Comparative analysis of the relationship between trichloroethylene metabolism and tissue-specific toxicity among inbred mouse strains: liver effects.

Trichloroethylene (TCE) is a widely used organic solvent. Although TCE is classified as carcinogenic to humans, substantial gaps remain in our understanding of interindividual variability in TCE metabolism and toxicity, especially in the liver. A hypothesis was tested that amounts of oxidative metabolites of TCE in mouse liver are associated with hepatic-specific toxicity. Oral dosing with TCE was conducted in subacute (600 mg/kg/d; 5 d; 7 inbred mouse strains) and subchronic (100 or 400 mg/kg/d; 1, 2, or 4 wk; 2 inbred mouse strains) designs. The quantitative relationship was evaluated between strain-, dose-, and time-dependent formation of TCE metabolites from cytochrome P-450-mediated oxidation (trichloroacetic acid [TCA], dichloroacetic acid [DCA], and trichloroethanol) and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione] in serum and liver, and various hepatic toxicity phenotypes. In subacute study, interstrain variability in TCE metabolite amounts was observed in serum and liver. No marked induction of Cyp2e1 protein levels in liver was detected. Serum and hepatic levels of TCA and DCA were correlated with increased transcription of peroxisome proliferator-marker genes Cyp4a10 and Acox1 but not with degree of induction in hepatocellular proliferation. In subchronic study, serum and liver levels of oxidative metabolites gradually decreased over time despite continuous dosing. Hepatic protein levels of CYP2E1, ADH, and ALDH2 were unaffected by treatment with TCE. While the magnitude of induction of peroxisome proliferator-marker genes also declined, hepatocellular proliferation increased. This study offers a unique opportunity to provide a scientific data-driven rationale for some of the major assumptions in human health assessment of TCE.

Comparative analysis of the relationship between trichloroethylene metabolism and tissue-specific toxicity among inbred mouse strains: kidney effects.

Trichloroethylene (TCE) is a well-known environmental and occupational toxicant that is classified as carcinogenic to humans based on the epidemiological evidence of an association with higher risk of renal-cell carcinoma. A number of scientific issues critical for assessing human health risks from TCE remain unresolved, such as the amount of kidney-toxic glutathione conjugation metabolites formed, interspecies and interindividual differences, and the mode of action for kidney carcinogenicity. It was postulated that TCE renal metabolite levels are associated with kidney-specific toxicity. Oral dosing with TCE was conducted in subacute (600 mg/kg/d; 5 d; 7 inbred mouse strains) and subchronic (100 or 400 mg/kg/d; 1, 2, or 4 wk; 2 inbred mouse strains) designs. The quantitative relationship was evaluated between strain-, dose, and time-dependent formation of TCE metabolites from cytochrome P-450-mediated oxidation (trichloroacetic acid [TCA], dichloroacetic acid [DCA], and trichloroethanol) and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione], and various kidney toxicity phenotypes. In subacute study, interstrain differences in renal TCE metabolite levels were observed. In addition, data showed that in several strains kidney-specific effects of TCE included induction of peroxisome proliferator-marker genes Cyp4a10 and Acox1, increased cell proliferation, and expression of KIM-1, a marker of tubular damage and regeneration. In subchronic study, peroxisome proliferator-marker gene induction and renal toxicity diminished while cell proliferative response was elevated in a dose-dependent manner in NZW/LacJ but not C57BL/6J mice. Overall, data demonstrated that renal TCE metabolite levels are associated with kidney-specific toxicity and that these effects are strain dependent.

Acetaminophen-induced acute liver injury in HCV transgenic mice.

The exact etiology of clinical cases of acute liver failure is difficult to ascertain and it is likely that various co-morbidity factors play a role. For example, epidemiological evidence suggests that coexistent hepatitis C virus (HCV) infection increased the risk of acetaminophen-induced acute liver injury, and was associated with an increased risk of progression to acute liver failure. However, little is known about possible mechanisms of enhanced acetaminophen hepatotoxicity in HCV-infected subjects. In this study, we tested a hypothesis that HCV-Tg mice may be more susceptible to acetaminophen hepatotoxicity, and also evaluated the mechanisms of acetaminophen-induced liver damage in wild type and HCV-Tg mice expressing core, E1 and E2 proteins. Male mice were treated with a single dose of acetaminophen (300 or 500 mg/kg in fed animals; or 200 mg/kg in fasted animals; i.g.) and liver and serum endpoints were evaluated at 4 and 24h after dosing. Our results suggest that in fed mice, liver toxicity in HCV-Tg mice is not markedly exaggerated as compared to the wild-type mice. In fasted mice, greater liver injury was observed in HCV-Tg mice. In fed mice dosed with 300 mg/kg acetaminophen, we observed that liver mitochondria in HCV-Tg mice exhibited signs of dysfunction showing the potential mechanism for increased susceptibility.

Glycine reduces platelet aggregation.

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl(-) influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1-10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.

Increased incidence of aflatoxin B1-induced liver tumors in hepatitis virus C transgenic mice.

Viral hepatitis and aflatoxin B1 (AFB1) exposure are common risk factors for hepatocellular carcinoma (HCC). The incidence of HCC in individuals coexposed to hepatitis C (HCV) or B virus and AFB1 is greater than could be explained by the additive effect; yet, the mechanisms are poorly understood because of the lack of an animal model. Our study investigated the outcomes and mechanisms of combined exposure to HCV and AFB1. We hypothesized that HCV transgenic (HCV-Tg; expressing core, E1, E2 and p7, nucleotides 342-2771) mice will be prone to hepatocarcinogenesis when exposed to AFB1. Neonatal (7 days old) HCV-Tg or C57BL/6J wild-type (WT) mice were exposed to AFB1 (6 µg/g bw) or tricaprylin vehicle (15 µl/g bw), and male offspring were followed for up to 12 months. No liver lesions were observed in vehicle-treated WT or HCV-Tg mice. Tumors (adenomas or carcinomas) and preneoplastic lesions (hyperplasia or foci) were observed in 22.5% (9 of 40) of AFB1-treated WT mice. In AFB1-treated HCV-Tg mice, the incidence of tumorous or pretumorous lesions was significantly elevated (50%, 18 of 36), with the difference largely due to a 2.5-fold increase in the incidence of adenomas (30.5 vs. 12.5%). Although oxidative stress and steatohepatitis were observed in both AFB1-treated groups, molecular changes indicative of the enhanced inflammatory response and altered lipid metabolism were more pronounced in HCV-Tg mice. In summary, HCV proteins core, E1, E2 and p7 are sufficient to reproduce the cocarcinogenic effect of HCV and AFB1, which is a known clinical phenomenon.

Sex-specific gene expression in the BXD mouse liver.

Differences in clinical phenotypes between the sexes are well documented and have their roots in differential gene expression. While sex has a major effect on gene expression, transcription is also influenced by complex interactions between individual genetic variation and environmental stimuli. In this study, we sought to understand how genetic variation affects sex-related differences in liver gene expression by performing genetic mapping of genomewide liver mRNA expression data in a genetically defined population of naive male and female mice from C57BL/6J, DBA/2J, B6D2F1, and 37 C57BL/6J x DBA/2J (BXD) recombinant inbred strains. As expected, we found that many genes important to xenobiotic metabolism and other important pathways exhibit sexually dimorphic expression. We also performed gene expression quantitative trait locus mapping in this panel and report that the most significant loci that appear to regulate a larger number of genes than expected by chance are largely sex independent. Importantly, we found that the degree of correlation within gene expression networks differs substantially between the sexes. Finally, we compare our results to a recently released human liver gene expression data set and report on important similarities in sexually dimorphic liver gene expression between mouse and human. This study enhances our understanding of sex differences at the genome level and between species, as well as increasing our knowledge of the molecular underpinnings of sex differences in responses to xenobiotics.

Evaluation of an in vitro toxicogenetic mouse model for hepatotoxicity.

Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to determine whether viable cells may be isolated from different mouse inbred strains, evaluate the reproducibility of cell yield, viability and functionality over subsequent isolations, and assess the utility of the model for toxicity screening. Hepatocytes were isolated from 15 strains of mice (A/J, B6C3F1, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, BALB/cByJ, AKR/J, MRL/MpJ, NOD/LtJ, NZW/LacJ, PWD/PhJ and WSB/EiJ males) and cultured for up to 7 days in traditional 2-dimensional culture. Cells from B6C3F1, C57BL/6J, and NOD/LtJ strains were treated with acetaminophen, WY-14,643 or rifampin and concentration-response effects on viability and function were established. Our data suggest that high yield and viability can be achieved across a panel of strains. Cell function and expression of key liver-specific genes of hepatocytes isolated from different strains and cultured under standardized conditions are comparable. Strain-specific responses to toxicant exposure have been observed in cultured hepatocytes and these experiments open new opportunities for further developments of in vitro models of hepatotoxicity in a genetically diverse population.

Time-course comparison of xenobiotic activators of CAR and PPARalpha in mouse liver.

Constitutive androstane receptor (CAR) and peroxisome proliferator activated receptor (PPAR)alpha are transcription factors known to be primary mediators of liver effects, including carcinogenesis, by phenobarbital-like compounds and peroxisome proliferators, respectively, in rodents. Many similarities exist in the phenotypes elicited by these two classes of agents in rodent liver, and we hypothesized that the initial transcriptional responses to the xenobiotic activators of CAR and PPARalpha will exhibit distinct patterns, but at later time-points these biological pathways will converge. In order to capture the global transcriptional changes that result from activation of these nuclear receptors over a time-course in the mouse liver, microarray technology was used. First, differences in basal expression of liver genes between C57Bl/6J wild-type and Car-null mice were examined and 14 significantly differentially expressed genes were identified. Next, mice were treated with phenobarbital (100 mg/kg by gavage for 24 h, or 0.085% w/w diet for 7 or 28 days), and liver gene expression changes with regards to both time and treatment were identified. While several pathways related to cellular proliferation and metabolism were affected by phenobarbital in wild-type mice, no significant changes in gene expression were found over time in the Car-nulls. Next, we determined commonalities and differences in the temporal response to phenobarbital and WY-14,643, a prototypical activator of PPAR alpha. Gene expression signatures from livers of wild-type mice C57Bl6/J mice treated with PB or WY-14,643 were compared. Similar pathways were affected by both compounds; however, considerable time-related differences were present. This study establishes common gene expression fingerprints of exposure to activators of CAR and PPARalpha in rodent liver and demonstrates that despite similar phenotypic changes, molecular pathways differ between classes of chemical carcinogens.

Metabolomic profiling of a modified alcohol liquid diet model for liver injury in the mouse uncovers new markers of disease.

Metabolomic evaluation of urine and liver was conducted to assess the biochemical changes that occur as a result of alcohol-induced liver injury. Male C57BL/6J mice were fed an isocaloric control- or alcohol-containing liquid diet with 35% of calories from corn oil, 18% protein and 47% carbohydrate/alcohol for up to 36 days ad libitum. Alcohol treatment was initiated at 7 g/kg/day and gradually reached a final dose of 21 g/kg/day. Urine samples were collected at 22, 30 and 36 days and, in additional treatment groups, liver and serum samples were harvested at 28 days. Steatohepatitis was induced in the alcohol-fed group since a 5-fold increase in serum alanine aminotransferase activity, a 6-fold increase in liver injury score (necrosis, inflammation and steatosis) and an increase in lipid peroxidation in liver were observed. Liver and urine samples were analyzed by nuclear magnetic resonance spectroscopy and electrospray infusion/Fourier transform ion cyclotron resonance-mass spectrometry. In livers of alcohol-treated mice the following changes were noted. Hypoxia and glycolysis were activated as evidenced by elevated levels of alanine and lactate. Tyrosine, which is required for l-DOPA and dopamine as well as thyroid hormones, was elevated possibly reflecting alterations of basal metabolism by alcohol. A 4-fold increase in the prostacyclin inhibitor 7,10,13,16-docosatetraenoic acid, a molecule important for regulation of platelet formation and blood clotting, may explain why chronic drinking causes serious bleeding problems. Metabolomic analysis of the urine revealed that alcohol treatment leads to decreased excretion of taurine, a metabolite of glutathione, and an increase in lactate, n-acetylglutamine and n-acetylglycine. Changes in the latter two metabolites suggest an inhibition of the kidney enzyme aminoacylase I and may be useful as markers for alcohol consumption.

Sustained formation of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts in mouse liver by peroxisome proliferators is dependent upon peroxisome proliferator-activated receptor-alpha, but not NADPH oxidase.

Reactive oxygen species are thought to be crucial for peroxisome proliferator-induced liver carcinogenesis. Free radicals have been shown to mediate the production of mitogenic cytokines by Kupffer cells and cause DNA damage in rodent liver. Previous in vivo experiments demonstrated that acute administration of the peroxisome proliferator di(2-ethylhexyl) phthalate (DEHP) led to an increase in production of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) radical adducts in liver, an event that was dependent on Kupffer cell NADPH oxidase, but not peroxisome proliferator-activated receptor (PPAR)alpha. Here, we hypothesized that continuous treatment with peroxisome proliferators will cause a sustained formation in POBN radical adducts in liver. Mice were fed diets containing either 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14,643, 0.05% w/w) or DEHP (0.6% w/w) for up to 3 weeks. Liver-derived radical production was assessed in bile samples by measuring POBN radical adducts using electron spin resonance. Our data indicate that WY-14,643 causes a sustained increase in POBN radical adducts in mouse liver and that this effect is greater than that of DEHP. To understand the molecular source of these radical species, NADPH oxidase-deficient (p47phox-null) and PPARalpha-null mice were examined after treatment with WY-14,643. No increase in radicals was observed in PPARalpha-null mice that were treated with WY-14,643 for 3 weeks, while the response in p47phox-nulls was similar to that of wild-type mice. These results show that PPARalpha, not NADPH oxidase, is critical for a sustained increase in POBN radical production caused by peroxisome proliferators in rodent liver. Therefore, peroxisome proliferator-induced POBN radical production in Kupffer cells may be limited to an acute response to these compounds in mouse liver.

WY-14,643 induced cell proliferation and oxidative stress in mouse liver are independent of NADPH oxidase.

Long-term exposure of rodents to peroxisome proliferators leads to increases in peroxisomes, hepatocellular proliferation, oxidative damage, suppressed apoptosis, and ultimately results in the development of hepatic adenomas and carcinomas. Peroxisome proliferators-activated receptor (PPAR)alpha was shown to be required for these pleiotropic responses; however, Kupffer cells, resident liver macrophages, were also identified as playing a role in peroxisome proliferators-induced effects, independently of PPARalpha. Previous studies showed that oxidants from NADPH (nicotinamide adenine dinucleotide phosphate, reduced) oxidase mediate acute effects of peroxisome proliferators in rodent liver. To determine if Kupffer cell oxidants are also involved in chronic effects, NADPH oxidase-deficient (p47(phox)-null) mice were fed 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14,643)-containing diet (0.1% wt/wt) for 1 week, 5 weeks, or 5 months along with Pparalpha-null and wild type mice. As expected, no change in liver size, cell replication rates, or other phenotypic effects of peroxisome proliferators were observed in Pparalpha-null mice. Through 5 months of treatment, the p47(phox)-null and wild type mice exhibited peroxisome proliferators-induced adverse liver effects, along with increased oxidative DNA damage and increased cell proliferation, a response that is potentially mediated through nuclear factor kappa B (NFkB). Suppressed apoptosis caused by WY-14,643 was dependent on both NADPH oxidase and PPARalpha. Collectively, these findings suggest that involvement of Kupffer cells in WY-14,643-induced parenchymal cell proliferation and oxidative stress in rodent liver is an acute phenomenon that is not relevant to long-term exposure, but they are still involved in chronic apoptotic responses. These results provide new insight for understanding the mode of hepatocarcinogenic action of peroxisome proliferators.

Multicenter study of acetaminophen hepatotoxicity reveals the importance of biological endpoints in genomic analyses.

Gene expression profiling is a widely used technique with data from the majority of published microarray studies being publicly available. These data are being used for meta-analyses and in silico discovery; however, the comparability of toxicogenomic data generated in multiple laboratories has not been critically evaluated. Using the power of prospective multilaboratory investigations, seven centers individually conducted a common toxicogenomics experiment designed to advance understanding of molecular pathways perturbed in liver by an acute toxic dose of N-acetyl-p-aminophenol (APAP) and to uncover reproducible genomic signatures of APAP-induced toxicity. The nonhepatotoxic APAP isomer N-acetyl-m-aminophenol was used to identify gene expression changes unique to APAP. Our data show that c-Myc is induced by APAP and that c-Myc-centered interactomes are the most significant networks of proteins associated with liver injury. Furthermore, sources of error and data variability among Centers and methods to accommodate this variability were identified by coupling gene expression with extensive toxicological evaluation of the toxic responses. We show that phenotypic anchoring of gene expression data is required for biologically meaningful analysis of toxicogenomic experiments.

Rates of ethanol metabolism decrease in sons of alcoholics following a priming dose of ethanol.

Rapid changes in rates of ethanol metabolism in response to acute ethanol administration have been observed in animals and humans. To examine whether this phenomenon might vary by risk for alcoholism, 23 young men with a positive family history of alcoholism (family history positive [FHP]) were compared to 15 young men without a family history of alcoholism (family history negative [FHN]). Rates of ethanol metabolism were measured in all subjects first after an initial ethanol dose (0.85 g/kg) and then, several hours later, a second dose (0.3 g/kg), and the two rates were compared. The two groups of subjects were similar in their histories of ethanol consumption. FHP subjects demonstrated faster initial rates of ethanol metabolism, 148+/-36 mg/kg/h, compared to FHN subjects, 124+/-18 mg/kg/h, P=.01. However, FHN subjects increased their rate of metabolism by 10+/-27% compared to a decrease of -15+/-24% in FHP subjects, P=.007. Fifty-two percent of the FHP and none of the FHN subjects exhibited a decline in metabolic rate of 20% or more, P=.0008. Since a significant proportion of FHP subjects exhibited a decrease in the second rate of ethanol metabolism, these preliminary data might help to partly explain why FHP individuals differ in their sensitivity to ethanol and are more likely to develop alcohol dependence.

Expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemical-induced chronic oxidative stress: identification of the molecular source of radicals responsible for DNA damage by peroxisome proliferators.

Oxidative stress to DNA is recognized as one of the mechanisms for the carcinogenic effects of some environmental agents. Numerous studies have been conducted in an attempt to document the fact that chemical carcinogens that are thought to induce production of oxidants also cause the formation of oxidative DNA lesions. Although many DNA adducts continue to be useful biomarkers of dose/effect, changes in gene expression have been proposed to be a practical novel tool for studying the role of chemically induced oxidative DNA damage. Here, we hypothesized that expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemically induced chronic oxidative stress. To test this hypothesis, mice were treated with a known rodent carcinogen and peroxisome proliferator, WY-14,643 (500 ppm, 1 month). A number of end points that are commonly used to assess oxidative DNA damage were considered. Our data demonstrate that no difference in 8-oxoguanine, the number of abasic sites, or single strand breaks can be detected in genomic DNA from livers of control or WY-treated animals. However, a concordant marked induction of genes specific for the long-patch base excision DNA repair, a predominant pathway that removes oxidized DNA lesions in vivo, was observed in livers of WY-treated mice. Kupffer cell NADPH oxidase, and peroxisomes in parenchymal cells have been proposed as the potential sources of peroxisome proliferator-induced oxidants. The analysis of expression of base excision DNA repair genes was used to assess whether this biomarker of oxidative stress can be used to determine the source of oxidants. The data suggest that DNA-damaging oxidants are generated by enzymes that are induced after activation of peroxisome proliferator activator receptor alpha, such as those involved in lipid metabolism in peroxisomes, and are not the result of activation of NADPH oxidase in Kupffer cells. We conclude that expression of base excision DNA repair genes is a sensitive in vivo biomarker for chemically induced oxidative stress to DNA that can be successfully used for the identification of the molecular source of radicals responsible for DNA damage in vivo.

Swift increase in alcohol metabolism (SIAM): understanding the phenomenon of hypermetabolism in liver.

The swift increase in alcohol metabolism (SIAM) is a phenomenon defined as a rapid increase in hepatic respiration and alcohol metabolism after administration of a bolus dose of alcohol. Continuous exposure to alcohol is known to produce adaptive changes in liver alcohol and oxygen metabolism. A considerable burst of hepatic respiration can also occur after administration of a single large dose of alcohol and results in a near doubling of alcohol metabolism, a high demand for oxygen, and downstream or pericentral hypoxia. These dramatic changes in rates of alcohol metabolism and tissue concentrations of oxygen are not due to induced enzyme activity in liver. This phenomenon depends on activation of mitochondrial function, an increase in co-factor supply for nicotinamide adenine dinucleotide-dependent alcohol metabolism, depletion of glycogen reserves, liberation of fatty acids through activation of an adrenergic response to alcohol providing substrate for catalase, and activation of Kupffer cells, the hepatic resident macrophages responsible for production of cytokines and prostaglandins. An understanding of the mechanisms of hypermetabolism in liver can have vital ramifications for knowledge of both alcohol-related and alcohol-unrelated liver injury because hypoxia that is a result of hypermetabolism can compound effects of pharmaceuticals and environmental agents on the liver. Swift increase in alcohol metabolism is an excellent example of the complexity of cell-cell interactions in liver and extrahepatic regulation of biochemical and molecular events in this organ, and this important phenomenon shall be considered in studies of liver disease and biochemistry.

The CYP inhibitor 1-aminobenzotriazole does not prevent oxidative stress associated with alcohol-induced liver injury in rats and mice.

Cytochrome P450 (CYP) 2E1 is induced by ethanol and is postulated to be a source of reactive oxygen species during alcoholic liver disease. However, there was no difference in liver pathology and radical formation between wild-type and CYP2E1 knockout mice fed ethanol. Other CYP isoforms may contribute these effects if CYP2E1 is inhibited or absent. The purpose of this study was, therefore, to determine if blocking most of the P450 isoforms with 1-aminobenzotriazole (ABT; 100 mg/kg i.g.), has any effect on liver damage and oxidative stress due to alcohol in rats and mice. Male C57BL/6 mice and Wistar rats were fed either high-fat control or ethanol-containing enteral diet for 4 weeks. ABT had a significant inhibitory effect on many P450 isoforms independent of concomitant alcohol administration. However, ABT did not protect against liver damage due to alcohol in either species. Indices of oxidative stress and inflammation were also similar in livers from vehicle-treated and ABT-treated animals fed ethanol. In summary, suppression of P450 activity with ABT had no apparent effect on oxidative stress caused by alcohol in both rats and mice. These data support the hypothesis that oxidative stress and liver damage can occur independently of CYP activities in both rats and mice during early alcohol-induced liver injury.

The role of gut-derived bacterial toxins and free radicals in alcohol-induced liver injury.

Previous research from this laboratory using a continuous enteral ethanol (EtOH) administration model demonstrated that Kupffer cells are pivotal in the development of EtOH-induced liver injury. When Kupffer cells were destroyed using gadolinium chloride (GdCl3 ) or the gut was sterilized with polymyxin B and neomycin, early inflammation due to EtOH was blocked. Anti-tumour necrosis factor (TNF)-α antibody markedly decreased EtOH-induced liver injury and increased TNF-mRNA. These findings led to the hypothesis that EtOH-induced liver injury involves increases in circulating endotoxin leading to activation of Kupffer cells. Pimonidazole, a nitro-imidazole marker, was used to detect hypoxia in downstream pericentral regions of the lobule. Following one large dose of EtOH or chronic enteral EtOH for 1 month, pimonidazole binding was increased significantly in pericentral regions of the liver lobule, which was diminished with GdCl3 . Enteral EtOH increased free radical generation detected with electron spin resonance (ESR). These radical species had coupling constants matching α-hydroxyethyl radical and were shown conclusively to arise from EtOH based on a doubling of the ESR lines when 13 C-EtOH was given. α-Hydroxyethyl radical production was also blocked by the destruction of Kupffer cells with GdCl3 . It is known that females develop more severe EtOH-induced liver injury more rapidly and with less EtOH than males. Female rats on the enteral protocol exhibited more rapid injury and more widespread fatty changes over a larger portion of the liver lobule than males. Plasma endotoxin, ICAM-1, free radical adducts, infiltrating neutrophils and transcription factor NFκB were approximately two-fold greater in livers from females than males after 4 weeks of enteral EtOH treatment. Furthermore, oestrogen treatment increased the sensitivity of Kupffer cells to endotoxin. These data are consistent with the hypothesis that Kupffer cells participate in important gender differences in liver injury caused by ethanol.

Molecular mechanisms of fibrosis-associated promotion of liver carcinogenesis.

Hepatocellular carcinoma (HCC) mostly develops in patients with advanced fibrosis; however, the mechanisms of interaction between a genotoxic insult and fibrogenesis are not well understood. This study tested a hypothesis that fibrosis promotes HCC via a mechanism that involves activation of liver stem cells. First, B6C3F1 mice were administered diethylnitrosamine (DEN; single ip injection of 1mg/kg at 14 days of age). Second, carbon tetrachloride (CCl(4); 0.2ml/kg, 2/week ip starting at 8 weeks of age) was administered for 9 or 14 weeks to develop advanced liver fibrosis. In animals treated with DEN as neonates, presence of liver fibrosis led to more than doubling (to 100%) of the liver tumor incidence as early as 5 months of age. This effect was associated with activation of cells with progenitor features in noncancerous liver tissue, including markers of replicative senescence (p16), oncofetal transformation (Afp, H19, and Bex1), and increased "stemness" (Prom1 and Epcam). In contrast, the dose of DEN used did not modify the extent of liver inflammation, fibrogenesis, oxidative stress, proliferation, or apoptosis induced by subchronic CCl(4) administration. This study demonstrates the potential role of liver stem-like cells in the mechanisms of chemical-induced, fibrosis-promoted HCC. We posit that the combination of genotoxic and fibrogenic insults is a sensible approach to model liver carcinogenesis in experimental animals. These results may contribute to identification of cirrhotic patients predisposed to HCC by analyzing the expression of hepatic progenitor cell markers in the noncancerous liver tissue.

Interstrain differences in the liver effects of trichloroethylene in a multistrain panel of inbred mice.

Trichloroethylene (TCE) is a widely used industrial chemical and a common environmental contaminant. It is a well-known carcinogen in rodents and a probable carcinogen in humans. Studies utilizing panels of mouse inbred strains afford a unique opportunity to understand both metabolic and genetic basis for differences in responses to TCE. We tested the hypothesis that strain- and liver-specific toxic effects of TCE are genetically controlled and that the mechanisms of toxicity and susceptibility can be uncovered by exploring responses to TCE using a diverse panel of inbred mouse strains. TCE (2100 mg/kg) or corn oil vehicle was administered by gavage to 6- to 8-week-old male mice of 15 mouse strains. Serum and liver were collected at 2, 8, and 24 h postdosing and were analyzed for TCE metabolites, hepatocellular injury, and gene expression of liver. TCE metabolism, as evident from the levels of individual oxidative and conjugative metabolites, varied considerably between strains. TCE treatment-specific effect on the liver transcriptome was strongly dependent on genetic background. Peroxisome proliferator-activated receptor-mediated molecular networks, consisting of the metabolism genes known to be induced by TCE, represent some of the most pronounced molecular effects of TCE treatment in mouse liver that are dependent on genetic background. Conversely, cell death, liver necrosis, and immune-mediated response pathways, which are altered by TCE treatment in liver, are largely genetic background independent. These studies provide better understanding of the mechanisms of TCE-induced toxicity anchored on metabolism and genotype-phenotype correlations that may define susceptibility or resistance.

Mechanism for prevention of alcohol-induced liver injury by dietary methyl donors.

Alcohol-induced liver injury (ALI) has been associated with, among other molecular changes, abnormal hepatic methionine metabolism, resulting in decreased levels of S-adenosylmethionine (SAM). Dietary methyl donor supplements such as SAM and betaine mitigate ALI in animal models; however, the mechanisms of protection remain elusive. It has been suggested that methyl donors may act via attenuation of alcohol-induced oxidative stress. We hypothesized that the protective action of methyl donors is mediated by an effect on the oxidative metabolism of alcohol in the liver. Male C57BL/6J mice were administered a control high-fat diet or diet enriched in methyl donors with or without alcohol for 4 weeks using the enteral alcohol feeding model. As expected, attenuation of ALI and an increase in reduced glutathione:oxidized glutathione ratio were achieved with methyl donor supplementation. Interestingly, methyl donors led to a 35% increase in blood alcohol elimination rate, and while there was no effect on alcohol metabolism in the stomach, a profound effect on liver alcohol metabolism was observed. The catalase-dependent pathway of alcohol metabolism was induced, yet the increase in CYP2E1 activity by alcohol was blunted, which may be mitigating production of oxidants. Additional factors contributing to the protective effects of methyl donors in ALI were increased activity of low- and high-K(m) aldehyde dehydrogenases leading to lower hepatic acetaldehyde, maintenance of the efficient mitochondrial energy metabolism, and promotion of peroxisomal beta-oxidation. Profound changes in alcohol metabolism represent additional important mechanism of the protective effect of methyl donors in ALI.