Pogostone inhibits the activity of CYP3A4, 2C9, and 2E1 in vitro.

CONTEXT: Pogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination. OBJECTIVE: To investigate the effect of pogostone on the activity of human CYP450s. MATERIALS AND METHODS: The effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0-100 µM pogostone and various concentrations of substrates. RESULTS: Pogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the IC50 values of 11.41, 12.11, and 14.90 µM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the Ki value of 5.69 µM and the KI/Kinact value of 5.86/0.056/(µM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the Ki value of 6.46 and 7.67 µM and was not affected by the incubation time. DISCUSSION AND CONCLUSIONS: The inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further in vivo investigations to validate the potential interaction.

Subchronic administration of mitoxantrone and the influence of enzyme inhibitors on its induced cardiotoxicity in mice: role of NRF-2/CYP2E1.

OBJECTIVE: Mitoxantrone (MTX)- induced cardiotoxicity is a clinical concern that is limiting its use. The aim of this paper, therefore, was to investigate the subchronic administration of MTX plus nonspecific/specific inhibitors of CYP450/2E1, to assess the extent of oxidative-induced injury by measuring levels of oxidative cardiac and injury biomarkers in mice and to evaluate the effects of CYP2E1 on caspase 3 activity and nuclear factor erythroid 2-related factor-2 (NRF-2). MATERIALS AND METHODS: Mice (n = 32) were divided into four treatment groups of eight: control, MTX, MTX + 4-methlypyrazole (4MP) and MTX + disulfiram (Disf). After 6 weeks of treatments, blood and heart samples were collected. RESULTS: Liquid chromatography-mass spectrometry (LCMS) analysis of MTX-treated plasma samples revealed several metabolites with different retention times. Cardiac antioxidant enzymes and creatine kinase (CK) levels were not significantly different among the groups. However, cardiac troponin and caspase 3 activity were significantly raised, with increased CYP2E1 expressions and reduced NRF-2 expression. Tissue damage was observed in all the treatment groups, including MTX, leading to the conclusion that MTX-induced cardiotoxicity was mediated by CYP2E1 activity, which initiated caspase 3 production, and decreased NRF-2 expression. CONCLUSIONS: Therefore, agents that inhibit CPY2E1 expression might attenuate MTX-induced cardiotoxicity by increasing NRF-2 expression.

Susceptibility to the acute toxicity of acrylonitrile in streptozotocin-induced diabetic rats: protective effect of phenethyl isothiocyanate, a phytochemical CYP2E1 inhibitor.

Diabetes mellitus is a significant global public health issue. The diabetic state not only precipitates chronic disease but also has the potential to change the toxicity of drugs and chemicals. Acrylonitrile (AN) is a potent neurotoxin widely used in industrial products. This study used a streptozotocin (STZ)-induced diabetic rat model to examine the role of cytochrome P450 2E1 (CYP2E1) in acute AN toxicity. The protective effect of phenethyl isothiocyanate (PEITC), a phytochemical inhibitor of CYP2E1, was also investigated. A higher incidence of convulsions and loss of the righting reflex, and decreased rates of survival, as well as elevated CYP2E1 activity, were observed in diabetic rats treated with AN when compared to those in non-diabetic rats, suggesting that diabetes confers susceptibility to the acute toxicity of AN. Pretreatment with PEITC (20-80 mg/kg) followed by AN injection alleviated the acute toxicity of AN in diabetic rats as evidenced by the decreased incidence of convulsions and loss of righting reflex, and increased rates of survival. PEITC pretreatment at 40 and 80 mg/kg decreased hepatic CYP2E1 activity in AN-exposed diabetic rats. PEITC pretreatment (20 mg/kg) increased the glutathione (GSH) content and glutathione S-transferase (GST) activity and further decreased ROS levels in AN-exposed diabetic rats. Collectively, STZ-induced diabetic rats were more sensitive to AN-induced acute toxicity mainly due to CYP2E1 induction, and PEITC pretreatment significantly alleviated the acute toxicity of AN in STZ-induced diabetic rats. PEITC might be considered as a potential effective chemo-preventive agent against AN-induced acute toxicity in individuals with an underlying diabetic condition.

Differential inhibition of naringenin on human and rat cytochrome P450 2E1 activity.

Cytochrome P450 2E1 (CYP2E1) has been proposed as a molecular target in oxidative stress-associated metabolic diseases. Rats are chosen as model organisms in most experiments studying CYP2E1-related toxicity; however, the human relevance of these results remains unclear. To describe differences in catalysis and inhibition between human and rat CYP2E1, recombinant human and rat CYP2E1 enzymes were treated with different concentrations of naringenin (NAR, 10 nM - 1 mM), and inhibition parameters were calculated. Interspecies differences in the catalytic efficiency for O-demethylation of 7-methoxy-4-(trifluoromethyl)coumarin were revealed (45-fold higher in human CYP2E1 than in the rat enzyme). Additionally, differences in the potency of inhibition of NAR were found (absolute half inhibitory concentration, IC50 = 204 ± 28 and 69 ± 4 µM; inhibition constant, Ki = 9 ± 2 and 161 ± 20 µM in human and rat CYP2E1, respectively). Although NAR exhibited a noncompetitive mechanism of inhibition of both CYP2E1 enzymes, this compound is an irreversible inhibitor of rat CYP2E1 and a reversible inhibitor of the human enzyme. Molecular docking suggested that differences in the potency of inhibition and time dependence between species could be attributable to the differential interactions of NAR with access channels to the CYP2E1 catalytic site. These results highlight the importance of finding the appropriate model to improve the predictability of animal-based assays for human risk assessment.

Drug targeting CYP2E1 for the treatment of early-stage alcoholic steatohepatitis.

BACKGROUND AND AIMS: Alcoholic steatohepatitis (ASH)-the inflammation of fatty liver-is caused by chronic alcohol consumption and represents one of the leading chronic liver diseases in Western Countries. ASH can lead to organ dysfunction or progress to hepatocellular carcinoma (HCC). Long-term alcohol abstinence reduces this probability and is the prerequisite for liver transplantation-the only effective therapy option at present. Elevated enzymatic activity of cytochrome P450 2E1 (CYP2E1) is known to be critically responsible for the development of ASH due to excessively high levels of reactive oxygen species (ROS) during metabolization of ethanol. Up to now, no rational drug discovery process was successfully initiated to target CYP2E1 for the treatment of ASH. METHODS: In this study, we applied a rational drug design concept to develop drug candidates (NCE) including preclinical studies. RESULTS: A new class of drug candidates was generated successfully. Two of the most promising small compounds named 12-Imidazolyl-1-dodecanol (abbr.: I-ol) and 1-Imidazolyldodecane (abbr.: I-an) were selected at the end of this process of drug discovery and developability. These new ω-imidazolyl-alkyl derivatives act as strong chimeric CYP2E1 inhibitors at a nanomolar range. They restore redox balance, reduce inflammation process as well as the fat content in the liver and rescue the physiological liver architecture of rats consuming continuously a high amount of alcohol. CONCLUSIONS: Due to its oral application and therapeutic superiority over an off-label use of the hepatoprotector ursodeoxycholic acid (UDCA), this new class of inhibitors marks the first rational, pharmaceutical concept in long-term treatment of ASH.

Alcohol consumption before pregnancy causes detrimental fetal development and maternal metabolic disorders.

Alcohol consumption before or during pregnancy poses serious health risks to the fetus; however, the underlying mechanisms involved remain obscure. Here, we investigated whether ethanol consumption before pregnancy affects maternal or fetal health and whether pharmacological inhibition of CYP2E1, a major ethanol oxidation enzyme, by 4-methylpyrazole (4-MP) has therapeutic effects. We found that ethanol consumption (5%) 2 weeks before pregnancy resulted in a decrease in the number of viable fetuses and abnormal fetal development, and these effects were accompanied by impaired maternal glucose homeostasis and hepatic steatosis during pregnancy. Neonates of ethanol-fed mice had postnatal macrosomia and significantly decreased growth rates during the lactation period. However, treatment with 4-MP, a CYP2E1 inhibitor, markedly ameliorated the reduction in insulin action and glucose disposal responsiveness in the livers of ethanol-fed mice. Blockage of CYP2E1 significantly reduced the alteration in hepatic lipid deposition, fatty acid oxidation, mitochondrial energy status, and macrophage infiltration observed in ethanol-fed mice. Finally, there was a positive correlation between postnatal macrosomia or growth retardation and increased inflammatory responses. Collectively, our study suggests that even moderate ethanol intake may be detrimental to fetal development and may cause growth retardation through maternal metabolic disorders.

Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2E1 Enzyme Activity: Inhibitory Effect of Cytochrome P450 Enzyme CYP2E1 by Salicylic Acid in Rat Liver Microsomes.

Inhibition of cytochrome P450 (CYP) alters the pharmacokinetic parameters of the drug and causes drug-drug interactions. Salicylic acid been used for the treatment of colorectal cancer (CRC) and chemoprevention in recent decades. Thus, the aim of this study was to examine the in vitro inhibitory effect of salicylic acid on CYP2E1 activity in rat liver microsomes (RLMs) using high-performance liquid chromatography (HPLC). High-performance liquid chromatography analysis of a CYP2E1 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 282 nm using 60% H2O, 25% acetonitrile, and 15% methanol as mobile phase. The CYP2E1 assay showed a good linearity (R2 > 0.999), good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80-120%), and low detection (4.972 µM and 1.997 µM) and quantitation limit values (15.068 µM and 6.052 µM), for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Salicylic acid acts as a mixed inhibitor (competitive and non-competitive inhibition), with Ki (inhibition constant) = 83.56 ± 2.730 µM and concentration of inhibitor causing 50% inhibition of original enzyme activity (IC50) exceeding 100 µM (IC50 = 167.12 ± 5.460 µM) for CYP2E1 enzyme activity. Salicylic acid in rats would have both low and high potential to cause toxicity and drug interactions with other drugs that are substrates for CYP2E1.

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.

Inhibition of CYP2E1 attenuates myocardial dysfunction in a murine model of insulin resistance through NLRP3-mediated regulation of mitophagy.

Insulin resistance leads to myocardial contractile dysfunction and deranged autophagy although the underlying mechanism or targeted therapeutic strategy is still lacking. This study was designed to examine the impact of inhibition of the cytochrome P450 2E1 (CYP2E1) enzyme on myocardial function and mitochondrial autophagy (mitophagy) in an Akt2 knockout model of insulin resistance. Adult wild-type (WT) and Akt2-/- mice were treated with the CYP2E1 inhibitor diallyl sulfide (100 mg/kg/d, i.p.) for 4 weeks. Cardiac geometry and function were assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate autophagy, mitophagy, inducible NOS (iNOS), and the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex. Akt2 deletion triggered insulin resistance, compromised cardiac contractile and intracellular Ca2+ property, mitochondrial ultrastructural damage, elevated O2- production, as well as suppressed autophagy and mitophagy, accompanied with elevated levels of NLRP3 and iNOS, the effects of which were significantly attenuated or ablated by diallyl sulfide. In vitro studies revealed that the NLRP3 activator nigericin nullified diallyl sulfide-offered benefit against Akt2 knockout on cardiomyocyte mechanical function and mitophagy (using Western blot and colocalization of GFP-LC3 and MitoTracker Red). Moreover, inhibition of iNOS but not mitochondrial ROS production attenuated Akt2 deletion-induced activation of NLRP3, substantiating a role for iNOS-mediated NLRP3 in insulin resistance-induced changes in mitophagy and cardiac dysfunction. In conclusion, these data depict that insulin resistance through CYP2E1 may contribute to the pathogenesis of myopathic changes including myocardial contractile dysfunction, oxidative stress and mitochondrial injury, possibly through activation of iNOS and NLRP3 signaling.

In vitro Inhibitory Effects of Isofraxidin on Human Liver Cytochrome P450 Enzymes.

Isofraxidin is a Coumarin compound widely distributed in plants, such as the Umbelliferae or Chloranthaceae, and it possesses numerous pharmacological activities. However, whether isofraxidin affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. In this study, the inhibitory effects of isofraxidin on the 8 human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes. The results showed that isofraxidin inhibited the activity of CYP1A2, 3A4, and 2E1, with IC50 values of 23.01, 15.49, and 15.98 µmol/L, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that isofraxidin was not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP1A2 and 2E1, with Ki values of 7.91, 10.14, and 9.30 µmol/L, respectively. In addition, isofraxidin is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.047/12.33 µmol/L-1min-1. The in vitro studies of isofraxidin with CYP isoforms indicate that isofraxidin has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by -CYP1A2, 3A4, and 2E1. Further clinical studies are needed to evaluate the significance of this interaction.

2,4-Dimethylaniline generates phosphorylated histone H2AX in human urothelial and hepatic cells through reactive oxygen species produced by cytochrome P450 2E1.

The Japanese Ministry of Health, Labour, and Welfare recently reported an outbreak of bladder cancer among workers who handled aromatic amines in Japan. 2,4-dimethylaniline (2,4-DMA) is one of the chemicals that workers are considered to have the most opportunities to be exposed. Genotoxic events are known to be crucial steps in the initiation of cancer. However, studies on the genotoxicity of 2,4-DMA are limited, particularly studies investigating the mechanism behind the genotoxicity by 2,4-DMA are completely lacking. We examined genotoxic properties of 2,4-DMA using phosphorylated histone H2AX (γ-H2AX), a sensitive and reliable marker of DNA damage, in cultured human urothelial and hepatic cells. Our results clearly showed that 2,4-DMA at a concentration range of 1-10 mM generates γ-H2AX in both cell lines, indicating that 2,4-DMA is genotoxic. During mechanistic investigation, we found that 2,4-DMA boosts intracellular reactive oxygen species, an effect clearly attenuated by disulfiram, a strong inhibitor of cytochrome P450 2E1 (CYP2E1). In addition, CYP2E1 inhibitors and the antioxidant, N-acetylcysteine, also attenuated γ-H2AX generation following exposure to 2,4-DMA. Collectively, these results suggest that γ-H2AX is formed following exposure to 2,4-DMA via reactive oxygen species produced by CYP2E1-mediated metabolism. Continuous exposure to genotoxic aromatic amines such as 2,4-DMA over a long period of time may have contributed to the development of bladder cancer. Our results provide important insights into the carcinogenicity risk of 2,4-DMA in occupational bladder cancer outbreaks at chemical plants in Japan.

Featured structure-activity relationships for some tri- and tetrachlorobiphenyls in human CYP2E1-activated mutagenicity - Impact of the extent of ortho-chlorination.

Polychlorinated biphenyls (PCBs) as a group of persistent organic pollutants are confirmed human carcinogens; however, their mutagenicity remains mostly unknown. We have reported the mutagenicity of some PCBs with one to four chlorines in mammalian cells expressing human CYP2E1. To further explore the structural requirements for the mutagenicity of PCBs, eight tri- and tetrachlorobiphenyls untested before were investigated for the induction of gene mutations and micronuclei in a V79-derived cell line expressing both human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1), with SULT1A1 activity inhibited by pentachlorophenol, a potent SULT1 inhibitor. 2,2',6-Tri-, 2,3',6-tri, 2,4',6-tri-, and 2,2',5-trichlorobiphenyls (PCBs 19, 27, 32, and 18, respectively) induced micronuclei and gene mutations in V79-hCYP2E1-hSULT1A1 cells, at potencies slightly higher than 2,6-dichlorobiphenyl, but one order of magnitude below that by 2,3,3'- and 2,3,4'-trichlorobiphenyls as reported recently; in the parental V79-Mz cells, they were nonmutagenic and weak in micronuclei induction. Among the four tetrachlorobiphenyls with varying number of ortho chlorines, 2,3,3',4'-tetrachlorobiphenyl (PCB 56) induced both micronuclei and gene mutations in V79-hCYP2E1-hSULT1A1 cells with a potency greater than the above compounds; however, 2,2',3,3'-tetrachlorobiphenyl was equivocal and 2,2',3,6'-tetra- and 2,2',6,6'-tetrachlorobiphenyls inactive in V79-hCYP2E1-hSULT1A1 cells. Immunofluorescent staining of micronuclei formed by PCBs 32 and 56 in V79-hCYP2E1-hSULT1A1 cells with centromere protein B antibodies indicated that they were predominantly whole chromosomes, implying aneugenic potentials. This study suggests that tri- and tetrachlorobiphenyls with a single ortho chlorine can be most mutagenic under activation by human CYP2E1, and greater numbers of ortho chlorines may cause a drastic decline in the activity, especially for tetrachlorobiphenyls.

Study on the potential way of hepatic cytotoxicity of N,N-dimethylformamide.

The intermediate metabolites and redox status imbalance were supported as the two major points for N,N-dimethylformamide (DMF)-induced hepatotoxicity. However, the potential mechanism has not yet been concerned. By applying two inhibitors, this study tried to seek the major role in DMF-induced toxicity on HL7702 cell. We observed that DMF induced cell apoptosis through mitochondrial-dependent and p53 pathway. Inhibition reactive oxygen species by catalase remarkably attenuated the mitochondrial transmembrane potential (MMP), apoptotic proteins, and apoptosis. On the contrary, it reduced the biodegradation rate of DMF by coincubation with CYP2E1 antagonist (DDC) partially reduced late apoptosis. However, the change in MMP, the ratio of Bax to Bcl-xl, and cleaved-caspase 9 was not attenuated by DDC. The pathway in DDC coincubation groups was related to the p53 rather than the mitochondrial pathway. Restoring the redox balance during biodegradation is much more effective than attenuating the metabolite rate of DMF. This study may provide a suitable prevention method to occupational workers.

Trichloroethylene exposure results in the phosphorylation of histone H2AX in a human hepatic cell line through cytochrome P450 2E1-mediated oxidative stress.

Trichloroethylene (TCE), a chlorinated hydrocarbon, was recently reclassified as a human carcinogen by the International Agency for Research on Cancer. Genotoxic events are known to be crucial steps in the initiation of cancer. The genotoxic properties of TCE have been examined in many studies using a standard battery of genotoxicity tests both in vitro and in vivo. However, consistent results have not been obtained, and studies investigating the mechanism behind the genotoxicity of this compound are lacking. In the present study, we examined the genotoxicity of TCE by assessing phosphorylated histone H2AX (γ-H2AX), a new sensitive and reliable marker of DNA damage, in WRL-68 cells, cultured human hepatocytes and mouse livers. Our results showed that TCE exposure results in the generation of γ-H2AX, both in vitro and in vivo. By investigating the in vitro mechanism, we found that TCE increases the levels of intracellular reactive oxygen species (ROS) and that this increase in ROS levels is attenuated in the presence of disulfiram, a specific cytochrome P450 2E1 (CYP2E1) inhibitor. Furthermore, γ-H2AX induced by TCE was also attenuated by CYP2E1 inhibitors and the antioxidant N-acetylcysteine. These results suggested that ROS, produced via cytochrome P450 2E1-mediated metabolic processing, is a major causal factor for γ-H2AX generation upon exposure to TCE.

In vitro evaluation of structural analogs of diallyl sulfide as novel CYP2E1 inhibitors for their protective effect against xenobiotic-induced toxicity and HIV replication.

Diallyl sulfide (DAS) has been shown to prevent xenobiotic (e.g. ethanol, acetaminophen) induced toxicity and disease (e.g. HIV-1) pathogenesis. DAS imparts its beneficial effect by inhibiting CYP2E1-mediated metabolism of xenobiotics, especially at high concentration. However, DAS also causes toxicity at relatively high dosages and with long exposure times. Therefore, the goal of the current study was to investigate the structural analogs of DAS for their improved toxicity profiles and their effectiveness in reducing xenobiotic-induced toxicity and HIV-1 replication. Previously, we identified commercially available analogs that possessed CYP2E1 inhibitory capacity greater than or equal to that of DAS. In this study, we evaluated the toxicity and efficacy of these analogs using hepatocytes, monocytes, and astrocytes where CYP2E1 plays an important role in xenobiotic-mediated toxicity. Our results showed that thiophene, allyl methyl sulfide, diallyl ether, and 2-prop-2-enoxyacetamide are significantly less cytotoxic than DAS in these cells. Moreover, these analogs reduced ethanol- and acetaminophen-induced toxicity in hepatocytes and HIV-1 replication in monocytes more effectively than DAS. Overall, our findings are significant in terms of using these DAS analogs as a tool in vitro and in vivo, especially to examine chronic xenobiotic-induced toxicity and disease pathogenesis that occurs through the CYP2E1 pathway.

Higher CYP2E1 Activity Correlates with Hepatocarcinogenesis Induced by Diethylnitrosamine.

Hepatocellular carcinoma (HCC) is one of the most common types of primary liver cancer and the third most frequent cause of cancer death worldwide. Diethylnitrosamine (DEN) is one of the recognized risk factors for hepatocarcinogenesis likely due to CYP2E1-mediated metabolic activation. However, CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients has not been determined; the role of CYP2E1 activity, in particular CYP2E1 constitutive activity and CYP2E1 inhibited activity, with respect to the hepatocarcinogenesis induced by DEN is not yet clear. Herein, we determined CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients and found that CYP2E1-mediated DEN metabolic activity was significantly elevated with a 43.3% positive rate, and clinicopathologic parameters did not affect the activity. Then, using a Sprague-Dawley rat liver tumor model induced by DEN, the relationship between CYP2E1 constitutive/inhibited activity and hepatocarcinogenesis was explored. The results showed that the CYP2E1 constitutive activity was strongly correlated with tumor incidence and severity of liver tumorigenesis (nodule numbers and size), whereas inhibition of CYP2E1 activity decreased hepatocyte proliferation, liver injury, and liver carcinogenesis in the presence of DEN. In conclusion, the higher CYP2E1 activity would lead to an increased incidence of HCC as a result of CYP2E1-mediated DEN activation. Therefore, higher CYP2E1 activity might be a risk factor for HCC induced by DEN.

The protective effect of diallyl trisulfide on cytopenia induced by benzene through modulating benzene metabolism.

It has been known that metabolism of benzene is necessary for its toxicity. The purpose of our study is to investigate the effect of diallyl trisulfide (DATS) on attenuating cytopenia in peripheral blood introduced by benzene through regulating benzene metabolism in rats. We established benzene poisoning model with benzene (1.3 g/kg), while the DATS treatment groups were treated with DATS plus benzene (15 or 30 mg/kg) for 28 days, respectively. The results of blood parameters and concentration of metabolites of benzene (t, t-MA and SPMA) determination in urine showed that DATS could effectively attenuate the cytopenia induced by benzene through regulating benzene metabolism. Western blot and chemical method were used to detect the activities and protein expression levels of enzymes CYP2E1 and GSTT1 in liver and enzymes MPO and NQO1 in bone marrow were tested. The results suggested that the inhibition of bioactivation in liver and bone marrow catalyzed by CYP2E1 and MPO and the activation of detoxification catalyzed by GSTT1 and NQO1 might be the critical mechanism, through which DATS modulated benzene metabolism to prevent benzene-induced cytopenia.

The effect of quercetin on the pharmacokinetics of chlorzoxazone, a CYP2E1 substrate, in healthy subjects.

PURPOSE: Previous in vitro studies have demonstrated that quercetin inhibits CYP2E1 enzyme, but there are no available data to indicate that quercetin inhibits CYP2E1 enzyme in humans. The purpose of the present study was to assess the effect of quercetin on CYP2E1 enzyme activity in healthy subjects using chlorzoxazone (CHZ) as a CYP2E1 substrate. METHODS: An open-label, two-period, sequential study was conducted in 12 healthy subjects. A single dose of CHZ 250 mg was given to subjects during control phase and after treatment phases. Quercetin at a dose of 500 mg was given to subjects twice daily for a period of 10 days. The blood samples were collected at predetermined time intervals after CHZ dosing and analyzed to determine the concentrations of CHZ and 6-hydroxychlorzoxazone (6-OHCHZ). RESULTS: Treatment with quercetin significantly enhanced the maximum plasma concentration (C max), area under the curve (AUC), and half-life (t 1/2) by 47.8, 69.3, and 36.4%, respectively, while significantly decreased the elimination rate constant (k el) and apparent oral clearance (CL/F) of CHZ by 25.1 and 41.6%, respectively, in comparison with the control. On the other hand, C max and AUC of 6-OHCHZ were decreased by 30.1 and 32.6%, respectively, after quercetin treatment when compared to control. In addition, geometric mean ratios and 90% confidence intervals for C max and AUC of CHZ and 6-OHCHZ were both out of the no-effect boundaries of 0.80-1.25, which indicates a significant pharmacokinetic interaction present between CHZ and quercetin. Furthermore, treatment with quercetin significantly decreased the metabolic ratios of C max and AUC by 57.1 and 60.1%, respectively, as compared to control suggesting that reduced formation of CHZ to 6-OHCHZ. CONCLUSIONS: The results suggest that altered pharmacokinetics of CHZ might be attributed to quercetin-mediated inhibition of CYP2E1 enzyme. Further, the inhibition of CYP2E1 by quercetin may represent a novel therapeutic approach for minimizing the ethanol-induced CYP2E1 enzyme activity and results in reduced hepatotoxicity of ethanol.

Quercetin prevents type 1 diabetic liver damage through inhibition of CYP2E1.

BACKGROUND: Increased CYP2E1 protein and activity levels can be the main cause of stress-mediated liver damage in diabetes. In this work we investigated the quercetin properties to prevent diabetic oxidative liver injury through inhibition of CYP2E1. METHODS: Animals were randomly divided into three groups (n=5 for each group): non-diabetic control, STZ-diabetic rats and STZ-diabetic rats administered with quercetin (50mg/kg bw, per day, during 30days). Markers of oxidative stress and liver injury, hepatocyte ultrastructure and levels of CYP2E1 protein and activity were examined using biochemical, electron microscopy and molecular biological methods. RESULTS: It was shown that symptoms of diabetes (hyperglycemia, bodyweight loss, damaged hepatocyte ultrastructure), signs of oxidative stress in liver (2-fold intensification of peroxide process and 2-fold depletion of antioxidants) and serum markers of liver damage (3.5-, 1.5- and 5-fold increase in levels of ALT, AST and GGT, respectively) were present in STZ-diabetic rats. We found 3- and 2.5-fold increase in levels of protein and activity of CYP2E1 in the liver of STZ-diabetic rats. We demonstrated that the administration of quercetin leads to significant decrease in CYP2E1 activity (5- and 2-times compared to STZ-diabetic and control group, respectively). That was accompanied by normalization of pro-oxidant-antioxidant balance, improving the ultrastructure of hepatocytes and rates of serum markers of liver injury. CONCLUSIONS: CYP2E1 can play a crucial role in stress-induced pathological processes in the liver in diabetes, and the inhibition of the enzyme by quercetin during the development of diabetes mainly prevents the oxidative damage in liver.

Kinetic characterizations of diallyl sulfide analogs for their novel role as CYP2E1 enzyme inhibitors.

Diallyl sulfide (DAS), a selective inhibitor of CYP2E1, has shown protective effects against alcohol- and acetaminophen-induced hepatotoxicity in many studies. However, DAS is also a CYP2E1 substrate that on metabolism produces toxic metabolites and causes cytotoxicity. The objective of this study was to find a potent DAS analog as a CYP2E1 inhibitor and has the characteristic of producing less toxic metabolites. We selected seven commercially available compounds that are similar to DAS (DAS analogs). First, we performed ligand-CYP2E1 docking study to determine the binding mode and binding energy. The analysis suggested a relative potential for these DAS analogs as CYP2E1 inhibitor. We then performed a comprehensive inhibition kinetics of DAS analogs and determined the relative IC50 , Ki , and types of inhibition compared to that of DAS. The results showed that compared to DAS, diallyl ether and allyl methyl sulfide have lower Ki values (3.1 and 4.4 µmol/L, respectively, vs. 6.3 µmol/L for DAS) and IC50 values (6.3 and 11.4 µmol/L, respectively, vs. 17.3 µmol/L for DAS). However, allyl methyl sulfide and thiophene showed similar inhibitory capacities to that of DAS, and four other DAS analogs showed lower potency than DAS. In conclusion, we have found relatively more potent inhibitors of CYP2E1, which have lower toxicity than DAS. These compounds can replace DAS not only as a tool for in vitro and in vivo studies that involve CYP2E1 inhibition, but also can lead the way for their use in preventing CYP2E1-mediated hepatic toxicity of alcohol and acetaminophen.