Mu-class GSTs are responsible for aflatoxin B(1)-8, 9-epoxide-conjugating activity in the nonhuman primate macaca fascicularis liver.
Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-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 (approximately 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.
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National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Dr., HFT-100, Jefferson, Arkansas 72079. Roosevelt Way NE, 100, Seattle, Washington 981.
Bammler
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Guo
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Kelly
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Eaton
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D016604:Aflatoxin B1 / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002273:Carcinogens / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004187:Disaccharides / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008252:Macaca fascicularis; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002273:Carcinogens; D018076:DNA, Complementary; D004187:Disaccharides; D012333:RNA, Messenger; C059555:O-(glucuronic acid 2-sulfate)-(1--3)-O-(2,5)-andydrotalitol 6-sulfate; C006426:aflatoxin B1-2,3-oxide; D002809:Chondroitin Sulfates; D016604:Aflatoxin B1