L-NAME, or after L-

NAME-induced hypertension was already established. The EPI effect

was dose-dependent, and disappeared with the removal of EPI

from the diet. The decrease in BP was associated with the presence

of EPI in plasma plus the improvement in markers of oxidative damage,

i.e. malondialdehyde and oxidized glutathione, and NF- κB.

Based on these results and previous work, we hypothesize that

the effects of EPI are consistent with the modulation of oxidant production

which allows a NO steady-state which finally result in the

antihypertensive effect. If this hypothesis is physiologically relevant,

it will provide an insight on how flavanol-containing nutrients are

involved in the regulation of tissue NO availability and cell redox

state, and subsequently in the prevention of the onset or progression

of hypertension and associated diseases.

109


Neuroblastoma sensitivity to Bortezomib:

role of Nrf2 transcription factor

ANNA LISA FURFARO 1 , SABRINA PIRAS 1 , MARIO PASSALACQUA 1 ,

CINZIA DOMENICOTTI 1 , MARIA ADELAIDE PRONZATO 1 ,

UMBERTO MARIA MARINARI 1 , LORENZO MORETTA 2 , NICOLA TRAVERSO 1 ,

AND MARIAPAOLA NITTI 1

1 Department of Experimental Medicine, University of Genoa, Genoa;

2 Giannina Gaslini Institute, Genoa

Bortezomib (BTZ), a selective inhibitor of proteasome approved

by US FDA for the treatment of some human malignant solid tumours,

has shown impressive clinical activity to overcome cancer

cell resistance to conventional therapy. However, the molecular mechanisms

of BTZ action are poorly understood. The transcription factor

Nrf2 plays a key role in the development of drug resistance and is

known to be regulated by the ubiquitin-proteasome system.

The purpose of this study is to point out the role of Nrf2 in neuroblastoma

cell response to BTZ treatment. In fact, neuroblastoma,

the most common solid tumour in childhood, is characterised by a

poor prognosis due to the gain of a chemoresistant phenotype.

We used two different neuroblastoma cell lines, SHSY-5Y and

HTLA, well known respectively as sensitive and resistant to therapy.

Evaluation of cell viability showed that SHSY-5Y cells were significantly

more sensitive than HTLA cells when exposed for 24h to

BTZ (20-40 nM). BTZ-treatments in HTLA cells were able to induce

a strong increase in mRNA expression of Nrf2 target genes

(HO-1, x-CT, NQO1), while only a slight gene induction of HO-1

and x-CT was observed in SHSY-5Y cells exposed to BTZ in comparison

to control cells. Moreover, in both cell lines BTZ-treatments

caused the nuclear translocation of Nrf2.

Our preliminary results lead to hypothesize that the crucial point

driving the different sensitivity of SHSY-5Y and HTLA cells to BTZ

might be the different ability of Nrf2 to bind DNA.

Grants: Genoa University, PRIN 2009M8FKBB_002

110


Potentiation of amyloid-β peptide neurotoxicity by

4-hydroxynonenal and 24-hydroxycholesterol

PAOLA GAMBA, GABRIELLA TESTA, SIMONA GARGIULO, BARBARA

SOTTERO, FEDERICA DI SCIPIO, ANDREA E. SPRIO, PAOLINA SALAMONE,

GIOVANNI N. BERTA, GABRIELLA LEONARDUZZI, AND GIUSEPPE POLI

Department of Clinical and Biological Sciences, University of Turin,

Orbassano (Turin), Italy

Alzheimer’s disease (AD) is the most prevalent form of mental

decline in developed countries. It is characterized by extracellular

deposits of amyloid-β (Aβ) (senile plaques) and intracellular inclusions

of hyperphosphorylated tau (neurofibrillary tangles). Moreover,

numerous data indicate that intraneuronal Aβ plays a primary

role in AD pathogenesis. A growing body of evidence suggests a

mechanistic link between lipid peroxidation and AD. The brain is

particularly vulnerable to oxidative stress, which is responsible for

the formation of highly reactive aldehydes, of which the most relevant

to brain pathophysiology appears to be 4-hydroxynonenal

(HNE), and cholesterol oxidation products, named oxysterols.

HNE production in the brain is stimulated by Aβ and, conversely,

Aβ production is up-regulated by this multifunctional aldehyde. Because

elevated levels of HNE have been found in the brain of AD

patients, it has been proposed as a biomarker of AD. Moreover, in

the brain, cholesterol is primarily converted into 24hydroxycholesterol

(24-OH) by CYP46A1. This oxysterol has been

shown to enhance the neurotoxic effect of the Aβ peptide in human

differentiated neuroblastoma cell lines, as well as augmenting ROS

generation.

We observed the ability of HNE and 24-OH to interact with Aβ,

with consequent potentiation of Aβ’s cytotoxicity as determined in

vitro using neuron-like cells derived from human dental-pulp progenitor

cells. Pre-incubation of cells with the aldehyde or the oxysterol

strongly enhanced Aβ uptake and intraneuronal accumulation, by

up-regulating a cluster of membrane receptors, composed by CD36,

β1-integrin and CD47. Consequently, the two lipid peroxidation products

markedly potentiate Aβ neurotoxicity, in terms of necrosis; this

event was confirmed by the employment of specific antibodies against

CD36 or β1-integrin. These data support a primary involvement of

altered brain lipid metabolism in the pathogenesis of AD.

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Cholesterol oxidation products contribute to the imbalance

between MMP-9 and TIMP-1/TIMP-2,

in the atherosclerotic plaque

SIMONA GARGIULO, PAOLA GAMBA, BARBARA SOTTERO, GABRIELLA

TESTA, GIUSEPPE POLI, AND GABRIELLA LEONARDUZZI

Department of Clinical and Biological Sciences, University of Turin, San

Luigi Gonzaga Hospital, Orbassano (Torino), Italy

The rupture of a vulnerable atherosclerotic plaque is a major cause

of thrombus formation leading to acute coronary syndrome. Connective

tissue integrity depends on a balance between degradation

and repair of the extracellular matrix. Its excessive degradation by

matrix metalloproteinases (MMPs), secreted mainly by macrophages,

contribute to plaque rupture. The MMPs are inhibited by tissue inhibitors

of metalloproteinases (TIMPs). Among MMPs, MMP-9 has

a prominent role in plaque destabilization and rupture. Oxysterols,

cholesterol oxidation products which are abundant in oxidized LDL,

are able to modulate several biochemical effects involved in the

pathogenesis of atherosclerosis.

We have studied the effect of an oxysterol mixture, whose composition

is similar to that found in the human plaques, on the MMP-

9 expression by human promonocytic U937 cells.

Oxysterols induced expression, synthesis and enzymatic activity

of MMP-9 while it didn’t affect TIMP-1 and TIMP-2 levels. Moreover,

oxysterol mixture induced the release of reactive oxygen species

(ROS) through NADPH-oxidase activation and mitochondria. By

using specific inhibitors and siRNAs we demonstrated the involvement

of classic and novel protein kinase C in NADPH-oxidase activation

and ROS overproduction as well as in MAPK activation. The

oxysterol mixture also up-regulated the nuclear levels of c-Fos and

c-Jun, both subunits of AP-1, and of p65, the catalytic subunit of

NF-kB.

Our data show that oxysterols accumulating in advanced atherosclerotic

lesions significantly contribute to plaque vulnerability by

promoting the imbalance between MMP-9 and TIMP-1/2 in phagocytic

cells.

112


Growth conditions and abiotic factors affect final

concentration of bioactive compounds in broccoli sprouts

ANNA MARIA GIUSTI 1 , MARIATERESA MALDINI 2 , SIMONA BAIMA 2 ,

FAUSTA NATELLA 2 , AND CRISTINA SCACCINI 2

1 Department of Experimental Medicine -Section of Medical Physiopathology,

Food Science and Endocrinology, University “Sapienza” of Rome, p.le Aldo

Moro, 5 00185 Rome Italy; 2 National Research Institute on Food and

Nutrition-INRAN, via Ardeatina 546, 00178 Rome, Italy

The beneficial effects of Brassica vegetables on human health have

been attributed to their complex mixture of phytochemicals such as

glucosinolates and anthocyanins.

Glucosinolates have attracted great interest from both toxicological

and pharmacological point of view, as they are able to induce

phase 2 detoxication enzymes and protect animals against chemically

induced cancer. Young broccoli plants are an especially good

source of chemoprotective glucosinolates, whose levels are greater

than in mature plants.

Anthocyanins, responsible for the red, purple and blue colours of

many vegetables and fruits, have been reported to be antioxidants

and vasoprotectors, inhibit inflammation and the growth of cancerous

cells and have anti-obesity effects.

The quali-quantitative composition of bioactive compounds in

vegetables is strongly affected by various factors, such as, growing

conditions and abiotic stresses.

In this work, the influence of different light and temperature regimes,

as well as, of some chemical and hormonal treatments on

glucosinolates and anthocyanins production in broccoli sprouts was

investigated.

A rapid and sensitive LC-MS/MS (MRM) method was developed

to analysed and quantify glucosinolates in broccoli sprouts. Total

anthocyanins content was determined by a spectrophotometric method

and by HPLC-DAD.

The results showed that glucosinolates and anthocyanins content

significantly increased in broccoli sprouts exposed to light respect to

sprouts grown in the dark. Also, abiotic stresses (sucrose, mannitol,

113


NaCl) and some hormones involved in plant stress responses were

able to increase accumulation of anthocyanins and glucosinolates,

with sucrose being the most effective one.

This work was supported by Italian Ministry of Agriculture, Food & Forestry

(MiPAAF) grant “NUTRIGEA” (DM 30281 23/12/2009).

114


KRIT 1 and the Reactive Oxygen Species.

A novel molecular pathway at the basis of cerebral cavernous

malformations?

LUCA GOITRE, MIGUEL VILLORIA-RECIO, VALENTINA CUTANO,

ROSALIA CANZONERI, ELIANA TRAPANI, ALESSANDRO MORINA, AND

SAVERIO FRANCESCO RETTA

Department of Clinical and Biological Sciences, University of Turin,

Orbassano (TO)

KRIT1 is a gene responsible for Cerebral Cavernous Malformations

(CCM), a major cerebrovascular disease characterized by abnormally

enlarged and leaky capillaries that predispose to seizures,

focal neurological deficits, and fatal intracerebral hemorrhages. Comprehensive

analysis of the KRIT1 gene in CCM patients has suggested

that KRIT1 functions need to be severely impaired for pathogenesis.

However, the molecular and cellular functions of KRIT1 as

well as CCM pathogenesis mechanisms are still research challenges.

We found that KRIT1 is involved in the maintenance of the intracellular

Reactive Oxygen Species (ROS) homeostasis to prevent

oxidative cellular damage. Specifically, KRIT1 loss/down-regulation

is associated with a significant increase in intracellular ROS levels.

Moreover, the modulation of intracellular ROS levels by KRIT1 is

strictly correlated with the modulation of the expression of proteins

involved in oxidative stress responses, including SOD2, FoxO1,

COX2 and c-Jun. Furthermore, the KRIT1-dependent maintenance

of low ROS levels facilitates the downregulation of cyclin D1 expression

required for cell transition from proliferative growth to quiescence.

Conversely, cell treatment with ROS scavenging agents

overcomes the upregulation of cyclin D1 and the reduced cell capacity

to exit from the proliferative cycle caused by KRIT1 loss. Finally,

the enhanced ROS levels in KRIT1-/- cells are associated with an

increased cell susceptibility to oxidative DNA damage.

Taken together, our results point to a mechanistic model whereby

KRIT1 limits the accumulation of intracellular oxidants and prevent

oxidative stress-mediated cellular dysfunction and DNA damage,

suggesting a novel mechanism for CCM pathogenesis.

115


Semi-targeted tandem mass spectrometry-based lipidomic

analysis of oxidized phosphatidylcholines induced by UVA

irradiation in dermal fibroblasts

FLORIAN GRUBER 1 , OLGA V. OSKOLKOVA 2 , ERWIN TSCHACHLER 1,3 ,

WOLFGANG BICKER 4 , AND VALERY N BOCHKOV 2

1 Departments of Dermoatology; 2 Vascular Biology of the Medical University

of Vienna, Austria; 3 CE.R.I.E.S, France; 4 FTC Forensisch-Toxikologisches

Labor BetriebsgmbH, Vienna, Austria

Oxidized phospholipids (OxPLs) are increasingly recognized as

molecules that are not only markers of oxidative stress but also mediators

involved in pathogenesis or resolution of disease states. We have

recently shown that UVA-generated non-fragmented OxPCs induce

adaptive responses in dermal fibroblasts (FB), while production of

fragmented species containing reactive carbonyl groups is correlated

to skin ageing and pathologies of the skin that develop upon solar

exposure such as actinic elastosis.

We report a procedure for analysis of a broad spectrum of molecular

species generated by UVA-1 oxidation (40 and 80 J/cm²) of four

most abundant species of polyunsaturated phosphatidyl-cholines in

dermal fibroblasts. The approach is based on two subsequent liquidliquid

extraction steps (acidic methanol/hexane extraction and Folch

phase separation) followed by reverse-phase core-shell HPLC coupled

to ESI-MS/MS.

More than 500 peaks corresponding in retention properties to polar

and oxidized PCs were detected within 8 minutes at 99 m/z values

in extracts from human dermal fibroblasts. Importantly, 217 of

these peaks were fluence-dependently and significantly increased

upon exposure of cells to UVA, suggesting that these are genuine

oxidized species.

We confirmed our previous findings that specific biologically active

OxPCs are dose dependently increased by UVA-1 and observed

accumulation of a plethora of non-fragmented and fragmented Ox-

PCs. This opens the possibility to identify novel OxPLs and OxPL

“signatures” that may either have detrimental effects in photoageing

and UV-induced pathologies, but also photoproducts that mediate

beneficial effects of UVA radiation, which is utilized for therapy of

inflammatory skin diseases.

116


The importance of inflammatory and pro-oxidant molecules in

monitoring colorectal carcinogenesis

TINA GUINA, MARCO MAINA, ALESSANDRO FALCONE, SIMONE

CALFAPIETRA, FIORELLA BIASI, AND GIUSEPPE POLI

Department of Clinical and Biological Sciences, University of Turin, Italy

The association among colorectal carcinoma (CRC) development,

inflammation and oxidative stress has been reported by many studies.

Macrophages are mainly involved in different phases of colorectal

carcinogenesis, expressing M1 pro-inflammatory phenotype in the

early stages of CRC, or M2 pro-tumoral phenotype in the later ones.

They are able to induce oxidative reactions involved in the production

of certain molecules engaged in carcinogenic process. Serum

levels of lipid peroxidation end product HNE (4-hydroxyl-nonenal)

was compared with M1-derived pro-inflammatory molecules

IL(interleukin)-8 and IL-6, and M2-derived pro-tumoral molecules

VEGF (vascular endothelial growth factor) and MMPs (matrix

metalloproteases) in patients with CRC at different stages of progression.

The results were then compared with the commonest inflammatory

marker CRP (C-reactive protein).

The serum levels of IL-8 increased in parallel with CRC malignancy,

reaching the significance at stages II/III. Similar behaviors of

MMP-9 activity and levels were observed. CRP showed similar trend,

but it was not correlated with IL-8 and MMP-9. Inversely, HNE significantly

decreased at stages II/III, in accordance with other studies

showing that malignant cells become less susceptible to lipid oxidation

anti-proliferative effect in order to ensure their survival. In conclusion,

combined evaluation of certain serum markers of macrophage

activation (M1/M2) during colorectal carcinogenesis, together

with oxidative stress marker HNE, might be considered as future

approach for monitoring colorectal tumor progression.

117


Development of hypoxia-sensitive fluorescent probes

for bioimaging

KENJIRO HANAOKA 1 , WEN PIAO 1 , KAZUKI KIYOSE 1 , YASUNOBU HIRATA 2 ,

AND TETSUO NAGANO 1

1 Graduate School of Pharmaceutical Sciences, the University of

Tokyo, Tokyo, Japan; 2 Department of Internal Medicine, the

University of Tokyo Hospital, Tokyo, Japan

Hypoxia, which is caused by an inadequate oxygen supply, is a

feature of several disease states, including cancer, cardiopathy, vascular

diseases, and ischemic diseases. One of the earliest cellular responses

is stabilization of HIF-1, a transcription factor of genes,

thereby, a series of proteins involved in glycolytic metabolism and

neovasculization are expressed. So, hypoxia-specific fluorescence

probes would be useful for elucidating biological systems, however,

there are few practical fluorescent probes for detecting hypoxia. A

decrease in oxygen causes an increase in reductive stress, and we

focused on hypoxia-selective bioreduction, which was occurred by

various reductases, and tried to design and synthesize fluorescent

probes which become fluorescent when they reduced by reductases

under hypoxic conditions. Many small molecule-based probes to detect

hypoxia (including probes for positron emission tomography (PET),

immunostaining, and phosphorescence imaging) take advantage of

this phenomenon, and most probes employ nitroaromatic or quinone

derivatives as hypoxia-sensitive moieties. Here, we focused on the

fluorescence quenching properties of azo compounds, and developed

a series of, i. e., green, red and NIR, fluorescent probes for detecting

hypoxia based on azo compound which was selectively reduced by

reductases under hypoxic conditions. Especially, near-infrared (NIR)

is relatively poorly absorbed by biomolecules such as hemoglobin,

water and lipids, and so it can penetrate deeply into tissues. We also

applied them to living cultured cells and/or mice, and achieved fluorescence

hypoxia imaging of biological living samples (JACS vol.132,

p15846 (2010).

118


Increased brain mitochondrial oxidative stress and genes

related to redox signalling induced by a high fat/high fructose

are not prevented by cinnamon polyphenols

ISABELLE HININGER-FAVIER 1,2 , THOMAS POYOT 3 , MIREILLE OSMAN 1,2 ,

CÉCILE BATANDIER 1,2 , KARINE COUTURIER 1,2 , FRÉDÉRIC CANINI 4 , AND

ANNE-MARIE ROUSSEL 1,2

1 Université Joseph Fourier, Laboratoire de Bioénergétique Fondamentale et

Appliquée, Grenoble, France ; 2 Inserm U1055, Grenoble, France;

3 Département de Radiobiologie et de Radiopathologie, Centre de

Recherches du Service de Santé des Armées, La Tronche, France;

4 Département des Facteurs Humains, Centre de Recherches du Service de

Santé des Armées La Tronche Cedex, France

Long term consumption of western diet leads to insulin resistance

(IR), oxidative stress and increased risk of metabolic syndrome and

diabetes type 2. More recently, brain has been associated with an

increased risk of cognitive impairment. Brain mitochondria dysfunction

related to an increased oxidative stress could be the missing link

between IR and cognitive impairment. Therefore, in this work, we

aim to study the impact of a high fat/high fructose diet (HF/HF),

inducing IR, on mitochondria oxidative stress and the potential protective

effects of cinnamon polyphenols, reported as antioxidants and

insulin sensitizers. Male Wistar rats were fed with a control diet or a

HFHF enriched or not with cinnamon polyphenols (CN) for 12 weeks.

After 12-week of HFHF diet we observed a mitochondrial oxidative

stress assessed by an increased TBARS level and a decreased of

total antioxidant capacity, while in contrast mitochondrial thioredoxine

reductase enzyme implicated against damaging protein oxidative

stress, was increased, suggesting an adaptative response. This effect

could be due to the effect of HFHF diet to lowered mRNA expression

of the inhibitory unit Keap1 implicated on KEAP1/Nrf2/ARE

pathway a chemical sensor of cellular redox tone. Glutaredoxine 2, a

redox sensor, was also decreased by HFHF diet. CN added to the

diet did not prevent oxidative stress and had no effect on redox

signaling. However, CN added to the diet reversed the increased

mRNA expression of IkB, observed in HFHF group, and brought

back these values to those of the control group, suggesting an

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antiinflammatory effect. Endly, in HF/HF diet, we reported a 20%

decreased of mRNA AMPK-a expression, whereas CN addition to

the diet led to an opposite effect and reversed the effect of HFHF on

AMPK signalling. These data show that HFHF consumption increases

brain mitochondrial oxidative stress and modulates related

brain redox signaling.

120


Oxidative stress related biomarkers in aged-related human

studies on vitamin intervention and healthy aging cohorts

EUGENE JANSEN 1 , PIET BEEKHOF 1 , JOHANNES CREMERS 1 ,

AND ABDONAS TAMOSIUNAS 2

1 Laboratory for Health Protection research, National Institute of Public

Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands;

2 Department of Population Studies, Institute of Cardiology, Lithuanian

University of Health Sciences, Sukileliu, av. 17, Kaunas LT-50161, Lithuania

Oxidative stress and redox status have been related to many chronic

diseases and ageing. To test whether biomarkers of oxidative stress

really can describe the underlying pathological processes, large-scale

screening is recommended in epidemiological cohorts. In addition

many components of the oxidation and antioxidant system must be

determined simultaneously to obtain an overall view of these processes.

In this study we present an automated method for the determination

of a large set of biomarkers of both oxidative processes and antioxidant

status, which is suitable for large-scale screening. The following

processes with representative biomarkers can be measured in

large series: lipid peroxidation, protein oxidation, glycation, inflammation,

tissue toxicity, iron status, total antioxidant status, anti oxidant

enzymes and general serum antioxidants.

The auto-analyser used for the measurements of oxidative stress

and antioxidant biomarkers is the LX-20 Pro (Beckman-Coulter).

The kits used on this auto-analyser are from various companies and

have been adjusted to the format of analyzer.

Applications will be shown of an age-related biomarker study of

a strategic intervention study with multivitamin and mineral supplements

(SOR/340006) and of a European cohort study on Health and

Aging (CHANCES, EC-FP7).

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Expression oxidative stress biomarkers in Pterygium

and normal conjunctiva

SIV JOHNSEN-SORIANO, RAUL MARTINEZ-BELDA,

CRISTINA PERIS-MARTINEZ, EMMA ARNAL,

AND FRANCISCO JAVIER ROMERO

FOM, Valencia, Spain

Purpose: To analyze the expression of oxidative stress markers

in pterygium tissue. Pterygium is a common opthalmological disease,

which has a fibrovascular neoformation characterized by a

triangular (wing-shaped) overgrowth of abnormal conjunctiva onto

the cornea and is composed of epithelium and a highly vascular loose

connective tissue.

Methods: 18 pterygium were surgically removed using the free

superior bulbar conjuctiva FLAP procedure and 15 normal bulbar

conjunctivas were also obtained. The research reported was conducted

in compliance with “Declaration of Helsinki”. Formalin-fixed,

paraffin-wax-embedded tissues were analyzed by immunohistochemistry

with GSH and HNE (Hydroxynonenal) antibodies.

Results: Our study revealed that the GSH positive cell density

significantly decreased (60,9 ± 2,7 Ki-67 positive cells/ total cells) in

the pterygium samples compared to the normal conjunctiva samples

(70,8 ± 5,0 Ki-67 positive cells/ total cells). Moreover, the HNE positive

cell number was significantly higher in the pterygium samples

(75,0± 3.5 HNE positive cells/ total cells) than in the conjunctiva

samples (66,9 ± 5.2 HNE positive cells/ total cells).

Conclusions: The finding of a lower GSH content and a higher

HNE content in pterygium samples suggest implication of oxidative

stress pathways in human pterygium pathogenesis.

122


Inhibition of myeloperoxidase-derived oxidant formation

by flavonoids

TRACEY KAJER 1 , CLARE L. HAWKINS 1,2 , SHANLIN FU 3 , DAVID

PATTISON 1 , GARRY GRAHAM 4 , AND MICHAEL J. DAVIES 1,2

1 The Heart Research Institute, Sydney, NSW 2042, Australia;

2 Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia;

3 University of Technology, Sydney, NSW, Australia; 4 Department of

Pharmacology, University of New South Wales and Department of Clinical

Pharmacology, St Vincent’s Hospital, Sydney, NSW, Australia

Leukocyte-derived myeloperoxidase (MPO) generates powerful

oxidants including hypochlorous (HOCl) and hypothiocyanous

acids (HOSCN)from H 2 O 2 and Cl - andSCN - ions respectively at sites

of inflammation. The damage induced by these species has been

implicated in a number of inflammatory pathologies including cardiovascular

disease. As MPO has two catalytic cycles, we hypothesised

that compounds that enhance the peroxidase activity of MPO

should reduce the formation of halogenating oxidants such as HOCl

and thus decrease oxidative damage. Studies with a range of model

phenols and flavonoids indicate that these materials act as effective

inhibitors of the formation of halogenating oxidants at low micromolar

concentrations. The effect of very low levels of these materials appears

to be additive, resulting in significant inhibition of oxidant formation

even when these are present at very low levels. In some cases

the radicals formed as a result of MPO-catalysed oxidation have been

detected, and resulting oxidation products characterised. A number

of these oxidation products are also effective MPO inhibitors. Overall,

we conclude that flavonoids and related materials can be effective

inhibitors of inflammatory damage induced by MPO, with this

arising primarily from inhibition of the halogenating enzymatic cycle

of MPO rather than as a result of scavenging of oxidants such as

HOCl and HOSCN once formed.

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Natural mutations leading to proteasomal degradation of

human Ncb5or, a novel oxidoreductase

FANNI S. KÁLMÁN, ÉVA KERESZTURI, ZSUZSA MOLNÁR, JÓZSEF MANDL,

AND MIKLÓS CSALA

Semmelweis University, Dept. of Medical Chemistry, Budapest

NADH cytochrome b5 oxidoreductase (Ncb5or) plays an as yet

unresolved role in the protection of β-cells against oxidative stress

and lipotoxicity. The predominant phenotype of lean Ncb5or-null

mouse is insulin-dependent diabetes due to β-cell death. The putative

role of human NCB5OR gene polymorphism in diabetes has

been poorly explored. Our aim was to investigate the effect of natural

exonic missense mutations on the expression of human NCB5OR

gene. Database search yielded five non-synonymous coding variants

in four exons. The mRNA and protein levels of exogenously

expressed wild type and mutant variants were analyzed in HEK293

and HepG2 cells. Although the mRNA levels were only slightly affected

by the mutations, two Glu-to-Gly replacements in the third

exon (p.E87G, p.E93G) caused a remarkable reduction in the expression

of Ncb5or protein. The repressing effect of these two mutations

was synergistic and could be attenuated by proteasome inhibitors.

Our results strongly indicate that p.E87G, p.E93G mutations

cause manifest conformational alterations in the b5 domain and lead

to enhanced proteasomal degradation of Ncb5or. These results provide

evidence for natural mutations leading to decreased Ncb5or protein

levels and also allow insight into determinants of the unique 3D

structure of the heme binding environment of this protein.

124


NADPH/NADP+ imbalance in the endoplasmic reticulum

results in mTOR pathway dependent autophagy

ORSOLYA KAPUY, JÓZSEF MANDL, AND GÁBOR BÁNHEGYI

Department of Medical Chemistry, Molecular Biology and Pathobiochemistry,

Semmelweis University, Budapest, Hungary

The surviving of living organisms are mainly dependent on the

ability of cells responding to external signals to make accurate decisions,

and to take the proper actions, such as cell growth and division

or cell death. Autophagy has an essential role in promoting cellularsurvival

by “self-eating” of parts of the cytoplasm and intracellular

organelles. A classical pathway controlling autophagy during starvation

involves the mTOR kinase. It has been recently shown that luminal

redox imbalance of pyridine nucleotides in the endoplasmic

reticulum (ER) together with oxidative stress results in autophagy.

Our goal is to reveal the regulatory mechanism of autophagy induction

caused by NADPH depletion at the ER, with a special emphasis

to explore its connection with mTOR signalling pathway. Luminal

NADPH reduction will be accomplished by silencing the NADPH

generating enzymes or by addition of pharmacological agent metyrapone

in HepG2 cells. The inhibition of mTOR is tested by following

the phosphorylation state of the main components of the pathway.

Autophagy was monitored by the widely used marker, LC3.

We show that depletion of luminal NADPH content has a significant

connection with mTOR pathway in mammalian cells. Due to ER

stress, FRAP the centre molecule of mTOR pathway gets inactivated

by dephosphorylation. Interestingly silencing the NADPH generating

enzymes or adding metyrapone has different effects on downstream

substrate of FRAP. Our data suggest that redox imbalance

caused ER stress is not as drastic inhibiting mTOR pathway as

rapamycin addition or starvation.

125


The effect of Salvia virgata on SOD and TBARS activities

of chronic myeloid leukemia cells

GÖKÇE KARATOPRAK, MÜKERREM BETÜL YERER, SELEN ERTÜRK,

AND MÜBERRA KOSAR

University of Erciyes

Turkey is a important country for Salvia species. The flora of Turkey

includes 88 species of the genus Salvia. Salvia virgata (Lamiaceae)

which has shown to be extremely rich with the phenolic compounds

that allows this species to be an important member of antioxidant

plants. The decoction of S. virgata herb is traditionally used in

leukemia in northwest Anatolia region. This study was performed to

investigate the effect of different Salvia extracts on superoxide

dismutase (SOD) activities and thiobarbituric acid reactive substances

(TBARS) levels of Jurkat (chronic myeloid leukemia) cells.

The 70% methanol and water extracts were prepared from the

aerial parts of S. virgata collected from Bursa, Turkey. Gallic acid,

rosmarinic acid, butylated hydroxytoluene and butylated

hydroxyanisole were used as positive controls. The cells at a number

of 2x107 cells/well were incubated for 24 h under 5% CO2 at 37°C

with the extracts and the positive controls. The SOD activities and

TBARS levels of the cells were than analysed spectrophotometrically

via a multifunctional microplate reader. The methanolic extract

was found as antioxidant whereas water extract was prooxidant in

0.5 mg/mL concentration. Therefore the water extract may have be

cytotoxic effect on leukemia. The composition of the extracts were

analysed by HPLC and rosmarinic acid was found to be main compound

in both extracts.

126


Effects of onion peel extracts on cell proliferation and LPSinduced

inflammatory stress in human colon carcinoma cells

JUNGMI KIM AND EUNJU PARK

Department of Food and Nutrition, Kyungnam University, Changwon,

Gyeongnam 631-701, Korea

The present study was aimed to determine the antiproliferative

activity of onion peel extracts (acetone, OPA; ethanol, OPE; methanol,

OPM) in HT-29 human colon carcinoma cells. To determine the

amount of phenolic acids and flavonoids in the studied extracts, HPLC

analysis was performed. In addition, the influence of OPE on antioxidant

and inflammation associated gene expression was also identified

in the model of lipopolysaccharide (LPS) stimulated HT-29

cells. High-performance liquid chromatography (HPLC) analysis

showed that the presence of well-known antioxidant compounds including

p-coumaric acid, epicatechin, and quercetin in onion peel

extracts and all of the antioxidant compounds measured in this study

was the highest in OPM. After incubation with onion peel extracts,

many HT-29 cells showed the loss of normal nuclear architecture

and either detachability from each other. The cytotoxic effects of onion

peel extracts on the HT-29 cells were confirmed by MTT and LDH

assay. Under LPS-induced oxidative conditions, we found effectively

down regulated TNF-α mRNA expression in OPE pretreated HT-

29 cells compared to in LPS only stimulated cells. In addition, upregulation

of heme oxiganase-1 (HO-1) and glutathione S-transferases

(GSTs) detoxification gene (GSTM1, GSTT1, GSTP1) expressions

were also found after treating with LPS at sublethal concentrations.

However, LPS-induced mRNA expressions of HO-1 and GSTs were

significantly attenuated by treatment with OPE. Therefore, onion peel

extracts might be a promising component of future nutraceuticals or

value-added products, although further investigations into underlying

molecular mechanism, including the TNF-α mediated NF-κB

signaling pathway are still necessary.

127


Effect of fermentation of pine needle juice on antioxidant

activity and α-glucosidase inhibitory activity

SO-YUN KIM 1 , HYUN-JEONG LEE 1 , JAE-HEE PARK 1 , RAE-YOUNG KIM 2 ,

AND EUNJU PARK 1

1 Department of Food and Nutrition, Kyungnam University, Changwon,

Gyeongnam 631-701, Korea; 2 Department of Hotel Culinary and Bakery,

Chang-shin College, Changwon, Gyeongnam 630-764, Korea

Pine needle, which includes lipid-lowering effect, antioxidant activity,

nitrate scavenging activity, anticancer and antibacterial activity,

has been used as folk medicine in Asia. Fermentaion enhances

the nutraceutical effects of fermented foods through producing

bioactive compounds with antioxidant activity. Therefore, the objective

of this study was to compare the total phenolic content (TPC),

antioxidant activity [DPPH radical scavenging activity (RSA), total

radical-trapping antioxidant parameter (TRAP), oxygen radical absorbance

capacity (ORAC), cellular antioxidant capacity (CAC), and

antigenotoxic activity], and α-glucosidase inhibitory activity in fresh

pine needle juice (PNJ) and fermented pine needle juice (FPNJ). The

TPC was shown as 22.98±0.67 and 27.36±1.05 μg GAE/mL in before

and after fermentation, respectively. DPPH RSA, TRAP, and

ORAC value in both PNJ and FPNJ increased in a dose-dependent

manner and these activities was significantly higher in PNJ than FPNJ.

PNJ and FPNJ had CAC in AAPH-induced HepG2 cells using

DCFH-DA and protective effect against H2O2-induced DNA damage.

The latter effect was higher in PNJ than FPNJ in all concentrations.

However, α-glucosidase inhibitory activity was significantly

higher in FPNJ than PNJ. Although TPC and α-glucosidase inhibitory

activity increased after fermentation, antioxidant activity decreased.

Therefore, this study could not find the additional beneficial

effects on health by the fermentation of PNJ. However, we suggest

that the consumption of PNJ and FPNJ are good sources, which might

provide antioxidative protection against free radicals in the human

body.

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Effect of melatonin and growth hormone on age-related

alteration in apoptosis and inflammation processes in the

dentate gyrus of male rats

ROMAN KIREEV 1 , SARA CUESTA 1 , CRUZ GARCIA 2 , ELENA VARA 2 ,

AND JESUS A.F. TRESGUERRES 1

1 Department Physiology, Medical School, University Complutense of Madrid;

2 Department Biochemistry and Molecular Biology, Medical School,

University Complutense of Madrid, Spain

The hippocampus is a target of age-related physiological and structural

changes. Alterations in this structure during aging are paralleled

by behavioural and functional deficits in hippocampus-dependent

learning and memory tasks. It has been suggested that the decrease

in the number of neurons could be due to apoptosis or neuro-inflammation.

In order to investigate the role of GH and melatonin in the

age-related changes, forty-six male Wistar rats of 22 months of age

were divided into three groups. One group remained untreated and

acted as control. The second was treated with growth hormone (GH)

for 10 weeks (2 mg/kg/d sc) and the third was submitted to melatonin

administration (1 mg/kg/d) in the drinking water for the same

time. A group of 2-months-old male rats was used as young controls.

All rats were killed by decapitation at 24 months of age and hippocampi

were collected. The level of TNFα, IL1β; and IL6 were analysed

by ELISA. The expression of Bax, Bad, Bcl2, NIAP, XIAP,

AIF, SIRT 1 and 2, TNFα, IL1β, IL10, GFAP genes were detected

by RT-PCR. The protein expressions of TNFα, IL1β, IL10, iNOS,

HO-1, NrF2, BVR, Bax, Bad, Bcl2 and Mcl-1 were studied by

Western blot.

Aging was associated with an increase in apoptosis promoting

markers (Bax, Bad, and AIF) and with the reduction of some antiapoptotic

ones (XIAP, NIAP). Expressions of SIRT1 and SIRT2 were

decreased in the hippocampus of old rats. GH treatment was able to

reduce the pro/anti-apoptotic ratio to levels observed in young animals

and also to increase SIRT2. Melatonin reduced also the expression

of pro-apoptotic genes and proteins, and increased levels of Mcl-

1 proteins and SIRT1. The levels of TNFα, IL1βand IL6 and the

expressions of mRNA of these pro-inflammatory cytokines were sig-

129


nificantly elevated during ageing and these results did correlate with

the increase in the expression of proteins. However, both mRNA

and protein expressions of IL10 were decreased. Melatonin treatment

decreased the level of all pro-inflammatory cytokines, whereas

GH significantly decreased TNFα and IL6 level, but did not affect

IL1β level. The expression of mRNA and protein of anti-inflammatory

cytokines (IL10) showed a significant enhancement after both

treatments. Aging induced also a significant elevation in the expression

of HO-1 and iNOS, both proteins and genes. Administration of

melatonin decreased mRNA and protein expression of HO-1 and

increased protein expression of BVR and NrF2. A significant decrease

in the expression of iNOS was observed after both treatments.

The hippocampus of aged rats showed a highly significant increase

in mRNA expression of GFAP. Administration of melatonin to old

males significantly lowered its mRNA expression.

These results suggest that aging in hippocampus (dentate gyrus)

is associated with an increase in pro-inflammatory, oxidative stress

substances, pro-apoptotic markers and increased expression of GFAP

genes. Melatonin and GH treatments are able to reduce inflammatory

parameters, apoptosis and to enhance survival markers.

This work was supported by grants: RETICEF RD 06/0013 (FIS) Instituto

Carlos III and SAF 2007, 66878-C02-01.

130


Markers of mitochondrial biogenesis are affected by aging and

exercise training in rat skeletal muscle

ERIKA KOLTAI, NIKOLETT HART, ALBERT W. TAYLOR, SATARO GOTO,

JENNY K. NGO, KELVIN J. A. DAVIES, AND ZSOLT RADAK

Semmelweis University, Faculty of Physical Education and Sport Sciences,

Budapest, Hungary

Impairments of the mitochondrial reticulum or network, and its

function have often been associated with aging. A decline in mitochondrial

biogenesis and mitochondrial protein quality control in

skeletal muscle, directly contributes to this problem, but exercise training

has been suggested as a possible cure.

In this report, we tested the effects of moderate intensity exercise

training on young and old rats, and assessed mitochondrial biogenesis

and mitochondrial protein quality control.

Twelve young (3 mo) and twelve old (26 mo) male Wistar rats

were used in the study and grouped into young control (YC), young

exercised (YE), old control (OC) and old exercised (OE). Exercised

rats were trained on treadmill for 6 weeks, 5 times per week, where

the running speed and duration of the exercise were gradually increased.

We examined in this study the gastrocnemius muscle by

Western blot and fluorometric assays. Statistical significance was

assessed by one-way ANOVA, followed by Tukey’s post hoc test.

Exercise training prevented or attenuated significant age-associated

(detrimental) declines in SIRT1 activity, AMPK, peroxisome

proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a)

and the Lon protease, in the gastrocnemius muscle of rats. Exercise

training also prevented the age-related (detrimental) increases in NRF1,

TFAM, Mfn1, Fis1 and polynucleotide phosphorylase (PNPase) levels.

Our data suggest that regular exercise training can help minimize

detrimental skeletal muscle aging deficits stimulating mitochondrial

biogenesis through the PGC1a system, a rejuvenating the mitochondrial

network via fission and fusion, and improving quality control

of mitochondrial proteins by the Lon protease. All of these properties,

working in conjunction with one another, would improve the

overall functionality of mitochondria in aged cells.

131


Proteomic profiling and post-transductional modifications

in human keratinocytes treated with Mucuna Pruriens

leaves extract

L. LAMPARIELLO 1 , A. CORTELAZZO 2 , C. STICOZZI 3 , G. BELMONTE 4 ,

R. GUERRANTI 2 , A. DI CAPUA 5 , M. ANZINI 5 , AND G. VALACCHI 3-6

1 Dipartimento di Chimica, Università di Siena, Siena, Italy; 2 Dipartimento di

Medicina Interna, Scienze Endocrino Metaboliche e Biochimica, Università

di Siena, Siena, Italy; 3 Dipartimento di Biologia ed Evoluzione, Università di

Ferrara, Ferrara, Italy; 4 Dipartimento di Scienze Biomediche, Università di

Siena, Siena, Italy; 5 Dipartimento Farmaco Chimico Tecnologico, Università

di Siena, Siena, Italy; 6 Department of Food and Nutrition, Kyung Hee

University, Seoul, South Korea

The plant-derived compounds have always been an important

source of drugs for various diseases and have received considerable

attention in recent years due to their numerous pharmacological proprieties.

The medicinal plant Mucuna pruriens (MP), belonging to

the family of Fabaceae, is the most popular drug in Ayurvedic system

of medicine. Its different preparations (from seeds), thanks, also,

to its free radical scavenger ability, have been used as therapeutic

medicine for several diseases such as rheumatoid arthritis, diabetes,

atherosclerosis, male infertility and nervous disorders. In addition, it

is also used in the management of Parkinsonism, as it is good source

of L-Dopa [1].

Plants from Nigeria seeds were grown in Botanical Garden, University

of Siena, and then the leaves were collected, dried under shade,

pulverized by a mechanical grinder and successively extracted with

methanol. The extracts were concentrated and lyophilized, then examined

in vitro for their antioxidant activity, probably due to the presence

of phenolic compounds. Antioxidant compounds like phenols,

polyphenols and flavonoids are able to scavenge free radicals such a

peroxide, hydroperoxide or lipid peroxyl and thus to inhibit the

oxidative mechanism [2].

Being the skin one of the main target of exogenous oxidative stress

(UV, O 3 , smoke etc) [3] and being several skin pathologies related to

increased oxidative stress (psoriasis, dermatitis, etc) [4], in this preliminary

study, cell viability by means of differently diluted samples

has been measured. Successively, human keratinocytes (HaCaT cell

132


line) have been treated with MP leaves methanolic extract, in order

to analyze changes in proteins expression. So, proteins from HaCaT

cells lysates treated with MP leaves extract where analyzed by SDS-

PAGE 12% according to Leammli method to determine the best conditions

for 2-DE analysis. Protein bands and spots evidenced by means

of silver stain were detected and analyzed using TotalLab and Image

Master 2D Platinum software. Protein spots were divided in 3 groups

based on the molecular weight as follows: Group 1 (100 - 250 KD),

Group 2 (36 - 100 KD) and Group 3 (10 - 37 KD). The analyses

revealed that after MP treatment there were 29, 26 and 7 new spots

in Group 1, Group2 and Group 3, respectively. On the other hand,

treatment with MP showed the lost of 39, 134 and 77 original in

Group 1, Group2 and Group 3, respectively. In addition, the treatment

with MP significantly decreased the levels of 4HNE protein

adducts especially for Group 2.

This preliminary study, suggests that the use of MP methanolic

extracts might be helpful in the treatment of oxidative stress-related

skin diseases.

References

1. Misra L. et al. Indian Journal of Biochemistry and Biophysics. 2007, 44,

56-60.

2. Kumar DS. et al. International Journal of PharmTech Research. 2010,

2, 2063-2070.

3. Valacchi G. et al. FEBS Lett . 2000, 466,165-168.

4. Briganti F. and Picardo M. J. Eur. Acad. Dermatol. Venereol. 2003,17,

663-669.

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Influence of an encapsulated fruit, berry and vegetable juice

concentrate and walking exercise on oxidative stress,

inflammation and skin microcirculation in overweight and

obese women

MANFRED LAMPRECHT 1,2 , GEORG OBERMAYER 1,2 , KURT STEINBAUER 3 ,

LIDIJA HOFMANN 4 , SETH HALLSTROEM 1 , GERHARD LEDINSKI 1 ,

AND JOACHIM F. GREILBERGER 1,5

1 Institute of Physiological Chemistry, Centre for Physiological Medicine,

Medical University of Graz, Graz, Austria; 2 Green Beat- Institute for Nutrient

Research and Sport Nutrition, Graz, Austria; 3 SportchirurgiePlus, Centre for

Individual Sport Medicine and Surgery, Graz, Austria; 4 FH Joanneum,

University of Applied Sciences, Graz, Austria; 5 Institute of Laboratory

Sciences, Dr Greilberger GmbH, Laßnitzhöhe, Austria

Design: Randomized, double-blind, placebo controlled clinical

trial to observe effects of supplementation with an encapsulated fruit,

vegetable and berry juice concentrate (Juice Plus+Ò) on markers of

oxidation, inflammation and skin microcirculation at rest and post

exercise.

Methods: We monitored 42 premenopausal (42±5 years, nonsmokers)

overweight and obese women for markers of oxidation and

inflammation both before and after a standardized 30 minute treadmill

walking test at 70% of VO2max and before and after 8 weeks of

supplementation.

Markers included: carbonyl proteins (CP), malondialdehyde

(MDA), total oxidation status (TOS), oxidized LDL (oxLDL),

interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Parameters

of skin microcirculation were also assessed at skin depths of 1

mm and 8 mm: oxygen saturation of hemoglobin (SO2Hb), relative

concentration of hemoglobin (rHb), blood flow through skin capillaries

(BF). Venous blood for oxidation markers was collected before

and immediately after 30 minutes of walking exercise. Skin microcirculation

measurements were conducted 15 min before and 15

min post exercise between the thumb and index finger on the back of

the hand. Statistical analysis used multifactorial analysis of variance

(p


and TNF-α due to supplementation. SO2Hb, rHb and BF in 1mm

skin depth increased significantly in the supplemented group. Exercise

affected the parameters of skin microcirculation significantly in

1mm as well as in 8mm depth.

Conclusion: These data indicate the nutraceutical decreased

plasma oxidation and inflammation status and improved microcirculation

at the skin surface. Exercise also improved skin microcirculation

and blood flow under the skin. The study also demonstrates a

better beneficial outcome on redox homeostasis and skin microcirculation

in overweight and obese women when both influencing factors,

supplementation and exercise, are combined.

135


Natural chemical screening identifies myricetin

and scutellarein as novel chemical inhibitors

of the SARS helicase, nsP13

JUNE LEE AND YOUNG-SAM KEUM

College of Pharmacy, Dongguk University, Republic of Korea

Severe acute respiratory syndrome (SARS) is an infectious disease

with a strong potential for transmission upon close personal contact

and is caused by the SARS-coronavirus (CoV). However, there

are no natural or synthetic compounds currently available that can

inhibit SARS-CoV. We examined the inhibitory effects of 64 purified

natural compounds against the activity of SARS helicase, nsP13,

and the Hepatitis C virus (HCV) helicase, NS3h, by conducting Fluorescence

Resonance Energy Transfer (FRET)-based double-strand

(ds) DNA unwinding assay or by using a colorimetry-based ATP

hydrolysis assay. While none of the compounds, examined in our

study inhibited the DNA unwinding activity or ATPase activity of

human HCV helicase protein, we found that myricetin and scutellarein

potently inhibit the SARS-CoV helicase protein in vitro by affecting

the ATPase activity, but not the unwinding activity, nsP13. In addition,

we observed that myricetin and scutellarein did not exhibit a

significant cytotoxicity against normal breast epithelial MCF10A

cells. Our study demonstrates for the first time that selected naturally-occurring

flavonoids, including myricetin and scultellarein might

serve as SARS-CoV chemical inhibitors.

136


Correlation between polyphenolic profile and antioxidant

activity of old and modern common wheat varieties

EMANUELA LEONCINI 1 , CECILIA PRATA 1 , MARCO MALAGUTI 1 ,

ANTONIO-SEGURA CARRETERO 3 , ILARIA MAROTTI 2 , PIETRO CATIZONE 2 ,

GIOVANNI DINELLI 2 , AND SILVANA HRELIA 1

1 Department of Biochemistry “G. Moruzzi” University of Bologna, via Irnerio,

48, 40126 Bologna, Italy; 2 Department of Agroenvironmental Science and

Technology, University of Bologna, viale Fanin, 44, 40127 Bologna, Italy;

3 Department of Analytical Chemistry, University of Granada, c/Fuentenueva

s/n, 18003 Granada, Spain

Phenolic compounds present in whole grains have gained attention

as they have strong antioxidant properties, potentially protective

against many degenerative diseases.

Aim of this study was to characterize the phytochemical profile of

modern and old common wheat (Triticum aestivum L.) varieties in

terms of antioxidant activity and phenolic composition and to evaluate

their antiproliferative or cytoprotective effects in different cell

culture systems: a leukemic cell line, HL60, and primary cultures of

neonatal rat cardiomyocytes.

The evaluation of the effect on cell viability and ROS intracellular

level in in vitro models allowed to highlight differences in

cyotoprotective/antiproliferative properties between cultivars and to

correlate them to peculiar phytochemical/ nutraceutical profile of the

extracts.

Data reported in this paper support the healthy role of cereals in

human nutrition due to the presence of nutraceutical components and

their possible synergistic effect and, above all, underline the importance

of a re-discovery of ancient varieties in the development of

functional food.

Thus, an increased intake of wheat grain derived products could

represent an effective strategy to achieve both chemoprevention and

protection against oxidative stress related diseases, thanks to their

antioxidant phytochemicals content.

Supported by Ministry of Economic Development (Project “Made in Italyover

50”) and Fondazione del Monte di Bo e Ra.

137


Oxidative stress activation of mir-125b involves Nrf2 and

targets Notch1 and p63

SONIA MANCA, GIANFRANCO BIOLCATI, FABIO VIRGILI,

CLAUDIO TALORA, AND ROBERTO AMBRA

National Institute of Nutrition and Food Research (INRAN)

MicroRNA are endogenous post-transcriptional modulators of gene

expression with critical functions in health and disease. Using a comprehensive

analysis of all human miRNAs registered in mirBase 12.0a

we recently identified miR-125b in the development of the Hailey-

Hailey disease (HHD), an autosomal dominant dermatosis HHD with

erosive and oozing skin lesions, characterized by increased production

of ROS.

By means of software prediction we identified Notch1 and p63 as

potential gene targets of miR-125b, which is consistent with their

previously involvement role in the pathogenesis of HDD. According

to the in silico prediction, exogenous expression of miR-125b strongly

suppressed both Notch1 and p63 proteins in HACAT human

keratinocyte cells. The ability of miR-125b to modulate both Notch1

and p63 expression was further confirmed by luciferase analysis.

We previously found that Notch1 expression is negatively regulated

by ROS induction in HACAT cells. We thus tested if also miR-

125b is affected by oxidative stress. Accordingly both t-butyl

hydroperoxide and arsenic induced strong expression of miR-125b

in HACAT cells, measured by means of qPCR.

Analysis of the promoter of miR-125b1 reveals potential signatures

for Nrf2, suggesting regulation of miR-125b expression by this

transcription factor. Accordingly, pre-treatment of HACAT cells with

the Nrf2 activator sulforaphane prevented the ROS-induced up-regulation

of miR-125b.

Results here reported give some new clues in miR-125b regulation

and effects, and point out new research to test sulforaphane as a

potential therapeutic approach for HDD.

138


Advanced glycation end-products promote lipogenesis in liver

of fructose-drinking mice

RAFFAELLA MASTROCOLA 1 , MASSIMO COLLINO 2 , DEBORA NIGRO 1 ,

CLAUDIO MEDANA 3 , AND MANUELA ARAGNO 1

1 Department of Experimental Medicine and Oncology, University of Turin,

Italy; 2 Department of Anatomy, Pharmacology and Forensic Medicine,

University of Turin, Turin, Italy; 3 Department of Analytical Chemistry,

University of Turin, Italy

Clinical studies have linked the rising consumption of fructose as

added sweetener instead of glucose to the present epidemic of obesity,

dyslipidemia and impaired glucose tolerance. In chemical experiments

fructose undergoes a non-enzymatic reaction from which advanced

glycation endproducts (AGEs) are derived, leading to the

formation of dysfunctional, fructosylated proteins, but the in vivo

formation of AGEs from fructose and their chemical structure are

still less known than those derived from glucose.

In the present study C57Bl/6J mice received 15% fructose or glucose

in water to drink for 30 weeks, resembling human habit to consume

sugary drinks. At the end of protocol both fructose (FRT) and

glucose (GLC) drinking mice had increased fasting glycaemia, glucose

intolerance, altered plasma lipid profile, and marked hepatic steatosis,

compared to water drinking mice.

Compared to GLC, FRT mice had higher hepatic triglycerides

deposition paralleled by greater increased expression and activity of

the transcription factor responsible for the de novo lipogenesis

SREBP1 and of its activating protein SCAP.

Moreover, MS-LC analysis showed a different pattern of AGEs

production in liver tissue between FRT and GLC mice. In detail,

FRT generated CML and CEL in a larger amount compared to GLC.

Double immunofluorescence and coimmunoprecipitation analysis

revealed an interaction between CML and SCAP that leads to

prolonged activation of SREBP1.

This new mechanism may contribute to the higher lipogenic power

of fructose with respect to glucose and may suggest more caution in

the wider employment of fructose as added sweetener in beverages

and foods.

139


Age related changes of Nrf2/Are signaling pathway by

Tocotrienol rich fraction, Piper betle and Chlorella vulgaris

in C57BL/6 Mice

YASMIN ANUM MOHD YUSOF, NORSYAHIDA ALIAHMAT, SUZANA

MAKPOL, AND WAN ZURINAH WAN NGAH

Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan

Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia

Supplementation of natural products with high antioxidant capacity

can alter the age related changes. The aim of this study was to

investigate the effects of antioxidants Tocotrienol Rich Fraction (TRF),

Piper betle (PB) and Chlorella vulgaris (CV) on modulating the Nrf2/

ARE signalling pathway associated with the production of endogenous

antioxidants (SOD, GPx, CAT) and phase II detoxifying enzymes

(HO1, Trx1, NQO1) during ageing in mice. The mice were

divided into three groups according to their age, young (6 months

old), middle (12 months old) and old age (18 months old). The mice

were sub-divided into control and treatment groups consisted of TRF

(30mg/kg), PB (10mg/kg and 50 mg/kg) and CV (50mg/kg). Blood

and heart tissues were collected for determination of genes expression

of antioxidants and phase II detoxifying enzymes via Nrf2/ARE

signalling pathway by Quantigene Plex 2.0 Assay. TRF down-regulated

Nrf2, Keap1, HO1 and GPx1 genes in young age group and

up-regulated HO1 genes in old age group (p


Organ and gender-dependent mitochondrial antioxidant

modulation by resveratrol

ANA C. MOREIRA 1,2,3 , ANA M. P. SILVA 1,3 , MARIA S. SANTOS 1,3 ,

AND VILMA A. SARDÃO 1

1 CNC-Center for Neuroscience and Cell Biology, University of Coimbra;

2 Doctoral Programme in Experimental Biology and Biomedicine, CNC,

University of Coimbra; and 3 Department of Life Sciences, University of

Coimbra, Portugal

Resveratrol is a phytoalexin recognized by its antioxidant capacity.

However, other beneficial properties, such as lifespan increase

make resveratrol an attractive compound. In the present work, we

used isolated brain and liver mitochondria from male and female

Wistar-Han rats to investigate the protective effects of resveratrol (RES)

against ADP/iron-induced lipid peroxidation and hydroperoxyde production

by the respiratory chain.

Five male and four female 8-10 weeks old Wistar-Han rats were

used to isolate brain and liver mitochondrial fractions. Under basal

conditions (absence of exogenous substrate), hydroperoxide generation

by brain and liver mitochondrial respiratory chain in female rats

is lower than in the male group (liver: 1600 pmol/mg protein (male)

vs 950 pmol/mg protein (female); brain: 1700 pmol/mg protein (male)

vs 650 pmol/mg protein (female)). In energized mitochondrial fractions

from female rat brain, RES increased the levels of

hydroperoxides (from 1500 to 2200 pmol/mg protein, and from 1700

to 2900 pmol/mg protein, male and female, respectively), but

thiobarbituric acid reactive substances (TBARS) are decreased in its

presence (2-fold lower). Similar results were observed in male rats

(3-fold lower). RES decreased mitochondrial lipid peroxidation induced

by ADP-Fe in both genders and organs.

The results suggest that RES may act through Complex I modulation

of hydrogen peroxide production and that the positive impact of

RES in many cellular signaling pathways may be mediated by increased

mitochondrial hydrogen peroxide redox signaling. Organ and

gender specific variations could be attributed to the differential levels

of antioxidant defenses.

141


Acknowledgements: PTDC/AGR-ALI/108326/2008 grant from Foundation for

Science and Technology (FCT). ACM, AMS and VAM are supported by FCT

PhD and Post-doc fellowships (SFRH/BD/33892/2009, SFRH/BD/76086/

2011 and SFRH/BPD/31549/2006, respectively).

142


Effect of caffeic acid on endothelial cell dysfunction caused by

high-glucose exposure

LUCIA NATARELLI, GIULIA RANALDI, MARIANNA ROSELLI,

RAFFAELLA COMITATO, FRANCESCO CIMINO 1 , FABIO VIRGILI,

AND RAFFAELLA CANALI

National Research Institute for Food and Nutrition (INRAN), Rome, Italy;

1 Dep. Farmaco-Biologico, School of Pharmacy, University of Messina, Italy

Diabetes mellitus is generally associated to vascular metabolic

dysfunctions and increased endothelial paracellular permeability.

Available reports suggest that a moderate and constant coffee consumption

is associated with a substantially lower risk of type 2 diabetes.

Caffeic acid and its metabolites derived from chlorogenic acid,

are the most abundant molecules present in circulation after coffee

consumption. However, their effects on endothelial cells and their

combinatory effects in the presence of high glucose (HG) concentration

are not totally known. In this study, the endothelial cells Ea.hy926

were treated with 25 mM of glucose to simulate an hyperglycemic

condition and the effects of 10 nM caffeic acid on high-glucose-dependent

increase of permeability and apoptosis was analyzed.

Our results indicate that caffeic acid is able to counteract the reduction

of trans-endothelial electrical resistance (TEER) associated

with HG incubation in a time-dependent manner. Differently, the effect

of caffeic acid on HG dependent FITC-dextran (40kDa) permeability,

used as measure of membrane integrity, was evident only after

72h of treatment. Moreover, caffeic acid countered after 48h of

treatment both HG-dependent decrease of cell proliferation, analyzed

by Bromo-Uridin incorporation and increase of Annexin V positive

cells. Similarly, caffeic acid totally reversed actin stress fiber formation

and cytosolic migration of p120 protein, after 48h of HG treatment.

Our results show that physiological concentration of caffeic

acid counteracts some of the effects triggered by the exposure to HG

on permeability and viability in endothelial cell.

143


Effect on skeletal muscle cells of medium conditioned by

human lung cancer cells treated with conjugated linoleic acid

ELENA PAIUZZI, MANUELA ORALDI, ROSA ANGELA CANUTO,

GIULIANA MUZIO, AND MARINA MAGGIORA

Department of Experimental Medicine and Oncology, University of Turin,

Corso Raffello 30, 10125 Turin, Italy

Conjugated linoleic acid (CLA) is thought to have anti-proliferative

and anti-inflammatory properties, but its effect on cancer cachexia

is unknown. This study investigated the effect of CLA on inflammatory

mediator production in human lung cancer cells, and the effect

of reduced mediators on myogenic differentiation of C2C12 murine

myoblasts. Myoblasts were grown in medium conditioned by A427

human lung-cancer cells, with or without CLA, to mimic only the

effect of molecules released from the tumour in vivo, excluding the

effect of host-produced cachectic factors.

CLA decreased lung cancer cell proliferation, and, at the same

time, reduced production of tumour necrosis factor (TNF)-α,

interleukin (IL)-1β and prostaglandin E2 (PGE2); there was no effect

on IL-6 production. Mechanisms underlining the effect of CLA

on cytokine or PGE2 release in A427 cells are probably mediated by

activation of peroxisome proliferator-activated receptor (PPAR)-α,

which increased at 24 hours of CLA treatment. In turn, the reduced

content of inflammatory mediators in medium conditioned by A427

cells in presence of CLA allowed the differentiation of muscle cells,

again through induction of PPARs.

In conclusion, the study evidenced the anti-inflammatory and

myogenic action of CLA and its possible application as novel dietary

supplement and therapeutic agent in inflammatory disease states,

such as cachexia.

144


Protection by caffeic and ferulic acids against UVA-induced

matrix metalloproteinase-1 through promotion of glutathione

in keratinocyte HaCaT cells

URAIWAN PANICH, THANYAWAN PLUEMSAMRAN, AND

TASANEE ONKOKSOONG

Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol

University, Bangkok 10700, Thailand

Ultraviolet A (UVA) plays a vital role in the pathogenesis of premature

skin aging through keratinocyte cytotoxicity and degradation

of collagen. Oxidative stress caused by UVA irradiation can mediate

induction of matrix metalloprotease-1 (MMP-1), a major enzyme responsible

for collagen damage. Protection against UV-mediated depletion

of glutathione (GSH), responsible for maintaining redox balance,

has been proposed as a possible mechanism by which botanical

compounds slow down skin aging process. This study therefore

aimed to assess inhibitory effects of caffeic acid (CA) and ferulic

acid (FA), powerful plant-based phenolic antioxidants, on UVA-induced

cytotoxicity and MMP-1 activity through modulation of GSH

in immortalized human keratinocyte (HaCaT) cells. The cytotoxicity,

MMP-1 activity and GSH levels were determined using MTT

reduction assay, zymography and spectrofluorometry, respectively.

In addition, mRNA levels of g-glutamate cysteine ligase (γ-GCL),

the rate-limiting enzyme in GSH synthesis, were assessed using realtime

reverse transcriptase-polymerase chain reaction. Pretreatment

of the cells with CA or FA prior to UVA (4 J/cm2) irradiation inhibited

cytotoxicity, induction of MMP-1 activity and oxidant formation.

Moreover, CA and FA were able to promote GSH content and

γ-GCL mRNA expression in irradiated cells. In conclusion, CA and

FA provided protective effects on UVA-mediated MMP-1 activation

in HaCaT cells possibly through upregulation of GSH. The mechanisms

by which phenolics regulate GSH synthesis at the transcriptional

level need further investigation.

145


RNA interference targeting cytosolic NADP+-dependent

isocitrate dehydrogenase exerts anti-obesity effect in vitro and

in vivo

JEEN-WOO PARK AND WOO SUK NAM

Kyungpook National University

A metabolic abnormality in lipid biosynthesis is frequently associated

with obesity and hyperlipidemia. NADPH is an essential reducing

equivalent for numerous enzymes require in fat and cholesterol

biosynthesis. Cytosolic NADP+-dependent isocitrate dehydrogenase

(IDPc) has been proposed as a key enzyme for supplying

cytosolic NADPH. We report here that knockdown of IDPc expression

by RNA interference (RNAi) inhibited adipocyte differentiation

and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc

expression by IDPc small interfering RNA (siRNA) resulted in a

reduction of differentiation and triglyceride level and adipogenic protein

expression as well as suppression of glucose uptake in cultured

adipocytes. In addition, the attenuation of Nox activity and ROS generation

accompanied with knockdown of IDPc was associated with

inhibition of adipogenesis and lipogenesis. The loss of body weight

and the reduction of triglyceride level were also observed in dietinduced

obese mice transduced with IDPc short-hairpin (shRNA).

Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis

and lipid biosynthesis is considered to be of therapeutic

value in the treatment and prevention of obesity and obesity-associated

metabolic syndrome.

146


Septic peritonitis-induced endothelial barrier dysfunction is

attenuated by ascorbate and dehydroascorbic acid

SURESH PENDEM AND JOHN X. WILSON

Department of Exercise and Nutrition Sciences, University at Buffalo

Sepsis is marked by oxidation of ascorbate and its depletion from

blood as well as by increased permeability of the microvascular endothelium

to plasma proteins. We investigated the recycling of

extracellular dehydroascorbic acid (DHAA) to intracellular ascorbate

by endothelial cells and its effect on the endothelial barrier in

experimental sepsis. We observed in primary cultures of murine microvascular

endothelial cells that the initial rate of DHAA recycling

was stimulated by septic insult (LPS and interferon-gamma), and it

was inhibited by glucose as well as by deficiency or pharmacological

inhibition of NADPH oxidase. To study the regulation of endothelial

permeability, mice were injected with ascorbate or DHAA

(200 mg/kg i.v.) either 1 h before (prophylactic injection) or 3-6 h

after (therapeutic injection) induction of polymicrobial septic peritonitis

by cecal ligation and puncture (CLP). CLP stimulated the extravasation

of plasma proteins in heart, liver, lung and skeletal muscle.

Ascorbate and DHAA did not alter extravasation in non-septic

mice but were equally effective in inhibiting the increase in extravasation

caused by CLP. We conclude that: (i) microvascular endothelial

cells respond to septic insult by NADPH oxidase-dependent activation

of a mechanism that accelerates the intracellular reduction of

DHAA, and (ii) parenteral ascorbate and DHAA are equally effective

for regulating the endothelial barrier in multiple organs during

experimental sepsis.

147


Cardiac oxidative modifications in fructose overload rats and

prevention by (-)-epicatechin

BARBARA PIOTRKOWSKI 1 , VALERIA CALABRO 1 , GIOVANNA ASCHETTINO 1 ,

LAURA FISCHERMAN 1 , MARCELA A. VAZQUEZ-PRIETO 2 ,

MONICA GALLEANO 1 , AND CESAR G. FRAGA 1,3

1 Physical Chemistry-Institute of Molecular Biochemistry and Medicine

(IBIMOL), UBA-CONICET, Buenos Aires, Argentina; 2 Department of

Pathology, School of Medicine, National University of Cuyo and Laboratory

of Cardiovascular Pathophysiology, Institute for Experimental Medical and

Biological Research (IMBECU)-CONICET, Mendoza, Argentina; 3 Department

of Nutrition, University of California, Davis, CA 95616, USA

The purpose of this study was to evaluate the effects of (-)epicatechin

(EC) supplementation on oxidative modifications in the

heart of rats subjected to a model of metabolic syndrome. Male

Sprague-Dawley rats were divided into three groups and received

for 8 w: i) control diet and tap water (C); ii) control diet and 10% (w/

v) fructose in the drinking water (F), and iii) 10% (w/v) fructose in

the drinking water, and the diet supplemented with EC (20 mg/kg

BW/d) (FEC). Systolic blood pressure in rats with fructose overload

was significantly higher than in control rats and EC prevented that

increase (C=130±4; F=142±3*; FEC=133±3 mmHg; *p


Hemozoin carrier of oxidised phospholipids: potential Trojan

horse in malaria infection

ANA REIS 1 , DENISE DAVALOS 2 , DAVID HARDY 1 , ANGELO ATTAMASIO 3 ,

EVELIN SCHWARZER 2 , AND CORINNE M SPICKETT 1

1 School of Life and Health Sciences, Aston University, Aston Triangle,

Birmingham, B47ET, UK; 2 Dipartimento di Genetica, Biologia e Biochimica,

Università di Torino, Via Santena 5 bis, 10126 Torino, Italy; 3 Dipartimento di

Scienze Biomediche e Oncologia Umana, Via Santena 7, 10126 Torino, Italy

Infection by Plasmodium falciparum is responsible for the death

of millions of people worldwide posing a serious health issue. The

parasite digests up to 80% of the host erythrocyte haemoglobin, except

the heme, which is dimerized to form crystalline insoluble

hemozoin and stored in its digestive vacuole. The hemozoin-containing

vacuole is expelled into circulation as a residual body (RB)

during schizogony. Targeted by phagocytes, the liberated hemozoin

is bioactive and modifies crucial immune functions. Previously, hydroxy

fatty acids were identified in saponified hemozoin and found

to inhibit monocyte functions, though information about their phospholipid

source is scarce. We investigated the phospholipid composition

of HZ from P. falciparum parasitized erythrocyte and analysed

lipids extracts from RB and hemozoin, obtained from synchronized

cultures of P. falciparum either immediately or 24h after schizogony,

by mass spectrometry (MS) using targeted scanning modes in Qtrap

instrument. The predominant phospholipids were phosphatidylcholines

(PC) and phosphatidylethanolamines (PE). Additionally,

oxidized phospholipids (OxPL) were found in hemozoin namely

hydroxy and hydroperoxy derivatives and aldehyde and carboxylic

acid derivatives esterified to PC and PE, resulting from oxidation in

omega-6 fatty acids. Confirmation of OxPL derivatives by tandem

MS (MS/MS) revealed modifications at various positions of the carbon

chain which are products of known bioactivity. The OxPL was

mainly observed after schizogony when the digestive vacuole’s integrity

and hemozoin partitioning was lost. The identification of

OxPL in hemozoin suggests that hemozoin may act as a Trojan horse

carrying OxPL into phagocytes, where it alters phagocyte functions

and modulates host immune responses.

149


GLP-1 receptor is involved in modulation of mitochondrial

bioenergetics in an acute model of cerebral ischaemia/

reperfusion

A. D. ROMANO 1 , M. LAURIOLA 1 , F. BELLANTI 1 , R. TAMBORRA 1 ,

T. ROLLO 1 , M. BLONDA 1 , G. IANNELLI 1 , G. SERVIDDIO 1 ,

AND G. VENDEMIALE 2

1 Institute of Geriatrics, Department of Medical and Occupational Sciences,

University of Foggia; 2 General Medicine, IRCCS Casa Sollievo della

Sofferenza, San Giovanni Rotondo, Foggia

Objectives: exenatide, an agonist of GLP-1 receptor (1), is a synthetic

peptide that is currently approved for the treatment of type 2

diabetes mellitus. Li et al. shown that exenatide reduces brain-infarcted

area, improving motor activity outcome in a rat model of cerebral

ischaemia/reperfusion (2). The aim of this study was to assess

both the effects of exenatide in terms of oxidative stress and mitochondrial

bioenergetics in animals subjected to brain acute ischemia/

reperfusion by ligation of bilateral common carotid arteries (2Vessel

Occlusion).

Material and Methods: 9 months old male Wistar rats were divided

into 3 groups: exenatide (Exe), 2Vessel Occlusion (2VO) and

sham (SHAM). Animals from Exe group were treated with exenatide

i.p. 10 µg/kg and subjected to cerebral ischemia according to the

2VO model; after 20 min of ischemia, carotid arteries were de-clamped

and rat brain reperfused for 20 min prior the sacrifice. Animals from

2VO group, were treated as previously explained except for the treatment.

Animals from SHAM group: the skin was cut as in the 2VO

model without clamping the artery.

Results: RCI from Exe group shows a significant improvement

when compared to 2VO group (t=4,39, p < 0,01). Mitochondrial

membrane potential was significantly reduced in the 2VO group compared

to Exe and SHAM groups (F = 94,04; p < 0,001). Discussion:

analysis of RCI indicates that the process of cellular respiration coupled

with ATP synthesis is altered in untreated animals compared to

controls; furthermore, treatment with exenatide was associated with

an improvement in mitochondrial respiration. These observations

pointed out the role of exenatide as a neuroprotectant in a mitochondrial-dependent

way.

150


Participation of nitric oxide and Src kinase in a signaling

pathway stimulated by 17β-estradiol (estrogen) in MCF-7

human breast cancer cells

HELOÍSA ROSA 1 , PEDRO PAULO MORAES MATHIAS 1 ,

WAGNER LUIZ BATISTA 2 , AND HUGO PEQUENO MONTEIRO 1

1 Departamento de Bioquímica, UNIFESP, São Paulo, Brazil; 2 Departamento

de Ciências Biológicas, UNIFESP, Diadema, Brazil

Tyrosine phosphorylation and redox-based modifications determine

the signaling pathways initiated and/or facilitated by Src, a nonreceptor

tyrosine kinase that plays pivotal role in numerous cellular

processes such as cell proliferation, migration and transformation.

We and others recently described S-nitrosylation of Src in mouse

fibroblasts and MCF-7 human breast cancer cells, by endogenous

and exogenous sources of nitric oxide (NO). The human estrogen

receptor is expressed in MCF-7 cells and plays a major role in human

breast cancers responsive to the hormone. To characterize the

components of a signaling pathway stimulated by estrogen and involving

the activation of Src in MCF-7 cells, we cloned and expressed

human recombinant Src with Histidine (His) tag (6xHis-Src). To investigate

the effects of estrogen on Src activation/expression, 6xHis-

Src/MCF-7 cells, which expresses two-fold increase of 6xHis-Src

when compared to wild type, cells were treated with 1, 10 or 100 nM

of 17b-estradiol (E2) during 2, 8, 16 or 24 hours. Western Blot analysis

showed that Src activity was stimulated in cells treated with 100 nM

E2 with maximum activities after 8 and 24 h incubation. Immunofluorescence

assay revealed an increase on the expression of Src

after treatment with E2. Intracellular levels of NO increased after 24

h incubation of 6xHis-Src/MCF-7 cells with 100 nM E2. Tyrosine

phosphorylation and s-nitrosylation of Src were stimulated after treatment

of 6xHis-Src/MCF-7 cells with 10 nM E2. Interestingly, PP1, a

potent and selective Src inhibitor, inhibited E2-stimulated Snitrosylation

of Src. Estrogen stimulates proliferation of MCF-7 cells

which is prevented by pre-incubation of the cells with 100 nM of

Fulvestrant (F), a selective estrogen receptor down-regulator. Our

findings suggest that the estrogen/NO/Src kinase signaling axis is of

major importance for the development of human breast cancers responsive

to estrogen.

151


Apoptosis-inducing Factor (AIF) role in anthracyclineinduced

cardiomyoblast cell death

SUSANA SAMPAIO, ANA C. MOREIRA, ANA F. BRANCO,

MARIA S. SANTOS, PAULO J. OLIVEIRAM, AND VILMA A. SARDÃO

Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Doxorubicin (DOX) is a broad-spectrum antineoplastic agent prescribed

for the treatment of several types of cancer. Despite its effectiveness,

long term therapy with DOX is associated with a cumulative

and irreversible cardiomyopathy. Several mechanisms for DOX

cardiotoxicity have been proposed, including production of reactive

oxygen species, disruption of mitochondrial function and calcium

homeostasis, as well as activation of p53/Bax pathway and caspase

activation. Nevertheless, caspase inhibitors do not entirely prevent

DOX-induced cell death. This suggests that both caspase-dependent

and independent mechanisms appear to be involved in DOX-induced

cell death. Mitochondria play an important role in apoptosis signaling

pathways through the release of different proapototic proteins. One

protein released from mitochondria upon apoptotic stimuli is the

apoptosis-inducing factor (AIF), which is translocated to the nucleus

promoting cell death independently of caspase activation. We hypothesize

that DOX causes cell death in cardiac cells through both

caspase-dependent and independent mechanism, the latter involving

AIF release from mitochondria. In the present work we demonstrate

that DOX-induced cell death in H9c2 cardiomyoblasts is not entirely

inhibited by a pan-caspase inhibitor. The data show a decrease in

AIF levels from mitochondrial fractions in H9c2 cells treated with

DOX and consequent increase in the nucleus following a 24 and 48h

incubation with clinical relevant DOX concentrations. The results

were confirmed by performing immunocytochemistry for AIF, and

co-localization with mitochondrial and nuclear markers. Pulse Field

Gel electrophoresis showed that DOX causes 50Kbp DNA fragmentation

which is also a marker for AIF cell death. Our results suggest

that DOX-induced cell death is in part due to AIF translocation in a

caspase independent process.

152


Pau d’Arco extract induces Nrf2-dependent gene expression

via ERK1/2

DIETER SCHMOLL, DIETMAR SCHUMMER, MARTIN GERLITZ,

AND MAIKE GLIEN

Sanofi-Aventis Deutschland, R&D, Industriepark Hoechst, 65926 Frankfurt,

Germany

Pau d’Arco, also called Red Lapacho, is a plant extract that has

been used in traditional medicine. Here, we studied the role of the

Nrf2 pathway in the biological action of Pau d’Arco. A water extract

of Pau d’Arco stimulated the expression of both a luciferase reporter

gene under control of a gene promoter containing Nrf2 binding sites

and of the Nrf2 target genes NQO1 and HO1 in HepG2 cells. RNA

interference experiments showed the dependency of Nrf2 for this

regulation. Pau d’Arco activated both ERK1/2 and p38MAPK. However,

only inhibitors of MEK1/2, which is upstream of ERK1/2, but

not of p38 MAPK prevented the induction of NQO1. HPLC-MS

analysis indicated that the active principle within the Pau d’Arco

extract is not based on flavonoids and quinoids such as lapachol.

Overall, the data suggest that the activation of Nrf2 via ERK1/2 could

contribute to the described anti-inflammatory effects of Pau d’Arco

in vivo.

153


Gender-dependent rat liver and brain mitochondrial

antioxidant defenses

ANA M. P. SILVA 1,3 , ANA C. MOREIRA 1,2,3 , MARIA S. SANTOS 1,3 ,

AND VILMA A. SARDÃO 1

1 CNC-Center for Neuroscience and Cell Biology, University of Coimbra;

2 Doctoral Programme in Experimental Biology and Biomedicine, CNC,

University of Coimbra; and 3 Department of Life Sciences, University of

Coimbra, Portugal

Several pathologies and drug-induced injury affect differently men

and women, with distinct outcomes in terms of mitochondrial injury.

The objective of this study was to perform a gender-comparison of

some mitochondrial antioxidant defenses including reduced and oxidized

glutathione (GSH/GSSG), glutathione peroxidase (GPx) and

manganese superoxide dismutase (MnSOD) in order to offer one

possible mechanism for the observed difference.

Five and four male and female 8-10 weeks old Wistar-Han rats,

respectively, were used to isolate brain and liver mitochondrial fractions

according to standard procedures. No differences between males

and females were found in liver mitochondria regarding GSH and

GSSG (20.00±3.97 and 15.39±2.77, in terms of GSH/GSSG ratio).

However, GPx (350±40 U/mg protein) and MnSOD activities in female

liver mitochondria were higher than in the equivalent male group

(220±12 U/mg protein for GPx, and 9.5±4.0 U/mg protein for

MnSOD). In brain mitochondria, MnSOD activity (32.5±3 U/mg

protein, male, and 16.5±9.1 U/mg protein, female) and GSH/GSSG

(1.01±0.17, male, and 1.57±0.26, female) is higher in males than in

females. No gender-related differences were found in the brain regarding

GPx activity.

The results show gender-related differences in some antioxidant

mitochondrial components, with the liver and brain presenting an

apparent contrast. These results may help explaining the different

effects in terms of production of mitochondrial reactive oxygen species

in both organs/genders which impact not only possible signaling

mechanisms and the effects of antioxidants but also the resistance to

drug- and pathology-induced mitochondrial injury.

154


Acknowledgements: This work is supported by PTDC/AGR-ALI/108326/2008

grant from Foundation for Science and Technology (FCT). ACM, AMS and

VAM are supported by FCT PhD and Post-doc fellowships (SFRH/BD/33892/

2009, SFRH/BD/76086/2011 and SFRH/BPD/31549/2006, respectively).

155


Anthocyanins in saskatoon berries

JIUXUE SONG AND JIM FANG

College of Pharmacy and Nutrition, University of Saskatchewan

Saskatoon (Amelanchier alnifolia Nutt., Rosaceae) is a fruit-producing

shrub or small tree of the Rosaceae family native to the North

American Great Plains. Saskatoon berries is also called the juneberry

or serviceberry and were traditional food used by aboriginal people.

Saskatoon berries have been grown on the Canadian prairies commercially

since the mid-1960s. Although saskatoon berry and blueberry

(Vaccinium corymbosum L., Ericaceae) belong to different

species, they share many similar characteristics that are important to

consumers, including fruit color, shape, size, texture and nutrition.

Both saskatoon berries and blue berries are rich in anthocyanins,

however, there are considerable differences in the content of individual

anthocyanins. For example, the saskatoon berries have more

cyanidin 3-galactoside, and cyanidin 3-glucoside than blueberries,

but has less malvidin 3-glucoside, and malvidin 3-galactoside. Compared

to blueberries, saskatoon berries have much higher contents of

protein, fat, fiber and calcium.

A comprehensive characterization of flavonoids was conducted

on saskatoon berries using a high performance liquid chromatography

(HPLC) method. Saskatoon fruits were grounded in a small food

processor and 0.1 g samples of the tissue were extracted with 1 ml

HPLC-grade methanol with hydrochloric acid (0.1%) for 3 times.

The extracts were combined and subjected to HPLC analysis. The

HPLC method employed a Synergi RP-Max column (250 x 4.6 mm,

4mm, Phenomenex, Torrance, CA) eluted with a 60min gradient of

5-25% acetonitrile in 1% aqueous formic acid at a flow rate of 1.0

ml/min. The peaks were identified by comparing their retention time

and HPLC-MS/MS fragmentation profiles to standard reference compounds.

Where no reference compounds were available, the identities

of the peaks were elucidated on the basis of their HPLC-MS/MS

fragmentation profiles.

This research is funded by Agriculture Development Fund, Saskatchewan

Ministry of Agriculture, Canada

156


The role of functional variants of phase I and II genes and

their contribution to oxidative stress in Serbian patients

with COPD

MARIJA STANKOVIC 1 , ALEKSANDRA NIKOLIC 1 , DJORDJE FRANCUSKI 1 ,

ALEKSANDRA TOPIC 2 , MARIJA MITIC-MILIKIC 3 ,

LJUDMILA NAGORNI-OBRADOVIC 3 , NATASA PETROVIC-STANOJEVIC 4 ,

MARINA ANDJELIC-JELIC 5 , AND DRAGICA RADOJKOVIC 1

1 Institute of Molecular Genetics and Genetic Engineering, University of

Belgrade, Belgrade; 2 Faculty of Pharmacy, University of Belgrade,

Department of Medical Biochemistry, Belgrade; 3 Institute for Tuberculosis

and Lung Disease, University Clinical Center of Serbia, Belgrade; 4 University

Clinical Center Zvezdara, Department of Pulmonology; Belgrade; 5 University

Clinical Center Zvezdara, Department of Endocrinology, Belgrade

Chronic obstructive pulmonary disease (COPD) is complex disorder

influenced by multiple genetic and environmental factors, as

well their interactins. Increased oxidative stress is prominent feature

of COPD. Variants of genes of phase I and II, implicated in oxidantsantioxidants

imbalance, might be involved in COPD.

We investigated the role of functional variants of cytochrome P450

(CYP), glutathione-S-transferase (GST) and microsomal epoxide

hydrolase (mEH) in COPD. The genotypes of 122 patients and 100

controls were determined by PCR-RFLP for CYP1A1*1A/*2A,

CYP2E1*1A/*5B, GSTP1 Ile105Val, mEPHX S (slow) and F (fast),

and by multiplex PCR for GSTM1 null and GSTT1 null alleles.

Analysis revealed that GSTM1 null variant was significantly higher

in patients than in controls (61.5% vs. 47.0%; OR=1.8; p=0.042).

Significant results were obtained for combinations of GSTM1 null

and GSTP1 105Val/(Val) (38.5% vs. 24.0%; OR=2.0; p=0.030), as

well CYP1A1*1A/*2A, GSTM1 null and mEH S/(S) (7.4% vs.

1.0%; OR=7.9; p=0.025).

In order to assess the contribution of combined genotype

CYP1A1*1A/*2A, GSTM1 null and mEH S/(S) to systemic

oxidative stress in patients, we measured 8-oxo-7,8-dihydro-2’deoxyguanosine

(8oxodG) in urine using ultra performance liquid

chromatography and tandem mass spectrometry (UHPLC-MS/MS).

A carrier of CYP1A1*1A/*2A, GSTM1 null and mEH S/(S)

157


combination had two times higher level of 8-oxodG adjusted to creatinine

(1.608 nmol/mmol) than carrier of all wild-type alleles (0.744

nmol/mmol).

In this study single and combined genotypes were associated with

COPD. Contribution of combined genotype to increased oxidative

stress was determined, that might be useful in COPD prevention by

antioxidant therapy in such carriers.

158


Resveratrol reverses SR-B1 and ABCA1 modulation by CS

in human keratynocites

C. STICOZZI 1 , G. BELMONTE 2 , A. PECORELLI 3 , F. PESSINA 4 ,

AND G. VALACCHI 1-5

1 Dept. of Evolutionary Biology, University of Ferrara, Ferrara, Italy; 2 Dept. of

Biomedical Sciences, University of Siena, Siena, Italy ; 3 Dept. of

Pathophysiology, Experimental Medicine and Public Heath, University of

Siena, Siena, Italy; 4 Dept. of Neuroscience, University of Siena, Siena, Italy;

5 Dept. of Food and Nutrition, Kyung Hee University, Seoul, Korea

Skin acts as a first line of defense against environmental insults

such as UV, ozone and cigarette smoke. Cigarette smoking has been

strongly linked to several health conditions like heart disease, lung

cancer, and other respiratory and circulatory ailments. However, the

effects of cigarette smoking in the skin are significant. It has been

shown that CS can affect oxygen skin flow, damage elastin and collagen,

can dehydrate and deprive the skin of necessary nutrients that

are necessary to retain the skin’s moisture and smoothness. Because

its composition (cholesterol fatty acids and ceramide) the upper layer

of the skin, the stratum corneoum, is very susceptible to free radicals

damage and therefore the alteration of the skin lipids composition by

the exposure to environmental stressors can affect the capacity of the

SC to protect us from dehydration and modify skin physiology. Scavenger

Receptor B1 (SR-B1) and ATP binding cassette A1 (ABCA1)

have been shown to be involved in the uptake and efflux of cholesterol,

respectively, therefore their role in skin homeostasis is crucial.

Previous data from our group have shown that CS exposure, via

its ability to induce oxidative stress, can modify the expression of

SR-B1 and ABCA1 levels by the formation of aldehydes (4HNE)

adducts. Based on the fact that resveratrol, the main active polyphenol

in red wine, has been demonstrated to show benefits against skin

disorders, in this study, we investigated the possible role of resveratrol

as protective agent against its ability to modify the expression of key

proteins involved in cholesterol intracellular levels such as SR-B1

and ABCA1.

Using immunoblotting analysis, we have demonstrated that

resveratrol is able to increased SRB1 and ABCA1 protein levels in

159


dose-dependent manner in human keratinocytes (HaCaT cells). When

the cells were exposed to CS, previously pretreated with different

doses of resveratrol, the lost of SR-B1 was reversed in a dose dependent

manner. As to concern ABCA1, there were not significant

effects of resveratrol on its expression. Resveratrol was also able to

modulate the levels of 4HNE protein adducts induced by CS.

Resveratrol thus appears to be a natural compound that can have

a role (either direct or indirect) on skin defense against CS exposure.

These properties make it a potential antioxidant that could be used to

prevent the alteration of skin lipid barrier induced by enviromental

stressors such us cigarettes smoke.

160


Protection of cells against myeloperoxidase-derived oxidants:

enzymatic removal by the NADPH / thioredoxin reductase /

thioredoxin system

ALDWIN SURYO RAHMANTO 1 AND MICHAEL J. DAVIES 1,2

1 2

The Heart Research Institute, Sydney, NSW 2042, Australia;

Faculty of Medicine, University of Sydney, Sydney, NSW 2006,

Australia

The NADPH-thioredoxin reductase (TrxR)-thioredoxin (Trx) system

is a major cellular antioxidant and reducing system, whose impairment

is implicated in cardiovascular disease and atherosclerosis.

Leukocyte-derived myeloperoxidase (MPO), a powerful and independent

diagnostic and prognostic risk factor for atherosclerosis, generates

powerful oxidants including hypochlorous (HOCl) and

hypothiocyanous acids (HOSCN). Reaction of these oxidants with

cells results in apoptosis and dysfunction. Compelling evidence exists

for the presence of MPO mRNA, protein, and MPO-mediated

damage in all grades of human atherosclerotic lesions. In this study,

the effects of HOCl, chloramines (from reaction of HOCl with Lys

residues and other amines) and HOSCN, on NADPH and TrxR individually,

or as an antioxidant system were investigated. Pathophysiological

levels of HOCl and chloramines (formed on taurine, N-Ac-

Lys, and Gly), but not HOSCN, directly depleted NADPH in vitro

and in cell lysates. Isolated TrxR was inactivated by HOCl and

HOSCN, as detected by a decrease in 5,52 -dithiobis(2-nitrobenzoic

acid) reduction. In contrast, exposure of TrxR to MPO-derived HOCl

or HOSCN, in the presence of NADPH, resulted in preservation of

TrxR activity, enhanced removal of the oxidants and an increased

rate of TrxR-mediated NADPH oxidation. Inclusion of Trx, the biological

partner for TrxR, into the reaction system enhanced both the

rate of NADPH oxidation and the removal of oxidants. Overall, these

data indicate that whilst the individual components of the NADPH-

TrxR-Trx system can be damaged by MPO-derived oxidants, the

complete enzyme system may act as catalytic protective mechanism

against MPO-derived oxidants under pathophysiological conditions.

161


Uncoupling of endothelial nitric oxide synthase by

myeloperoxidase-derived oxidants

JIHAN TALIB 1 AND MICHAEL J. DAVIES 1,2

1 The Heart Research Institute, Sydney, NSW 2042, Australia; 1,2 Faculty

of Medicine, University of Sydney, Sydney, NSW 2006, Australia

Decreased bioavailability of nitric oxide (NO . ) associated with

endothelial dysfunction, a key early, and defining, feature of cardiovascular

disease and atherosclerosis. NO . is generated by endothelial

nitric oxide synthase (eNOS), a dimeric protein containing a zincthiolate

cluster. Disruption of this cluster induces eNOS uncoupling

and decreased NO . generation. The leukocyte-derived heme enzyme

myeloperoxidase (MPO) is a potent inducer of vascular injury, with

this enzyme detected in all grades of atherosclerotic lesions. This

enzyme mediates damage via the generation of potent oxidants including

hypochlorous acid (HOCl) and hypothiocyanous acid

(HOSCN) from H 2 O 2 and chloride (Cl - ) and thiocyanate ions (SCN)

respectively. As SCN - is present at elevated levels in smokers

(≤ 250 μM vs ~50 μM in controls) and is the preferred substrate for

MPO, we hypothesised that the enhanced formation of HOSCN, a

thiol-specific oxidant, by MPO in smokers may result in increased

disruption of the zinc-thiolate centre and inactivation of eNOS in

human coronary endothelial arterial cells (HCAEC). Western blotting

experiments indicate that exposure of recombinant or HCAEC

eNOS to 50-200 mM HOSCN or HOCl induces uncoupling of the

eNOS dimer. Similar damage was induced by a MPO/H 2 O 2 /SCN - /

±Cl - system. Increasing HOSCN levels, or SCN - (50-200 mM) for

the enzyme system, caused enhanced eNOS uncoupling, and decreased

formation of citrulline (a product of NO . formation) from

arginine by eNOS. These findings suggest that elevated levels of

SCN - detected in smokers may enhance eNOS inactivation and endothelial

dysfunction during the development of atherosclerosis in

smokers.

162


Prevention of high fat induced liver steatosis: role of

eicosapentaenoic and docosahexaenoic acids supplementation

GLADYS TAPIA 1 , VIRGINIA FERNÁNDEZ 1 , LUIS VIDELA 1 ,

RODRIGO VALENZUELA 2 , CYNTHIA BARRERA 2 ,

AND AMANDA DESPESSAILLES 1

1 Program of Pharmacology, ICBM; 2 Nutrition and Dietetics School, Faculty

of Medicine, University of Chile, Santiago, Chile

The development of insulin resistance is a critical step in the onset

of the diseases that constitute the metabolic syndrome, including abnormal

and pathological accumulation of triglycerides in the liver

(liver steatosis). Eicosapentaenoic (EPA) and docosaexaenoic acids

(DHA) increases insulin sensitivity, have anti-inflammatory properties,

stimulate lipolysis and inhibit hepatic lipogenesis, and are considered

protective agents against these nutritional alterations.

Objective: To evaluate the effect of EPA+DHA dietary supplementation

in the prevention of insulin resistance, inflammation and

hepatic steatosis induced by high fat diet (HFD).

Methods: Male C57BL/6J mice (n=9 for each group) were fed

for 12 week with the following diets: (i) control diet (20% protein,

70% carbohydrate, 10% fat), (ii) control diet plus EPA+DHA (200

mg/kg), (iii) high fat diet (20% protein, 20% carbohydrate, 60% fat)

or (d) high fat diet plus EPA and DHA. Weight increase, insulin

resistance (HOMA), liver steatosis (histology and liver total fat) and

inflammation (serum TNFa; levels and histology) were analyzed.

Results: (iii) group showed a significant increase in final weight

(p


Sulforaphane protects SH-SY5Y cells against amyloid-beta

peptide oligomer-induced oxidative stress and neuronal

apoptosis

ANDREA TAROZZI, CECILIA BOLONDI, FABIANA MORRONI,

GIORGIO CANTELLI-FORTI, AND PATRIZIA HRELIA

Department of Pharmacology, Alma Mater Studiorum,University of Bologna,

via Irnerio 48, 40126 Bologna, Italy

The oxidative stress induced by amyloid-beta (Ab) peptide aggregation

forms such as soluble oligomers have a casual role in the

neuronal dysfunction and death associated with Alzheimer’s disease

(AD). Among dietary isothiocyanates, present in cruciferous vegetables,

sulforaphane (SF) is known as a strong inducer of antioxidant

endogenous molecules and potential neuroprotective agents. In this

study, we found that pretreatment of human neuroblastoma SH-SY5Y

cells with SF showed a dose-dependent inhibitory effect of Ab oligomer-induced

reactive oxygen species (i.e. hydrogen peroxide and

superoxide anion) formation and neuronal apoptosis. These

neuroprotective effects of SF were also observed after co-treatment

of SH-SY5Y cells with SF and Ab oligomers. Last, in both experimental

conditions, SF induced the translocation of Nrf-2 into the

nucleus and subsequent GSH induction. The antioxidant and

antiapoptotic effects observed with SF were abolished by the addition

of buthionine sulfoximine, supporting the main role of GSH in

the neuroprotective effects displayed by SF. These findings suggest

that SF may prevent and counteract the neurotoxicity induced by Ab

oligomers.

Supported by MIUR-FIRB project RBAP11HSZS (2011) and Fondazione di

Cassa di Risparmio di Imola (Italy)

164


Interaction between 24-hydroxycholesterol and amyloid-β

in amplifying neuronal damage in Alzheimer’s disease

GABRIELLA TESTA 1 , PAOLA GAMBA 1 , ELENA TAMAGNO 2,3 ,

MICHELA GUGLIELMOTTO 2,3 , SIMONA GARGIULO 1 , BARBARA SOTTERO 1 ,

GABRIELLA LEONARDUZZI 1 , AND GIUSEPPE POLI 1

1 Department of Clinical and Biological Sciences, University of Turin, Italy;

2 Department of Experimental Medicine and Oncology, General Pathology

Section, University of Turin, Turin, Italy; 3 Neuroscience Institute of the

Cavalieri Ottolenghi Foundation (NICO), University of Turin, Turin, Italy

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized

by the extracellular accumulation of amyloid-beta (Aβ) in

neuritic plaques. Several studies show that elevated cholesterol levels

increase Aβ but the failure to demonstrate a transfer of cholesterol

from the circulation into the brain makes difficult to explain the

link between hypercholesterolemia and AD. In contrast to cholesterol

itself, its oxidized metabolites 24-hydroxycholesterol (24-OH),

27-hydroxycholesterol (27-OH) and 7-hydroxycholesterol (7β-OH)

are able to pass the blood-brain barrier.

We have investigated the potential implication of these oxysterols

in the pathogenesis of AD. Our studies show that 24-OH, 7β-OH

and 27-OH enhance the binding of Aβ to human differentiated neuronal

cell lines (SK-N-BE and NT-2) by up-regulating expression

and synthesis of CD36 and β1-integrin receptors, which both form,

with CD47 receptor, the complex that mediates the Aβ binding to

neuronal cells. However, only 24-OH markedly potentiates the proapoptotic

and pronecrogenic effects of Ab on cells: this peculiar behaviour

of 24-OH depends on its strong enhancement of the generation

of reactive oxygen species (ROS). Cell incubation with antioxidants

quercetin or genistein prevents 24-OH’s pro-oxidant effect and

potentiation of Aβ-induced necrosis and apoptosis. Thus, the presence

of 24-OH appears to enhance the adhesion of Aβ to the neuronal

cells, then to amplify the neurotoxic action of Aβ by increasing

ROS levels.

These results support a potential role of the oxysterol 24-OH in the

pathogenesis of AD.

165


Palm oils offer myocardial protection against ischaemia/

reperfusion injury despite significant variation in antioxidant

and fatty acid composition

E. TRUTER, D.J. BESTER, AND A.J. ESTERHUYSE

Department of Biomedical Sciences, Faculty of Health and Wellness, Cape

Peninsula University of Technology, South Africa

Background: It has been shown that dietary supplementation with

Malaysian produced red palm oil (RPO) improves functional recovery

and reduces infarct size, following ischaemia/reperfusion (IR) in

rat hearts. We subsequently investigated the composition of several

other palm oils. South American palm oil (Buruti oil) varied significantly

in composition from other palm oils.

Materials and Methods: Oils were analyzed for fatty acid composition

and antioxidant content. RPO and Buriti oil were then subjected

to ex-vivo myocardial functional recovery testing in a working

heart perfusion, ischaemia/reperfusion model.

Results: Fatty acid and antioxidant composition of palm oils from

Africa were similar to that of Malaysian RPO. Buriti oil however,

showed some differences in fatty acid composition, as well as significant

differences in antioxidant content, especially with respect to

vitamin E. Both RPO and Buriti oil offered significant cardiac protection

against ischaemia/reperfusion injury when compared to control

hearts.

Conclusion: We previously hypothesized that dietary RPO supplementation

reduces myocardial ischaemia/ reperfusion injury and

infarct size due to its natural antioxidant and fatty acid content. Based

on their composition, African palm oils may induce similar effects as

Malaysian RPO with regards to cardiovascular protection. Our functional

studies with Buriti oil demonstrate that the presence of

tocotrienols is not essential for cardiovascular protection. Furthermore,

the variation in fatty acids may indicate that fatty acid composition

has a less significant role in cardiovascular protection against

ischaemia/reperfusion injury than previously hypothesized.

166


Postconditioning (PostC) cardioprotection in experimental

models of cardiac hypertrophy: spontaneously hypertensive

(SHR) and nandrolone (ND)-abuse rats

FRANCESCA TULLIO, CARMELINA ANGOTTI, MARIA-GIULIA PERRELLI,

VITINA CARRIERO, PASQUALE PAGLIARO, AND CLAUDIA PENNA

Department of Clinical and Biological Sciences, University of Turin, Italy

Background: Cardiac PostC reduces ischemia/reperfusion (I/R)

injury via a redox-sensitive mechanism. However in the presence of

comorbidities the effectiveness of PostC is reduced. We studied

whether PostC can reduce I/R injury in the presence of ventricular

hypertrophy induced by hypertension or ND, an anabolic-androgenic

steroid.

Methods: A) Hypertension model: hearts isolated from SHR and

Wistar-Kyoto (WKY) rats were subjected to: 30-min ischemia and

120-min reperfusion (I/R); I/R+PostC (5 cycles 10s I/R); 4-weeks

pretreatment with an angiotensin-converting enzyme inhibitor (ACE-

I) before to subject the hearts to I/R with or without PostC maneuvers;

ACE-I infusion during early reperfusion (20 or 40 min) with or without

PostC maneuvers. B) Drug-abuse: hearts isolated from ND

pretreated (14-days or 10-weeks) rats and untreated rats underwent

to I/R and PostC protocols. Infarct size and left ventricular pressure

were evaluated. WB studies were also performed.

Results and Conclusions: Data confirm PostC effectiveness in

normotensive WKY and PostC ineffectiveness in SHR in limiting

infarct size. Chronic ACE inhibition favors infarct size reduction in

SHR; yet in WKY ACE-I reduces infarct size, but attenuates infarctsize

limiting effects of PostC. ACE-I given in reperfusion does not

reduce infarct size and does not recover PostC protection in SHR. In

ND groups data show that 14-days ND does not induce hypertrophy

and improves the post-ischemic cardiac function via b 2 -adrenoreceptor

involvement with and without PostC. However, 10-weeks ND treatment

induces cardiac hypertrophy, increases myocardial susceptibility

to I/R injury and abolishes cardioprotection by PostC. ND alters

the responses of survival kinases to PostC.

167


Antiproliferative effect of COX-2 inhibitors of two 1,5diarylpyrrole-3-alkoxyethyl

ethers on human keratinocytes:

possible use in skin cancer

G. VALACCHI 1,2 , C. STICOZZI 1 , G. BELMONTE 3 , A. DI CAPUA 4 ,

A. CAPPELLI 4 , A. GIORDANI 5 , M. BIAVA 6 , AND M.ANZINI 4

1 Dept. of Evolutionary Biology, University of Ferrara, Ferrara, Italy; 2 Dept. of

Food and Nutrition, Kyung Hee University, Seoul, Korea; 3 Dept. of

Biomedical Sciences, University of Siena, Siena, Italy; 4 Dept. of Medicinal

and Pharmaceutical Chemistry, University of Siena, Siena, Italy; 5 Rottapharm

Madaus, Monza, Italy; 6 Dip. di Studi di Chimica e Tecnologie del Farmaco,

Università degli Studi di Roma,”La Sapienza”, Roma, Italy

Selective COX-2 inhibitors belonging to the class of diaryl

heterocycles (e.g., celecoxib, rofecoxib, valdecoxib, eterocoxib, etc.),

are devoid of the undesirable effects (mainly gastrointestinal) due to

their capacity to inhibit both isoforms of cyclooxygenases (constitutive

COX-1, responsible for cytoprotective effects and inducible

COX-2, responsible for inflammatory effects). In addition, because

several reports have identified an increased risk for cardiovascular

events associated with the use of COXib some of these compounds

have been withdrawn from the market.

In this way, being interested into the development of new antiinflammatory

agents a series of substituted 1,5-diarylpyrrole-3alkoxyethyl

esters[1,2] and ethers[3] was synthesized with the aim

to obtain new, highly selective, potent, and safer COX-2 inhibitors.

On the basis of our previous results obtained in “in vitro” and “in

vivo” anti-inflammatory systems, in the current study we evaluated

the effect of compounds two 3-alkoxyethyl ethers, VA441 and VA428

(up to 100 µM), in comparison to well known COX-2 inhibitors such

as celecoxib and rofecoxib on HaCaT cell (keratinocytes). In particular,

we analyzed cell proliferation along with crucial molecules in

cell cycle progression, i.e. NFkB and ERK as targets D mediators

and cyclin D1 and p21 Cip1 D Kip as final effectors.

Both compounds VA441 and VA428 showed a strong inhibition

of cell proliferation and this was via the strong induction of ERK that

in this condition is able to activate the cell cycle inhibitor p21 and

decrease therefore the cell cycle regulator cyclin D1. Worth of note

168


was the effect of VA428, which had also an inhibitory effect on the

transcription factor NFkB, that regulates cyclin D1 expression. These

effects were not appreciated in cells treated with celecoxib and

rofecoxib in the range of no toxic doses. These data showed that

compounds VA441 and VA428, along with their role in inhibiting

COX-2 have a possible therapeutic use against pro-proliferative disorders

such as skin cancer.

References:

[1] Cappelli, A. et al. 3-Substituted-1,5-diarly-2-alkyl-pyrroles Highly Selective

and Orally Effective COX-2 Inhibitors. WO/2008/014821

[2] Biava, M. et al. Cyclooxygenase-2 Inhibitors. 1,5-Diarylpyrrol-3-acetic Esters

with Enhanced Inhibitory Activity toward Cyclooxygenase-2 and Improved

Cyclooxygenase-2/Cyclooxygenase-1 Selectivity J. Med. Chem. 2007, 50,

5403-5411

[3] Anzini, M. et al. Synthesis, Biological Evaluation, and Enzyme Docking

Simulations of 1,5-Diaryl pyrrole-3-alcoxyethyl Ethers as Highly Selective

COX-2 Inhibitors Endowed with Anti-inflammatory and AntinociceptiveActivity

J. Med. Chem., 2008, 51, 4476-4481

169


Selenium can prevent aluminium-induced inflammation and

decrease in hepatic redox status in mice

DALE VIEZELIENE 1 , PIET BEEKHOF 2 , ERIC GREMMER 2 ,

HILIARAS RODOVICIUS 3 , ILONA SADAUSKIENE 1 , EUGENE JANSEN 2 ,

AND LEONID IVANOV 2

1 Department of Biochemistry, Medical Academy, Lithuanian University of

Health Sciences, A.Mickeviciaus 9, LT-44307, Kaunas, Lithuania and

Neuroscience Institute, Medical Academy, Lithuanian University of Health

Sciences, Eiveniu 4, LT-50009, Kaunas, Lithuania; 2 Laboratory for Health

Protection Research, National Institute for Public Health and the

Environment, PO Box 1, 3720 BA Bilthoven, the Netherlands; 3 Department of

Drug Chemistry, Medical Academy, Lithuanian University of Health Sciences,

A.Mickeviciaus 9, LT-44307 Kaunas, Lithuania

Trace element selenium (Se) can increase antioxidant capacity of

intracellular systems, because of its involvement as a cofactor in

glutathion peroxidase catalyzed reaction.

Recent studies demonstrated that aluminium (Al) ions interfere

with metabolism of selenium and therefore systemic Al toxicity may

directly (or indirectly) affect Se homeostasis. Since Se status in European

population is generally rather low, exposure to Al may interfere

with Se-driven protection against environmental factors that evoke

oxidative stress.

To gain a deeper insight into these complex interactions the present

study was carried out to investigate a possible protective effect of Se

on Al-induced toxicity in mouse organs under short-term exposure

to Al. It was focused on the systemic effects of (co)exposure to Al

and Se on the redox status in the liver, kidney and brain.

Short term exposure (16 hrs) to Al resulted in an increase of the

systemic inflammation parameter IL-6, whereas serum levels of TNFa

remained unaffected.

This indicated an increased intracellular oxidative stress and altered

redox status in the liver since the selective increase of IL-6

concentration serves as a protective intrahepatocellular process driven

by oxidative stress. As a result, intracellular redox parameters of glutathione

status were also affected, because GSHtot decreased significantly

upon Al exposure.

Both the increase of IL-6 and decrease of glutathion status could

170


e prevented by co-exposure to Se. The redox status of kidney and

brain was not markedly affected.

Therefore it can be concluded that short-term exposure to Al causes

adverse effects on intracellular oxidative stress processes in liver, as

reflected by the increase of serum IL-6 concentration, which can be

restored by co-administration of trace element Se as a part of the

glutathione redox system.

171


Effects of age and ethnicity on oxidative status and influence of

fasting calorie restriction in men’s health

WAN ZURINAH WAN NGAH, MOHAMAD HANAPI JOHARI,

AND NORWAHIDAH ABDUL KARIM

Department of Biochemistry, Faculty of Medicine, University Kebangsaan,

Malaysia

Oxidative status may contribute to susceptibility towards degenerative

diseases which can be reduced by calorie restriction (FCR)

resulting in increased longevity. This study examined the effects of

age and ethnicity where 329 Malay and Chinese men aged 20-80

years were recruited. Plasma protein carbonyl levels was higher in

Chinese compared to Malay men (p


UVA-1 and UVA-1 oxidized phospholipids elicit an autophagic

stress response in primary keratinocytes

YI ZHAO 1 , CHENGFENG ZHANG 1 , HEIDEMARIE ROSSITER 1 , ULRICH

KÖNIG 1 , LEOPOLD ECKHART 1 , SUSANNE KARNER 1 , ERWIN TSCHACHLER 2 ,

AND FLORIAN GRUBER 1

1 Department of Dermatology, Medical University of Vienna, Austria and

2 CE.R.I.E.S, Neuilly, France

Oxidant stress generates potentially detrimental modified proteins.

Macroautophagy is a candidate mechanism for degradation of damaged

cell content, and its role in the stress response of epidermal

keratinocytes (KC) is not known. We investigated the role of autophagy

after UVA-1 irradiation and UVA-1 oxidized phospholipids

(OxPL).

We studied in vitro whether exposure to UVA-1 or OxPL initiates

autophagy in primary KC by LC3 conversion assays. We used primary

KC of LC3-GFP transgenic autophagy marker mice to investigate

autophagosome formation. Finally, we investigated the stress

responses in KCs from epidermal autophagy deficient mice (ATG7 -

/-). We found that UVA-1 irradiation, treatment with OxPL and the

autophagy inducer rapamycin induced LC3 conversion after 4h, indicative

of autophagosome initiation. In LC3-GFP transgenic KC

we found strongly enhanced formation of autophagosomes 6 hours

after treatment. The autophagy marker p62 however was not degraded,

rather we found high molecular weight complexes positive

for p62 upon oxidant stress. Importantly, these complexes were

strongly accumulated in stressed ATG7 -/- KC, as were baseline p62

levels. p62 is both, transcriptional target and inducer of the Nrf2 antioxidant

response (that is activated by UVA-1 and OxPL). Indeed,

mRNA and protein levels of Nrf2 dependent genes were significantly

elevated in ATG7 -/- keratinocytes exposed to oxidant stressors.

In conclusion, our data show that autophagy is initiated in KC by

UVA-1 and OxPL and may be necessary for the clearance of oxidized

protein complexes and that autophagy amplifies the Nrf2-antioxidant,

an effect that was reported as detrimental in other epithelia.

173


A novel neurotrophic drug for cognitive enhancement and

Alzheimer’s disease

MARGUERITE PRIOR, JENNIFER EHREN, QI CHEN, PAMELA MAHER,

AND DAVID SCHUBERT

Cellular Neurobiology Laboratory, Salk Institute For Biological Studies,

La Jolla, CA 92037

At present, there are few drugs that improve the memory deficits

associated with normal aging and none that prevent cognitive decline

in chronic neurodegenerative conditions such as Alzheimer’s

disease (AD), which is the most common cause of dementia in the

elderly affecting more than 24 million people worldwide. Historically,

the search for a treatment for AD has been focused on the amyloid

beta peptide (Aβ) that mediates familial Alzheimer’s disease pathology.

However, given that age is the greatest risk factor for AD,

our laboratory has explored an alternative drug discovery paradigm

to select drug candidates for neurodegenerative disease that is based

on efficacy in cell models of multiple age-associated pathologies rather

than exclusively amyloid metabolism. This scheme has identified an

exceptionally potent, orally active, neurotrophic compound (J147)

that facilitates memory in normal rodents, prevents behavioral and

synaptic protein loss in AD transgenic mice, and reverses cognitive

loss in aged transgenic AD mice. J147 is also neurogenic in both

very old and young mice and reduces the significant loss in dendritic

spines that occurs with age. Strikingly, we have found that the neurotrophic

and memory-enhancing activities of J147 are associated

with the induction of brain derived neurotrophic factor (BDNF), a

growth factor that is reduced with age and in AD brain, that is required

for normal cognitive function, and is implicated in

neurogenesis. J147 has the medicinal chemical properties of a good

CNS drug. Together, our data so far suggests that J147 has potential

for the treatment of AD.

174


175

AUTHOR INDEX


A

Adams, A.F. 95

Adams, M. 95

Agüera, E. 58

Ai-Ping, W. 95

Aliahmat, N. 140

Aman, C. 83

Ambra, R. 138

Ambrosini, G. 60, 62

Anderson, R.M. 6

Andjelic-Jelic, M. 157

Andrade, S. 86

Angelone, T. 65

Angeloni, C. 64

Angotti, C. 65, 167

Anwar, S. 84

Anzini, M. 132, 168

Aquilano, K. 66

Aragno, M. 139

Armagan, G. 67, 68

Arnal, E. 69, 122

Aschettino, G. 148

Asghar, M. 70

Attamasio, A. 149

Aung, H.H. 71

Avellana, J.A. 46

B

Baima, S. 113

Baldelli, S. 66

Bamonti, F. 83, 87, 89

Bánhegyi, G. 125

Barella, L. 101

Barger, Jamie 6

Barrera, C. 163

Batandier, C. 119

Batista, W.L. 151

Beekhof, P. 121

Beekhof, P. 170

176

Bellanti, F. 72, 150

Belmonte, G. 132, 159, 168

Berta, G.N. 111

Bester, D.J. 73, 166

Bettega Felipe, K. 103

Biasi, F. 117

Biava, M. 168

Bicker, W. 116

Biolcati, G. 138

Birringer, M. 101

Blanco, C. 55

Blonda, M. 72, 150

Bochkov, V.N. 116

Bolondi, C. 76, 164

Bonara, P. 87, 89

Bordignon-Luiz, M.T. 106 107

Borrás Juan Gambini , C. 46

Bozaykut, P. 79

Branco, A.F. 152

Brewer, G.J. 43

Brigelius-Flohé, R. 13

Brown, L.J. 77

Buonocore, D. 92

C

Cadiz, N. 102

Calabrese, E.J. 48

Calabro, V. 48, 148

Calfapietra, S. 117

Campolo, J. 91

Canali, R. 84, 143

Canini, F. 119

Cantelli-Forti, G. 76, 164

Canuto, R.A. 144

Canzoneri, R. 115

Cappelli, A. 168

Carcaillon, L. 55

Carlson, D.A. 27

Carranza, A. 78


Carretero, A.S. 137

Carriero, V. 167

Casteilla, L. 25

Castillo, V. 102

Castro, E. 86

Catalgol, B. 79

Catizone, P. 137

Cervellati, F. 80

Cervellati, C. 80

Chartoumpekis, D. 82

Chen, Q. 174

Chugh, G. 70

Cighetti, G. 83

Cillard, J. 99

Cimino, F. 84, 143

Ciriolo, M.R. 26, 66

Collino, M. 139

Colman, R.J., 6

Comitato, R. 143

Cornelius, C. 48

Cortelazzo, A. 132

Cortopassi, G. 85

Couturier, K. 119

Cremers, J. 121

Csala, M. 124

Cuervo, A.M. 45

Cuesta, S. 129

Curcio, M. 86

Curi Pedrosa, R. 103, 107

Cutano, V. 115

D

Dagci, T. 68

D’arc Castania Darin , J. 104

D’Archivio, M. 60, 62

Davalos, D. 149

Davies, K.J.A. 52, 99, 131

Davies, M.J. 123, 161, 162

de Liso, F. 83, 87, 89

177

De Giuseppe, R. 83, 87, 89

Del Bo’, C. 91, 108

Delamarche, A. 99

Despessailles, A. 163

Di Capua, A. 132, 168

Di Scipio, F. 111

Dian-Yuan, Z. 95

Dinelli, G. 137

Domann, F.E. 18

Domenicotti, C. 110

Doria, E. 92

Dovinova, I. 93

E

Èaèányiová, S. 93

Ece, A. 79

Eckhart, L. 173

Edeas, M. 95

Ehren, J. 97, 174

Emrani, R. 99

Ermak, G. 99

Ernst, I.M.A. 101

Ertürk, S. 126

Esterhuyse, A.J. 73, 166

F

Fabbri, D. 64

Falcone, A. 117

Fang, J. 156

Fauzi, N.M. 39

Fernandez, V. 102, 163

Ferreira, E.A. 103, 106, 107

Ferro Aissa, A. 104

Filesi, C. 60, 62

Filho, D.W. 106

Filho, D.F. 103

Fischer, S.J. 27

Fischerman, L. 148

Fortes Gris, E. 103, 106, 107


Fracassetti, D. 91, 108

Fraga, C. 109

Fraga, C.G. 78, 148

Fraga, M.F. 42

Francuski, D. 157

Fratantonio, D. 84

Frei, B. 28

Fu, S. 123

Furfaro, A.L. 110

G

Galaris, D. 95

Galleano, M. 78, 109, 148

Gamba, P. 111, 112, 165

Garcia-Garcia, F.J. 55

Garcia, C. 129

Gargiulo, S. 111, 112, 165

Gendelman, R. 27

Geréz, E. 78

Gerlitz, M. 153

Ghosh, D. 43

Gilbert, B. 98

Giordani, A. 168

Giovannini, C. 60, 62

Giraldo, A.I. 58

Giusti, A.M. 113

Glien, M. 153

Goitre, L. 115

Gomes Correia, J.F. 103

Goto, S. 131

Graham, G. 123

Gravina, S. 44

Greggi Antunes, L.M. 104

Greilberger, J.F. 134

Gremmer, E. 170

Gruber, F. 116, 173

Grune, T. 40, 79

Guerranti, R. 132

Guglielmetti, S. 108

178

Guglielmotto, M. 165

Guina, T. 117

Gutiérrez-Avila, G. 55

H

Hagen, T. 28

Hallstroem, S. 134

Hanaoka, K. 118

Hardy, D. 149

Hart, N. 131

Hawkins, C.L. 123

Hayes, J.D. 77

He, D.Y. 27

Heber, D. 22

Hermogenes Gomes, T.D.U. 104

Hernandes, L.C. 104

Hininger-Favier, I. 119

Hirata, Y. 118

Ho, E. 20

Hocht, C. 78

Hofmann, L. 134

Hrelia, P. 76, 164

Hrelia, S. 64, 137

Hui, S. 95

I

Iannelli, G. 72, 150

Iuliano, L. 35

Ivanov, L. 170

J

Jackson, M.J. 54

Jang, A.H.K.D. 95

Janini, M. 86

Jansen, E. 121, 170

Jiang, B. 95

Jida, E.H. 98

Johari, M.H. 172

Johnsen-Soriano, S. 69, 122


K

Kajer, T. 123

Kalashnikova, E. 27

Kálmán, F.S. 124

Kanit, L. 67

Kapuy, O. 125

Karatoprak, G. 126

Karim, N.A. 172

Karner, S. 173

Kayani, A. 54

Kensler, T. 82

Kensler, T.W. 9

Kereszturi, É. 124

Keum, Y.S. 136

Kim, J. 127

Kim, S.Y. 128

Kim, R.Y. 128

Kipp, A.P. 13

Kireev, R. 129

Kiyose, K. 118

Klimis-Zacas, D. 91, 108

Koltai, E. 131

König, U. 173

Konyalioglu, S. 68

Kosar, M. 126

Kristek, F. 93

L

Lafontaine, L. 95

Lamé, M.W. 71

Lampariello, L. 132

Lamprecht, M. 134

Lauriola, M. 150

Ledinski, G. 134

Lee, J. 136

Lee, H.J. 128

Lefeuvre, L. 99

Leonarduzzi, G. 34, 111, 112, 165

Leoncini, E. 137

179

Levault, K.R. 43

Levy, A.P. 24

Li, L. 28

Li, Z. 22

Limão, S. 86

Litterio, C. 109

Lokhandwala, M. 70

M

Maggiora, M. 144

Maher, P. 97, 174

Maia, W. 86

Maina, M. 117

Majzúnová, M. 93

Makpol, S. 140

Malaguti, M. 137

Maldini, M. 113

Manca, S. 138

Mand, J. 124, 125

Mann, G.E. 10

Mariani, E. 76

Marinar, U.M. 110

Marotti, I. 137

Martinez-Belda, R. 122

Marzatico, F. 92

Masella, R. 60, 62

Mastrocola, R. 139

Mattivi, F. 106, 107

Mayer, M. 78

Mazlan, M.B. 75

McArdle, A. 54

McNulty, H. 19

Mecocci, P. 56

Medana, C. 139

Mian, T.S. 75

Mitic-Milikic, M. 157

Mohamed, A. 98

Mohamed, K. 46

Molnár, Z. 124


Monteiro, H. 86

Moraes Mathias, P.P. 151

Moreira, A.C. 141, 152, 154

Moretta, L. 110

Morina, A. 115

Morroni, F. 76, 164

Moscow, P. 95

Motohashi, H. 8

Müller, M. 13

Murley, H. 95

Muzio, G. 144

N

Nagano, T. 118

Nagorni-Obradovic, L. 157

Nagy, L. 33

Nam, W.S. 146

Natarelli, L. 143

Natella, F. 113

Nelson, M.B. 95

Ngo, J.K. 131

Nigro, D. 139

Niki, E. 32

Nikolic, A. 157

Nitti, M. 110

Novembrino, C. 83, 87, 89

Nuttall, J. 23

Nyunt, T. 71

O

Obermayer, G. 134

O’Connor, M.S. 50

Oliveiram, P.J. 152

Omar, N.S. 75

Onkoksoong, T. 145

Oraldi, M. 144

Oskolkova, O.V. 116

Osman, M. 119

Oteiza, P.I. 23

180

Ozaltin, E. 79

Ozer, N.K. 79

P

Packer, L. 27

Pagliaro, P. 65, 167

Pagliei, B. 66

Paiuzzi, E. 144

Pakanová, Z. 93

Panich, U. 145

Park, E. 127, 128

Park, J.W. 146

Park, J.H. 128

Passalacqua, M. 110

Pattison, D. 123

Pavan, B. 80

Pearson, T. 54

Pecorelli, A. 159

Pedrosa, R.C. 106

Pelletier, G. 95

Pendem, S. 147

Penna, C. 65, 167

Pequeno Monteiro, H. 151

Pérez-Herrera, A. 58

Peris-Martinez, C. 122

Perrelli, M.G. 167

Perrelli, M.G. 65

Pessina, F. 159

Petrlova, J. 71

Petrovic-Stanojevic, N. 157

Piao, W. 118

Pientka, L. 56

Piotrkowski, B. 148

Piras, S. 110

Pires Bianchi, M.L. 104

Pitt, R.A. 39

Pluemsamran, T. 145

Poli, G. 34, 111, 112, 117, 165

Polidori, M.C. 56


Polizio, A. 78

Pook, M. 85

Porrini, M. 91, 108

Poyo, T. 119

Prata, C. 137

Prior, M. 174

Prolla, T. 6

Pronzato, M.A. 110

Puyó, A.M. 78

Q

Qing-Yu, S. 95

R

Radak, Z. 131

Radojkovic, D. 157

Rahman, I. 29

Ranaldi, G. 143

Rattan, S. 51

Rébillard, A. 99

Reis, A. 39, 149

Retta, S.F. 115

Ribeiro de Almeida, M. 104

Ricciardi, E. 84

Rimbach, G. 101

Riso, P. 91, 108

Rodovicius, H. 170

Rodriguez-Mañas, L. 46, 55

Rollo, T. 72, 150

Romano, A.D. 72, 150

Romero, F.J. 69, 122

Rosa, H. 151

Roselli, M. 143

Rossiter, H. 173

Roussel, A.M. 119

Rutledge, J.C. 71

S

Sadauskiene, I. 170

181

Sahdeo, S. 85

Saija, A. 84

Sakellariou, G. 54

Salamone, P. 111

Sampaio, S. 152

Sancak, Ö. 101

Sánchez-López, F. 58

Santangelo, C. 60, 62

Santos, M.S. 141, 152, 154

Sardão, V.A. 141, 152, 154

Sartori, A. 86

Scaccini, C. 113

Scazzocchio, B. 60, 62

Schmoll, D. 153

Schoenfeld, R. 85

Schopfer, F.J. 36

Schubert, D. 97, 174

Schummer, D. 153

Schwarzer, E. 149

Scullion, S. 54

Serna, E. 46

Serviddio, G. 38, 72, 150

Shan, Y. 85

Skoko, J. 82

Silva, A.M. P. 141, 154

Slocum, S. 82

Smith, A. 28

Soane, M. 86

Song, J. 156

Sottero, B. 111, 112, 165

Sozen, E. 79

Speciale, A. 84

Spickett, C.M. 39, 149

Sprio, A.E. 111

Stankovic, M. 157

Steinbauer, K. 134

Stewart, D. 77

Sticozzi, C. 80, 132, 159, 168

Strain, J.J. 19


Strumilo, S. 86

Supasai, S. 23

Surh, Y.J. 12

Suryo Rahmanto, A. 161

Suzuki, T. 8

T

Taguchi, K. 8

Taira, C.A. 78

Talib, J. 162

Talora, C. 138

Tamagno, E. 165

Tamborra, R. 72, 150

Tamosiunas, A. 121

Tapia, G. 102, 163

Tarozzi, A. 76, 164

Taverniti, V. 108

Taylor, A.W. 131

Testa, G. 111, 112, 165

Tirosh, O. 37

Topic, A. 157

Trapani, E. 115

Traverso, N. 110

Tresguerres, J.A.F. 55, 129

Trovato Salinaro, A. 48

Truter, E. 73, 166

Tschachler, E. 116, 173

Tullio, F. 167

Túnez, I. 58

V

Valacchi, G. 80, 132, 159, 168

Valenzuela, R. 163

van Ommen, B. 16

Vara, E. 129

Vargas, R. 102

Varì, R. 60, 62

Vasilaki, A. 54

Vazquez-Prieto, M.A. 148

182

Vendemiale, G. 150

Vicinanza, R. 22

Videla, L.A. 102

Videla, L. 163

Viezeliene, D. 170

Vigna, L. 87, 89

Vijg, J. 44

Villoria-Recio, M. 115

Viña, J. 46

Virgili, F. 84, 138, 143

Vrhovsek3, U. 106, 107

W

Wagner, A.E. 101

Wakabayashi, N. 82

Walker, M.P. 43

Wan Ngah, W.Z. 75, 140, 172

Weindruch, R. 6

Ward, M. 19

Wilson, J.X. 147

Wilson, D.W. 71

Y

Yalcin, A. 67, 68

Yamamoto, M. 8

Yanshu, Z. 95

Yerer, M.B. 126

Yusof, Y.A.M. 140

Z

Zeisel, S.H. 17

Zhang, C. 173

Zhang, W. 28

Zhang, Y. 22

Zhao, Y. 173

Zho-Sheng, L. 95

Zhong-Li, Z. 95

Zorad, Š. 93

Zurinah, W. 75, 140


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