An improved in vitro method for screening toxin and medicine targeting CYP2E1.

The cytochrome P450 enzyme 2E1 (CYP2E1) presents in both microsome and mitochondrion, which influences the metabolism of many xenobiotics. The mice active liver homogenate was prepared for the medicinal incubation and mitochondrion was extracted for chemical screening targeting CYP2E1 enzyme. Representative CYP2E1 inducers (ethanol and pyrazole) and inhibitors (diallyldisulfide and kaempferol) were applied to evaluate the effectiveness of homogenate-mitochondrial system. In parallel, the in-vitro microsomal method targeting CYP2E1 was also operated for comparison. The results showed that in homogenate-mitochondrial method, the protein level and activity of CYP2E1 were increased by ethanol and pyrazole; reduced by diallyldisulfide and kaempferol, and this homogenate-mitochondrial method is convenient with good repeatability and reproducibility in screening chemicals targeting CYP2E1, especially for the inducers. Thus, the homogenate-mitochondrial method might be effective in screening both CYP2E1 inhibitor and inducer.

[Induction of rat hepatic CYP2E1 expression by arecoline in vivo].

The regulation mechanism of arecoline on rat hepatic CYP2E1 was studied in vivo. After oral administration of arecoline hydrobromide (AH; 4, 20 and 100 mg x kg(-1) x d(-1)) to rats for one week, the hepatic CYP2E1 mRNA level remained unchanged, but the hepatic CYP2E1 protein content was dose-dependently increased. Additionally, although the hepatic CYP2E1 activity was induced by AH treatment, the induction was attenuated with the increase in dosage. The results indicate that the effect of arecoline on rat hepaticdoes not involve transcriptional activation of the gene, but largely involves the stabilization of CYP2E1 protein against degradation or increased efficiency of CYP2E1 mRNA translation, and additionally involve the post- ranslational modification of CYP2E1 protein. Furthermore, the CYP2E1 response is fairly equal among the different species, the induction of rat hepatic CYP2E1 by arecoline suggests that there is a risk of metabolic interaction among the substrate drugs of CYP2E1 in betel-quid use human.

Chemical characteristics for optimizing CYP2E1 inhibition.

Cytochrome P450 2E1 (CYP2E1) expression and activity in the liver is associated with the degree of liver damage in patients with alcoholic steatohepatitis (ASH) as well as non-alcoholic steatohepatitis (NASH). CYP2E1 is known to generate reactive oxygen species, which leads to oxidative stress, one of the hallmarks of both diseases. Apart from ROS, toxic metabolites can be formed by CYP2E1 metabolism, further potentiating liver injury. Therefore, CYP2E1 is implicated in the pathogenesis of ASH and NASH. The aim of this study was to determine the chemical characteristics of compounds that are important to inhibit CYP2E1. To this end, structurally related analogs that differed in their lipophilic, steric and electronic properties were tested. In addition, homologues series of aliphatic primary alcohols, secondary alcohols, aldehydes, ketones and carboxylic acids were tested. It was found that inhibition of the CYP2E1 activity is primarily governed by lipophilicity. The optimal log D7.4 (octanol/water distribution coefficient at pH 7.4) value for inhibition of CYP2E1 was approximately 2.4. In the carboxylic acids series the interaction of the carboxylate group with polar residues lining the CYP2E1 active site also has to be considered. This study sketches the basic prerequisites in the search for inhibitors of CYP2E1, which would strengthen our therapeutic armamentarium against CYP2E1 associated diseases, such as ASH and NASH.

[Influence of omega-3 polyunsaturated fatty acids on oxidative stress and cytochrome P450 2E1 expression in rat liver].

Polyunsaturated fatty acids (PUFAs), omega-3 ones in particular, form phospholipid layer of biological membranes, which provides normal functioning of membrane-associated complexes of enzymes and transmembrane transport. Free omega-3 PUFAs regulate the transcription of many genes, and thereby have an effect on the level of metabolic processes, particularly control of lipid and carbohydrate metabolism in the liver. Cytochrome P450 2El (1.14.14.1) causes the transformation of lipophilic exogenous and endogenous substances, as well as involvement in homeostasis, both at the cellular and systemic levels. The aim is to study changes in expression of cytochrome P450 2E1, and to assess the antioxidant system and the level of peroxidation processes in the liver of experimental animals under the chronic action omega-3 PUFAs. During experiment more than two-fold increase in the content of cytochrome P450 2El was observed in the liver of rats which additionally received omega-3 PUFAs for 4 weeks in the standard daily diet. At the same time, such changes in the enzyme expression did not lead to an imbalance of pro- and antioxidant processes in the liver.

CYP2E1 induction leads to oxidative stress and cytotoxicity in glutathione-depleted cerebellar granule neurons.

Increasing evidence suggests that brain cytochrome P450 (CYP) can contribute to the in situ metabolism of xenobiotics. In the liver, some xenobiotics can be metabolized by CYPs into more reactive products that can damage hepatocytes and induce cell death. In addition, normal CYP activity may produce reactive oxygen species (ROS) that contribute to cell damage through oxidative mechanisms. CYP2E1 is a CYP isoform that can generate ROS leading to cytotoxicity in multiple tissue types. The aim of this study was to determine whether CYP2E1 induction may lead to significant brain cell impairment. Immunological analysis revealed that exposure of primary cerebellar granule neuronal cultures to the CYP inducer isoniazid, increased CYP2E1 expression. In the presence of buthionine sulfoximine, an agent that reduces glutathione levels, isoniazid treatment also resulted in reactive oxygen species (ROS) production, DNA oxidation and cell death. These effects were attenuated by simultaneous exposure to diallyl sulfide, a CYP2E1 inhibitor, or to a mimetic of superoxide dismutase/catalase, (Euka). These results suggest that in cases of reduced antioxidant levels, the induction of brain CYP2E1 could represent a risk of in situ neuronal damage.

Alcoholic liver disease: a synopsis of the Charles Lieber's Memorial Symposia 2009-2012.

This paper is based upon the 'Charles Lieber Satellite Symposia' organized by Manuela G. Neuman at each of the 2009-2012 Research Society on Alcoholism (RSA) Annual Meetings. The presentations represent a broad spectrum dealing with alcoholic liver disease (ALD). In addition, a literature search (2008-2013) in the discussed area was performed in order to obtain updated data. The presentations are focused on genetic polymorphisms of ethanol metabolizing enzymes and the role of cytochrome P4502E1 (CYP2E1) in ALD. In addition, alcohol-mediated hepatocarcinogenesis, immune response to alcohol and fibrogenesis in alcoholic hepatitis as well as its co-morbidities with chronic viral hepatitis infections in the presence or absence of human deficiency virus are discussed. Finally, emphasis was led on alcohol and drug interactions as well as liver transplantation for end-stage ALD.

Cryopreserved porcine hepatocytes: expression and induction of cytochrome P450, isoform CYP2E1.

Cryopreservation of porcine hepatocytes for their use in bioartificial liver devices may result in reduced cytochrome P450 (CYP) enzyme activity. The aim of this study was to assess the effects of several CYP inducers on the isoform CYP2E1 protein expression in cryopreserved porcine hepatocytes. Isolated porcine hepatocytes were cryopreserved for 1 month, thawed, and cultured for 3 days. During medium culture, the hepatocytes were exposed to the following CYP inducers: dimethyl sulfoxide, rifampicin, phenobarbital, 3-methylcholanthrene, and dexamethasone. CYP2E1 protein expression was determined by immunoblotting. CYP2E1 protein levels were constantly detected in cryopreserved porcine hepatocytes. CYP inducers did not modify CYP2E1 protein levels. Long-term cryopreserved porcine hepatocytes preserved their capacity for CYP2E1 protein expression, although exposure of these hepatocytes to CYP inducers did not modify the CYP2E1 protein expression.