The Toxicologist - Society of Toxicology

frequently studied. In the current study, we tested the efficacy of substituted quinolines
(code name = PQ1) in combination with tamoxifen in T47D cells. Previous
studies conducted by our group shows that PQ1 is a gap junctional activator. Gap
junctions are intercellular channels allowing the passage of small molecules from
one cell to another. Loss of gap junctional intercellular communication has been
observed in different kinds of cancer. Therefore, in our study we proposed to determine
the combinational efficacy of PQ1 and tamoxifen on breast cancer. The results
show that combinational treatment of PQ1 and tamoxifen cause a 55% decrease
in the colony growth assay compared to control. Combination of 10 μM
tamoxifen and PQ1 200 nM or 500 nM resulted in only 16% cell viability compared
to controls at 48 hr in T47D cells by MTT assay. We found a significant increase
in BAX protein at 1 hr in the presence of 500 nM PQ1 alone, 10 μM tamoxifen
alone and combination of PQ1 and tamoxifen. A 2-fold increase was
observed in active caspase 3 in the presence of combinational treatment of 10 μM
tamoxifen and 200 or 500 nM PQ1. Also, flow cytometric analysis showed a 50%
increase in the number of apoptotic cells in the presence of combination of tamoxifen
and PQ1 compared to the control. Furthermore, the results showed that combinational
treatment of tamoxifen and PQ1 significantly reduces the expression of
survivin in T47D cells. In conclusion, the present study demonstrates that combinational
treatment of tamoxifen and PQ1 (gap junctional activator) can be used to
potentiate apoptosis of T47D human breast cancer cells. Thus, gap junctional activator,
PQ1, could potentially alter either the length or dose of tamoxifen clinically
used for breast cancer patients.
1086 ALTERATION OF CYTOCHROME P450 GENE
EXPRESSION IN THE KIDNEY AND LIVER OF MALE
SPRAGUE-DAWLEY RATS BY ACUTE DOXORUBICIN
TOXICITY.
B. Zordoky and A. El-Kadi. Pharmaceutical Sciences, University of Alberta,
Edmonton, AB, Canada.
Doxorubicin (DOX) is a potent anti-neoplastic antibiotic used to treat a variety of
malignancies; however, its use is limited by significant cardiotoxicity, nephrotoxicity,
and hepatotoxicity. We have previously shown that DOX cardiotoxicity induces
several cardiac cytochrome P450 (CYP) enzymes with subsequent alteration in
CYP-mediated arachidonic acid metabolism. CYP-mediated arachidonic acid
metabolites play pivotal roles in the heart, kidney, and liver physiology.
Nevertheless, the effect of acute DOX toxicity on the expression of renal and hepatic
CYP genes was not examined previously. Therefore, in the current study, we
have investigated the effect of acute DOX toxicity on CYP gene expression in the
kidney and the liver of male Sprague Dawley rats. Acute DOX toxicity was induced
by a single intraperitoneal injection of 15 mg/kg of the drug. After 24 hours, the
kidney and the liver were harvested and the expression of different CYP genes was
determined by real time-PCR. Our results showed that acute DOX toxicity caused
a significant induction of CYP1B1 and CYP4A3 in both the kidney and the liver.
However, CYP2E1, CYP4A1, CYP4A2, and CYP4F1 were significantly induced in
the kidney but not in the liver of DOX-treated rats. On the other hand, CYP2C11
gene expression was significantly inhibited in both the kidney and the liver, whereas
CYP2B1 and CYP2J3 were inhibited significantly in the liver but not in the kidney
of DOX-treated rats. The expression of CYP1A1, CYP4F4, CYP4F5, and CYP4F6
was not significantly altered in both the kidney and liver. In conclusion, acute
DOX toxicity alters the expression of several CYP genes in an organ-specific manner.
The changes in CYP gene expression may result in altered arachidonic acid metabolism
which may contribute to DOX-induced nephrotoxicity and hepatotoxicity.
In addition, inhibition of several hepatic CYP enzymes may account for drug
interactions with DOX due to inhibition of CYP-mediated drug metabolism. (This
work was supported by the Heart and Stroke Foundation of Alberta, NWT, and
Nunavut).
1087 REACTIVE METABOLITES OF BISPHENOL A FORMED
BY RAT LIVER MICROSOMES AND TRAPPED BY
DANSYL GLUTATHIONE.
T. Buranachokpaisan, B. Winnik, B. Buckley and P. E. Thomas. Rutgers, The
State University of NJ, and Environmental & Occupational Health Sciences Institute,
Piscataway, NJ.
Bisphenol A (BPA) is an environmental contaminant that has attracted considerable
concern for its possible role as an endocrine disruptor of estrogen action.
However, metabolism-derived toxicity of BPA is inadequately explored. We examined
metabolism of BPA with rat liver microsomes (Mx) by trapping of possible reactive
metabolites with dansyl glutathione (dGSH). Experimental procedures: BPA
was incubated with 5 different Mx: β-Naphthoflavone-treated (NF), phenobarbital-treated
(PB), pregnenolone 16α-carbonitrile-treated (PCN), vehicle-treated
male (CM), and vehicle-treated female (CF), which represent the following P450s
respectively: 1A1/2; 2B1/2; 3A1/2; 2C11, 2C13, and 3A2; and 2C12. The incubations
were performed in phosphate buffer pH 7.4 at 37C in the presence of
NADPH and dGSH as a trapping agent. At the end of the incubation the reactions
were quenched with 2 volumes of ice-cold DTT in methanol, centrifuged and the
supernatants were analyzed by HPLC with fluorescence detection. The adduct
peaks were further analyzed by LC-MS. Results: Four dGSH adducts were found,
with the highest activity per mg protein, in PB and in the decreasing order: CM,
PCN, NF, and CF. The adducts were also inhibited by their corresponding P450
chemical and antibody inhibitors. The kinetics of adduct formation of the two
most abundant adducts, BPA and phenol BPA, followed Michaelis-Menten kinetics.
The 5-hydroxy metabolite was found unstable and formed at the lowest rate,
while 4-isopropyl adduct peak was incompletely resolved from dGSH, hence, its kinetics
could not be reliably determined at this time. Conclusion: Four reactive
metabolites of BPA were formed by rat liver P450 enzymes in the order of decreasing
activity: 2B1/2, 2C11, 3A1, 1A1/2.
1088 A KEY ROLE FOR CYP3A4 IN MDMA (ECSTASY)-
INDUCED HEPATOTOXICITY AND IMPLICATIONS
FOR ACUTE MDMA TOXICITY TREATMENT.
I. Antolino Lobo 1 , S. Nijmeijer 1 , I. Meijerman 2 , J. Meulenbelt 3 , M. van den
Berg 1 and M. van Duursen 1 . 1 Institute for Risk Assessment Sciences, Utrecht
University, Utrecht, Netherlands, 2 Department of Pharmaceutical Sciences, Utrecht
University, Utrecht, Netherlands and 3 National Institute for Public Health and the
Environment (RIVM), Bilthoven, Netherlands.
Over the last years, an increasing number of reports on adverse, toxic effects after
recreational use of MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) have
been published in the literature. Large interindividual variations in susceptibility
toward MDMA (hepato)toxicity have been observed, which can largely be attributed
to differences in both phase I and phase II metabolism. We have used the
human liver epithelial (THLE) cell line transfected with a single cytochrome P450
to study the implications of metabolism on MDMA toxicity. Inhibition of the
phase II enzymes catechol-O-methyltransferase (COMT) and especially glutathione
(GSH) significantly increased MDMA cytotoxicity after CYP3A4 and
CYP2D6-mediated MDMA metabolism by about 50% and 20%, respectively.
This indicates a major detoxification role for GSH in cells after CYP3A4 or
CYP2D6-mediated metabolism of a physiologically relevant concentration of
MDMA. These results were confirmed by experiments showing a 40% reduction of
GSH level after MDMA exposure in THLE-CYP3A4, but not THLE-CYP2D6
cells. These data suggest that CYP3A4 is the main enzyme responsible for cytotoxic
metabolite formation and GSH depletion after MDMA exposure, which might
lead to hepatotoxic effects. CYP3A4 is also a major enzyme involved in the metabolism
of therapeutic drugs. The potential interaction of uncontrolled use of
MDMA with these prescription drugs, e.g. to control psychiatric disorders or in the
treatment of acute clinical signs in MDMA toxicity, is of relevant concern.
Moreover, many of these pharmaca can induce CYP3A4 via activation of the pregnane
X receptor (PXR) thus potentially increasing MDMA-mediated toxicity.
Further studies are being performed in our laboratory to determine this potential
drug-drug interaction through PXR-mediated CYP3A4 expression using a reporter
gene assay.
1089 THE MITOCHONDRIAL TRANSPORTER ABCB6
REGULATES CYTOCHROME P450 ENZYME
EXPRESSION.
H. Chavan, M. Oruganti and P. Krishnamurthy. Pharmacology, Toxicology, and
Therapeutics, Kansas University Medical Center, Kansas City, KS. Sponsor: U. Apte.
Cytochrome P450 (CYP450) enzymes belong to a superfamily of hemoprotein that
play a pivotal role in the metabolism and clearance of various xenobiotics and environmental
toxins from the body. Haem is an important structural module for
CYP450 and is essential for its activity. Further, haem is also known to regulate the
expression of CYP450 enzymes. In this study we investigated whether the mitochondrial
half transporter ABCB6, which regulates cellular haem biosynthesis and
cellular haemoprotein pool, plays a role in the expression and/or function of
CYP450s. To investigate this question, we developed mice deficient in Abcb6
(Abcb6+/- mice) and evaluated the expression and activity of two liver specific
CYP450s; Cyp3a11 and Cyp2e1. These mice demonstrate decreased porphyrin levels
compared to Abcb6+/+ mice (50% decrease in porphyrin levels in Abcb6+/-
mice compared to Abcb6 +/+ mice). Interestingly, inspite of decreased porphyrin
levels, we found that the expression of both Cyp3a11 and Cyp2e1 was induced in
Abcb6 +/- mice. However, this increase in expression did not translate into increased
activity when tested with substrates specific for Cyp3a11 (midazolam) and
Cyp2e1 (chlorzoxazone). Cellular synthesis of CYP450s is thought to be tightly coordinated
with haem synthesis to sustain adequate supply of haem for newly syn-
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