Formation and persistence of sterigmatocystin--DNA adducts in rat liver determined via 32P-postlabeling analysis.
A 32P-postlabeling method has been employed to detect the in vitro and in vivo modification of DNA by the mycotoxin sterigmatocystin (ST). ST-modified DNA was initially incubated under buffered alkaline conditions to convert unstable ST-N7-guanine moieties to stable, putative ST-formamidopyrimidine derivatives. DNA was subsequently digested with micrococcal nuclease and spleen phosphodiesterase, and the resulting ST-modified nucleotides, purified by reverse-phase thin-layer chromatography (TLC), were labeled at the 5' position via incubation with [gamma-32P]ATP and T4 polynucleotide kinase. 32P-labeled ST-nucleotides were separated by reverse-phase and anion-exchange TLC. Cerenkov quantitation of excised TLC fractions indicated that ST-DNA moieties could be detected with a sensitivity of 1 ST adduct in 3-5 X 10(7) nucleotides. Initial enzymatic digestion of ST-modified DNA was found to yield ST-modified di- and trinucleotides which, upon 32P-labeling followed by incubation with nuclease P1, liberated unmodified 5'-terminal nucleotides suggesting that ST-formamidopyrimidine-modified DNA was a poor substrate for micrococcal nuclease and spleen phosphodiesterase. Dose-dependent ST-DNA adduct formation was detected in the liver of male Fischer 344 rats over a 27-fold range of ST administered (0.33-9 mg/kg). In addition, ST-DNA adducts, formed in rats given a 9 mg/kg dose, were found to persist up to 105 days after treatment at a level of 0.5% of the 2-h value. Loss of these adducts from liver DNA was observed to exhibit a triphasic profile: rapid loss during the first 24 h (t 1/2 = 12 h) followed by a slower decline from 1 to 14 days post dosing (t 1/2 = 7 days) and an extremely slow decline from days 14 to 105 post treatment (t 1/2 = 109 days). This experimental approach to the study of mycotoxin-DNA interactions permits the quantitative description of DNA modification in ST-treated animals. Further refinement of this approach may be useful in defining the precise relationship between ST exposure and tumorigenesis in ST-exposed human populations.
D000818:Animals; D001345:Autoradiography / Q000379:methods; D002273:Carcinogens / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D008099:Liver / Q000032:analysis*; D008297:Male; D009841:Oligonucleotides / Q000032:analysis; D010761:Phosphorus Radioisotopes; D051381:Rats; D011916:Rats, Inbred F344; D013241:Sterigmatocystin / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism*
D002273:Carcinogens; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D014966:Xanthenes; D013241:Sterigmatocystin; D004247:DNA