cDNA cloning, expression and activity of a second human aflatoxin B-1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3

The aflatoxin B-1 (AFB(1)) aldehyde metabolite of AFB(1) may contribute to the cytotoxicity of this hepatocarcinogen via protein adduction, Aflatoxin B-1 aldehyde reductases, specifically the NADPH-dependent aldo-keto reducta ses of rat (AKR7A1) and human (AKR7A2), are known to metabolize the AFB(1) dihydrodiol by forming AFB1 dialcohol, Using a rat AKR7A1 cDNA, we isolated and characterized a distinct aldo-keto reductase (AKR7A3) from an adult hu man liver cDNA library. The deduced amino acid sequence of AKR7A3 shares 80 and 88% identity with rat AKR7A1 and human AKR7A2, respectively. Recombina nt rat AKR7A1 and human AKR7A3 were expressed and purified from Escherichia colt as hexa-histidine tagged fusion proteins. These proteins catalyzed th e reduction of several model carbonyl-containing substrates. The NADPH-depe ndent formation of AFB(1) dialcohol by recombinant human AKR7A3 was confirm ed by liquid chromatography coupled to electrospray ionization mass spectro metry, Rabbit polyclonal antibodies produced using recombinant rat AKR7A1 p rotein were shown to detect nanogram amounts of rat and human AKR7A protein . The amount of AKR7A-related protein in hepatic cytosols of 1,2-dithiole-3 -thione-treated rats was 18-fold greater than in cytosols from untreated an imals, These antibodies detected AKR7A-related protein in normal human live r samples ranging from 0.3 to 0.8 mu g/mg cytosolic protein. Northern blot analysis showed varying levels of expression of AKR7A RNA in human liver an d in several extrahepatic tissues, with relatively high levels in the stoma ch, pancreas, kidney and liver. Based on the kinetic parameters determined using recombinant human AKR7A3 and AFB(1) dihydrodiol at pH 7.4, the cataly tic efficiency of this reaction (k(2)/K, per M/s) equals or exceeds those r eported for other enzymes, for example cytochrome P450s and glutathione S-t ransferases, known to metabolize AFB(1) in vivo. These findings indicate th at, depending on the extent of AFB(1) dihydrodiol formation, AKR7A may cont ribute to the protection against AFB(1)-induced hepatotoxicity.