Hepatitis: Gonzalez FJ

Gonzalez FJ, Shah YM. · National Cancer Research, National Institutes of Health, Bethesda, MD 20892, USA. · Toxicology. · Pubmed #18006136 No free full text.

Abstract: Peroxisome proliferator chemicals are classic non-genotoxic carcinogens. These agents cause liver cancers when chronically administered to rats and mice. Peroxisome proliferators include the widely prescribed lipid and cholesterol lowering fibrate drugs. In contrast to the results in rodents, there is no evidence that fibrates are associated with elevated risk of liver cancer or any other neoplasms in humans thus indicating a species difference in the hepatocarcinogenic response. The biological effects of peroxisome proliferators are mediated by the peroxisome proliferator-activated receptor (PPAR)alpha. Pparalpha-null mice are resistant to all of the pleiotropic effects of peroxisome proliferators, including cell proliferation and hepatocarcinogenesis. The mechanism of hepatocellular proliferation involves downregulation of the microRNA let-7c gene by PPARalpha. Let-7c controls levels of proliferative c-myc by destabilizing its mRNA. Thus, upon suppression of let-7c, c-myc mRNA and protein are elevated resulting in enhanced hepatocellular proliferation. In contrast, PPARalpha-humanized mice, that respond to Wy-14,643 by lower serum triglycerides and induction of genes encoding fatty acid metabolizing enzymes, are resistant to peroxisome proliferator-induced cell proliferation and cancer. These mice do not exhibit downregulation of let-7c gene expression thus forming the basis for the resistance to hepatocellular carcinogenesis.

Gonzalez FJ. · Laboratory of Metabolism, National Cancer Institute, Bethesda, MD 20892, USA. · Mutat Res. · Pubmed #15603755 No free full text.

Abstract: Cytochromes P450 are responsible for metabolism of most xenobiotics and are required for the efficient elimination of foreign chemicals from the body. Paradoxically, these enzymes also metabolically activate biologically inert compounds to electrophilic derivatives that can cause toxicity, cell death and sometimes cellular transformation resulting in cancer. To establish the role of these enzymes in toxicity and carcinogenicity in vivo, gene knockout mice have been developed. To illustrate the role of P450s in toxicity, CYP2E1-null mice were employed with the commonly used analgesic drug acetaminophen. CYP2E1 is the rate-limiting enzyme that initiates the cascade of events leading to acetaminophen hepatotoxicity; in the absence of this P450, toxicity will only be apparent at high concentrations. Other enzymes and nuclear receptors are also involved in activation or inactivating chemicals. CYP2E1 is induced by alcohol and the primary P450 that carries out ethanol oxidation that can lead to the production of activated oxygen species and oxidative stress that elevate ERK1/2 phosphorylation through EGRF/c-Raf signaling. Paradoxically, activation of this pathway inhibits apoptotic cell death stimulated by reactive oxygen generating chemicals but accelerates necrotic cell death produced by polyunsaturated fatty acids. CYP2E1 is thought to contribute to liver pathologies that result from alcoholic liver disease and non-alcoholic steatohepatitis.

Li L, Oropeza CE, Sainz B, Uprichard SL, Gonzalez FJ, McLachlan A. · Department of Microbiology and Immunology College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America. · PLoS One. · Pubmed #19424486 links to  free full text

Abstract: The host cellular factors that promote persistent viral infections in vivo are, in general, poorly understood. Utilizing the hepatitis B virus (HBV) transgenic mouse model of chronic infection, we demonstrate that the nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1), is essential for viral biosynthesis in the liver. The dependency of HBV transcription on HNF4alpha links viral biosynthesis and persistence to a developmentally regulated transcription factor essential for host viability.

Okiyama W, Tanaka N, Nakajima T, Tanaka E, Kiyosawa K, Gonzalez FJ, Aoyama T. · Department of Metabolic Regulation, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Japan. · J Hepatol. · Pubmed #19398233 No free full text.

Abstract: BACKGROUND/AIMS: Alcoholic liver disease (ALD) is one of the leading causes of cirrhosis and yet efficient therapeutic strategies are lacking. Polyenephosphatidylcholine (PPC), a major component of essential phospholipids, prevented alcoholic liver fibrosis in baboons, but its precise mechanism remains uncertain. We aimed to explore the effects of PPC on ALD using ethanol-fed peroxisome proliferator-activated receptor alpha (Ppara)-null mice, showing several similarities to human ALD. METHODS: Male wild-type and Ppara-null mice were pair-fed a Lieber-DeCarli control or 4% ethanol-containing diet with or without PPC (30 mg/kg/day) for 6 months. RESULTS: PPC significantly ameliorated ethanol-induced hepatocyte damage and hepatitis in Ppara-null mice. These effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ROS)-generating enzymes, including cytochrome P450 2E1, acyl-CoA oxidase, and NADPH oxidases, in addition to restoration of increases in Toll-like receptor 4 and CD14. PPC also decreased Bax and truncated Bid, thus inhibiting apoptosis. Furthermore, PPC suppressed increases in transforming growth factor-beta1 expression and hepatic stellate cell activation, which retarded hepatic fibrogenesis. CONCLUSIONS: PPC exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ALD as a result of inhibition of the overexpression of ROS-generating enzymes. Our results demonstrate detailed molecular mechanisms of the anti-oxidant action of PPC.

Holdener M, Hintermann E, Bayer M, Rhode A, Rodrigo E, Hintereder G, Johnson EF, Gonzalez FJ, Pfeilschifter J, Manns MP, Herrath MG, Christen U. · Pharmazentrum Frankfurt/Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit, Johann Wolfgang Goethe University, 60590 Frankfurt am Main, Germany. · J Exp Med. · Pubmed #18474629 links to  free full text

Abstract: Autoimmune liver diseases, such as autoimmune hepatitis (AIH) and primary biliary cirrhosis, often have severe consequences for the patient. Because of a lack of appropriate animal models, not much is known about their potential viral etiology. Infection by liver-tropic viruses is one possibility for the breakdown of self-tolerance. Therefore, we infected mice with adenovirus Ad5 expressing human cytochrome P450 2D6 (Ad-2D6). Ad-2D6-infected mice developed persistent autoimmune liver disease, apparent by cellular infiltration, hepatic fibrosis, "fused" liver lobules, and necrosis. Similar to type 2 AIH patients, Ad-2D6-infected mice generated type 1 liver kidney microsomal-like antibodies recognizing the immunodominant epitope WDPAQPPRD of cytochrome P450 2D6 (CYP2D6). Interestingly, Ad-2D6-infected wild-type FVB/N mice displayed exacerbated liver damage when compared with transgenic mice expressing the identical human CYP2D6 protein in the liver, indicating the presence of a stronger immunological tolerance in CYP2D6 mice. We demonstrate for the first time that infection with a virus expressing a natural human autoantigen breaks tolerance, resulting in a chronic form of severe, autoimmune liver damage. Our novel model system should be instrumental for studying mechanisms involved in the initiation, propagation, and precipitation of virus-induced autoimmune liver diseases.

Tanaka N, Moriya K, Kiyosawa K, Koike K, Gonzalez FJ, Aoyama T. · Department of Metabolic Regulation, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan. · J Clin Invest. · Pubmed #18188449 links to  free full text

Abstract: Transgenic mice expressing HCV core protein develop hepatic steatosis and hepatocellular carcinoma (HCC), but the mechanism underlying this process remains unclear. Because PPARalpha is a central regulator of triglyceride homeostasis and mediates hepatocarcinogenesis in rodents, we determined whether PPARalpha contributes to HCV core protein-induced diseases. We generated PPARalpha-homozygous, -heterozygous, and -null mice with liver-specific transgenic expression of the core protein gene (Ppara(+/+):HCVcpTg, Ppara(+/-):HCVcpTg, and Ppara(-/-):HCVcpTg mice. Severe steatosis was unexpectedly observed only in Ppara(+/+):HCVcpTg mice, which resulted from enhanced fatty acid uptake and decreased mitochondrial beta-oxidation due to breakdown of mitochondrial outer membranes. Interestingly, HCC developed in approximately 35% of 24-month-old Ppara(+/+):HCVcpTg mice, but tumors were not observed in the other genotypes. These phenomena were found to be closely associated with sustained PPARalpha activation. In Ppara(+/-):HCVcpTg mice, PPARalpha activation and the related changes did not occur despite the presence of a functional Ppara allele. However, long-term treatment of these mice with clofibrate, a PPARalpha activator, induced HCC with mitochondrial abnormalities and hepatic steatosis. Thus, our results indicate that persistent activation of PPARalpha is essential for the pathogenesis of hepatic steatosis and HCC induced by HCV infection.

Shan W, Nicol CJ, Ito S, Bility MT, Kennett MJ, Ward JM, Gonzalez FJ, Peters JM. · Department of Veterinary and Biomedical Sciences and The Center for Molecular Toxicology and Carcinogenesis, The Huck Institute of Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA. · Hepatology. · Pubmed #18038451 No free full text.

Abstract: Potential functional roles for the peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) in skeletal muscle fatty acid catabolism and epithelial carcinogenesis have recently been described. Whereas PPARbeta/delta is expressed in liver, its function in this tissue is less clear. To determine the role of PPARbeta/delta in chemically induced liver toxicity, wild-type and PPARbeta/delta-null mice were treated with azoxymethane (AOM) and markers of liver toxicity examined. Bile duct hyperplasia, regenerative hyperplasia, and increased serum alanine aminotransferase (ALT) were found in AOM-treated PPARbeta/delta-null mice, and these effects were not observed in similarly treated wild-type mice. Exacerbated carbon tetrachloride (CCl(4)) hepatoxicity was also observed in PPARbeta/delta-null as compared with wild-type mice. No differences in messenger RNAs (mRNAs) encoding cytochrome2E1 required for the metabolic activation of AOM and CCl(4) were observed between wild-type or PPARbeta/delta-null mice in response to CCl(4). Significant differences in the expression of genes reflecting enhanced nuclear factor kappa B (NF-kappaB) activity were noted in PPARbeta/delta-null mice. Conclusion: Results from these studies show that PPARbeta/delta is protective against liver toxicity induced by AOM and CCl(4), suggesting that this receptor is hepatoprotective against environmental chemicals that are metabolized in this tissue.

Yang Q, Nagano T, Shah Y, Cheung C, Ito S, Gonzalez FJ. · Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. · Toxicol Sci. · Pubmed #17690133 links to  free full text

Abstract: To determine the impact of the species difference between rodents and humans in response to peroxisome proliferators (PPs) mediated by peroxisome proliferator-activated receptor (PPAR)alpha, PPAR alpha-humanized transgenic mice were generated using a P1 phage artificial chromosome (PAC) genomic clone bred onto a ppar alpha-null mouse background, designated hPPAR alpha PAC. In hPPAR alpha PAC mice, the human PPAR alpha gene is expressed in tissues with high fatty acid catabolism and induced upon fasting, similar to mouse PPAR alpha in wild-type (Wt) mice. Upon treatment with the PP fenofibrate, hPPAR alpha PAC mice exhibited responses similar to Wt mice, including peroxisome proliferation, lowering of serum triglycerides, and induction of PPAR alpha target genes encoding enzymes involved in fatty acid metabolism in liver, kidney, and heart, suggesting that human PPAR alpha (hPPAR alpha) functions in the same manner as mouse PPAR alpha in regulating fatty acid metabolism and lowering serum triglycerides. However, in contrast to Wt mice, treatment of hPPAR alpha PAC mice with fenofibrate did not cause significant hepatomegaly and hepatocyte proliferation, thus indicating that the mechanisms by which PPAR alpha affects lipid metabolism are distinct from the hepatocyte proliferation response, the latter of which is only induced by mouse PPAR alpha. In addition, a differential regulation of several genes, including the oncogenic let-7C miRNA by PPs, was observed between Wt and hPPAR alpha PAC mice that may contribute to the inherent difference between mouse and human PPAR alpha in activation of hepatocellular proliferation. The hPPAR alpha PAC mouse model provides an in vivo platform to investigate the species difference mediated by PPAR alpha and an ideal model for human risk assessment PPs exposure.

Miyata M, Tozawa A, Otsuka H, Nakamura T, Nagata K, Gonzalez FJ, Yamazoe Y. · Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai, 980-8578, Japan. · J Pharmacol Exp Ther. · Pubmed #15466244 links to  free full text

Abstract: Mice lacking the farnesoid X receptor (FXR) involved in the maintenance of hepatic bile acid levels are highly sensitive to cholic acid-induced liver toxicity. Serum aspartate aminotransferase (AST) activity was elevated 15.7-fold after feeding a 0.25% cholic acid diet, whereas only slight increases in serum AST (1.7- and 2.5-fold) were observed in wild-type mice fed 0.25 and 1% cholic acid diet, respectively. Bile salt export pump mRNA and protein levels were increased in wild-type mice fed 1% cholic acid diet (2.1- and 3.0-fold) but were decreased in FXR-null mice fed 0.25% cholic acid diet. The bile acid output rate was 2.0- and 3.7-fold higher after feeding of 0.25 and 1.0% cholic acid diet in wild-type mice, respectively. On the other hand, no significant increase in bile acid output rate was observed in FXR-null mice fed 0.25% cholic acid diet in contrast to a significant decrease observed in mice fed a 1.0% cholic acid diet in spite of the markedly higher levels of hepatic tauro-conjugated bile acids. Unconjugated cholic acid was not detected in the bile of wild-type mice fed a control diet, but it was readily detected in wild-type mice fed 1% cholic acid diet. The ratio of biliary unconjugated cholic acid to total cholic acid (unconjugated cholic acid and tauro-conjugated cholic acid) reached 30% under conditions of hepatic taurine depletion. These results suggest that the cholic acid-induced enhancement of canalicular bile acid output rates and excretion of unconjugated bile acids are involved in adaptive responses for prevention of cholic acid-induced toxicity.

Leclercq IA, Farrell GC, Field J, Bell DR, Gonzalez FJ, Robertson GR. · Storr Liver Unit, Department of Medicine and Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead NSW 2145, Australia. · J Clin Invest. · Pubmed #10772651 links to  free full text

Abstract: Nonalcoholic steatohepatitis (NASH) and alcoholic liver disease have similar pathological features. Because CYP2E1 plays a key role in alcoholic liver disease with its ability to stimulate lipid peroxidation, we tested the proposal that CYP2E1 could also be a factor in the development of NASH. In a dietary model - mice fed a methionine- and choline-deficient (MCD) diet - liver injury was associated with both induction of CYP2E1 and a 100-fold increase in hepatic content of lipid peroxides. Microsomal NADPH-dependent lipid oxidases contributed to the formation of these lipid peroxides, and in vitro inhibition studies demonstrated that CYP2E1 was the major catalyst. To further define the role of CYP2E1 as an initiator of oxidative stress in NASH, Cyp2e1(-/-)mice were administered the MCD diet. CYP2E1 deficiency neither prevented the development of NASH nor abrogated the increased microsomal NADPH-dependent lipid peroxidation, indicating the operation of a non-CYP2E1 peroxidase pathway. In Cyp2e1(-/-) mice with NASH (but not in wild-type mice), CYP4A10 and CYP4A14 were upregulated. Furthermore, hepatic microsomal lipid peroxidation was substantially inhibited by anti-mouse CYP4A10 antibody in vitro. These results show that experimental NASH is strongly associated with hepatic microsomal lipid peroxidation. CYP2E1, the main enzyme associated with that process in wild-type mice, is not unique among P450 proteins in catalyzing peroxidation of endogenous lipids. We have now identified CYP4A enzymes as alternative initiators of oxidative stress in the liver.

Kono H, Bradford BU, Yin M, Sulik KK, Koop DR, Peters JM, Gonzalez FJ, McDonald T, Dikalova A, Kadiiska MB, Mason RP, Thurman RG. · Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina, Chapel Hill 27599, North Carolina. · Am J Physiol. · Pubmed #10600824 links to  free full text

Abstract: The continuous intragastric enteral feeding protocol in the rat was a major development in alcohol-induced liver injury (ALI) research. Much of what has been learned to date involves inhibitors or nutritional manipulations that may not be specific. Knockout technology avoids these potential problems. Therefore, we used long-term intragastric cannulation in mice to study early ALI. Reactive oxygen species are involved in mechanisms of early ALI; however, their key source remains unclear. Cytochrome P-450 (CYP)2E1 is induced predominantly in hepatocytes by ethanol and could be one source of reactive oxygen species leading to liver injury. We aimed to determine if CYP2E1 was involved in ALI by adapting the enteral alcohol (EA) feeding model to CYP2E1 knockout (-/-) mice. Female CYP2E1 wild-type (+/+) or -/- mice were given a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. All mice gained weight steadily over 4 wk, and there were no significant differences between groups. There were also no differences in ethanol elimination rates between CYP2E1 +/+ and -/- mice after acute ethanol administration to naive mice or mice receiving EA for 4 wk. However, EA stimulated rates 1.4-fold in both groups. EA elevated serum aspartate aminotransferase levels threefold to similar levels over control in both CYP2E1 +/+ and -/- mice. Liver histology was normal in control groups. In contrast, mice given ethanol developed mild steatosis, slight inflammation, and necrosis; however, there were no differences between the CYP2E1 +/+ and -/- groups. Chronic EA induced other CYP families (CYP3A, CYP2A12, CYP1A, and CYP2B) to the same extent in CYP2E1 +/+ and -/- mice. Furthermore, POBN radical adducts were also similar in both groups. Data presented here are consistent with the hypothesis that oxidants from CYP2E1 play only a small role in mechanisms of early ALI in mice. Moreover, this new mouse model illustrates the utility of knockout technology in ALI research.

Guidotti LG, Eggers CM, Raney AK, Chi SY, Peters JM, Gonzalez FJ, McLachlan A. · Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. · J Virol. · Pubmed #10559356 links to  free full text

Abstract: The role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARalpha. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARalpha but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice.