The developing murine lung is susceptible to acetaminophen toxicity.

Acetaminophen (n-acetyl-p-aminophenol, APAP) use in the neonatal intensive care unit is rapidly increasing. Although APAP-related hepatotoxicity is rarely reported in the neonatal literature, other end-organ toxicity can occur with toxic exposures. APAP-induced lung injury has been reported with toxic exposures in adults, but whether this occurs in the developing lung is unknown. Therefore, we tested whether toxic APAP exposures would injure the developing lung. Neonatal C57BL/6 mice (PN7, early alveolar stage of lung development) were exposed to a dose of APAP known to cause hepatotoxicity in adult mice (280 mg/kg, IP). This exposure induced significant lung injury in the absence of identifiable hepatic toxicity. This injury was associated with increased pulmonary expression of Cyp2e1, the xenobiotic enzyme responsible for the toxic conversion of APAP. Exposure was associated with increased pulmonary expression of antioxidant response genes and decreased pulmonary glutathione peroxidase activity level. Furthermore, we observed an increase in pulmonary expression of proinflammatory cytokines and chemokines. Lastly, we were able to demonstrate that this toxic APAP exposure was associated with a shift in pulmonary metabolism away from glycolysis with increased oxidative phosphorylation, a finding consistent with increased mitochondrial workload, potentially leading to mitochondrial toxicity. This previously unrecognized injury and metabolic implications highlight the need to look beyond the liver and evaluate both the acute and long-term pulmonary implications of APAP exposure in the perinatal period.

Benzene-induced mouse hematotoxicity is regulated by a protein phosphatase 2A complex that stimulates transcription of cytochrome P4502E1.

Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hematotoxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene-induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A Aα subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against benzene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation analysis revealed more exhaled benzene but fewer benzene metabolites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening disclosed that PP2A complexes containing the B56α subunit participate in regulating Cyp2e1 expression. Notably, PP2A-B56α suppression in HepG2 cells resulted in persistent ß-catenin phosphorylation at Ser33-Ser37-Thr41 in response to CYP2E1 agonists. In parallel, nuclear translocation of ß-catenin was inhibited, concomitant with a remarkable decrease of Cyp2e1 expression. These findings support the notion that a regulatory cascade comprising PP2A-B56α, ß-catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals.

Transforming growth factor-ß signaling confers hepatic stellate cells progenitor features after partial hepatectomy.

Liver regeneration involves not only hepatocyte replication but progenitor aggregation and scarring. Partial hepatectomy (PH), an established model for liver regeneration, reactivates transforming growth factor-ß (TGF-ß) signaling. Hepatic stellate cells (HSCs) are primarily responding cells for TGF-ß and resident in stem cell niche. In the current study, PH mice were treated with SB-431542, an inhibitor of TGF-ß Type I receptor, aiming to address the role of TGF-ß signaling on the fate determination of HSCs during liver regeneration. After PH, control mice exhibited HSCs activation, progenitor cells accumulation, and a fraction of HSCs acquired the phenotype of hepatocyte or cholangiocyte. Blocking TGF-ß signaling delayed proliferation, impaired progenitor response, and scarring repair. In SB-431542 group, merely no HSCs were found coexpressed progenitor makers, such as SOX9 and AFP. Inhibition of TGF-ß pathway disturbed the epithelial-mesenchymal transitions and diminished the nuclear accumulation of ß-catenin as well as the expression of cytochrome P450 2E1 in HSC during liver regeneration. We identify a key role of TGF-ß signaling on promoting HSC transition, which subsequently becomes progenitor for generating liver epithelial cells after PH. This process might interact with an acknowledged stem cell function signaling, Wnt/ß-catenin.

SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing toxic reactive oxygen species (ROS) and is regulated at the posttranslational level. Small ubiquitin-like modifier (SUMO)ylation is a posttranslational modification that involves the addition of SUMOs, which modulate protein stability, activity, and localization. We demonstrated that ubiquitin-conjugation enzyme 9, the SUMO-conjugating enzyme, is induced in the livers of an intragastric ethanol mouse model. Our aim is to examine whether SUMOylation could regulate ethanol-induced CYP2E1 expression in ALD and to elucidate the molecular mechanism(s). CYP2E1 and UBC9 expression in vitro and in vivo was detected by real-time PCR and immunoblotting/immunostaining. SUMOylation was assayed by mass spectrometry and coimmunoprecipitation. Ubc9 expression was induced in ethanol-fed mouse livers, and silencing inhibited ethanol-mediated CYP2E1 microsomal retention and enzymatic activity. CYP2E1 SUMOylation was found to be induced by ethanol in vitro and in vivo. Ubc9 silencing prevents ethanol-induced lipid accumulation and ROS production. UBC9 was highly expressed in human ALD livers. Finally, we found that lysine 410 is a key SUMOylated residue contributing to CYP2E1 protein stability and activity preventing CYP2E1 SUMOylation. Ethanol-mediated up-regulation of CYP2E1 via SUMOylation enhancing its protein stability and activity and may have important implications in ALD.-Tomasi, M. L., Ramani, K., Ryoo, M., Cossu, C., Floris, A., Murray, B. J., Iglesias-Ara, A., Spissu, Y., Mavila, N. SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

miRNA-122 Protects Mice and Human Hepatocytes from Acetaminophen Toxicity by Regulating Cytochrome P450 Family 1 Subfamily A Member 2 and Family 2 Subfamily E Member 1 Expression.

Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i.p. with an acetaminophen dose not lethal to the control. LKO livers exhibited higher basal expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 family 1 subfamily A member 2 (CYP1A2) that convert acetaminophen to highly reactive N-acetyl-p-benzoquinone imine. Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of aryl hydrocarbon receptor (AHR) and mediator 1 (MED1), two transactivators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE (Encyclopedia of DNA Element) database identified association of CCCTC-binding factor (CTCF) with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1, or Ctcf in Mir122-/- hepatocytes reduced Cyp1a2 expression. Pulse-chase studies found that CYP2E1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to acetaminophen toxicity that correlated with upregulation of AHR, MED1, and CYP1A2 expression. Collectively, these results reveal a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in patients with ALF.

A dual inhibition: microRNA-552 suppresses both transcription and translation of cytochrome P450 2E1.

MicroRNAs (miRNAs) can direct post-transcriptional or transcriptional gene silencing. Here, we report that miR-552 is in the nucleus and cytosol and inhibits human cytochrome P450 (CYP) 2E1 expression at both transcriptional and post-transcriptional levels. MiR-552 via its non-seed sequence forms hybrids with a loop hairpin of the cruciform structure in CYP2E1 promoter region to inhibit SMARCE1 and RNA polymerase II binding to the promoter and CYP2E1 transcription. Expressing SMARCE1 reverses the inhibitory effects of miR-552 on CYP2E1 mRNA expression. MiR-552 with mutations in non-seed region losses its transcriptional, but retains its post-transcriptional repression to CYP2E1. In contrast, mutation in miR-552 seed sequence suppresses its inhibitory effects on CYP2E1 expression at protein, but not at mRNA, levels. Our results suggest that miR-552 is a miRNA with a dual inhibitory ability at transcriptional and post-transcriptional levels leading to an effective inhibition.

Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice.

BACKGROUND: Liver damage is a serious and sometimes fatal consequence of long-term alcohol intake, which progresses from early-stage fatty liver (steatosis) to later-stage steatohepatitis with inflammation and fibrosis/necrosis. However, very little is known about earlier stages of liver disruption that may occur in problem drinkers, those who drink excessively but are not dependent on alcohol. METHODS: We examined how repeated binge-like alcohol drinking in C57BL/6 mice altered liver function, as compared with a single binge-intake session and with repeated moderate alcohol consumption. We measured a number of markers associated with early- and later-stage liver disruption, including liver steatosis, measures of liver cytochrome P4502E1 (CYP2E1) and alcohol dehydrogenase (ADH), alcohol metabolism, expression of cytokine mRNA, accumulation of 4-hydroxynonenal (4-HNE) as an indicator of oxidative stress, and alanine transaminase/aspartate transaminase as a measure of hepatocyte injury. RESULTS: Importantly, repeated binge-like alcohol drinking increased triglyceride levels in the liver and plasma, and increased lipid droplets in the liver, indicators of steatosis. In contrast, a single binge-intake session or repeated moderate alcohol consumption did not alter triglyceride levels. In addition, alcohol exposure can increase rates of alcohol metabolism through CYP2E1 and ADH, which can potentially increase oxidative stress and liver dysfunction. Intermittent, excessive alcohol intake increased liver CYP2E1 mRNA, protein, and activity, as well as ADH mRNA and activity. Furthermore, repeated, binge-like drinking, but not a single binge or moderate drinking, increased alcohol metabolism. Finally, repeated, excessive intake transiently elevated mRNA for the proinflammatory cytokine IL-1B and 4-HNE levels, but did not alter markers of later-stage liver hepatocyte injury. CONCLUSIONS: Together, we provide data suggesting that even relatively limited binge-like alcohol drinking can lead to disruptions in liver function, which might facilitate the transition to more severe forms of liver damage.

Establishment of an alcoholic fatty liver disease model in mice.

BACKGROUND: Alcoholic fatty liver disease (AFLD) defines an important stage in the progression of alcoholic liver disease (ALD), which is a major cause of morbidity and mortality worldwide. OBJECTIVE: To establish a mouse model of AFLD. METHODS: Male C57BL/6 mice were divided into the following two groups: (i) a control group, which was allowed free access to food and water and (ii) an alcohol-treated group, which was administered a 15% (v/v) alcohol solution instead of water. After 8-9 months of treatment, serum biochemical indexes, histopathological changes, liver triglyceride content, iron storage, and ferritin light chain protein expression were measured using an automatic biochemical analyzer, hematoxylin-eosin (HE) staining, a commercially available kit, Prussian blue staining, and Western blot analysis, respectively. RESULTS: Compared with the control group, the alcohol-treated group displayed increased levels of serum LDH, ALT, and AST, decreased levels of ALB, and no significant change in levels of TP. Additionally, increased levels of serum TG, T-CHO, and LDL and decreased levels of serum GLU and HDL were observed in the alcohol-treated mice. HE staining showed that lipid vacuolization occurred in the livers of alcohol-treated mice. The alcohol-treated mice also exhibited increased liver triglyceride content. Moreover, Prussian blue staining and Western blot analysis demonstrated that chronic alcohol administration caused iron overloading of the liver. CONCLUSIONS: Chronic administration of 15% (v/v) alcohol in the drinking water over 8-9 months caused AFLD in mice. Our results establish an AFLD model that represents a promising tool for the future study of the progression of ALD.

Sparstolonin B attenuates early liver inflammation in experimental NASH by modulating TLR4 trafficking in lipid rafts via NADPH oxidase activation.

Although significant research data exist on the pathophysiology of nonalcoholic steatohepatitis (NASH), finding an efficient treatment regimen for it remains elusive. The present study used sparstolonin B (SsnB), a novel TLR4 antagonist derived from the Chinese herb Sparganium stoloniferum, as a possible drug to mitigate early inflammation in NASH. This study used an early steatohepatitic injury model in high-fat-fed mice with CYP2E1-mediated oxidative stress as a second hit. SsnB was administered for 1 wk along with bromodichloromethane (BDCM), an inducer of CYP2E1-mediated oxidative stress. Results showed that SsnB administration attenuated inflammatory morphology and decreased elevation of the liver enzyme alanine aminotransferase (ALT). Mice administered SsnB also showed decreased mRNA expression of proinflammatory cytokines TNF-α, IFN-γ, IL-1ß, and IL-23, while protein levels of both TNF-α and IL-1ß were significantly decreased. SsnB significantly decreased Kupffer cell activation as evidenced by reduction in CD68 and monocyte chemoattractant protein-1 (MCP1) mRNA and protein levels with concomitant inhibition of macrophage infiltration in the injured liver. Mechanistically, SsnB decreased TLR4 trafficking to the lipid rafts, a phenomenon described by the colocalization of TLR4 and lipid raft marker flotillin in tissues and immortalized Kupffer cells. Since we have shown previously that NADPH oxidase drives TLR4 trafficking in NASH, we studied the role of SsnB in modulating this pathway. SsnB prevented NADPH oxidase activation in vivo and in vitro as indicated by decreased peroxynitrite formation. In summary, the present study reports a novel use of the TLR4 antagonist SsnB in mitigating inflammation in NASH and in parallel shows a unique molecular mechanism of decreasing nitrative stress.

A non-hepatotropic parasite infection increases mortality in the acetaminophen-induced acute liver failure murine model: possible roles for IL-5 and IL-6.

We evaluated the effects of a non-hepatotropic parasite infection (Taenia crassiceps) on the outcome of acetaminophen-induced acute liver failure in mice. Uninfected and T. crassiceps infected mice orally received either 300 mg/kg acetaminophen or water as vehicle (n = 5 per group). Survival analysis, hepatocyte necrosis, alanine aminotransferase (ALT) levels, CYP2E1 protein, interleukin (IL-) 5, and IL-6 were assessed for all groups. All infected mice died within 16 h after exposure to acetaminophen (Tc+APAP group), whereas only one-third of uninfected animals exposed to acetaminophen (APAP group) died. Uninfected (Control group) and infected (Tc group) mice that received the vehicle showed no liver damage. Tc+APAP mice exhibited massive liver necrosis characterised by marked balloning degeneration of hepatocytes and higher serum ALT compared to Control, Tc, and APAP animals. Liver tissue from Tc+APAP mice also displayed increased expression of CYP2E1 protein and higher mRNA and protein levels of IL-5 and IL-6 compared to the other groups. These findings suggest that non-hepatotropic parasite infections may increase mortality following acute liver failure by promoting hepatocyte necrosis via IL-5 and IL-6-dependent CYP2E1 overproduction. This study identifies new potential risk factors associated with severe acute liver failure in patients.

Amomum cardamomum L. ethyl acetate fraction protects against carbon tetrachloride-induced liver injury via an antioxidant mechanism in rats.

BACKGROUND: Medicinal herb-derived drug development has become important in the relief of liver pathology. Amomun cardamomum is traditionally used therapeutically in Korea to treat various human ailments including dyspepsia, hiccupping, and vomiting. We investigated to assess the protective effect of A. cardamomum on carbon tetrachloride (CCl4)-induced liver damage through antioxidant activity in hepatic tissues of Sprague-Dawley rats. METHODS: Antioxidant properties of different fractions from A. cardamomum from ethanol extracts were evaluated by an in vitro free radical scavenging systems. The protective effect of the ethyl acetate fraction from A. cardamomum (EAAC) against CCl4-induced cytotoxicity was determined by a cell viability assay using HepG2 hepatocarcinoma cells. In vivo study, the influence of EAAC concentrations of 100 and 200 mg/kg following CCl4-induced hepatic injury was assessed. Serum levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and alkaline phosphatase (ALP) were determined, as was lipid peroxidation (malondialdehyde, MDA). Effect of EAAC on liver detoxification enzymes including superoxide dismutase (SOD), total glutathione (GSH), and glutathione S-transferase (GST) activity was measured in rat liver homogenates. Liver cytochrome P450 (CYP2E1) expression level was determined by quantification of mRNA. RESULTS: Phytochemical analysis of A. cardamomum indicated that EAAC was enriched in total polyphenol and total flavonoid. Most of the tannins were confined to the hexane fraction. Hepatoprotective properties of EAAC were evident, with significantly reduced serum levels of GOT, GPT, and ALP compared with the control group. Improved hepatic antioxidant status was evident by increased SOD, GSH, and GST enzymes in rat liver tissue. Liver lipid peroxidation induced by CCl4 was apparent by increased intracellular MDA level. EAAC suppressed lipid peroxidation as evidenced by the significant decrease in MDA production. Expression of CYP2E1 was also significantly decreased at the higher concentration of EAAC, indicating the hepatoprotective efficacy of EAAC on acute liver damage. CONCLUSION: These results indicated that EAAC has a significant hepatoprotective activity on CCl4-induced acute hepatic injury in rats, which might be derived from its antioxidant properties and CYP2E1 downregulation.

Effects of Flavonoids in Lysimachia clethroides Duby on the Activities of Cytochrome P450 CYP2E1 and CYP3A4 in Rat Liver Microsomes.

Incubation systems were established to investigate the effects of quercetin, kaempferol, isoquercitrin and astragalin in Lysimachia clethroides Duby on the activities of CYP2E1 and CYP3A4 in rat liver microsomes in vitro. Probe substrates of 4-nitrophenol and testosterone as well as flavonoids at different concentrations were added to the incubation systems. After incubation, a validated high performance liquid chromatography (HPLC) method was applied to separate and determine the relevant metabolites. The results suggested that kaempferol exhibited a weak inhibition of CYP2E1 activity with an IC50 of 60.26 ± 2.54 µM, while quercetin and kaempferol caused a moderate inhibition of CYP3A4 activity with IC50 values of 18.77 ± 1.69 µM and 32.65 ± 1.32 µM, respectively. Isoquercitrin and astragalin had no effects on the activities of either CYP2E1 or CYP3A4. It could be speculated from these results that the inhibitory effects of quercetin and kaempferol on the activities of CYP2E1 and CYP3A4 could be the mechanisms underlying the hepatoprotective effects of L. clethroides.

Dietary tomato powder inhibits alcohol-induced hepatic injury by suppressing cytochrome p450 2E1 induction in rodent models.

Chronic and excessive alcohol consumption leads to the development of alcoholic liver disease (ALD) and greatly increases the risk of liver cancer. Induction of the cytochrome p450 2E1 (CYP2E1) enzyme by chronic and excessive alcohol intake is known to play a role in the pathogenesis of ALD. High intake of tomatoes, rich in the carotenoid lycopene, is associated with a decreased risk of chronic disease. We investigated the effects of whole tomato (tomato powder, TP), partial tomato (tomato extract, TE), and purified lycopene (LYC) against ALD development in rats. Of the three supplements, only TP reduced the severity of alcohol-induced steatosis, hepatic inflammatory foci, and CYP2E1 protein levels. TE had no effect on these outcomes and LYC greatly increased inflammatory foci in alcohol-fed rats. To further support the protective effect of TP against ALD, TP was supplemented in a carcinogen (diethylnitrosamine, DEN)-initiated alcohol-promoted mouse model. In addition to reduced steatosis and inflammatory foci, TP abolished the presence of preneoplastic foci of altered hepatocytes in DEN-injected mice fed alcohol. These reductions were associated with decreased hepatic CYP2E1 protein levels, restored levels of peroxisome proliferator-activated receptor-α and downstream gene expression, decreased inflammatory gene expression, and reduced endoplasmic reticulum stress markers. These data provide strong evidence for TP as an effective whole food prevention strategy against ALD.

Polymorphisms in NEIL-2, APE-1, CYP2E1 and MDM2 Genes are Independent Predictors of Gastric Cancer Risk in a Northern Jiangsu Population (China).

China is one of the countries with the highest incidence of gastric cancer, and accounts for over 40% of all new gastric cancer cases in the world. Genetic factors as well as environmental factors play a role in development of gastric cancer. To investigate the independent roles of single nucleotide polymorphisms (SNPs) in base excision repair (BER) genes (APE1 and NEIL2), carcinogen metabolism gene (CYP2E1) and tumor suppressor pathway gene (MDM2) for gastric cancer susceptibility in a Chinese population, we conducted a hospital based case-control study to evaluate the potential association between these polymorphisms and susceptibility to gastric cancer in a Northern Jiangsu population. We also associated the NEIL-2 mRNA expression with the studied NEIL2 SNP genotypes to assess whether the genotypes have influence on the NEIL2 mRNA (hence protein) expression. Five SNPs, APE 1 (rs2275008), NEIL 2 (rs804270), MDM2 (rs2279744), and CYP 2E1 (rs2480256 and rs2031920), were genotyped by TaqMan assays in 105 gastric cancer cases and 118 controls. Genotype frequency distribution showed that the APE 1 SNP (rs2275008), NEIL 2 SNP (rs804270), MDM2 SNP (rs2279744), and CYP 2E1 SNP (rs2031920) had more mutant alleles in gastric cancer cases than controls (76.19, 68.57, 54.29, and 43.81%, respectively), while CYP 2E1 SNP (rs2480256) had large percentage of both alleles (43.81%). Risk analysis revealed that there was increased risk for gastric cancer in subjects with mutant alleles in APE 1 (rs2275008: OR 5.49, 95% CI = 2.6-5.7, p <.0001), NEIL 2 (rs804270: OR 2.3, 95% CI = 1.22-4.3, p=0.01), MDM2 (rs2279744: OR 14.65, 95% CI = 5.63-8.15, p < .0001), and CYP 2E1 (rs2031920: OR 8.385, 95% CI = 3.2-5.3, p < .0001) SNPs. Moreover, the NEIL2 mRNA expression analysis showed that there was significant differential expression of NEIL2 mRNA among the randomly tested NEIL2 genotypes (p = 0.005), with low expression seen in variant genotypes than in other genotypes. In conclusion, variant alleles in the NEIL2 (rs804270), APE1 (rs2275008), CYP2E1 (rs2031920) and MDM2 (rs2279744) SNPs may independently influence susceptibility to gastric cancer in a Northern Jiangsu Chinese population. The genotypes may also independently influence their respective gene mRNA expression, as seen in our study, where there was differential expression of the NEIL2 mRNA among the genotypes, with low NEIL2 mRNA expression seen in the variant genotype.

Human placental lactogen induces CYP2E1 expression via PI 3-kinase pathway in female human hepatocytes.

The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes.

Aging and alcohol interact to alter hepatic DNA hydroxymethylation.

BACKGROUND: Aging and chronic alcohol consumption are both modifiers of DNA methylation, but it is not yet known whether chronic alcohol consumption also alters DNA hydroxymethylation, a newly discovered epigenetic mark produced by oxidation of methylcytosine. Furthermore, it has not been tested whether aging and alcohol interact to modify this epigenetic phenomenon, thereby having an independent effect on gene expression. METHODS: Old (18 months) and young (4 months) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18% of energy) or an isocaloric Lieber-DeCarli control diet for 5 weeks. Global DNA hydroxymethylation and DNA methylation were analyzed from hepatic DNA using a new liquid chromatography-tandem mass spectrometry method. Hepatic mRNA expression of the Tet enzymes were measured via quantitative real-time polymerase chain reaction. RESULTS: In young mice, mild chronic alcohol exposure significantly reduced global DNA hydroxymethylation compared with control mice (0.22 ± 0.01 vs. 0.29 ± 0.06%, p = 0.004). Alcohol did not significantly alter hydroxymethylcytosine levels in old mice. Old mice fed the control diet showed decreased global DNA hydroxymethylation compared with young mice fed the control diet (0.24 ± 0.02 vs. 0.29 ± 0.06%, p = 0.04). This model suggests an interaction between aging and alcohol in determining DNA hydroxymethylation (pinteraction  = 0.009). Expression of Tet2 and Tet3 was decreased in the old mice relative to the young (p < 0.005). CONCLUSIONS: The observation that alcohol alters DNA hydroxymethylation indicates a new epigenetic effect of alcohol. This is the first study demonstrating the interactive effects of chronic alcohol consumption and aging on DNA hydroxymethylation.

Sex steroid hormones regulate constitutive expression of Cyp2e1 in female mouse liver.

CYP2E1 is of paramount toxicological significance because it metabolically activates a large number of low-molecular-weight toxicants and carcinogens. In this context, factors that interfere with Cyp2e1 regulation may critically affect xenobiotic toxicity and carcinogenicity. The aim of this study was to investigate the role of female steroid hormones in the regulation of CYP2E1, as estrogens and progesterone are the bases of contraceptives and hormonal replacement therapy in menopausal women. Interestingly, a fluctuation in the hepatic expression pattern of Cyp2e1 was revealed in the different phases of the estrous cycle of female mice, with higher Cyp2e1 expression at estrus (E) and lower at methestrus (ME), highly correlated with that in plasma gonadal hormone levels. Depletion of sex steroids by ovariectomy repressed Cyp2e1 expression to levels similar to those detected in males and cyclic females at ME. Hormonal supplementation brought Cyp2e1 expression back to levels detected at E. The role of progesterone appeared to be more prominent than that of 17ß-estradiol. Progesterone-induced Cyp2e1 upregulation could be attributed to inactivation of the insulin/PI3K/Akt/FOXO1 signaling pathway. Tamoxifen, an anti-estrogen, repressed Cyp2e1 expression potentially via activation of the PI3K/Akt/FOXO1 and GH/STAT5b-linked pathways. The sex steroid hormone-related changes in hepatic Cyp2e1 expression were highly correlated with those observed in Hnf-1α, ß-catenin, and Srebp-1c. In conclusion, female steroid hormones are clearly involved in the regulation of CYP2E1, thus affecting the metabolism of a plethora of toxicants and carcinogenic agents, conditions that may trigger several pathologies or exacerbate the outcomes of various pathophysiological states.

Chronic free-choice drinking in crossed high alcohol preferring mice leads to sustained blood ethanol levels and metabolic tolerance without evidence of liver damage.

BACKGROUND: Crossed high alcohol preferring (cHAP) mice were selectively bred from a cross of the HAP1 × HAP2 replicate lines, and we demonstrate blood ethanol concentrations (BECs) during free-choice drinking that are reminiscent of those observed in alcohol-dependent humans. Therefore, this line may provide an unprecedented opportunity to learn about the consequences of excessive voluntary ethanol (EtOH) consumption, including metabolic tolerance and liver pathology. Cytochrome p450 2E1 (CYP2E1) induction plays a prominent role in driving both metabolic tolerance and EtOH-induced liver injury. In this report, we sought to characterize cHAP drinking by assessing whether pharmacologically relevant BEC levels are sustained throughout the active portion of the light-dark cycle. Given that cHAP intakes and BECs are similar to those observed in mice given an EtOH liquid diet, we assessed whether free-choice exposure results in metabolic tolerance, hepatic enzyme induction, and hepatic steatosis. METHODS: In experiment 1, blood samples were taken across the dark portion of a 12:12 light-dark cycle to examine the pattern of EtOH accumulation in these mice. In experiments 1 and 2, mice were injected with EtOH following 3 to 4 weeks of access to water or 10% EtOH and water, and blood samples were taken to assess metabolic tolerance. In experiment 3, 24 mice had 4 weeks of access to 10% EtOH and water or water alone, followed by necropsy and hepatological assessment. RESULTS: In experiment 1, cHAP mice mean BEC values exceeded 80 mg/dl at all sampling points and approached 200 mg/dl during the middle of the dark cycle. In experiments 1 and 2, EtOH-exposed mice metabolized EtOH faster than EtOH-naïve mice, demonstrating metabolic tolerance (p < 0.05). In experiment 3, EtOH-drinking mice showed greater expression of hepatic CYP2E1 than water controls, consistent with the development of metabolic tolerance (p < 0.05). EtOH access altered neither hepatic histology nor levels of alcohol dehydrogenase and aldehyde dehydrogenase. CONCLUSIONS: These results demonstrate that excessive intake by cHAP mice results in sustained BECs throughout the active period, leading to the development of metabolic tolerance and evidence of CYP2E1 induction. Together, these results provide additional support for the cHAP mice as a highly translational rodent model of alcoholism.

ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes.

AIM: Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress. METHODS: Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH). RESULTS: ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity. CONCLUSION: We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.

Protective effect of L-theanine on carbon tetrachloride-induced acute liver injury in mice.

We studied effects of L-theanine, a unique amino acid in tea, on carbon tetrachloride (CCl(4))-induced liver injury in mice. The mice were pre-treated orally with L-theanine (50, 100 or 200 mg/kg) once daily for seven days before CCl(4) (10 ml/kg of 0.2% CCl(4) solution in olive oil) injection. L-theanine dose-dependently suppressed the increase of serum activity of ALT and AST and bilirubin level as well as liver histopathological changes induced by CCl(4) in mice. L-theanine significantly prevented CCl(4)-induced production of lipid peroxidation and decrease of hepatic GSH content and antioxidant enzymes activities. Our further studies demonstrated that L-theanine inhibited metabolic activation of CCl(4) through down-regulating cytochrome P450 2E1 (CYP2E1). As a consequence, L-theanine inhibited oxidative stress-mediated inflammatory response which included the increase of TNF-α and IL-1ß in sera, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in livers. CCl(4)-induced activation of apoptotic related proteins including caspase-3 and PARP in mouse livers was also prevented by L-theanine treatment. In summary, L-theanine protects mice against CCl(4)-induced acute liver injury through inhibiting metabolic activation of CCl(4) and preventing CCl(4)-induced reduction of anti-oxidant capacity in mouse livers to relieve inflammatory response and hepatocyte apoptosis.