IN VITRO METABOLISM OF 6-THIOGUANINE IN RAT SUBCELLULAR FRACTIONS

The metabolism of the antitumor agent 6-thioguanine (TG, NSC-752) by rat liver was studied in vitro. Livers from adult male Sprague-Dawley rats were homogenized and the "liver homogenate" was subjected to differential centrifugation to obtain the "10,000 x g pellet", the "post-mitochondrial fraction", the "cytosol fraction", and the "microsomes". The homogenity of each fraction was estimated by appropriate marker enzyme assays. To delineate the in vitro metabolism of TG by rat liver, 0.2 mM of {8-('14)C}TG was incubated with different subcellular fractions in KCl-Tris-MgCl(,2) buffer, pH 7.4 at 37(DEGREES). The metabolites formed were identified by chromatography, UV spectrometry, as well as mass spectrometry. After a 1 hr incubation, TG was metabolized by the liver homogenate, the 10,000 x g pellet and the post-mitochondrial fraction mainly to 6-thioguanosine (TGR), accompanied by varying lesser amounts of 6-thiouric acid (TUA), allantoin, guanine-6-sulfinic acid (G-SO(,2)H) and an unknown product. In comparison, the cytosal fraction converted TG almost entirely to TGR and TUA in equal amounts. The formation of TGR from TG was limited by the endogenous supply of ribose-1-phosphate. With the microsomal fraction, however, TG was metabolized significantly to G-SO(,2)H and the unknown, accompanied with some TGR. After a 5 hr incubation the metabolism of TG was changed to favor the catabolic route, yielding mostly TUA in the post-mitochondrial and cytosol fractions; but mainly allantoin in the liver homogenate fraction. The kinetic studies of TG metabolism by the subcellar fractions indicated that the formation of TGR served as a depot form of TG. The level of TGR decreased when the catabolism of TG became prominent. The oxidation of TG to GSO(,2)H mediated by the hepatic microsomes represented a new catabolic pathway of TG. This GSO(,2)H, under acidic conditions, readily decomposes to guanine and inorganic sulfate. In the presence of reduced glutathione in Tris buffer, pH 7.8 at 25(DEGREES), GSO(,2)H is adducted to glutathione chemically to form S-(2-amino-purin-6-yl) glutathione and conceivably, inorganic sulfate. Therefore, the formation of GSO(,2)H from TG might have implication in the desulfuration mechanism of TG. On the other hand, the unknown formed from TG by the action of the microsomal enzymes appeared to be a TG conjugate. However, it is neither a glutathione, a glucuronide, nor a ribose conjugate. Additionally, the deamination of TG by guanine deaminase (E.C.3.5.4.3) isolated from rat liver was also investigated. TG is a poorer substrate (Km = 4.8 x 10('-3)M) for guanine deaminase than that of guanine (Km = 4.7 x 10('-6)M) at pH 7.25, optimal pH for TG as a substrate. TG is also a competitive inhibitor of guanine for guanine deaminase, with a ki of 2.2 x 10('-4)M. ^