Search

1. Pentachlorophenyl methyl sulphoxide and pentachlorophenyl methyl sulphone were found to be substrates for microsomal and cytosolic glutathione-S-transferase of rabbit, monkey, chicken and human liver, covalently immobilized on beaded sepharose. 2. Protein was immobilized with greater than 95% transferase activity, measured by dinitrochlorobenzene. Immobilized rabbit liver microsomal transferase activity was more stable than immobilized cytosolic activity. 3. The sulphoxide moiety was displayed by glutathione in the presence of chicken liver microsomal protein. The sulphone moiety was displayed by glutathione in the formation of a diglutathione under catalysis by rhesus monkey liver cytosolic and microsomal protein. 4. Chlorine was displaced by transferases from all species to form regioisomeric monoglutathiones. 5. Qualitative and quantitative differences were observed in product distributions between species and between microsomal and cytosolic protein.

1. Radioactivity from oral doses of N-isopropyl[1-14C]acetanilide was excreted in urine (53.5%), faeces (8.1%) and expired air (17.0%) of rat. 2. Enterohepatic circulation occurred during formation of approximately 34% of the metabolites. N-isopropylacetanilide was metabolized by oxidation in all moieties of the molecule with subsequent conjugation with glucuronic and sulphuric acids. 3. The sulphate ester of 4'-hydroxyacetanilide (acetaminophen) was the major metabolite (28 % of the dose).

Cardiac transplantation is an increasingly important treatment for patients with end-stage heart failure. Rejection is one of the major limitations, and currently, serial endomyocardial biopsies are required to diagnose rejection. In the year after transplantation, patients routinely undergo 12, 14, or more biopsies. Infiltration of lymphocytes into the graft is a central feature of rejection. Previous studies from our laboratory have demonstrated the feasibility of detecting early rejection noninvasively with gamma scintigraphy after administration of autologous lymphocytes labeled with 111In.Eight patients were studied at the time of routine biopsy an average of 4.5 months after cardiac transplantation. Autologous lymphocytes were isolated and labeled with 111In. Forty-eight to 72 hours later, patients underwent planar scintigraphic imaging. Myocardial accumulation of labeled lymphocytes was quantified (indium excess, IE) with a previously described and validated technique. Animal studies have shown that an IEor = 0.07 is associated with rejection. Two of four patients with biopsy grade 0 or 1A rejection had no excess accumulation of labeled lymphocytes. The other two patients with biopsy grade 0 or 1A had an average IE of 0.13 +/- 0.04 (SD), which may actually represent the higher sensitivity of the scintigraphic approach, since the whole myocardium is interrograted. All four patients with biopsy grade 1B rejection had increased accumulation of labeled lymphocytes (IE = 0.18 +/- 0.06, P = .06 compared with all patients with grade 0 or 1A biopsies).The development of a sensitive, specific, and noninvasive method of diagnosing cardiac allograft rejection in humans might obviate the need for endomyocardial biopsy as well as improve the accuracy of diagnosis. The results suggest that scintigraphic detection of labeled lymphocytes is a promising approach for the noninvasive detection of cardiac transplant rejection. In addition, the approach should permit the assessment of the efficacy of antirejection therapy.

1. [1-14C]Propachlor-GSH was perfused at 0.34, 3.4 and 34 mumol/h through the portal vein or the right renal artery of anaesthetized rats with biliary and ureteral cannulas. Urine was the predominant route for elimination of the 14C regardless of the route of perfusion. 2. Mercapturic acid conjugates were found to be the end-products of metabolism of the 14C-propachlor-GSH by rat kidneys and liver, indicating that all enzymes necessary for the catabolism of GSH conjugates to mercapturates were present in each of these tissues. 3. Perfusion of 14C-propachlor-GSH at 34 mumol/h appeared to exceed the metabolic capacity of both liver and kidneys for this substrate. At this dose, parent compound was detected in the bile of portally perfused rats, and the amounts of 14C recovered in bile, kidneys and carcasses of renally perfused rats were greater than when smaller dosages were perfused.

Conventional rats metabolize 2-chloro-N-isopropylacetanilide (propachlor) mainly to various 2-methylsulphonylacetanilides and to residues in the faeces that are unextractable. Mercapturic acid pathway (MAP) metabolites of propachlor are the source of the methylsulphonyl sulphur and the insoluble faecal residues. Germ-free and antibiotic-treated rats quantitatively metabolized propachlor only to MAP metabolites, which were excreted in the urine and faeces (all water-soluble). Germ-free rats given enemas of caecal contents from conventional rats were qualitatively similar to conventional rats with respect to the metabolism of propachlor within 56 days of the inoculations i.e. they excreted 2-methylsulphonyl acetanilides in the urine and insoluble residues in the faeces. They approached quantitative similarity within 180 days. Antibiotic-treated rats spontaneously recovered the ability to metabolize propachlor as conventional rats, qualitatively within 6 days and quantitatively within 21 days. While differences occurred in the faecal metabolites of propachlor, biliary secretion of metabolites by germ-free rats was not different from that of conventional or antibiotic-treated rats.

Rats excreted the 14C from a single oral dose of N‐isopropyl‐N‐[14C]phenyloxamic acid [I, a soil metabolite from 2‐chloro‐N‐isopropylacetanilide (propachlor)] in approximately equal quantities in the urine (49.2%) and feces (48.2%). A milking goat given daily oral doses of [ 14C]‐I (1 mg of I three times daily) excreted more 14C in the feces (53.6%) than it excreted in the urine. From both species, I accounted for 97 to 100% of the urinary 14C, and all of the 14C that was extractable from the feces (73 to 75% of the 14C in feces was extractable with methanol). Goat milk samples collected 16 hr after the last dose contained no detectable 14C. Tissue residues of 14C were determined.

Sheep and rats metabolized single oral doses of O-O-dimethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate (I) to three major metabolites that were excreted in the urine (approximately 70% of the 14C). These were the glucuronide of 3,5,6-trichloro-2-pyridinol, O-methyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate, and 3,5,6-trichloro-2-pyridinol. The latter two metabolites and the parent compound were isolated from sheep feces. Sheep plasma contained the same metabolites that were found in sheep urine, and no parent compound was detected in the plasma. Tissue residues from I were determined. Visceral fat contained the highest concentration of I-equivalents (11.8 ppm). Sheep excreted a single oral dose of 3,5,6-trichoropyridinol (II) unchanged in the feces and as II-glucuronide in the urine.

A sheep dosed topically with C‐crufomate (4‐tert‐butyl‐2‐chlorophenyl methyl methylphosphoramidate) excreted 45.5% of the 14C dose in the urine within 9 days. The feces contained 1.2% and the carcass 40.4% (this included the 37.7% of the dose remaining on the skin in the dosing area) of the dose. At sacrifice, the fat, liver, kidney, lung, and skin (where the dose was applied) contained the highest concentrations of 14C. Fourteen urinary metabolites were isolated and characterized by mass spectrometry. The metabolic reactions involved were oxidations of the t‐butyl moiety, O‐demethylation, replacement of the H‐N‐CH3 moiety with a hydroxyl group, oxidation of the N‐methyl group to yield N‐formyl phosphoramidates, hydrolysis of the phosphoramidate moiety to yield phenols, conjugation with glucuronic acid and combinations of these reactions.

Procedures are described for extracting glucuronic acid and sulfuric acid conjugates from animal urine by using a nonionic type of adsorption chromatography. A chromatographic column (Porapak Q coated with trioctylamine) was developed to take advantage of nonspecific adsorption characteristics of these compounds on anion-exchange columns. The separation procedure provided sufficient cleanup of the conjugates to allow for their chromatography by other chromatographic methods.

Methanesulfinic and methanesulfonic acids were shown to be present in urine from rats fed laboratory rat diet. The daily excretions of each acid were equivalent at 0.47 mumol day-1. The excretion of methanesulfinic acid increased about ninefold and that of methanesulfonic doubled when rats were dosed with 17 mumol of pentachlorothioanisole. The excretion of methanesulfinic acid above control levels in urine may be a method for detecting methylthio displacements that occur during the intermediary metabolism of xenobiotics.

Metabolites in which the chlorine from propachlor has been replaced by a cysteine group or a methylsulfonyl group [-S(O2) CH3] are present in the urine of rats dosed orally with propachlor. In the present study, urine from rats given single oral doses of 35S-labeled cysteine conjugate of propachlor contained metabolites having the methylsulfonyl groups labeled with 35S. No metabolites containing 14C-labeled methylsulfonyl groups were isolated from urine of rats given single oral doses of the cysteine conjugate of propachlor in which the cysteine group was uniformly labeled with 14C. These findings show that the cysteine conjugate of propachlor is the source of sulfur in the methylsulfonyl-containing metabolites. Therefore, we suggest that a C-S lyase present in the animal cleaves the cysteine conjugate of propachlor and thus allows further metabolism of the sulfur to a methylsulfonyl moiety.

Fifteen metabolites of crufomate (4‐tert‐butyl‐2‐chlorophenyl methyl methylphosphoramidate, I) were identified in the excreta from rats given single oral doses of I. Compound I was not detected in either the urine or the feces. The metabolic reactions observed were N‐and O‐demethylation, oxidations of the t‐butyl moiety, replacement of the H‐N‐CH3 with an OH moiety, hydrolysis of the phosphoramidate moiety to yield the phenol, conjugation with glucuronic acid, and combinations of these reactions. No ring dehalogenation or ring substitution was observed.

Sheep metabolized a single oral dose of 2‐chloro‐N‐isopro‐pylacetanilide (propachlor) to four urinary metabolites. These were 2‐(S‐cysteinyl)‐N‐isopropylacetanilide and 2‐[S‐(N‐acetyl) cysteinyl]‐N‐isopropylacetanilide and the glucuronide conjugates of 4'‐hydroxy‐N‐isopropyl‐2‐methylsulfonylacetanilide and N‐(1‐hydroxyisopropyl)‐2‐methylsulfonylacetanilide. Residues (ppb equivalents of propachlor) from [14C]‐propachlor in the milk from a goat given daily oral doses (1.3 mg of propachlor three times daily for 15 days) plateaued at about 2 to 4 ppb equivalents of propachlor. In goat tissue, residues ranged from 1 ppb (fat) to 20 ppb (liver). Fecal and tissue metabolites were not identified.

During enterohepatic circulation conjugated steroids are hydrolyzed by intestinal bacteria, further metabolized mainly by anaerobic bacteria, reabsorbed, reconjugated and delivered to the blood for renal excretion. 21-Dehydroxlylation of tetrahydrodeoxycorticosterone (THCOD) to pregnanolone is performed exclusively by Eubacterium lentum, a normal inhabitant of the intestinal flora. The concentration of E. lentum in feces can be determined from the highest dilution of fresh voided stool capable of 21-dehydroxylating THDOC. Such studies revealed that the concentration increases from 106/g wet feces during the first year of life to 108/g feces in the 7th and 8th decade. Colonization appears to be independent of diet since the organisms are present in babies whether breast-fed or on formula, in subjects on a “Western diet” and in Africans consuming a diet low in meat and fat.

Germfree (GF) rats and antibiotic-treated rats with cannulated bile ducts (ABC) were given single oral doses of 2-(S-(N-[2H3]acetyl)cysteine)-N-isopropyl[1-14C]acetanilide. The GF rats excreted the dose in about equal quantities in the urine and feces; and the ABC rats excreted the dose in about equal quantities in the urine, feces, and bile. The mercapturate (60-75% of the dose in both cases) was isolated and the amount of exchange of N-acetyl deuterium to N-acetyl hydrogen was determined for all samples by mass spectrometry. The percentages of exchange were: ABC rats, bile 6.5%, urine 13%, feces 0.0%; GF rats, urine 15%, feces 4.7%. The remainder of the doses was present as the mercapturic acid sulfoxide. ABC rats dosed with 2-(S-[3,3-3H]cysteine)-N-isopropyl[1-14C]acetanilide (14C/3H = 0.50) excreted 42% of the dose in the urine and bile as the mercapturic acid that had a 14C/3H ratio the same as the original cysteine conjugate. The results of these studies show that a mercapturic acid and a cysteine conjugate can be absorbed from the gastrointestinal tract and resecreted in the bile or excreted by the kidney without having undergone metabolism other than acetylation of the cysteine nitrogen atom and oxidation of the sulfur to a sulfoxide. ABC rats were also dosed with 2-methylthio-N-isopropyl[1-14C]acetanilide, a suspected metabolic intermediate in the metabolism of propachlor (2-chloro-N-isopropylacetanilide). The dose was excreted in about equal quantities in the bile (48%) and urine (46%) as metabolites with a methylsulfonyl group in the 2-position. All are known metabolites of propachlor in conventional rats.

The mechanism for the formation of a class of sulfur-containing conjugates of xenobiotics was further investigated in this report. The major biliary metabolites of 2-acetamido-4-(chloromethyl)thiazole in the rat were found to be the mercapturic acid conjugate of 2-acetamido-4-methylthiazole and the glucuronic acid conjugate of 2-acetamido-4-(mercaptomethyl)thiazole. When these two compounds were introduced directly into the cecum of the rat, 2-acetamido-4-[(methylsulfinyl)methyl]thiazole and 2-acetamido-4-[(methylsulfonyl)methyl]thiazole were found as urinary metabolites. These results give strong support to a proposed mechanism in which intestinal microfloral metabolism of biliary metabolites, together with enterohepatic circulation, is necessary for the formation and urinary excretion of the 4-(methylthiomethyl), 4-(methylsulfinyl-methyl), and 4-(methylsulfonylmethyl) analogs of 2-acetamido-4-(chloromethyl)thiazole in the rat.

Twenty-five metabolites were isolated from the urine, feces or plasma of sheep given single oral doses of [14C]crufomate (4-t-butyl-2-chlorophenyl methyl methylphosphoramidate). These metabolites resulted from one or more of the following transformations: oxidatin of a methyl group in the t-butyl moiety to yield either an alcohol or a carboxylic acid; hydrolysis of the phosphate and phosphate and phosphoramidate bonds; oxidatin of the N-methyl group to yield N-formyl phosphoramidates; methylation of the N-formyl group to yield N-methyl-N-formyl phosphoramidates; oxidative N-demethylation; conjugation with glucuronic acid. No ring-hydroxylation of dechlorination was observed, and no crufonate was isolated from the urine, feces or plasma.