The Keratinocytes Apoptosis Induced By Trichloroethylene Through The Mitochondrial Pathway

Objective The purposes of this study is to observe the activity of caspase-3, caspase-8 and caspase-9 and the rate of apoptosis during the apoptosis process, as well as the inhibitive effects of the specific caspase inhibitor and the protective effect of ginkgo biloba extract. Not only with intention to elucidate the possible pathway of apoptosis of Keratinocytes induced by trichloroethylene, but also to throw light on prevention and cure of related occupational dermatitis.Methods Normal human KC were isolated from foreskins of healthy donors and cultured in serum-free medium. Cells were treated with 0.125, 0.25,0.5,1.0 or 2.0 mmol/L TCE, the KC cultured in medium was set as the blank control group; The inhibitive groups were pretreated with 100μmol/l Z-DEVD-FMK (inhibitor of caspase-3)or Z-LEHD-FMK (inhibitor of caspase-9)for 1 h, then were stimulated with 2.0 mmol/l TCE; To observe EGb's protective effect, cells were pre-incubated with different concentrations of (50,100,150 mg /L)EGb for 2 hours, then treated with 2.0 mmol/L TCE. Changes of the following indicators were observed:①MTT assay was used to detect the viability of different cells;②The activity of caspase-3, caspase-8 and caspase-9 were calculated according to spectrophotometry;③Change of the apoptotic rate was detected by flow cytometer (FCM) after double-stained with Annexin V-FITC and propidium iodide (PI).Results Our study found that:①Changes of cell viability: The minimum effective concentration for cell viability reduction was 0.125 mmol/L at 12 h and the shortest time required to produce a chang was 4 h at a concentration of 2.0 mmol/L(compared with control group, P<0.01). Cell viability in all the groups markedly decreased while been stimulated from 12 h to 24 h(P<0.05).As the concentration of TCE increased, the cell viability decreased in a dose-dependent manner.②Changes of caspase-3 activity: At 8 h, 1.0 and 2.0 mmol/L TCE groups can significantly enhanced caspase-3 activity (P<0.05). The caspase-3 activity in all the groups showed differences and was significantly higher than those of control cells when time was over 12h(P<0.05). The activity of caspase-3 reached its peak at 12 h in all groups.③Changes of caspase-9 and caspase-8 activity: When exposure for 8 h, the caspase-9 activity of 2.0 mmol/L TCE stimulative group significantly increased (compared with control group, P<0.01). The caspase-9 activity in all the groups showed differences when exposure time is over 12 h(compared with control group, P<0.01). The activity of caspase-9 reached its peak at 12 h in all groups. The activity of caspase-8 in the various dose groups at different times had no statistical difference (compared with the control group, P>0.01).④Changes of the apoptosis rate: After exposing to different doses of TCE for 12 h, the rate of apoptosis enhanced with the increase of dose, which showed a dose- effect relationship.⑤Effects of caspase-3 inhibitor: The cells pretreated with caspase-3 inhibitor resulted in a decrease in the caspase-3 activity and apoptosis rates (compared with 2.0 mmol/L TCE exposed group, P<0.01), while there was no significant difference in caspase-9 activity (compared with 2.0 mmol/L TCE stimulate group, P>0.01).⑥Effects of caspase-9 inhibitor: the group pretreated with caspase-9 inhibitor showed a difference in caspase-9 and caspase-3 activity and apoptosis rate in comparison to 2.0 mmol/L TCE exposed group(P<0.01), while there was no statistical significance compared with the control group(P>0.01).⑦The protective effect of EGb: 50 mg /L EGb pretreatment had a protective effect, the caspase-3, caspase-9 activity and apoptosis rate was significantly decreased (compared with 2.0 mmol/L TCE group, P<0.01). When the concentration was over 100 mg /L, there was no significant difference on the above indicators(compared with control group, P>0.05).Conclusion Our results suggested that during the apoptosis of keratinocytes induced by trichloroethylene, death receptor pathway maybe not involved in the apoptosis process while the mitochondrial pathway plays an important role. This pathway might be related to upregulation of caspase-9 activation which subsequently activate caspase-3 mediated apoptosis at last. Ginkgo biloba extract(EGb) can prevent cell apoptosis through inhibition of the activation of caspase-9. This study partly throw light on the skin toxicity mechanism of TCE, and provide an experimental basis for the prevention and treatment of the skin damage caused by TCE and the development of effective protective gear of skin.