Abstract
The covalent binding of the activated forms of several aflatoxins to N-7 of guanine residues on purified DNA has been studied. The aflatoxins include aflatoxin B1 (AFB1) and two human metabolites, aflatoxicol and aflatoxin M1, along with aflatoxicol M1, a rabbit and trout metabolite. DNA binding studies using tritiated [3H]aflatoxins indicate that equimolar solutions of each aflatoxin upon activation with chloroperoxybenzoic acid readily react to produce covalently bound adducts. These reactions produce alkali-labile sites which can be identified using a simple variation of the Maxam-Gilbert sequencing procedure. Two DNA fragments were exposed to each aflatoxin, and the reaction intensities at 33 guanine residues were determined. As much as 10-fold variation in reaction intensities was observed for various guanyl sites. Data indicate that none of the aflatoxins had identical reaction profiles, although AFB1 and aflatoxicol M1 were similar, as were aflatoxicol and aflatoxin M1. Hence, the frequency with which the various aflatoxin epoxides might damage specific sites critical for tumor initiation in vivo would not be predictable from total covalent binding indices. The frequency of occurrence of modifications at particular sites for AFB1 was also compared with the empirical "rules" established for AFB1 by Misra et al. (Misra, R. P., Muench, K. F., and Humayun, M. Z. (1983) Biochemistry 22, 3351-3359). Identical sites within fragments were compared for each aflatoxin, and the data showed that the attacking frequency for some such sites varied significantly. These results indicate that binding intensity rules based on nearest neighbor nucleotides do not reliably predict guanyl-AFB1 binding frequencies.
Highlights
From the Departments of $Food Science and Technology and §Biochemistry and Biophysics, Oregon State University, Coruallis, Oregon97331
The frequency as a result of AFB1-AFM, interconversion,hasnotbeen with which the various aflatoxinepoxides might dam- reported from anyspecies
Theoretical treatmentssuggest that lipophilicity and the datashowed that the attacking frequency for some such sites varied significantly. These results indicate that binding intensity rules based on nearest neighbor nucleotides do not reliably predict guanylAFBl binding frequencies
Summary
From the Departments of $Food Science and Technology and §Biochemistry and Biophysics, Oregon State University, Coruallis, Oregon97331. There is no experimental age specific sites critical for tumor initiation in vivo information available as to the relative efficiencies of AFBl, would not be predictable from total covalent binding AFL, AFL-MI, andAFM, epoxides, once formed, to interact indices. The frequencyof occurrence of modifications covalently with DNA It is possible, for example, that AFB,, at particular sites for AFBl was compared with AFL,AFL-M,, and AFM, are all good substrates for cytothe empirical urules”established for AFBl by Misra et chrome P-450epoxidation but that theepoxides formed have al.(Misra, R. Theoretical treatmentssuggest that lipophilicity and the datashowed that the attacking frequency for some such sites varied significantly These results indicate that binding intensity rules based on nearest neighbor nucleotides do not reliably predict guanylAFBl binding frequencies. Steric interactiondifferences with the cytochrome P-450activating enzyme may contribute to the varying mutagenic activities observed (Loew and Poulsen, 1981; Garner et al, 1979).3Finally, the differing physicochemical characteristics which render AFB,, AFM,, AFL-M1, and AFL readily separable on HPLC couldsignificantly impact the genomic sequence with which their epoxides preferentially interact so
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