Morse HR et al. (1996), The identification and repair of DNA adducts in...

XB-ART-18822

Mutagenesis 1996 Jan 01;111:101-9.

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

Morse HR , Jones NJ , Peltonen K , Harvey RG , Waters R .

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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.

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Species referenced: Xenopus laevis

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	Fig. 1. Typical autoradiographs obtained from 32P-post-labelling. Calf thymus DNA treated in vivo with 0.5 mg/1 B[a]P in the presence of S9 mix (A). Xenopus laevis larvae DNA treated m vivo with B[a]P at stage 38 (B), stage 45 (C) and stage 50 (D) of development Arrows numbered 1-3 indicate the main three adducts induced over all stages of development investigated. Adduct 3, franj-{ + )-am/-B[a]PDE-N2 -guanine; adduct 4, cw-( + )-an;/-B[a]PDE-Af2-guanine; adduct 5, m-{-)-deoxyguanosine//ran5-<-)-deoxyadenosine.
	Fig. 2. Levels of total B[a]P-DNA adducts induced in larvae exposed to 0.01, 0.05 and O.I mg/l B[a]P for 24 h. Filled/front blocks, stage 38; hatched/middle blocks, stage 45; open/back blocks, stage 50.
	Fig. 3. Induction of individual B[a]P-DNA adducts at different stages. Individual adduct types were quantified by the excision of a single adduct spot from the TLC plate and calculating amol adduct/ug DNA after scintillation counting. The data represent induction of adducts 1-3 indicated in Figure I after 24 h exposure of larvae to 0.1 mg/1 B[a]P. Stage 38, filled/front blocks; stage 45, hatched/middle blocks; stage 50, open/back blocks.
	Fig. 4. Repair of total B[a]P-DNA adducts during a 6 day (144 h) recovery period following 24 h exposure to 0.01 (•), 0.05 (•) and O.I mg/l (A) B[a]P at stages 38 (A), 45 (B) and 50 (C) of development
	Fig 5. Autoradiographs illustrating differential repair of B[a]P-DNA adducts at stage 50 of development. Individual adducts were monitored over a 6 day recovery period after 24 h exposure to B[a]P. Plates indicate total B[o]P-DNA adduct induction in X.laevis larvae DNA after 24 h exposure to O.I mg/l B[a]P in vivo at stage 50 (A) and adducts persisting up to 6 days post-exposure (B).
	Fig. 6. Repair of the major individual B[a]P-DNA adducts over 144 h following a 24 h exposure to 0.1 mg/1 B[o]P at stage 50. Adduct 1 (•), adduct 2 (A) and adduct 3 (•) as indicated in Figure 1.
	Fig. 7. Autoradiographs obtained following 32P-post-labelling all possible B[a]PDE-DNA adducts Plates indicate the cis and trans products resulting from the reaction of racemic anli-B[a]PDE with deoxyguanosine 3'-monophosphate (A) and deoxyadenosine 3'-monophosphate (B).