CYP2E1 Content Research Articles

Schisandrin C improves acetaminophen-induced liver injury in mice by regulating Nrf2 signaling pathway

Excess acetaminophen(APAP) can be converted by the cytochrome P450 system to the toxic metabolite N-acetyl-p-benzoquinoneimine(NAPQI), which consumes glutathione(GSH). When GSH is depleted, NAPQI covalently binds with proteins, inducing mitochondrial dysfunction and oxidative stress and thereby leading to hepatotoxicity. Schisandrin C(SinC) is a dibenzocyclooctadiene derivative isolated from Schisandra chinensis. Although there is some evidence showing that SinC has hepatoprotective activity, its protective effect and mechanism on APAP-induced liver injury remain unclear. In this paper, an acute liver injury mouse model was established by intraperitoneal injection of APAP at a dose of 400 mg·kg~(-1) to evaluate the effect of SinC administration on the APAP-induced liver injury and its mechanism through an animal experiment. At the same time, a potential candidate drug was provi-ded for traditional Chinese medicine(TCM) prevention and treatment of overdose APAP-induced liver injury. In the APAP-induced liver injury mouse model, we found that SinC can relieve hepatic histopathological lesions and significantly reduce the activities of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP). It was also capable of increasing the content of GSH and superoxide dismutase(SOD) and decreasing the levels of total bilirubin(TBIL), direct bilirubin(DBIL), malondialdehyde(MDA), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α). Further analysis showed that SinC decreased the content of CYP2 E1 in liver tissues at protein and mRNA levels and increased nuclear factor erythroid 2-related factor 2(Nrf2) and the expression of its downstream targets(including HO-1, NQO1 and GCLC). Taken together, the above results indicate that SinC can alleviate APAP-induced liver injury by reducing the expression of CYP2 E1, suppressing apoptosis, improving inflammatory response and activating the Nrf2 signaling pathway to inhibit oxidative stress.

Time-dependent regulation of hepatic cytochrome P450 mRNA in male liver-specific PGC-1α knockout mice

The circadian rhythm has profound effect on the body, exerting effects on diverse events like sleep-wake patterns, eating behavior and hepatic detoxification. The cytochrome p450 s (Cyps) is the main group of enzymes responsible for detoxification. However, the underlying mechanisms behind circadian regulation of the Cyps are currently not fully clarified. Therefore, the aim of the present study was to investigate the requirement of hepatic peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) for the circadian regulation of the hepatic expression of Cyp1−4 using liver-specific PGC-1α knockout (LKO) mice and littermate controls. The circadian regulator genes Bmal1 and Clock displayed decreased mRNA content at zeitgeber time (ZT) 12, compared to ZT-2 and the mRNA content of Cyp2a4 and Cyp2e1 was higher at ZT-12 than at ZT-2. Moreover, the increase in Cyp2e1 mRNA content was not observed in the PGC-1α LKO mice and hepatic PGC-1α deficiency tended to blunt the rhythmic expression of Clock and Bmal1. However, no circadian regulation was evident at the protein level for the investigated Cyps except for a change in Cyp2e1 protein content in the LKO mice. Of the measured transcription factors, only, the mRNA content of peroxisome proliferator-activated receptor α, showed rhythmic expression. To further analyze the difference between the control and LKO mice, principal component analysis were executed on the mRNA data. This demonstrated a clear separation of the experimental groups with respect to ZT and genotype. Our finding provides novel insight into the role of hepatic PGC-1α for basic and circadian expression of Cyps in mouse liver. This is important for our understanding of the molecular events behind circadian Cyp regulation and hence circadian regulation of hepatic detoxification capacity.

Probing functional interactions between cytochromes P450 with principal component analysis of substrate saturation profiles and targeted proteomics

We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics. This analysis, complemented with the assays with specific inhibitors of CYP3A4 and CYP2C19, the primary C152 metabolizers, demonstrated a significant contrast between different HLM samples. To unveil the source of these differences, we implemented Principal Component Analysis (PCA) of the SSP series obtained with HLM samples with a known composition of the P450 pool. Our analysis revealed that the parameters of C152 metabolism are primarily determined by the content of CYP2A6, CYP2B6, CYP2C8, CYP2E1, and CYP3A5 of those only CYP2B6 and CYP3A5 can metabolize C152. To validate this finding, we studied the effect of enriching HLM with CYP2A6, CYP2E1, and CYP3A5. The incorporation of CYP3A5 into HLM decreases the rate of C152 metabolism while increasing the role of CYP2B6 in its turnover. In contrast, incorporation of CYP2A6 and CYP2E1 reroutes the C152 demethylation towards some P450 enzyme with a moderate affinity to the substrate, most likely CYP3A4. Our results reveal a sharp non-additivity of the individual P450 properties and suggest a pivotal role of P450–P450 interactions in determining drug metabolism routes. This study demonstrates the high potential of our new PCA-based approach in unveiling functional interrelationships between different P450 species.

Why Hepatic CYP2E1-Elevation by Itself Is Insufficient for Inciting NAFLD/NASH: Inferences from Two Genetic Knockout Mouse Models.

Simple SummaryCytochrome P450 CYP2E1 is an enzyme engaged in the breakdown of various compounds (drugs, carcinogens, dietary nutrients and toxins) as well as endogenous compounds (steroids and fatty acids), resulting in both detoxification and elimination of these agents. Often, it can also convert these agents, i.e., Acetaminophen, into more toxic products. Elevated liver CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While liver CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings of elevated liver CYP2E1 content in two genetic mouse models with impaired degradation and thus a disrupted disposal of CYP2E1 when fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, despite comparable hepatic CYP2E1 elevation and functional stabilization in these two models, NAFLD/NASH was only observed in the mouse livers that exhibited concurrently enhanced liver fat production. These findings reinforce the notion that in addition to elevated liver CYP2E1 content, CYP2E1-mediated NAFLD/NASH requires liver fat accumulation derived from either enhanced liver fat-production or ingestion of a high fat/high carbohydrate diet.Hepatic cytochrome P450 CYP2E1 is an enzyme engaged in the metabolic biotransformation of various xenobiotics and endobiotics, resulting in both detoxification and/or metabolic activation of its substrates to more therapeutic or toxic products. Elevated hepatic CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While hepatic CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings in two mouse models of E3 ubiquitin ligase genetic ablation fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, albeit comparable hepatic CYP2E1 elevation and functional stabilization in these two models upon E3 ubiquitin ligase genetic ablation and consequent disruption of its ubiquitin-dependent proteasomal degradation, NAFLD/NASH was only observed in the mouse livers that exhibited concurrent SREBP1c-transcriptional upregulation of hepatic lipogenesis. These findings reinforce the critical complicity of an associated prolipogenic scenario induced by either an inherently upregulated hepatic lipogenesis or a high fat/high carbohydrate diet in CYP2E1-mediated NAFLD/NASH.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Crosstalk between CYP2E1 and CYP3A enzymes and its Possible Involvement in Alcohol‐Drug Interactions.

The core of the drug‐metabolizing ensemble in human liver is formed by the ensemble of different P450 enzymes co‐localized in the membrane of endoplasmic reticulum (ER). There is an emerging recognition of the fact that multiple P450 species interact with each other with the formation of heteromeric complexes, where the catalytic properties of the individual enzymes are modified. Functional consequences of these interactions are likely to be responsible for the lack of a straight correlation between the composition of the P450 ensemble and the profile of drug metabolism. Of particular importance are the changes in P450‐P450 crosstalk induced by alcohol consumption. Although the multi‐fold increase in the content of CYP2E1 in liver of alcohol consumers is well documented, the involvement of CYP2E1 in alcohol‐drug interactions is considered insignificant due to a minor role of this enzyme in drug metabolism. However, the effects of induction of CYP2E1 may also involve its effects on the functional properties of other drug‐metabolizing enzymes.In order to probe the crosstalk between CYP2E1 and other P450 species and elucidate its role in alcohol‐drug interactions we established a model of alcohol‐induced increase in CYP2E1 content that implements incorporation of purified CYP2E1 into human liver microsomes (HLM). The adopted CYP2E1 becomes a fully‐functional member of the drug‐metabolizing ensemble and interacts with other P450 enzymes with the formation of heteromeric complexes (Davydova et al. 2019, https://doi.org/10.1101/685545). Investigating the impact of CYP2E1 on CYP3A enzymes, we found that the enrichment of HLM with CYP2E1 results in an increased rate of metabolism of 7‐benzyloxyquinoline (BQ) and eliminates the homotropic cooperativity observed with this CYP3A‐specific substrate. To probe the physiological relevance of these effects we compared three pulled preparations of HLM from normal donors (N‐HLM) with a preparation obtained from heavy alcohol consumers (A‐HLM). The content of major P450 species in the samples was characterized with mass‐spectroscopic determination of their specific peptides. The content of CYP2E1 in N‐HLM samples was from 2 to 5 lower than that found in A‐HLM. In contrast, the content of CYP3A in A‐HLM was the lowest among all four preparations. Despite the lower content of CYP3A, A‐HLM exhibited much higher rate of metabolism and lower homotropic cooperativity with BQ, similar to what is observed in CYP2E1‐enriched N‐HLM. Incorporation of additional CYP2E1 into A‐HLM results in a further increase in the rate of BQ metabolism that makes it an order of magnitude higher than in the N‐HLM sample with a similar CYP3A content. Importantly, the CYP2E1‐dependent activation observed with BQ, a highly specific substrate of CYP3A, is not observable with 7‐dimethylamino‐4‐trifluoromethyl coumarin (Coumarin 152), a substrate preferentially metabolized by CYP3A, but capable of high‐affinity interactions with several other P450 species. This observation corroborates our hypothesis that the mechanisms of substrate‐dependent activation of a particular P450 enzyme through P450‐P450 crosstalk involve a reorganization of P450 heterooligomers induced by the interactions of the target enzyme with its specific substrate.Support or Funding InformationThis research was supported by the grant R21‐AA024548 from NIH.

Roles of Two Major Alcohol Dehydrogenases, ADH1 (Class I) and ADH3 (Class III), in the Adaptive Enhancement of Alcohol Metabolism Induced by Chronic Alcohol Consumption in Mice.

It is still unclear which enzymes contribute to the adaptive enhancement of alcohol metabolism by chronic alcohol consumption (CAC). ADH1 (Class I) has the lowest Km for ethanol and the highest sensitivity for 4-methylpyrazole (4MP) among ADH isozymes, while ADH3 (Class III) has the highest Km and the lowest sensitivity. We investigated how these two major ADHs relate to the adaptive enhancement of alcohol metabolism. Male mice with different ADH genotypes (WT, Adh1-/- and Adh3-/-) were subjected to CAC experiment using a 10% ethanol solution for 1month. Alcohol elimination rate (AER) was measured after ethanol injection at a 4.0g/kg dose. 4MP-sensitive and -insensitive AERs were measured by the simultaneous administration of 4MP at a dose of 0.5mmol/kg in order to estimate ADH1 and non-ADH1 pathways. AER was enhanced by CAC in all ADH genotypes, especially more than twofold in Adh1-/- mice, with increasing ADH1 and/or ADH3 liver contents, but not CYP2E1 content. 4MP-sensitive AER was also increased by CAC in WT and Adh3-/- strains, which was greater in Adh3-/- than in WT mice. The sensitive AER was increased even in Adh1-/- mice probably due to the increase in ADH3, which is semi-sensitive for 4MP. 4MP-insensitive AER was also increased in WT and Adh1-/- by CAC, but not in Adh3-/- mice. ADH1 contributes to the enhancement of alcohol metabolism by CAC, particularly in the absence of ADH3. ADH3 also contributes to the enhancement as a non-ADH1 pathway, especially in the absence of ADH1.

Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse liver

Fasting has been shown to regulate the expression of the cytochrome p450 (CYP) enzyme system in the liver. However, the exact mechanism behind the fasting-induced regulation of the CYP’s remains unknown. In the present study we tested the hypothesis that the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), which is a key-regulator of energy metabolism, is responsible for the fasting-induced regulation of the CYP’s. Lox/lox and liver specific PGC-1α (LKO) mice of both sexes, fasted for 18 h and the content of the CYP’s as well as the hepatic metabolome was assessed. Fasting increased the mRNA content of Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10. The fasting-induced response in Cyp4a10 mRNA content was different between lox/lox and LKO mice, while the absence of PGC-1α had no effect on the fasting-induced response for the other Cyp’s. Moreover, the fasting-induced response in mRNA content of Sirtinus 1 and Perilipin 2 was different between lox/lox and LKO mice. Only the CYP1A isoform showed a fasting-induced response at the protein level. Absence of hepatic PGC-1α had no effect on the apparent metabolome, where fasting vs fed was the only discriminate in the following multivariate analysis. In conclusion, hepatic PGC-1α is not essential for the fasting-induced regulation of hepatic CYP’s.

Towards a System Approach to the Human Cytochrome P450 Ensemble: A New Strategy for Studying the Network of P450‐P450 Interactions in Human Liver Microsomes

The composition of the human cytochrome P450 ensemble is commonly recognized as a key factor determining the interindividual variability in drug metabolism. However, recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a direct correlation between the composition of the cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of interactions between multiple cytochrome P450 species co‐localized in the microsomal membrane. A change in the content of any particular P450 enzyme alters the landscape of protein‐protein interactions and thus affects drug metabolism in a complex, hard‐to‐predict manner. Therefore, a systematic exploration of the network of P450‐P450 interactions and identification of their functional consequences represents an issue of emerging importance for revealing the interrelationships between the profile of P450 expression and the profile of drug metabolism. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into the microsomes. Here we demonstrate high potential of this approach for creating preparations of HLM with altered composition of the P450 ensemble. An important characteristic of this strategy is its adaptability for incorporating genetically or chemically modified proteins. This includes the incorporation of proteins bearing fluorescent probes and light‐activating crosslinkers. Therefore, this strategy may be used for monitoring protein‐protein interactions in HLM with a variety of biophysical techniques, as well as for searching the partners of P450 protein‐protein interactions with the technique of molecular fishing.Here we report the use of our new approach for studying the effect of enriching the HLM with ethanol‐inducible CYP2E1, one of the the most populated P450 species in human liver. Incorporation of CYP2E1 into HLM results in an increase in the rate of metabolism of CYP2E1‐specific substrates, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Our studies with a series of substrates of drug‐metabolizing P450s showed that CYP2E1 incorporation results in pronounced alterations in the metabolism of substrates of several other P450 species present in HLM. In particular, we observed over 2‐fold increase in the rate of metabolism and a substantial decrease in the apparent KM observed with 3‐cyano‐7‐etoxycoumarin (CEC), the substrate of CYP1A2 and CYP2C19. This finding may indicate that the increase in CYP2E1 concentration results in redirecting the main route of CEC metabolism from CYP2C19 to CYP1A2, which is characterized with lower KM and higher turnover numbers with this substrate. Similarly, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax, observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5.Support or Funding InformationThis research was supported by grant R01‐GM114369 from NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes.

Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

Dietary Omega-3 Polyunsaturated Fatty Acids Alter Fatty Acid Composition of Lipids and CYP2E1 Expression in Rat Liver Tissue.

Omega-3 polyunsaturated fatty acids (PUFAs) are used for the treatment and prevention of numerous pathologies in humans. As recently found, PUFAs play significantly protective roles in liver, cardiovascular system and kidney. They also are widely used in total parenteral nutrition. We evaluated the effect of omega-3 PUFA consumption on liver fatty acid composition and the expression of CYP2E1, one of the key enzymes in detoxification and prooxidant systems of liver cells. To estimate the oxidative stress in liver tissue, the antioxidant status and the level of lipid peroxidation were determined in a rodent model. Animals were divided into two groups: control (n = 10) and experimental (n = 10). Epadol-containing omega-3 PUFA fish oil capsules were administered to Wistar rats within 4 weeks (0.1 mL/100 g b.w./day). The consumption of omega-3 PUFAs resulted in changes of fatty acid composition of liver tissue. A significant increase was detected in the α-linolenic, eicosapentaenoic and docosahexaenoic acid content (5.1-, 16-, and 1.3-fold, respectively, p < 0.05), while the content of linoleic and arachidonic acid was reduced (1.7- and 3.2-fold, respectively, p < 0.05). This caused significant increases in the omega-3:omega-6 ratio. Consumption of omega-3 PUFAs led to a 3-fold (p < 0.05) increase in CYP2E1 content, which could entail enhanced Nrf2 expression levels and increases in the HO-1 content in rat liver. The alteration in CYP2E1 expression did not have an impact on the level of lipid peroxidation and on the prooxidant/antioxidant balance.

Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity.

Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT), CYP2E1 knockout (KO) or CYP2E1 humanized transgenic knockin (KI), mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA), an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These results suggest that autophagy is protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. We speculate that autophagy-dependent processes such as mitophagy and lipophagy help to minimize ethanol-induced CYP2E1-dependent oxidative stress and therefore the subsequent liver injury and steatosis. Attempts to stimulate autophagy may be helpful in lowering ethanol and CYP2E1-dependent liver toxicity.

Effect of ammonium pyrrolidine dithiocarbamate (PDTC) on NF-κB activation and CYP2E1 content of rats with immunological liver injury

Context: Ammonium pyrrolidine dithiocarbamate (PDTC) is a potent inhibitor of nuclear factor-κB (NF-κB). Recent studies have shown that NF-κB plays an essential role in the regulation of genes whose products are involved in the pathogenesis of immunological liver injury.Objective: To study the function of NF-κB in immunological liver injury of rat model and its effect on CYP2E1 content and metabolic activity.Materials and methods: The present study investigated the effect of passivating NF-κB activation on CYP2E1 using Bacillus calmette Guérin (BCG)-induced immunological liver injury in Sprague–Dawley rats measured in terms of enzyme levels. The degree of hepatic injury of rats was measured by using biochemical parameters, hepatic tissue pathological changes, and physiological parameters. Protein localization of liver NF-κB was detected by immunohistochemical assay. Western blot analysis was used to detect the protein expression of NF-κB, IκBα, iNOS, and CYP2E1. The content of CYP2E1 of homogenate in the rat liver was detected by ELISA assay and the enzyme kinetics of CYP2E1 probe drug chlorzoxazone was evaluated by high-performance liquid chromatography (HPLC) assay.Results: The results showed that BCG-pretreatment (125 mg/kg) significantly (p < 0.01) increased the weight of liver and spleen (increased by 70% and 248%, respectively), serum levels of alanine transarninase (ALT) and aspartate aminotransferase (AST) (increased by 200% and 75.8%, respectively), the expression of NF-κB and iNOS (increased by 228% and 303%, respectively), and decreased CYP2E1 content and metabolic activity (p < 0.05). Administration of PDTC (50, 100, and 200 mg/kg) reversed above hepatic injury stimulated by BCG in vivo. Moreover, PDTC (ED50: 76 mg/kg) dose dependently inhibited down-regulation of CYP2E1 (p < 0.05).Conclusion: Passivation of NF-κB can inhibit the down-regulation of CYP2E1 and iNOS to induce in rat liver tissue with immunological liver injury; NF-κB may be involved in the CYP2E1 regulation through iNOS.

Restoration of the normal Clara cell phenotype after chronic allergic inflammation

Bronchiolar Clara cells play a critical role in lung homoeostasis. The main goal of this study was to evaluate the effects of chronic allergy on these cells and the efficacy of budesonide (BUD) and montelukast (MK) in restoring their typical phenotypes after ovalbumin-induced chronic allergy in mice. Chronic allergy induced extensive bronchiolar Alcian blue-periodic acid-Schiff (AB/PAS)-positive metaplasia. In addition, cells accumulated numerous big electron-lucent granules negative for Clara cell main secretory protein (CC16), and consequently, CC16 was significantly reduced in bronchoalveolar lavage. A concomitant reduction in SP-D and CYP2E1 content was observed. The phenotypic changes induced by allergy were pharmacologically reversed by both treatments; MK was more efficient than BUD in doing so. MK decreased AB/PAS reactivity to control levels whereas they remained persistently elevated after BUD. Moreover, most non-ciliated cells recovered their normal morphology after MK, whereas for BUD normal cells coexisted with 'transitional' cells that contained remnant mucous granules and stained strongly for CC16 and SP-D. Glucocorticoids were also less able to reduce inflammatory infiltration and maintained higher percentage of neutrophils, which may have contributed to prolonged mucin expression. These results show that chronic allergy-induced mucous metaplasia of Clara cells affects their defensive mechanisms. However, anti-inflammatory treatments were able to re-establish the normal phenotype of Clara cell, with MK being more efficient at restoring a normal profile than BUD. This study highlights the role of epithelial cells in lung injuries and their contribution to anti-inflammatory therapies.

Cytochrome P4502E1 inhibitor, chlormethiazole, decreases lipopolysaccharide‐induced inflammation in rat Kupffer cells with ethanol treatment

To investigate the role of Cytochrome P4502E1 in sensitizing Kupffer cells to lipopolysaccharide (LPS)-mediated inflammation after ethanol induction. Sprague-Dawley rats were fed a liquid ethanol diet, control diet or ethanol diet supplemented with CYP2E1 inhibitor, chlormethiazole (CMZ), for 4 weeks. Hepatic CYP2E1 protein, nuclear factor-kappa B (NF-κB) p65 protein and tumor necrosis factor (TNF)-α mRNA were measured. In vitro, isolated Kupffer cells from control rats were exposed to ethanol with different CMZ concentration; CYP2E1 expression and reactive oxygen species (ROS) generation were compared. The identified CMZ concentration was further utilized to evaluate the role of CYP2E1 on the sensitization of ethanol-induced Kupffer cell to LPS. The effect of LPS alone was tested in controlled Kupffer cells without ethanol. TNF-α, nuclear NF-κB p65 and cytoplasm IκB-α were monitored for all groups. Ethanol feeding increased hepatic CYP2E1 level, nuclear accumulation of NF-κB p65 and TNF-α expression in rats. These changes were inhibited by CMZ supplementation. In cultured Kupffer cells, increased CYP2E1 content and ROS production by in vitro ethanol induction were dose-dependently inhibited by CMZ. Compared with LPS alone, the ethanol induction group produced significantly more TNF-α, nuclear NF-κB p65 and less cytoplasm IκB-α under LPS stimuli. CMZ abolished the effects of ethanol on LPS-stimulated NF-κB translocation and TNF-α generation in Kupffer cells. In cultured Kupffer cell, using CMZ as inhibitor, ethanol-induced CYP2E1 overexpression was proved to contribute to the sensitization of Kupffer cells to LPS stimuli, with amplification of ROS production and activation of NF-κB, resulting in increased TNF-α production.

Assessing the impact of the duration and intensity of inhalation exposure on the magnitude of the variability of internal dose metrics in children and adults

The objective of this study was to assess the impact of the exposure duration and intensity on the human kinetic adjustment factor (HKAF). A physiologically based pharmacokinetic model was used to compute target dose metrics (i.e. maximum blood concentration (Cmax) and amount metabolized/L liver/24 h (Amet)) in adults, neonates (0–30 days), toddlers (1–3 years), and pregnant women following inhalation exposure to benzene, styrene, 1,1,1-trichloroethane and 1,4-dioxane. Exposure scenarios simulated involved various concentrations based on the chemical’s reference concentration (low) and six of U.S. EPA’s Acute Exposure Guideline Levels (AEGLs) (high), for durations of 10 min, 60 min, 8 h, and 24 h, as well as at steady-state. Distributions for body weight (BW), height (H), and hepatic CYP2E1 content were obtained from the literature or from P3M software, whereas blood flows and tissue volumes were calculated from BW and H. The HKAF was computed based on distributions of dose metrics obtained by Monte Carlo simulations [95th percentile in each subpopulation/median in adults]. At low levels of exposure, ranges of Cmax-based HKAF were 1–6.8 depending on the chemical, with 1,4-dioxane exhibiting the greatest values. At high levels of exposure, this range was 1.1–5.2, with styrene exhibiting the greatest value. Neonates were always the most sensitive subpopulation based on Cmax, and pregnant women were most sensitive based on Amet in the majority of the cases (1.3–2.1). These results have shown that the chemical-specific HKAF varies as a function of exposure duration and intensity of inhalation exposures, and sometimes exceeds the default value used in risk assessments.

The role of CYP2E1 in the protection of garlic oil's from n-hexane-induced neurotoxicity

To study the role of CYP2E1 in the protective effects and mechanism of garlic oil (GO) on the peripheral nerve injuries induced by n-hexane. Fifty male Wistar rats were randomly divided into five groups (n = 10): the control, the GO (80 mg/kg) control, the n-hexane (2000 mg/kg) model, the low dose GO (40 mg/kg) plus n-hexane, and the high dose GO (80 mg/kg) plus n-hexane groups. All rats were treated by intragastric administration 6 times a week for 10 weeks. The gait scores were determined every two weeks for monitoring the peripheral neurotrosis. All rats were sacrificed in 10 weeks, the activities and expression levels of hepatic CYP2E1 and 2, 5-HD in serum were examined. As compared with control group, the content and activity of hepatic CYP2E1 in GO control group reduced by 83.1% and 48.3% respectively (P < 0.01), the content and activity of hepatic CYP2E1 in model group increased by 112.5% and 72.2% respectively (P < 0.01). As compared with model group, the contents of hepatic CYP2E1 in low dose and high dose GO groups reduced by 32.9% and 39.1% respectively, the activities of hepatic CYP2E1 in low dose and high dose GO groups reduced by 27.4% and 44.5% respectively (P < 0.01); the contents of serum 2,5-HD in low dose and high dose GO groups reduced by 47.7% and 78.7% respectively (P < 0.01). The gait scores in model, low dose and high dose GO groups were significantly lower than that in control group, but the gait scores in low dose and high dose GO groups were significantly lower than that in model group (P < 0.05). Garlic oil can effectively reduce the peripheral neurotrosis induced by n-hexane due to the decreased content and activity of hepatic CYP2E1, resulting in the reduced formation of 2, 5-HD from n-hexane.

酒肝宁煎剂对酒精性肝损伤大鼠CYP2E1及白介素6、肿瘤坏死因子-α含量的影响

：Objective To explore theinfluences of Jinganning decoction on the content of CYP2E1 and cytokine IL-6,TNF in ratsmodels of alcohdic hepatic injury.Methos 72 male Wistar clean level rats were randomlydivided into 6 groups:blank control group,Jiuganning drug toxicity test group,alcoholichepatic disease model group,Yiganling treatment group,Jiuganning low dose group,andJiuganning high dose group.Blank control group was treated with physiologicalsaline;Jiuganning drug toxicity test group was treated with Jiuganning decoction toobserve toxicity and side effects.Jiuganning low and high dose treatment group weretreated with 17.5 g/kg and 23.5 g/kg crude drug.Yiganning treatment group were treatedwith 0.206 g/kg crude drug.After 4 weeks'treatment,the content of TNF,IL-6,MDA as well asthe activity of CYP2E1 and SOD were measured.Results Compared with the alcoholic hepaticdisease model group,the content of IL-6(F=46.281,9.101),TNF and the activity (F=62.537)ofCYP2E1 of all treatment group were significantly improved(P＜0.01).The content of SOD(F=19.210)in the rats serum of alltreatment group was significantly increased while the MDA(F=34.952)was decreased.All theindexes in the treatment group had significant differences with the alcoholic hepaticdisease and model group(P＜0.01).Conclusions The mechanism of Jiuganning decoction can effectively reducethe content of CYP2E1 and IL-6,TNF-a,with the changes of cell physiopathology and theinfluences of relevant enzymes,reveals the functional routines of the decoction treatinghepatic injury:anti-lipid peroxidative,adjusting cytokine's activity,protectinghepatocyte,palliating the hepatocyte inflammation,and easing fibration. Key words: Jiuganning decoetion; Alcoholic hepatitis; Oxidative stress; Lipid peroxidation

Study of cytochrome P450 2E1 and its allele variants in liver injury of nondiabetic, nonalcoholic steatohepatitis obese women.

CYP2E1 enzyme is related to nonalcoholic steatohepatitis (NASH) due to its ability for reactive oxygen species production, which can be influenced by polymorphisms in the gene. The aim of this study was to investigate hepatic levels, activity, and polymorphisms of the CYP2E1 gene to correlate it with clinical and histological features in 48 female obese NASH patients. Subjects were divided into three groups: (i) normal; (ii) steatosis; and (iii) steatohepatitis. CYP2E1 protein level was assayed in microsomes from liver biopsies, and in vivo chlorzoxazone hydroxylation was determined by HPLC. Genomic DNA was isolated for genotype analysis through PCR. The results showed that liver CYP2E1 content was significantly higher in the steatohepatitis (45%; p=0.024) and steatosis (22%; p=0.032) group compared with normal group. Chlorzoxazone hydroxylase activity showed significant enhancement in the steatohepatitis group (15%, p=0.027) compared with the normal group. c2 rare allele of RsallPstl polymorphisms but no C allele of Dral polymorphism was positively associated with CHZ hydroxylation, which in turn is correlated with liver CYP2E1 content (r=0.59; p=0.026). In conclusion, c2 allele is positively associated with liver injury in NASH. This allele may determine a higher transcriptional activity of the gene, with consequent enhancement in pro-oxidant activity of CYP2E1 thus affording liver toxicity.

S-adenosyl- l-methionine decreases the elevated hepatotoxicity induced by Fas agonistic antibody plus acute ethanol pretreatment in mice

The current study was designed to investigate the effect and potential mechanism of exogenous administration of S-adenosyl- l-methionine (SAM) on the enhanced hepatotoxicity induced by the Fas agonistic Jo2 antibody plus acute ethanol pretreatment in C57BL/6 mice. Acute ethanol plus Jo2 treatment produces liver toxicity under conditions in which ethanol alone or Jo2 alone do not. SAM significantly attenuated this elevated hepatotoxicity in mice as manifested by a decrease of serum aminotransferases and morphological amelioration. Levels of SAM and activity of methionine adenosyltransferase were lowered by the ethanol plus Jo2 treatment but restored by administration of SAM. The ethanol plus Jo2 treatment increased activity and content of CYP2E1, iNOS content and TNF-α levels; these increases were blunted by SAM. SAM also protected against the elevated oxidative and nitrosative stress found after ethanol plus Jo2, likely due to the decreases in CYP2E1, iNOS and TNF-α. Calcium-induced swelling of mitochondria was enhanced by the ethanol plus Jo2 treatment and this was prevented by SAM. JNK and P38 MAPK were activated by the ethanol plus Jo2 treatment; JNK activation was partially prevented by SAM. It is suggested that SAM protects against the ethanol plus Jo2 toxicity by restoring hepatic SAM levels, preventing the increase in iNOS, CYP2E1 and TNF-α and there by lowering the elevated oxidative/nitrosative stress and activation of the JNK signal pathway, ultimately preventing mitochondrial damage.