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When Escherichia coli were incubated with the strong mutagen 2[14C]2-amino-4-(5-nitro-2 furyl)-thiazole (ANFT) radioactivity became tightly and presumably covalently bound to DNA. Hydrolysis of the DNA with nucleases yielded low molecular weight radioactive material. The bound radioactivity was associated with at least two functionally and chemically distinct adducts. One of these was rapidly removed in uvr+ E. coli while the other was more persistent. Analysis of enzymatic hydrolysates on a Dowex AG 50W-X4 column showed that the 'excisable adducts' were chromatographically different from most of the persistent ones. ANFT caused daughter-strand gaps when the DNA of treated cells was replicated, provided this DNA contained excisable adducts. In situations where removal of these adducts was complete no gaps were found in newly synthesized DNA. 3-[14C]2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) (another strongly mutagenic nitrofuran) became bound to the DNA of E. coli WP2 uvrA to a slightly greater extent than did [14C]ANFT. In contrast, [14C]nitrofurazone (the semicarbazide of 5-nitro-2-furaldehyde) a much weaker mutagen, gave considerably less binding. With AF2 and ANFT there was roughly the same relation between the amount of adduct formed and the subsequent yield of daughter strand gaps when the DNA replicated while with nitrofurazone the yield of gaps per adduct was somewhat lower. Incubation in vitro of [14C]ANFT with DNA in the presence of an E. coli nitrofuran reductase preparation also resulted in the binding of 14C to DNA.

Reductive metabolism of the aromatic nitro group of 5-nitrofurans is thought to be an important step in the mechanism of their toxicity. Microsomal nitroreductase activity with p-nitrobenzoic acid and N-[4-(5-nitro-2-furyl)-2-thiazolyl[formamide (FANFT) as substrates was assessed in the renal cortex, outer medulla, inner medulla, bladder transitional epithelial and non-epithelial bladder tissue. Cortex and transitional epithelial tissue contained the most p-nitrobenzoic acid reductase activity. However, FANFT reductase activity was similar in all areas tested except nonepithelial bladder tissue, which was 10% of the others. FANFT reduction was inhibited by oxygen, but not by carbon monoxide, allopurinol or aspirin and required NADPH. These results are consistent with NADPH-cytochrome c reductase catalyzed FANFT reduction. In medullary microsomes, the apparent Km and Vmax were 0.125 mM and 0.84 nmol/mg of protein per min, respectively. Transitional epithelial microsomes incorporated approximately 1 and 10% of the total [2-14C]FANFT metabolized into t-RNA and trichloroacetic acid-precipitable material, respectively. Two products of FANFT reduction were demonstrated by high-pressure liquid chromatography. One product was reversibly oxidized to FANFT and the other was tentatively identified by mass spectral analysis as an open chain nitrile. In view of the relatively low oxygen tension in the renal inner medulla and bladder mucosa, these results suggest that medullary and transitional epithelial nitro-reductases may be involved in the pathogenesis of 5-nitrofuran toxicity.

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.