Reduction of aflatoxin B₁ dialdehyde by rat and human aldo-keto reductases

Oxidation of the mycotoxin aflatoxin (AF) B₁ yields the 8,9-epoxide, which nonenzymatically hydrolyzes rapidly to a dihydrodiol that in turn undergoes slow, base-catalyzed ring opening to a dialdehyde [Johnson, W. W., Harris, T. M., and Guengerich F. P. (1996) J. Am. Chem. Soc. 118, 8213-8220]. AFB₁ dialdehyde does not bind to DNA but can react with protein lysine groups. One enzyme induced by cancer chemopreventive agents is AFB₁ aldehyde reductase (AFAR), which catalyzes the NADPH-dependent reduction of the dialdehyde to a dialcohol. AFB₁ dialdehyde is known to convert nonenzymatically to AFB₁ dihydrodiol at neutral pH, and we reinvestigated the enzymatic reaction by preparing AFB₁ dialdehyde at pH 10 and then used this to initiate reactions (at neutral pH) with rat and human AFAR isozymes. Two monoalcohols were identified as products, and their identities were established by NaB²H₄ reduction, chemical cleavage, and mass spectrometry. The monoalcohol corresponding to reduction at C-8 formed first in reactions catalyzed by either the rat or the human AFAR. This C-8 monoalcohol was further reduced to AFB₁ dialcohol by AFAR. The other monoalcohol (C-6a) was formed but not reduced to the dialcohol rapidly. Steady-state kinetic parameters were estimated for the reduction of AFB₁ dialdehyde by rat and human AFAR to the monoalcohols. The apparent k_cat and K_m values were not adequate to rationalize the observed ΔA₃₄₀ spectral changes in a kinetic model. Simulation fitting was done and yielded parameters indicative of greater enzyme efficiency. A survey of 12 human liver cytosol samples showed a variation of 2.3-fold in AFAR activity. Rats treated with AFB₁ excreted the dialcohol and a monoalcohol in urine. The results of these studies are consistent with a role of (rat and human) AFAR in protection against AFB₁ toxicity.