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.

ω-Imidazolyl-alkyl derivatives as new preclinical drug candidates for treating non-alcoholic steatohepatitis.

Cytochrome P450 2E1 (CYP2E1)-associated reactive oxygen species production plays an important role in the development and progression of inflammatory liver diseases such as alcoholic steatohepatitis. We developed two new inhibitors for this isoenzyme, namely 12-imidazolyl-1-dodecanol (I-ol) and 1-imidazolyldodecane (I-an), and aimed to test their effects on non-alcoholic steatohepatitis (NASH). The fat-rich and CYP2E1 inducing Lieber-DeCarli diet was administered over 16 weeks of the experimental period to induce the disease in a rat model, and the experimental substances were administered simultaneously over the last four weeks. The high-fat diet (HFD) pathologically altered the balance of reactive oxygen species and raised the activities of the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP) and γ-glutamyl-transferase (γ-GT); lowered the level of adiponectine and raised the one of tumor necrosis factor (TNF)-α; increased the hepatic triglyceride and phospholipid content and diminished the serum HDL cholesterol concentration. Together with the histological findings, we concluded that the diet led to the development of NASH. I-ol and, to a lesser extent, I-an shifted the pathological values toward the normal range, despite the continued administration of the noxious agent (HFD). The hepatoprotective drug ursodeoxycholic acid (UDCA), which is used off-label in clinical practice, showed a lower effectiveness overall. I-ol, in particular, showed extremely good tolerability during the acute toxicity study in rats. Therefore, cytochrome P450 2E1 may be considered a suitable drug target, with I-ol and I-an being promising drug candidates for the treatment of NASH.

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.

The Effect of 4-Methylpyrazole on Oxidative Metabolism of Acetaminophen in Human Volunteers.

INTRODUCTION: Acetaminophen (APAP) is commonly ingested in both accidental and suicidal overdose. Oxidative metabolism by cytochrome P450 2E1 (CYP2E1) produces the hepatotoxic metabolite, N-acetyl-p-benzoquinone imine. CYP2E1 inhibition using 4-methylpyrazole (4-MP) has been shown to prevent APAP-induced liver injury in mice and human hepatocytes. This study was conducted to assess the effect of 4-MP on APAP metabolism in humans. METHODS: This crossover trial examined the ability of 4-MP to inhibit CYP2E1 metabolism of APAP in five human volunteers. Participants received a single oral dose of APAP 80 mg/kg, both with and without intravenous 4-MP, after which urinary and plasma oxidative APAP metabolites were measured. The primary outcome was the fraction of ingested APAP excreted as total oxidative metabolites (APAP-CYS, APAP-NAC, APAP-GSH). RESULTS: Compared with APAP alone, co-treatment with 4-MP decreased the percentage of ingested APAP recovered as oxidative metabolites in 24-hour urine from 4.48 to 0.51% (95% CI = 2.31-5.63%, p = 0.003). Plasma concentrations of these oxidative metabolites also decreased. CONCLUSIONS: These results show 4-MP effectively reduced oxidative metabolism of APAP in human volunteers ingesting a supratherapeutic APAP dose. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03878693.

CYPs-mediated drug-drug interactions on psoralidin, isobavachalcone, neobavaisoflavone and daidzein in rats liver microsomes.

The incubation system of CYP2E1 and CYP3A4 enzymes in rat liver microsomes was established to investigate the effects of psoralidin, isobavachalcone, neobavaisoflavone and daidzein from Fructus Psoraleae in vitro. The relevant metabolites were measured by the method of high performance liquid chromatography (HPLC), after probe substrates of 4-nitrophenol, testosterone and the drugs at different concentrations were added to the incubation systems. In addition, real-time RT-PCR was performed to determine the effect of psoralidin, neobavaisoflavone and daidzein on the mRNA expression of CYP3A4 in rat liver. The results suggested that psoralidin, isobavachalcone and neobavaisoflavone were Medium-intensity inhibitors of CYP2E1 with Ki values of 2.58, 1.28 and 19.07 µM, respectively, which could inhibit the increase of CYP2E1 and reduce diseases caused by lipid peroxidation. Isobavachalcone (Ki = 37.52 µM) showed a weak competitive inhibition on CYP3A4. Psoralidin and neobavaisoflavone showed obvious induction effects on CYP3A4 in the expression level of mRNA, which could accelerate the effects of drug metabolism and lead to the risk of inducing DDIs and serious adverse reactions. The results could be used for guideline of Fructus Psoraleae in clinic, which aimed to calculate the drug toxicity by studying the drug-drug interactions caused by the induction and inhibition of CYP450.

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.

End-product inhibition of skatole-metabolising enzymes CYP1A, CYP2A19 and CYP2E1 in porcine and piscine hepatic microsomes.

The hepatic cytochrome p450 enzymes 1 A, 2A19 and 2E1 is very important for the elimination of skatole from the body of pigs. Impaired skatole metabolism, results in skatole accumulation, which give rise to off flavor of the meat. Several metabolites of skatole has been identified, however the role of these metabolites in the inhibition of the skatole metabolizing enzymes are not documented. Using microsomes from pigs and fish, we determined the ability of several skatole metabolites to inhibit CYP1 A, CYP2A19 and CYP2E1 dependent activity. Our results show that 2-aminoacetophenone is an inhibitor of porcine CYP2A19 and CYP2E1 activity, but not the piscine orthologues. In conclusion, there is species specific differences in the inhibition of CYP1 A and CYP2A19 dependent metabolism of probe substrates. This is relevant to the evaluation of different model systems and to the reduction of off flavor of meat.

Mutagenic Activity of N-Nitrosodiethylamine in Cell Lines Expressing Human CYP2E1-Adequacy of Dimethylsulfoxide as Solvent.

Human CYP2E1 metabolizes many xenobiotics of low-molecular weight, thereby activating various promutagens/procarcinogens. In toxicological studies in vitro, dimethylsulfoxide (DMSO) is a common vehicle for organic compounds. However, it was observed to potently inhibit CYP2E1 activity. We were interested in whether it affects CYP2E1-dependent mutagenic responses. In this study, N-nitrosodiethylamine (NDEA), which is soluble in both water and DMSO, was used as a model promutagen. It induced Hprt gene mutations and micronuclei in a Chinese hamster V79-derived cell line expressing both human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) even at low-micromolar concentrations, but was inactive in parental V79 cells. Mutagenicity of NDEA was also observed in a recombinant V79-hCYP2E1 cell line that expresses human CYP2E1 at a lower level. NDEA induced micronuclei in human L-02 hepatocytes which expressed CYP2E1 even more weakly. DMSO did not modify NDEA-induced gene mutations or micronuclei, up to 0.2% (v:v, the highest noncytotoxic concentration) in V79-hCYP2E1-hSULT1A1 cells. In parental V79-Mz cells, NDEA induced micronuclei with Aroclor 1254-induced rat liver S9 mix, and this effect was unaffected by DMSO up to 0.2%. However, it inhibited the effect of NDEA in L-02 (by 44%) and V79-hCYP2E1 cells (by 70%) at 0.2%, with the effects of NDEA remaining statistically significant. No effect of DMSO was observed on CYP2E1 protein expression in V79-hCYP2E1-hSULT1A1 or its mRNA transcripts in each cell line. We conclude that DMSO may not significantly affect CYP2E1-dependent mutagenic effects, at concentrations up to 0.2% in cells with relatively high CYP2E1 expression. Environ. Mol. Mutagen. 60:214-226, 2019. © 2018 Wiley Periodicals, Inc.

Inhibition of CYP2E1 With Propylene Glycol Does Not Protect Against Hepatocellular Injury in Human Acetaminophen Daily-Dosing Model.

Acetaminophen (APAP)-induced liver injury is initiated by metabolism of APAP by the cytochrome P-450 (CYP) system, primarily CYP2E1. We previously demonstrated CYP inhibition following administration of a liquid APAP formulation containing propylene glycol, a CYP2E1 inhibitor, and other excipients. This study was undertaken to determine if propylene glycol specifically inhibits production of CYP-derived metabolites and if propylene glycol reduces the rise in alanine aminotransferase (ALT) seen following prolonged APAP dosing. Human subjects were randomized to receive 4 g of APAP daily in one arm of the study or 4 g of APAP with 5 mL of 99% propylene glycol in the other arm, both for 14 days. After a washout period of at least 14 days, subjects were crossed over between arms. Outcomes were rise of ALT greater than 2 times baseline (responders) and proportion of randomly sampled CYP-derived metabolites relative to total metabolites produced. There was no difference in percentage of responders between treatment groups: 6 of 21 in the APAP group (29%) compared with 8 of 20 in the APAP + propylene glycol group (40%); chi-square, P = .59. For all subjects, the mean percentage of CYP-derived metabolites produced was 5.8% (APAP) versus 4.3% (APAP + propylene glycol); P = .018. This effect was solely attributable to the responders: the mean percentage of CYP metabolites of responders was 7.7% (APAP) versus 4.6% (APAP + propylene glycol), P = .050, whereas there was no difference for the nonresponders. Five subjects were responders in both arms (2% probability of random occurrence). Our data indicates that propylene glycol inhibits CYP2E1 metabolism of APAP in some subjects but does not effect hepatocellular indury at the dose given.

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.