BACKGROUND: Previously we have shown that acute exposure to thimerosal (Thi) can induce oxidative stress and DNA damage in a human conjunctival cell line. However, the long-term effect of Thi on Chang conjunctival cells is not clear. Therefore, the aim of this study was to further investigate the fate of the cells after acute exposure to Thi. METHOD: Cells were first exposed to various concentrations of Thi (0.00001 % ∼ 0.001 %) for 30 min, and then cells were assessed after a 24-h recovery period. Morphologic changes were observed under a light microscope and cell viability was evaluated. Cell apoptosis, cell cycle distribution and mitochondrial membrane potential (MMP) (rhodamine 123 assay) were detected by flow cytometry analysis. Poly (ADP-ribose) polymerase (PARP), activation of caspase-3 and microtubule-associated protein light chain 3 (LC-3) were examined by western blot analysis. RESULTS: DNA strand breaks were significantly increased in a dose-dependent manner with 30 min exposure to Thi, although no significant cell death was detected. However, after 24-h recovery, the ratio of apoptotic cells was significantly increased to 0.0005 % and 0.001 % in Thi treated groups (p < 0.001 compared to the control group). Apoptosis was confirmed by the cleavage of PARP and caspase-3 activation. In addition, G2/M cell cycle arrest and decrease of MMP were recorded. Finally, the LC-3 results indicated the occurrence of autophagy in Thi-treated cells. CONCLUSION: Acute exposure to Thi can induce DNA damage, and eventually can lead to cell death, probably through the caspase-dependent apoptosis pathway, while autophagy might also be involved.
BACKGROUND: Previously we have shown that acute exposure to thimerosal (Thi) can induce oxidative stress and DNA damage in a human conjunctival cell line. However, the long-term effect of Thi on Chang conjunctival cells is not clear. Therefore, the aim of this study was to further investigate the fate of the cells after acute exposure to Thi. METHOD: Cells were first exposed to various concentrations of Thi (0.00001 % ∼ 0.001 %) for 30 min, and then cells were assessed after a 24-h recovery period. Morphologic changes were observed under a light microscope and cell viability was evaluated. Cell apoptosis, cell cycle distribution and mitochondrial membrane potential (MMP) (rhodamine 123 assay) were detected by flow cytometry analysis. Poly (ADP-ribose) polymerase (PARP), activation of caspase-3 and microtubule-associated protein light chain 3 (LC-3) were examined by western blot analysis. RESULTS: DNA strand breaks were significantly increased in a dose-dependent manner with 30 min exposure to Thi, although no significant cell death was detected. However, after 24-h recovery, the ratio of apoptotic cells was significantly increased to 0.0005 % and 0.001 % in Thi treated groups (p < 0.001 compared to the control group). Apoptosis was confirmed by the cleavage of PARP and caspase-3 activation. In addition, G2/M cell cycle arrest and decrease of MMP were recorded. Finally, the LC-3 results indicated the occurrence of autophagy in Thi-treated cells. CONCLUSION: Acute exposure to Thi can induce DNA damage, and eventually can lead to cell death, probably through the caspase-dependent apoptosis pathway, while autophagy might also be involved.
Authors: N Zamzami; P Marchetti; M Castedo; D Decaudin; A Macho; T Hirsch; S A Susin; P X Petit; B Mignotte; G Kroemer Journal: J Exp Med Date: 1995-08-01 Impact factor: 14.307