[Changes of caspase-8 and caspase-9 activity during apoptosis of keratinocytes induced by trichloroethylene].
OBJECTIVE
To observe the change of caspase-8, caspase-9 activity and apoptosis rates in the process of trichloroethylene-induced damage in keratinocytes, and explore the tentative mechanism of apoptosis.


METHODS
Human keratinocytes were exposed to 0.125, 0.250, 0.500, 1.000 and 2.000 mmol/L trichloroethylene for 4, 8, 12 and 24 h. The inhibitive groups were pretreated with 100 micromol/L Z-LEHD-FMK (a specific inhibitor of caspase-9) for 1 h, and were stimulated with 2.000 mmol/l TCE for 12 h. MTT assay was used to detect the viability of different cells; The activity of caspase were calculated according to spectrophotometry; Change of the apoptotic rates was assessed by flow cytometer (FCM) after double-stained with Annexin V-FITC and propidium iodide (PI).


RESULTS
(1) The minimum effective concentration for cell viability reduction was 0.125 mmol/L at 12 h and the shortest time required to produce a change was 4 h at a concentration of 2.000 mmol/L (compared with control group, P < 0.01). Cell viability in all the groups markedly decreased from 12 h to 24 h (P < 0.05). (2) The activity of caspase-8 in the various dosage groups at different times had no statistical difference compared with the control group, P > 0.01. (3) At 8 h, 1.000 and 2.000 mmol/L TCE groups could significantly enhance caspase-9 activity (P < 0.05). The caspase-9 activity in all the groups showed differences and was significantly higher than those of control cells when time was over 12 h (P < 0.05). (4) After exposing to different dosages of TCE for 12 h, the rate of apoptosis rose to (80.43 +/- 4.21)% with the increase of dosage, compared with the control group, (9.40 +/- 2.98)%, which showed a dose-effect relationship. (5) The cells pre-treated with caspase-9 inhibitor resulted in a decrease in the caspase-9 activity and apoptosis rates (compared with 2.000 mmol/L TCE exposed group, P < 0.01). However, there was no statistical significance in comparison with the control group (P > 0.05).


CONCLUSIONS
Caspase-9 may be an important mediator of apoptosis in keratinocytes induced by trichloroethylene.
Zhu
Qi-xing
QX
Department of Occupational and Environmental Health, School of Public Health, Anhui Medical University, Hefei, 230032, China.
Ye
Liang-ping
LP
Wang
Li-jie
LJ
Shen
Tong
T
D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D014241:Trichloroethylene / Q000633:toxicity*
D014241:Trichloroethylene; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9