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Characterization of Mutagenic Glucuronide Formation from Benzo(a)pyrene in the Nonrecirculating Perfused Rat Liver¹

Departments of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27514 [F. W. K., R. G. T.], and University of Maryland, Baltimore, Maryland 21201 [F. C. K.]

Excretion of mutagenic metabolites of benzo(a)pyrene into bile from livers of com oil- or 3-methylcholanthrene-treated Sprague-Dawley rats perfused with a nonrecirculating perfusion system was quantitated. Mutagenic benzo(a)pyrene metabolites were detected using Salmonella typhimurium (strain TA 98) grown in the presence of limiting amounts of histidine. Microsomes were not included in the bacterial assay since metabolic activation was carried out by the perfused liver. Mutagenic activity was detected only if ß-glucuronidase was added to the assay mixture or if bile was treated with acid to hydrolyze glucuronides prior to assay. When livers were perfused with 20 µM benzo(a)pyrene, stable, mutagenic glucuronides were exported from com oil-treated livers at maximal rates of 149 ± 24 (S.E.) revertants/g/hr and at rates of 225 ± 22 revertants/g/hr in livers from 3-methylcholan-threne-treated rats.

Chromatography of bile by high-performance liquid chromatography demonstrated that two peak areas contained phenolic glucuronides which were hydrolyzed by ß-glucuronidase. These two peaks, one which cochromatogaphed with authentic 3-benzo()pyrenyl-ß-D-glucuronide, accounted for all of the mutagenic activity in bile from livers perfused with benzo(a)pyrene. A good correlation (r = 0.86) between rates of mutagen production and rates of formation of phenolic glucuronides was observed under a variety of experimental conditions. The mutagenic activity observed with pure 3-benzo(a)pyrenyl-ß-D-glucuronide exposed to ß-glucuronidase was 4 revertants/nmol. When the rate of mutagen production was divided by the rate of production of 3-benzo(a)pyrenyl-ß-D-glucuronide by the perfused liver, a value of 4 revertants/nmol was also obtained. Therefore, it is concluded that mutagens exported in bile from livers perfused with benzo(a)pyrene can be accounted for predominantly by hydrolysis products of phenolic glucuronides.

¹ Supported in part by Grants AA-00033, CA-23080, CA-20807, and ES-02759.

² Recipient of a National Cancer Institute Postdoctoral Fellowship CA-07217.

³ To whom requests for reprints should be addressed.

Received 11/ 8/83. Accepted 7/30/84.