Plasma Oxylipin Profile Discriminates Ethnicities in Subjects with Non-Alcoholic Steatohepatitis: An Exploratory Analysis.

Non-alcoholic fatty liver disease (NAFLD) is a common liver pathology that includes steatosis, or non-alcoholic fatty liver (NAFL), and non-alcoholic steatohepatitis (NASH). Without a clear pathophysiological mechanism, it affects Hispanics disproportionately compared to other ethnicities. Polyunsaturated fatty acids (PUFAs) and inflammatory lipid mediators including oxylipin (OXL) and endocannabinoid (eCB) are altered in NAFLD and thought to contribute to its pathogenesis. However, the existence of ethnicity-related differences is not clear. We employed targeted lipidomic profiling for plasma PUFAs, non-esterified OXLs and eCBs in White Hispanics (HIS, n = 10) and Caucasians (CAU, n = 8) with biopsy-confirmed NAFL, compared with healthy control subjects (HC; n = 14 HIS; n = 8 CAU). NAFLD was associated with diminished long chain PUFA in HIS, independent of histological severity. Differences in plasma OXLs and eCBs characterized ethnicities in NASH, with lower arachidonic acid derived OXLs observed in HIS. The secondary analysis comparing ethnicities within NASH (n = 12 HIS; n = 17 CAU), confirms these ethnicity-related differences and suggests lower lipoxygenase(s) and higher soluble epoxide hydrolase(s) activities in HIS compared to CAU. While causes are not clear, these lipidomic differences might be with implications for NAFLD severity and are worth further investigation. We provide preliminary data indicating ethnicity-specific lipidomic signature characterizes NASH which requires further validation.

Ethnicity-specific alterations of plasma and hepatic lipidomic profiles are related to high NAFLD rate and severity in Hispanic Americans, a pilot study.

Nonalcoholic fatty liver disease (NAFLD) is a progressive condition that includes steatosis (NAFL) and nonalcoholic steatohepatitis (NASH). In the U.S., Hispanics (HIS) are afflicted with NAFLD at a higher rate and severity compared to other ethnicities. To date, the mechanisms underlying this disparity have not been elucidated. In this pilot study, we compared untargeted plasma metabolomic profiles for primary metabolism, complex lipids, choline and related compounds between a group of HIS (n = 7) and White Caucasian (CAU, n = 8) subjects with obesity and biopsy-characterized NAFL to ethnicity-matched lean healthy controls (n = 14 HIS and 8 CAU). We also compared liver and plasma metabolomic profiles in a group of HIS and CAU subjects with obesity and NASH of comparable NAFLD Activity Scores, to BMI-matched NASH-free subjects in both ethnicities. Results highlight signs of metabolic dysregulation observed in HIS, independent of obesity, including higher plasma triglycerides, acylcarnitines, and free fatty acids. With NASH progression, there were ethnicity-related differences in the hepatic profile, including higher free fatty acids and lysophospholipids seen in HIS, suggesting lipotoxicity is involved in the progression of NASH. We also observed greater hepatic triglyceride content, higher plasma triglyceride concentrations and lower hepatic phospholipids with signs of impaired hepatic mitochondrial ß-oxidation. These findings provide preliminary evidence indicating ethnicity-related variations that could potentially modulate the risk for progression of NALD to NASH.

IL28B genotype and the expression of ISGs in normal liver.

BACKGROUND & AIMS: Both polymorphisms in the IL28B gene locus and ISG expression levels are associated with the outcome of hepatitis C virus (HCV) infection. The two are also interrelated, although the mechanism is unknown. Favourable CC genotype at rs12979860 expresses lower baseline ISG levels and responds better to treatment than unfavourable CT and TT genotypes. Little is known about this relationship in normal, uninfected liver. This study sought to explore this relationship. METHODS: Normal human liver specimens (64) and HCV positive human liver specimens (95) were genotyped for IL28B rs12979860 C > T. mRNA levels of ISGs and other relevant genes were studied by qPCR. RESULTS: Most studied ISGs had significantly different expression by IL28B genotype in normal liver. CC genotype expressed the highest levels, CT intermediate and TT the lowest. This is opposite to the pattern seen in HCV patients. Principal component analysis of IL28B genotype and ISG expression further revealed a distinct set of genes correlated with the C allele (ISG15, HTATIP2, LGALS3BP, IRF2 and BCL2) and T allele (IFNα, ß, γ, λ3 and CD80). CONCLUSION: A subset of ISGs was found to be differentially expressed in normal liver by IL28B genotype. This suggests a relationship between IL28B genotype and gene expression before HCV infection.

The association between the SLC6A3 VNTR 9-repeat allele and alcoholism-a meta-analysis.

BACKGROUND: Dopamine transporter gene (SLC6A3) represents a promising candidate involved in the development of alcoholism. This study aimed to explore the association between the 9-repeat allele (A9) of a 40-bp variable number tandem repeat (VNTR) polymorphism in the 3' un-translated region (3' UTR) of the SLC6A3 gene and alcoholism. METHODS: The SLC6A3 VNTR was genotyped by PCR in unrelated Mexican Americans including 337 controls and 365 alcoholics. Pearson's chi-square test or Fisher's exact test was used to compare the genotype and allele distribution. Meta-analyses were performed for population-based case-control association studies of the SLC6A3 VNTR polymorphism with alcoholism. Data were analyzed under random effect models with the Comprehensive Meta-analysis (v.2) statistical software package. RESULTS: In Mexican Americans, no significant difference was found in allele and genotype distribution between controls and alcoholics or between controls and alcoholics with alcohol withdrawal seizure (AWS) or delirium tremens (DT) (unadjusted p > 0.05). A total of 13 research articles were included in the meta-analyses. No significant difference of the SLC6A3 VNTR A9 was noted between controls and alcoholics at the genotypic and allelic level when all ethnic populations, only Caucasian populations, or only Asian populations were considered (p > 0.05). Significant associations were observed between SLC6A3 VNTR A9 and alcoholics with AWS or DT at the genotypic as well as allelic level when all ethnic populations or only Caucasian populations were considered (p < 0.05, OR 1.5-2.1). CONCLUSIONS: Meta-analyses suggest a possible association between the SLC6A3 VNTR A9 and alcoholic subgroup with AWS or DT.

Retinoid X receptor alpha Regulates the expression of glutathione s-transferase genes and modulates acetaminophen-glutathione conjugation in mouse liver.

Nuclear receptors, including constitutive androstane receptor, pregnane X receptor, and retinoid X receptor (RXR), modulate acetaminophen (APAP)-induced hepatotoxicity by regulating the expression of phase I cytochrome P450 (P450) genes. It has not been fully resolved, however, whether they regulate APAP detoxification at the phase II level. The aim of the current study was to evaluate the role of RXRalpha in phase II enzyme-mediated detoxification of APAP. Wild-type and hepatocyte-specific RXRalpha knockout mice were treated with a toxic dose of APAP (500 mg/kg i.p.). Mutant mice were protected from APAP-induced hepatotoxicity, even though basal liver glutathione (GSH) levels were significantly lower in mutant mice compared with those of wild-type mice. High-performance liquid chromatography analysis of APAP metabolites revealed significantly greater levels of APAP-GSH conjugates in livers and bile of mutant mice compared with those of wild-type mice. Furthermore, hepatocyte RXRalpha deficiency altered the gene expression profile of the glutathione S-transferase (Gst) family. Basal expression of 13 of 15 Gst genes studied was altered in hepatocyte-specific RXRalpha-deficient mice. This probably led to enhanced APAP-GSH conjugation and reduced accumulation of N-acetyl-p-benzoquinone imine, a toxic electrophile that is produced by biotransformation of APAP by phase I P450 enzymes. In conclusion, the data presented in this study define an RXRalpha-Gst regulatory network that controls APAP-GSH conjugation. This report reveals a potential novel strategy to enhance the detoxification of APAP or other xenobiotics by manipulating Gst activity through RXRalpha-mediated pathways.

Retinoid X receptor alpha regulates glutathione homeostasis and xenobiotic detoxification processes in mouse liver.

Retinoid X receptor alpha (RXRalpha) plays a pivotal role in regulating liver metabolism. RXRalpha-mediated gene expression involved in amino acid metabolism was examined using the NIA Mouse 15K cDNA microarray containing 15,000 different expressed sequence tags. Seven amino acid metabolic genes, three of which encode enzymes involved in phase II detoxification process, were identified as RXRalpha target genes in mouse liver. Glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferasemu, and glutathione peroxidase 1 were down-regulated in the liver of hepatocyte RXRalpha-deficient mice. The down-regulation of GCLC in RXRalpha-deficient mice led to 40% and 45% reductions in the rate of glutathione (GSH) synthesis and level of hepatic GSH, respectively. Primary hepatocytes from RXRalpha-deficient mice were more sensitive to t-butylhydroperoxide-induced oxidative stress. However, GSH diminished RXRalpha-deficient mice were resistant to acetaminophen (APAP)-induced hepatotoxicity. Analysis of phase I detoxification genes revealed that CYP1A2 and CYP3A11 were up-regulated in wild-type mice but down-regulated in RXRalpha-deficient mice after APAP administration. Taken together, the data indicate that RXRalpha centrally regulates both phase I and phase II drug metabolism and detoxification. Regulation of hepatic GSH levels by RXRalpha is essential to protect hepatocytes from oxidative stress, whereas up-regulation of phase I drug metabolism genes by RXRalpha may render the liver more sensitive to APAP-induced toxicity.

RXRalpha mRNA expression is associated with cell proliferation and cell cycle regulation in Hep3B cell.

Retinoids are well-characterized differentiation and anti-proliferation agents. The functional role of retinoid x receptor alpha (RXRalpha) in cell proliferation and cell cycle regulation is not well understood. Using human Hep3B cell line, we showed that the mRNA level of RXRalpha was closely associated with cell growth. RXRalpha mRNA expression elevated along with the proliferation of Hep3B cells. This association was most evident in RXRalpha and was also noted with retinoic acid (RA) receptor alpha (RARalpha), but not found in other RARs and RXRs. The expression of RXRalpha and cyclin A mRNA was co-regulated when Hep3B cells were cultured in serum-free medium. The mRNA levels of RXRalpha and cyclin A appeared to be highest in G1/S phase in Hep3B cells treated by aphidicolin. Taken together, our data suggest that RXRalpha may be actively involved in cell proliferation and cell cycle regulation in Hep3B cells.

RXRalpha-regulated liver SAMe and GSH levels influence susceptibility to alcohol-induced hepatotoxicity.

Retinoids influence the pathogenesis of alcohol liver disease (ALD). To analyze the impact of retinoid X receptor alpha (RXRalpha) on ALD, alcohol-induced hepatotoxicity was studied using mice fed ethanol intragastrically for 25 days. Alcohol-induced microvesicular fat around the central vein and drug-induced morphological changes (loss of rough endoplasmic reticulum, pinkish cytoplasm, and enlarged hepatocyte) in the pericentral area were observed in the liver of wild-type mice. In the hepatocyte RXRalpha-deficient mouse liver, alcohol induced fat accumulation, mitosis, acute inflammation, and necrosis. The histology score after alcohol treatment was significantly higher in mutant mice than in wild-type mice. However, drug-induced morphological changes were not apparent in alcohol-treated hepatocyte RXRalpha-deficient mice. Northern analysis showed that the basal and alcohol-induced CYP2B, CYP2A, and CYP3A mRNA levels were lower in hepatocyte RXRalpha-deficient mice than in wild-type mice, which in turn may protect mutant mice from morphological changes. Compared with wild-type mice, hepatocyte RXRalpha-deficient mice have significant lower levels of S-adenosylmethionine and glutathione, which is further reduced after alcohol treatment, and that may account for severe liver injury induced by alcohol. The overall result suggests an important role of RXRalpha in preventing alcohol-induced liver injury.

Long-term, low-dose lead exposure alters the gonadotropin-releasing hormone system in the male rat.

Lead is a male reproductive toxicant. Data suggest that rats dosed with relatively high levels of lead acetate for short periods of time induced changes in the hypothalamic gonadotropin-releasing hormone (GnRH) at the molecular level, but these changes were attenuated with increased concentration of exposure. The current study evaluated whether exposure to low levels of lead acetate over longer periods of time would produce a similar pattern of adaptation to toxicity at the molecular and biologic levels. Adult 100-day-old Sprague-Dawley male rats were dosed with 0, 0.025, 0.05, 0.1, and 0.3% lead acetate in water. Animals were killed after 1, 4, 8, and 16 weeks of treatment. Luteinzing hormone (LH) and GnRH levels were measured in serum, and lead levels were quantified in whole blood. Hypothalamic GnRH mRNA levels were also quantified. We found no significant differences in serum LH and GnRH among the groups of animals treated within each time period. A significant dose-related increase of GnRH mRNA concentrations with lead dosing occurred in animals treated for 1 week. Animals treated for more than 1 week also exhibited a significant increase in GnRH mRNA, but with an attenuation of the increase at the higher concentrations of lead with increased duration of exposure. We conclude that the signals within and between the hypothalamus and pituitary gland appear to be disrupted by long-term, low-dose lead exposure.

Activin Receptor Type II Gene Expression is Induced During Embryonic Development and Differentiation of Murine F9 Embryonal Carcinoma Cells: (activin receptor/development/endoderm/embryonal carcinoma cell/retinoic acid).

The functional role of activins in mesoderm induction has been shown in early amphibian embryos; however, its role in early mammalian embryogenesis is still unknown. The protein and mRNA for activin subunits are present in mouse preimplantation embryos. In this study we report on the expression of activin receptor type II (ActR-II) gene in pre- and post-implantation mouse embryos and the regulation of ActR-II mRNA in F9 embryonal carcinoma cells that served as a model for murine embryonic development. The results show that ActR-II gene is expressed in ovulated mature oocytes, becomes decreased at 2-celll stage, and is substantially activated in day-6 embryo at the stage when ectoderm and endoderm are formed. When F9 cells are treated with retinoic acid (RA) and undergo endodermal differentiation, ActR-II mRNA is induced. The 6.0 kb transcript is enhanced more than the 3.0 kb transcript. suggesting that these two mRNA species are controlled by different promotors. Addition of cyclic AMP analogue, which further induces differentiation of F9 cells into parietal endoderm, does not further enhance ActR-II gene expression. The effect of RA on ActR-II mRNA levels is direct and does not require ongoing protein synthesis. Furthermore, all-trans-RA is more effective than 9-cis-RA in the induction of ActR-II gene expression. The results of this study demonstrate that the ActR-II gene is activated during endodermal differentiation and suggest that activin action in mammalian embryonic development is mediated through increased activin receptor gene expression.

Differential Expression of Retinoic Acid Receptor mRNA during Mouse Embryogenesis.

Retinoic Acid, mediated through its receptors (RAR), exhibits striking effects on cell proliferation, differentiation, and pattern formation. At least three types of RARs (α, ß, and γ) have been cloned. The ontogeny and semi-quantitated amount of three RAR mRNA were studied in mouse embryos and placentas using reverse transcriptase-polymerase chain reaction technique. The amplified cDNA products were confirmed by correct sizes, Southern blot hybridization and restriction enzyme digestion. The results show that RAR-α mRNA is constitutively expressed in the oocytes and embryos of all stages, suggesting that RAR-α is a housekeeping gene. RAR-ß mRNA is present in the oocytes, becomes undetectable at the 2-cell stage when the maternal RNA is degraded, and re-expresses at morula and blastocyst stages when the embryonic genome is activated. RAR-γ mRNA is not expressed until balstocyst stage. In the postimplantation embryos, no significant change in all three RAR mRNA levels was detected from day 10 through the late gestational stage (day 18). Placental tissue expressed all three RAR transcripts throughout gestation. The differential activation of the these three RAR genes in the preimplantation embryos suggests RARs as the important regulators during early embryogenesis.