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N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide (NFTA) metabolism was examined in vitro using microsomes prepared from rat liver and renal cortex and from rabbit liver and renal cortex and outer and inner medulla. NFTA nitroreduction was observed with each tissue. Three mol of NADPH were used per mol of NFTA reduced. Substrate and inhibitor specificity suggested that the microsomal nitroreduction was due to NADPH:cytochrome c reductase. Metabolite(s) formed bound to protein, RNA, DNA, and synthetic polyribonucleotides. Maximum covalent binding was seen with polyguanylic acid. A guanosine-NFTA adduct was isolated. Binding was inhibited by sulfhydryl compounds and vitamin E. The [14C]NFTA:glutathione or [3H]glutathione:NFTA conjugates obtained from microsomal incubations showed identical chromatographic properties as the product obtained by the reaction of synthetic N-hydroxy-NFTA with [3H]glutathione. Structures of synthetic N-hydroxy-NFTA and the microsomal reduction product 1-[4-(2-acetylaminothiazolyl)]-3-cyano-1-propanone were established by mass spectrometry. The latter reduction product did not bind macromolecules. These results suggest that renal NADPH:cytochrome c reductase reduces NFTA to an N-hydroxy-NFTA intermediate that binds nucleophilic sites on macromolecules.

Understanding the role of prostaglandin H synthase (PHS) in the carcinogenic process and the metabolic steps involved in the activation of carcinogens will facilitate experiments using pharmacological agents to prevent carcinogenesis. This study assesses the relative amounts of PHS-catalyzed binding of the urinary tract carcinogens [2-14C]-N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) and [2-14C]-2-amino-4-(5-nitro-2-furyl) thiazole (ANFT). Binding to protein and nucleic acid was assessed using PHS prepared from ram seminal vesicle, dog bladder transitional epithelium and rabbit renal inner medulla. PHS-catalyzed binding of ANFT was significantly greater than FANFT in each tissue. Substrate and inhibitor experiments were consistent with the prostaglandin hydroperoxidase activity of PHS catalyzing the binding of FANFT and ANFT. Oxygen was required for metabolism with arachidonic acid but not with peroxide as cosubstrate. The amount of PHS-catalyzed ANFT binding to protein was at least 4-fold greater than FANFT. Whereas a significant amount of FANFT was bound to protein, no FANFT binding to DNA could be detected. By contrast, PHS catalyzed the binding of ANFT to both protein and DNA. A PHS-catalyzed metabolite of ANFT was tentatively identified as the 4-keto analog by mass spectral analysis. The lower rate of PHS-catalyzed metabolism of FANFT compared to ANFT and the lack of detectable FANFT binding to DNA suggest that the metabolic steps involved in the initiation of FANFT-induced bladder cancer include 1) deformylation of FANFT to ANFT, 2) PHS-catalyzed activation of ANFT and 3) binding of an activated ANFT metabolite(s) to DNA.

It has been demonstrated that N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA), when fed with the diet, causes transitional carcinomas in rats. An important step in the mechanism of NFTA-induced carcinogenesis is endogenous metabolic activation to an ultimate carcinogen. We have proposed that the enzyme complex prostaglandin H synthase (PHS) is involved in the activation of certain renal and urinary tract carcinogens. This proposal was assessed by examining the activation of the 5-nitrofuran renal carcinogen NFTA and its deacetylated analogue 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) by PHS. Ram seminal vesicular and rabbit renal inner medullary microsomes were used as a source of PHS. Both NFTA and ANFT were activated by PHS to bind microsomal protein. Both microsomal preparations activated ANFT to bind DNA. However, only ram seminal vesicular microsomes activated NFTA to bind DNA. The rate of ANFT binding to macromolecules was considerably greater than NFTA with both microsomal preparations. Although activated ANFT was shown to bind several different homopolynucleotides, a preference for binding polyguanylic acid was demonstrated. Glutathione inhibition of carcinogen binding to macromolecules was shown to be due to the formation of a thioether conjugate. Deacetylation of NFTA was demonstrated in both tissues with deacetylation significantly exceeding acetylation of ANFT to NFTA in the kidney. Thus, renal PHS activation of both NFTA and ANFT was demonstrated with the rate of ANFT activation being considerably greater than NFTA. The conversion of NFTA to ANFT by intact tissue suggests that ANFT may contribute to NFTA renal carcinogenesis.