Cytochrome P450 2E1 Inhibitor Research Articles

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

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. In this study, we applied a rational drug design concept to develop drug candidates (NCE) including preclinical studies. 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. 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.

Assessment of CYP2A6 and CYP2C19 Genetic Variation on Cotinine‐N‐Oxide Formation and their Potential Effect on the Nicotine Metabolic Ratio (NMR)

SignificanceThe nicotine metabolic ratio (NMR) of 3‐hydroxycotinine (3HC)/cotinine in smokers is a determinant of CYP2A6 activity and is widely used in clinical practice to help select smoking cessation pharmacotherapy. The major mode of metabolism of nicotine is via the formation of cotinine and subsequently to 3HC by the enzyme CYP2A6. Cotinine is also metabolized to cotinine‐N‐glucuronide and to cotinine‐N‐oxide (COX), accounting for ~7 and 5% of urinary nicotine metabolites, respectively, in smokers. The goal of the present study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway.MethodsWe used specific inhibitors of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in cotinine‐N‐oxidation reactions using pooled human liver microsomes (HLM). COX formation was monitored by UPLC‐MS/MS, and enzyme kinetic analysis was performed using microsomal fractions from CYP‐overexpressing HEK293 cell lines. Additionally, genotype‐phenotype analysis was performed for a panel of 113 HLM specimens.ResultsInhibition of COX formation was only observed when using inhibitors of CYPs 2A6, 2B6, 2C19, 2E1, and 3A4. CYPs 2A6 and 2C19 exhibited similar oxidation activity against cotinine with Vmax/KM values of 4.4, and 4.2 nL•min−1•mg−1, respectively; the Vmax/KM value for the 2B6 isoform was of 2.2 nL•min−1•mg−1. The activity observed for both CYPs 2E1 and 3A4 were low, with KM’s greater than 10 mM. We measured the levels of COX formation in 113 HLM specimens and genotyped variants in CYPs 2A6 (*2, *9, and *14), 2B6 (*2, *5, *9, rs4803419 and rs8109525) and 2C19 (*2 and *17) using qPCR. An association between decreased levels of COX formation and CYP variants in the 2C19 (up to 35% reduction) and 2A6 (up to 38% reduction) isoforms was observed. No statistically significant correlation was observed with variants in the 2B6 isoform.ConclusionsThese results demonstrate that genetic variants in CYPs 2A6 and 2C19 are associated with decreased COX formation, potentially affecting the levels of free cotinine and consequently the calculated NMR in smokers and clinical practices surrounding smoking cessation therapies.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program and CONACyT I2T2 (to Y. Perez‐Paramo), Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)

Cryptotanshinone from the Salvia miltiorrhiza Bunge Attenuates Ethanol-Induced Liver Injury by Activation of AMPK/SIRT1 and Nrf2 Signaling Pathways.

Cryptotanshinone (CT), a diterpene that is isolated from Salvia miltiorrhiza Bunge, exhibits anti-cancer, anti-oxidative, anti-fibrosis, and anti-inflammatory properties. Here, we examined whether CT administration possess a hepatoprotective effect on chronic ethanol-induced liver injury. We established a chronic alcohol feeding mouse model while using C57BL/6 mice, and examined the liver sections with hematoxylin-eosin (H&E) and Oil Red O (ORO) staining. Further, we analyzed the lipogenesis, fatty acid oxidation, oxidative stress, and inflammation genes by using quantitative polymerase chain reaction (qPCR) and immunoblotting in in vivo, and in vitro while using HepG2 and AML-12 cells. CT treatment significantly ameliorated ethanol-promoted hepatic steatosis, which was consistent with the decreased hepatic triglyceride levels. Interestingly, CT activated the phosphorylation of AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and nuclear factor E2-related factor 2 (Nrf2) proteins. Importantly, compound C (AMPK inhibitor) significantly blocked the CT-mediated reduction in TG accumulation, but not Ex52735 (SIRT1 inhibitor), which suggested that CT countering ethanol-promoted hepatic steatosis is mediated by AMPK activation. Furthermore, CT significantly inhibited cytochrome P450 2E1 (CYP2E1) and enhanced both the expression of antioxidant genes and hepatic glutathione levels. Finally, CT inhibited the ethanol-induced inflammation in ethanol-fed mice and HepG2 cells. Overall, CT exhibits a hepatoprotective effect against ethanol-induced liver injury by the inhibition of lipogenesis, oxidative stress, and inflammation through the activation of AMPK/SIRT1 and Nrf2 and the inhibition of CYP2E1. Therefore, CT could be an effective therapeutic agent for treating ethanol-induced liver injury.

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

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. 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). 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. These results show 4-MP effectively reduced oxidative metabolism of APAP in human volunteers ingesting a supratherapeutic APAP dose. ClinicalTrials.gov Identifier: NCT03878693.

Upregulation of CYP2E1 expression causes oxidative damage induced by 2-chloroethanol in primary cultured rat astrocytes

Brain edema caused by subacute poisoning with 1,2-dichloroethane (1,2-DCE) has gained much attention during recent years, but its underlying mechanism is poorly understood. As an intermediate metabolite of 1,2-DCE in vivo, 2-chloroethanol (2-CE) can be transformed into chloroacetaldehyde and reactive oxygen species (ROS) through cytochrome P450 2E1 (CYP2E1) mediated metabolism. In previous studies, it was found that CYP2E1 expression is enhanced in the brain of mice treated with 1,2-DCE. This study was designed to verify the roles of CYP2E1 overexpression in 2-CE induced cytotoxicity in rat astrocytes, and the contribution of specific signaling molecules to the upregulation of CYP2E1 expression caused by 2-CE. The results of this study demonstrate that treatment with 2-CE can enhance CYP2E1 protein and mRNA levels, cause an increase in ROS and MDA levels, and higher percentages of apoptotic cells in rat astrocytes. Pretreatment with either diallyl sulfide or vitamin C, the inhibitor of CYP2E1 or scavenger of ROS, respectively, can suppress the levels of CYP2E1 expression, ROS and MDA, ameliorate cell apoptosis, and attenuate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Additionally, pretreatment with the inhibitor of either ERK1/2 or transcriptional factor specificity protein 1 (SP1) can suppress the CYP2E1 expression, and alleviate the oxidative damage caused to these cells. In conclusion, our findings demonstrate that CYP2E1 overexpression plays a crucial role in 2-CE induced oxidative damage of rat astrocytes, and that CYP2E1 expression is upregulated partially through the activation of the ERK1/2 and SP1 signaling pathways by ROS generated during CYP2E1-mediated 2-CE metabolism. This study provides novel information that can be used in elucidating the mechanism by which 1,2-DCE induces brain edema.

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.

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 (80mg/kg) was administered orally without or with silymarin (100mg/kg), a known inhibitor of CYP2E1, CYP3A4 and quercetin (10 and 20mg/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 20mg/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.

Dihydromyricetin alleviates acetaminophen-induced liver injury via the regulation of transformation, lipid homeostasis, cell death and regeneration

AimsWe previously reported that Hovenia dulcis Thunb. extract, a traditional Chinese medicine rich in dihydromyricetin (DHM), exhibited a significant hepatoprotective effect against acetaminophen (APAP)-induced liver injury. However, whether DHM plays a protective role in APAP hepatotoxicity and what mechanisms are involved remain unclear. In this study, we evaluated the hepatoprotective effects of DHM against APAP-induced liver injury. Main methodsMale C57BL/6 mice were used for the experiment. LC-MS, q-PCR, immunochemistry and western blot analysis were employed to mechanism analysis. Key findingsDHM exhibited a protective effect against APAP-induced liver injury. Further mechanistic investigations revealed that the protective effect of DHM against APAP hepatotoxicity had multi-target and multi-pathway characteristics involving APAP metabolism, lipid regulation, and hepatocyte death and regeneration. DHM pretreatment resulted in cytochrome P450 2E1 inhibition and UDP-glucuronosyltransferase 1A1 activation, affecting APAP biotransformation. Moreover, DHM pretreatment significantly ameliorated lipid dysregulation via peroxisome proliferator-activated receptor and sterol regulatory element-binding protein-1c (SREBP-1c) signalling pathways. Furthermore, DHM regulated the expression of cell death- and liver regeneration-associated proteins. SignificanceThese results suggested that DHM alleviated APAP-induced liver injury in mice by inhibiting hepatocyte death, promoting p53-related regeneration, and regulating lipid homeostatic imbalance and APAP transformation. Based on these findings, DHM provides a potential and novel approach for preventing and treating APAP-induced liver damage, and SREBP-1c signalling might be a new therapeutic target for APAP hepatotoxicity.

Cytochrome P450 Enzyme Contributions to the N‐oxide Detoxification Pathway of Tobacco‐Specific Nitrosamines; a Possible Role in Tobacco‐Related Cancer Risk

Tobacco‐specific nitrosamines (TSNAs) including 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) are among the most potent carcinogens found in tobacco products. The major mode of metabolism of NNK is by reduction to form 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), which, like other TSNAs including N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), and N′‐nitrosoanabatine (NAT), is detoxified primarily by UDP‐glucuronosyltransferases (UGTs) to their glucuronide products, but also by N‐oxidation of the common pyridine ring. TSNA‐N‐oxides were identified previously in the urine of rodents exposed to TSNAs, and NNAL‐N‐oxide was found in the urine of smokers. The enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors (furafylline, tranylcypromine, clopidrogrel, montelukast, sulfaphenazole, quinidine, chlomethiazole, and ketoconazole) of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in oxidation reactions using pooled human liver microsomes. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors of CYPs 2A6, 2C8, 2C19, 2E1 and 3A4; no detectable levels of NNN‐, NAT‐ or NAB‐N‐oxide formation were observed in human liver microsomes. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation were examined using microsomal fractions from CYP over‐expressing HEK293 cell lines. CYPs 2A13, 2C8 and 2E1 exhibited the highest oxidation activity against NNK, with Vmax/KM values of 675, 610 and 559 nL•min−1•mg−1, respectively, and against NNAL, with Vmax/KM values of 105, 67 and 86 nL•min−1•mg−1, respectively. These data suggest that several cytochrome P450 enzymes play an important role in the in situ detoxification of NNK and NNAL and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk assessment.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program (to Y. Perez‐Paramo), and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of Cytochrom P4502E1 in Alcoholic Liver Disease and alcohol mediated carcinogenesis

Various factors are involved in the pathogenesis of alcoholic liver disease (ALD) and ethanol-mediated carcinogenesis. In addition to genetic, epigenetic and immunologic mechanisms, acetaldehyde-associated toxicity, oxidative stress as well as cytokine-mediated inflammation are of major importance. Oxidative stress, with the generation of reactive oxygen species (ROS), develops either in inflammation (alcoholic hepatitis) or during oxidation of ethanol via cytochrome P4502E1 (CYP2E1). CYP2E1 is induced by ethanol, oxidizes ethanol to acetaldehyde, and generates ROS during this process. ROS results in protein damage, enhanced fibrogenesis and DNA lesions. Furthermore, CYP2E1 induction results in an enhanced activation of various procarcinogens and an increased degradation of retinol and retinoic acid (RA), a compound responsible for cell differentiation and proliferation. An inhibition of CYP2E1 results in an improvement of ALD and chemically induced carcinogenesis in animal experiments. In humans, CYP2E1 is induced following the consumption of 40grams of ethanol per day after one week. However, the induction varies inter-individually. The mechanism for this is still unclear. Patients with ALD show a significant correlation between CYP2E1, the occurrence of highly carcinogenic etheno DNA adducts and the severity of fibrosis. First results on the effect of CYP2E1 inhibition by chlormethiazole, a specific CYP2E1 inhibitor on ALD, can be expected soon.

Antioxidant, Anti-inflammatory, and Analgesic Activities of Citrus reticulata Blanco Leaves Extracts: An In Vivo and In Vitro Study

Citrus species are cultivated and consumed widely. Citrus have been investigated for their pharmacological activity and human health. Their beneficial effects include antibacterial, analgesic, anti-inflammatory, and antitumoral effects. This studywas designed to evaluate the analgesic effect and the antioxidant and anti-inflammatory activities of Citrus reticulata Blanco leaves extracts (ECR) in cell and animal models. Antioxidant, anti-inflammatory, and antinociceptive activities were evaluated in mice using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition, xylene-induced ear edema, formalin assay and acetic acid-writhing response. Expression of antiinflammatory genes was measured in lipopolysaccharide (LPS)-treated Huh7 cells. ECR showed a significant DPPH radical scavenging activity. No behavioral changes or deaths were observed in mice at doses less than 2,000 mg/kg body weight. Different concentrations of methanolic and aqueous extracts (100–500 mg/kg body wt.) reduced the duration of linking behavior in the second phase of the formalin chemical nociception assay and decreased the number of acetic acidinduced writhing responses in mice, indicating significant analgesic activity. ECR also diminished xylene-induced ear swelling in mice, suggesting an In Vivo anti-inflammatory action. No toxicity of ECR in the range of 0.1–10 μg/ml was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Cell treatment with LPS-induced oxidative/ nitrosative stress as assessed by flow cytometry as the fluorescence of 2′,7′-dichlorofluorescein. This effect was significantly inhibited in a dose-dependent manner by ECR. Administration of ECR caused a dose-dependent inhibition of cytochrome P450 2E1, inducible nitric oxide synthase, tumor necrosis factor α, and interleukin-6 expression in LPS-treated cells. The present study demonstrates that extracts of Citrus reticulata leaves are safe, having antioxidant, anti-inflammatory, and analgesic effects both In Vivo and In Vitro.

Allyl Sulfide Counteracts 1-Bromopropane-Induced Neurotoxicity by Inhibiting Neuroinflammation and Oxidative Stress

Chronic exposure to 1-bromopropane (1-BP), an alternative to ozone-depleting solvents, produces potential neurotoxicity in occupational populations. However, no therapeutic strategy is available currently. Accumulating evidence suggests that cytochrome P4502E1 (CYP2E1) is critical for the active metabolism of 1-BP. The purpose of this study is aimed to test whether inhibition of CYP2E1 by allyl sulfide, a specific inhibitor of CYP2E1, could be able to protect against 1-BP-induced neurotoxicity. Male Wistar rats were intoxicated with 1-BP for 9 continuous weeks with or without allyl sulfide pretreatment. Results clearly demonstrated that 1-BP exposure induced decrease in NeuN+ cells and increase in cleaved caspase-3 expression and TUNEL+ cells in motor cortex of rats, which was significantly ameliorated by allyl sulfide. Allyl sulfide treatment also recovered the motor performance of rats treated with 1-BP. Mechanistically, allyl sulfide-inhibited 1-BP-induced expression of CYP2E1 in microglia, which was associated with suppression of microglial activation and M1 polarization in motor cortex of rats. Reduced oxidative stress was also observed in rats treated with combined allyl sulfide and 1-BP compared with 1-BP alone group. Furthermore, we found that allyl sulfide abrogated 1-BP-induced activation of Nuclear factor(NF)-κB and GSH/Thioredoxin/ASK1 pathways, the key factor for the maintenance of M1 microglial inflammatory response and oxidative stress-related neuronal apoptosis, respectively. Thus, our results showed that allyl sulfide exerted neuroprotective effects in combating 1-BP-induced neurotoxicity through inhibition of neuroinflammation and oxidative stress. Blocking CYP2E1 activity by allyl sulfide might be a promising avenue for the treatment of neurotoxicity elicited by 1-BP and other related neurotoxicants.

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 1-10mM 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.

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.

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.

Kaempferol’s Protective Effect on Ethanol-Induced Mouse Primary Hepatocytes Injury Involved in the Synchronous Inhibition of SP1, Hsp70 and CYP2E1

The mechanism of ethanol-induced hepatotoxicity was complicated, accompanied by the over-expressions of the cytochrome P450 2E1 (CYP2E1), heat shock protein 70 (Hsp70) and the nuclear factor specificity protein 1 (SP1). Kaempferol (Kaem) could protect the ethanol-induced hepatotoxicity likely by inhibiting the CYP2E1 expression and activity. This study investigated the protective mechanism(s) of kaempferol on ethanol-induced toxicity by dynamic alteration of SP1, Hsp70 and CYP2E1 among the nucleus and different organelles in hepatocytes. After ethanol treatment alone and co-incubation hepatocytes with kaempferol, protein levels of CYP2E1, Hsp70, and SP1 were determined in vitro (western blotting and immunofluorescence). Hepatocytes' viability was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods. Glutathione (GSH) levels were evaluated for ethanol-induced oxidative stress. In the ethanol-treated hepatocytes, kaempferol decreased protein levels of CYP2E1 in both microsome and mitochondria, cytosolic Hsp70 and SP1 in nuclear and cytosol, and the oxidative stress and increased the cell viability compared to those of ethanol group. Collectively, our findings propose that the protective mechanism of kaempferol is involved in the synchronous, early and persistent inhibitions of mitochondrial and microsomal CYP2E1, cytosolic Hsp70 and nuclear and cytosolic SP1 in mouse primary hepatocytes' injury induced by ethanol.

Carcinogenic Etheno DNA Adducts in Alcoholic Liver Disease: Correlation with Cytochrome P-4502E1 and Fibrosis.

One mechanism by which alcoholic liver disease (ALD) progresses is oxidative stress and the generation of reactive oxygen species, among others due to the induction of cytochrome P-4502E1 (CYP2E1). Experimental data underline the key role of CYP2E1 because ALD could be partially prevented in rats by the administration of the specific CYP2E1 inhibitor chlormethiazole. As CYP2E1 is linked to the formation of carcinogenic etheno DNA adducts in ALD patients, a causal role of alcohol-induced CYP2E1 in hepatocarcinogenesis is implicated. The purpose of this study was to investigate CYP2E1 induction in ALD, and its correlation with oxidative DNA lesions and with hepatic histology. Hepatic biopsies from 97 patients diagnosed with ALD were histologically scored for steatosis, inflammation, and fibrosis. CYP2E1 and the exocyclic etheno DNA adduct 1,N6 -etheno-2'deoxyadenosine (εdA) were determined immunohistochemically. In addition, in 42 patients, 8-hydroxydeoxyguanosine (8-OHdG) was also evaluated using immunohistochemistry. A significant positive correlation was found between CYP2E1 and εdA (p<0.0001) as well as between CYP2E1 and 8-OHdG (p=0.039). Both CYP2E1 (p=0.0094) and ɛdA (p<0.0001) also correlated significantly with the stage of hepatic fibrosis. Furthermore, a significant correlation between the fibrosis stage and the grade of lobular inflammation (p<0.0001) was observed. However, the amount of alcohol consumed did not correlate with any of the parameters determined. These data suggest an important role of CYP2E1 in the generation of εdA, in the fibrotic progression of ALD, and thus in alcohol-mediated hepatocarcinogenesis. CYP2E1 may be a target in the treatment of ALD and a potential prognostic marker for disease progression.

High CYP2E1 activity correlates with hepatofibrogenesis induced by nitrosamines.

Hepatofibrosis, which leads to cirrhosis and eventual hepatocellular carcinoma, is a common response to chronic toxin-mediated liver injury. Nitrosamines are potent hepatotoxic agents that cause necrosis and subsequent fibrosis in the liver as a result of cytochrome P450 2E1 (CYP2E1)-dependent metabolism, which generates toxic metabolites that form adducts with nucleic acids, leading to hepatotoxicity and mutagenesis. Herein, CYP2E1 activity and content were determined in fibrotic liver tissue from patients with hepatocellular carcinoma. The relationship between CYP2E1 innate activity and hepatofibrogenesis was evaluated, the effect of inhibition of CYP2E1 activity on hepatofibrosis was determined in a Sprague-Dawley rat model of diethylnitrosamine-induced hepatofibrosis. The results demonstrated that the CYP2E1 activities in human fibrotic tissues are significantly higher than that in normal liver tissues. In rats treated with diethylnitrosamine, the livers demonstrated various degree of fibrotic changes and collagen deposition in individual rats. The Ishak score, which determines the stage of fibrosis, correlated with CYP2E1 innate activity, with greater fibrosis in rat livers with higher CYP2E1 innate activity. Inhibition of CYP2E1 during diethylnitrosamine treatment decreased hepatofibrosis and there was an inverse correlation between the degree of inhibition and the extent of hepatofibrosis. Therefore, high CYP2E1 activity is a risk factor for hepatofibrogenesis induced by nitrosamines.

Impairment of Akt activity by CYP2E1 mediated oxidative stress is involved in chronic ethanol-induced fatty liver

Protein kinase B (PKB/Akt) plays important roles in the regulation of lipid homeostasis, and impairment of Akt activity has been demonstrated to be involved in the development of non-alcoholic fatty liver disease (NAFLD). Previous studies suggest that cytochrome P4502E1 (CYP2E1) plays causal roles in the pathogenesis of alcoholic fatty liver (AFL). We hypothesized that Akt activity might be impaired due to CYP2E1-induced oxidative stress in chronic ethanol-induced hepatic steatosis. In this study, we found that chronic ethanol-induced hepatic steatosis was accompanied with reduced phosphorylation of Akt at Thr308 in mice liver. Chronic ethanol exposure had no effects on the protein levels of phosphatidylinositol 3 kinase (PI3K) and phosphatase and tensin homologue deleted on chromosome ten (PTEN), and led to a slight decrease of phosphoinositide-dependent protein kinase 1 (PDK-1) protein level. Ethanol exposure resulted in increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)-Akt adducts, which was significantly inhibited by chlormethiazole (CMZ), an efficient CYP2E1 inhibitor. Interestingly, N-acetyl-L-cysteine (NAC) significantly attenuated chronic ethanol-induced hepatic fat accumulation and the decline of Akt phosphorylation at Thr308. In the in vitro studies, Akt phosphorylation was suppressed in CYP2E1-expressing HepG2 (CYP2E1-HepG2) cells compared with the negative control HepG2 (NC-HepG2) cells, and 4-HNE treatment led to significant decrease of Akt phosphorylation at Thr308 in wild type HepG2 cells. Lastly, pharmacological activation of Akt by insulin-like growth factor-1 (IGF-1) significantly alleviated chronic ethanol-induced fatty liver in mice. Collectively, these results indicate that CYP2E1-induced oxidative stress may be responsible for ethanol-induced suppression of Akt phosphorylation and pharmacological modulation of Akt in liver may be an effective strategy for the treatment of ethanol-induced fatty liver.

CYP2E1 regulates the development of radiation-induced pulmonary fibrosis via ER stress- and ROS-dependent mechanisms.

Radiation-induced pulmonary fibrosis (RIPF) is one of the most common side effects of lung cancer radiotherapy. This study was conducted to identify the molecular mechanism responsible for RIPF. We revealed that the transcriptional level of cytochrome P450 2E1 (CYP2E1) was elevated by examining expression profile analysis of RIPF mouse models. We also confirmed that CYP2E1 regulated levels of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in alveolar epithelial type II (AE2) cells and lung fibroblasts. Inhibition of CYP2E1 via its siRNA or inhibitor significantly attenuated epithelial-to-mesenchymal transition and apoptosis of AE2 cells, as well as myofibroblast formation induced by radiation. Finally, the effects of a CYP2E1 inhibitor on development of RIPF were evaluated by in vivo studies. Taken together, the results of the present study suggest that CYP2E1 is an important mediator of RIPF development that functions by increasing cellular ER stress and ROS levels.