Effects Of Folic Acid On The Damage Of HaCaT Cells Induced By Cr(?)

Occupational exposures of hexavalent chromium [Cr(?)] are mainly from chromate production,electroplating,stainless steel welding and ferrochrome production related industries.Cr(?)can cause serious harm to the human which could induce lung cancer,nasopHaryngeal carcinoma,and has been classified as human class ? carcinogens by IARC.Studies have shown that Cr(?)could induce human skin cytotoxicity,chromosome aberration,DNA double-strand breaks and so on.Folic acid involves in the transfer of a carbon unit for the methylation of DNA methyl in the process of human biochemistry,and maintains the stability of the genome,and plays an important role in the process of DNA synthesis,maintenance in cells.The aims of this study were to investigate the effects of Cr(?)on HaCaT cells and the effects of folic acid on Cr(?)-induced HaCaT cells damage in order to provide theoretical basis for the further study of the protective effects of folic acid on chromium occupational exposure population.The main contents are as follows:(1)MTT assay was used to detect the survival rate of different concentrations of Cr(?)and folic acid on HaCaT cells,and obtained IC50,and selected appropriate concentration for next experiment.(2)SCGE / comet assay was used to detect the DNA damage of Cr(?)on HaCaT cells and effects of folic acid on DNA damage induced by Cr(?).The DNA damage was evaluated by Tail Length,Tail moment and Tail DNA %.(3)5-mC immunofluorescence assay was used to detect the the total methylationlevels of different doses of Cr(?)on of HaCaT cells and the changes of total DNAmethylation levels with folic acid pretreatment.(4)The expression of hMLH1 mRNA and protein in HaCaT cells with Cr(?)treatment and folic acid pretreatment in HaCaT cells were detected by RT-PCR and Western blot.The results are as follows:(1)With the increase of Cr(?)concentration,the cells survival rate decreased.When the concentrations of Cr(?)were 10.00 ?M and above,the cells survival rates were significantly different from the control group(P<0.05).IC 50 was 15.88 ?M.The folic acid had no effects on cells viability in the concentrations ranges of 2.5-320.0 n M(P>0.05).(2)The Kruskal Wallis test showed the results that the DNA damage index(Tail Length,Tail DNA%,Tail Moment and Olive Tail Moment)of the control group and Cr(?)expouesed groups were statistically different(P<0.05).When the concentrations of Cr(?)were 10,20,40 and 80 ?M,the Tail Length,Tail DNA%,Tail Moment and Olive Tail Moment were higher than the control group(P<0.05).There were significant differences in the average levels of the folic acid pretreatment groups and control group and Cr(?)exposured group(P<0.05).When the folic acid concentration was 320 nM,there was no significant difference in Tail Length,Tail DNA%,Tail Moment,Olive Tail Moment with the control group(P>0.05).Spearman correlation analysis showed that Cr(?)doses were positively correlated with Tail Length,Tail DNA%,Tail Moment and Olive Tail Moment(r=0.915,P<0.01;r=0.846,P<0.01;r=0.879,P<0.01;r=0.869,P<0.01),and folic acid doses were negative correlated with Tail Length,Tail DNA%,Tail Moment,Olive Tail Moment(r=-0.797,P<0.01;r=-0.740,P<0.01;r=-0.783,P<0.01;r=-0.751,P<0.01).(3)The results of variance analysis showed that the average optical density of Cr(?)groups and control group were significantly different(F=11.367,P<0.01).LSD test showed that the average optical density was lower than the control group when concentrations of Cr(?)were above 5.00 ?M(P<0.05).The average optical density of cells which were pretreated by different concentrations of folic acid(20.0,40.0,80.0,160.0,320.0 nM)were significantly different from those in the control group and Cr(?)exposured group(F=9.928,P<0.01).When the pretreatment concentration of folic acid were 160.0 nM and 320.0 nM,the average optical density were higher than Cr(?)exposured group(P<0.05).Pearson correlation analysis showed that Cr(?)doses were negatively correlated with cells average optical density(r =-0.633,P<0.01).The doses of folic acid were positively correlated with the cells average optical density(r = 0.865,P <0.01).(4)?2 test showed that when the concertrations of Cr(?)were 5.00 ?M and above,hMLH1 mRNA levels were lower than the control group(P<0.01).When the concentrations of Cr(?)were 10.00 ?M and more,the expression rates of mRNA were less than 50%.The expression levels of hMLH1 protein were lower than the control group(P<0.01)when the Cr(?)at the medium or high concentrations(10.00,20.00,40.00,80.00 ?M).?2 test showed that the levels of h MLH1 mRNA with folic acid pretreatment were statistically different from that the control group(P<0.01).When the folic acid concentrations were 160.0 nM and 320.0 nM,the mRNA expression levels were higher than Cr(?)expoursed group alone.When folic acid were at higher concentrations(80.0 nM,160.0 nM),the protein expression levels were higher than the control group(P<0.01).