Effect of Microsomal Enzyme Inducers on the Urinary Excretion Pattern of Mutagenic Metabolites of the Carcinogen 2,4-Toluenediamine

2,4-Toluenediamine [(TDA) CAS: 95-80-7] was administered to rats pretreated with the microsomal enzyme inducers phenobarbital (PB), β-naphthoflavone (βNF), or 3-methyl-cholanthrene (MCA). The 24-hour urines of male F344 rats were examined for their mutagenic potency by means of the Salmonella assay, with the Aroclor® 1254-pretreated rat liver S-9 fraction as an activating system. No revertants were found with TDA or its urinary metabolites in the absence of the S-9 fraction. In the presence of S-9, the number of revertants increased as the concentration of TDA or its urinary metabolites increased. The urinary metabolites, generated after the microsomal enzyme inducers (PB, βNF, MCA), had increased mutagenic activity as compared with the controls (saline, corn oil). In the presence of β-glucuronidase (βG), increased numbers of TA98 revertants were noted in the urine of rats pretreated with PB, saline, or corn oil. Addition of sulfatase did not alter the number of TA98 revertants. Conversely, βG treatment of urine from rats pretreated with MCA or βNF led to a decrease in the number of TA98 revertants as compared to levels in urine without βG. Addition of known urinary metabolites of TDA, such as 4-acetylamino-2-aminobenzoic acid or 2,4-diacetyl-aminobenzoic acid, to βNF-pretreated rat urine had no inhibitory effect on the mutagenicity in the absence of βG. However, in the presence of βG, the inhibitory effect was similar to that noted with βNF-pretreated rat urine. Upon separation of urinary metabolites (βNF-pretreated rat urine) into free, conjugated, and water-soluble forms, the maximum number of TA98 revertants was associated with the free ethyl acetate-extractable fraction, which accounted for the total mutagenic activity associated with the original volume of urine. Conjugated metabolites showed much less mutagenic activity, and an inhibitory principle was associated with the water-soluble fraction.