Changes of caspase-8 and caspase-9 activity during apoptosis of keratinocytes induced by trichloroethylene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSION</b>Caspase-9 may be an important mediator of apoptosis in keratinocytes induced by trichloroethylene.</p>

Cytotoxicity of trichloroethylene in keratinocytes involving alterations of mitochondrial function and ultrastructure / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore mechanism of dermal toxicity of trichloroethylene(TCE).</p><p><b>METHODS</b>Normal human keratinocytes (KC) were isolated from foreskins of healthy donors undergoing circumcision by two-step trypsin digestion and cultured in serum-free medium. Cells were treated with medium, 1% acetone (volume fraction) 0.125, 0.500 or 2.000 mmol/L TCE for different time (4, 8, 12 or 24) hours. After treatment, MTT assay and ATPase activity detected, inhibition ratio of mitochondrial enzyme was calculated according to optical density (A) value of MTT assay. Mitochondrial membrane potential (MMP) was detected by flow cytometry FCM after being stained with Rhodamine123 (Rh123). Morphological changes were also observed through transmission electron microscope (TEM).</p><p><b>RESULTS</b>Cellular viability and ATPase activity declined with dose of TCE, while inhibition ratio of mitochondrial enzyme increased with dose of TCE. FCM results showed that after treatment with 2.000 mmol/L TCE, fluorescence density of Rh123 decreased quickly from 18.73 +/- 0.45(0 h) to 8.20 +/- 0.66(8 h) (P < 0.01). After 8 h, fluorescence density maintained at the level equal to that of 8 h (fluorescence density of Rh123 were 8.20 +/- 0.36 and 8.20 +/- 0.40 for 12 and 24 h respectively, compared with that for 8 h group, P > 0.05). The results also showed that MMP diminished with dose of TCE. Under TEM, mitochondria in TCE-treated group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae but in control group, mitochondrial structure was integrated, with uniform matrix and visible mitochondria cristae.</p><p><b>CONCLUSIONS</b>TCE could inhibit mitochondrial metabolic enzyme, reduce ATP production, diminish MMP, and destroy ultrastructure of mitochondria in KC, all these contributing to the cytotoxicity of TCE.</p>