Epoxidation of perillyl alcohol by engineered bacterial cytochrome P450 BM3.

Perillyl alcohol (POH) is a secondary metabolite of plants. POH and its derivatives are known to be effective as an anticancer treatment. In this study, oxidative derivatives of POH, which are difficult to synthesize chemically, were synthesized using the engineered bacterial cytochrome P450 BM3 (CYP102A1) as a biocatalyst. The activity of wild-type (WT) CYP102A1 and 29 engineered enzymes toward POH was screened using a high-performance liquid chromatography. They produced one major product. Among them, the engineered CYP102A1 M601 mutant with seven mutations (R47L/F81I/F87V/E143G/L150F/L188Q/E267V) showed the highest conversion, 6.4-fold higher than the WT. Structure modeling using AlphFold2 and PyMoL suggests that mutations near the water channel may be responsible for the increased catalytic activity of the M601 mutant. The major product was identified as a POH-8,9-epoxide by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis. The optimal temperature and pH for the product formation were 35 °C and pH 7.4, respectively. The kcat and Km of M601 were 540 min-1 and 2.77 mM, respectively. To improve POH-8,9-epoxide production, substrate concentration and reaction time were optimized. The optimal condition for POH-8,9-epoxide production by M601 was 5.0 mM POH, pH 7.4, 35 ℃, and 6 h reaction, which produced the highest concentration of 1.72 mM. Therefore, the biosynthesis of POH-8,9-epoxide using M601 as a biocatalyst is suggested to be an efficient and sustainable synthetic process that can be applied to chemical and pharmaceutical industries.

3-OH Phloretin Inhibits High-Fat Diet-Induced Obesity and Obesity-Induced Inflammation by Reducing Macrophage Infiltration into White Adipose Tissue.

Phloretin and its glycoside phlorizin have been reported to prevent obesity induced by high-fat diet (HFD), but the effect of 3-OH phloretin, a catechol metabolite of phloretin, has not been investigated. In this study, we investigated the anti-obesity effects of phloretin and 3-OH phloretin in HFD-fed mice. The body weight gain induced by HFD was more inhibited by administration of 3-OH phloretin than by phloretin. The increases in fat mass, white adipose tissue (WAT) weight, adipocyte size, and lipid accumulation by HFD were also remarkably inhibited by 3-OH phloretin and, to a lesser extent, by phloretin. The HFD-induced upregulation of chemokines and pro-inflammatory cytokines was suppressed by 3-OH phloretin, preventing M1 macrophages from infiltrating into WAT and thereby reducing WAT inflammation. 3-OH phloretin also showed a more potent effect than phloretin on suppressing the expression of adipogenesis regulator genes, such as PPARγ2, C/EBPα, FAS, and CD36. Fasting blood glucose and insulin levels increased by HFD were diminished by the administration of 3-OH phloretin, suggesting that 3-OH phloretin may alleviate obesity-induced insulin resistance. These findings suggested that 3-OH phloretin has the potential to be a natural bioactive compound that can be used in the prevention or treatment of obesity and insulin resistance.

Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration.

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP+ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.

P450-driven plastic-degrading synthetic bacteria.

Plastic contamination currently threatens a wide variety of ecosystems and presents damaging repercussions and negative consequences for many wildlife species. Sustainable plastic waste management is an important approach to environmental protection and a necessity in the current life cycle of plastics in nature. Plastic biodegradation by microorganisms is a notable possible solution. This opinion article includes a proposal to use hypothetical P450 enzymes with an engineered active site as potent trigger biocatalysts to biodegrade polyethylene (PE) via in-chain hydroxylation into smaller products of linear aliphatic alcohols and alkanoic acids based on cascade enzymatic reactions. Furthermore, we propose the adoption of P450 into plastic-eating synthetic bacteria for PE biodegradation. This strategy can be applicable to other dense plastics, such as polypropylene (PP) and polystyrene (PS).

Enzymatic Production of 3-OH Phlorizin, a Possible Bioactive Polyphenol from Apples, by Bacillus megaterium CYP102A1 via Regioselective Hydroxylation.

Phlorizin is the most abundant glucoside of phloretin from the apple tree and its products. Phlorizin and its aglycone phloretin are currently considered health-beneficial polyphenols from apples useful in treating hyperglycemia and obesity. Recently, we showed that phloretin could be regioselectively hydroxylated to make 3-OH phloretin by Bacillus megaterium CYP102A1 and human P450 enzymes. The 3-OH phloretin has a potent inhibitory effect on differentiating 3T3-L1 preadipocytes into adipocytes and lipid accumulation. The glucoside of 3-OH phloretin would be a promising agent with increased bioavailability and water solubility compared with its aglycone. However, procedures to make 3-OH phlorizin, a glucoside of 3-OH phloretin, using chemical methods, are not currently available. Here, a biocatalytic strategy for the efficient synthesis of a possibly valuable hydroxylated product, 3-OH phlorizin, was developed via CYP102A1-catalyzed regioselective hydroxylation. The production of 3-OH phlorizin by CYP102A1 was confirmed by HPLC and LC-MS spectroscopy in addition to enzymatic removal of its glucose moiety for comparison to 3-OH phloretin. Taken together, in this study, we found a panel of mutants from B. megaterium CYP102A1 could catalyze regioselective hydroxylation of phlorizin to produce 3-OH phlorizin, a catechol product.

Cyclophilin A is an endogenous ligand for the triggering receptor expressed on myeloid cells-2 (TREM2).

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor expressed on macrophages, microglial cells, and pre-osteoclasts, and that participates in diverse cellular function, including inflammation, bone homeostasis, neurological development, and coagulation. In spite of the indispensable role of the TREM2 protein in the maintenance of immune homeostasis and osteoclast differentiation, the exact ligand for TREM2 has not yet been identified. Here, we report a putative TREM2 ligand which is secreted from MC38 cells and identified as a cyclophilin A (CypA). A specific interaction between CypA and TREM2 was shown at both protein and cellular levels. Exogenous CypA specifically interacted and co-localized with TREM2 in RAW264.7 cells, and the physical interactions were shown to regulate TREM2 signaling transduction. The Pro144 residue in the extracellular domain of TREM2 was found to be the specific binding site of CypA. When considered together, this provides evidence that CypA interacts specifically with TREM2 as a potent ligand.

Promoted ABA Hydroxylation by Capsicum annuum CYP707As Overexpression Suppresses Pollen Maturation in Nicotiana tabacum.

Abscisic acid (ABA) is a key signaling molecule that mediates plant response to stress. Increasing evidence indicates that ABA also regulates many aspects of plant development, such as seed germination, leaf development, and ripening. ABA metabolism, including ABA biosynthesis and degradation, is an essential aspect of ABA response in plants. In this study, we identified four cytochrome P450 genes (CaCYP707A1, 2, 3, and 4) that mediate ABA hydroxylation, which is required for ABA degradation in Capsicum annuum. We observed that CaCYP707A-mediated ABA hydroxylation promotes ABA degradation, leading to low levels of ABA and a dehydration phenotype in 35S:CaCYP707A plants. Importantly, seed formation was strongly inhibited in 35S:CaCYP707A plants, and a cross-pollination test suggested that the defect in seed formation is caused by improper pollen development. Phenotypic analysis showed that pollen maturation is suppressed in 35S:CaCYP707A1 plants. Consequently, most 35S:CaCYP707A1 pollen grains degenerated, unlike non-transgenic (NT) pollen, which developed into mature pollen grains. Together our results indicate that CaCYP707A mediates ABA hydroxylation and thereby influences pollen development, helping to elucidate the mechanism underlying ABA-regulated pollen development.

Regioselective Hydroxylation of Phloretin, a Bioactive Compound from Apples, by Human Cytochrome P450 Enzymes.

Phloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min-1, respectively. However, CYP3A4 has a 3.3-fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h-1 productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6ß-hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.

Axl is a key regulator of intestinal γδ T-cell homeostasis.

Gut-homing γδ T cells are induced by chemokines and cell adhesion molecules and play a critical role in homeostasis and mucosal immunity; however, little is known regarding their upstream regulators. We investigated the role of Axl as a specific regulator of chemokines and cell adhesion molecule in the distribution of intestinal γδ T cells. The population of γδ T-cell receptor-positive cells including Vγ1 and Vγ7 subsets was remarkably increased in the intraepithelial lymphocytes of Axl-/- mice compared with those of wild-type (WT) mice. An increased number of migrated γδ T cells were observed in the coculture with intraepithelial cells from Axl-/- mice. The mRNA expression level of chemokine (C-C motif) ligand (CCL) 25 was specifically higher in the small intestine of Axl-/- mice than in WT mice. In adoptive transfer, the migration of both thymic and extrathymic γδ T cells was increased in Axl-/- mice. The activation of Axl signaling down-regulated CCL25 expression via ERK signaling pathway and reduced the population of γδ T cells. Systemic dissemination was suppressed in Axl-/- mice infected with Salmonella typhimurium. Thus, our findings suggest that Axl plays a critical role in regulating the migration of γδ T cells for the maintenance of homeostasis and bacterial resistance.-Kim, S.-M., Park, M., Yee, S.-M., Ji, K.-Y., Lee, E.-H., Nguyen, T.-V., Nguyen, T. H.-L., Jang, J., Kim, E.-M., Choi, H.-R., Yun, C.-H., Kang, H.-S. Axl is a key regulator of intestinal γδ T-cell homeostasis.

TREM2 Acts as a Tumor Suppressor in Colorectal Carcinoma through Wnt1/ß-catenin and Erk Signaling.

TREM2 (triggering receptor expressed on myeloid cells) is involved in the development of malignancies. However, the function of TREM2 in colorectal cancer has not been clearly elucidated. Here, we investigated TREM2 function for the first time in colorectal epithelial cancer cells and demonstrated that TREM2 is a novel tumor suppressor in colorectal carcinoma. Blockade of TREM2 significantly promoted the proliferation of HT29 colorectal carcinoma cells by regulating cell cycle-related factors, such as p53 phosphorylation and p21 and cyclin D1 protein levels. HT29 cell migration was also increased by TREM2 inhibition via MMP9 (matrix metalloproteinase 9) expression upregulation. Furthermore, we found that the tumor suppressor effects of TREM2 were associated with Wnt/ß-catenin and extracellular signal-regulated kinase (ERK) signaling. Importantly, the effect of TREM2 in the suppression of tumor development was demonstrated by in vivo and in vitro assays, as well as in human colon cancer patient tissue arrays. Overall, our results identify TREM2 as a potential prognostic biomarker and therapeutic target for colorectal cancer.

Extracts from Erythronium japonicum and Corylopsis coreana Uyeki reduce 1,3-dichloro-2-propanol-mediated oxidative stress in human hepatic cells.

In this study, it was demonstrated that 1,3-dichloro-2-propanol (1,3-DCP) induced oxidative stress and cell death in HuH7, human hepatocytes. The protective effects of Erythronium japonicum (E. japonicum) and Corylopsis coreana Uyeki (C. coreana Uyeki) extracts against 1,3-DCP-treated cells were also investigated. First, the activities of superoxide dismutase (SOD) and catalase (CAT) were diminished by the treatment of 1,3-DCP. Moreover, 1,3-DCP stimulated the expression and catalytic activity of cytochrome P450 2E1 (CYP2E1), an enzyme that generates reactive oxygen species in the liver. In contrast, co-treatment of 1,3-DCP with the extracts significantly decreased ROS generation and inhibited CYP2E1 activity without affecting its expression. The co-administration of extracts also restored the activities of SOD and CAT reduced by 1,3-DCP and protected against 1,3-DCP-mediated cell death. In conclusion, these results suggest that 1,3-DCP induces oxidative stress through the elevated CYP2E1 level, which is inhibited by the extracts, protecting cells against the effects of 1,3-DCP.

Structural and biochemical analyses reveal ubiquitin C-terminal hydrolase-L1 as a specific client of the peroxiredoxin II chaperone.

Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.

Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition.

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.

Cargo-Free Nanoparticles Containing Cationic Lipids Induce Reactive Oxygen Species and Cell Death in HepG2 Cells.

Nanoparticles (NPs) containing cationic monovalent lipids such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA), have been widely used for the delivery of nucleic acid such as small-interfering RNA and polypeptide to cells as cancer therapies and vaccine development. Several previous reports have suggested that cationic liposomes induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. Here, we systematically investigated the effects of DOTAP- or DOTMA-containing NPs without any cargo on the human carcinoma cells, HepG2. Treatment with NPs containing DOTAP or DOTMA increased the production of cellular ROS, such as H2O2 and lipid peroxidation, in HepG2 cells and concomitantly decreased cell viability. These effects were dependent on the lipid concentration, surface density of cationic lipids, and particle size of NPs. However, neutral NPs consisting of 1,2-dioleoyl-3-phosphocholine did not elicit the effective ROS generation or cell death regardless of the lipid concentration and particle size. The present study suggests that DOTAP- and DOTMA-NPs are able to induce cancer cell death through production of ROS in the absence of any therapeutic cancer reagents. These results also provide a rational background for the design of delivery systems using cationic lipid-based NP formulations.

Cofactor-free light-driven whole-cell cytochrome P450 catalysis.

Cytochromes P450 can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. Here, we have designed a novel light-driven platform for cofactor-free, whole-cell P450 photo-biocatalysis using eosin Y (EY) as a photosensitizer. EY can easily enter into the cytoplasm of Escherichia coli and bind specifically to the heme domain of P450. The catalytic turnover of P450 was mediated through the direct transfer of photoinduced electrons from the photosensitized EY to the P450 heme domain under visible light illumination. The photoactivation of the P450 catalytic cycle in the absence of cofactors and redox partners is successfully conducted using many bacterial P450s (variants of P450 BM3) and human P450s (CYPs 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) for the bioconversion of different substrates, including marketed drugs (simvastatin, lovastatin, and omeprazole) and a steroid (17ß-estradiol), to demonstrate the general applicability of the light-driven, cofactor-free system.

Bacterial ß-(1,3)-glucan prevents DSS-induced IBD by restoring the reduced population of regulatory T cells.

Bacterial ß-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield ß-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial ß-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial ß-(1,3)-glucan at daily doses of 2.5 or 5mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial ß-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1ß, IL-6 and IL-17A/F, were markedly decreased in the colon of ß-(1,3)-glucan-pretreated mice. ß-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, ß-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial ß-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production.

Regioselective hydroxylation of 17ß-estradiol by mutants of CYP102A1 from Bacillus megaterium.

A large set of mutants of CYP102A1 from Bacillus megaterium have human cytochrome P450-like activities and the ability to metabolize a number of marketed drugs and steroids. Here, we tested whether the CYP102A1 mutants could be used to produce hydroxylated human metabolites of 17ß-estradiol (E2). A set of the mutants, which were generated by site-directed and random mutagenesis, was used to produce hydroxylated human metabolites of E2 in this study. Some of the tested mutants could regioselectively generate 2-OH E2 as a major metabolite but not other hydroxylated products. These results suggest that CYP102A1 mutants would be useful for the bioconversion of steroid hormones to hydroxylated products, which can be used for industrial applications.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Decreased level of albumin in peripheral blood mononuclear cells of streptozotocin-induced diabetic rats.

We investigated the phenotypic level of albumin in peripheral blood mononuclear cells (PBMC) of streptozotocin (STZ)-induced diabetic rats. A specific reduction of albumin was identified by 2-dimensional electrophoresis and mass spectrometry. Decreased albumin content was also confirmed by immunoblotting and quantitative real-time PCR. Since albumin is a major and predominant antioxidant in plasma, the PBMC albumin may also contribute to their antioxidant activity. By measuring the amount of H2O2, lipid peroxidation and the redox form of glutathione, it was found that the production of the oxidative stress was elevated in STZ-diabetic rats compared to that of normal control. We suggest, therefore, that decreased albumin content may lead to the decreased antioxidant activity in the PBMC of type 1 diabetic rats.

Estrogen-related receptor γ controls hepatic CB1 receptor-mediated CYP2E1 expression and oxidative liver injury by alcohol.

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.