The Toxicologist - Society of Toxicology

events in the liver. AZD9056 was evaluated in vitro in the Ames test and the mouse
lymphoma assay and in vivo in a rat micronucleus study and UDS study. There was
no evidence of genotoxicity seen in any of these studies. In further studies rats
dosed with AZD9056 were evaluated for cellular proliferation in hepatic foci by co
administration of BrdU via osmotic minipumps. A marginal increase in hepatocyte
proliferation, as determined by BrdU incorporation was detected in animals dosed
at 125 mg/kg/day. There were no effects on hepatocyte cell proliferation at doses up
to 75 mg/kg/day. Foci of altered hepatocyte morphology and histopathological
staining are a naturally occurring age related finding in the rat. AZD9056 at doses
of 75 mg/kg/day for 6 months exacerbates the formation of these foci in a dose dependent
manner. There is no evidence of a genotoxic change that may be related to
these findings and cellular proliferation has a clear no effect level. It was concluded
that these findings have no impact on clinical safety of AZD9056.
1099 THE EFFECT OF VITAMIN E DEFICIENCY ON DCA-
AND TCA-INDUCED OXIDATIVE STRESS IN THE
LIVERS OF MALE B6C3F1 MICE FOLLOWING
SUBCHRONIC EXPOSURE.
J. Cearfoss and E. Hassoun. Pharmacology, University of Toledo, Toledo, OH.
Dichloroacetate (DCA) and Trichloroacetate (TCA) are water disinfection by products
that are known to induce hepatotoxicity/ hepatocarcinogenicity in rodents.
Previous studies in our lab have indicated induction of various biomarkers of oxidative
stress (OS) in the livers of mice after subchronic exposure. To assess the effect
of marginal Vitamin E deficiency on TCA- and DCA-induced OS, two groups
of male B6C3F1 mice were fed either normal diet supplemented with vitamin E
(E-normal group), or diet that was not supplemented with the vitamin( E-deficient).
The two groups were subdivided into subgroups that were treated subchronically,
by gavage, with the following: water (control), 77 mg DCA/kg/day, or 77 mg
TCA/kg/day. Mice were sacrificed and the livers were collected and assayed for biomarkers
of OS, such as production of superoxide anion (SA), lipid peroxidation
(LP) and DNA single strand breaks (SSBs), as well as for antioxidant enzyme activities,
including the activities of superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GSH-PX). DCA and TCA produced significant increases
in the production of SA, LP and DNA-SSBs in the two groups when compared
with the corresponding controls of each groups. However, the increases in those
biomarkers were less significant in the E-deficient group when compared with the
E-normal group. While DCA and TCA treatment resulted in SOD and GSH-Px
inhibition in the E-normal group, they induced those enzyme activities in the Edeficient
group. CAT activity on the other hand was not significantly changed
when comparing various treatments in the E-deficient with the E-normal group.
These results suggest that marginal vitamin E deficiency in mice results in compensatory
increases in antioxidant enzyme activities to provide partial protection
against DCA- and TCA-induced OS. (Supported by NIH/NIEHS grant #
R15ES013706-01A2)
1100 O- AND N-DEMETHYLATION OF LEVOMETHORPHAN
BY HUMAN CYTOCHROME P450 ENZYMES.
M. Sunouchi 1 , A. Miyajima-Tabata 2 , R. Kikura-Hanajiri 3 and Y. Goda 3 .
1 Division of Pharmacology, National Institute of Health Sciences, Tokyo 158-8501,
Japan, 2 Division of Medical Devices, National Institute of Health Sciences, Tokyo
158-8501, Japan and 3 Division of Pharmacognosy and Phytochemistry, National
Institute of Health Sciences, Tokyo 158-8501, Japan. Sponsor: A. Hirose.
Levomethorphan is the enantiomer of dextromethorphan. Dextromethorphan is an
antitussive medicine and its metabolism has been reported well, whereas levomethorphan
is an opioid analgesic with few metabolism studies. We have established
the chiral analytical method for dextromethorphan, levomethorphan and
their metabolites in biological samples using LC-MS/MS. In this study, we investigated
the metabolic properties of levomethorphan by human and rat drug metabolizing
enzymes and compared with the metabolism of dextromethorphan.
These drugs were incubated with rat or pooled human liver microsomes in
NADPH generating system. Recombinant cytochrome P450 (CYP) 2D6 and
CYP3A4 were also used for the particular analyses of the enantioselective metabolisms.
The separation of these drugs and their metabolites was achieved on a Chiral
CD-Ph column in a 0.1% formic acid-acetonitrile by a linear gradient program.
MRM was used in the positive mode of an ESI-MS/MS for the quantitative analysis.
After 20-min incubation with liver microsomes, levomethorphan was metabolized
to O-demethyl and N-demethyl form by 63% and 15% (rat), 11% and 7.1%
(human), respectively. The total amount of these metabolites from levomethorphan
was higher than that from dextromethorphan both in rat (2.0 times) and human
(3.1 times) microsomes. In the case of the incubation with CYP2D6, O-demethylation
was the major metabolism pathway from levomethorphan as well as from
dextromethorphan. O-demethylation of levomethorphan by CYP2D6 showed 2.3
times higher than dextromethorphan. In the case of CYP3A4, only N-demethyled
metabolites of these drugs were detected and N-demethylation of these drugs was
showed almost similar degree. These results suggest that the enantioselective metabolism
of levomethorphan is mainly related to the O-demethylation by CYP2D6.
1101 EFFECT OF ACARBOSE ON ALANINE
AMINOTRANSFERASE 1 AND 2 PROTEINS AND
GLUCONEOGENESIS IN RAT TISSUES.
K. Kumagai, S. Arakawa, T. Matsuyama, K. Kai, M. Teranishi and A.
Sanbuissho. Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd.,
Shizuoka, Japan.
Acarbose, an inhibitor of α-glucosidase and α-amylase, causes the elevation of
serum alanine aminotransferase (ALT) activity without any histopathological
changes in rats. The mechanism for serum ALT elevation is still unclear, although it
is assumed to be due to increased gluconeogenesis by the pharmacological effect of
acarbose. Rat ALT isozymes, ALT1 and ALT2, have been recently identified and
have been reported that the two isozymes are expressed differently in tissues. The
present study investigated the effect of acarbose on rat ALT1 and ALT2 proteins in
the serum and in the isozyme-expressed tissues: liver, heart, skeletal muscle, jejunum,
and white and brown adipose tissues. The effect of acarbose on gluconeogenesis
in the liver was also evaluated. Acarbose dissolved in distilled water was administered
by gavage to male F344 rats at dose levels of 0 or 2000 mg/kg/day for 7
days. The acarbose-treated rats had a significant increase in serum ALT activity
compared with the control. Acarbose also induced increased serum levels in aspartate
transferase and non-esterified fatty acid, and decreased serum levels in triacylglycerol
and glucose. No histopathological changes by the acarbose treatment were
observed in the liver, heart, skeletal muscle, jejunum, white or brown adipose tissues.
Western blot analysis revealed that ALT1 protein levels in the serum and liver
of acarbose-treated rats were significantly higher than those in the control. On the
other hand, compared to the control, acarbose-administered rats had no increases
in ALT1 proteins in other tissues or ALT2 proteins in any tissues. Moreover, serum
ALT2 protein was not detected in either control or acarbose-administered rats.
Phosphoenolpyruvate carboxykinase protein in the acarbose-treated rat livers was
significantly increased compared to the control. In conclusion, the results of the
present study suggest that acarbose causes increases in gluconeogenesis and ALT1
protein in the liver, resulting in the elevation of ALT1 protein and ALT activity in
the serum.
1102 ENZYMES AND OXIDATIVE DAMAGE INDICATORS IN
RATS FOLLOWING SUBACUTE OR SUBCHRONIC
MANGANESE CHLORIDE EXPOSURE.
P. Huang 1 , G. Li 2 and Z. Sun 1 . 1 Department of Toxicology and Sanitary Chemistry,
School of Public Health and Family Medicine, Capital Medical University, Beijing,
China and 2 Beijing Research Center for Preventive Medicine, Beijing, China.
Sponsor: W. Zheng.
Manganese (Mn) toxicity is most often found in mining and welding industry
workers. Accumulation of manganese in the brain can result in a syndrome similar
to that of Parkinson’s disease. Observations on former Mn-alloy workers suggested
that residual effects could last for years after exposure. The objective of this study
was to assess effects of Mn in the liver of rats following subacute or subchronic exposure
and after recovery. Male Sprague-Dawley rats weighing 110 to 120 g were
exposed to 6.0 mg Mn/kg b.w. using MnCl2 for 30 days, 90 days, or for 90 days
followed by a 30-day post-exposure recovery period. Results showed that serum
AST and ALT increased compared to controls. Mn concentrations in the liver of
treated animals increased 1.5 to 2.5-fold compared to controls. Similarly, concentrations
of Ca and Cu increased in treated animals, while concentrations of Fe, Mg
and Zn decreased in liver tissues after subacute or subchronic exposure. Subacute or
subchronic Mn exposure also inhibited GPx activity, decreased GSH levels and increased
MDA levels in liver tissues. All of the indicators returned to normal after
the recovery period. We further evaluated the effects of manganese on rat hepatocyte
mitochondria, membranes, and nuclei in the three periods. The results showed
significant inhibition of mitochondrial SDH, cellular Na+,K+-ATPase,
Ca2+,Mg2+-ATPase, Cu2+-ATPase, and GPx activities in hepatocytes. In addition,
GSH levels decreased while MDA levels increased in hepatocyte mitochondria,
membranes, and nuclei. All of these markers were normal after the recovery period.
Taken together, these data suggest that MnCl2 exposure injured rat liver tissue, that
the extent of injury is positively correlated with exposure time, and that the effect
on mitochondria is presumably more serious than that on nuclei and membranes.
Most biomarker alterations returned to pre-exposure levels by themselves after stopping
Mn exposure.
SOT 2011 ANNUAL MEETING 235