Characterization of neutral metabolites of benzo[a]pyrene in urine from germfree rats.

Abstract

[14C]Benzo[a]pyrene (BP) was administered to male germfree rats. Urinary metabolites, constituting 9% of the administered radioactivity, were fractionated by lipophilic ion exchange chromatography. More than 80% of the urinary metabolites were conjugated, while neutral metabolites constituted 13-18%. The latter group was characterized by reversed-phase HPLC, ultraviolet spectrometry (UV) and gas chromatography/mass spectrometry (GC/MS). Relative quantities of BP metabolites were estimated from the distribution of radioactivity upon HPLC fractionation. Some coeluted peaks were further quantitated from the total ion current chromatograms obtained in the GC/MS analysis. Two 7,8,9,10-tetrols which might be produced from the ultimate carcinogen r-7-t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-BP (anti-7,8-diol-9,10-epoxide) were detected at trace levels and indicated to be the r-7-t-8,9-c-10-(7,10/8,9-) and r-7-t-8,9,10-(7/8,9,10-) isomers. A trans-11,12-dihydrodiol was characterized as a major metabolite, while a trans-7,8-dihydrodiol was present at trace levels. Three additional quantitatively important metabolites were identified as isomeric trihydroxy-BPs. Two metabolites coeluting with BP quinones on HPLC were detected with relatively high abundance and tentatively identified as carboxylic methyl ester derivatives of BP quinones. Three quinones were detected with 1,6-, 3,6- and 6,12-substitutions. The 6,12- and 11,12-dihydroxy-BPs were also found at trace levels. A group of quinone-like metabolites were tentatively identified as trioxo-BPs. No monohydroxy-BP was detected in the neutral fraction of the urine extract. The time course of excretion was also studied and found to differ between individual metabolites.