Objective: To explore the potential mechanism of trichloroethylene (TCE)-induced apoptosis in normal human epidermis keratinocyte (NHEK) by assaying the Caspase activities, mitochondrial membrane potential (DeltaPsim) and apoptosis in vitro.
Methods: NHEK was exposed to TCE and Caspases-3, 8 and 9 activities were determined using a commercial assay kit. Apoptosis and DeltaPsim were detected by flow cytometry (FCM) after double-stained with annexin-V and PI, Rh123 and PI respectively. NHEK was pretreated with inhibitor of Caspase-3 or 9 to verify the activation of Caspases by TCE treatment.
Results: Various dose of TCE exposure could increase the Caspases-3 and 9 activities in dose- and time-dependent way. There was marked difference between TCE-treated group and control at 12 or 24 h. But no significant influence of Caspase-8 activity was evoked. 0.125, 0.25, 0.5, 1.0 and 2.0 mmol/L TCE treated NHEK 4 h then cultured for 12 h. Annexin-V(+)/PI(-) proportion were (20.1 +/- 4.1)%, (30.0 +/- 7.5)%, (42.1 +/- 8.2)%, (56.0 +/- 6.1)% and (79.1 +/- 4.3)% respectively. There was marked difference between TCE-treated group except for 0.125 mmol/L and control (9.4 +/- 3.0)% (all P < 0.05). FITC(+)/PI(-) proportion were marked positive correlation with Caspase-3 and 9 activities, r = 0.786, 0.736 (both P < 0.05). Caspase-3 activities had also a marked positive correlation with Caspase-9 activities, r = 0.845 (P < 0.05). There was no correlation with Caspase-8 activities. Pretreatment for 1 h with 100 micromol/L Z-DEVD-FMK decreased the Caspase-3 activities from (0.963 +/- 0.043) to (0.349 +/- 0.045) nmol pNA * min(-1) * ml(-1), annexin-V(+)/PI(-) proportion decreased from (80.0 +/- 5.5)% to (16.3 +/- 3.2)% in 2.0 mmol/L TCE treated NHEK with a significant difference (P < 0.01), but there was no change of Caspase-9 activities. 100 micromol/L Z-LEHD-FMK pretreatment decreased the Caspase-3 activities to (0.338 +/- 0.011) nmol pNA * min(-1) * ml(-1), annexin-V(+)/PI(-) proportion decreased to (16.1 +/- 1.7)% in 2.0 mmol/L TCE treated NHEK. And the Caspase-9 activities decreased from (0.821 +/- 0.031) to (0.240 +/- 0.043) nmol pNA * min(-1) * ml(-1) with a significant difference (P < 0.01). NHEK was cultured for 4, 8, 12, 24 h after a 4-hour treatment with 2.0 mmol/L TCE. Rhod123 fluorescence intensity (FI) were respectively with a marked decrease as compared with 0 h (18.7 +/- 0.5, all P < 0.01). At 0.125, 0.5 and 2.0 mmol/L TCE treated NHEK for 4 h then cultured 8 h, Rh123 FI were 16.1 +/- 0.5, 12.1 +/- 0.6 and 8.1 +/- 0.6 with a marked decrease as compared with control (18.1 +/- 0.5, all P < 0.01).
Conclusion: TCE-induced NHEK apoptosis is mediated intrinsically through the mitochondrial pathway of the decrease of DeltaPsim and the Caspase-9 dependent activation of Caspase-3.