Quercetin reduced the formation of N-acetyl-p-benzoquinoneimine, a toxic metabolite of paracetamol in rats and isolated rat hepatocytes.

N-acetyl-p-benzoquinoneimine (NAPQI) is toxic metabolite of paracetamol formed primarily by cytochrome P4502E1 (CYP2E1) metabolic pathway when administered at therapeutic doses or overdose. The influence of quercetin (flavonoid) on the bioactivation of paracetamol to NAPQI was investigated using rat liver microsomes and rats in vivo. Paracetamol (80 mg/kg) was administered orally without or with silymarin (100 mg/kg), a known inhibitor of CYP2E1, CYP3A4 and quercetin (10 and 20 mg/kg) to rats for 15 consecutive days. Area under the plasma concentration-time curve (AUC0-∞ ) and the peakplasma concentration (Cmax ) of paracetamol were dose-dependently increased with quercetin (10 and 20 mg/kg) compared to paracetamol control group (p < 0.001). On the other hand, the AUC0-∞ and Cmax of NAPQI were decreased significantly with quercetin. The same results were observed with silymarin also. The elevated liver and kidney functional enzymes/compounds were significantly reduced by quercetin and silymarin compared to paracetamol control group. The formation of NAPQI was reduced in the incubation samples in presence of quercetin in experiment using isolated rat hepatocytes. The presentstudy results revealed that quercetin might be inhibited the CYP2E1-mediated metabolism of paracetamol; thereby decreased the formation of NAPQI and protected the liver and kidney.

Total sesquiterpene glycosides from Loquat (Eriobotrya japonica) leaf alleviate high-fat diet induced non-alcoholic fatty liver disease through cytochrome P450 2E1 inhibition.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by hepatic steatosis, which affects 20-40% of the population in the world. Loquat (Eriobotrya japonica) Leaf possesses several pharmacological actions. Many sesquiterpene glycosides were reported to be isolated exclusively from the Loquat Leaf, however, their biological activity has been rarely investigated. The present study was designed to evaluate the pharmacological effect of total sesquiterpene glycosides (TSG) in high-fat diet (HFD) induced NAFLD mice with its related mechanisms of action. Mice were fed with a normal diet or HFD for 8 weeks. TSG (25 and 100mg/kg/day), simvastatin (10mg/kg/day) or vehicle were orally administered for last 4 weeks of the 8-week HFD feeding period. From the result, it was showed that TSG significantly reduced the body weight and fat deposition in the liver of NAFLD mice. It also decreased total cholesterol (TC) and triglyceride (TG) contents in the serum. Compared with NAFLD mice, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were increased and decreased after the administration of TSG in a dose of 100mg/kg, respectively. TSG reduced alanine aminotransferase (ALT) activity as well. Finally, TSG was found to suppress the expression of cytochrome P450 2E1 (CYP2E1) and the phosphorylation of c-jun terminal kinase (JNK) in NAFLD mice. In summary, this study demonstrates that TSG reduces oxidative stress by downregulating of CYP2E1 expression and JNK phosphorylation in NAFLD, and alleviates NAFLD ultimately. TSG potentially serves as bioactive compounds for the treatment of NAFLD.

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.

Persimmon vinegar and its fractions protect against alcohol-induced hepatic injury in rats through the suppression of CYP2E1 expression.

CONTEXT: Medical therapies for alcohol-induced liver disease are often difficult to handle and limited in efficacy. OBJECTIVE: In an attempt to find natural therapeutics, here, we investigate the preventive effect of persimmon vinegar (PV) and its fractions against alcohol-induced hepatic injury, in addition to the underlying mechanism, in rats chronically administered with alcohol. MATERIALS AND METHODS: Forty male Wistar rats were randomized into five groups (n = 8 per group); normal control (NC), ethanol control (EC), ethanol + PV, ethanol + water-insoluble PV fraction (PI) and ethanol + water-soluble PV fraction (PS). PV, PI or PS was orally administrated at the level of 100 mg/kg B.W by oral gavage every day for 4 weeks prior to ethanol administration. The liver sections were stained with hematoxylin & eosin and gene expression was assessed by real-time polymerase chain reaction. RESULTS: After a 4-week treatment, histological observation revealed that PV and its fractions mitigated alcohol-induced changes in the liver. CYP2E1 expression was significantly increased in the EC group compared with the NC group, but was significantly suppressed in the PV group compared with the EC group (p = 0.044). We also found significant decreases in hepatic mRNA expression of interleukin (IL)-1ß, IL-12ß, toll-like receptor (TLR)-4 and cyclooxygenase (COX)-2 in the PV-, PI- and PS-treated groups compared with those of the EC group. DISCUSSION AND CONCLUSION: Taken together, the present findings suggest that PV and its fractions hold great promise as natural remedies with anti-inflammatory activities that alleviate alcohol-induced liver damage.

OMEGA-3 POLYUNSATURATED FATTY ACIDS NORMALIZE THE FUNCTION OF MITOCHONDRIA, ACTIVITY OF ENZYMES OF PROOXIDANT-ANTIOXIDANT SYSTEM AND THE EXPRESSION OF CYTOCHROME Р450 2Е1 AFTER ISOPROTERENOLINDUCED MYOCARDIAL INJURY.

We have studied the influence of dietary ω-3 polyunsaturated fatty acids (ω-3 PUFA) on the functioning of subsarcolemmal and interfibrillar mitochondrial fractions of rat myocardium, changes in expression of cytochrome P450 (CYP2E1) and the activity of enzymes of prooxidant-antioxidant system after isoproterenol-induced myocardial injury. It has been found that in vivo administration of ω-3 PUFA (Epadol 0.1 ml/100 gr of weight for 4 weeks) significantly reduced the swelling of subsarcolemmal and interfibrillar mitochondrial fractions by 65.52% 54.84% respectively, pointing for a decrease of damage of the mitochondrial function evoked by in vivo administration of isoproterenol. In vivo administration of ω-3 PUFAs prevents a decrease in the activity of antioxidant enzymes catalase and superoxide dismutase (2.65 and 7.1- fold, respectively) after isoproterenol-induced myocardial injury. We suggest that the development of oxidative stress after isoproterenol-induced myocardial injury can be caused by a significant increase in the expression of cytochrome P450 2E1 (73.3%), and administration of ω-3 PUFAs prevents such changes.

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.

Schisandrol B protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of liver regeneration.

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra sphenanthera is a traditional hepato-protective Chinese medicine and Schisandrol B (SolB) is one of its major active constituents. In this study, the protective effect of SolB against APAP-induced acute hepatotoxicity in mice and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated a protective effect of SolB against APAP-induced liver injury. SolB pretreatment significantly attenuated the increases in alanine aminotransferase and aspartate aminotransferase activity, and prevented elevated hepatic malondialdehyde formation and the depletion of mitochondrial glutathione (GSH) in a dose-dependent manner. SolB also dramatically altered APAP metabolic activation by inhibiting the activities of CYP2E1 and CYP3A11, which was evidenced by significant inhibition of the formation of the oxidized APAP metabolite NAPQI-GSH. A molecular docking model also predicted that SolB had potential to interact with the CYP2E1 and CYP3A4 active sites. In addition, SolB abrogated APAP-induced activation of p53 and p21, and increased expression of liver regeneration and antiapoptotic-related proteins such as cyclin D1 (CCND1), PCNA, and BCL-2. This study demonstrated that SolB exhibited a significant protective effect toward APAP-induced liver injury, potentially through inhibition of CYP-mediated APAP bioactivation and regulation of the p53, p21, CCND1, PCNA, and BCL-2 to promote liver regeneration.

Glucosamine enhances paracetamol bioavailability by reducing its metabolism.

Paracetamol has an extensive first-pass metabolism that highly affects its bioavailability (BA); thus, dose may be repeated several times a day in order to have longer efficacy. However, hepatotoxicity may arise because of paracetamol metabolism. Therefore, this project aimed to increase paracetamol BA in rats by glucosamine (GlcN). At GlcN-paracetamol racemic mixture ratio of 4:1 and paracetamol dose of 10 mg/kg, paracetamol area under the curve (AUC) and maximum concentration (Cmax ) were significantly increased by 99% and 66%, respectively (p < 0.05). Furthermore, paracetamol AUC and Cmax levels were increased by 165% and 88% in rats prefed with GlcN for 2 days (p < 0.001). Moreover, GlcN significantly reduced phase Ι and phase I/ΙΙ metabolic reactions in liver homogenate by 48% and 54%, respectively. Furthermore, GlcN molecule was found to possess a good in silico binding mode into the CYP2E1 active site-forming bidentate hydrogen bonding with the Thr303 side chain. Finally, serum ALT and AST levels of rats-administered high doses of paracetamol were significantly reduced when rats were prefed with GlcN (p < 0.01). In conclusion, GlcN can increase the relative BA of paracetamol through reducing its metabolism. This phenomenon is associated with reduction in hepatocytes injury following ingestion of high doses of paracetamol.

Fennel and raspberry leaf as possible inhibitors of acetaminophen oxidation.

In addition to CYP2E1, several CYP isoenzymes, notably CYP1A2, 2D6, and 3A4, are suggested to contribute in acetaminophen oxidation and formation of the hepatotoxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). The in vitro CYP2E1 inhibitory potentials of fennel and raspberry leaf, herbs previously found to inhibit CYP1A2, 2D6, and 3A4 activities in vitro, were investigated. Extracts from commercially available herbal products were incubated with recombinant cDNA-expressed human CYP2E1. A validated LC/MS/MS methodology was applied for determination of 6-hydroxychlorzoxazone formation with disulfiram used as a positive inhibitory control. CYP2E1 IC50 inhibition constants were found to be 23 ± 4 and 27 ± 5 µg/ml for fennel and raspberry leaf, respectively, constants significantly lower than those presented in the literature for other herbal extracts. Together with previous findings, the presented in vitro data for CYP2E1 inhibition suggest that fennel and raspberry leaf have a significant potential of inhibiting all the major metabolic pathways for acetaminophen oxidation and NAPQI formation. Both herbs should be further investigated for their in vivo ability of inhibiting acetaminophen oxidation and NAPQI formation.

ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes.

AIM: Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress. METHODS: Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH). RESULTS: ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity. CONCLUSION: We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.

Garlic-derived S-allylmercaptocysteine is a hepato-protective agent in non-alcoholic fatty liver disease in vivo animal model.

PURPOSE: To investigate the hepato-protective properties and underlying mechanisms of SAMC in a non-alcoholic fatty liver disease (NAFLD) rat model. METHODS: Female rats were fed with a diet comprising highly unsaturated fat diet (30% fish oil) for 8 weeks to develop NAFLD with or without an intraperitoneal injection of 200 mg/kg SAMC three times per week. After euthanasia, blood and liver samples of rats were collected for histological and biochemical analyses. RESULTS: Co-treatment of SAMC attenuated NAFLD-induced liver injury, fat accumulation, collagen formation and free fatty acids (FFAs). At the molecular level, SAMC decreased the lipogenesis marker and restored the lipolysis marker. SAMC also reduced the expression levels of pro-fibrogenic factors and diminished liver oxidative stress partly through the inhibition in the activity of cytochrome P450 2E1-dependent pathway. NAFLD-induced inflammation was also partially mitigated by SAMC treatment via reduction in the pro-inflammatory mediators, chemokines and suppressor of cytokine signaling. The protective effect of SAMC is also shown partly through the restoration of altered phosphorylation status of FFAs-dependent MAP kinase pathways and diminished in the nuclear transcription factors (NF-κB and AP-1) activity during NAFLD development. CONCLUSIONS: SAMC is a novel hepato-protective agent against NAFLD caused by abnormal liver functions. Garlic or garlic derivatives could be considered as a potent food supplement in the prevention of fatty liver disease.

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes.

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20µM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7µM) and CYP3A4-mediated metabolism of testosterone to 6ß-hydroxytestosterone (IC(20) 7.06µM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4µM). Inhibition of testosterone to 6ß-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8µM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6µM and 11.9µM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.

Lycium barbarum polysaccharides protect mice liver from carbon tetrachloride-induced oxidative stress and necroinflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum has been used as a traditional Chinese medicine to nourish liver, kidneys and the eyes. AIM OF THE STUDY: We investigated the protective mechanisms of Wolfberry, Lycium barbarum polysaccharides (LBP) in carbon tetrachloride (CCl(4))-induced acute liver injury. MATERIALS AND METHODS: Mice were intraperitoneally injected with a 50 µl/kg CCl(4) to induce acute hepatotoxicity (8h) and were orally fed with LBP 2 h before the CCl(4) injection. There were six experimental groups of mice (n=7-8 per group), namely: control mice (vehicle only; 1 mg/kg LBP or 10 mg/kg LBP), CCl(4)-treated mice and CCl(4)+LBP treated mice (1 mg/kg LBP or 10 mg/kg LBP). RESULTS: Pre-treatment with LBP effectively reduced the hepatic necrosis and the serum ALT level induced by CCl(4) intoxication. LBP remarkably inhibited cytochrome P450 2E1 expression and restored the expression levels of antioxidant enzymes. It also decreased the level of nitric oxide metabolism and lipid peroxidation induced by CCl(4). LBP attenuated hepatic inflammation via down-regulation of proinflammatory mediators and chemokines. Furthermore, LBP promoted liver regeneration after CCl(4) treatment. The protective effects of LBP against hepatotoxicity were partly through the down-regulation of nuclear factor kappa-B activity. CONCLUSION: LBP is effective in reducing necroinflammation and oxidative stress induced by a chemical toxin, thus it has a great potential use as a food supplement in the prevention of hepatic diseases.

Hepatoprotective effects of chestnut (Castanea crenata) inner shell extract against chronic ethanol-induced oxidative stress in C57BL/6 mice.

This study was carried out to evaluate the protective effects of chestnut inner shell extract (CISE) on chronic ethanol-induced oxidative stress in liver. Mice were fed a control liquid diet (Normal-control), liquid diet containing ethanol alone (EtOH+Vehicle), or were administered CISE and ethanol (EtOH+CISE) for 6 weeks. Administration of ethanol induced liver damage with significant increase of plasma GOT, GPT, hepatic triglyceride (TG) and thiobarbituric acid reactive substance (TBARS) levels. By contrast, co-treatment of CISE with ethanol significantly decreased the activities of GOT and GPT in the plasma, and hepatic TG and TBARS levels. Histological observations were consistent with the result obtained from hepatic lipid quantification. Moreover, CISE treatment with ethanol decreased CYP2E1 expression and increased activities of catalase and superoxide dismutase, which were significantly inhibited by treatment with ethanol alone. To determine the active compound of CISE, fractionation of CISE was conducted and scoparone and scopoletin were identified as main compounds. These compounds were also shown to inhibit the ethanol-induced reduction in antioxidant enzyme activity in an in vitro model system. These results suggest that CISE has protective effects against ethanol-induced oxidative damage, possibly by inhibition of lipid accumulation, peroxidation and increase of antioxidant defense system in the liver.

Amelioration of oxidative stress by dandelion extract through CYP2E1 suppression against acute liver injury induced by carbon tetrachloride in Sprague-Dawley rats.

The protective effects of common dandelion leaf water extract (DLWE) were investigated by carbon tetrachloride (CCl4) induced hepatitis in Sprague-Dawley rats. The animals were divided into five groups: normal control, DLWE control, CCl4 control, and two DLWE groups (0.5 and 2 g/kg bw). After 1 week of administering corresponding vehicle or DLWE, a single dose of CCl4 (50% CCl4/olive oil; 0.5 mL/kg bw) was administered 24 h before killing in order to produce acute liver injury. The DLWE treatment significantly decreased CCl4-induced hepatic enzyme activities (AST, ALT and LDH) in a dose dependent manner. Also, the obstructed release of TG and cholesterol into the serum was repaired by DLWE administration. Hepatic lipid peroxidation was elevated while the GSH content and antioxidative enzyme activities were reduced in the liver as a result of CCl4 administration, which were counteracted by DLWE administration. Furthermore, the hepatocytotoxic effects of CCl4 were confirmed by significantly elevated Fas and TNF-α mRNA expression levels, but DLWE down-regulated these expressions to the levels of the normal control. Highly up-regulated cytochrome P450 2E1 was also lowered significantly in the DLWE groups. These results indicate that DLWE has a protective effect against CCl4-induced hepatic damage with at least part of its effect being attributable to the attenuation of oxidative stress and inflammatory processes resulting from cytochrome P450 activation by CCl4.

Major tanshinones of Danshen (Salvia miltiorrhiza) exhibit different modes of inhibition on human CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities in vitro.

This study investigated the effects of tanshinones on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6beta-hydroxylase) activities in vitro using pooled human liver microsomes and specific human CYP isoforms. Tanshinone I, tanshinone IIA, and cryptotanshinone were potent competitive CYP1A2 inhibitors (K(i)=1.5-2.5 microM); medium competitive inhibitors of CYP2C9 (K(i)=22-62 microM); medium competitive inhibitors of CYP2E1 (K(i)=3.67 microM) for tanshinone I and 10.8 microM for crytotanshinone; but weak competitive inhibitors of CYP3A4 (K(i)=86-220 microM). Dihydrotanshinone was a competitive inhibitor of human CYP1A2 (K(i)=0.53 microM) and CYP2C9 (K(i)=1.92 microM), a noncompetitive inhibitor of CYP3A4 (K(i)=2.11 microM) but an uncompetitive CYP2E1 inhibitor. In conclusion, these results showed that tanshinones inhibited the metabolism of various CYP probe substrates in human liver microsomes and specific human CYP isoforms in vitro. Given that CYP1A2, 2C9, 2E1 and 3A4 are responsible for the metabolism and disposition of a large number of drugs currently used, the potential herb-drug interactions of Danshen preparations containing the major tanshinones with drugs which are substrates of these CYPs may be important.

Mechanism for prevention of alcohol-induced liver injury by dietary methyl donors.

Alcohol-induced liver injury (ALI) has been associated with, among other molecular changes, abnormal hepatic methionine metabolism, resulting in decreased levels of S-adenosylmethionine (SAM). Dietary methyl donor supplements such as SAM and betaine mitigate ALI in animal models; however, the mechanisms of protection remain elusive. It has been suggested that methyl donors may act via attenuation of alcohol-induced oxidative stress. We hypothesized that the protective action of methyl donors is mediated by an effect on the oxidative metabolism of alcohol in the liver. Male C57BL/6J mice were administered a control high-fat diet or diet enriched in methyl donors with or without alcohol for 4 weeks using the enteral alcohol feeding model. As expected, attenuation of ALI and an increase in reduced glutathione:oxidized glutathione ratio were achieved with methyl donor supplementation. Interestingly, methyl donors led to a 35% increase in blood alcohol elimination rate, and while there was no effect on alcohol metabolism in the stomach, a profound effect on liver alcohol metabolism was observed. The catalase-dependent pathway of alcohol metabolism was induced, yet the increase in CYP2E1 activity by alcohol was blunted, which may be mitigating production of oxidants. Additional factors contributing to the protective effects of methyl donors in ALI were increased activity of low- and high-K(m) aldehyde dehydrogenases leading to lower hepatic acetaldehyde, maintenance of the efficient mitochondrial energy metabolism, and promotion of peroxisomal beta-oxidation. Profound changes in alcohol metabolism represent additional important mechanism of the protective effect of methyl donors in ALI.

Phyllanthus urinaria extract attenuates acetaminophen induced hepatotoxicity: involvement of cytochrome P450 CYP2E1.

Acetaminophen is a commonly used drug for the treatment of patients with common cold and influenza. However, an overdose of acetaminophen may be fatal. In this study we investigated whether mice, administered intraperitoneally with a lethal dose of acetaminophen, when followed by oral administration of Phyllanthus urinaria extract, may be prevented from death. Histopathological analysis of mouse liver sections showed that Phyllanthus urinaria extract may protect the hepatocytes from acetaminophen-induced necrosis. Therapeutic dose of Phyllanthus urinaria extract did not show any toxicological phenomenon on mice. Immunohistochemical staining with the cytochrome P450 CYP2E1 antibody revealed that Phyllanthus urinaria extract reduced the cytochrome P450 CYP2E1 protein level in mice pre-treated with a lethal dose of acetaminophen. Phyllanthus urinaria extract also inhibited the cytochrome P450 CYP2E1 enzymatic activity in vitro. Heavy metals, including arsenic, cadmium, mercury and lead, as well as herbicide residues were not found above their detection limits. High performance liquid chromatography identified corilagin and gallic acid as the major components of the Phyllanthus urinaria extract. We conclude that Phyllanthus urinaria extract is effective in attenuating the acetaminophen induced hepatotoxicity, and inhibition of cytochrome P450 CYP2E1 enzyme may be an important factor for its therapeutic mechanism.

Effect of polyphenolic compounds from Solanum torvum on plasma lipid peroxidation, superoxide anion and cytochrome P450 2E1 in human liver microsomes.

Previous studies presented evidence that plants contain antioxidants that have free radical-scavenging properties. Overproduction of free radicals leads to oxidative stress, a factor associated with a variety of diseases, such as diabetes. Cytochrome P450 2E1 enzymes (CYP2E1) are involved in drug metabolism in the liver and metabolism of DNA-reaction generating intra-mitochondrial ROS, which leads to micro- and macro-vascular pathology in diabetes. Plant-based chemicals can affect CYP2E1 enzymes and related defense mechanisms, possibly leading to protection against oxidative stress. We investigated the effect of Solanum torvum (ST) extracts on the inhibition of CYP2E1 activity in human liver microsomes. ST extract was analyzed for antioxidant activity by the ABTS method. Polyphenolic compounds were measured by the total phenol content using the Folin-Ciocalteau reagent. Flavonoid and tannin content were analyzed by standard methods. Oxidative stress was evaluated by measuring lipid peroxidation by TBARS and superoxide anion scavenging levels in plasma from diabetic patients. Results showed that 10 mg/ml of ST had CYP2E1 catalytic inhibiting activity (57.16 %). The IC50 value of CYP2E1 catalytic inhibiting activity level was 5.14 mg/ml by concentration in a dependent manner. One gram of concentrated ST extract had an antioxidant activity index of 3.68 mg of trolox and 360.53 mg of ascorbic acid equivalent. Effects on free radical-scavenging, as measured by TBARS and superoxide anion, showed IC50 values of 20.60 and 10.26 microg/ml, respectively. Polyphenolic compounds found included phenol, flavonoid and tannin, measuring 160.30, 104.36 and 65.91 mg/g, respectively. These results imply that ST is a natural source of polyphenolic antioxidants, which have cytochrome P450 2E1 enzyme inhibiting and free radical scavenging properties, as related to lipid peroxidation and superoxide anion activity. ST could potentially be used for reducing oxidative stress in diabetes. Study for usage as a conventional medicine, including dosage and chronic toxicity in humans, should be pursued.

Protective effects of diallyl sulfide, a garlic constituent, on the warm hepatic ischemia-reperfusion injury in a rat model.

PURPOSE: The aim of this study is to evaluate the effects of diallyl sulfide (DAS) on the warm hepatic ischemia-reperfusion (IR) injury in a rat model. METHODS: Rats (n = 8-10/group) were subjected to sham operation or warm ischemia (1 h)-reperfusion (3 h) preceded by a single intraperitoneal dose (1.75 mmol/kg) of DAS or vehicle, and relevant biochemical parameters were monitored. RESULTS: Warm IR injury caused a significant increase in the plasma markers of liver injury, which was attenuated by DAS. The hepatoprotective effects of DAS were associated with significant reductions in lipid peroxidation markers and in situ generation of superoxide in the liver and increases in the glutathione levels of the liver and bile, suggestive of an antioxidant effect for DAS. Additionally, DAS caused an almost twofold increase in the protein expression of the liver heme oxygenase-1, an enzyme that confers cytoprotection against oxidative stress. Whereas the total cytochrome P450 remained unchanged, the protein levels and activity of CYP2E1, which plays an important role in the generation of reactive oxygen species, significantly decreased by DAS pretreatment. CONCLUSIONS: DAS protects the liver from warm IR injury by reducing oxidative stress through, at least in part, induction of heme oxygenase-1 and inhibition of CYP2E1.