Induction Of CYP2E1 Research Articles

Induction of Cyp2e1 contributes to asparaginase-induced hepatocyte sensitization to lipotoxicity

One of the leading therapies for acute lymphoblastic leukemia (ALL) is the chemotherapeutic agent PEGylated E. coli-derived-l-asparaginase (PEG-ASNase). Due to the high risk of dose-limiting liver injury, characterized by clinically elevated levels of hepatic transaminases, PEG-ASNase therapy is generally avoided in adult patients. Our preclinical investigations have indicated that PEG-ASNase-induced liver injury is associated with the release of free fatty acids (FFAs) from white adipose tissue (WAT), suggesting potential lipotoxic effects. However, it remains uncertain whether PEG-ASNase directly induces hepatotoxicity or sensitizes hepatocytes to FFA-induced toxicity. Our results show that PEG-ASNase treatment results in hepatocyte apoptosis and lipid peroxidation. Ex vivo and in vitro studies in mouse and human WAT suggest that PEG-ASNase induces the expression of adipose triglyceride lipase (ATGL), activates the lipase, and stimulates adipose tissue lipolysis, suggesting that the FFAs from WAT may contribute to the observed liver injury. Moreover, treatment with PEG-ASNase sensitizes hepatocytes to FFA-induced lipotoxicity. Mechanistically, our RNA-sequencing (RNA-seq) analyses reveal that PEG-ASNase-induced sensitization to lipotoxicity is accompanied by the induction of Cyp2e1. We demonstrated that this sensitization effect is attenuated by both pharmacological and genetic inhibition of Cyp2e1. Our findings suggest that PEG-ASNase therapy induces WAT lipolysis and sensitizes hepatocytes to hepatic lipotoxicity in a Cyp2e1-dependent manner.

Combinatory Effects of Acrylamide and Deoxynivalenol on In Vitro Cell Viability and Cytochrome P450 Enzymes of Human HepaRG Cells.

Acrylamide (AA) can be formed during the thermal processing of carbohydrate-rich foods. Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., contaminates many cereal-based products. In addition to potential co-exposure through a mixed diet, co-occurrence of AA and DON in thermally processed cereal-based products is also likely, posing the question of combinatory toxicological effects. In the present study, the effects of AA (0.001-3 mM) and DON (0.1-30 µM) on the cytotoxicity, gene transcription, and expression of major cytochrome P450 (CYP) enzymes were investigated in differentiated human hepatic HepaRG cells. In the chosen ratios of AA-DON (10:1; 100:1), cytotoxicity was clearly driven by DON and no overadditive effects were observed. Using quantitative real-time PCR, about twofold enhanced transcript levels of CYP1A1 were observed at low DON concentrations (0.3 and 1 µM), reflected by an increase in CYP1A activity in the EROD assay. In contrast, CYP2E1 and CYP3A4 gene transcription decreased in a concentration-dependent manner after incubation with DON (0.01-0.3 µM). Nevertheless, confocal microscopy showed comparably constant protein levels. The present study provided no indication of an induction of CYP2E1 as a critical step in AA bioactivation by co-occurrence with DON. Taken together, the combination of AA and DON showed no clear physiologically relevant interaction in HepaRG cells.

CYP2E1 in 1,4-dioxane metabolism and liver toxicity: insights from CYP2E1 knockout mice study.

1,4-Dioxane (DX), an emerging water contaminant, is classified as a Group 2B liver carcinogen based on animal studies. Understanding of the mechanisms of action of DX liver carcinogenicity is important for the risk assessment and control of this environmental pollution. Previous studies demonstrate that high-dose DX exposure in mice through drinking water for up to 3 months caused liver mild cytotoxicity and oxidative DNA damage, a process correlating with hepatic CYP2E1 induction and elevated oxidative stress. To access the role of CYP2E1 in DX metabolism and liver toxicity, in the current study, male and female Cyp2e1-null mice were exposed to DX in drinking water (5000 ppm) for 1 week or 3 months. DX metabolism, redox and molecular investigations were subsequently performed on male Cyp2e1-null mice for cross-study comparisons to similarly treated male wildtype (WT) and glutathione (GSH)-deficient Gclm-null mice. Our results show that Cyp2e1-null mice of both genders were resistant to DX-induced hepatocellular cytotoxicity. In male Cyp2e1-null mice exposed to DX for 3 months, firstly, DX metabolism to β-hydroxyethoxyacetic acid was reduced to ~ 36% of WT levels; secondly, DX-induced hepatic redox dysregulation (lipid peroxidation, GSH oxidation, and activation of NRF2 antioxidant response) was substantially attenuated; thirdly, liver oxidative DNA damage was at a comparable level to DX-exposed WT mice, accompanied by suppression of DNA damage repair response; lastly, no aberrant proliferative or preneoplastic lesions were noted in DX-exposed livers. Overall, this study reveals, for the first time, that CYP2E1 is the main enzyme for DX metabolism at high dose and a primary contributor to DX-induced liver oxidative stress and associated cytotoxicity. High dose DX-induced genotoxicity may occur via CYP2E1-independent pathway(s), potentially involving impaired DNA damage repair.

Human quad liver-on-chip system as a tool toward bridging the gap between animals and humans regarding toxicology and pharmacology of a cannabidiol-rich cannabis extract

Cannabidiol (CBD) is a major phytocannabinoid from Cannabis sativa. It is currently widely available and widely used in the USA, but despite its rapid progress to market, the pharmacology and toxicology of both CBD and cannabidiol-rich cannabis extracts (CRCE) remain largely unknown. The goals of this study were to investigate the potential of a novel human microphysiological system to emulate CRCE-induced hepatotoxicity and pharmacological properties demonstrated in animal models. For this purpose, C57BL6/J male mice were subjected to dosing with either 0, 61.5, 184.5, or 615 mg/kg of CRCE for 10 days. The liver-on-chip system, incorporating human primary hepatocytes, sinusoidal endothelial cells, as well as Kupffer and stellate cells was subjected to 0, 300, 1,200, or 4,400 ng/mL of CRCE (8 h exposure followed by 16 h washout) for 5 days. Administration of CRCE in mice resulted in nearly 4-fold elevations of plasma ALT at 615 mg/kg (p < 0.01) and a dose-dependent decrease in intrahepatic miR-122. Elevated levels of ALT, paralleled by decreased intrahepatic and increased effluent levels of miR-122, were also observed in the liver-on-chip, although these results were not statistically significant. Exposure to CRCE resulted in a robust and dose-dependent induction of key cytochrome P450 enzymes, namely Cyp1a2, Cyp2b6 (CYP2B10), Cyp2e1, and Cyp2c9 (CYP2C19) in both mouse livers and liver-on-chip. The results of this study demonstrate the congruence between the responses observed in mouse and human liver-on-chip experimental systems and provide evidence of the potential microphysiological systems hold for translating animal data into clinical practice.

Hydrogen Peroxide Induces Ethanol-inducible CYP2E1 via the NFkB-classical Pathway: CYP2E1 mRNA Levels are not High in Alcoholic Hepatitis.

The aim of the present study is to elucidate the mechanism of CYP2E1 induction as a causative factor of alcoholic hepatitis (AH) and its relationship with inflammation. Chronic alcohol consumption induces CYP2E1, which is involved in the development of alcoholic hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H2O2), the concentration of which is elevated under inflammatory conditions. The mechanisms underlying the induction of CYP2E1 by H2O2 were examined with a focus on Keap1, a target factor of H2O2. We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H2O2, and evaluated changes in the expression of the NFkB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH. The induction of CYP2E1 by H2O2 was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H2O2, markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients. We herein demonstrated that the expression of CYP2E1 was induced by H2O2. The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H2O2 did not after the knockdown of RelA. These results suggest that H2O2 acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H2O2-Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AHH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH.

Metabolism-dependent mutagenicity of two structurally similar tobacco-specific nitrosamines (N-nitrosonornicotine and N-nitrosoanabasine) in human cells, partially different CYPs being activating enzymes

N-nitrosonornicotine (NNN) and N-nitrosoanabasine (NAB) are both tobacco-specific nitrosamines bearing two heterocyclic amino groups, NAB bearing an extra -CH2- group (conferring a hexa- rather than penta-membered cycle) but with significantly decreased carcinogenicity. However, their activating enzymes and related mutagenicity remain unclear. In this study, the chemical-CYP interaction was analyzed by molecular docking, thus the binding energies and conformations of NNN for human CYP2A6, 2A13, 2B6, 2E1 and 3A4 appeared appropriate as a substrate, so did NAB for human CYP1B1, 2A6, 2A13 and 2E1. The micronucleus test in human hepatoma (HepG2) cells with each compound (62.5–1000 μM) exposing for 48 h (two-cell cycle) was negative, however, pretreatment with bisphenol AF (0.1–100 nM, CYPs inducer) and ethanol (0.2% v:v, CYP2E1 inducer) potentiated micronucleus formation by both compounds, while CITCO (1 μM, CYP2B6 inducer) selectively potentiated that by NNN. In C3A cells (endogenous CYPs enhanced over HepG2) both compounds induced micronucleus, which was abolished by 1-aminobenzotriazole (60 μM, CYPs inhibitor) while unaffected by 8-methoxypsoralen (1 μM, CYP2A inhibitor). Consistently, NNN and NAB induced micronucleus in V79-derived recombinant cell lines expressing human CYP2B6/2E1 and CYP1B1/2E1, respectively, while negative in those expressing other CYPs. By immunofluorescent assay both compounds selectively induced centromere-free micronucleus in C3A cells. In PIG-A assays in HepG2 cells NNN and NAB were weakly positive and simply negative, respectively; however, in C3A cells both compounds significantly induced gene mutations, NNN being slight more potent. Conclusively, both NNN and NAB are mutagenic and clastogenic, depending on metabolic activation by partially different CYP enzymes.

ERRγ-inducible FGF23 promotes alcoholic liver injury through enhancing CYP2E1 mediated hepatic oxidative stress

Fibroblast growth factor 23 (FGF23) is a member of endocrine FGF family, along with FGF15/19 and FGF21. Recent reports showed that under pathological conditions, liver produces FGF23, although the role of hepatic FGF23 remains nebulous. Here, we investigated the role of hepatic FGF23 in alcoholic liver disease (ALD) and delineated the underlying molecular mechanism. FGF23 expression was compared in livers from alcoholic hepatitis patients and healthy controls. The role of FGF23 was examined in hepatocyte-specific knock-out (LKO) mice of cannabinoid receptor type 1 (CB1R), estrogen related receptor γ (ERRγ), or FGF23. Animals were fed with an alcohol-containing liquid diet alone or in combination with ERRγ inverse agonist. FGF23 is mainly expressed in hepatocytes in the human liver, and it is upregulated in ALD patients. In mice, chronic alcohol feeding leads to liver damage and induced FGF23 in liver, but not in other organs. FGF23 is transcriptionally regulated by ERRγ in response to alcohol-mediated activation of the CB1R. Alcohol induced upregulation of hepatic FGF23 and plasma FGF23 levels is lost in ERRγ-LKO mice, and an inverse agonist mediated inhibition of ERRγ transactivation significantly improved alcoholic liver damage. Moreover, hepatic CYP2E1 induction in response to alcohol is FGF23 dependent. In line, FGF23-LKO mice display decreased hepatic CYP2E1 expression and improved ALD through reduced hepatocyte apoptosis and oxidative stress. We recognized CBIR-ERRγ-FGF23 axis in facilitating ALD pathology through hepatic CYP2E1 induction. Thus, we propose FGF23 as a potential therapeutic target to treat ALD.

Quercetin inhibits the expression of MYC and CYP2E1 and reduces oxidative stress in the myocardium of spontaneously hypertensive rats.

Oxidative stress is one of the most important pathological processes in chronic heart failure caused by hypertension. These processes involve MYC-regulated mechanisms, including the induction of CYP2E1 as a potent prooxidant factor. In this work, we used qPCR, Western blot analysis, and biochemical markers of oxidative stress to investigate the ability of quercetin to inhibit oxidative stress by modulating MYC expression. We studied spontaneously hypertensive rats (SHRs) in which the onset of cardiac pathology was observed at least at 4 months of age and the development of pathology occurred during life up to 22 months of age. Wistar rats were used as normotensive controls. We observed overexpression of the transcription factor MYC (p=0.0024) in the myocardium of SHRs compared to normotensive controls, and an increased expression of MYC-target gene, CYP2E1, (p=0.0001) in the old SHR group compared to young SHRs. This probably contributed significantly to the development of oxidative stress in the cardiac tissue of old SHRs. We demonstrated that long-term treatment of old SHRs with quercetin resulted in dramatic inhibition of MYC (p=0.0000), and a significant decrease inCYP2E1 (p=0.0001)expression and CYP2E1 protein levels (p=0.0136). This probably contributed significantly to the decrease in lipid peroxidation (p=0.0000). Quercetin was also able to activate antioxidant activity, resulting in a significant improvement in the prooxidant-antioxidant balance in the heart. In turn, the elimination of oxidative stress could contribute to a decrease in blood pressure (p=0.0000) and relative heart weight (p=0.0071) in quercetin-treated old SHRs compared to the untreated old SHR group.

Triphenyl phosphate induces clastogenic effects potently in mammalian cells, human CYP1A2 and 2E1 being major activating enzymes

As a new-type flame retardant and toxic substance, triphenyl phosphate (TPP) is a ubiquitous pollutant present even in human blood. TPP is transformed by human CYP enzymes to oxidized/dealkylated metabolites. The impact of TPP metabolism on its toxicity, however, remains unclear. In this study, the genotoxicity of TPP in several mammalian cell lines and its relevance to CYP/sulfortransferase (SULT) activities were investigated. The results indicated that TPP induced micronucleus formation at ≥1 μM concentrations in a human hepatoma (C3A, endogenous CYPs being substantial) cell line, which was abolished by 1-aminobenzotriazole (CYPs inhibitor). In cell line HepG2 (parental to C3A with lower CYP expression) TPP was inactive up to 10 μM, while pretreatment with ethanol (CYP2E1 inducer), PCB 126 (CYP1A inducer), or rifampicin (CYP3A inducer) led to micronucleus formation by TPP. In V79-Mz and V79-derived cells expressing human CYP1A1 TPP was inactive (up to 32 μM), and in cells expressing human CYP1B1, 2B6 and 3A4 it induced micronucleus weakly (positive only at 32 μM). However, TPP induced micronucleus potently in V79-derived cells expressing human CYP1A2, while this effect was drastically reduced by human SULT1A1 co-expression; likewise, TPP was inactive in cells expressing both human CYP2E1 and SULT1A1, but became positive with pentachlorophenol (inhibitor of SULT1) co-exposure. Moreover, in C3A cells TPP selectively induced centromere-free micronucleus (immunofluorescent assay), and TPP increased γ-H2AX (by Western blot, indicating double-strand DNA breaks). In conclusion, this study suggests that TPP is potently clastogenic, human CYP1A2 and 2E1 being major activating enzymes while SULT1A1 involved in detoxification.

S1213 Predictors of Hepatotoxicity in Patients Receiving Immune Checkpoint Inhibitors: A Large Population-Based Study

Introduction: Immune checkpoint inhibitors (ICPI) are monoclonal antibodies that interrupt co-inhibitory signaling pathways and promote immune-mediated eradication of cell tumors. ICPI has emerged in recent years as an efficacious treatment for advanced malignancies. Hepatotoxicity is an established adverse event associated with ICPI. The aim of this study is to determine the predictors of hepatotoxicity in patients receiving ICPI. Methods: We reviewed data from a large database (Explorys IBM) that aggregates health records from 26 large healthcare systems. We identified adults who received ICPI from 2011-2021. We excluded patients diagnosed with alcoholism, viral hepatitis, other drug induced liver injury & biliary disease. Of this cohort, we collected data on hepatocellular injury after the initiation of ICPI. Demographic information & data on potential risk factors were collected including malnutrition, chronic kidney disease (CKD), metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), smoking, concomitant use of cytochrome P450-CYP2E1 inducers & use of ICPI combination. Univariable & multivariable logistic regression analyses were performed. Results: Out of 69 million adults in the database, 21060 received ICPI (0.03%) of whom 1780 had ICPI-induced hepatotoxicity (8.4%). Men received more ICPI than women (57.4% vs 42.5%). The majority who received ICPI were > 65 yr old (63.7%). Table summarizes the characteristics of patients received ICPI. In multivariable model, NAFLD & the use of ICPI combination were most associated with hepatotoxicity (OR 4.29 [ 95% CI: 4.15-4.43)] & (OR 4.30 [95% CI: 4.18-4.43]), respectively. These factors were closely followed by metabolic syndrome & malnutrition (OR 3.16 [95% CI: 3.10-3.22]) & (OR 3.07 [95% CI: 2.97-3.16]), respectively. Moreover, ICPI-induced hepatotoxicity was significantly associated with CKD (OR 2.46 [95% CI: 2.39-2.52]), smoking (OR 2.33 [95% CI: 2.28-2.38]), concomitant CYP2E1 inducers (OR 2.85 [95% CI: 2.77-2.93]), and to a lesser extent, with age >65 (OR 1.39 [95% CI: 1.33-1.44]). No significant association with gender was noted (OR 1.00 [95% CI: 0.93-1.07]), Figure. Conclusion: This large study shows that the highest predictors of ICPI-induced hepatotoxicity were underlying NAFLD & the use of ICPI combination. Other significant factors were age >65, metabolic syndrome, malnutrition, CKD, smoking, & concomitant use of CYP2E1 inducers. Further studies are needed to evaluate pathophysiology and molecular aspects of these relationships. Table 1. - Characteristics and conditions of patients in cohort ICPI ICPI + Hepatotoxicity Total 21060 1780 Number (Percentage) Number (Percentage) Age18-65>65 7630 (36.3%)13430 (63.7%) 630 (35.4%)1150 (64.6%) GenderFemaleMale 8960 (42.5%)12100 (57.4%) 760 (42.7%)1020 (57.3%) NAFLD 1530 (7.2%) 270 (15.1%) Malnutrition 6080 (28.8%) 770 (43.2%) Metabolic Syndrome 230 (1.09%) 50 (2.8%) CKD 5140 (24.4%) 550 (30.8%) Smoking 6400 (30.3%) 640 (35.9%) CYP2E1 inducers 2140 (10.1%) 330 (18.5%) Footnote: ICPI: Immune checkpoint inhibitor; NAFLD: Non-alcoholic fatty liver disease; CKD: Chronic Kidney disease. Figure 1.: Forest plot of predictors of ICPI-induced hepatotoxicity

A PanFzd receptor agonist induces β‐catenin activation to promote hepatic repair after surgical and acetaminophen‐induced injury

Background and AimsWnt/β‐catenin signaling pathway regulates hepatocyte proliferation and supports some hepatic functions during liver regeneration (LR) following either partial hepatectomy (PH) or acetaminophen‐induced liver injury (AILI). However, its therapeutic modulation in clinically relevant scenarios remains to be directly investigated. CM0011, a PanFzd agonist, is a tetravalent antibody that can recruit Frizzled receptor and LRP5/6 co‐receptor in a manner that phenocopies the activity of canonical Wnts. Here, we validate the mechanism by which PanFzd agonist induces β‐catenin activity and investigate its use in various models of hepatic injury to determine the translational applicability.MethodsTo validate the mechanism of action of CM0011, we assessed its use in hepatocyte‐specific Wnt co‐receptors LRP5‐6 double knockout (LRP5‐6‐DKO) and in endothelial cell‐specific Wntless knockout (EC‐Wls‐KO) mice, since both models show impaired zone‐3 target genes expression and delayed LR due to interruption of Wnt/β‐catenin signaling. We next assessed any impact of CM0011 on LR after PH or in AILI in C57/BL6 mice. For all studies, 5 mg/kg CM0011 or CM0156 (control) were injected intraperitoneally (i.p) in mice every other day for 7 days. When applicable, PH was performed on day 8 and regenerating livers were harvested at 24h post PH for analysis. 1 mg/ml BrdU was given in drinking water to label proliferating cells. For AILI, a single dose of 600 mg/kg APAP dissolved in 0.9% saline was injected i.p followed by 5 mg/kg of CM0011 or CM0156 injected i.p at 12h or 32h post injury. Livers and sera were collected at 48h or 60h, respectively, for analysis.ResultsLRP5/6‐dKO mice showed no rescue of zone‐3 gene expression after CM0011 treatment validating a requirement of Wnt co‐receptors. CM0011 treatment rescued zone‐3 expression of GS and Cyp2e1 in EC‐Wls‐KO. CM0011 also induced Cyclin D1 and hepatocyte proliferation, rescuing LR in the EC‐Wls‐KO mice. Pretreatment with CM0011 for 7 days induced expression of Wnt pathway downstream target genes GS and Cyp2e1, periportal expression of Cyclin D1 and hepatocyte proliferation, as shown by BrdU incorporation at baseline. 24h after PH, CM0011 treated mice had significantly higher liver weight to body weight ratio (LW/BW) and profound hepatocyte proliferation around the periportal region. In AILI model, early CM0011 treatment at 12h post‐injury enhanced necrosis, increased serum ALT level, delayed hepatocyte proliferation and increased mortality, likely due to Cyp2e1 induction. However, treatment with CM0011 at 32h post‐injury accelerated liver repair shown by decreased serum ALT level, profound panzonal hepatocyte proliferation at 60h, leading to a dramatic decrease in necrotic area.ConclusionCM0011 specifically and efficiently activates the canonical Wnt pathway in murine livers and has therapeutic benefits following surgical resection or after APAP overdose.

Triptolide regulates oxidative stress and inflammation leading to hepatotoxicity via inducing CYP2E1.

Triptolide (TP), the main active compound extracted from medicine-tripterygium wilfordii Hook f. (TWHF). It has anti-tumor and immunomodulatory properties. Our study aimed to investigate the mechanisms of hepatotoxicity treated with TP in vivo and in vitro, as well as their relationship with the NF-κB (p65) signal pathway; and to assess TP-induced hepatotoxicity after CYP2E1 modulation by the known inhibitor, clomethiazole, and the known inducer, pyrazole. Mice were given TP to cause liver injury and IHHA-1 cells were given TP to cause hepatocyte injury. The enzyme activity and hepatotoxicity changed dramatically when the CYP2E1 inhibitor and inducer were added. In comparison to the control group, the enzyme inducer increased the activity of CYP2E1, whereas the enzyme inhibitor had the opposite effect. Our findings suggest that TP is an inducer of CYP2E1 via a time-dependent activation mechanism. In addition, TP can promote oxidative stress, inflammatory and involving the NF-κB (p65) signal pathway. Therefore, we used triptolide to stimulate C57 mice and IHHA-1 cells to determine whether TP can promote oxidative stress and inflammation by activating CYP2E1 in response to exacerbated liver damage and participate in NF-κB (p65) signaling pathway.

Human CYP enzyme-activated genotoxicity of 2,2′,4,4′-tetrabromobiphenyl ether in mammalian cells

Polybrominated biphenyl ethers (PBDEs) are a group of persistent organic pollutants with endocrine-disrupting, neurotoxic, tumorigenic and DNA-damaging activities. They are hydroxylated by human liver microsomal CYP enzymes, however, their mutagenicity remains unknown. In this study, 2,2′,4,4′-tetrabromobiphenyl ether (BDE-47, relatively abundant in human tissues) was investigated for micronuclei induction and DNA damage in mammalian cells. The results indicated that BDE-47 up to 80 μM under a 6 h/18 h (exposure/recovery, covering 2 cell cycles) regime did not induce micronuclei in V79-Mz and V79-derived cell lines expressing human CYP1A1 or 1A2, while it was moderately positive in human CYP2B6-, 2E1-and 3A4-expressing cell lines (V79-hCYP2B6, V79-hCYP2E1-hSULT1A1 and V79-hCYP3A4-hOR, respectively). Following 24 h exposure, BDE-47 induced micronuclei in V79-hCYP2E1-hSULT1A1 and V79-hCYP3A4 cells at increased potencies. In the human hepatoma (HepG2) cells BDE-47 (48 h exposure) was inactive up to 40 μM, however, pretreatment of the cells with ethanol (0.2%, v:v, inducer of CYP2E1) or rifampicin (10 μM, inducer of CYP3A4) led to significant micronuclei formation by BDE-47; pretreatment with bisphenol AF (100 nM) also potentiated BDE-47-induced micronuclei formation (which was blocked by a CYP2E1 inhibitor trans-1,2-dichloroethylene or a CYP3A inhibitor (ketoconazole). Immunofluorescent staining of centromere protein B with the micronuclei formed by BDE-47 in HepG2 cells pretreated with ethanol or rifampicin demonstrated selective formation of centromere-containing micronuclei. The increased phosphorylation of both histones H2AX and H3 in HepG2 by BDE-47 also indicated an aneugenic potential. Therefore, this study suggests that BDE-47 is an aneugen activated by several human CYP enzymes.

Oxidative stress and genotoxicity in 1,4-dioxane liver toxicity as evidenced in a mouse model of glutathione deficiency

1,4-Dioxane (DX) is a synthetic chemical used as a stabilizer for industrial solvents. Recent occurrence data show widespread and significant contamination of drinking water with DX in the US. DX is classified by the International Agency for Research on Cancer as a group 2B carcinogen with the primary target organ being the liver in animal studies. Despite the exposure and cancer risk, US EPA has not established a drinking water Maximum Contaminant Level (MCL) for DX and a wide range of drinking water targets have been established across the US and by Health Canada. The DX carcinogenic mechanism remains unknown; this information gap contributes to the varied approaches to its regulation. Our recent mice study indicated alterations in oxidative stress response accompanying DNA damage as an early change by high dose DX (5000 ppm) in drinking water. Herein, we report a follow-up study, in which we used glutathione (GSH)-deficient glutamate-cysteine ligase modifier subunit (Gclm)-null mice to investigate the role of redox homeostasis in DX-induced liver cytotoxicity and genotoxicity. Gclm-null and wild-type mice were exposed to DX for one week (1000 mg/kg/day by oral gavage) or three months (5000 ppm in drinking water). Subchronic exposure of high dose DX caused mild liver cytotoxicity. DX induced assorted molecular changes in the liver including: (i) a compensatory nuclear factor erythroid 2-related factor 2 (NRF2) anti-oxidative response at the early stage (one week), (ii) progressive CYP2E1 induction, (iii) development of oxidative stress, as evidenced by persistent NRF2 induction, oxidation of GSH pool, and accumulation of the lipid peroxidation by-product 4-hydroxynonenal, and (iv) elevations in oxidative DNA damage and DNA repair response. These DX-elicited changes were exaggerated in GSH-deficient mice. Collectively, the current study provides additional evidence linking redox dysregulation to DX liver genotoxicity, implying oxidative stress as a candidate mechanism of DX liver carcinogenicity.

Ethanol-induced intestinal miRNAs in liver injury and early development of liver cancer

While binge alcohol-induced gut leakage has been studied extensively in the context of ROS-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol-inducible CYP2E1 is a key enzyme in ethanol metabolism, and promotes alcohol-induced hepatic steatosis and inflammatory liver disease at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that ethanol promotes CYP2E1 induction and activation, the mechanisms by which CYP2E1 expression is regulated in the context of intestinal damage remain poorly defined. Specific miRNAs have been shown to repress the expression of CYP2E1, supporting our hypothesis that these miRNAs contribute to ethanol-induced intestinal injury and early development of liver cancer. We utilized in vitro cell model and in vivo mouse model to study intestinal and hepatic cell death and growth after modulation of specific miRNA levels directly or indirectly by depletion or overexpression of microRNA-binding proteins. We made two key observations that 1) the RNA-binding protein AUF1 binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs; 2) the Serine/Threonine kinase MST1 mediates oxidative stress-induced phosphorylation of RNA-binding proteins such as AUF1. This finding suggests that ROS-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation for developing liver cancer. Thus, our study demonstrated the critical functions of AUF1 phosphorylation by MST1 in intestine injury and liver cancer development.

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

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

2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside alleviated the acute hepatotoxicity and DNA damage in diethylnitrosamine-contaminated mice

Aims2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside (TSG) is the key bioactive ingredient extracted from Polygonum multiflorum Thumb. Pharmacological studies suggest that it exerts numerous biological effects, including anti-oxidant, anti-aging, and anti-inflammation. This study aimed at investigating the effect of TSG on diethylnitrosamine (DEN)-induced acute hepatotoxicity and DNA damage. Main methodsFifty male C57BL/6 mice were randomly divided into 5 groups (n = 10 each): control, DEN, DEN+TSG (low), DEN+TSG (high) and TSG (high) groups. DEN (100 mg/kg) was injected intraperitoneally (i.p.) alone or with TSG (30 or 60 mg/kg, i.p.) for 5 consecutive days. Key findingsTSG inhibited liver injury and inflammatory cell infiltration in DEN-treated mice. It also attenuated DEN-induced accumulation of reactive oxygen species (ROS), proinflammatory cytokines, and DNA damage. Moreover, TSG promoted the expression of nuclear erythroid 2-related factor 2 (Nrf2) target antioxidant genes by enhancing Nrf2 protein phosphorylation and nuclear translocation. As major phase I detoxification enzymes, cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 1 subfamily A member 1 (CYP1A1) are responsible for the metabolic activation of DEN. We found that TSG administration inhibited CYP2E1 and CYP1A1 induction in DEN-treated mice. SignificanceThese results indicate that TSG can alleviate DEN-induced acute hepatotoxicity by modulating the Nrf2-related antioxidant system and metabolic activation of DEN. Therefore, TSG might be a promising medication for DEN-induced liver injury treatment.

Kinetic study of the expression of genes related to hepatic steatosis, glucose and lipid metabolism, and cellular stress during overfeeding in mule ducks.

Induced by overfeeding, hepatic steatosis is a process exploited for the "foie gras" production in mule ducks. To better understand the mechanisms underlying its development, the physiological responses of mule ducks overfed with corn for a duration of 11 days were analyzed. A kinetic analysis of glucose and lipid metabolism and cell protection mechanisms was performed on 96 male mule ducks during overfeeding with three sampling times (after the 4th, the 12th, and the 22nd meal). Gene expression and protein analysis realized on the liver, muscle, and abdominal fat showed an activation of a cholesterol biosynthetic pathway during the complete overfeeding period mainly in livers with significant correlations between its weight and its cholesterolemia (r = 0.88; P < 0.0001) and between the liver weight and the hmgcr and soat1 expression (r = 0.4, P < 0.0001 and r = 0.67; P < 0.0001, respectively). Results also revealed an activation of insulin and amino acid cells signaling a pathway suggesting that ducks boost insulin sensitivity to raise glucose uptake and use via glycolysis and lipogenesis. Cellular stress analysis revealed an upregulation of key autophagy-related gene expression atg8 and sqstm1(P < 0.0001) during the complete overfeeding period, mainly in the liver, in contrast to an induction of cyp2e1(P < 0.0001), suggesting that autophagy could be suppressed during steatosis development. This study has highlighted different mechanisms enabling mule ducks to efficiently handle the starch overload by keeping its liver in a nonpathological state. Moreover, it has revealed potential biomarker candidates of hepatic steatosis as plasma cholesterol for the liver weight.

1114 The Effect of Metabolic Syndrome on Acetaminophen-Induced Acute Liver Injury/Failure - Morbidity and Mortality: A Single Liver Transplant Center Experience

INTRODUCTION: Acetaminophen (N-acetyl-p-aminophenol; APAP) toxicity is a major cause of acute liver injury in USA. Several risk factors could increase the risk and severity of APAP-induced acute liver injury/failure (APAP-ALI/ALF), including malnutrition, chronic hepatitis C, alcoholism, hepatic cytochrome P450-inducing medications, and pre-existing liver disease. Whether metabolic syndrome affects the risk or the severity of APAP-ALI/ALF has not been definitively confirmed. Clinical and animal studies show conflicting results. We aim to assess the effect of pre-existing metabolic syndrome on the natural history of APAP-ALI/ALF, including morbidity and mortality METHODS: This was a retrospective study performed at a tertiary care liver transplant center. A collective database of all patients with APAP-ALI/ALF from 2001-2012 was reviewed. Primary outcomes were 28-day mortality and the need for liver transplantation. Secondary outcomes were the degree of liver injury and elevation of biochemical markers RESULTS: A total of 327 patients were evaluated with APAP-ALI/ALF. A subset of 219 patients who had a recorded BMI in the medical record were included for analysis. There were 151 females and 68 males, with a mean age of 39.1 years old. Benzodiazepine or opioid co-ingestion occurred in 116 patients. A total of 60 patients were obese (BMI &gt; 30 Kg/m2) with a mean BMI of 38.3 Kg/m2, and 21 were morbidly obese (BMI &gt; 40 Kg/m2). 58 patients died (24 obese and 34 non-obese). Liver transplantation was performed in 4 out of the 7 patients who were listed. There was a statistically significant association between obesity and the 28-day mortality, OR 2.451 (95% CI 1.291-4.651) and P 0.005. No significant association was found between obesity and either listing for liver transplantation or liver transplantation (P 0.351 and 0.916 respectively). Maximum total bilirubin and INR were slightly higher in obese patients; however, this was not statistically significant (P 0.08 and 0.823, respectively). Pearson correlation showed a weak association between rising BMI and a higher maximum total bilirubin and INR, but this was not statistically significant (r 0.08, P 0.201 for total bilirubin and r 0.07, P 0.307 for INR) CONCLUSION: Our study demonstrates that obesity is associated with a higher mortality in patients with APAP-ALI/ALF. Preexisting hepatocyte lipid accumulation, CYP2E1 induction, reduced hepatic glutathione stores as well as mitochondrial dysfunction have been proposed as risk factors that may be contributing.

Simultaneous phenotyping of CYP2E1 and CYP3A using oral chlorzoxazone and midazolam microdoses.

Chlorzoxazone is the paradigm marker substrate for CYP2E1 phenotyping in vivo. Because at the commonly used milligram doses (250-750mg) chlorzoxazone acts as an inhibitor of the CYP3A4/5 marker substrate midazolam, previous attempts failed to combine both drugs in a common phenotyping cocktail. Microdosing chlorzoxazone could circumvent this problem. We enrolled 12 healthy volunteers in a trial investigating the dose-exposure relationship of single ascending chlorzoxazone oral doses over a 10,000-fold range (0.05-500mg) and assessed the effect of 0.1and 500mg of chlorzoxazone on oral midazolam pharmacokinetics (0.003mg). Chlorzoxazone area under the concentration-time curve was dose-linear in the dose range between 0.05and 5mg. A nonlinear increase occurred with doses ≥50mg, probably due to saturated presystemic metabolic elimination. While midazolam area under the concentration-time curve increased 2-fold when coadministered with 500mg of chlorzoxazone, there was no pharmacokinetic interaction between chlorzoxazone and midazolam microdoses. The chlorzoxazone microdose did not interact with the CYP3A marker substrate midazolam, enabling the simultaneous administration in a phenotyping cocktail. This microdose assay is now ready to be further validated and tested as a phenotyping procedure assessing the impact of induction and inhibition of CYP2E1 on chlorzoxazone microdose pharmacokinetics.