Changes of nitric oxide and nitric oxide synthase in cultured human keratinocytes after treated with trichloroethylene

Objective To observe the changes of nitric oxide(NO)and nitric oxide synthase(NOS)in cultured normal human keratinocytes after treated with trichloroethylene(TCE)and further explore the dermatotoxicity mechanism of TCE.Methods Normal human keratinocytes were cultured in serum-free medium and treated with 0.125、0.25、0.5、1.0 or 2.0 mmol/L of TCE for 4,12,24,48 and 72 hours after stimulation,the content of NO and the activity of NOS(inducible-NOS and constitutive-NOS)were detected to analysis time-dose-effect relationship.Meanwhile,RT-PCR was applied to determine the expression of iNOS mRNA.Results Both NO production and iNOS activity did not change at the concentration of 0.125 mmol/L and 0.25 mmol/L in any time point after stimulation,they began to elevate 48 hours after stimulated with 0.5mmol/L of TCE[(32.19±4.75)μmol/L compared with the control group:(23.70±3.11)μmol/L,P0.05,and(1.15±0.02)×103U/L compared with the control group:(0.77±0.06)×103U/L,P0.05,respectively],displaying a trend of increase and ahead of time along with the increase of TCE concentration.NO release was consistent with the elevation of iNOS activity but not cNOS;RT-PCR also showed an over-expression of iNOS mRNA after TCE stimulation.Conclusion Dose-dependently and time-relatedly,TCE can induce keratinocytes to generate large amount of NO which attributes to the up-regulation of iNOS,and all this may contribute to the cytotoxicity and dermato-toxicity of TCE.