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Use of the Isolated Perfused Rat Liver to Study Carcinogen-DNA Adduct Formation from Aflatoxin B₁ and Sterigmatocystin¹

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Isolated rat livers were perfused under controlled conditions with two hepatocarcinogens, aflatoxin B₁ (AFB₁) and sterigmatocystin. Both were metabolically activated to molecular forms that bound to DNA yielding carcinogen-DNA component adducts that were released by acidic hydrolysis and analyzed by high-pressure liquid chromatography. Binding to DNA was dose dependent at 1 and 3 mg/liver doses for sterigmatocystin and over a range of 0.05 to 3 mg/liver for AFB₁. The major DNA adduct detected for sterigmatocystin was 1,2-dihydro-2-(N⁷-guanyl)-1-hydroxysterigmatocystin, which presumably was formed by the reaction of the sterigmatocystin 1,2-oxide with DNA. Qualitatively and quantitatively, the binding of AFB₁ to DNA was essentially the same in perfused liver as in the livers of rats given the carcinogen i.p. As in in vivo models described previously, phenobarbital pretreatment of rats serving as donors of livers used in perfusion resulted in reduction in the level of hepatic AFB₁-DNA adducts to one-third of the control value. The major DNA adduct formed was 2,3-dihydro-2-(N⁷-guanyl)-3-hydroxyaflatoxin B₁; this adduct was derived from the aflatoxin B₁ 2,3-oxide and accounted for approximately 80% of the carcinogen-derived radioactivity incorporated into DNA. The putative imidazole ring-opened derivative of this adduct was also formed and represented approximately 6.5% of the binding. One of the important characteristics of the isolated liver perfusion model was the fact that minor aflatoxin adducts were observed that do not form in significant quantities in vitro. The technique was demonstrated to have significant potential as a tool for isolation of these adducts in view of the extraordinarily high levels of DNA binding observed (>1 AFB₁ adduct/10³ DNA bases) at carcinogen doses that would be severely toxic if administered to animals by conventional protocols. One of the minor AFB₁ adducts was chromatographically identical to an adduct formed by reaction of an epoxide of aflatoxin P₁, an AFB₁ metabolite, with guanyl residues in DNA. A second minor adduct was formed by the reaction of an activated metabolite of aflatoxin M₁ with the guanine base.

¹ This work was supported by Grants T32 CA 09112 and 5 P01 ES 00597-06 from the NIH.

² To whom requests for reprints should be addressed.

Received 2/ 1/80. Accepted 7/18/80.