title: [Effects of trichloroethylene on hepatotoxicity in cytochrome 2E1-silenced hepatocytes].
abstract: OBJECTIVE
  To prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes.
  
  
  METHODS
  Short hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2E1-silenced hepatocytes were selected; real-time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1-silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE (0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L). The cell viability and half maximal inhibitory concentration (IC50) of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR.
  
  
  RESULTS
  The IC50s of TCE for L02 hepatocytes and CYP2E1-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2E1-silenced hepatocytes hada significantly lower apoptotic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE (P < 0.05 or P < 0.01). The mRNA expression level of bcl-2 (anti-apoptosis gene) in CYP2E1-silenced hepatocytes was 15% ∼ 60% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of caspase-3 and caspase-9 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30% ∼ 60% lower than those in L02 hepatocytes (P < 0.01). The mRNA expression level of p53 (cancer suppressor gene) in CYP2E1-silenced hepatocytes was 81 - 278% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of c-fos and k-ras (oncogenes) in CYP2E1-silenced hepatocytes were 20-68% lower than those in L02 hepatocytes (P < 0.01).
  
  
  CONCLUSIONS
  CYP2E1-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology. Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes, suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.
authors.affiliation: Shenzhen Center for Disease Control and Prevention, Shenzhen, Guangdong Province, China.
authors.forename: Xin-yun
authors.initials: XY
authors.lastname: Xu
authors.forename: Ji-yan
authors.initials: JY
authors.lastname: Mao
authors.forename: Kan-lang
authors.initials: KL
authors.lastname: Mao
authors.forename: Guo-hong
authors.initials: GH
authors.lastname: Liu
authors.forename: Jie-yuan
authors.initials: JY
authors.lastname: Ci
authors.forename: Xi-fei
authors.initials: XF
authors.lastname: Yang
authors.forename: De-sheng
authors.initials: DS
authors.lastname: Wu
authors.forename: Hai-yan
authors.initials: HY
authors.lastname: Huang
authors.forename: Ran
authors.initials: R
authors.lastname: Zhang
authors.forename: Xin-feng
authors.initials: XF
authors.lastname: Huang
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D034622:RNA Interference; D014241:Trichloroethylene / Q000633:toxicity*
chemical_list: D014241:Trichloroethylene; D019392:Cytochrome P-450 CYP2E1