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Cytochrome P450 2E1 (CYP2E1) gene expression is known to be induced by interleukin-4 (IL4) and repressed by inflammatory cytokines, such as interleukin-1β3 (IL1β3) in human hepatocytes. The mechanisms involved in these transcriptional regulations remain elusive. In order to study these mechanisms, various constructs of the human CYP2E1 promoter were prepared and transfected into the human HepG2 hepatoma cell line. Our findings revealed that an IL4-responsive region of 128 bp (-671/-544) was required to mediate induction by IL4. IL1β caused moderate but significant decrease of the promoter activity, which was abolished when the two cytokines were combined. The IL1β inhibitory effect is mediated through a regulatory sequence independent of that of IL4. Furthermore, by using specific signaling pathway inhibitors, we demonstrated that IL4 activation required protein kinase C (PKC) activation. In addition, our results suggest that induction by IL4 was not dependent on a single binding site but rather on a complex region which includes putative binding sites for signal transducer and activator of transcription (STAT)6, activator protein (AP)-1, nuclear factor kappa-B (NFκB), nuclear factor of activated T cells (NFAT) and CCAAT enhancer binding protein (C/EBP). Electrophoretic mobility shift assays suggest that AP1 and NFAT transcription factors are able to bind to three sites in the IL4-responsive region. [Copyright &y& Elsevier]

Individuals, typically, are exposed to mixtures of environmental xenobiotics affecting multiple organs and acting through different xenosensors and pathways in species and cell-type specific manners. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and α-endosulfan are Persistent Organic Pollutants (POPs) and endocrine disruptors which act through different xenosensors and accumulate in the liver. Our objective in this HEALS study was to investigate the effects of the mixture of these POPs on gene expression in a human-derived hepatocyte cell line, HepaRG. We found that, in spite of having largely uncorrelated effects, TCDD and α-endosulfan, when mixed, alter the expression of genes. The combined effects of the mixture of the POPs significantly altered the expression of 100 genes (42 up- and 58 down-regulated) whereas the same concentration of either POP alone did not alter significantly the expression of these genes. For 32 other genes, selective inhibitory crosstalk between TCDD and α-endosulfan was observed. One of the POPs inhibited the effect, on gene expression, of the other in the mixture although, when used alone, that POP did not affect expression. The expression of another 82 genes was significantly altered (up- or down-regulated) by a single POP. The addition of the second POP either increased, in the same direction, the effect on gene expression or had no further effect. At low concentrations (0.2 nM TCDD and 1 μM α-endosulfan), the POPs still had significant effects and the levels of expression of the corresponding proteins were found to be affected for some genes. Particularly striking was the 80–90% inhibition, by the mixture, of the expression of a number of genes of several hepatic intermediary metabolic pathways (glycerolipid metabolism, FXR/RXR activation, glycolysis/gluconeogenesis, retinoid and bile acid biosynthesis), whereas each pollutant alone had only a moderate effect. [ABSTRACT FROM AUTHOR]

Abstract: In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) α agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPARα agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at −574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum. [Copyright &y& Elsevier]

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) activator, increases the expression of the cytosolic aspartate aminotransferase (cAspAT) gene in human liver cells, which may partially explain the increase of this enzyme in the serum of individuals undergoing fenofibrate treatment. Conversely, in rodents, fenofibrate represses the expression of the cAspAT gene. We compared the mechanisms of fenofibrate action in human and rat hepatoma cells. Transfection assays of the wild-type and mutated rat promoters in Fao and H4IIEC3 cells established a critical role for sequences similar to nuclear receptor responsive elements in the −404/−366 bp region. Nuclear proteins bound to these sequences and the amounts of protein bound were decreased by fenofibrate treatment, probably accounting for the decreased gene expression. Pharmacological studies confirmed the involvement of PPARα. However, this receptor did not bind directly to these sequences. The human promoter was cloned and the regulatory region localized between −2663/−706 bp. Co-transfection assays suggested that, in humans, the PPARα was also involved in the increase in expression of the cAspAT gene due to fibrates, without the presence of a canonical PPAR responsive element. [Copyright &y& Elsevier]

Individuals as well as entire ecosystems are exposed to mixtures of Persistent Organic Pollutants (POPs). Previously, we showed, by a non-targeted approach, that the expression of several genes involved in carbohydrate metabolism was almost completely inhibited in the human hepatic cell line HepaRG following exposure to a mixture of the organochlorine insecticide alpha-endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin. In this European HEALS project, which studies the effects of the exposome on human health, we used a Physiologically Based BioKinetic model to compare the concentrations previously used in vitro with in vivo exposures for humans. We investigated the effects of these POPs on the levels of proteins, on glycogen content, glucose production and the oxidation of glucose into CO 2 and correlated them to the expression of genes involved in carbohydrate metabolism as measured by RT-qPCR. Exposure to individual POPs and the mixture decreased the expression of the proteins investigated as well as glucose output (up to 82%), glucose oxidation (up to 29%) and glycogen content (up to 48%). siRNAs that specifically inhibit the expression of several xenobiotic receptors were used to assess receptor involvement in the effects of the POPs. In the HepaRG model, we demonstrate that the effects are mediated by the aryl hydrocarbon receptor and the estrogen receptor alpha, but not the pregnane X receptor or the constitutive androstane receptor. These results provide evidence that exposure to combinations of POPs, acting through different signaling pathways, may affect, more profoundly than single pollutants alone, metabolic pathways such as carbohydrate/energy metabolism and play a potential role in pollutant associated metabolic disorders. • TCDD and α-endosulfan mixture decreases carbohydrate metabolism in liver cells. • PBBK model of TCDD and α-endosulfan in human liver. • α-endosulfan acts via estrogen α receptor in HepaRG cells. • Glucose oxidation and production and glycogen content are decreased by the mixture. [ABSTRACT FROM AUTHOR]