Protection against aflatoxin B1-induced cytotoxicity by expression of the cloned aflatoxin B1-aldehyde reductases. rat AKR7A1 and human AKR7A3

The reduction of the aflatoxin B-1 (AFB(1)) dialdehyde metabolite to its corresponding mono and dialcohols, catalyzed by aflatoxin B-1 -aldehyde reductase (AFAR, rat AKR7A1, and human AKR7A3), is greatly increased in livers of rats treated with numerous chemoprotective agents. Recombinant human AKR7A3 has been shown to reduce the AFB(1)-dialdehyde at rates greater than those of the rat AKR7A1. The activity of AKR7A1 or AKR7A3 may detoxify the AFB(1)-dialdehyde, which reacts with proteins, and thereby inhibits AFB(1)-induced toxicity; however, direct experimental evidence of this hypothesis was lacking. Two human B lymphoblastoid cell lines, designated pMF6/1A2/AKR7A1 and pMF6/1A2, were genetically engineered to stably express AKR7A1 and/or cytochrome P4501A2 (1A2). The pMF6/1A2/AKR7A1 cells were refractory to the cytotoxic effects of 3 ng/mL AFB(1), in comparison to pM6/ 1A2 cells, which were more sensitive. Diminished protection occurred at higher concentrations of AFB(1) in pMF6/1A2/AKR7A1 cells, suggesting that additional factors were influencing cell survival. COS-7 cells were transfected with either vector control, rat AKR7A1, or human AKR7A3, and the cells were treated with AFB(1) -dialdehyde. There was a 6-fold increase in the dialdehyde LC50, from 66 mu M in vector-transfected cells to 400 mu M in AKR7A1-transfected cells, and an 8.5-fold increase from 35 mu M in vectortransfected cells to 300 mu M in AKR7A3-transfected cells. In both cases, this protective effect of the AFAR enzyme was accompanied by a marked decrease in protein adducts. Fractionation of the cellular protein showed that the mitochondria/nuclei and microsomal fractions contained the highest concentration of protein adducts. The levels of human AKR7A3 and AKR7A2 were measured in 12 human liver samples. The expression of AKR7A3 was detectable in all livers and lower than those of AKR7A2 in 11 of the 12 samples. Overall, these results provide the first direct evidence of a role for rat AKR7A1 and human AKR7A3 in protection against AFB(1)-induced cytotoxicity and protein adduct formation.