The Toxicologist - Society of Toxicology

The peppermint oil caused a concentration dependent increase in hepatocyte cytotoxicity.
After one-day treatment, a significant, ~40%, reduction of cellular ATP
and total glutathione content, along with ~10-fold increase in ALT/AST release
were detected at concentration of 0.01% (v/v). No further increase in cytotoxicity
was observed with time till the end of the treatment. Application of pulegone and
mentofurane from the total oil on human cadaver skin showed that both significantly
penetrated the dermis and the epidermis.
In conclusion, the present study demonstrated that toxic components of peppermint
oil can cross the skin barrier and its in vitro toxicity in rat hepatocytes.
Although the toxic concentrations were greater (~20 fold) than those recommended
by authorities for human use, attention still needs to be taken to avoid potential
liver damage resulting from overdosing.
1108 MOLECULAR MECHANISM OF ALTERED EZETIMIBE
DISPOSITION IN NON-ALCOHOLIC
STEATOHEPATITIS.
R. N. Hardwick, C. D. Fisher, S. M. Street, M. J. Canet and N. J. Cherrington.
Pharmacology and Toxicology, University of Arizona, Tucson, AZ.
Ezetimibe (EZE) lowers serum lipid levels by blocking Neimann-Pick C1-Like 1mediated
cholesterol uptake in the intestine. Disposition of EZE and its pharmacologically
active glucuronide metabolite (EZE-G) to the intestine is dependent on
biliary efflux from the liver primarily via ABCC2. Because it has been suggested
that ABCC2 and other hepatobiliary transporters are altered during non-alcoholic
steatohepatitis (NASH), the purpose of the current study was to determine the effect
of experimental NASH on EZE disposition in vivo. Rats were fed a methionine-choline
deficient (MCD) diet for 8 weeks and then administered 10 mg/kg
EZE either by intravenous bolus or oral gavage. Plasma and bile samples were collected
over a 2 hour period followed by terminal urine, liver and intestinal tissue
collection. EZE and EZE-G concentrations were determined by LC-MS/MS.
Hepatic expression of the sinusoidal Abcc3 transporter was induced in MCD animals.
This correlated with increased plasma concentrations of EZE-G in both i.v.
and p.o. dosed MCD animals. Hepatic expression of the biliary Abcc2 and Abcb1
transporters was also increased in MCD; however, the biliary efflux of EZE-G was
diminished in MCD animals. Interestingly, the cellular localization of Abcc2 appeared
to be internalized away from the canalicular membrane in MCD liver. This
phenomenon may account for the decreased efflux of EZE-G observed in MCD.
Expression of the EZE metabolizing enzyme Ugt1a1 was not altered in MCD liver
or intestine. Importantly, induction of ABCC2, 3 and ABCB1 protein was also
found in human NASH liver samples compared to normal. Similarly, ABCC2 is
also internalized in human NASH as evidenced by immunohistochemical staining
and reduced glycosylation status of the protein. The combination of induced expression
and altered localization of key efflux drug transporters in rodent and
human NASH samples shifts the disposition profile of EZE toward plasma retention,
thereby diminishing drug delivery to the active site.
1109 DIETARY N-ACETYLCYSTEINE (NAC) REDUCES
HEPATOCELLULAR LIPID ACCUMULATION
FOLLOWING 3, 3’, 4, 4’, 5-PENTACHLOROBIPHENYL
(PCB 126) EXPOSURE.
I. Lai 1, 2 , A. Olivier 3 , M. Li 1, 2 , K. Dhakal 1, 2 and L. Robertson 1, 2 . 1 Occupation
and Environmental Health, University of Iowa, Iowa City, IA, 2 Interdisciplinary
Program in Human Toxicology, University of Iowa, Iowa City, IA and 3 Department of
Pathology, University of Iowa, Iowa City, IA.
Glutathione (GSH) is a key intracellular antioxidant because of its thiol-containing
cysteine group. However, due to difficulty in direct uptake, an alternative cysteine
source, such as N-acetylcysteine, NAC, has been widely used as a thiol-donor to
protect against reactive species. NAC as a supplement is of particular interest since
previous studies have shown that exposure to aryl hydrocarbon agonists, including
the most potent polychlorinated biphenyl (PCB) congener, PCB 126, caused liver
pathology and reduced glutathione levels. We hypothesized that dietary NAC supplementation
in rats will reduce PCB 126-induced toxicity. Male Sprague-Dawley
rats were fed a standard AIN-93G diet or a modified diet supplemented with 1.0%
NAC. After one week, rats from each dietary group were given a single ip injection
of corn oil (control), 1, or 5 μmol/kg body weight PCB126 in corn oil, followed
two weeks later by euthanization. Growth rate was slowed up to 20% by PCB 126
in a dose-dependent manner. Relative liver weight was increased in a dose-dependent
manner (42-52%) by PCB 126, while NAC had no effect. Hepatic CYP1A1 activity
was maximally-induced by PCB 126, indicating potent AhR activation. Total
GSH levels in the liver were diminished in a dose-dependent manner (4-34%) by
PCB 126, but not increased by NAC supplementation. Histological examination of
liver tissue showed hepatocellular swelling and increased lipid accumulation
(steatosis) caused by PCB 126. Interestingly, based on lipid quantification by os-
mium staining, hepatocellular lipid accumulation was diminished in rats supplemented
with NAC. Based on these results, we conclude that NAC supplementation
is a promising treatment in protecting against PCB 126-induced toxicity.
(Supported by NIEHS P42 ES 013661)
1110 SERUM CYTOKERATIN 18 AND CYTOKINE
ELEVATIONS SUGGEST A HIGH PREVELENCE OF
OCCUPATIONAL LIVER DISEASE IN
ELASTOMER/POLYMER WORKERS HIGHLY-EXPOSED
TO ACRYLONITRILE, BUTADIENE, AND STYRENE.
M. Cave 1, 2 , K. Falkner 1 , B. Costello 1 , B. Gregory 1 , L. Henry 1 and C. McClain 1,
2 . 1 Department of Medicine/GI, University of Louisville, Louisville, KY and 2 Robley
Rex Veterans Administration Medical Center, Louisville, KY.
Objective: Occupational liver disease is likely to be under-recognized because, in
many cases, routine serologic liver chemistries are not effective biomarkers.
Cytokeratin 18 (CK18) is a novel serologic biomarker for occupational liver disease.
We recently demonstrated that serum CK18 and pro-inflammatory cytokines
were elevated in chemical workers with toxicant-associated steatohepatitis (TASH)
due to high-level vinyl chloride exposures. The purpose of this study is to determine
the prevalence of CK18 elevation in elastomer/polymer workers exposed to mixtures
of acrylonitrile, 1,3 butadiene, and styrene. Methods: 82 chemical workers
were evaluated. CK18 was determined by ELISA, and pro-inflammatory cytokines
were measured by multi-analyte chemiluminescent detection. Results: Mean routine
liver chemistries (aspartate aminotransferase, alanine aminotransferase, total
bilirubin, albumin, and alkaline phosphatase) were in the normal range. In fact,
only 3 of 82 total subjects had any single lab abnormality detected by these tests.
However, 39% (32 of 82) had elevated CK18 levels which were not explained by alcohol
or obesity, except potentially in 4 cases. The pattern of CK18 elevation was
consistent with TASH in the majority of cases (78%). TNFα, IL-6, IL-8, MCP-1,
and PAI-1 were increased in these workers compared to those with normal CK18
levels. Conclusions: These results suggest a high prevalence of occupational liver
disease and TASH in elastomer/polymer workers with elevated CK18 and pro-inflammatory
cytokines.
Funding Information: NIEHS (P30ES014443-01A1, T35ES014559), the NIAAA
(K23AA18399-01A, 1P01AA017103-01, R37AA010762, RC2AA019385), and
NCRR (5P20RR024489-02).
1111 MITOCHONDRIAL TOXICITY OF
CHLOROACETALDEHYDE IN HEPG2 CELLS.
K. Falkner 1 , B. Hill 1 , B. Sansbury 1 , J. Gaurdiola 1 , C. McClain 2 and M. Cave 2 .
1 Department of Medicine/GI, University of Louisville, Louisville, KY and 2 Louisville
VA Medical Center, Louisville, KY.
Background: We first described a high prevalence of toxicant-associated steatohepatitis
in vinyl chloride workers. The steatohepatitis was atypical in that serum markers
indicated a non-apoptotic cell death mechanism. Vinyl chloride toxicity is associated
with its metabolites including chloroacetaldehyde (ClAc). We hypothesized
that ClAc may disrupt mitochondrial function causing ATP depletion leading to
necrotic cell death. Furthermore, nrf2 inducers may be protective against ClAc toxicity.
Methods: HepG2 were grown in DMEM with 10% FBS in a 5% CO2 at
37OC. Cell death was determined by MTT assay. Protein sulfhydryl group modification
was determined by the ability of cells to reduce BODIPY-IAM labeling.
Respiration was determined using a Seahorse XF24 Extracellular Flux Analyzer.
ATP levels were determined using a kit from Sigma. Results ClAc was toxic to
HepG2 cells with an LD50 of 108.5 ± 2.1 uM and 89.7 ± 0.7 uM for serum-containing
and serum free media, respectively. Treatment of cells for 1 hr with either 50
or 100 uM ClAc reduced the labeling of protein thiols by 24 and 41%, respectively.
ClAc reduced oxygen consumption in a dose dependent manner by up to 69%,
similar to that observed with 40 uM 4-hydroxynonenal. The ATP-dependent oxygen
consumption was reduced by up to 86%. In HepG2 cells treated with ClAc for
4 h, ATP was depleted by 15% or 75% for treatments with 50 uM and 100 uM, respectively.
Pretreatment with the nrf-2 activators, oltipraz and sulforaphane, decreased
the toxicity of ClAc. Conclusions: ClAc is thiol reactive and capable of severely
compromising mitochondrial respiration at concentrations in the toxic
range. Near the LD 50, ATP depletion is significant and may result in necrotic cell
death rather than ATP-dependent apoptosis. Nrf2 activators were protective
against ClAc toxicity. NIH Funding Sources: P30ES014443-01A1,
1P01AA017103-01, K23AA18399-01A, R37AA010762, RC2AA019385,
P20RR024489 and R01AA18869.
SOT 2011 ANNUAL MEETING 237