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Aflatoxin Q1 8,9-oxide was synthesized and found to yield lower levels of N7-guanyl adducts than obtained from aflatoxin B1 8,9-oxide when mixed with calf thymus DNA or Salmonella typhimurium TA 98 cells. However, when S. typhimurium TA 98 was treated with the (analogous) epoxides of aflatoxin B1, aflatoxin G1, aflatoxin Q1, or sterigmatocystin, the ratios of revertants to N7-guanyl DNA adducts were similar. Aflatoxin Q1 and aflatoxin B1 8,9-oxide (trapped here as the glutathione conjugate) are the major oxidative products formed from aflatoxin B1 at all substrate concentrations in human liver microsomes, and cytochrome P-450 (P-450) 3A4 appears to be the dominant enzyme involved in both oxidations, as judged by studies involving correlation of activities in different liver samples, chemical inhibition, immunoinhibition, and reconstitution with purified hepatic and yeast recombinant P-450 3A4. Aflatoxin Q1 is not appreciably oxidized in human liver microsomes and is not very genotoxic. The postulated formation of both aflatoxin Q1 and aflatoxin 8,9-oxide from aflatoxin B1 can be rationalized by a model in which P-450 3A4 binds the substrate in either of two different configurations. This is further demonstrated by the dichotomous effect of 7,8-benzoflavone--this flavone stimulates 8,9-epoxidation while inhibiting the 3 alpha-hydroxylation reaction to form aflatoxin Q1. Thus, the 3 alpha-hydroxylation of aflatoxin B1 to aflatoxin Q1 is viewed as a potentially significant detoxication pathway.

The regio- and stereospecificity exhibited by reaction of aflatoxin B1 8,9-epoxide with DNA as well as the efficiency of reaction are remarkable and suggests that a specific orientation of bound epoxide facilitates formation of the transition state leading to guanine N7 adducts. We have compared aflatoxins B1 and B2 with aflatoxins G1 and G2 as to their binding with calf thymus DNA, d(ATGCAT)2, d(GCATGC)2, and plasmid pBR322. Aflatoxins B1 and B2 contain a cyclopentenone ring fused to the lactone ring of the coumarin. They have similar DNA association constants and intercalate with B-DNA, as demonstrated by NMR analysis of association with d(ATGCAT)2 and d(GCATGC)2, alteration of pBR322 electrophoretic mobility, and flow dichroism using linearly oriented calf thymus DNA. The less planar delta-lactone ring of aflatoxins G1 and G2 reduces DNA binding affinity by approximately 1 order of magnitude. Nevertheless, binding studies with d(ATGCAT)2 and d(GCATGC)2 suggest that aflatoxins G1 and G2 also bind B-DNA by intercalation. To establish the existence of a relationship between the association of these aflatoxins with DNA and adduct formation induced by aflatoxin epoxides, we compared the yield of guanine N7 adduct from aflatoxin B1 8,9-epoxide and from aflatoxin G1 9,10-epoxide at three concentrations of calf thymus DNA. As DNA concentration is decreased, two observations are made: (1) the number of adducts formed by either aflatoxin B1 8,9-epoxide or aflatoxin G1 9,10-epoxide is reduced with a concomitant increase in formation of the respective dihydrodiols, and (2) the ratio of adducts formed by aflatoxin G1 9,10-epoxide to those formed by an equivalent concentration of aflatoxin B1 8,9-epoxide decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Much evidence supports the view that the rate of conjugation of glutathione (GSH) with aflatoxin B1 (AFB1) exo-epoxide is an important factor in determining the species variation in risk to aflatoxins and that induction of GSH S-transferases can yield a significant protective effect. An assay has been developed in which the enzymatic formation of the conjugates of GSH and AFB1 exo-epoxide and the recently described AFB1 endo-epoxide is measured directly. 1H NMR spectra are reported for both the AFB1 exo- and endo-epoxide-GSH conjugates. Structural assignments were made by comparison with AFB1 exo- and endo-epoxide-ethanethiol conjugates, for which nuclear Overhauser effects were measured to establish relative configurations. The endo-epoxide was found to be a good substrate for GSH conjugate formation in rat liver cytosol while mouse liver cytosol conjugated the exo-epoxide almost exclusively. Human liver cytosol conjugated both epoxide isomers to much lower extents than did cytosols prepared from rats or mice. Purified rat GSH S-transferases catalyzed the formation of the AFB1 exo-epoxide-GSH conjugate in the order 1-1 approximately 4-4 approximately 3-3 greater than 2-2 greater than 4-6 (7-7 and 8-8 did not form the exo-epoxide-GSH conjugate at levels above the nonenzymatic rate). The only rat GSH S-transferases that conjugated the endo-epoxide were 4-4 and 4-6, with 4-4 being the more active.(ABSTRACT TRUNCATED AT 250 WORDS)

The genotoxicity of the dihydrofurans aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), and sterigmatocystin (STG) was examined in a bacterial system in which the induction of SOS repair is monitored with the umuC gene linked to a lacZ reporter gene in plasmid pSK1002. Human liver microsomal cytochrome P-450NF oxidized the dihydrofurans (in the presence of calf thymus DNA) to give guanyl-N7 adducts in the order AFB1 greater than STG greater than AFG1. The order of the umu response seen was STG greater than AFB1 greater than AFG1, when either the dihydrofurans were activated enzymatically or the synthetic epoxides of the dihydrofurans were added directly to the bacteria. Thus, the umu response per molecule of guanyl-N7 DNA adduct follows the order STG greater than AFB1 greater than AFG1. A similar pattern has been reported in the literature for Salmonella typhimurium base substitution dependent his reversions, but the pattern AFB1 greater than STG greater than AFG1 has been found for bacterial frame-shift-dependent mutagenesis and hepatocarcinogenesis. The guanyl-N7 adduct derived from AFG1 has considerably less of all of these biological activities per molecule. Neither guanine imidazole ring opening nor apurinic site formation appears to be a factor involved in the differential biological responses seen with the three guanyl-N7 adducts. These findings indicate that these structurally related guanyl-N7 DNA adducts have intrinsic differences which give rise to divergent biological responses.