Effect Of The Impairment And Apoptosis Of Oxidative Hair Dyes On Human Kidney Cells

Hair dyes has been used to decorate our life for hundreds of years. There are reports that many patients suffer from diseases such as kidney disease and inhere kidney disease becoming more serious because of staining hair color, and acute renal failure because of taking the wrong medicine of hair dyes. Our experiments uses two key components in oxidative hair dyes as intervention materials to discuss the effects of the proliferation, impairment and apoptosis of oxidative hair dyes on the human kidney cells(HK-2 cells).ObejectTo make sure the effects of the proliferation, impairment and apoptosis of oxidative hair dyes on HK-2 by the two key components in oxidative hair dyes.MethodThe HK-2 cells in the logarithmic growth period were incubated by oxidative hair dyes at different doses for 12 hours and 24 hours. There are 14 groups:1. Control group (RPMI-1640)2. H2O2 group(0.03%)3. PAP group(divided into 6 groups by different concentration and whether have H2O2)(1) PAP1(p-aminophenol 40 ug/ml)(2) PAP2(p-aminophenol 4 ug/ml)(3) PAP3(p-aminophenol 0.4 ug/ml)(4) PAP1H(p-aminophenol 40 ug/ml+0.03%H₂O₂)(5) PAP2H(p-aminophenol 4 ug/ml+0.03%H₂O₂)(6) PAP3H(p-aminophenol 0.4 ug/ml+0.03%H₂O₂)4. PPD group(divided into 6 groups by different concentration and whether have H2O2)(1) PPD1(paraphenylenediamine 40 ug/ml) (2) PPD2(paraphenylenediamine 4 ug/ml)(3) PPD3(paraphenylenediamine 0.4 ug/ml)(4) PPD1H(paraphenylenediamine 40 ug/ml+0.03%H₂O₂)(5) PPD2H(paraphenylenediamine 4 ug/ml+0.03%H₂O₂)(6) PPD3H(paraphenylenediamine 0.4 ug/ml+0.03%H₂O₂)The capacity of proliferation of HK-2 cells was assessed by tetrazolium salt colorimetry assay (MTT assay) at 12 hours and 24 hours after culture, in order to observe the growth inhibiting effect of oxidative hair dyes at different doses and different culture time. The levels of LDH in the supernatants were detected in four groups at 12 hours and 24 hours after culture in order to observe the impairment effect of oxidative hair dyes at different doses and different culture time. The rate of cell apoptosis were analysed by FCM. The method of reverse transcriptase PCR (RT-PCR) was used to detect the mRNA expression levels of cysteine-containing aspartate-specific protease-3(Caspase-3). Scanning electron microscope was used to observe the HK-2 cellular appearance intervened by different substances.Result:1. The effect of the proliferation of HK-2 cells incubated by oxidative hair dyes for 12 hours and 24 hours(1) The A₄₉₂ numbers of the experiment groups were significant higher than those of control group (P＜0.01) and with time dependent.(2) Contrasting to PAP2 and PAP3, A₄₉₂ numbers of PAP1 declined obviously (P＜0.01). There were no differences of the A492 numbers between PAP2 and PAP3 in-12h (P＞0.05). Contrasting to PAP3, A₄₉₂ numbers of PAP2 declined obviously (P＜0.01) in 24h. A₄₉₂ numbers of PAP1H, PAP2H, PAP3H increased one by one (P＜0.01). Contrasting to same concentration alone PAP or 0.03% H₂O₂, the commixture of PAP and 0.03% H₂O₂ had the more growth inhibiting effect on HK-2 cells (P＜0.01)(3) Contrasting to PPD2 and PPD3, A₄₉₂ numbers of PPD1 decreased obviously (P＜0.01). There were no differences of the A₄₉₂ numbers between PPD2 and PPD3 in 12h and 24h (P＞0.05). A₄₉₂ numbers of. PPD1H, PPD2H, PPD3H increased one by one (P＜0.01). Contrasting to same concentration alone PPD or 0.03% H₂O₂, the commixture of PPD and 0.03% H₂O₂ had the more growth inhibiting effect on HK-2 cells (P＜0.01).(4) Contrasting to the alone PPD, the same concentration of PAP had the more effect of the A₄₉₂ numbers decrease on HK-2 (P＜0.01). Be admixed with 0.03% H₂O₂, contrasting to PPD, the same concentration of PAP had the more growth inhibiting effect on HK-2 cells (P＜0.01)2. The effect of the levels of LDH of HK-2 cells incubated by oxidative hair dyes for 12 hours and 24 hours(1) The LDH values of HK-2 cells incubated by oxidative hair dyes were higher than those of control group (P＜0.01) and with time dependent.(2) LDH of PAP1, PAP2, PAP3 increased one by one (P＜0.01) LDH of PAP1H, PAP2H, PAP3H increased one by one (P＜0.01 ). PAP1H contrasted PAP1, PAP2H contrasted PAP2, PAP3H contrasted PAP3, the LDH values increased obviously (P＜0.01). Contrasting to alone 0.03% H₂O₂, the commixture of PAP and 0.03% H₂O₂ had the more effect on the LDH increasing in HK-2 (P＜0.01)(3) Contrasting to PPD2 and PPD3, LDH values of PPD1 increased obviously (P＜0.01). There were no differences of LDH between PPD2 and PPD3 (P＞0.05). LDH ofPAP1H, PAP2H, PAP3H increased one by one (P＜0.01). PPD1H contrasted PPD1, PPD2H contrasted PPD2, PPD3H contrasted PPD3, the LDH increased obviously (P＜0.01) Contrasting to alone 0.03% H₂O₂, the commixture of PPD and 0.03% H₂O₂ had the more effect on the LDH increasing in HK-2 (P＜0.01)(4) Contrasting to the alone PPD, the same concentration of PAP had the more effect on the LDH increasing in HK-2 (P＜0.01). Be admixed with 0.03% H₂O₂, contrasting to PPD, the same concentration of PAP had the more effect on the LDH increasing (P＜0.01)3. The effect of apoptosis rate of HK-2 cells incubated by oxidative hair dyes for 12 hours(1) The apoptosis rate of HK-2 cells incubated by oxidative hair dyes were higher than those of control group (P＜0.05) (2) Apoptosis rate of PAP1, PAP2, PAP3 decreased one by one (P＜0.05). Apoptosis rate of PAP1H, PAP2H, PAP3H decreased one by one (P＜0.05). PAP1H contrasted PAP1, PAP2H contrasted PAP2, PAP3H contrasted PAP3, the apoptosis rate increased obviously (P＜0.05)(3) Apoptosis rate of PPD1H, PPD2H, PPD3H decreased one by one (P＜0.05). PPD1H contrasted PPD1, PPD2H contrasted PPD2, PPD3H contrasted PPD3, the apoptosis rate increased obviously (P＜0.05)(4) Contrasting to the alone PPD, the same concentration of PAP had the more effect on the apoptosis rate increasing in HK-2 (P＜0.05). Be admixed with 0.03% H₂O₂, contrasting to PPD, the same concentration PAP had the more effect on the apoptosis rate increasing (P＜0.05)4. The effect of the mRNA expression levels of Caspase-3 of HK-2 cells incubated by oxidative hair dyes for 12h(1) The mRNA expression levels of Caspase-3 of HK-2 cells incubated by oxidative hair dyes were higher than those of control group (P＜0.01).(2) The mRNA expression levels of Caspase-3 of PAP1, PAP2, PAP3 decreased one by one (P＜0.01). The mRNA expression levels of Caspase-3 of PAP1H, PAP2H, PAP3H decreased one by one (P＜0.01). PAP1H contrasted PAP1, PAP2H contrasted PAP2, PAP3H contrasted PAP3, the mRNA expression levels of Caspase-3 increased obviously (P＜0.01).(3) The mRNA expression levels of Caspase-3 of PPD1, PPD2, PPD3 decreased one by one (P＜0.01). The mRNA expression levels of Caspase-3 of PPD1H, PPD2H, PPD3H decreased one by one (P＜0.01). PPD1H contrasted PPD1, PPD2H contrasted PPD2, PPD3H contrasted PPD3, the mRNA expression levels of Caspase-3 increased obviously (P＜0.01).(4) Contrasting to the alone PPD, the same concentration PAP had the more effect on the mRNA expression levels of Caspase-3 increasing in HK-2 (P＜0.01). Be admixed with 0.03% H₂O₂, contrasting to PPD, the same concentration PAP had the more effect on the apoptosis rate increasing (P＜0.01)5. The effect of appearance of HK-2 cells incubated by oxidative hair dyes for 12hContrasting to control group, appearance of HK-2 cells of PAP1, PAP1H, PPD1, PPD1H all had changed. The microvillus of cell surface disappeared in different extent. PAP1H have changed mostly above all, then the PAP1 and PPD1H, the last PPD1.Conclusion:1. The capacities of proliferation of HK-2 cells incubated for 12 hours and 24 hours were inhibited by PAP and PPD at the doses between 0.4～40μg/ml. Admixed with 0.03% H2O2, the inhibition of PAP and PPD were more obvious with concentration dependent and time dependent. In the same condition(whether admixed with H2O2 or not), the same concentration PAP had more inhibition than those of PPD.2. The impairment effects on HK-2 cells incubated for 12 hours and 24 hours are enhanced by oxidative hair dyes PAP and PPD at the doses between 0.4～40μg/ml. Admixed with 0.03% H2O2, the impairment effects of PAP and PPD were more obvious with concentration dependent and time dependent. In the same condition(whether admixed with H2O2 or not), the same concentration of PAP had more impairment effects than those of PPD.3. The apoptosis effects on HK-2 cells incubated for 12 hours are enhanced by oxidative hair dyes PAP and PPD at the doses between 0.4～40μg/ml. Admixed with 0.03% H202, the apoptosis effects of PAP and PPD were more obvious with concentration dependent. In the same condition(whether admixed with H2O2 or not), the same concentration of PAP had more apoptosis effects than those of PPD.4. PAP and PPD in oxidative hair dyes incubated for 12 hours had bad effect on cells', appearance. Admixed with 0.03% H2O2, the appearance effects of PAP and PPD were more obvious. In the same condition(whether admixed with H2O2 or not), the same concentration of PAP had more appearance effects than those of PPD.