The Toxicologist - Society of Toxicology

191 Influence of Genetics on Paraoxonase 1 Activity in
Monozygotic and Dizygotic Twins.
L. Podolefsky 1 , K. M. Kelly 2 , J. C. Murray 3 , T. J. Raife 4 and G. Ludewig 1, 2 .
1 Grad Program in Human Toxicology, University of Iowa, Iowa City, IA;
2 Occupational and Environmental Health, University of Iowa, Iowa City, IA;
3 Pediatrics, University of Iowa, Iowa City, IA; 4 Pathology, University of Iowa, Iowa
City, IA.
Paraoxonase 1 (PON1) is an important HDL-associated endogenous antioxidant
found to play a major role in susceptibility to health effects from pesticides and oxidative
stress. A number of gene polymorphisms influence both protein concentration
and substrate specificity, as do certain lifestyle factors. However, reports about
the influence of PON1 genetics have varied widely in the literature. The goal of this
study was to examine the influence of genes and health data in a group of monozygotic
and dizygotic twins to better understand the effect of genetics on PON1 activity
levels. DNA, serum, and standard blood donation information was obtained
from 6 sets of dizygotic twins and 13 sets of monozygotic twins. DNA was genotyped
for polymorphisms within PON1, PON2, C-reactive protein (CRP), and
tumor necrosis factor (TNF). PON1 activity was determined using phenyl acetate
(PA) and CMPA [4-(Chloromethyl)phenyl acetate] as substrates, and genotyping
was performed using the TaqMan-Applied Biosystems 7900 HT System. Using a
general linear model, PON1 Q192R, L55M and C-108T were significantly associated
with both PA and CMPA activity, with CMPA activity showing a stronger association
with genetic variants. PON2 S311C was significantly associated with PA
activity, but not CMPA. Activity for either substrate was not found to be associated
with CRP or TNF polymorphisms. BMI was found to significantly correlate with
CMPA activity, but not PA activity (correlation = -.34), while gender did not correlate
with either substrate. Pair-wise analysis of all twins with identical PON genotypes
showed no difference in PON activity variance between siblings, regardless of
zygosity. Though other shared or variant genetic and lifestyle factors may influence
PON1 activity, the findings in this twin population suggest a strong link between
PON activity and specific PON polymorphisms.
192 Contribution of Environmental and Genetic Factors to
Pancreatic Cancer.
S. Chittiboyina, A. A. Bond, L. M. Kamendulis and B. A. Hocevar.
Environmental Health, Indiana University School of Public Health, Bloomington, IN.
Pancreatic cancer is the fourth leading cause of cancer deaths in the United States
with a five year survival rate of less than 6%. Several environmental risk factors have
been identified for pancreatic cancer, including dietary factors. In particular, high
dietary intake of folate has been associated with a decreased incidence of pancreatic
cancer, while low plasma folate levels are associated with an increased cancer risk. In
conjunction with diet and lifestyle determinants, an individual’s folate pathway status
is determined by their genetic makeup. In the present study, we determined the
expression of selected SNPs in the folate metabolic pathway in a cohort of pancreatic
cancer patients and healthy related and unrelated control groups. In agreement
with other cancer studies, we show that cancer cases were more likely to express the
TT allele of the methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism
compared to controls. Expression of this allele has been associated with low
folate levels and elevated homocysteine (Hcy) levels. In support of this, we found
that pancreatic cancer patients display elevated serum Hcy levels in comparison to
both control groups. In addition, 48% of the pancreatic cancer patients exhibited
hyperhomocysteinemia, as defined by a value >15 μM/L, (range 11.15 - 26.17
μM/L), while no subjects in either control group exhibited hyperhomocysteinemia.
In addition, we found that SNPs in betaine hydroxymethyltransferase (BHMT; rs
3733890) and serine hydroxymethyl transferase (SHMT; rs 1979277) exhibited
significantly different distributions between the case and control groups (p < 0.05).
While a clear association was not seen between cancer risk and alcohol or smoking
use in our population, self reported environmental exposure data suggested an association
between pancreatic cancer and exposure to welding fumes. These findings
underlie the necessity to investigate further the interaction between environmental
exposures and genetic factors which could provide further insight into the etiology
of pancreatic cancer.
193 The Impact of CYP2S1 Single Nucleotide Polymorphisms on
the Metabolic Activation of the Anticancer Prodrug, AQ4N.
N. Bajaj, N. M. Singh and A. M. Rowland. Chemistry and Biochemistry, NMSU,
Las Cruces, NM.
Cytochrome P450 2S1 (CYP2S1) is one of the most recent additions to the P450
superfamily of enzymes. Although its physiological role has not yet been defined, it
has been shown to influence metabolism of bioactive lipids, including
40 SOT 2013 ANNUAL MEETING
prostaglandins and retinoids. CYP2S1 is predominantly expressed in extra-hepatic
epithelial cells. Its expression is elevated in cancer and catalyzes the metabolic activation
of the anticancer prodrug, AQ4N, under hypoxic conditions. Interestingly
our lab has also demonstrated that increased CYP2S1 expression may protect
against AQ4 cytotoxicity under normoxic (21% O2) conditions. The main objective
of this study is to determine whether individual variability in the CYP2S1 enzyme
alters sensitivity of human lung cells to AQ4N and AQ4-mediated cytotoxicity.
Five published CYP2S1 allelic variants have been published: CYP2S1*2
(R380C), CYP2S1*3 (P466L), CYP2S1*4 (S61N), CYP2S1*5 (L230R).
According to the NCBI database, two additional non-synonymous variants have
been identified in cancer (A205T and L189F) and L189F is restricted to African
American populations. We generate each of the polymorphisms in a CYP2S1-Flag
mammalian expression vector, using site directed mutagenesis. Thus far, two stable
lines (S61N and L189F) in bronchial epithelial cells (BEAS-2B) and four stable
(S61N, R166H, A205T, and P466L) alveolar carcinoma cell lines (A549) have
been made. Examination of AQ4N and AQ4 cytotoxicity in BEAS-2B cells revealed
that both mutants (S61N and L189F) exhibit significantly increased cytotoxicity
in response to AQ4N compared with wild type CYP2S1-Flag and
pcDNA3.1 controls. Interestingly, the S61N polymorphism was significantly more
sensitive to AQ4 than any of the other cell lines. This effect appears to be selective
because cytotoxicity is not altered in response to a similar topoisomerase II inhibitor,
mitoxantrone. We are currently testing the effects of these polymorphisms
on AQ4N and AQ4 levels, using HPLC. Research funded by NIH NIGMS Grant
# R25GM061222.
194 The Remarkable Genotoxic Effect of Exposure to Ethyl
Tertiary Butyl Ether in ALDH2 Knockout Mice.
R. Wang, Z. Weng, K. Ohtani, M. Suda, Y. Yanagiba and T. Suzuki. Japan
National Institute of Occupational Safety and Health, Kawasaki, Japan.
Ethyl Tertiary Butyl Ether (ETBE) is used in gasoline for vehicles as a biofuel.
ETBE exposure induced liver damage and other health effects only at high concentrations
in our previous studies, and its No Observed Adverse Effect Level
(NOAEL) was calculated to be 500 ppm. However, in mice without ALDH2 enzyme
activity, ETBE could induce DNA damage even at the NOAEL. To find out
how low concentration at which ETBE shows its genotoxic effect, we did the exposure
experiment with mice at low range of ETBE concentrations. METHODS:
Male Aldh2-/- (KO), Aldh2+/- (HT) as well as C57BL/6 strain (WT), at 8 weeks
old were exposed to ETBE at 0, 50, 200 and 500 ppm, 6 hr/day and 5 days/week,
for 9 weeks. Blood, liver, epididymides were sampled 20 hr after the last exposure,
and DNA damages were analyzed with comet assay in these tissues. RESULTS: The
tail intensity (TI) was used to evaluate the degree of DNA damage. In the leukocytes
of WT mice, the TI value was not affected in any exposure group as compared
to the control. However, the TI was significantly increased in 200 and 500 ppm
groups of KO mice. Similar results were also obtained in HT mice, but there was
no difference between the two types of mice. In liver cells and sperm, ETBE also induced
DNA damage, but this effect was only observed in KO and HT mice as in
the leukocytes. The NOAEL was 50 ppm in these types of mice. These results suggest
that ALDH2 deficiency may increase the susceptibility to the health effect of
ETBE exposure. We thank Ms. S. Watanabe for her assistance in the manipulation
of the animals.
195 MGMT Haplotypes Alter MGMT Expression and Can Thus
Affect Response to Alkylating Agents.
M. Xu, I. Nekhayeva, C. E. Cross, C. M. Rondelli and S. Z. Abdel-Rahman.
OB/Gyn, UTMB, Galveston, TX.
Glioblastoma (GB) is rapidly fatal. However, treatment with temozolomide (TMZ)
and radiation is beneficial, but only for some patients. TMZ alkylates tumor DNA
to form O6-alkylguanine (O6-AG) DNA adducts, inducing apoptosis. Because
O6-AG is repaired by O6-methylguanine-DNA methyltransferase (MGMT), levels
of MGMT are critical in determining tumor response to TMZ. Single nucleotide
polymorphisms (SNPs) in the promoter/enhancer (P/E) region of the MGMT gene
can alter its transcription and thus alter MGMT protein levels. Genetic variants are
not arrayed as individual SNPs but as combinations forming specific “haplotypes”.
To date, no studies have determined the haplotypes structure of the P/E region of
MGMT or their effect on its transcription. We sequenced 104 DNA samples from
healthy individuals and identified 8 SNPS in this region (7/Y, 135/K, 290 /R,
485/M, 575/M, 666/R, 777/M and 1099/Y). Using bioinformatics, we inferred
the haplotypes encompassing these SNPs. We identified 21 potential haplotypes
ranging in frequency from 0.39 to 0.00005, of which 10 were identified in our
sample population as 20 paired haplotype combinations. We hypothesized that
these haplotypes alter the regulation of MGMT transcription. Luciferase-reporter
constructs containing different MGMT haplotypes were transfected into a GB cell