The Toxicologist - Society of Toxicology

1103 THE ROLE OF 3, 3’, 4, 4’, 5-
PENTACHLOROBIPHENYL (PCB 126) IN COPPER
DISPOSITION IN RODENT LIVER.
M. Li 1, 2 , I. Lai 1, 2 , B. Wels 3 , D. Simmons 3 , G. Ludewig 1, 2 and L. Robertson 1, 2 .
1 Interdisciplinary Program in Human Toxicology, University of Iowa, Iowa City, IA,
2 Occupation and Environmental Health, University of Iowa, Iowa City, IA and 3 State
Hygienic Laboratory, University of Iowa, Iowa City, IA.
Copper, an essential trace element and ubiquitous in animals and humans, is essential
for the proper functioning of the electron transport chain in the mitochondria
as well as an antioxidant in copper-zinc superoxide dismutase (CuZnSOD).
However, in its free form, copper can also participate in Fenton-like reactions that
result in the production of reactive hydroxyl radicals. In previous studies copper
levels in rodent liver were increased following exposure to aryl-hydrocarbon receptor
agonists, including the most potent polychlorinated biphenyl (PCB) congener,
PCB 126. Increased hepatic copper was accompanied by several histological
changes, including steatosis. In an effort to assess the effect of dietary copper on
these changes, male Sprague-Dawley rats were fed an AIN-93G diet with one of
three dietary copper levels: low (2 ppm), adequate (6 ppm), and high (10 ppm).
After three weeks, 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 by PCB 126 in a dose-dependent
manner, significantly at the highest dose (40-75%). Relative liver weight
was increased in a dose-dependent manner (30-65%) by PCB 126, while there was
no effect on growth or liver weight by dietary copper. Hepatic cytochrome P450 activity
was maximally-induced by 1 μmol/kg PCB 126. Increasing dietary copper
and increasing dose of PCB 126 both increased hepatic copper levels. Blood copper
and serum ceruloplasmin levels were biphasic, diminishing with increasing PCB
126 dose at low dietary copper, while increasing in rats fed high dietary copper.
Hepatic CuZnSOD activity was not significantly affected by dietary copper. We
conclude that while reducing dietary copper reduced hepatic copper levels, the
function of essential copper enzymes were not negatively affected. (Supported by
NIEHS P42 ES 013661)
1104 ENTEROHEPATIC CIRCADIAN RHYTHM IN MICE:
CONTRIBUTIONS OF BILE ACIDS.
Y. J. Zhang, G. L. Guo and C. D. Klaassen. Pharmacology, Toxicology, and
Therapeutics, University of Kansas Medical Center, Kansas City, KS.
Diurnal fluctuation of bile acid (BA) concentration in the enterohepatic system of
mammals has been known for a long time. Recently, BAs have been shown as signaling
molecules beyond their well-established roles in dietary lipid absorption and
cholesterol homeostasis. The current study depicts diurnal variation of individual
BAs detected by ultra-performance liquid chromatography/mass spectrometry
(UPLC/MS) in serum and livers collected from C57BL/6 mice fed regular chow or
chow containing cholestyramine (resin). Circadian rhythms of mRNA of vital BArelated
nuclear receptors, enzymes, and transporters in livers and ilea were determined
in control- and resin-fed mice, as well as in farnesoid X receptor (FXR) null
mice. The circadian profiles of BAs show enhanced bacterial dehydroxylation during
the fasting phase and efficient hepatic reconjugation of BAs in the fed phase.
The resin removes more than 90% of BAs with β-hydroxy groups, such as muricholic
acids and ursodeoxycholic acid, from serum and livers, but markedly increases
deoxycholic acid and lithocholic acid in both compartments. Data from
both resin-fed and FXR-null mouse models indicate that BAs regulate their own
biosynthesis through the FXR-regulated ileal fibroblast growth factor 15. BA flux
also influences the daily mRNA levels of multiple BA transporters. In conclusion:
BA concentration and composition exhibit circadian variations in mouse liver and
serum, which influences the circadian rhythms of BA metabolizing genes in liver
and ileum. The diurnal variations of BAs appear to serve as a signal that coordinates
daily nutrient metabolism in mammals. This study is supported by NIH grants
DK-081461, ES-009716, ES-009649, ES-013714, and RR-021940.
1105 MYELOPEROXIDASE-DEPENDENT METABOLISM OF
P-CRESOL: A NOVEL PATHWAY FOR XENOBIOTIC
AND ENDOBIOTIC BIOTRANSFORMATION DURING
INFLAMMATION.
J. Houghton 1 , K. Hayakawa 1 , D. DeGroot 2 , B. Zhao 2 , M. Denison 2 and J. P.
Eiserich 1 . 1 Department of Internal Medicine, University of California, Davis, Davis,
CA and 2 Department of Environmental Toxicology, University of California, Davis,
Davis, CA.
Myeloperoxidase (MPO) is a heme protein abundantly expressed in neutrophils
that is traditionally regarded as a critical host defense enzyme that facilitates bacterial
killing. Based upon its oxidizing P450-like behavior, we speculated that MPO
236 SOT 2011 ANNUAL MEETING
could play an unexpected role in xenobiotic and endobiotic metabolism. p-Cresol
(4-methylphenol) is a natural product that humans are exposed to both exogenously
(ie. cigarette smoke) and endogenously (ie. tyrosine metabolism by gut microflora).
Herein we demonstrate that MPO is capable of converting p-cresol into
nitrated and oxidized metabolites, the latter with bioactivity not shared with the
parent compound. Enzymatic experiments with MPO revealed that p-cresol was
converted primarily to 4α,9β-dihydro-8,9β-dimethyl-3(4H)-dibenzofuranone,
otherwise known as “Pummerer’s ketone” (PK), with smaller yields of 2-nitro-4methylphenol,
and 2,2’-dihydroxy-5,5’-dimethyldiphenyl but no chlorinated products.
In vitro and cell culture approaches revealed that PK is a ligand for the AhR
and activates its ability to bind to the dioxin-response element (DRE), and induces
a 5-fold induction in the expression of cytochrome P450 1A1 mRNA.
Additionally, PK induces the activation and nuclear translocation of the transcription
factor Nrf-2, and consequently induces the expression of heme oxygenase-1
(HO-1) and glutathione cysteine ligase catalytic (GCLC) subunit. Chemical reactions,
coupled with LC/MS analyses, revealed that PK can react with the model
thiol compounds glutathione (GSH) and N-acetylcysteine (NAC) to form covalent
Michael addition products, suggesting that the cellular bioactivity of PK may be
dictated by the formation of adducts with cysteine residues in critical proteins essential
to transcriptional responses. Our data illustrate that MPO may play a previously
unrecognized and important function in xenobiotic and endobiotic metabolism
during inflammation.
1106 TOXICITY, TOXICOGENOMIC, AND METABOLOMIC
STUDY OF PENTAMETHYLCHROMANOL.
T. Parman 1 , D. Bunin 1 , H. H. Ng 1 , T. Harrison 1 , J. McDunn 2 , I. M.
Kapetanovic 3 and C. E. Green 1 . 1 Biosciences, SRI International, Menlo Park, CA,
2 Metabolon, Inc., Durham, NC and 3 National Cancer Institute, Bethesda, MD.
Pentamethyl-6-chromanol (PMCol) is a candidate chemopreventive agent against
androgen dependent cancers. PMCol has been shown to cause hepatotoxicity,
nephrotoxicity and hematological effects. The objectives of this study were to determine
the mechanisms of toxicity and identify sensitive early markers of hepatic
and renal injury using renal biomarkers (albumin, TIM-1, lipocalin-2, osteopontin,
clusterin), toxicogenomics and metabolomic approaches. PMCol was administered
to male Sprague-Dawley rats, 200 and 2000 mg/kg daily for 7 or 28 days. Changes
in clinical chemistry included elevated alanine aminotransferase, total bilirubin,
cholesterol and triglycerides, indicative of liver toxicity that was confirmed by microscopic
findings (periportal hepatocellular hydropic degeneration and cytomegaly)
in treated rats. Metabolomic evaluations of liver tissue revealed time and
dose dependent changes including depletion of glutathione and glutathione conjugates,
decreased methionine, and increased S-adenosylhomocysteine, cysteine, and
cystine. PMCol treatment also decreased cofactor levels, e.g. FAD+ and NAD(P)+.
Cholesterol and sphingomyelin were markedly higher in plasma from treated rats.
Microarray analysis found that differentially expressed genes were enriched in the
glutathione and cytochrome P450 pathways in liver by PMcol treatment. RTqPCR
of six upregulated genes (Akr7a3, Cyp1a1, Gpx2, Gsr, Gstp1, and Hmox1)
and one down regulated gene (Acnat2) confirmed the microarray results.
Metabolomic, urine biomarkers and histopathologic changes in kidneys were also
observed but were minor compared to hepatic effects. In conclusion, metabolomics
and toxicogenomics demonstrate that chronic exposure to high doses of PMCol induces
liver damage and dysfunction, probably due to both inhibition of synthesis
and depletion of liver glutathione, as well as modification of other drug metabolism
pathways. (Supported by NCI Contract HHSSN261200433005C)
1107 EVIDENCE OF PEPPERMINT OIL SKIN
PENETRATION AND CYTOTOXICITY ON RAT
HEPATOCYTES IN VITRO.
M. Dong 1 , O. Weber 1 , E. Di Lenarda 1 , P. End 2 , S. Chibout 1 , A. Wolf 1 and F.
Pognan 1 . 1 Preclinical Safety, Novartis, Basel, Switzerland and 2 Drug Metabolism and
Pharmacokinetics, Novartis, Basel, Switzerland.
Peppermint oil is commonly used for digestive disorders taken orally and the relief
of muscle pain by topical application. However, it has been suspected of potential
adverse effects, including liver toxicity. The aim of the present study was to examine
the acute effects of peppermint oil on rat hepatocytes in vitro.
Freshly isolated rat hepatocytes were cultured in collagen-sandwich configuration.
Cells were treated daily with peppermint oil (China Oil) at the concentrations
ranging from 0.001% to 0.3% (v/v) for up to three days. GC/MS analysis determined
that this peppermint oil contained 1.18 % of pulegone and 0.78% of its
main metabolite, mentofurane. Both have been described as hepatotoxicants in
vivo in rat. Cellular ATP content, total glutathione, as well as ALT and AST release
were determined.