Enhanced macromolecular binding of N-[4-(5-nitro-2-furyl)-2-thiazolyl]- formamide in germfree versus conventional rats.

To investigate the binding of a nitrofuran to tissue macromolecules in vivo, the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)- 2-[35S]thiazolyl]formamide [[35S]FANFT; 94 mCi/mmol] was given p.o. to conventional [n = 4; 115 +/- 8 g (S.D.)] and germfree [n = 4; 105 + 5 g] female CD rats [1.23 mCi/rat]. After 18 hr, organs were removed, and macromolecules were then isolated from individual livers and kidneys and from pooled urinary bladders. A hydroxylapatite isolation procedure was followed (Beland et al., J. Chromatogr., 174: 177-186, 1979), and the nucleic acids obtained were further purified by digestion with appropriate nucleases and/or centrifugation (105,000 X g). The results are as follows and are given in pairs (conventional/germ-free) expressed as pmol FANFT bound per mg macromolecule. Protein binding levels were: liver, 165 +/- 40/307 +/- 31; kidney, 72 +/- 19/88 +/- 24; bladder, 272/322. RNA levels were: liver, 217 +/- 184/413 +/- 196; kidney, 219 +/- 60/617 +/- 196; bladder, 448/1373. DNA levels were: liver, 9.8 +/- 7.5/17.0 +/- 7.5; kidney, 0.69 +/- 0.38/4.5 +/- 1.0. The quantity of bladder DNA was insufficient for accurate measurement. Diethylaminoethyl cellulose chromatography of liver RNA from a germfree rat, either before or after RNase digestion, showed that the majority of the radioactivity was associated with a polynucleotide fraction that appeared to be RNase resistant and accounted for only a small portion of the total RNA but that also permitted the intercalation of ethidium bromide. The deformylated FANFT metabolite, 2-amino-4-(5-nitro-2-fury)thiazole, reacted with transfer RNA upon reduction with sodium dithionite in vitro to give adduct(s) that also appeared to be RNase resistant. Thus, these results show that the urinary bladder carcinogen FANFT or its metabolites react in vivo with protein and nucleic acid of both target and nontarget organs and that binding levels are elevated in germfree rats.