Background The hepatic endocannabinoid system and cytochrome P450 2E1 (CYP2E1), a key enzyme causing alcohol-induced reactive oxygen species (ROS) generation, are major contributors to the pathogenesis of alcoholic liver disease. The nuclear hormone receptor oestrogen-related receptor γ (ERRγ) is a constitutively active transcriptional activator regulating gene expression. Objective To investigate the role of ERRγ in the alcohol-mediated regulation of CYP2E1 and to examine the possibility to control alcohol-mediated oxidative stress and liver injury through an ERRγ inverse agonist. Design For chronic alcoholic hepatosteatosis study, C57BL/6J wild-type and CB1−/− mice were administered alcohol for 4 weeks. GSK5182 and chlormethiazole (CMZ) were given by oral gavage for the last 2 weeks of alcohol feeding. Gene expression profiles and biochemical assays were performed using the liver or blood of mice. Results Hepatic ERRγ gene expression induced by alcohol-mediated activation of CB1 receptor results in induction of CYP2E1, while liver-specific ablation of ERRγ gene expression blocks alcohol-induced expression of CYP2E1 in mouse liver. An ERRγ inverse agonist significantly ameliorates chronic alcohol-induced liver injury in mice through inhibition of CYP2E1-mediated generation of ROS, while inhibition of CYP2E1 by CMZ abrogates the beneficial effects of the inverse agonist. Finally, chronic alcohol-mediated ERRγ and CYP2E1 gene expression, ROS generation and liver injury in normal mice were nearly abolished in CB1−/− mice. Conclusions ERRγ, as a previously unrecognised transcriptional regulator of hepatic CB1 receptor, controls alcohol-induced oxidative stress and liver injury through CYP2E1 induction, and its inverse agonist could ameliorate oxidative liver injury due to chronic alcohol exposure.

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Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in E. coli each of the woodrat proteins gave the characteristic maximum at 450 nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.

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The titrated extract of Centella asiatica (TECA) is a reconstituted mixture comprising of asiatic acid, madecassic acid, asiaticoside and madecassoside, and is used as a therapeutic agent in wound healing and also as an anti-microbial, anticancer and anti-aging agent. Although these properties and the associated cell signaling pathways have been elucidated, the cellular mechanism of anti-photoaging upon ultraviolet (UV) exposure in normal human dermal fibroblasts (NHDFs) remains unknown. In this study, we investigated the photoprotective role of TECA via microRNA (miRNA) expression profiling analysis. Low dose of TECA did not exhibit toxicity and showed a protective effect against UVB irradiation in NDHFs. miRNA microarray experiments revealed that specific miRNAs were altered by TECA stimulation in UVB-irradiated NHDFs. Functional bioinformatic analysis showed that the putative target genes of the altered miRNAs were associated with the positive regulation of cell proliferation, anti-apoptosis, small GTPase- and Ras-mediated signal transduction and activation of MAPKK. Therefore, these results suggest that TECA may serve as a potential natural chemoprotective agent against UVB-mediated damage in NHDFs through changes in the expression of specific miRNAs.

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1·CPR complex and inactive (CYP2B1) 2 ·CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a nonbinding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners.

The aim of this study was to investigate the mechanisms by which troxerutin protects cells against ultraviolet B (UVB) radiation. First, we demonstrate that pre-treatment with troxerutin protects normal human dermal fibroblasts (nHDFs) against UVB-induced cytotoxicity. As shown by migration assay and DNA repair analysis, troxerutin increased cell migration and DNA repair activity in the nHDFs. Subsequently, we analyzed microRNA (miRNA) expression profiles in the nHDFs. miRNAs are 19- to 24-nucleotide (nt) non-coding RNA molecules that regulate the translation of target genes through RNA interference. In UVB-exposed cells, miRNAs act on crucial functions, such as apoptosis and cellular senescence. miRNA expression is significantly altered during the protective process induced by phytochemicals. Therefore, understanding changes that occur in miRNA expression profiles may help to elucidate the protective mechanisms of troxerutin. We identified 11 miRNAs that were significantly (>2-fold) upregulated and 12 that were significantly downregulated (>2-fold) following treatment of the nHDFs with troxerutin. In addition, we investigated the biological functions of these miRNAs through the prediction of miRNA targets and Gene Ontology analysis of the putative targets. Overall, our findings indicate that pre-treatment with troxerutin increases the viability of UVB-exposed nHDFs through the alteration of the miRNA expression profiles.

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