Hepatoprotective effects of S-adenosyl-L-methionine against alcohol-and cytochrome P450 2E1-induced liver injury

S-adenosyl-L-methionine (SAM) acts as a methyl donor for methylation reactions and participates in the synthesis of glutathione. SAM is also a key metabolite that regulates hepatocyte growth, differentiation and death. Hepatic SAM levels are decreased in animal models of alcohol liver injury and in patients with alcohol liver disease or viral cirrhosis. This review describes the protection by SAM against alcohol and cytochrome P450 2E1-dependent cytotoxicity both in vitro and in vivo and evaluates mechanisms for this protection.

Cytochrome P450 2E1 RsaI/PstI and DraI Polymorphisms Are Risk Factors for Lung Cancer in Mongolian and Han Population in Inner Mongolia

Objective： To explore the relationship between cytochrome P450 2E1 （CYP2E1） RsaI/PstI and DraI polymorphism and lung cancer susceptibility in Mongolian and Han population in Inner Mongolia of China. Methods： CYP2E1 RsaI/PstI and DraI polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in 64 lung cancer patients, 150 healthy Mongolian and 150 healthy Han individuals. The distribution of genotype and allele frequencies of CYP2E1 RsaI/PstI and DraI polymorphisms were studied. Results： The risk of lung cancer was increased in individuals with CYP2E1 （cl/cl） and CYP2E1 （DD） with OR values of 2.431 （95%CI=1.082-5.460） and 2.778 （95%CI=1.358-5.683） respectively （P0.05）. When CYP2E1 RsaI/PstI and DraI polymorphisms were combined, the risk of lung cancer was reduced in individuals with CYP2E1 （cl/c2＋c2/c2 and DD＋CC） with OR values of 0.233 （95%CI=0.088-0.615, P0.05）. In smokers, the susceptibility to lung cancer was higher in the individuals with CYP2E1 （c1/c1） and CYP2E1 （DD） than in the individuals with c2 and C allele （P0.05, OR=2.643 and 4.308 respectively）. There was no significant difference in distribution of CYP2E1 genotype frequency between healthy Mongolian, Han population and lung cancer patients, healthy controls in Inner Mongolia. Conclusion： CYP2E1 （c1/c1） and CYP2E1 （DD） are predisposing factors of lung cancer in population in Inner Mongolia. CYP2E1 （c2﹢C） co-mutation may decrease the risk of lung cancer. Smoking exerts synergetic effect with CYP2E1 （c1/c1） and CYP2E1 （DD） on the occurrence of lung cancer.

Long-term modifications of blood pressure in normotensive and spontane-ously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase

Arachidonic acid cytochrome P-450 （CYP） hydroxylase 4A isoforms, including 4A1, 4A2, 4A3 and 4A8 in the rat kidney, catalyze arachidonic acid to produce 19/20-Hydroxyeicosatetraenoic acids （20-HETE）, a biologically active metabolite, which plays an important role in the regulation of blood pressure. However, controversial results have been reported regarding the exact role of 20-HETE on blood pressure. In the present study, we used recombinant adenoassociated viral vector （rAAV） to deliver CYP 4A1 cDNA and antisense 4A1 cDNA into Sprague-Dawley （SD） rats and spontaneously hypertensive rats （SHR）, respectively, to investigate the effects of long-term modifications of blood pressure and the potential for gene therapy of hyperténsion. The mean systolic pressure increased by 14.2±2.5 mm Hg in rAAV.4A 1-treated SD rats and decreased by 13.7±2.2 mm Hg in rAAV.anti4A l-treated SHR rats 5 weeks after the injection compared with controls and these changes in blood pressure were maintained until the experiments ended at 24 weeks. In 4A1 treated animals CYP4A was overexpressed in various tissues, but preferentially in the kidney at both mRNA and protein levels. In anti-4Al-treated SHR, CYP4A mRNA in various tissues was probed, especially in kidneys, but 4A l protein expression was almost completely inhibited. These results suggest that arachidonic acid CYP hydroxylases contribute not only to the maintenance of normal blood pressure but also to the development of hypertension. rAAV-mediated anti4A administration strategy has the potential to be used as targeted gene therapy in human hypertension by blocking expression of CYP 4A in kidneys.

Cytochrome P450 family 17 subfamily A member 1 mutation causes severe pseudohermaphroditism: A case report

BACKGROUND 17α-Hydroxylase deficiency(17-OHD)is a rare form of congenital adrenal hyperplasia,characterized by hypertension,hypokalemia,and gonadal dysplasia.However,due to the lack of a comprehensive understanding of this disease,it is prone to misdiagnosis and missed diagnosis,and there is no complete cure.CASE SUMMARY We report a female patient with 17-OHD.The patient was admitted to the Department of Neurology of our hospital due to limb weakness.During treatment,it was found that the patient’s condition was difficult to correct except for hypokalemia,and her blood pressure was difficult to control with various antihypertensive drugs.She was then transferred to our department for further treatment.On physical examination,the patient's gonadal development was found to be abnormal,and chromosome analysis demonstrated karyotype 46,XY.Considering the possibility of 17-OHD,the cytochrome P450 family 17 subfamily A member 1(CYP17A1)test was performed to confirm the diagnosis.CONCLUSION The clinical manifestations of 17-OHD are complex.Hormone determination,imaging examination,chromosome determination and CYP17A1 gene test are helpful for early diagnosis.

Functionalized selenium nanoparticles ameliorated acetaminophen-induced hepatotoxicity through synergistically triggering PKCδ/Nrf2 signaling pathway and inhibiting CYP 2E1

Selenium nanoparticles(SeNPs)have been demonstrated potential for use in diseases associated with oxidative stress.Functionalized SeNPs with lower toxicity and higher biocompatibility could bring better therapeutic activity and clinical application value.Herein,this work was conducted to investigate the protective effect of Pleurotus tuber-regium polysaccharide-protein complex funtionnalized SeNPs(PTR-SeNPs)against acetaminophen(APAP)-induced oxidative injure in HepG2 cells and C57BL/6J mouse liver.Further elucidation of the underlying molecular mechanism,in particular their modulation of Nrf2 signaling pathway was also performed.The results showed that PTR-SeNPs could significantly ameliorate APAP-induced oxidative injury as evidenced by a range of biochemical analysis,histopathological examination and immunoblotting study.PTR-SeNPs could hosphorylate and activate PKCδ,depress Keap1,and increase nuclear accumulation of Nrf2,resulting in upregulation of GCLC,GCLM,HO-1 and NQO-1 expression.Besides,PTR-SeNPs suppressed the biotransformation of APAP to generate intracellular ROS through CYP 2E1 inhibition,restoring the mitochondrial morphology.Furthermore,the protective effect of PTR-SeNPs against APAP induced hepatotoxicity was weakened as Nrf2 was depleted in vivo,indicating the pivotal role of Nrf2 signaling pathway in PTR-SeNPs mediated hepatoprotective efficacy.Being a potential hepatic protectant,PTR-SeNPs could serve as a new source of selenium supplement for health-promoting and biomedical applications.

Hepatoprotective and antioxidant effects of lycopene on non-alcoholic fatty liver disease in rat

AIM To evaluate the hepatoprotective effect of lycopene(Ly) on non-alcoholic fatty liver disease(NAFLD) in rat. METHODS A rat model of NAFLD was first established by feeding a high-fat diet for 14 wk. Sixty-five rats were randomly divided into normal group, model group and Ly treatment groups. Alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglycerides(TG), total cholesterol(TC) in serum and low density lipoproteincholesterol(LDL-C), high density lipoprotein-cholesterol(HDL-C), free fatty acid(FFA), malondialdehyde(MDA), superoxide dismutase(SOD), glutathione(GSH) in liver tissue were evaluated, respectively. While the hepatoprotective effect was also confirmed by histopathological analysis, the expression levels of TNF-α and cytochrome P450(CYP) 2E1 in rat liver were determined by immunohistochemistry analysis.RESULTS A significant decrease was observed in the levels of serum AST(2.07-fold), ALT(2.95-fold), and the blood lipid TG(2.34-fold) and TC(1.66-fold) in the dose of 20 mg/kg Ly-treated rats(P < 0.01), compared to the model group. Pretreatment with 5, 10 and 20 mg/kg of Ly significantly raised the levels of antioxidant enzyme SOD in a dose-dependent manner,to 90.95 ± 9.56, 109.52 ± 11.34 and 121.25 ± 10.68(P < 0.05, P < 0.01), as compared with the model group. Similarly, the levels of GSH were significantly increased(P < 0.05, P < 0.01) after the Ly treatment. Meanwhile, pretreatment with 5, 10 and 20 mg/kg of Ly significantly reduced MDA amount by 30.87, 45.51 and 54.49% in the liver homogenates, respectively(P < 0.01). The Ly treatment group showed significantly decreased levels of lipid products LDL-C(P < 0.05, P < 0.01), improved HDL-C level and significantly decreased content of FFA, compared to the model group(P < 0.05, P < 0.01). Furthermore, the Ly-treated group also exhibited a down-regulated TNF-α and CYP2E1 expression, decreased infiltration of liver fats and reversed histopathological changes, all in a dosedependent manner(P < 0.05, P < 0.01). CONCLUSION This study suggests that Ly has a protective effect on NAFLD, down-regulates expression of TNF-α, and that CYP2E1 may be one of the action mechanisms for Ly.

Coexistence of hyperlipidemia and acute cerebral ischemia/reperfusion induces severe liver damage in a rat model

AIM:To investigate the correlation of hyperlipemia(HL) and acute cerebral ischemia/reperfusion(I/R) injury on liver damage and its mechanism.METHODS:Rats were divided into 4 groups:control,HL,I/R and HL+I/R.After the induction of HL via a high-fat diet for 18 wk,middle cerebral artery occlusion was followed by 24 h of reperfusion to capture I/R.Serum alanine transaminase(ALT) and aspartate aminotransferase(AST) were analyzed as part of liver function tests and liver damage was further assessed by histological examination.Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL) assay.The expression of genes related to apoptosis(caspase-3,bcl-2) was assayed by immunohistochemistry and Western blotting.Serum tumor necrosis factor-(TNF-),interleukin-1(IL-1) and liver mitochondrial superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA) and Ca 2+ levels were measured to determine inflammatory and oxidative/antioxidative status respectively.Microsomal hydroxylase activity of the cytochrome P450 2E1(CYP2E1)-containing enzyme was measured with aniline as the substrate,and CYP2E1 expression in the liver tissue and microsome was determined by immunohistochemistry and Western blotting respectively.RESULTS:HL alone induced by high-fat diet for 18 wk resulted in liver damage,indicated by histopathological analysis,and a considerable increase in serum ALT(25.13 ± 16.90 vs 9.56 ± 1.99,P < 0.01) and AST levels(18.01 ± 10.00 vs 11.33 ± 4.17,P < 0.05) compared with control.Moreover,HL alone induced hepatocyte apoptosis,which was determined by increased TUNEL-positive cells(4.47 ± 0.45 vs 1.5 ± 0.22,P < 0.01),higher caspase-3 and lower bcl-2 expression.Interestingly,compared with those in control,HL or I/R groups,massive increases of serum ALT(93.62 ± 24.00 vs 9.56 ± 1.99,25.13 ± 16.90 or 12.93 ± 6.14,P < 0.01) and AST(82.32 ± 26.92 vs 11.33 ± 4.17,18.01 ± 10.00 or 14.00 ± 6.19,P < 0.01) levels in HL+I/R group were observed suggesting severe liver damage,which was confirmed by liver histology.In addition,HL combined with I/R also caused significantly increased hepatocyte apoptosis,as evidenced by increased TUNEL-positive cells(6.20 ± 0.29 vs 1.5 ± 0.22,4.47 ± 0.45 or 1.97 ± 0.47,P < 0.01),elevated expression of caspase-3 and lower expression of bcl-2.Furthermore,when compared to HL or I/R alone,HL plus I/R enhanced serum TNF-,IL-1,liver mitochondrial MDA and Ca 2+ levels,suppressed SOD and GSH-Px in liver mitochondria,and markedly up-regulated the activity(11.76 ± 2.36 vs 4.77 ± 2.31 or 3.11 ± 1.35,P < 0.01) and expression(3.24 ± 0.38 vs 1.98 ± 0.88 or 1.72 ± 0.58,P < 0.01) of CYP2E1 in liver.CONCLUSION:The coexistence of HL and acute cerebral I/R induces severe liver damage,suggesting that cerebral ischemic stroke would exaggerate the damage of liver caused by HL.This effect is possibly due to en-hanced CYP2E1 induction which further promotes oxidative damage,inflammation and hepatocyte apoptosis.

Evaluation in vinyl chloride monomer-exposed workers and the relationship between liver lesions and gene polymorphisms of metabolic enzymes

AIM： To analyze occupational health hazards exposure to doses lower than the Chinese occupational health standard in a selected VC polymerization plant in China, and also to elucidate the relationship between genetic polymorphisms and genetic susceptibility on liver lesions of workers exposed to vinyl chloride monomer （VCM）. METHODS： In order to explore the mechanism of VCM- related health effects, we used a case-control design to investigate the association between the genetic polymorphisms of metabolic enzymes and liver lesions in workers occupationally exposed to VCM. Genotypes of CYP2E1, GSTT1, GSTM1, ALDH2 and ADH2 were identified using PCR and PCR-RFLP. RESULTS： Even when the concentration of VCM was lower than the current Chinese occupational health standard, neurasthenia, pharyngeal irritation, liver ultrasonography abnormalities and hemoglobin disorders were significantly higher in exposure subjects compared to non-exposure subjects, and the relative risks （RRand 95% C1） were 1.74 （1.06-2.85）, 1.97 （1.56-2.48）, 10.69 （4.38-26.12）, and 2.07 （1.20-3.57）. CYP2E1 c1c2/c2c2 genotype was significantly associated with liver damages （OR 3.29, 95% CI 1.51-7.20, P〈0.01）. CONCLUSION： The incidences of neurasthenia and liver ultrasonography abnormalities significantly increase when the cumulative exposure dose increases. The genotypes of metabolic enzymes （CYP2E1 c1c2/c2c2, null GSTT1 and ADH2 1-1） play important roles in VCM metabolism. Polymorphisms of CYP 2E1, GSTT1 and ADH2 may be a major reason of genetic susceptibility in VCM-induced hepatic damage.

Detection of CYP2E1,a Genetic Biomarker of Susceptibility to Benzene Metabolism Toxicity in Immortal Human Lymphocytes Derived from the Han Chinese Population

Objective Cytochrome P450 2E1 （CYP2E1） is an important metabolizing enzyme involved in oxidative stress responses to benzene, a chemical associated with bone marrow toxicity and leukemia, We aimed to identify the CYP2E1 genetic biomarkers of susceptibility to benzene toxicity in support of environmental and occupational exposure prevention, and to test whether a model using immortal human lymphocytes might be an efficient tool for detecting genetic biomarkers. Methods Immortalized human lymphocyte cell lines with independent genotypes on four CYP2E1 SNP sites were induced with 0.01% phenol, a metabolite of benzene. CYP2E1 gene function was evaluated by mRNA expression and enzyme activity. DNA damage was measured by Single-Cell Gel Electrophoresis （SCGE）. Results Among the four SNPs, cells with rs2070673TT and rs2030920CC showed higher levels of ~YP2E1 transcription and enzymatic activity than the other genotypes in the same SNP site. Cells with higher gene expression genotypes also showed higher comet rates compared with lower gene expression genotypes. Conclusion These results suggest that CYP2E1 rs2070673 and rs2030920 might be the genetic biomarkers of susceptibility to benzene toxicity and that the immortalized human lymphocytes model might be an efficient tool for the detection of genetic biomarkers of susceptibility to chemicals.

Role of CYP2E1 gene polymorphisms association with hepatitis risk in Northeast India

AIM:To investigate hepatitis virus, genetic and environmental factors, and their interactions in predisposing patients to liver diseases in Northeast India. METHODS:A total of 104 jaundice patients and 124 community controls were included. Serological analysis was performed by routine enzyme-linked immunosorbent assay, and nucleic acid testing for hepatitis viruses was done by polymerase chain reaction (PCR), followed by PCR direct sequencing for viral genotyping. Cytochrome P450 2E1 (CYP2E1) polymorphism was studied by PCR-restriction fragment length polymorphism. Nitrite and volatile nitrosamines in indigenous foods consumed routinely by the Northeast Indian ethnic population were estimated by Griess’s reagent and GC-MS, respectively.RESULTS: Hepatitis A virus (HAV) infection was predominantly prevalent (36.5%) in our cohort, followed by hepatitis B virus (HBV), hepatitis E virus (HEV) andhepatitis C virus. HBV genotype D and HEV genotype 1 were the most dominant. CYP2E1 c1/c2 genotype frequency was comparatively higher in alcoholic (P<0.0001,OR =30.5) and cryptogenic (P=0.014, OR=8.714) patients, and was associated with significantly higher hepatitis risk (P=0.0.007,OR=6.489). Mutant C allele of Cyp2E1 DraⅠ frequency was comparatively higher in HAV (P=0.006), alcoholic (P =0.003) and cryptogenic (P=0.014) cases, and was associated with overall hepatitis risk (P=0.026, OR=5.083). Indigenous foods, Gundruk, Kharoli, betel leaf and nuts were found to have the highest nitrite content. CONCLUSION: Apart from viral factors, CYP2E1 polymorphism might be associated with increased risk of liver diseases in Northeast India. Indigenous foods that contain nitrite and nitrosamine might be an associated risk factor.

CYP2E1 Pst Ⅰ/Rsa Ⅰ polymorphism and colorectal cancer risk:A meta-analysis

AIM:To clarify the association between CYP2E1 PstⅠ/RsaⅠ polymorphism and susceptibility to colorectal cancer.METHODS:A meta-analysis based on 10 eligible casecontrol studies involving 4979 cases and 6012 controls was carried out to summarize the data on the association between CYP2E1 RsaⅠ/PstⅠ polymorphism and colorectal cancer risk.RESULTS:In comparison of the homozygote c2c2 and c2 carriers(c1c2 + c2c2) and the homozygous wild-type genotype(c1c1),no association was found between CYP2E1 RsaⅠ/PstⅠ polymorphism and colorectal cancer risk [odds ratio(OR)=1.24(95% CI:0.93-1.66) for c2c2;OR = 1.02(95% CI:0.88-1.19) for c2 carriers].In stratif ied analysis,Caucasians with c2c2 homozygote appeared to have an increased risk of colorectal cancer(OR=2.67,95% CI:1.03-6.89,P=0.043),no signif icant associations were found in other groups.CONCLUSION:c2c2 homozygote of CYP2E1 PstⅠ/RsaⅠ polymorphism may be associated with the increased risk of colorectal cancer in Caucasians,which needs further investigations.

Association of combined CYP2E1 gene polymorphism with the risk for esophageal squamous cell carcinoma in Huai'an population,China

Background Cytochrome P450 2E1 （CYP2E1） has an important role in the metabolic activation of precarcinogens such as N-nitrosoamines and other low relative molecular mass, organic compounds. This study examined whether CYP2E1 Rsal and Dral polymorphism are associated with susceptibility to esophageal squamous cell carcinoma and the correlation between the genotypes and expression levels of CYP2E1 mRNA.Methods Seventy-seven patients with newly diagnosed, untreated esophageal squamous cell carcinoma and 79 healthy controls matched in age, gender and residence were recruited for the control study. An Rsal polymorphism in the 5＇-flanking region and a Dral polymorphism in the sixth intron of the CYP2E1 gene, which could possibly affect its transcription, were determined in this study by polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） and mRNA level of CYP2E1 was measured by quantitative real-time reverse transcription PCR.Results No significant association of Rsal or Dral polymorphism of CYP2E1 with susceptibility of esophageal squamous cell carcinoma were demonstrated （OR=1.67, 95%CI： 0.89-3.15, P=0.11; OR=1.11, 95%CI： 0.59-2.09, P=0.74, respectively）. With SHEsis software, no linkage disequilibrium was detected between Rsal and Dral polymorphism （D＇=0.528, r^2=0.27）. When combined Rsal polymorphism with Dral polymorphism, the association between that carrying c2 allele and DD genotype and the risk for esophageal squamous cell carcinoma were found （OR=5.77, 95%CI： 1.65-20.22）. Compared with the normal controls, the mRNA levels with Rsal polymorphism, Dral polymorphism, or any combined genotypes in cases showed no statistical difference.Conclusions This study suggests that carrying c2 allele and DD genotype conferreded an elevated risk for esophageal squamous cell carcinoma. There was no significant statistical relationship between the genotypes c1/c2, D/C, or the combined allele and mRNA expression.

The Physiological Mechanism of Improved Formaldehyde Resistance in Petunia hybrida Harboring a Mammalian cyp2e1 Gene

Cytochrome P450 CYP2E1 is mainly present in hepatocytes in the livers of mammals,where it plays an important role in the metabolism of xenobiotic organic substances. Previous studies showed that transgenic petunia(Petunia hybrid) plants harboring a mammalian cyp2e1 gene(designated cyp2e1-transgenic petunia) exhibited increased resistance to formaldehyde stress. In this study,we used cyp2e1-transgenic petunia plants to analyze physiological indexes related to formaldehyde stress responses. The results indicated that under formaldehyde stress,the malondialdehyde content in cyp2e1-transgenic petunia plants was lower than in β-glucuronidase gene(gus)-transgenic and wild-type petunia plants. The activities of both superoxide dismutase and peroxidase in the cyp2e1-transgenic plants were higher than in gus-transgenic and wild-type plants. The alcohol dehydrogenase activity was slightly increased and more glutathione was consumed. Additionally,under formaldehyde stress,the levels of plant hormones including indole-3-acetic acid,zeatin and abscisic acid in cyp2e1-transgenic petunia plants displayed decreasing trends,whereas the level of gibberellic acid displayed an increasing trend. In contrast,the indole-3-acetic acid,zeatin and abscisic acid levels in gus-transgenic and wild-type petunia plants displayed increasing trends,whereas the gibberellic acid level displayed a decreasing trend. At 72 h after incubation of 0.5 g of cyp2e1-transgenic petunia plants in 40 mL of treatment solution containing formaldehyde at 50 mg·L^(-1),the formaldehyde content remaining in the treatment solution was close to zero while approximately half of original formaldehyde remained in the treatment solutions containing gus-transgenic and wild-type petunia plants.

Ginsenosides from Stems and Leaves of Ginseng Prevent Ethanol-Induced Lipid Accumulation in Human L02 Hepatocytes

Objective： To investigate the effect of ginsenosides from stems and leaves of ginseng on ethanolinduced lipid deposition in human LO2 hepatocytes. Methods： LO2 cells were exposed to ethanol for 36 h and treated with or without ginsenosides. The viability of LO2 cells was evaluated by methylthiazolyldiphenyl- tetrazolium bromide assay and the triglyceride （TG） content was detected. Lipid droplets were determined by oil red O staining. Intracellular reactive oxygen species （ROS） production and the mitochondrial membrane potential were tested by flow cytometry. The ATP level was measured by reverse phase high performance liquid chromatography. The expression of cytochrome p450 2E1 （CYP2E1） and peroxisome proliferator- activated receptor （PPAR α） was detected by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Results： Ethanol exposure resulted in the increase of TG level, lipid accumulation and ROS generation, and the decrease of mitochondrial membrane potential and ATP production in the cells. However, ginsenosides significantly reduced TG content （9.69 ± 0.22 μg/mg protein vs. 4.93 ± 0.49 μg/mg protein, P〈0.01）, and ROS formation （7254.8± 385.7 vs. 5825.2± 375.9, P〈0.01）. Meanwhile, improvements in mitochondrial membrane potential （10655.33± 331.34 vs. 11129.52 ± 262.35, P〈0.05） and ATP level （1.20±0.18 nmol/mg protein vs. 2.53±0.25 nmol/mg protein, P〈0.01） were observed by treatment with ginsenosides. Furthermore, ginsenosides could down-regulate CYP2E1 expression （P〈0.01） and upregulate PPAR α expression （P〈0.01） in ethanol-treated cells. Conclusions： Ginsenosides could prevent ethanol-induced hepatocyte steatosis in vitro related to the inhibition of oxidative stress and the improvement of mitochondrial function. In addition, the modulation of CYP2E1 and PPAR α expression may also play an important role in the protective effect of ginsenosides against lipid accumulation.