Selenite (SeL) or selenomethionine (SeM) are the most common selenium (Se) compounds taken as dietary antioxidants to reduce oxidative stress. Because the public may frequently supplement Se compounds at high doses, the possible pro-oxidant effect of Se becomes a concern. SeL and SeM have entirely different pharmacokinetic effects based on dose-related cytotoxicity. Our laboratory has shown previously that high doses of SeL resulted in cytotoxicity and induction of 8-hydroxydeoxyguanosine (8-OHdG) in DNA of primary human keratinocytes (NHK), compared with those treated with the same doses of SEM: Besides Se compounds, other dietary antioxidants, such as vitamin (Vit) C or Vit E, are often supplemented and taken together with Se compounds. However, the cellular effects of these interactions of Se with antioxidants are still unknown. In addition, copper is commonly present in drinking water, food, soil, or the environment to increase the possibility of subchronic toxicity. Copper has been shown to inhibit SeL-induced cytotoxicity and apoptosis in human colonic carcinoma cells. The present study was designed to investigate the interactive effects of SeL or SeM plus Vit C, trolox (a water-soluble Vit E), or copper sulfate (CuSO(4)) on cell viability and induction of 8-OHdG adduct formation in DNA of NHK. NHK cells were treated with no Se, SeL (126.6 microM Se), or SeM (316.6 microM Se) plus two doses each of Vit C (2.27 and 4.45 microM), trolox (40 and 80 microM), or CuSO(4) (7.85 and 15.7 microM) for 24 h. Coincubation of Vit C or CuSO(4) with SeL appeared to protect NHK against SeL-induced cytotoxicity. However, synergistic effects were observed between SeL and trolox resulting in enhanced cytotoxicity. On the other hand, SeM + Vit C, SeM + trolox, and SeM + CuSO(4) did not affect cell viability. In the absence of Se supplementation, Vit C, trolox, or CuSO(4) alone did not induce 8-OHdG adduct formation, regardless of dose. When NHK cells were coincubated with SeL (126.6 microM Se) and Vit C or CuSO(4), they protected NHK from SeL-induced DNA damage with a reduction in 8-OHdG generation. In contrast, treatment of SeL + trolox elevated generation of 8-OHDG: Furthermore, treatments of SeM plus trolox or CuSO(4) elevated 8-OHdG adduct formation. In terms of apoptosis measured as internucleosomal DNA fragmentation, copper protected NHK against SeL-induced apoptosis in cultured NHK. These data suggest that the use of CuSO(4) may play a protective role in SeL-induced cytotoxicity, DNA oxidative damage, and apoptosis and that there may be potentially deleterious interactions among common high-dose antioxidant supplements taken by the public.