Mu-Class GSTs are responsible for aflatoxin B1-8,9-epoxide-conjugating activity in the nonhuman primate Macaca fascicularis liver

Abstract

Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B1 (AFB1) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (~200,000 pmol/min/mg) activity toward aflatoxin B1-8,9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB1- induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha- class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (< 2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (~300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, Mf GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession AF200709) and mfaGSTM2 (GenBank accession AF200710), were generated by RT-PCR. CDNA- derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.