J. E. Bakke

Irreversible binding and toxicity of the herbicide dichlobenil (2,6-dichlorobenzonitrile) in the olfactory mucosa of mice

Following a single ip injection (12, 25, 50 mg/kg) of the herbicide dichlobenil (2,6-dichlorobenzonitrile) into C57Bl mice or Sprague-Dawley rats, an extensive destruction of the glands of Bowman and in the neuroepithelium of the olfactory region was observed. In mice, necrosis of the Bowmans glands was evident 8 hr after the lowest dose (12 mg/kg). Degeneration and/or necrosis of the neuroepithelium developed less rapidly but appeared at all doses examined. The mucosal lesions were most severe in the dorsal meatus and in the medial aspects of the ethmoturbinates. Three to seven days after dosing, the olfactory region was covered by an attenuated surface epithelium or by a respiratory-like epithelium. Seven to twenty days after dosing, there was fibrosis of the olfactory region. Partial regeneration of the olfactory epithelium and scattered intact Bowmans glands were observed after 20 days. Autoradiograms of mice given a single iv injection of 14C-labeled dichlobenil showed a high irreversible binding of radioactivity in Bowmans glands, whereas the binding in the olfactory epithelium was insignificant. In mice pretreated with metyrapone the binding decreased markedly, indicating that the reactive metabolite was formed by a cytochrome P450-dependent mechanism. The metyrapone treatment also resulted in a decreased or completely inhibited toxicity of dichlobenil to the olfactory mucosa. Hence, the tissue-specific toxicity of dichlobenil seems to be mediated by a reactive, tissue-binding metabolite. We propose that dichlobenil induces a primary lesion in the glands of Bowman, resulting from the pronounced binding of a metabolite in these glands. The toxicity to the olfactory neuroepithelium may be secondary to the destruction of the glands of Bowman.

Metabolism of mercapturic acid-pathway metabolites of 2-chloro-N-isopropylacetanilide (propachlor) by gastrointestinal bacteria

1. Mercapturic acid pathway metabolites of propachlor, labelled with 14C, were incubated with pig caecal contents, small and large intestinal contents, and pure cultures of gastrointestinal bacteria. 2. The glutathione, cysteine, N-acetylcysteine, and the S-oxide of the N-acetylcysteine conjugates of propachlor (2-chloro-N-isopropylacetanilide) were each metabolized by mixed pig caecal micro-organisms to 2-mercapto-N-isopropylacetanilide. 3. The extent of formation of 2-mercapto-N-isopropylacetanilide by mixed pig caecal micro-organisms from mercapturic acid-pathway metabolites of 14C-propachlor was as follows: glutathione (43.4%), cysteine (32.6%), N-acetylcysteine (5.2%), and the S-oxide of the N-acetylcysteine conjugate of propachlor (6.1%). The S-oxide of the N-acetylcysteine conjugate of propachlor was converted by the mixed pig caecal micro-organisms to N-acetylcysteine and cysteine conjugates of propachlor. 4. Small intestinal contents metabolized the glutathione conjugate of propachlor to the cysteine conjugate; little C-S lyase activity was present in the small intestinal contents. 5. The cysteine conjugate of propachlor was metabolized to 2-mercapto-N-isopropylacetanilide by Fusobacterium necrophorum, Bacteroides vulgatus, Megasphaera elsdenii and Eubacterium aerofaciens.

Mercapturic acid pathway metabolites of xenobiotics: generation of potentially toxic metabolites during enterohepatic circulation

Abstract The body detoxifies many xenobiotics via the mercapturic acid pathway (MAP). Unfortunately, in some cases this process provides intermediate metabolites that are substrates for mutagen/toxin-forming reactions - in particular reactive thiols. Jerome Bakke and Jan-Ake Gustafsson described this process of methylthiolation and the novel metabolic pathway involved. They highlight the increasing load of xenobiotics imposed by industrial processes and the toxicological consequences of their methylthiolated metabolites in individual organisms and the biosphere in general.

Enterohepatic circulation in formation of propachlor (2-chloro-N-isopropylacetanilide) metabolites in the rat

1. Bile secreted from rats given single oral doses of 2-chloro-N-isopropylacetanilide (propachlor) contained 58% dose as metabolites from the mercapturic acid pathway (glutathione, mercapturate, cysteine conjugates, and a sulphoxide of the mercapturate). 2. Bile secreted from rats given single oral doses of the cysteine conjugate of propachlor contained glucuronide conjugates of hydroxylated 2-methylsulphonyl-N-isopropylacetanilides. 3. In contrast, when the intestinal microflora were bypassed by intravenous administration of the cysteine conjugate of propachlor, the bile contained only the mercapturate and the sulphoxide of the mercapturate. 4. Rats fed an antibiotic-containing diet and given single oral doses of either propachlor or the cysteine conjugate of propachlor excreted only mercapturic acid pathway metabolites in the urine, bile, and faeces, and no methylsulphonyl-containing metabolites. Faecal 14C from the antibiotic-fed rats given either propachlor or the cysteine conjugate of propachlor was extractable, in contrast to previously reported unextractable faecal 14C residues from untreated rats given propachlor orally. 5. From these results, we conclude that metabolism by the microflora was necessary for production of the methylsulphonyl-containing metabolites excreted by the rat. Enterohepatic circulation of the xenobiotic moiety of these mercapturic acid pathway metabolites is influenced by the presence of a microbial C-S lyase.

Metabolism of 2,6-Dichlorobenzonitrile, 2,6-Dichlorothiobenzamide in Rodents and Goats

1. Twelve 14C-labelled metabolites were isolated from either urine or bile from either rats (11 metabolites) or goats (7 metabolites) given single oral doses of 2,6-dichlorobenzo[14C]nitrile (DCBN). Five of these metabolites were also excreted in urine from rats dosed orally with 2,6-dichlorothiobenz[14C]-amide (DCTBA). 2. All metabolites from either DCBN or DCTBA were benzonitriles with the following ring substituents: Cl2, OH (three isomers); Cl2, (OH)2; Cl, (OH)2; Cl, OH, SH; Cl, OH, SCH3; SCH3, SOCH3, OH; Cl2, S-(N-acetyl)cysteine; Cl, S-(N-acetyl)cysteine; Cl, OH, S-(N-acetyl)cysteine. 3. The thiobenzamide moiety of DCTBA was converted to the nitrile in all the excreted urinary metabolites. No hydrolysis of the nitrile in DCBN to either an amide or an acid was detected. 4. Urine was the major route for excretion; however, enterohepatic circulation occurred. 5. Whole-body autoradiography of 14C-DCBN and 14C-DCTBA in mice showed the presence of bound residues in the mucosa of the nasal cavity, trachea, tongue, oesophagus, the kidney, liver and the intestinal contents.

The metabolism of pentachloromethylthiobenzene in germ-free and conventional rats

1. Both germfree and conventional rats excreted over 80% of oral doses of pentachloromethylthio[14C]benzene in the faeces.2. The faeces from germ-free rats contained mainly N-acetyl-S-(methylthiotetrachlorophenyl)cysteine.3. The faeces from conventional rats contained bis-(methylthio)tetrachlorobenzene and non-extractable residues in about equal amounts.

Metabolism of 2-acetamido-4-(chloromethyl)thiazole in germfree and conventional rats.

Abstract In contrast with conventional rats, 2-acetamido-4-(chloromethyl)thiazole was not metabolized to the 4-(methylthiomethyl)-, 4-(methylsulfinylmethyl)- and 4-(methylsulfonylmethyl) analogues by germfree rats. Mechanisms for the formation of these metabolites from the mercapturate and the S-glucuronide are proposed. These mechanisms involve the biliary excretion of a mercapturic acid conjugate and an S-glucuronide conjugate which are metabolized in the intestine to metabolites that are reabsorbed, metabolized and excreted with the urine.

Metabolism of bis-methylthiotetrachlorobenzene in rats

Abstract Oral doses of bis-methylthiotetrachlorobenzene (bis-MTTCB) given to control rats and rats with cannulated bile ducts showed that at least 50% of the dose, although excreted mainly as bis-MTTCB in the feces, was metabolized. The metabolism involved replacement of one of the methylthio groups with glutathione, biliary excretion of the mercapturic acid pathway metabolites, and subsequent reformation of bis-MTTCB which is excreted with the feces.

Role of intestinal flora in metabolism of agrochemicals conjugated with glutathione

Intestinal processes which mediate the metabolic fate of glutathione conjugates of xenobiotics are reviewed. The role of the intestinal microflora in the metabolism of premercapturic acid pathway metabolites is discussed. The possible significance of mercapturic acid pathway metabolite catabolism in the bioaccumulation of methylthio-containing residues in the environment is presented.

Biliary secretion, retention and excretion of five 14C-labelled polychlorinated biphenyls in the rat

Abstract Five 14 C-labelled polychlorinated biphenyls: 2,4′,5-trichlorobiphenyl, 2,2′,4,5′-tetrachlorobiphenyl, 2,2′,4,5,5′-pentachlorobiphenyl, 2,2′,3,4,4′-pentachlorobiphenyl and 2,2′,4,4′,5,5′-hexachlorobiphenyl were administered orally to bile-cannulated rats. The activity secreted in the bile, excreted in the feces and the urine was determined. Residues of radioactivity in certain tissues and the carcass were also measured. The trichlorobiphenyl showed the highest absorption (93.8%±5.4) from the gastrointestinal tract and biliary secretion of radioactivity (87.6%±6.1 of the dose). The hexachlorobiphenyl showed the lowest absorption and biliary secretion, 28.2%±1.4 and 18.6%±1.3, respectively. The urinary excretion was low and the radioactive residues in the eviscerated carcasses increased with the chlorine content of the biphenyls.