The reaction of trans-4-hydroxy-2-nonenal (4-HNE) with primary amines was investigated to elucidate chemistry that may clarify the nature of its physiological covalent binding with protein-based primary amino groups. Such binding of 4-HNE, generated endogenously from lipid peroxidation, appears to be a pathophysiologic factor in the modification of low-density lipoprotein and perhaps other instances. We now show that 4-HNE reacts with primary amines in aqueous acetonitrile at pH 7.8 to afford after workup, in 14-23% yield, the corresponding pyrroles, which were characterized by independent synthesis from 4-oxononanal. Additional, mostly unstable adducts are also formed, some of which eventually "age" to the pyrrole. Hydride reduction after initial adduct formation permits the isolation of more stable materials, one of which has been identified as the reduced amine Michael addition product. Pyrrole formation may constitute a physiologically important reaction of 4-hydroxyalkenals.

Aflatoxin B1, a potently carcinogenic fungal metabolite, is converted to the biologically active form by chemical oxidation using dimethyldioxirane and enzymatically by cytochrome P450 mixed-function oxidases. Both processes give rise to mixtures of the exo-and endo-8,9-epoxides. Methanolysis studies reveal exclusive trans opening of both epoxides under neutral conditions in CHjOH and CH30H/H20 mixtures; an S N~ mechanism is postulated. Under acidic conditions, the ex0 isomer gives mixtures of trans and cis solvolysis products, suggesting that the reaction is, at least in part, S N~; the endo isomer gives only the trans product. The ex0 isomer reacts with DNA by attack of the nitrogen atom at the 7 position of guanine on C8 of the epoxide to give the trans adduct; the endo epoxide fails to form an adduct at this or any other site in DNA. The exo isomer is strongly mutagenic in a base-pair reversion assay employing Salmonella typhimurium; the endo isomer is essentially nonmutagenic. Aflatoxin Bl and its derivatives intercalate in DNA. These results are consistent with a mechanism in which intercalation of the exo epoxide optimally orients the epoxide for an sN2 reaction with guanine but intercalation of the endo isomer places the epoxide in an orientation which precludes reaction. Thus, while the exo epoxide is a potent mutagen, the endo epoxide fails to react with DNA.

Aflatoxin B 1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [

The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFBI-N7-Gua of 4%. The predominant mutation was G --T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G -> T mutations of AFB,-N7-Gua, unlike those of the AFBI-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin. It is concluded that the AFB,-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face ofthe guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.The fungal metabolite aflatoxin B1 (AFB1) contaminates the food supply in eastern Asia and sub-Saharan Africa, where it is associated with an increased incidence of hepatocellular carcinoma (HCC) (1). AFB1 requires metabolic conversion to its exo-8,9-epoxide (2, 3) in order to cause damage to DNA (4), the event that presumably initiates the genetic changes resulting in HCC (5). The AFB1 epoxide reacts with guanine to form a population of adducts (6), the principal of which both in vitro (3, 7-9) and in vivo (8,(10)(11)(12)) is 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFBl-N7-Gua). The positively charged imidazole ring of this adduct promotes depurination, resulting in apurinic (AP) site formation. Alternatively, under slightly basic conditions the imidazole ring of AFB1-N7-Gua opens to form the chemically and biologically stable AFB1 formamidopyrimidine (AFBI-FAPY) (1). The initial AFB,-N7-Gua adduct, the AFB1-FAPY, and the AP site, individually or collectively, represent the likely chemical precursors to the genetic effects of AFB1.The nature of mutations induced by electrophilic derivatives of AFB1 has been studied in several forward mutation assays.Upon examining AFB1 mutagenesis in the endogenous Escherichia coli lacI gene, Foster et al. (13) (20,21). The same mutations are observed in human hepatocytes grown in culture (22).The genetic studies cited above indicate that several mutations occur in DNA globally modified by AFB1, but the predominant mutation induced or selected in vivo appears to be the GC -> TA transversion. At least three DNA lesio...

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