Male Hepatocytes Research Articles

Sirt2 Regulates Liver Metabolism in a Sex-Specific Manner.

Sirtuin-2 (Sirt2), an NAD+-dependent lysine deacylase enzyme, has previously been implicated as a regulator of glucose metabolism, but the specific mechanisms remain poorly defined. Here, we observed that Sirt2-/- males, but not females, have decreased body fat, moderate hypoglycemia upon fasting, and perturbed glucose handling during exercise compared to wild type controls. Conversion of injected lactate, pyruvate, and glycerol boluses into glucose via gluconeogenesis was impaired, but only in males. Primary Sirt2-/- male hepatocytes exhibited reduced glycolysis and reduced mitochondrial respiration. RNAseq and proteomics were used to interrogate the mechanisms behind this liver phenotype. Loss of Sirt2 did not lead to transcriptional dysregulation, as very few genes were altered in the transcriptome. In keeping with this, there were also negligible changes to protein abundance. Site-specific quantification of the hepatic acetylome, however, showed that 13% of all detected acetylated peptides were significantly increased in Sirt2-/- male liver versus wild type, representing putative Sirt2 target sites. Strikingly, none of these putative target sites were hyperacetylated in Sirt2-/- female liver. The target sites in the male liver were distributed across mitochondria (44%), cytoplasm (32%), nucleus (8%), and other compartments (16%). Despite the high number of putative mitochondrial Sirt2 targets, Sirt2 antigen was not detected in purified wild type liver mitochondria, suggesting that Sirt2's regulation of mitochondrial function occurs from outside the organelle. We conclude that Sirt2 regulates hepatic protein acetylation and metabolism in a sex-specific manner.

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

The effects of dark deprivation and chronic alcohol intoxication on the liver of rats

BACKGROUND: Nighttime exposure to artificial lighting promotes epiphyseal melatonin deficiency and disrupts the circadian cycle of most body functions in mammals. Several studies have associated these factors with the development of many liver pathologies. A considerable lack of melatonin, along with a mismatch in the daily cycle of vital processes, increases the sensitivity of the liver to alcohol-induced damage and the severity of alcoholic illness.
 AIM: This study aimed to identify the combined effect of constant lighting and chronic alcohol intoxication (CAI) on the liver structure of both sexes of rats.
 MATERIALS AND METHODS: TThe study was conducted on 120 male and 80 female outbred Wistar rats aged 6 months. The trial lasted for 3 weeks. Four groups of rats of each sex were formed (control group, CAI group, dark deprivation group, and group exposed to both factors). Preparations stained with hematoxylin and eosin were evaluated. Sudan III staining was used to assess the severity of fatty degeneration, and the expression of Ki-67 and p53 as markers of proliferation and apoptosis, respectively, was measured immunohistochemically.
 RESULTS: Chronic alcohol intoxication and dark deprivation cause similar morphological changes in the livers of male and female rats, resulting in fatty degeneration, necrosis, and increased hepatocyte apoptotic activity. The combined effect of these factors causes more dramatic changes in the structure of the liver of males, resulting in the development of cirrhotic changes in 13.3% of them and toxic hepatitis in 20%, whereas only 5% of females show signs of alcoholic hepatitis. Increased Ki-67 expression in male hepatocytes is found only when CAI and dark deprivation are combined. However, in females, an increase in Ki-67 expression is observed both alone and when these factors are combined.
 CONCLUSION: The findings allow us to conclude that female rats adapt more successfully to the independent and combined effects of CAI and dark deprivation than male rats.

Gender dimorphism in hepatocarcinogenesis-DNA methylation modification regulated X-chromosome inactivation escape molecule XIST.

Sex disparities constitute a significant issue in hepatocellular carcinoma (HCC). However, the mechanism of gender dimorphism in HCC is still not completely understood. 5-Hydroxymethylcytosine (5hmC)-Seal technology was utilised to detect the global 5hmC levels from four female and four male HCC samples. Methylation of XIST was detected by Sequenom MassARRAY methylation profiling between HCC tissues (T) and adjacent normal liver tissues (L). The role of Tet methylcytosine dioxygenase 2 (TET2) was investigated using diethylnitrosamine (DEN)-administered Tet2-/- female mice, which regulated XIST in hepatocarcinogenesis. All statistical analyses were carried out by GraphPad Prism 9.0 and SPSS version 19.0 software. The results demonstrated that the numbers of 5hmC reads in the first exon of XIST from female HCC tissues (T) were remarkably lower than that in female adjacent normal liver tissues (L). Correspondingly, DNA methylation level of XIST first exon region was significantly increased in female T than in L. By contrast, no significant change was observed in male HCC patients. Compared to L, the expression of XIST in T was also significantly downregulated. Female patients with higher XIST in HCC had a higher overall survival (OS) and more extended recurrence-free survival (RFS). Moreover, TET2 can interact with YY1 binding to the promoter region of XIST and maintain the hypomethylation state of XIST. In addition, DEN-administered Tet2-/- mice developed more tumours than controls in female mice. Our study provided that YY1 and TET2 could interact to form protein complexes binding to the promoter region of XIST, regulating the methylation level of XIST and then affecting the expression of XIST. This research will provide a new clue for studying sex disparities in hepatocarcinogenesis. XIST was significantly downregulated in HCC tissues and had gender disparity. Methylation levels in the XIST first exon were higher in female HCC tissues, but no significant change in male HCC patients. The TET2-YY1 complex regulate XIST expression in female hepatocytes. Other ways regulate XIST expression in male hepatocytes.

Differences in hepatocellular iron metabolism underlie sexual dimorphism in hepatocyte ferroptosis

Males show higher incidence and severity than females in hepatic injury and many liver diseases, but the mechanisms are not well understood. Ferroptosis, an iron-mediated lipid peroxidation-dependent death, plays an important role in the pathogenesis of liver diseases. We determined whether hepatocyte ferroptosis displays gender difference, accounting for sexual dimorphism in liver diseases. Compared to female hepatocytes, male hepatocytes were much more vulnerable to ferroptosis by iron and pharmacological inducers including RSL3 and iFSP1. Male but not female hepatocytes exhibited significant increases in mitochondrial Fe2+ and mitochondrial ROS (mtROS) contents. Female hepatocytes showed a lower expression of iron importer transferrin receptor 1 (TfR1) and mitochondrial iron importer mitoferrin 1 (Mfrn1), but a higher expression of iron storage protein ferritin heavy chain 1 (FTH1). It is well known that TfR1 expression is positively correlated with ferroptosis. Herein, we showed that silencing FTH1 enhanced while knockdown of Mfrn1 decreased ferroptosis in HepG2 cells. Removing female hormones by ovariectomy (OVX) did not dampen but rather enhanced hepatocyte resistance to ferroptosis. Mechanistically, OVX potentiated the decrease in TfR1 and increase in FTH1 expression. OVX also increased FSP1 expression in ERK-dependent manner. Elevation in FSP1 suppressed mitochondrial Fe2+ accumulation and mtROS production, constituting a novel mechanism of FSP1-mediated inhibition of ferroptosis. In conclusion, differences in hepatocellular iron handling between male and female account, at least in part, for sexual dimorphism in induced ferroptosis of the hepatocytes.

Combined effects of arsenic and 2,2-dichloroacetamide on different cell populations of zebrafish liver

Arsenic (As) and disinfection by-products are important health risk factors in the water environment. However, their combined effects on different cell populations in the liver are not well known. Here, zebrafish were exposed to 100 μg/L As, 300 μg/L 2,2-dichloroacetamide (DCAcAm), and their combination for 23 days. Then transcriptome profiles of cell populations in zebrafish liver were analyzed by single-cell RNA sequencing (scRNA-seq). A total of 13,563 cells were obtained, which were identified as hepatocytes, hepatic duct cells, endothelial cells and macrophages. Hepatocytes were the main target cell subtype of As and DCAcAm exposures. DCAcAm exposure induced higher toxicity in male hepatocytes, which specifically changed amino acid metabolism, response to hormone and cofactor metabolism. However, As exposure caused higher toxicity in female hepatocytes, which altered lipid metabolism, carbon metabolism, and peroxisome. Combined exposure to As and DCAcAm decreased toxicities in hepatocytes compared to each one alone. Female hepatocytes had higher tolerance to co-exposure of As and DCAcAm than male hepatocytes. Further, combined exposure to As and DCAcAm induced functional changes in macrophages similar to As alone groups, which mainly altered the transfer of sterol and cholesterol. Hepatic duct cells and endothelial cells were not influenced by exposures to As and DCAcAm. This study for the first time highlights the cell-specific combined responses of As and DCAcAm in zebrafish liver, which provide useful information for their health risk assessment in a co-exposure environment.

The Effect of CMCE Propolis Extract Administrations on Interleukin-1 (IL-1) Levels and the Hepatocyte HistopathologicalFindings of Rats� Induced with CCl4

Introduction:Liver exposure to toxic substances will disrupt the metabolic system. Carbon tetrachloride (CCl4) is a hepatotoxic substance causing a liver damage. Continuous multi-stage countercurrent extraction(CMCE) propolis extract contains active balsamic substances, caffeic acid, phenethyl esters and flavonoids.Objective: to prove the effect of CMCE propolis extract on IL-1 levels and the hepatocyte histopathology findingsof male wistar rats�induced by CCl4.Methods:In the posttest only control group design, the total sample of 28 rats was divided into 4 groups: positive control group (K-0; standard feed); group P-1 (propolis3.6 mg/200 g); P-2 (propolis7.2mg/200gr and P-3 (propolis14.4mg/200g) were respectively given for 14 days,yet those on the 14thday were given CCl4. The IL-1 level was measured with the ELISA method, while the male wistar rats�hepatocyte histopathology findings was determined by the HE staining methods. IL-1 levelswere analyzed usingOne Way Anova test,followed by the Post Hoc LSD test. Hepatic cell histopathology was analyzed usingKruskal Wallis, followed by Mann-Whitney U.Results:Post Hoc LSD analysis shows that the levels of IL-1 in P-1 (983.54 �19.67), P-2 (841.58�19.47), and P-3 (755.50�31.63) are significantlylower thanthosein K-0 (1131.69�14.91) withp<0.05. Mann-Whitney U analysis shows that the hepatocyte histopathology in the P-1 group (2.55�0.39), P-2 (1.24�0.94) and P-3 (1.20�0.93) is significantlylower thanthatinK-0 (2.94�0.16) with p<0.05.Conclusion: The administration of CMCE propolis extract may reduce IL-1 levels and improve the male wistar rats�hepatic cell histopathologyinduced with carbon tetrachloride (CCl4).

Hepatic Transcript Profiles of Cytochrome P450 Genes Predict Sex Differences in Drug Metabolism.

Safety assessments of new drug candidates are an important part of the drug development and approval process. Often, possible sex-associated susceptibilities are not adequately addressed, and better assessment tools are needed. We hypothesized that hepatic transcript profiles of cytochrome P450 (P450) enzymes can be used to predict sex-associated differences in drug metabolism and possible adverse events. Comprehensive hepatic transcript profiles were generated for F344 rats of both sexes at nine ages, from 2 weeks (preweaning) to 104 weeks (elderly). Large differences in the transcript profiles of 29 drug metabolizing enzymes and transporters were found between adult males and females (8–52 weeks). Using the PharmaPendium data base, 41 drugs were found to be metabolized by one or two P450 enzymes encoded by sexually dimorphic mRNAs and thus were candidates for evaluation of possible sexually dimorphic metabolism and/or toxicities. Suspension cultures of primary hepatocytes from three male and three female adult rats (10–13 weeks old) were used to evaluate the metabolism of 11 drugs predicted to have sexually dimorphic metabolism. The pharmacokinetics of the drug or its metabolite was analyzed by liquid chromatography/tandem mass spectrometry using multiple reaction monitoring. Of those drugs with adequate metabolism, the predicted significant sex-different metabolism was found for six of seven drugs, with half-lives 37%–400% longer in female hepatocytes than in male hepatocytes. Thus, in this rat model, transcript profiles may allow identification of potential sex-related differences in drug metabolism.SIGNIFICANCE STATEMENTThe present study showed that sex-different expression of genes coding for drug metabolizing enzymes, specifically cytochrome P450s, could be used to predict sex-different drug metabolism and, thus, provide a new tool for protecting susceptible subpopulations from possible adverse drug events.

Specific diacylglycerols generated by hepatic lipogenesis stimulate the oncogenic androgen receptor activity in male hepatocytes.

Obesity-induced hepatocellular carcinoma (HCC) is more prevalent in males than in females, but the underlying mechanism remains unclear. The influence of hepatic androgen receptor (AR) pathway on the gender difference of HCC has been well documented. Here we investigated the role of hepatic lipogenesis, which is elevated in the livers of obese and nonalcoholic fatty liver disease (NAFLD) patients, in stimulating the AR pathway for the male preference of obesity induced HCC. Male C57BL/6J mice were fed a fructose-rich high carbohydrate diet (HCD) to induce hepatic lipogenesis. The effect of hepatic lipogenesis on AR was examined by the expression of hydrodynamically injected AR reporter and the endogenous AR target gene; the mechanism was delineated in hepatoma cell lines and validated in male mice. The hepatic lipogenesis induced by a fructose-rich HCD enhanced the transcriptional activity of hepatic AR in male mice, which did not happen when fed a high fat diet. This AR activation was blocked by sh-RNAs or inhibitors targeting key enzymes in lipogenesis, either acetyl-CoA carboxylase subunit alpha (ACCα), or fatty acid synthase (FASN), in vivo and in vitro. Further mechanistic study identified that specific unsaturated fatty acid, the oleic acid (C18:1 n-9), incorporated DAGs produced by hepatic lipogenesis are the key molecules to enhance the AR activity, through activation of Akt kinase, and this novel mechanism is targeted by metformin. Our study elucidates a novel mechanism underlying the higher risk of HCC in obese/NAFLD males, through specific DAGs enriched by hepatic lipogenesis to increase the transcriptional activity of hepatic AR, a confirmed risk factor for male HCC.

Improving engraftment of hepatocyte transplantation using alpha-1 antitrypsin as an immune modulator

For patients with non-cirrhotic liver-based metabolic disorders, hepatocyte transplantation can be an effective treatment. However, long-term function of transplanted hepatocytes following infusion has not been achieved due to insufficient numbers of hepatocytes reaching the liver cell plates caused by activation of the instant blood-mediated inflammatory reaction (IBMIR). Our aim was to determine if the natural immune modulator, alpha-1 antitrypsin (AAT), could improve engraftment of transplanted hepatocytes and investigate its mechanism of action. A tubing loop model was used to analyse activation of the IBMIR when human hepatocytes were in contact with ABO-matched blood and 4 mg/ml AAT. Platelet and white cell counts, complement and cytokine expression were analysed. To determine if AAT could improve short-term engraftment, female rats underwent tail vein injection of AAT (120 mg/kg) or water (control) prior to the intrasplenic transplantation of 2 × 107 male hepatocytes. At 48 h and 1 week, livers were collected for analysis. In our loop model, human hepatocytes elicited a significant drop in platelet count with thrombus formation compared to controls. Loops containing AAT and hepatocytes showed no platelet consumption and no thrombus formation. Further, AAT treatment resulted in reduced IL-1β, IL-6 and IFN-γ and increased IL-1RA compared to untreated loops. In vivo, AAT significantly improved engraftment of rat hepatocytes compared to untreated at 48 h. AAT infusion may inhibit the IBMIR, thus improving short-term engraftment of donor hepatocytes and potentially improve the outcomes for patients with liver-based metabolic disease.Key messages• Alpha-1 antitrypsin (AAT) acts as an immune modulator to improve the efficacy of hepatocyte transplantation.• Treatment with AAT decreased thrombus formation and pro-inflammatory cytokine expression in a tubing loop model.• AAT significantly improved engraftment of donor hepatocytes within the first 48 h post transplantation.

Erratum to: Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells.

During manuscript proofing, the following sentence was not deleted in the section "Results" at the end of the paragraph: "Both male and female hepatocytes responded in a similar fashion to cotinine, whereas male hepatocyte function was more sensitive to chrysene, fluorene and naphthalene than female hepatocytes".

Differential Proteomic Analysis of Gender-dependent Hepatic Tumorigenesis in Hras12V Transgenic Mice

Male prevalence is an outstanding characteristic of hepatocellular carcinoma (HCC), and the underlying mechanisms for this have remained largely unknown. In the present study, Hras12V transgenic mice, in which hepatocyte-specific expression of the ras oncogene induces male-biased hepatic tumorigenesis, were studied, and altered proteins were detected by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Protein samples from hepatic tumor tissues (T) and peritumor tissues (P) of transgenic males and females and the corresponding normal liver tissues (Wt) of nontransgenic males and females were subjected to pairwise comparisons based on proteomic analysis. Among 2381 autodetected protein spots, more than 1600 were differentially expressed based on a pairwise comparison (|ratio| > = 1.5, p < = 0.05). Of these, 180 spots were randomly selected for matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) identification; finally, 89 distinct proteins were obtained. Among these 89 proteins, 7 and 50 proteins were further validated by Western blotting and literature investigation, respectively. Intriguingly, compared with Wt, the altered proteins were relatively concentrated in T in transgenic females but in P in transgenic males. Consistently, the levels of p-ERK and p-mTOR were significantly higher in the T of females compared with that of males. The pathway enrichment assay showed that 5 pathways in males but only 1 in females were significantly altered in terms of the upregulated proteins in T compared with Wt. These data indicate that female hepatocytes are disturbed by oncogenes with great difficulty, whereas male hepatocytes readily do so. In addition, 33 proteins were gender-dependently altered in hepatic tumorigenesis. Moreover, 4% DNA packaging and 4% homeostasis-related functional proteins were found in females but not in males, and more nucleus proteins were found in females (8%) than in males (3%). In conclusion, the proteomic data and comparative analysis presented here offer crucial clues for elucidating the mechanisms that underlie the male prevalence in HCC.

Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells

The liver is a dynamic organ which is both multifunctional and highly regenerative. A major role of the liver is to process both endo and xenobiotics. Cigarettes are an example of a legal and widely used drug which can cause major health problems for adults and constitute a particular risk to the foetus, if the mother smokes during pregnancy. Cigarette smoke contains a complex mixture of thousands of different xenobiotics, including nicotine and polycyclic aromatic hydrocarbons. These affect foetal development in a sex-specific manner, inducing sex-dependant molecular responses in different organs. To date, the effect of maternal smoking on the foetal liver has been studied in vitro using cell lines, primary tissue and animal models. While these models have proven to be useful, poor cell phenotype, tissue scarcity, batch-to-batch variation and species differences have led to difficulties in data extrapolation toward human development. Therefore, in this study we have employed hepatoblasts, derived from pluripotent stem cells, to model the effects of xenobiotics from cigarette smoke on human hepatocyte development. Highly pure hepatocyte populations (>90%) were produced in vitro and exposed to factors present in cigarette smoke. Analysis of ATP levels revealed that, independent of the sex, the majority of smoking derivatives tested individually did not deplete ATP levels below 50%. However, following exposure to a cocktail of smoking derivatives, ATP production fell below 50% in a sex-dependent manner. This was paralleled by a loss metabolic activity and secretory ability in both female and male hepatocytes. Interestingly, cell depletion was less pronounced in female hepatocytes, whereas caspase activation was ~twofold greater, indicating sex differences in cell death upon exposure to the smoking derivatives tested.

The direction of cross affects [corrected] obesity after puberty in male but not female offspring.

BackgroundWe investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl (B6N).ResultsWe found that F1-males with a BFMI mother developed 1.8 times more fat mass on a high fat diet at 10 weeks than F1-males of a BFMI father. The phenotype was detectable from six weeks on and was preserved after cross-fostering. RNA-seq data of liver provided evidence for higher biosynthesis and elongation of fatty acids (p = 0.00635) in obese male offspring of a BFMI mother versus lean offspring of a BFMI father. Furthermore, fatty acid degradation (p = 0.00198) and the peroxisome pathway were impaired (p = 0.00094). The circadian rhythm was affected as well (p = 0.00087). Among the highest up-regulated protein coding genes in obese males were Acot4 (1.82 fold, p = 0.022), Cyp4a10 (1.35 fold, p = 0.026) and Cyp4a14 (1.32 fold, p = 0.012), which hydroxylize fatty acids and which are known to be increased in liver steatosis. Obese males showed lower expression of the genetically imprinted and paternally expressed 3 (Peg3) gene (0.31 fold, p = 0.046) and higher expression of the androgen receptor (Ar) gene (2.38 fold, p = 0.068). Allelic imbalance was found for expression of ATP-binding cassette transporter gene Abca8b. Several of the differentially expressed genes contain estrogen response elements.ConclusionsParent-of-origin effects during gametogenesis and/or fetal development in an obese mother epigenetically modify the transcription of genes that lead to enhanced fatty acid synthesis and impair β-oxidation in the liver of male, but not female F1 offspring. Down-regulation of Peg3 could contribute to trigger this metabolic setting. At puberty, higher amounts of the androgen receptor and altered access to estrogen response elements in affected genes are likely responsible for male specific expression of genes that were epigenetically triggered. A suggestive lack of estrogen binding motifs was found for highly down-regulated genes in adult hepatocytes of obese F1 males (p = 0.074).Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2164-2) contains supplementary material, which is available to authorized users.

Synchronized necrotic death of attached hepatocytes mediated via gap junctions.

Extensive studies have unveiled the intracellular molecular signaling pathways of cell death. To better understand cell death in tissues, it is important to investigate the influence of neighboring cells on the response to death stimuli. By time-lapse microscopy, we found that cells in couplets (two hepatocytes attached to each other) died independently when stimulated with anti-Fas antibody and staurosporine, whereas acetaminophen (APAP) and aryl alcohol caused synchronized cell death although its timing varied among different couplets. Synchronized death of couplets was not caused by APAP when hepatocytes were deficient in both Connexin26 and Connexin32, indicating a crucial role of gap junctions in the synchronized death process. We also demonstrated that APAP-sensitive male hepatocytes were protected by attachment to APAP-insensitive female hepatocytes, with this protection being dependent on gap junctions. These findings indicate that APAP-induced and aryl alcohol-induced necrotic death of hepatocytes is modulated by attached neighboring cells via gap junctions.

935 OVEREXPRESSION OF CREM ALPHA IN CD4+ T CELLS ACCELERATES ACUTE ConA-INDUCED HEPATITIS BUT DOES NOT INFLUENCE FIBROSIS

Introduction: Aquaporins (AQPs) are a family of channel proteins that facilitate the osmotic movement of water and small solutes across biological membranes. In the hepatobiliary tract, AQPs participate in formation and flow of bile. We have previously shown that Liver X Receptor b (LXRb), a nuclear receptor activated by oxysterols, controls the expression of AQP-1 in pancreas and kidney. Aim and Methods: In the present study we aimed to investigate the role of LXRb in controlling water channel homeostasis in the hepatobiliary tract, in-vivo. Both male and female WT and LXRb−/− mice were studied at the age of 12 months. Results: In WT mice, LXRb protein was strongly expressed in the nuclei of intrahepatic cholangiocytes while a weak expression was detected in hepatocytes. Male LXRb−/− mice demonstrated a significant increase of serum levels of both total bilirubin and alkaline phosphatase, indicating the presence of a mild cholestasis. In the whole liver, there was a significant reduction in the levels of both the mRNA of AQP-1 and AQP-4 in male LXRb−/− mice. Interestingly, AQP-4 protein expression was detected on the plasma membrane of male WT hepatocytes while in LXRb−/− mice, the immunoreactivity was identified in the cytosol, suggesting a possible defect in its transport to the membrane. No AQP-4 was detected in female mice either WT or LXRb−/−. In the kidney, AQP-2 trafficking is controlled by caveolin-1, a major component of caveolae, that is involved in transcytosis, endocytosis and signal transduction. In prostate cancer cells, caveolin-1 is upregulated by testosterone and acts as Androgen Receptor (AR) co-regulator being involved in non-genomic effects of AR. In LXRb−/− male mice, strong immunoreactivity of caveolin-1 was detected in the cytosol while in WT mice it was on the cell surface. Thus there is impaired translocation of caveolin-1 in the mutant mice. Interestingly, in LXRb−/− male mice, serum testosterone levels were in normal range. Conclusion: LXRb regulates the level of AQP-1 and AQP-4 mRNA as well as the cellular localization of AQP-4, via caveolin-1, in male mice.

The genotoxic potential of methapyrilene using the alkaline Comet assay in vitro and in vivo

The genotoxicity of methapyrilne (MP) has been evaluated in a number of assays since it was found to be a rat hepatocarcinogen with subsequent withdrawal as an over-the-counter antihistamine. Whilst it has not been classified as a genotoxin, there are reports of positive findings from mammalian cell gene mutation and transformation assays. To investigate further the genotoxic potential of MP, the alkaline Comet assay was used to evaluate DNA damage both in primary hepatocytes in culture and in vivo in the rat. To confirm bioactivation was required to induce the hepatotoxic mechanism, aminobenzotriazole, a broad spectrum cytochrome P450 enzyme inhibitor was used as a pre-treatment. The levels of glutathione and glutathione disulfide were determined in both hepatocytes in culture and in the liver following in vivo exposure. MP showed significant increases in DNA damage in freshly isolated male rat hepatocyte suspensions that could be significantly reduced by pre-incubation of aminobenzotriazole (ABT). DNA damage showed a marked sex difference, with male hepatocytes being more susceptible, and showing a concurrent depletion of glutathione (GSH) compared with female hepatocytes. Modulation of the GSH levels by diethylmaleate and γ-glutamylcysteinylethyl ester, elevated and reduced the levels of DNA damage, respectively. In the in vivo Comet assay, there was no evidence of DNA damage following MP (150 mg/kg p.o) treatment for three consecutive days, although histological and liver enzyme changes were seen. Total protein GSH content was elevated in MP-treated animals and superoxide dismutase levels were increased specifically in periportal regions. Taken together, these data support the potential for MP to induce oxidative stress. The differences in DNA damage detected by the Comet assay in vitro, and in rat liver in vivo, could be attributed to differences in metabolism and response to oxidant insult or the inability of the assay to discriminate damage in a small number of individual cells in the whole liver.

Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

l-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 °C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl- l-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher l-methionine- l-sulfoxide levels relative to control hepatocytes, whereas the increases in l-methionine- d-sulfoxide (Met- d-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met- d-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine- dl-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

L-Methionine-dl-sulfoxide Metabolism and Toxicity in Freshly Isolated Mouse Hepatocytes: Gender Differences and Inhibition with Aminooxyacetic Acid

L-methionine-dl-sulfoxide (MetO) is an L-methionine (Met) metabolite, but its role in Met metabolism and toxicity is not clear. In this study, MetO uptake, metabolism to Met, cytotoxicity, and glutathione (GSH) and glutathione disulfide (GSSG) status were characterized in freshly isolated mouse hepatocytes incubated at 37 degrees C with 0 to 30 mM MetO for 0 to 5 h. In male hepatocytes, dose-dependent cytotoxicity concomitant with GSH depletion without GSSG formation occurred after exposure to 20 or 30 mM MetO but not after exposure to 10 mM MetO. Interestingly, female hepatocytes exposed to 30 mM MetO showed no cytotoxicity and exhibited increased intracellular GSH levels compared with control hepatocytes. Male hepatocytes had approximately 2-fold higher levels of intracellular Met-d-O or Met-l-O after MetO (30 mM) exposure for 0 to 1.5 h compared with female hepatocytes. In hepatocytes of both genders, Met-l-O was detected at nearly 5-fold higher levels than Met-d-O, and no significant increase in cellular Met levels was detected. Addition of aminooxyacetic acid (AOAA), an inhibitor of transamination reactions, to MetO-exposed male hepatocytes resulted in higher cellular Met-d-O and Met-l-O levels and decreased the cytotoxicity of MetO. Interestingly, exposure of control male hepatocytes to AOAA selectively increased cellular Met-d-O levels to levels similar to those observed after exposure to MetO (30 mM). Analysis of MetO transamination activity by glutamine transaminase K in mouse liver cytosol revealed similar rates of MetO transamination in cytosol of both genders. Taken together, these results provide evidence for stereoselective oxidation of Met to Met-d-O under physiological conditions and suggest a major role for MetO transamination in MetO metabolism and toxicity.

Inherent Sexually Dimorphic Expression of Hepatic CYP2C12 Correlated with Repressed Activation of Growth Hormone-Regulated Signal Transduction in Male Rats

Because of its myriad physiologic functions, it is not surprising that the actions of growth hormone (GH) are mediated by recruiting/activating dozens of signaling molecules involved in numerous transduction pathways. The particular signal transduction pathway activated by the hormone is determined by the affected target cell, the sexually dimorphic secretory GH profile (masculine episodic or feminine continuous) to which the cell is exposed, and the individual's sex. In this regard, expression of female-specific CYP2C12, the most abundant cytochrome P450 in female rat liver, is solely regulated by the feminine GH profile. Sex is a modulating factor in this response in that males are considerably less responsive than females to the CYP2C12-induction effects of continuous GH. Using primary hepatocytes derived from male and female hypophysectomized rats, we have identified several factors in a transduction pathway activated by the feminine GH regime and associated with the induction of hepatic CYP2C12. Elements in the proposed pathway, in their likely order of activation, are the growth hormone receptor, extracellular signal-regulated kinases, the cAMP-response element-binding protein, and hepatocyte nuclear factors 4alpha and 6, which subsequently bind and activate the CYP2C12 promoter. Recruitment and/or activation levels of all of the component factors in the pathway were highly suppressed in male hepatocytes, possibly explaining the dramatically lower induction levels of CYP2C12 in males exposed to the same continuous GH profile as females.