Fecapentaene-12 (FP-12), a fecal unsaturated, ether-linked lipid excreted by most human individuals in Western populations, has been found to be a potent genotoxin in mammalian cells. Its mechanism of genotoxicity may be mediated by oxygen radical-induced DNA damage or by direct DNA alkylation, of which the relative importance remains to be determined. In the present study, induction of oxidative genetic damage by FP-12 has been investigated, in combination with the biological inactivation of single-stranded bacteriophage phi X-174 DNA. It was shown that formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), a marker for oxidative DNA damage, is induced dose dependently by FP-12 in 2'-deoxyguanosine (dG). It was demonstrated by application of radical scavengers that production of both the superoxide anion and singlet oxygen may be involved in the induction of 8-oxodG. The effect of OH radical scavenging appeared to be less pronounced. Enzymatic peroxidation of FP-12, which has been demonstrated to stimulate oxygen radical formation, was found to increase the hydroxylation ratio in dG, an effect which was less pronounced in single-stranded DNA and even absent in double-stranded DNA. No induction of 8-oxodG was observed after exposure of human skin fibroblasts to 60 microM FP-12 for 3 h in vitro. It was concluded that the induction of 8-oxodG by FP-12 is determined by the accessibility of the guanine molecule rather than the rate of oxygen radical formation. Although free radical formation is known to be stimulated by enzymatic peroxidation of FP-12, the inactivation of phi X-174 DNA spontaneously induced by FP-12 was found to be reduced by application of peroxidases. This furthermore demonstrates that the increased formation of reactive oxygen species by enzymatic peroxidation of FP-12 does not directly relate to increased induction of genotoxic effects. The fact that addition of radical scavengers shows limited effects on the inactivation of phi X-174 DNA suggests that the contribution of oxidative DNA damage to the genotoxic potential of FP-12 is only of minor importance.