Biliary excretion and microfloral transformation of major conjugated metabolites of 2,4-dinitrotoluene and 2,6-dinitrotoluene in the male Wistar rat

1. Major biliary conjugates of the male Wistar rat dosed orally with 2,4-dinitrotoluene (2,4-DNT) or 2,6-dinitrotoluene (2,6-DNT) were examined by hplc using potassium 2,4-dinitrobenzyl glucuronide (potassium 2,4-DNB-G), potassium 2,6-dinitrobenzyl glucuronide (potassium 2,6-DNB-G), pyridinium 2,4-dinitrobenzyl sulphate (pyridinium 2,4-DNB-S) and pyridinium 2,6- dinitrobenzyl sulphate (pyridinium 2,6-DNB-S) as authentic compounds. Other metabolites were also examined by hplc. In addition, metabolites formed by incubation of potassium 2,4-DNB-G and potassium 2,6-DNB-G with rat intestinal microflora under nitrogen were examined by hplc. 2. Conjugates detected directly from bile following administration of 2,4-DNT and 2,6-DNT were 2,4- DNB-G and 2,6-DNB-G, which accounted for 35.0 and 51.5% of the administered dose respectively. No peaks corresponding to pyridinium 2,4-DNB-S and pyridinium 2,6-DNB-S were detected in bile samples. 3. 2-Amino-4- nitrotoluene, 4-amino-2-nitrotoluene, 2,4-diaminotoluene and 4-acetylamino- 2-nitrobenzoic acid (0.02-0.12% of the dose excreted in 24 h), in addition to the known metabolites 2,4-dinitrobenzyl alcohol (2,4-DNB), 2,4- dinitrobenzaldehyde and 2,4-dinitrobenzoic acid (0.09-0.14%), were detected in ether extracts of bile of rat given 2,4-DNT. 2,6-Dinitrobenzyl alcohol (2,6-DNB), 2-amino-6-nitrotoluene and 2,6-dinitrobenzaldehyde (0.02-0.03%), which are known metabolites, were detected in ether extracts of bile from rat given 2,6-DNT. 4. Potassium 2,4-DNB-G was transformed by the anaerobic incubation of rat intestinal microflora into 2,4-DNB, 4-amino-2-nitrobenzyl alcohol and 2-amino-4-nitrobenzyl alcohol. Potassium 2,6-DNB-G was transformed into 2,6-DNB and 2-amino-6-nitrobenzyl alcohol by the anaerobic incubation. Time-course studies showed that 2,4-DNB, 4-amino-2-nitrobenzyl alcohol, 2-amino-4-nitrobenzyl alcohol and 2,6-DNB, 2-amino-6-nitrobenzyl alcohol peaked at 30, 75, 120 and 10, 50 min respectively. 5. These results, together with previous findings, show that 2,4-dinitrobenzaldehyde and 2,6- dinitrobenzaldehyde, which are potent mutagens, are formed either by the hepatic metabolism of 2,4-DNB and 2,6-DNB formed by the intestinal metabolism of 2,4-DNB-G and 2,6-DNB-G excreted in bile or by the direct hepatic metabolism of 2,4-DNT and 2,6-DNT.