CYP2E1 Protein Research Articles

Honokiol attenuates diet‐induced non‐alcoholic steatohepatitis by regulating macrophage polarization through activating peroxisome proliferator‐activated receptor γ

Non-alcoholic steatohepatitis (NASH) may develop into hepatic cirrhosis. This study aimed to investigate whether honokiol could prevent NASH induced by high-cholesterol and high-fat (CL) diet in mice and the possible mechanism involved. Mice were fed with CL diet for 12weeks to establish a NASH model; honokiol (0.02% w/w in diet) was added to evaluate its effect on NASH. Murine peritoneal macrophages, RAW264.7 and ANA-1 cells, were used to explore the possible mechanisms of honokiol on macrophage polarization. Mice developed NASH after fed with CL diet for 12weeks. Honokiol supplementation alleviated insulin resistance, hepatic steatosis, inflammation, and fibrosis induced by CL diet. Immunohistochemistry showed that honokiol induced more M2 macrophages in livers compared with CL diet alone. Honokiol decreased M1 marker genes (TNFα and MCP-1) and increased M2 marker gene (YM-1, IL-10, IL-4R and IL-13) expression in mice liver compared with CL diet. Moreover, treatment with honokiol lowered alanine aminotransferase and aspartate aminotransferase in serum and preserved liver from lipid peroxidation, evidenced by lowered hepatic malondialdehyde level. Honokiol has antioxidant function, as honokiol upregulated hepatic glutathione and superoxide dismutase level and downregulated hepatic CYP2E1 protein level. Hepatic peroxisome proliferator-activated receptor γ (PPARγ) and its target genes were upregulated by honokiol. Furthermore, honokiol (10μM) treatment in mouse peritoneal cells, RAW264.7 cells and ANA-1 cells, led to M2 macrophage polarization, whereas a PPARγ antagonist, GW9662, abolished this effect of honokiol. Honokiol can attenuate CL diet-induced NASH and the mechanism in which possibly is polarizing macrophages to M2 phenotype via PPARγ activation.

SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing toxic reactive oxygen species (ROS) and is regulated at the posttranslational level. Small ubiquitin-like modifier (SUMO)ylation is a posttranslational modification that involves the addition of SUMOs, which modulate protein stability, activity, and localization. We demonstrated that ubiquitin-conjugation enzyme 9, the SUMO-conjugating enzyme, is induced in the livers of an intragastric ethanol mouse model. Our aim is to examine whether SUMOylation could regulate ethanol-induced CYP2E1 expression in ALD and to elucidate the molecular mechanism(s). CYP2E1 and UBC9 expression in vitro and in vivo was detected by real-time PCR and immunoblotting/immunostaining. SUMOylation was assayed by mass spectrometry and coimmunoprecipitation. Ubc9 expression was induced in ethanol-fed mouse livers, and silencing inhibited ethanol-mediated CYP2E1 microsomal retention and enzymatic activity. CYP2E1 SUMOylation was found to be induced by ethanol in vitro and in vivo. Ubc9 silencing prevents ethanol-induced lipid accumulation and ROS production. UBC9 was highly expressed in human ALD livers. Finally, we found that lysine 410 is a key SUMOylated residue contributing to CYP2E1 protein stability and activity preventing CYP2E1 SUMOylation. Ethanol-mediated up-regulation of CYP2E1 via SUMOylation enhancing its protein stability and activity and may have important implications in ALD.-Tomasi, M. L., Ramani, K., Ryoo, M., Cossu, C., Floris, A., Murray, B. J., Iglesias-Ara, A., Spissu, Y., Mavila, N. SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Hepatic and hippocampal cytochrome P450 enzyme overexpression during spontaneous recurrent seizures.

Available evidence points to a role of cytochrome P450 (Cyp) drug biotransformation enzymes in central nervous system diseases, including epilepsy. Deviations in drug pharmacokinetic profiles may impact therapeutic outcomes. Here, we ask whether spontaneous recurrent seizure (SRS) activity is sufficient to modulate the expression of major Cyp enzymes in the liver and brain. Unilateral intrahippocampal (IH) kainic acid (KA) injections were used to elicit nonconvulsive status epilepticus (SE), epileptogenesis, and SRS, as monitored by video-electroencephalography. Intraperitoneal (IP) KA injection was used to trigger generalized tonic-clonic SE. KA-injected mice and sham controls were sacrificed at 24-72 hours and 1 week post-SE (IH or IP KA), and during the chronic stage (SRS; 6 weeks post-IH KA). Liver and brain tissues were processed for histology, real-time quantitative polymerase chain reaction, Western blot, or microsomal enzymatic assay. Cyp2e1, Cyp3a13, glial fibrillary acidic protein (GFAP), IBA1, xenobiotic nuclear receptors nr1i2 (PXR), nr1i3 (CAR) and nr3c1 (glucocorticoid receptor [GR]) expression was examined. Serum samples were obtained to assay corticosterone levels, a GR activator. A significant increase of Cyp3a13 and Cyp2e1 transcript level and protein expression was found in the liver and hippocampi during SRS, as compared to control mice. In the ipsilateral hippocampus, Cyp2e1 and Cyp3a protein upregulation during SRS positively correlated to GFAP expression. GFAP+ , and not IBA1+ , cells colocalized with Cyp2e1 or Cyp3a expression. In the liver, a trend increase in Cyp3a microsomal activity was found during SRS as compared to control mice. The transcript levels of the Cyp upstream regulators GR, xenobiotic nr1i2, and nr1i3 receptors were unchanged at SRS. Corticosterone levels, a GR ligand, were increased in the blood post-SE. SRS modifies Cyp expression in the liver and the hippocampus. Nuclear receptors or inflammatory pathways are candidate mechanisms of Cyp regulation during seizures.

Herba Gelsemii elegantis is detoxified by ramulus et folium Mussaendae pubescentis extract by modulating hepatic cytochrome P450 and glutathione S-transferase enzymes in rats.

Herba Gelsemii elegantis (GE) has been frequently used as a Chinese folk medicine but has high acute toxicity. In Traditional Chinese Medicine, it may be detoxified by Ramulus et Folium Mussaendae pubescentis (MP), but the detoxification mechanism has remained elusive. The present study aimed to evaluate the detoxification mechanisms by which MP modulates the effect of GE in rats, including the inhibition of hepatic cytochrome P (CYP)450 and glutathione S-transferase (GST) enzymes. Male Sprague Dawley rats were orally administered GE at three doses (0.36, 0.43 or 0.54 g/kg) alone and, at the highest dose, in combination with MP (21.6 g/kg) every day for 7 consecutive days. The control group of animals received the same volume of saline. The mRNA and protein expression of hepatic CYPs representative of two subfamilies (CYP2E1 and CYP1A2) were separately assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), western blot and immunohistochemistry assays. The mRNA and protein expression as well as enzyme activity of hepatic GST were assessed by RT-qPCR, western blot and colorimetric assays, respectively. The results indicated that GE significantly inhibited CYP2E1 mRNA and protein expression in a dose-dependent manner. Co-administration of MP increased CYP2E1 mRNA and protein expression as compared with the high GE dose alone. Cells expressing CYP2E1, located around the hepatic vascular plexus under a clear background, were identified by immunohistochemical staining. The results for CYP1A2 were similar to those for CYP2E1. At all concentrations used, GE significantly inhibited GST mu 1 (GSTm1) mRNA and protein expression in a dose-dependent manner, as compared with the control. Combination of GE and MP increased the mRNA and protein expression of GSTm1 as compared with the high dose of GE. However, the differences in GST-pi mRNA and protein expression between the GE and GE + MP groups were not significant. Of note, rats co-treated with MP were significantly protected from the decrease in GST activity produced by GE. The present study indicated that co-administration of GE and MP upregulated the activities of CYP450 and GST enzymes when compared with GE alone. This modulation may explain for the effect of MP in reducing the acute toxicity of GE.

MiRNA-122 Protects Mice and Human Hepatocytes from Acetaminophen Toxicity by Regulating Cytochrome P450 Family 1 Subfamily A Member 2 and Family 2 Subfamily E Member 1 Expression

Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i.p. with an acetaminophen dose not lethal to the control. LKO livers exhibited higher basal expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 family 1 subfamily A member 2 (CYP1A2) that convert acetaminophen to highly reactive N-acetyl-p-benzoquinone imine. Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of aryl hydrocarbon receptor (AHR) and mediator 1 (MED1), two transactivators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE (Encyclopedia of DNA Element) database identified association of CCCTC-binding factor (CTCF) with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1, or Ctcf in Mir122-/- hepatocytes reduced Cyp1a2 expression. Pulse-chase studies found that CYP2E1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to acetaminophen toxicity that correlated with upregulation of AHR, MED1, and CYP1A2 expression. Collectively, these results reveal a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in patients with ALF.

Glycyrrhetinic acid prevents acetaminophen-induced acute liver injury via the inhibition of CYP2E1 expression and HMGB1-TLR4 signal activation in mice

Acetaminophen (APAP) is a widely used antipyretic and analgesic drug, which is safe and effective at the therapeutic dose. Unfortunately, excessive dosage of APAP could cause severe liver injury due to lack of effective therapy. Successful therapeutic strategies are urgently requested in clinic. Glycyrrhetinic acid (GA), derived from a traditional medicine licorice, has been shown to exert anti-inflammatory and antioxidant actions. In this study, the effect and the underlying mechanism of GA on APAP-induced hepatotoxicity were explored. Our results showed that pretreatment with GA significantly reduced serum ALT and AST activities, alleviated hepatic pathological damages with hepatocellular apoptosis, down-regulated expression of CYP2E1 mRNA and protein, increased GSH levels, and reduced reactive oxygen species (ROS) productions in the liver of APAP-exposed mice. Furthermore, GA obviously inhibited APAP-induced HMGB1-TLR4 signal activation, as evaluated by reduced hepatic HMGB1 release, p-IRAK1, p-MAPK and p-IκB expression as well as the productions of TNF-α and IL-1β. In addition, GA attenuated hepatic neutrophils recruitment and macrophages infiltration caused by APAP. These findings reflected that GA could alleviate APAP-induced hepatotoxicity, the possible mechanism is associated with down-regulation of CYP2E1 expression and deactivation of HMGB1-TLR4 signal pathway.

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

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

Effects of antrosterol from Antrodia camphorata submerged whole broth on lipid homeostasis, antioxidation, alcohol clearance, and anti-inflammation in livers of chronic-alcohol fed mice

Ethnopharmacological relevanceAntrodia camphorata is a functional fungus in Taiwan and owns several pharmacological functions. Antrosterol, a bioactive constitute of sterols in edible Antrodia camphorata submerged whole broth, can protect liver from CCl4 damage via enhancing antioxidant and anti-inflammatory capacities. Aim of the studyThe aim of this study was to investigate the hepatoprotection of antrosterol (named as EK100) against alcohol consumption. Materials and methodsA Lieber-DeCarli regular EtOH diet (EtOH liquid diet, 5% (v/v) alcohol) was applied to induce alcoholic liver damage. Mice were randomly divided into 5 groups: (1) Control: control liquid diet; (2) EtOH: EtOH liquid diet; (3) EK100_1X: EtOH liquid diet and 1mg EK100 (Antrosterol)/Kg body weight (bw); (4) EK100_5X: EtOH liquid diet and 5mg EK100/Kg bw; (5) EK100_10X: EtOH liquid diet and 10mg EK100/Kg bw. At the end of experiment, the livers were collected for histo-pathological analyses, RNA and protein extraction, and enzymatic activities. ResultsAntrosterol reduced serum/liver lipids of alcohol-diet fed mice which highly related to upregulated fatty acid β-oxidation and downregulated lipogenesis, and increased fecal lipid/bile-acid outputs. Antrosterol enhanced hepatic antioxidant capabilities in alcohol-diet fed mice while it also lowered serum alcohol level, as well as increased alcohol dehydrogenase (ADH) and catalase (CAT) activities and decreased CYP2E1 protein expression in livers of alcohol-diet fed mice. Besides, antrosterol lowered hepatic inflammation and fibrosis related gene expressions, as well as serum AST/ALT values and TNF-α/IL-1β contents in alcohol-diet fed mice. ConclusionBased on the results, hepatoprotection of antrosterol is mostly attributed to its regulations of lipid homeostasis, antioxidant capability, alcohol metabolism, and anti-inflammation.

Effects of tetrahydroberberine and tetrahydropalmatine on hepatic cytochrome P450 expression and their toxicity in mice

The aim of this study was to investigate the effects of tetrahydroberberine (THB) and tetrahydropalmatine (THP) on the expression of mouse liver cytochrome P450s, and evaluate their liver toxicity in mice. Real-time polymerase chain reaction (PCR) and western blot analyses were used to analyze the expression of major P450 isoforms. Liver toxicity was evaluated by measuring serum biochemical parameters and performing histopathological analysis. The real-time PCR results showed that THB induced Cyp1a2 (1.66 ± 0.34 fold, P < 0.05), Cyp3a11 (1.57 ± 0.24 fold, P < 0.05), and Cyp2e1 (1.75 ± 0.97 fold, P < 0.05) mRNA expression, while THP inhibited Cyp1a2 (0.66 ± 0.12 fold, P < 0.05) mRNA expression. The western blot results confirmed that the expression of CYP1A2, CYP3A, and CYP2E1 proteins in the mouse liver was induced by THB, whereas that of CYP1A2 was inhibited by THP. Toxicological studies showed that THB (40 mg/kg, oral gavage) increased mouse serum aspartate transaminase and total bilirubin, and liver malondialdehyde levels, and induced liver edema. No obvious changes in serum and liver tissue biochemical parameters were found and no significant pathological changes were detected in liver tissues after THP administration. Our results provide more information on the toxicity of THB and THP, and their related drug-drug interactions.

Long-term administration of tacrolimus and everolimus prevents high cholesterol-high fructose-induced steatosis in C57BL/6J mice by inhibiting de-novo lipogenesis

AimTo investigate the effects of tacrolimus (TC) and everolimus (EV) on non-alcoholic steatohepatitis (NASH) induced by high fat, high cholesterol and fructose (fast food) diet in C57BL/6J mice.Materials and MethodsC57BL/6J mice were divided into four groups (n=8). 1) Standard Chow (SC); 2) Fast food (FF) diet; 3) FF + Tacrolimus (TC, 1mg/kg) and; 4) FF + Everolimus (EV, 1mg/kg) and treated for 16 weeks. Serum and tissue samples were analyzed for evidence of inflammation, fibrosis, lipogenesis, and apoptosis.ResultsTC and EV treatments significantly reduced the hepatic lipid accumulation, improved liver-body weight ratio, blood biochemistry, and insulin resistance in mice fed with FF diet. However, inflammation, enlarged portal tracts, and fibrosis were pronounced in EV treated group. The lipogenic parameters, Peroxisome proliferator-activated receptor gamma (PPAR-γ), Sterol regulatory element-binding protein 1(SREBP-1), mammalian target of rapamycin (m-TOR), Stearoyl-CoA desaturase-1 (SCD-1) and fatty acid translocase (CD36) were significantly down-regulated in livers of TC and EV treated groups as compared to FF group. TC improved Bcl2/Bax ratio, decreased apoptosis, CYP2E1 protein expression and liver fibrosis levels, however, EV offered no such protection. Further, in an In-vitro model of lipotoxicity using the mouse hepatocyte (AML-12) cell line, treatment with TC and EV significantly reduced lipid accumulation and lipogenic and apoptotic markers induced with palmitic acid.ConclusionIn FF diet induced model of NASH, both TC and EV inhibited hepatic lipid accumulation and improved metabolic parameters such as insulin resistance and dyslipidemia. However, mice administered with EV exhibited inflammatory and fibrotic responses despite reduced hepatic steatosis.

Progressive obesity alters ovarian insulin, phosphatidylinositol-3 kinase, and chemical metabolism signaling pathways and potentiates ovotoxicity induced by phosphoramide mustard in mice†

Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1Ser302, pAKTThr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A phosphoramide mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.

Resveratrol Ameliorates Experimental Alcoholic Liver Disease by Modulating Oxidative Stress

The aim of this study was to investigate the hepatoprotective effects of resveratrol in alcoholic liver disease (ALD). Alcohol was administered to healthy female rats starting from 6% (v/v) and gradually increased to 20% (v/v) by the fifth week. After 16 weeks of intervention, liver enzymes (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were analyzed using a chemistry analyzer, while hepatic antioxidant enzymes, oxidative stress markers, and caspase 3 activity were assessed using ELISA kits. Furthermore, hepatic CYP2E1 protein levels and mRNA levels of antioxidant and inflammation-related genes were determined using western blotting and RT-PCR, respectively. The results showed that resveratrol significantly attenuated alcohol-induced elevation of liver enzymes and improved hepatic antioxidant enzymes. Resveratrol also attenuated alcohol-induced CYP2E1 increase, oxidative stress, and apoptosis (caspase 3 activity). Moreover, genes associated with oxidative stress and inflammation were regulated by resveratrol supplementation. Taken together, the results suggested that resveratrol alleviated ALD through regulation of oxidative stress, apoptosis, and inflammation, which was mediated at the transcriptional level. The data suggests that resveratrol is a promising natural therapeutic agent against chronic ALD.

Development of an IgY Antibody-Based Immunoassay for the Screening of the CYP2E1 Inhibitor/Enhancer from Herbal Medicines.

Cytochrome P450 (CYP) 2E1 is an important enzyme involved in the metabolism of many endogenous and exogenous compounds. It is essential to evaluate the expression of CYP2E1 in the studies of drug–drug interactions and the screening of drugs, natural products, and foodstuffs. The present work is a feasibility study on the development of immunoassays using a specific and sensitive chicken-sourced anti-CYP2E1 IgY antibody. Cloning, expression, and purification of a recombinant CYP2E1 (mice origin) protein were carried out. Anti-CYP2E1 IgY antibodies were generated by immunizing white Leghorn chickens with purified recombinant CYP2E1 protein and were purified by immune affinity chromatography. The IgY titer attained a peak level (≥1:128,000) after the fifth booster injection. For evaluation of the expression of CYP2E1 in different herbal treatment samples, the mice were treated by oral gavage for 3 days with alcohol (50% 15 mL/kg), acetaminophen (APAP, 300 mg/kg), Cornus officinalis extract (100 mg/kg), Alhagi-honey extract (100 mg/kg), Apocynum venetum extract (100 mg/kg), hyperoside (50 mg/kg), isoquercetin (50 mg/kg), 4-hydroxyphenylacetic acid (50 mg/kg), 3-hydroxyphenylacetic acid (50 mg/kg), and 3,4-hydroxyphenylacetic acid (50 mg/kg). The expression of CYP2E1 was determined by Western blot analysis, immunohistochemistry, ELISA, and immunomagnetic beads (IMBs) using anti-CYP2E1 IgY in liver tissue. The results showed that C. officinalis extract, Alhagi-honey extract, A. venetum extract, hyperoside, isoquercetin, and their xenobiotics 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3,4-hydroxyphenylacetic acid significantly decreased CYP2E1 levels. Alcohol and APAP treatments significantly increased CYP2E1 levels as analyzed with Western blot analysis, immunohistochemistry, and ELISA. The IMB method is suitable for large-scale screening, and it is a rapid screening (20 min) that uses a portable magnet and has no professional requirements for the operator, which makes it useful for on-the-spot analysis. Considering these results, the anti-CYP2E1 IgY could be applied as a novel research tool in screening for the CYP2E1 inhibitor/enhancer.

Global Deletion of Glutathione S-Transferase A4 Exacerbates Developmental Nonalcoholic Steatohepatitis

We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor α (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We used it to probe the importance of lipid peroxidation in progression of NASH beyond simple steatosis. Feeding Gsta4-/-/Ppara-/- double-knockout (dKO) mice liquid diets containing corn oil resulted in a percentage fat-dependent increase in steatosis and necroinflammatory injury (P<0.05). Increasing fat to 70% from 35% resulted in increases in formation of 4-hydroxynonenal protein adducts accompanied by evidence of stellate cell activation, matrix remodeling, and fibrosis (P<0.05). Comparison of dKO mice with wild-type (Wt) and single knockout mice revealed additive effects of Gsta4-/-and Ppara-/- silencing on steatosis, 4-hydroxynonenal adduct formation, oxidative stress, serum alanine amino transferase, expression of tumor necrosis factor alpha, Il6, interferon mRNA, and liver pathology (P<0.05). Induction of Cyp2e1 protein by high-fat diet was suppressed in Gsta4-/- and dKO groups (P<0.05). The dKO mice had similar levels of markers of stellate cell activation and matrix remodeling as Ppara-/- single KO mice. These data suggest that lipid peroxidation products play a role in progression of liver injury to steatohepatitis in NASH produced by high-fat feeding during development but appear less important in development of fibrosis.

A non-hepatotropic parasite infection increases mortality in the acetaminophen-induced acute liver failure murine model: possible roles for IL-5 and IL-6.

We evaluated the effects of a non-hepatotropic parasite infection (Taenia crassiceps) on the outcome of acetaminophen-induced acute liver failure in mice. Uninfected and T. crassiceps infected mice orally received either 300 mg/kg acetaminophen or water as vehicle (n = 5 per group). Survival analysis, hepatocyte necrosis, alanine aminotransferase (ALT) levels, CYP2E1 protein, interleukin (IL-) 5, and IL-6 were assessed for all groups. All infected mice died within 16 h after exposure to acetaminophen (Tc+APAP group), whereas only one-third of uninfected animals exposed to acetaminophen (APAP group) died. Uninfected (Control group) and infected (Tc group) mice that received the vehicle showed no liver damage. Tc+APAP mice exhibited massive liver necrosis characterised by marked balloning degeneration of hepatocytes and higher serum ALT compared to Control, Tc, and APAP animals. Liver tissue from Tc+APAP mice also displayed increased expression of CYP2E1 protein and higher mRNA and protein levels of IL-5 and IL-6 compared to the other groups. These findings suggest that non-hepatotropic parasite infections may increase mortality following acute liver failure by promoting hepatocyte necrosis via IL-5 and IL-6-dependent CYP2E1 overproduction. This study identifies new potential risk factors associated with severe acute liver failure in patients.

Chronic Acrylamide Exposure in Male Mice Results in Elevated DNA Damage in the Germline and Heritable Induction of CYP2E1 in the Testes.

Acute acrylamide exposure in male rodents results in reduced reproductive performance and dominant lethality. However, the reproductive effects of low dose chronic exposure, which better reflects the nature of human exposure, remain far less certain. Human dietary consumption of acrylamide has been estimated at an average of 1-4 µg/kg bw/day. In order to simulate this exposure, male mice were provided with acrylamide (1 µg/ml) via their drinking water continuously for six months, which was equivalent to a human dose of 10.5 µg/ kg bw/day. This exposure regime increased DNA damage in the spermatozoa, without affecting a concomitant reduction in overall fertility. The offspring of acrylamide treated mice did not have an increased incidence of skin papilloma formation following the two-stage tumor induction protocol. However, the male offspring of acrylamide treated fathers had significantly increased levels of DNA damage in their spermatozoa, despite having had no direct toxicant exposure. It was also found that the F0, and most crucially, F1 mice had increased levels of CYP2E1 protein in their germ cells. This is significant as CYP2E1 is the sole enzyme responsible for conversion of acrylamide to its harmful metabolite glycidamide. This altered expression may be due to epigenetic alterations. Additionally, the F0 and F1 mice had increased oxidative adducts in the DNA of their germ cells, which was hypothesized to arise as a byproduct of increased CYP2E1 activity. Therefore, chronic paternal acrylamide exposure in mice has consequences for their offspring, and raises concerns for the effects of acrylamide exposure in the human population and the accumulated effects with multiple generations of exposure.

Meta-analysis of CYP2E1 polymorphisms in liver carcinogenesis

BackgroundThe CYP2E1 protein is a monooxygenase with certain polymorphisms linked to liver cancer. However, results from individual studies remain controversial. AimsTo evaluate CYP2E1 polymorphisms in liver carcinogenesis through meta-analysis. MethodsAll studies about CYP2E1 polymorphisms and liver cancer were retrieved from seven major databases. Original data from each study were pooled and re-analyzed. ResultsTotal of 16 articles with 4862 cases were selected, including 1820 cases of liver cancer and 3042 cases of controls. The c1 allelic frequency in the cases and controls was 83.3% and 85.3%, respectively. Five genetic variations were compared: dominant c1c2/c2c2 vs. c1/c1 (OR=0.987 (0.853, 1.141)), homozygous c2c2 vs. c1c1 (OR=0.767 (0.526, 1.119)), heterozygous c1c2 vs. c1c1 (OR=1.005 (0.854, 1.182)), recessive c2c2 vs. c1c2/c2c2 (OR=0.771 (0.530, 1.122)), and different alleles c2 vs. c1 (OR=0.947 (0.828, 1.082)). Pooled data were further analyzed based on ethnicity, control sources, and HWE (Hardy–Weinberg equilibrium). These results from stratified groups were similar to that of nonstratified groups. ConclusionsOur meta-analysis results suggest that there is no evidence for a major role of CYP2E1 polymorphism in liver carcinogenesis, but do not rule out the possibility in certain cases.

The immature rat as a potential model for chemical risks to children: Ontogeny of selected hepatic P450s

Concern about potential susceptibilities of infants and children to chemicals has led to the consideration of immature rodents as potential test surrogates. Maturation of some hepatic microsomal cytochrome P450s (CYPs), that participate in metabolic activation of organic solvents and polycyclic aromatic hydrocarbons (PAHs), may differ significantly between humans and rodents. The present investigation was undertaken to delineate the ontogeny of selected hepatic CYPs in male and female Sprague-Dawley (S-D) rats, and to contrast them with developmental profiles in humans. Microsomes were prepared from the liver of sexed and unsexed postnatal day (PND) 1–90 rats, and total CYP450 levels, as well as CYP1A1/2, CYP2E1 and CYP2B1/2 activities and protein, were quantified. CYP1A1/2 and CYP2E1 activity and expression rose rapidly after birth, peaked from PND 21–40/50, and declined substantially to adult values by PND 90. The same ontogenic profiles were manifested when the enzyme activities were expressed per entire liver or liver normalized to body weight. CYP1A1/2 and CYP2E1 activity and protein expression were well correlated. CYP2B1/2 activity peaked abruptly on PND 21 and declined irregularly to adult values. These patterns are in contrast to human CYP1A2 and CYP2E1, which are reported to progressively increase in liver during the first few months to years of life. The three CYP protein developmental profiles were largely gender independent in rats. The immature rat does not appear to be a suitable model for assessing risks posed to infants and children by chemicals metabolically activated by CYP2E1, based on the findings of greater carbon tetrachloride hepatotoxicity in preweanlings and weanlings than in adult animals. Additional studies are required to determine whether immature S-D rats may be used as an animal model for substrates of other CYPs, as total CYP450 levels in the liver progressively rose during maturation, similarly to humans.

Targeted CYP2E1 quantification and its correlation to currently acceptable clinical biochemical indices.

BackgroundThe Cytochrome P450 enzymes are commonly known for their major role in metabolism. Besides its metabolic role, CYP2E1 gene expression has been associated with the onset of diabetic nephropathy. CYP2E1 protein elevation has also been reported to be responsible for the production of reactive oxygen species. The aims of this study were (i) to optimize and validate a targeted proteomic approach for quantitating CYP2E1 and validating it as a suitable clinical test, (ii) to investigate the concurrency between ESI-LCMS-MS quantitated circulating CYP2E1 and gold standard indices in the context of outpatient point-of-care clinical settings involving various groups of diabetic patients and (iii) to investigate the concurrency profile of circulating CYP2E1 protein, CYP2E1 gene expression and reactive oxygen species (ROS). This is a cross sectional study involving three groups of subjects (n = 166): control, pre-diabetes, and diabetes. We optimized a targeted proteomic approach for absolute quantification of CYP2E1. “YPEIEEK” and “GTVVVPTLYDNQEFPDPEK” were the representative peptides of CYP2E1 for our analytical method. Deuterated forms of “YPEIEEK” and “GTVVVPTLYDNQEFPDPEK” were used as internal standards. Lymphocytes were isolated from whole blood, microsomes were prepared, followed by in-solution digestion for production of tryptic peptides. Amounts of “YPEIEEK” and “GTVVVPTLYDNQEFPDPEK” from patients’ samples were calculated from a calibration curve.Results“YPEIEEK” is a unique and reliable representative peptide for CYP2E1 quantification. “GTVVVPTLYDNQEFPDPEK” showed poor reproducibility and sensitivity. Incremental amounts of CYP2E1 protein in the peripheral circulation clearly showed concurrency with CYP2E1 gene expression and ROS levels in our study population. Elevations of CYP2E1 were observed even when gold standard clinical indicator for glycemic control (HbA1c) was within normal reference limits. Quantitated amounts of CYP2E1 protein in the pre-diabetes and diabetes groups showed significant difference relative to control group (p < 0.001). No significant differences were observed between the medians of pre-diabetes and diabetes groups (p = 0.870).ConclusionsCYP2E1 protein in peripheral blood can be reliably quantitated by the validated targeted proteomic approach method. Quantifiable amounts of CYP2E1 preceded abnormal HbA1C levels which indicates quantitation of CYP2E1 could be useful as an additional tool for early indication of diabetic risks and it complications.

Differential contribution of complement receptor C5aR in myeloid and non-myeloid cells in chronic ethanol-induced liver injury in mice.

Complement is implicated in the development of alcoholic liver disease. C3 and C5 contribute to ethanol-induced liver injury; however, the role of C5a receptor (C5aR) on myeloid and non-myeloid cells to progression of injury is not known. C57BL/6 (WT), global C5aR-/-, myeloid-specific C5aR-/-, and non-myeloid-specific C5aR-/- mice were fed a Lieber-DeCarli diet (32%kcal EtOH) for 25 days. Cultured hepatocytes were challenged with ethanol, TNFα, and C5a. Chronic ethanol feeding increased expression of pro-inflammatory mediators in livers of WT mice; this response was completely blunted in C5aR-/- mice. However, C5aR-/- mice were not protected from other measures of hepatocellular damage, including ethanol-induced increases in hepatic triglycerides, plasma alanine aminotransferase and hepatocyte apoptosis. CYP2E1 and 4-hydroxynonenal protein adducts were induced in WT and C5aR-/- mice. Myeloid-specific C5aR-/- mice were protected from ethanol-induced increases in hepatic TNFα, whereas non-myeloid-specific C5aR-/- displayed increased hepatocyte apoptosis and inflammation after chronic ethanol feeding. In cultured hepatocytes, cytotoxicity induced by challenge with ethanol and TNFα was completely eliminated by treatment with C5a in cells from WT, but not C5aR-/- mice. Further, treatment with C5a enhanced activation of pro-survival signal AKT in hepatocytes challenged with ethanol and TNFα. Taken together, these data reveal a differential role for C5aR during ethanol-induced liver inflammation and injury, with C5aR on myeloid cells contributing to ethanol-induced inflammatory cytokine expression, while non-myeloid C5aR protects hepatocytes from death after chronic ethanol feeding.