Student Research: Changhong Wang

PhD, , 1999
Faculty Advisor: David L. Eaton

Identification of Hepatic Glutathione S-transferase(s) Involved in Aflatoxin B1-8,9-Epoxide Conjugating Activity in the Non-human Primate Macaca Fascicularis


Abstract

Large species differences exist in sensitivity to aflatoxin B1 (AFB1)-induced liver cancer. Mice are resistant to AFB1-induced liver cancer because they express an alpha-calss GST (mGSTA3-3) that has high activity toward the reacrtive intermediate aflatoxin B1-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO activity and are sensitive to AFB1-induced hepatocarcinogenesis, although an inducible, high AFBO-activity alpha-class GST enzyme (rGSTA5-5) can confer resistance in rats. In contrast to rodents, constitutively expressed human hepatic aplha-class GSTs have little or no AFBO detoxifying activity. Recently, we found that the non-human primate Macaca fascicularis (Mf), has significant constitutive hepatic GST activity toward AFBO. To determine which specific form of GST(s) is (are) responsible for this activity: (1) a cDNA library from a male Macaca fascicularis liver was constructed and screened using the human alpha-class GSTA1 cDNA as a probe; (2) hepatic Mf GSTs were purified and characterized; (3) Mf mu-class GST cDNAs were cloned by RT-PCR. cDNA cloning from a Mf hepatic cDNA library identified three different alpha-class GSTs, but when expressed, none had measurable AFBO activity even though they were active toward other substrates. Purification of GST proteins by glutathione agarose )GSHA) affinity chromatography indentified a mu-class GST, GSHA-GST, which had relatively high AFBO conjugating activity from Mf liver. Western blotting and enzymatic activity analyses of these proteins revealed that the GSTs with AFBO activity are mu-class GSTs. Further cDNA cloning isolated two distinct mu-class Mf GSTs: mfGSTM1 and mfGSTM2. Sequence analysis revealed that mfGSTM1 shares 97% homology with hGSTM4 and mfGSTM2 shares 96% homology with hGSTM2. The recombinant protein mfGSTM1-1 had no AFBO activity whereas mfGSTM2-2 have an AFBO activity of 333 pmol/min/mg. The cDNA-expressed mfGSTM2-2 had a slightly different CNBr digestion pattern and different AFBO-exo vs. endo activity from the purified Mf mu-class GST protein, suggesting that the cloned GSMT2 was not identical to the liver purified mu-class GST. Northern blot results demonstrated that mfGSTM2 is expressed in liver but not in lung or kidney. These results indicate that, unlike rodents, mu-class GSTs are responsible for the hepatic cytosolic GST activity toward AFBO in Macaca fascicularis.