The Toxicologist - Society of Toxicology

comparable to in vivo disposition studies, which suggests that precision-cut tissue
slices are a useful model for studying the (enantioselective) metabolism of PCB136
in different tissues (supported by ES05605, ES013661 and ES017425).
1831 STRUCTURAL CHARACTERIZATION OF O-
METHYLATED-CATECHOL METABOLITE OF
BENZO[A]PYRENE-7, 8-DIONE IN THREE HUMAN
LUNG CELLS.
M. Huang, L. Zhang, I. A. Blair and T. M. Penning. Centers of Excellence in
Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology,
University of Pennsylvania, Philadelphia, PA.
Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon, is a
ubiquitous environmental pollutant occurring in tobacco smoke and residues of
fossil fuel combustion. Metabolic activation of the proximate carcinogen B[a]P-
7,8-trans-dihydrodiol by aldo-keto reductases (AKRs) leads to B[a]P-7,8-dione that
is redox-active and generates reactive oxygen species resulting in oxidative DNA
damage in human lung cells. O-methylation of the corresponding catechol by
cactechol-O-methyltransferase (COMT) is predicted as one pathway for detoxification
of B[a]P-7,8-dione. We investigated the occurrence of this pathway in human
bronchoalveolar H358 cells, human lung adenocarcinoma A549 cells, and immortalized
human bronchial epithelial HBEC-KT cells following treatment of B[a]P-
7,8-dione for 24 hours. After acidification of the culture medium, a single omethyl-B[a]P-7,8-catechol
product was detected in the organic phase of medium
from each cell line using HPLC-UV and LC-MS/MS. An authentic metabolite
standard was subsequently produced by enzymatic synthesis and purified by semipreparative
HPLC and characterized by [1H]- NMR. The definite structure of the
cellular metabolite was identified to be o-8-methyl-B[a]P-7,8-catechol. It is concluded
that human COMT may play a critical role in the detoxification of B[a]P-
7,8-dione in lung cells [Supported by P30-ES013508 and 1R01-CA-39504
awarded to TMP].
1832 A MEANS TO STUDY CRITICAL ADDUCT PROTEIN
TARGETS: COMPARISON OF TOXIC NAPHTHALENE
AND NON-TOXIC DIETHYL MALEATE SHOWS
EQUIVALENT IN VIVO COVALENT BINDING AND
GLUTATHIONE DEPLETION IN AIRWAY EPITHELIUM.
D. Krawiec1 , D. Morin1 , L. Van Winkle2 and A. Buckpitt1 . 1Molecular Biosciences, UC Davis, Davis, CA and 2Anatomy Physiology and Cell Biology, UC
Davis, Davis, CA.
Reactive metabolites have the potential to bind covalently to cellular macromolecules
(protein and/or DNA) and these interactions have been associated with the
carcinogenic and toxic actions of some chemicals. Not all reactive metabolite protein
binding results in cytotoxicity. This suggests that either overall adduct levels are
not important or there are critical proteins that are adducted by cytotoxic reactive
metabolites whereas these are not modified by reactive, non-cytotoxic metabolites.
To test the hypothesis that critical protein adducts can be discriminated from noncritical
protein targets, we have evaluated the formation and persistence of reactive
metabolite protein adducts from naphthalene, which causes necrosis of Clara cells,
and diethyl maleate which produces transient swelling of airway epithelial cells but
in no necrosis (Phimister et al., JPET 314: 506, 2005). In vivo comparison of protein
binding in airway epithelium for both NA (300 mg/kg) and DEM (1000
mg/kg), doses which produce equivalent depletion of airway epithelial glutathione
levels, have shown an average covalent binding which is identical for the two compounds:
1.42 ± 0.13 nmoles/mg protein for NA and 1.43 ± 0.15 nmoles/mg protein
for DEM between 1 and 8 hours post-dose. Adducts are stable within the
measured time frame, indicating that the differences in cellular response are not related
to differential persistence of adducts on protein. GSH depletion was also similar
for the two compounds at the doses used in the protein adduct studies: 32.8 ±
17.3 percent control for NA and 41.3 ± 17.5 percent control for DEM between 1
and 4 hours. Identification of proteins targeted by DEM and comparison to known
NA adducts provides a novel approach to identifying critical and non-critical protein
adducts in the lung. Supported by NIEHS 04311 and 04699
1833 EFFECT OF DDT ON TESTOSTERONE
BIOTRANSFORMATION BY RAT BRAIN MICROSOMES.
A. Sierra-Santoyo1 , O. Horacio2 and M. L. Lopez-Gonzalez1 . 1Departamento de
Toxicologia, CINVESTAV-IPN, Mexico, D.F., Mexico and 2Departamento de
Nefrologia, Inc., Mexico, D.F., Mexico.
Dichlorodiphenyltrichloroethane (DDT) is a liver cytochrome P-450 (CYP) inducer
and an endocrine disruptor. Information about the effect of DDT on CYP
expression in extrahepatic tissues is limited. The objective of this study was to de-
392 SOT 2011 ANNUAL MEETING
termine the effect of DDT on CYP-dependent testosterone hydroxylase activities
by rat brain microsomes. Wistar adult male rats were administered with an oral
dose of DDT technical grade (0.1, 1, 5, 10 and 100 mg/kg) dissolved in corn oil.
Animals were sacrificed 24 h later and brain was removed and processed to obtain
microsomes. Brain microsomes were incubated with [4- 14 C]-testosterone and
metabolites were separated by thin layer chromatography and quantified by radio
scanning and CYP2B1/2, 2A, 2C11 and 3A2 levels were determined by Western
blot analysis. DDT altered testosterone biotransformation and modified the profile
of metabolites. A significant reduction on 2α-hydroxytestosterone (OHT), 6α-
OHT and 7α-OHT was observed at all doses. At the doses of 0.1 and 1 mg/kg a
significant decrease on 15α-OHT, 6β-OHT and androstenedione formation was
detected. At the dose of 100 mg/kg the 6-dehydrotestosterone formation increased
2-fold while at the doses of 5 and 10 mg/kg 16α-OHT formation increased more
than 2-fold. CYP2A and 2B1/2 levels increased at the doses from 0.1 to 5 mg/kg
and decreased at the two highest doses respect to the control group. CYP2C11 levels
decreased in a dose-dependent manner at all doses. These results indicate that a
single dose of DDT modifies brain CYP-dependent testosterone-hydroxylating activities
and represent another endocrine disruption mechanism.
1834 TWO MECHANISMS INVOLVED IN ECSTASY-
INDUCED HEPATOTOXICITY.
I. Antolino Lobo 1, 2 , W. Strach 1 , D. A. Fiechter 1 , I. S. Ludwig 1 , M. van den
Berg 1 , J. Meulenbelt 1, 2 and M. van Duursen 1 . 1 Endocrine Toxicology, IRAS,
Utrecht, Netherlands and 2 National Poisons Information Centre, National Institute
for Public Health and the Environment., Bilthoven, Netherlands.
3,4-methylenedioxymethamphetamine (Ecstasy, MDMA) is a widely used recreational
drug, which is known to induce hepatotoxicity in humans. Yet, the mechanism
of MDMA-induced hepatotoxicity is still unclear. We have investigated to
role of MDMA metabolism and immunological responses in vitro, two suggested
pathways which could lead to MDMA-induced liver injury. To study the role of
specific metabolic pathways in MDMA toxicity, a human liver epithelial (THLE)
cell line transfected with a single cytochrome P450 enzyme (CYP450) was used.
We observed major MDMA-induced toxic events after CYP3A4 and CYP2D6-mediated
metabolism with a reduced cell viability of 50% and 25% at 4mM MDMA,
respectively. In addition, MDMA exposure resulted in a 40% decrease of GSH level
in THLE-CYP3A4, but not THLE-CYP2D6 cells. This indicates that CYP3A4formed
MDMA metabolites and subsequent GSH depletion play a key role in
MDMA–induced cytotoxicity. To evaluate the possible contribution of immune responses
in MDMA-induced liver toxicity, we established a co-culture model with a
classical human liver cell line (HepG2) and a human monocyte cell line (THP-1).
When cell viability of mono-cultured HepG2 cells was compared with co-cultured
HepG2 and THP-1 cells, an increased cell viability of 10% at 4 mM MDMA exposure
was observed in HepG2 cells. This indicates a moderately protective role of
monocytes on liver cells upon MDMA exposure. Cytokine levels (TNF-α, IL-1β)
and the chemokine IL-8 are being evaluated to clarify these findings. Taken together,
these data suggest that both CYP3A4-mediated metabolism and immune
responses can affect MDMA-induced cytotoxicity in liver cells in vitro. Studies are
currently being performed to elucidate the suggested mechanisms and to determine
the clinical relevance of these findings.
1835 TOXICITY OF AMINONITRILES IN MALE SPRAGUE-
DAWLEY RATS.
M. Y. Farooqui and T. Lacy. Biology, University of Texas Pan American, Edinburg, TX.
Aminonitriles (AN) are especially potent representatives of bifunctional compounds.
Importance of Amino moiety for drug design is hard to overestimate due
to a set of intrinsic properties and nitrile functional group can be involved into hydrogen-bonding
and can be selectively transformed to primary amine, aldehyde,
ketone, or carboxylic acid (hydrolysis). Some toxicity symptoms of AN appear in
the literature. We have studied toxicities and metabolism of 4 AN namely
aminoacetonitrile, β-aminopropionitrile, N,N- dmethylaminopropionitrile and
β,β-Imminodipropionitrile in male Sprague-Dawley rats using 0.25 LD50 oral
doses. Following treatment rats were observed for visible signs of toxicity and concentrations
of cyanide, thiocyanate, cyanoacetic acid and glutathione were determined
at various time intervals. All of the AN studied produced significant cholinomimetic
effects including vasodilation in ears, bloated abdomens, disorientation,
ataxia and visual impairment. Additional significant effects observed were severe
cerebral hemorrhage and urinary retention in the bladders. The concentration of
cyanide increased 4 to 7 fold in plasma and organs. The concentrations of thiocyanate
increased 2-4 fold in plasma and urine. Levels of glutathione significantly
decreased 30-60% of controls in organs. The concentrations of another metabolite