DNA Damage and Telomere Shortening in Embryonic Stem Cells Induced by Cigarette Smoke.

Cigarette smoke is associated with high risk of lung, cardiovascular, degenerative diseases, and reducing developmental abilities of embryo. Stem cells are important for functional maintenance of tissues by replenishing damaged cells, which maybe very sensitive to toxic factors. Embryonic stem cells (ESCs) provide a valuable in vitro model, alternative to whole animal models, for testing toxicity of chemicals and environmental pollution including cigarette smoke. To test the hypothesis that cigarette smoke affects the pluripotency and chromosome stabilization of stem cells. Mouse ESCs were exposed to smoke components, cigarette smoke condensate (CSC) at the concentration of 0.2 mg/ml for 20 h (acute treatment) and then cultured for two weeks, or 0.02 mg/ml for two weeks (chronic treatment). In addition, cadmium, a component of CSC, was also tested its effect on ESCs at concentration of 5µM or 20µM. ESCs were evaluated for their pluripotency by Oct4, Nanog, SSEA1 and AP immunofluorescence microscopy, proliferation by morphology and colony number, apoptosis by Tunel staining, and particularly DNA damage by rH2AX staining and telomere function by Q-FISH measurement. DNA damage and apoptosis were increased in ESCs treated with CSC 0.2 mg/ml for 20 h and the antioxidant N-acetyl-L-cysteine (NAC) failed to rescue the damaging effects by CSC. Telomere length did not show any difference by further Q-FISH quantification compared to controls without exposure to CSCs or cadmium. Number of ESC colonies was also significantly reduced when ESCs were cultured with CSC 0.15mg/ml for two weeks. Surprisingly, when ESCs were treated with CSC 0.2 mg/ml for only 20 h and then cultured for two weeks without exposure to CSC, no differences were found between CSC treated group and control. ESCs treated with cadmium at 20 µM for 20 h also showed similar results. Low doses of CSC (0.02 mg/ml) and cadmium (5 µM) did not negatively affect pluripotency and proliferation of ESCs following treatments of two weeks. However, telomere length was shorter in CSC group and the shortest in cadmium group, which also had more DNA damage, comparing with controls. These results demonstrate that acute smoke negatively affects the pluripotency and chromosome stabilization of ESCs. However, authentic ESCs have inherent strong mechanisms, or ESCs select best quality stem cells, to maintain genomic stability for their indefinite self-renewal in long term culture without cigarette smoke. Chronic smoke did not negatively affect pluripotent markers expression and proliferation of ESCs but shorten the telomere length of ESCs after long term culture which will reduce the authentic pluripotency of ESCs indeed. These data also indicate that ESCs might not be used as a sensitive model for toxicological tests, in contrast to early embryos. Research supported by: James and Esther King Biomedical Research Program, NSFC (31000611), Fundamental Research Funds for the Central Universities (JH).

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