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ABSTRACT: CYP2A5 metabolizes xenobiotics and activates hepatocarcinogens, and induction occurs in response to hepatic damage and cellular stress. We evaluated whether ethanol can elevate CYP2A5 and whether CYP2E1 plays a role in the ethanol induction of CYP2A5. Wild-type (WT), CYP2E1 knockout (KO), and CYP2E1 knockin (KI) mice were fed ethanol for 3 weeks. Ethanol increased CYP2E1 and CYP2A5 protein and activity in WT mice but not in the KO mice. Induction of CYP2A5 (and CYP2E1) was restored in the KI mice. Ethanol induction of CYP2A5 occurred only after CYP2E1 was first induced. Immunohistochemical staining revealed that CYP2E1 and CYP2A5 colocalize to the same zones in the liver. Ethanol also elevated CYP2A5 mRNA levels in WT and KI mice but not in KO mice. Induction of CYP2A5 by cadmium was partially decreased in KO mice compared with WT or KI mice. Ethanol elevated CYP2A4 mRNA levels in all mice although the extent of induction was lowest in the KO mice. In summary, ethanol elevated mouse hepatic CYP2A5 levels, which may be of toxicological significance because CYP2A5 metabolizes nicotine and other drugs and activates hepatocarcinogens. Induction of CYP2A5 by ethanol is potentiated by the induction of CYP2E1. We speculate that ethanol induction of CYP2E1 followed by increases in reactive oxygen species and activation of Nrf2 are important steps in the mechanism by which ethanol induces CYP2A5. The possibility that induction of CYP2E1 is permissive for the induction of CYP2A5 may reflect a new contribution by CYP2E1 to the actions of ethanol.
Drug metabolism and disposition: the biological fate of chemicals 11/2010; 39(2):330-6. · 3.74 Impact Factor
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Clinica chimica acta; international journal of clinical chemistry 10/2009; 411(1-2):122-3. · 2.54 Impact Factor
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Jian Zhuge
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ABSTRACT: Previous studies show that treatment with a polyunsaturated fatty acid, arachidonic acid (AA), or high concentrations of cycloleucine, an inhibitor of methionine adenosyltransferase (MAT), which lowers levels of S-adenosyl-L-methionine (SAM), increased toxicity in hepatocytes from pyrazole-treated rats which expressed high levels of cytochrome P450 2E1 (CYP2E1). In this study, I used concentrations of cycloleucine or AA, which by themselves do not produce any toxicity, to evaluate whether a decrease in SAM sensitizes hepatocytes to AA toxicity, especially in hepatocytes enriched in CYP2E1. Levels of SAM were lower by 50% in hepatocytes from pyrazole- compared to saline-treated rats. Cycloleucine treatment caused a 50% decline in SAM levels with both hepatocyte preparations and SAM levels were lowest in the pyrazole-treated hepatocytes. The combination of cycloleucine plus AA produced some toxicity and apoptosis in hepatocytes from saline-treated rats but increased toxicity and apoptosis was found in the hepatocytes from pyrazole-treated rats. Cytotoxicity could be prevented by incubation with SAM, the antioxidant trolox, and the mitochondrial permeability transition inhibitor trifluoperazine. The enhanced cytotoxicity could also be protected by treating rats with chlormethiazole, a specific inhibitor of CYP2E1, thus validating the role of CYP2E1. Cycloleucine plus AA treatment elevated production of reactive oxygen species (ROS) and lipid peroxidation to greater extents with the hepatocytes from pyrazole-treated rats than that from the saline-treated rats. I hypothesize that increased production of ROS by hepatocytes enriched in CYP2E1 potentiates AA-induced lipid peroxidation and toxicity when hepatoprotective levels of SAM are lowered. Such interactions, e.g. induction of CYP2E1, decline in SAM and polyunsaturated fatty acid-induced lipid peroxidation, may contribute to alcohol-induced liver injury.
Molecular and Cellular Biochemistry 08/2008; 314(1-2):105-12. · 2.06 Impact Factor
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Jian Zhuge
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ABSTRACT: 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a phylogenetically conserved serine/threonine protein kinase. AMPK may inhibit cell growth and proliferation and also regulates apoptosis. 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) is a cell-permeable AMPK activator. Activation of AMPK with AICAR has been shown to induce apoptosis of the rat hepatoma cell line FTO2B cells and almost completely inhibited HepG2 cells growth. In this study, a HepG2 cell line, which was transfected with a vector containing human CYP2E1 cDNA (E47 cells), was treated with AICAR. Cell proliferation was blocked, and apoptosis and necrosis were elevated as assessed by cellular morphology, DNA content assay, and lactate dehydrogenase leakage. AICAR treatment significantly increases CYP2E1 activity (20-fold) and expression (5.5-fold) in E47 cells. Iodotubericidin, which inhibits the conversion of AICAR to its activated form AICAR monophosphate, the antioxidants trolox and MnTMPyP, and 4-methylpyrazole, an inhibitor of CYP2E1, all can protect the E47 cells from AICAR-induced necrosis. Production of intracellular reactive oxygen species was increased by AICAR treatment in E47 cells. The cytotoxicity mechanism of AICAR in E47 cells is suggested to include AMPK activation, p53 phosphorylation, p21 expression, overexpression of CYP2E1, and intracellular ROS accumulation.
Cell Biology and Toxicology 06/2008; 25(3):253-63. · 2.51 Impact Factor
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ABSTRACT: Cytochrome P450 2E1 (CYP2E1) is suggested to play a role in alcoholic liver disease, which includes alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. In this study, we investigated whether CYP2E1 plays a role in experimental alcoholic fatty liver in an oral ethanol-feeding model. After 4 weeks of ethanol feeding, macrovesicular fat accumulation and accumulation of triglyceride in liver were observed in wild-type mice but not in CYP2E1-knockout mice. In contrast, free fatty acids (FFAs) were increased in CYP2E1-knockout mice but not in wild-type mice. CYP2E1 was induced by ethanol in wild-type mice, and oxidative stress induced by ethanol was higher in wild-type mice than in CYP2E1-knockout mice. Peroxisome proliferator-activated receptor alpha (PPARalpha), a regulator of fatty acid oxidation, was up-regulated in CYP2E1-knockout mice fed ethanol but not in wild-type mice. A PPARalpha target gene, acyl CoA oxidase, was decreased by ethanol in wild-type but not in CYP2E1-knockout mice. Chlormethiazole, an inhibitor of CYP2E1, lowered macrovesicular fat accumulation, inhibited oxidative stress, and up-regulated PPARalpha protein level in wild-type mice fed ethanol. The introduction of CYP2E1 to CYP2E1-knockout mice via an adenovirus restored macrovesicular fat accumulation. These results indicate that CYP2E1 contributes to experimental alcoholic fatty liver in this model and suggest that CYP2E1-derived oxidative stress may inhibit oxidation of fatty acids by preventing up-regulation of PPARalpha by ethanol, resulting in fatty liver.
Hepatology 06/2008; 47(5):1483-94. · 11.66 Impact Factor
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ABSTRACT: S-Adenosyl-l-methionine (SAM) is the principal biological methyl donor. Methionine adenosyltransferase (MAT) catalyzes the only reaction that generates SAM. Hepatocytes were treated with cycloleucine, an inhibitor of MAT, to evaluate whether hepatocytes enriched in cytochrome P450 2E1 (CYP2E1) were more sensitive to a decline in SAM. Cycloleucine decreased SAM and glutathione (GSH) levels and induced cytotoxicity in hepatocytes from pyrazole-treated rats (with an increased content of CYP2E1) to a greater extent as compared to hepatocytes from saline-treated rats. Apoptosis caused by cycloleucine in pyrazole hepatocytes appeared earlier and was more pronounced than control hepatocytes and could be prevented by incubation with SAM, glutathione reduced ethyl ester and antioxidants. The cytotoxicity was prevented by treating rats with chlormethiazole, a specific inhibitor of CYP2E1. Cycloleucine induced greater production of reactive oxygen species (ROS) in pyrazole hepatocytes than in control hepatocytes, and treatment with SAM, Trolox, and chlormethiazole lowered ROS formation. In conclusion, lowering of hepatic SAM levels produced greater toxicity and apoptosis in hepatocytes enriched in CYP2E1. This is due to elevated ROS production by CYP2E1 coupled to lower levels of hepatoprotective SAM and GSH. We speculate that such interactions e.g. induction of CYP2E1, decline in SAM and GSH may contribute to alcohol liver toxicity.
Archives of Biochemistry and Biophysics 11/2007; 466(2):177-85. · 2.93 Impact Factor
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ABSTRACT: Ethanol treatment causes an increase in expression of TGF-beta1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-beta1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-beta1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-beta1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-beta1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-beta1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-beta1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-beta1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-beta1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease.
Free Radical Biology and Medicine 11/2006; 41(7):1100-12. · 5.42 Impact Factor
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ABSTRACT: Induction of oxidative stress plays a key role in serum deprivation-induced apoptosis. CYP2E1 plays an important role in toxicity of many chemicals and ethanol and produces oxidant stress. We investigated whether CYP2E1 expression can sensitize HepG2 cells to toxicity as a consequence of serum deprivation. The models used were HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells which do not express CYP2E1. E47 cells showed greater growth inhibition and enhanced cell death after serum deprivation, as compared to the C34 cells. DNA ladder and flow cytometry assays indicated that apoptosis occurred at earlier times after serum deprivation in E47 than C34 cells. Serum withdrawal-induced E47 cell death could be rescued by antioxidants, the mitochondrial permeability transition inhibitor cyclosporine A, z-DEVD-fmk, and a CYP2E1 inhibitor 4-methylpyrazole. Increased production of reactive oxygen species (ROS) and lipid peroxidation occurred in E47 cells after serum deprivation, and there was a corresponding decline in the E47 cell mitochondrial membrane potential and reduced glutathione (GSH) levels. We propose that the mechanism of this serum withdrawal plus CYP2E1 toxicity involves increased production of intracellular ROS, lipid peroxidation, and decline of GSH levels, which results in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of serum deprivation-induced cell death by CYP2E1 may contribute to the sensitivity of the liver to alcohol-induced ischemia and growth factor deprivation.
Free Radical Biology and Medicine 02/2006; 40(1):63-74. · 5.42 Impact Factor
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ABSTRACT: The rapid development of molecular biology in recent decades has dramatically changed the way we practice medicine. With the
help of an impressive arsenal of new technologies, including high-throughput sequencing and microarrays, we are now well-equipped
to probe into the molecular nature of diseases. Which set of genes are involved? What are the genetic and epigenetic alterations
associated with these genes? In this chapter, we will describe the basic concepts of molecular biology, including genes, types
of mutations, and gene expression.
01/1970: pages 13-25;
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ABSTRACT: Previous results showed that pyrazole potentiates lipopolysaccharide (LPS)-induced liver injury in mice. Mechanisms involved the overexpression of cytochrome P450 2E1 (CYP2E1), oxidative stress, and activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). The current study was carried out to test the hypothesis that the mitochondria permeability transition (MPT) plays a role in this pyrazole plus LPS toxicity. Mice were injected intraperitoneally with pyrazole for 2 days, followed by a challenge with LPS with or without treatment with cyclosporin A (CsA), an inhibitor of the MPT. Serum alanine aminotransferase and aspartate aminotransferase were increased by pyrazole plus LPS treatment, and CsA treatment could attenuate these increases. CsA also prevented pyrazole plus LPS-induced hepatocyte necrosis. Formation of 4-hydroxynonenal protein adducts and 3-nitrotyrosine protein adducts in liver tissue was increased by the pyrazole plus LPS treatment, and CsA treatment blunted these increases. Swelling, cytochrome c release from mitochondria to the cytosol, and lipid peroxidation were increased in mitochondria isolated from the pyrazole plus LPS-treated mice, and CsA treatment prevented these changes. CsA did not prevent the increased levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), pp38 MAPK, and p-JNK2. In conclusion, although CsA does not prevent elevations in upstream mediators of the pyrazole plus LPS toxicity (iNOS, TNF-α, CYP2E1, MAPK), it does protect mice from the pyrazole plus LPS-induced liver toxicity by preventing the MPT and release of cytochrome c and decreasing mitochondrial oxidative stress. These results indicate that mitochondria are the critical targets of pyrazole plus LPS in mediating liver injury.
Free Radical Biology and Medicine.
