Epidemiological evidence suggests that chewing betel quid and smoking have synergistic potential in the development of oral squamous-cell carcinoma in Taiwan. Chewing betel quid produces alkalization of saliva. This study investigated the response of human oral cancer OEC-M1 cells to nicotine in different pH environments (6.5 and 8) by examining its effects on DNA damage as evidenced by single-cell gel electrophoresis. Nicotine (1 and 10 muM) significantly induced DNA strand breakage when cultured at pH 8 for 6 h but did not induce DNA damage at pH 6.5. Nicotine-induced DNA damage was also time dependent. When cells were pretreated with catalase or N-acetylcysteine, a significant reduction in nicotine-induced DNA damage was observed. Flow cytometric analyses showed that the production of 8-oxoguanine was significantly increased following nicotine (10 muM) treatment. Posttreatment of nicotine-damaged DNA by endonuclease III and formamidopyrimidine-DNA glycosylase, recognizing oxidized DNA bases, increased the extent of DNA damage. These results suggest that nicotine-induced DNA strand breakage is pH dependent, and oxidative stress might be involved in nicotine-induced DNA damage. Finally, cigarette smoke condensate (equivalent to 8 muM nicotine) induced significant DNA strand breaks in OEC-M1 cells at pH 8 and correlated with the generation of oxidative DNA damage. Thus, alkaline saliva generated by chewing betel quid plays an important role in cigarette-related nicotine-induced DNA damage, and reactive oxygen species may be involved in generating this DNA damage.