V. J. Feil

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 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.

Propachlor detoxication in the small intestine: Cysteine conjugation

14C-labeled 2-(S-cysteinyl)-N-isopropylacetanilide was recovered from the media of everted sacs of rat small intestine incubated in media that contained [14C] propachlor (2-chloro-N-isopropyl-[1(-14) C]acetanilide). The metabolite was identified on the basis of chromatographic characteristics and mass spectra of the butyl ester-trifluoro-acetamide derivative. This evidence shows that the cysteinyl conjugate was formed during incubations of the small intestine. Although the glutathione conjugate has not been isolated from the intestine, it is a likely precursor of the cysteinyl metabolite. Data from experiments conducted with 10(-5) and 10(-4) M propachlor in the media showed that the capacity to metabolize propachlor to polar metabolites was approximately the same throughout the length of the small intestine.

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.

Tissue distribution, excretion, and metabolism of 1,2,7,8-tetrachlorodibenzo-p-dioxin in the rat.

A tissue distribution, excretion, and metabolism study was conducted using a relatively non-toxic dioxin congener, i.e., 1,2,7,8-tetrachlorodibenzo-p-dioxin (1278-TCDD), to gain a better understanding of mammalian metabolism of dioxins. Conventional, bile duct cannulated, and germ free male rats were administered mg/kg quantities as a single oral dose. Elimination of 1278-TCDD was largely complete by 72 h. Distribution of [14C]1278-TCDD was low in all tissues examined. Metabolites were identified in urine, bile, and feces by negative ion FAB-MS and 1H-NMR, or GC/MS. The major fecal metabolite was a NIH-shifted hydroxylated TCDD. The bile contained a glucuronide conjugate of this hydroxy TCDD, and a diglucuronide conjugate of a dihydroxy-triCDD. The major metabolites in urine were glucuronide and sulfate conjugates of 4,5-dichlorocatechol.

Identification of NIH-shifted metabolites of 1,3,7,8-tetrachlorodibenzo-p-dioxin in the rat by NMR comparison with synthesized isomers.

In the rat, 1,3,7,8-tetrachlorodibenzo-p-dioxin was oxidatively metabolized to the NIH-shifted products 2-hydroxy-1,4,7,8-tetrachlorodibenzo-p-dioxin and 3-hydroxy-1, 2,7,8-tetrachlorodibenzo-p-dioxin. The chlorine substitution patterns were determined by comparison with 1H NMR spectra of six synthesized isomers in CDCl3, CD3OD, and acetone-d6. Glucuronide and glucuronide-sulfate conjugates of the monohydroxy dioxins were identified in the bile by FAB-MS. The dominance of the NIH-shift products in the metabolism of tetrachlorodibenzo-p-dioxins indicates that the same isomers may be produced from differently substituted chlorinated dioxins.

Metabolism of 2,6-dichlorobenzamide in rats and mice

1. Oral doses of 2,6-dichlorobenzamide (DCB) were excreted by rats as DCB, two monohydroxy-DCBs, 2-chloro-5-hydroxy-6-(methylthio)benzamide and 2-chloro-5-hydroxy-6-[S-(N-acetyl)cysteinyl]benzamide (mercapturic acid). 2. Biliary excretion (33% of the dose), enterohepatic circulation and intestinal micro-floral metabolism were involved in formation of 2-chloro-5-hydroxy-6-(methylthio)benzamide, and the mercapturic acid served as a precursor. 3. Whole body autoradiography and microautoradiography showed the accumulation of non-extractable residues from DCB in the nasal mucosa and contents of the large intestines of rats and mice dosed with 14C-labelled DCB.

Dichlorovinyl cysteine (DCVC) in the mouse kidney: tissue-binding and toxicity after glutathione depletion and probenecid treatment

The kidney binding of dichloro[14C]vinyl cysteine (14C-DCVC, 8 mg/kg body wt) and the kidney histopathology of DCVC (5 mg/kg body wt) were examined and compared in female C57BL mice subjected to various treatments. To evaluate the roles of organic anion transport and glutathione (GSH) status, mice were pretreated with probenecid (inhibitor of organic anion transport), l-buthionine-S,R-sulfoximine (BSO; inhibitor of GSH synthesis) or with diethyl maleate (DEM; GSH-depleting agent). In addition, the sites of 14C-DCVC binding in BSO-treated and control mice were monitored by microautoradiography. Probenecid was found to inhibit both kidney binding and toxicity of DCVC. In BSO-treated mice, DCVC binding remained roughly unchanged, whereas nephrotoxicity was severely increased and topographically extended to the subcapsular region. Microautoradiography showed that the site of DCVC binding in the straight portion of the proximal tubule was not changed by BSO. In DEM-treated mice, a clearly decreased DCVC binding was observed, while the effect on nephrotoxicity was minute. The effects of probenecid on DCVC binding and toxicity support a role for carriermediated transport of DCVC equivalents into the target cells. The BSO result suggests a protective function of GSH towards the nephrotoxicity of DCVC. Moreover, they support our previous contention that a primary lesion occurs at the site of DCVC binding, followed by a secondary, dose-dependent lesion localized outside the DCVC-binding region. In the case of DEM it is proposed that a DEM-GSH conjugate might compete for the uptake and/or activation of DCVC in the target cells.

Studies on the displacement of methylthio groups by glutathione

1. 14C-Methylthio-labelled 2-methylthio-4-ethylamino-6-tert-butylamino-sym-triazine (terbutryn), pentachlorothioanisole (PCTA), and 1,4-bis(methylthio)tetrachloro-benzene (bis-MTTCB) and their methylthio-oxidation congeners were reacted with glutathione (GSH) in the presence and absence of immobilized liver microsomal enzymes. 2. 13C-Methylthio-labelled terbutryn sulphoxide and terbutryn sulphone were used to study displacement of the methylthio moiety by GSH using 13C-n.m.r. 3. Methanesulphinic acid was identified as the group displaced by GSH from the methyl sulphones in vitro. 4. Methanesulphenic acid is proposed to be the group displaced by GSH from methyl sulphoxides forming methyl mercaptan, methyl glutathionyl disulphide and methane-sulphinic acid in vitro. 5. Rats given 14C-methylthio-labelled terbutryn, PCTA, bis-MTTCB, and their methylthio-oxidation congeners excreted 14CO2 and 14C-methanesulphinic acid in varying amounts. These results were compared to the in vitro data.

Biliary excretion and intestinal metabolism in the intermediary metabolism of pentachlorothioanisole

1. Biliary metabolites from rats dosed with pentachlorothioanisole (PCTA) were characterized by fast atom bombardment mass spectrometry and electron impact mass spectrometry. 2. Most of the biliary metabolites from PCTA were mercapturic acid pathway metabolites of methylsulphinyltetrachlorobenzene (51% of the dose); the remaining characterized biliary metabolites (20%) were mainly methylsulphinyltetrachlorothiophenols excreted as unknown conjugates. 3. Pathways are proposed for the intermediary metabolism of PCTA to bis-(methylthio)tetrachlorobenzene (bis-MTTCB) involving glutathione conjugation, biliary excretion, intestinal metabolism, and enterohepatic circulation.