Metabolism of the dyestuff intermediate 2,4-diaminoanisole in the rat

Abstract

1. 2,4-Diamino[ring-U-¹⁴C] administered intraperitoneally to rats is excreted chiefly via the urine (79 and 85% of the dose in 24 and 48h, respectively). The isotope in the faeces was 2·1 and 8·9% of the dose at 24 and 48 h.

2. The major metabolic pathway was acetylation of the amine group(s), resulting in 4-acetylamino-2-aminoanisole and 2,4-diacetylaminoanisole.

3. Oxidative pathways yielded 2,4-diacetylaminophenol (O-demethylation), 5-hydroxy-2,4-diacetylaminoanisole (ring hydroxylation), and 2-methoxy-5-(glycolamido)acetanilide or its isomer (ω-oxidation).

4. These major metabolites were excreted in the urine both as free and glucuronic acid conjugates.

1. A single oral dose of desmethylimipramine (80mg/kg) administered to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (45%), aniline (30%), biphenyl (30%) and ethylmorphine (15%) in vitro at 5 h after dosage; there was no decrease in cytochrome P-450 or b₅.

2. A single oral dose of ethoxyquin (200mg/kg) to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (65%), aniline (40%) and biphenyl (40%) in vitro at 1 h after dosage; inhibition was less at 5 h. There were no changes in the contents of cytochromes P-450 and b₅.

3. The max. plasma concn. of thiabendazole occurred 2-4h after oral dosing (50-200mg/kg) to rats. Thiabendazole (100mg/kg) administered orally 30min after oral ethoxyquin (400 mg/kg) or thiabendazole (200 mg/kg) administered orally 30 min after oral desmethylimipramine (80 mg/kg) delayed absorption of the thiabendazole and resulted in markedly decreased plasma concentration of the anthelmintic.

4. Simultaneous administration of ethoxyquin (300 mg/kg) potentiated the anthelmintic effect of thiabendazole (750 mg/kg) on the helminth parasite, Nematospiroides dubius, the mouse. Desmethylimipramine showed no similar potentiation.

1. The α-isomer of 1,2,3,4,5,6-hexachlorocyclohexane (HCH) is converted, on incubation with the 100 000g supernatant fraction (cytosol) of rat liver homogenates, to four positional isomers of S-(dichlorophenyl)glutathione (DCPG).

2. Radiochemical evidence shows that primary conjugates formed are subject to rapid aromatization.

3. The GSH-conjugates produced from γ-HCH (lindane), Δ-HCH, and three stereoisomers of 1,3,4,5,6-pentachlorocyclohex-1-ene (PCCH) have been characterized by g.l.c. of their aromatic moieties. All compounds were exclusively converted to at least two positional isomers of DCPG. An isomer of HCH and its trans-dehydrochlorination product yielded DCPG of almost identical composition.

4. β-HCH was not entirely unreactive, but the identity of the product remained uncertain.

5. DCPG-formation from HCH-d₆ exhibited a significant deuterium isotope effect (5·8 at 310 K for the α-configuration), while none was found for the conversion of PCCH-d₅ (1·2 at 298 K for the 3,4,6/5-isomer).

6. In the absence of GSH, liver cytosol protein mediated a trans-dehydrochlorination of lindane and of α-HCH to PCCH with a catalytic factor of 15 and 25, respectively. Addition of GSH raised HCH conversion by a factor of 3 to 4 and resulted in the formation of DCPG.

7. GSH-conjugation of PCCH is shown to be enzymic.

8. It is concluded that the rate of formation of DCPG from HCH in rat liver cytosol depends on gradual monodehydrochlorination, and that the enzymic transfer of GSH onto PCCH is not preceded by a second dehydrochlorination. The transfer and elimination reactions involved in DCPG formation from PCCH are considered taking into account (a) differences between stereoisomers in reaction rate and product composition and (b) the observation that a purified soluble GSH-S-transferase (E.C. 2.5.1.18) converted 3,4,6/5-PCCH-and α-HCH-to the same set of four isomeric DCPGs as did the entire cytosol fraction.

9. In corroboration of earlier evidence, transferase activity associated with liver microsomes and mitochondria also converts α-HCH and 3,4,6/5-PCCH each to four positional isomers of DCPG.

10. The result of the study is discussed with reference to earlier work in mammals and in insects and in relation to HCH-biotransformation by rats in vivo.

1. The disposition of spirohydantoin mustard (SHM) has been examined in rats and dogs after i.v. administration of [hydantoin-4-¹⁴C] SHM and [2-chloroethyl-U-¹⁴C] SHM.

2. Four hours after dosing to rats and dogs, renal clearance of the ethyl-¹⁴C moiety (14-21%) is lower than that of the hydantoin-¹⁴C moiety (29-35%). In contrast, biliary excretion of the ethyl-¹⁴C in rats is greater, and there appears to be enterohepatic circulation of the ethyl-¹⁴C.

3. Less than 1% of the radioactivity appearing in rat bile is unchanged SHM. Two major metabolites containing the ethyl-¹⁴C moiety are conjugates of glutathione or cysteine.

4. Levels of ¹⁴C in plasma decline in a biphasic manner. No difference in the initial plasma disappearance of the two labelled moieties is observed, but disappearance of the ethyl-¹⁴C during the second phase is slower.

5. Concentrations of ethyl-¹⁴C and hydantoin-¹⁴C in the brains of rats and dogs, 4h after i.v. administration, are equivalent to those in plasma.

6. Radioactivity is also distributed to the brain of rats after oral administration of [2-chloroethyl-U-¹⁴C]SHM, but at a concn. less than half that of plasma.