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Title: Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite

Abstract

Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

Authors:
 [1];  [2];  [2];  [1];  [2];  [2];  [1];  [2];  [3];  [1];  [2];  [2];  [4]
  1. Veterans Administration Medical Center, White River Junction, VT 05009 (United States)
  2. (United States)
  3. Lilly Research Laboratories, Indianapolis, IN 46285 (United States)
  4. Veterans Administration Medical Center, White River Junction, VT 05009 (United States) and Department of Microbiology/Immunology, Dartmouth College, Hanover, NH 03755 (United States) and Department of Medicine, Dartmouth College, Hanover, NH 03755 (United States). E-mail: ralph.c.nichols@dartmouth.edu
Publication Date:
OSTI Identifier:
20850498
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 217; Journal Issue: 3; Other Information: DOI: 10.1016/j.taap.2006.09.007; PII: S0041-008X(06)00318-8; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACUTE EXPOSURE; ARSENIC; BIOSYNTHESIS; BROMIDES; DEXAMETHASONE; HYDROXYLATION; INHIBITION; LIVER CELLS; NITROPHENOL; PROTEINS; RATS; TOXICITY

Citation Formats

Noreault-Conti, Trisha L., Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755, Vermont Forensic Laboratory, Waterbury, VT 05671, Jacobs, Judith M., Department of Microbiology/Immunology, Dartmouth College, Hanover, NH 03755, Department of Biochemistry, Dartmouth College, Hanover, NH 03755, Trask, Heidi W., Department of Biochemistry, Dartmouth College, Hanover, NH 03755, Wrighton, Steven A., Sinclair, Jacqueline F., Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755, Department of Biochemistry, Dartmouth College, Hanover, NH 03755, and Nichols, Ralph C. Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite. United States: N. p., 2006. Web. doi:10.1016/j.taap.2006.09.007.
Noreault-Conti, Trisha L., Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755, Vermont Forensic Laboratory, Waterbury, VT 05671, Jacobs, Judith M., Department of Microbiology/Immunology, Dartmouth College, Hanover, NH 03755, Department of Biochemistry, Dartmouth College, Hanover, NH 03755, Trask, Heidi W., Department of Biochemistry, Dartmouth College, Hanover, NH 03755, Wrighton, Steven A., Sinclair, Jacqueline F., Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755, Department of Biochemistry, Dartmouth College, Hanover, NH 03755, & Nichols, Ralph C. Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite. United States. doi:10.1016/j.taap.2006.09.007.
Noreault-Conti, Trisha L., Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755, Vermont Forensic Laboratory, Waterbury, VT 05671, Jacobs, Judith M., Department of Microbiology/Immunology, Dartmouth College, Hanover, NH 03755, Department of Biochemistry, Dartmouth College, Hanover, NH 03755, Trask, Heidi W., Department of Biochemistry, Dartmouth College, Hanover, NH 03755, Wrighton, Steven A., Sinclair, Jacqueline F., Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755, Department of Biochemistry, Dartmouth College, Hanover, NH 03755, and Nichols, Ralph C. Fri . "Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite". United States. doi:10.1016/j.taap.2006.09.007.
@article{osti_20850498,
title = {Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite},
author = {Noreault-Conti, Trisha L. and Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755 and Vermont Forensic Laboratory, Waterbury, VT 05671 and Jacobs, Judith M. and Department of Microbiology/Immunology, Dartmouth College, Hanover, NH 03755 and Department of Biochemistry, Dartmouth College, Hanover, NH 03755 and Trask, Heidi W. and Department of Biochemistry, Dartmouth College, Hanover, NH 03755 and Wrighton, Steven A. and Sinclair, Jacqueline F. and Department of Pharmacology/Toxicology, Dartmouth College, Hanover, NH 03755 and Department of Biochemistry, Dartmouth College, Hanover, NH 03755 and Nichols, Ralph C.},
abstractNote = {Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.},
doi = {10.1016/j.taap.2006.09.007},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 217,
place = {United States},
year = {Fri Dec 15 00:00:00 EST 2006},
month = {Fri Dec 15 00:00:00 EST 2006}
}
  • Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (CYP) levels by arsenic. In rat and human hepatocyte cultures, arsenic, in the form of arsenite, decreases the induction of several CYPs. The present study investigated whether arsenite utilizes transcriptional or post-transcriptional mechanisms to decrease CYP3A23 in primary cultures of rat hepatocytes. In these cultures, a 6-h treatment with 5 {mu}M arsenite abolished dexamethasone (DEX)-mediated induction of CYP3A23 protein and activity, but didmore » not inhibit general protein synthesis. However, arsenite treatment only reduced DEX-induced levels of CYP3A23 mRNA by 30%. The effects of arsenite on CYP3A23 transcription were examined using a luciferase reporter construct containing 1.4 kb of the CYP3A23 promoter. Arsenite caused a 30% decrease in DEX-induced luciferase expression of this reporter. Since arsenite abolished induction of CYP3A23 protein, but caused only a small decrease in CYP3A23 mRNA, the effects of arsenite on translation of CYP3A23 mRNA were investigated. Polysomal distribution analysis showed that arsenite decreased translation by decreasing the DEX-mediated increase in CYP3A23 mRNA association with polyribosomes. Arsenite did not decrease intracellular glutathione or increase lipid peroxidation, suggesting that the effect of arsenite on CYP3A23 does not involve oxidative stress. Overall, the results suggest that low-level arsenite decreases both transcription and translation of CYP3A23 in primary rat hepatocyte cultures.« less
  • Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured ratmore » hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.« less
  • Growth studies were done on a cultured rat liver cell line (RLC-GAI) grown in a chemically defined medium in the presence of lead nitrate. Lead reversibly inhibited the growth of these cells even after 6 d of exposure to the heavy metal. To compare lead sensitivity in various cell lines, G150 and LD50 values were determined in the RLC-GAI cells as well as two glioma cell lines (B82 and C/sub 6/) and a neuroblastoma cell line (N18). The LD50 values paralleled but were consistently lower than the G150 values. Since lead is known to affect heme synthesis, hemin was addedmore » to test the possibility of preventing the growth-inhibitory effect of the lead. The growth capacity of lead-treated cells did not change with the addition of hemin. It is thought that differential cultured cell lines such as these could be useful in examining the molecular mechanism of lead toxicity.« less
  • The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role formore » metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.« less
  • Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by β-oxidation. Another biotransformation pathway involves β-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2′,7′-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C{sub 12}), necrosis (release of lactatemore » dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it contributes to the toxicity of (E)-2-ene-VPA in PB-pretreated rat hepatocytes. -- Highlights: ► (E)-2,4-diene-valproic acid is a reactive and toxic metabolite of valproic acid (VPA). ► In situ, this metabolite is not responsible for VPA toxicity in rat hepatocytes. ► This metabolite enhances (E)-2-ene-VPA toxicity in PB-pretreated hepatocytes.« less