The identification and repair of DNA adducts induced by waterborne benzo[a]pyrene in developing Xenopus laevis larvae

Abstract

We report on the formation and subsequent repair of benzo[a]pyrene-induced DNA adducts in Xenopus laevis larvae in vivo, as monitored by 32P-post labelling. In vivo benzo[a]pyrene is metabolized by the cytochrome P450 family of enzymes to metabolites, of which the 7,8-diol-9,10-epoxides have been implicated as causing potentially tumourigenic lesions. Larvae were exposed to waterborne benzo[a]pyrene (0.01, 0.05 and 0.1 mg/l) for 24 h at stages 38, 45 and 50 of development (24 h, 5 days and 2 weeks post-hatching, respectively) and allowed to recover for up to 6 days. A wide range of adduct lesions were observed at stage 50, three of which were observed at all stages investigated. Adduct repair was biphasic, with an initial rapid repair over the first 24 h post exposure, followed by a much slower decline, resulting in persistence of adducts for at least 6 days post exposure. The individual lesions were repaired at different rates, with some being almost completely repaired after 6 days recovery, whereas one of the main adducts showed restricted repair at stage 50 and another no repair at all. Identification of some adducts has been achieved, by the inclusion of isomeric standards of (+)- or (-)-anti-benzo[a]pyrene diol epoxide reacted with deoxyguanosine and adenosine 3'-monophosphates prepared in vitro. The non-repairable lesion at stage 50 has been shown to be the (+)-trans-anti-benzo[a]pyrene diol epoxide-N2-guanine adduct. This adduct was observed at all stages, but was only maximally repaired at stages 38 and 45.