Aflatoxin B1 (AFB1) is a fungal toxin that has been implicated as a causative agent in human hepatic and extrahepatic carcinogenesis. In this review, the mechanisms involved in AFB1 toxicity are delineated, in order to describe the features that make a specific cell, tissue, or species susceptible to the mycotoxin. Important considerations include: (i) different mechanisms for bioactivation of AFB1 to its ultimate carcinogenic epoxide metabolite; (ii) the balance between bioactivation to and detoxification of the epoxide; (iii) the interaction of AFB1 epoxide with DNA and the mutational events leading to neoplastic transformation; (iv) the role of cytotoxicity in AFB1 carcinogenesis; (v) the significance of nonepoxide metabolites in toxicity; and (vi) the contribution of mycotoxin-unrelated disease processes. Although considerable controversy remains about the importance of specific events, a great deal has been learned about biochemical and molecular actions of AFB1.

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In order to study the mechanism of cancer production by aflatoxin B1 (AFB1) in extrahepatic tissues which have relatively low cytochrome P450 monooxygenase (P450) activity, we have examined prostaglandin H synthase (PHS)-mediated AFB1 activation [( 3H]AFB1-DNA binding). [3H]AFB1 was activated by both purified PHS and microsomal PHS from guinea-pig kidney and liver, as well as by P450 in lung, kidney and liver microsomes, though P450-mediated [3H]AFB1-DNA binding in lung and liver was much higher than that catalyzed by PHS. Arachidonic acid (AA)-dependent [3H]AFB1-DNA binding could be inhibited by the PHS inhibitor indomethacin (0.1 mM), but was enhanced by the P450 inhibitor SKF-525A (3 mM), confirming that the reaction was independent of P450. Pulmonary PHS-mediated [3H]AFB1--DNA binding was less than 0.1 pmol [3H]AFB1/mg protein/min. HPLC analysis showed only minimal formation of [3H]AFM1 and [3H]AFQ1 by PHS, confirming that the low rate of PHS-dependent [3H]AFB1-DNA adduct formation was not due to conversion of AFB1 to other metabolites by PHS. The omission of AA did not diminish [3H]AFB1-DNA binding. In AA-free incubates, indomethacin inhibited, and SKF-525A enhanced, [3H]AFB1-DNA binding to a similar degree as in complete incubates, indicating that DNA binding in AA-free incubates was catalyzed by PHS. This reaction was also inhibited by 4-bromophenacyl bromide, a phospholipase A2 inhibitor, by 92%. These data are consistent with previous reports indicating the ability of AFB1 to stimulate the release of endogenous AA from membranes, presumably by stimulating phospholipase A2 activity, which may lead to enhanced bioactivation of AFB1 by PHS in vivo.

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Aflatoxin B1 (AFB1) is a potent hepatotoxic and hepatocarcinogenic mycotoxin that requires bioactivation to AFB1-2,3-oxide for activity. In addition to epoxidation, microsomal monooxygenases biotransform AFB1 to the less toxic metabolites, aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1). The lung is at risk from AFB1 both via inhalation and via the circulation. In the present study, we have characterized rabbit lung and liver microsomal AFB1-DNA binding (an index of AFB1-2,3-oxide formation), AFM1 formation and AFQ1 formation. Vmax values for AFB1-DNA binding were not different between lung and liver when expressed per mg microsomal protein (1.06 +/- 0.13 and 2.12 +/- 1.30 nmol/mg/h for lung and liver respectively), but lung values were greater than liver when expressed per nmol cytochrome P450 (3.64 +/- 0.31 and 1.29 +/- 0.70 nmol/nmol P450/h for lung and liver respectively). Km values for this reaction were not different between lung and liver. Vmax values for AFM1 formation in liver microsomes were greater than in lung when expressed per mg protein, but not when expressed per nmol P450. No differences were detected for the Km for AFM1 formation between lung and liver microsomes. For AFQ1 formation, no differences were detected between Vmax values of lung and liver, regardless of whether results were expressed per mg protein or per nmol P450, while the Km for AFQ1 formation was lower in liver. SKF-525A inhibited these reactions by 63-74% in lung microsomes and 90-96% in liver microsomes. These results indicate that the lung is capable of activating AFB1, and that rabbit lung microsomes contain high activity for this reaction. Furthermore, little AFM1 and AFQ1 are formed in lung microsomes, leading to minimal shunting of AFB1 from the activation pathway.

The abilities of different rabbit lung cell types to bioactivate aflatoxin B1 (AFB1) to a DNA-binding and mutagenic metabolite have been examined. Microsomes were prepared from centrifugal elutriation-enriched preparations of isolated rabbit lung cell types. The activation of [3H]AFB1 (5.0 or 200 microM), measured indirectly as covalent binding to calf thymus DNA, was concentrated in microsomes from the non-ciliated bronchiolar epithelial (Clara) cell-rich fractions (13-22 times the activity of whole lung microsomes). Microsomes from type II cell-rich fractions had minimal activity. Significant correlations were detected between the rates of microsomal DNA binding and the percentages of Clara cells in the fractions. Prior treatment of rabbits with the cytochrome P450 class 1A inducer beta-naphthoflavone had no significant effect on the microsomal activation of AFB1. In other experiments, intact, enriched isolated rabbit lung cells were incubated with AFB1 (0-1.5 microM) in a modification of the Ames mutagenicity assay, using Salmonella typhimurium strain TA100. The ability to activate AFB1 to mutagenic metabolite(s) in this system was localized in Clara cell-rich fractions, with no significant activity being detected in other fractions. The results of these studies indicate that the biotransformation of AFB1 to DNA-binding and mutagenic metabolite(s) in rabbit lung is heterogeneous, and that the Clara cell is specifically implicated in this ability.

Amiodarone is a potent and efficacious antiarrhythmic agent, yet associated with its use are life-threatening pulmonary fibrosis and hepatotoxicity. We have investigated the susceptibility of the male Sprague-Dawley rat to pulmonary and hepatic toxicity after repeated exposure to amiodarone and the effects of such exposure on hepatic and extrahepatic drug metabolizing enzymes. Animals received amiodarone (200 mg.kg-1.day-1 i.p., 5 days/week) for 1 week followed by 150 mg.kg-1.day-1 (5 days/week) for 3 additional weeks. No signs of pulmonary fibrosis or hepatotoxicity were observed, based on histological examination, lung hydroxyproline content, and plasma alanine aminotransferase activity. Analysis of tissues revealed extensive accumulation of amiodarone and desethylamiodarone in lung and liver, but concentrations were significantly lower in animals treated for 4 weeks than for 1 week. In a separate experiment, rats received amiodarone 150 mg.kg-1.day-1 i.p. (5 days/week) for 1 or 4 weeks. No differences in tissue concentrations of amiodarone and desethylamiodarone were detected between animals treated for 1 or 4 weeks. This regimen did not affect hepatic or extrahepatic monooxygenase activities. These results indicate that, in the male Sprague-Dawley rat, there is no observable pulmonary or hepatic toxicity following short-term amiodarone exposure, and there is enhanced elimination of amiodarone and desethylamiodarone when the daily dose of amiodarone is decreased after 1 week from 200 to 150 mg/kg.