Metabolism of N-isopropylacetanilide in rat.

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).

Identification of the major urinary and fecal metabolites of 3,5-dinitrobenzamide in chickens and rats.

Colostomized chickens given oral doses of 3,5-dinitrobenzamide (nitromide) cleared nitromide predominantly through the urine (58% of dose) and feces (21% of dose). Rats cleared 52% of nitromide via urinary excretion and 44% via feces. Major urinary metabolites for both chickens and rats include: 3-amino-5-nitrobenzamide, 3-acetamido-5-nitrobenzamide, 3-acetamide-5-aminobenzamide, and 3,5-diacetamidobenzamide. The major fecal metabolite in chickens was 3-acetamido-5-nitrobenzamide (67% of fecal 14C) and 3-acetamido-5-aminobenzamide in rats (approximately 50%).

Intermediary metabolism of pentachloronitrobenzene in the control and germ-free rat and rat with cannulated bile ducts.

1. Nearly 70% of single oral doses of 14C-labelled pentachloronitrobenzene (PCNB) was excreted in bile within 24 h. 2. The characterized biliary metabolites of PCNB were either mercapturic acid pathway metabolites or catabolites thereof (thiols, methylthiols, S-glucuronides). 3. A major biliary metabolite was S-(aminotetrachlorophenyl)glutathione. 4. Conjugation with glutathione with subsequent catabolism to bis-methylthiotetrachlorobenzene was the major pathway in the control rat. 5. Germ-free experiments showed that only nitro- group displacement occurred, and no nitro- group reduction was detected.

Detection of cardiac transplant rejection with 111In-labeled lymphocytes and gamma scintigraphy.

BACKGROUND: 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. METHODS AND RESULTS: 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 IE > or = 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). CONCLUSIONS: 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.

Rate of glycolysis during ischemia determines extent of ischemic injury and functional recovery after reperfusion.

The efficacy of increasing glycolysis during ischemia for enhancing the salutary effects of reperfusion was evaluated in isolated perfused rabbit hearts subjected to low-flow ischemia followed by reperfusion. Control hearts were perfused with buffer containing 0.4 mM palmitate, 5 mM glucose, and 70 mU/l insulin. Additional groups of hearts were perfused with double glucose/insulin and 1 mM dichloroacetate or were subjected to substrate priming to increase preischemic glycogen content. Ischemic contracture was completely prevented in hearts perfused with high glucose/insulin and was delayed markedly by either dichloroacetate or enhanced preischemic glycogen [45 +/- 14 and 31 +/- 20 min, respectively; P < 0.01 each vs. control (11 +/- 10 min)] and inversely related to the rate of lactate production. With reperfusion, recovery of developed pressure was 56 +/- 23% of baseline in control hearts, 90 +/- 8% in hearts receiving high glucose/insulin, 92 +/- 5% in hearts receiving dichloroacetate, and 79 +/- 19% in hearts with increased glycogen (P < 0.05 each vs. control hearts). Creatine kinase release was reduced by > 55% in treated hearts. Thus enhancement of glycolysis by diverse mechanisms during ischemia decreased ischemic damage and improved the recovery of contractile function with reperfusion.

Effect of AT-125 on the metabolism of propachlor and the glutathione conjugates of propachlor and bromobenzene in rat.

1. Dosing rats with the gamma-glutamyl-transpeptidase inhibitor AT-125 results in the excretion of free glutathione in the urine of rat: this treatment did not lead to the excretion of glutathione conjugates of orally dosed xenobiotics, neither did AT-125 increase the biliary excretion of glutathione conjugates. 2. Dosing rat with AT-125 prior to dosing with 2-chloro-N-isopropylacetanilide decreased the excretion of 2-methylsulphonylacetanilide metabolites from 23% of the dose to < 0.5%. 3. We conclude that glutathione and glutathione-xenobiotic conjugates are probably not processed in vivo by the same pathway, and that AT-125 can alter the in vivo transport of mercapturic acid pathway metabolites.

Glutathione-mediated methylthio-turnover and sex differences in the metabolism of pentachlorothioanisole by rat.

1. Sex differences observed in the metabolism of pentachlorothioanisole in rat were due to: (1) greater excretion in urine by females, and greater biliary excretion by males; (2) formation of pentachlorophenyl mercapturic acid pathway metabolites by females; and (3) redox-cycling between methylthio and methylsulphoxyl oxidation congeners in intermediary metabolites by females. 2. Three methylthio-turnover processes are proposed in the intermediary metabolism of pentachlorothioanisole.

Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats.

1. 14C-Cysteinyl- and homocysteinylpropachlor were metabolized to their respective mercapturic acids by rat kidneys in situ. First-pass elimination of 14C in urine was 47.5% for the cysteine conjugate and 36% for the homocysteine conjugate. 2. About half of the perfused 14C-labelled material isolated from urine from kidneys perfused with homocysteinylpropachlor was unchanged homocysteinylpropachlor and about half was the corresponding mercapturic acid. However, only the corresponding mercapturic acid and the S-oxide of this mercapturic acid (31.4% and 1.7% of the dose) were found in urine from kidneys perfused with cysteinylpropachlor, indicating that rat kidneys more efficiently acetylated the natural substrate, the cysteine conjugate.

Conjugation of polychlorinated agrochemical sulphoxides and sulphones by glutathione.

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.

Metabolism of 1,2,4-trichlorobenzene in rats: examination of thiol formation.

1. More than 60% of oral doses of 14C-1,2,4-trichlorobenzene (ca. 21 mg/kg) administered to rats were excreted in bile as S-trichlorophenyl-mercapturic acid pathway metabolites. 2. The biliary metabolites were ultimately excreted in urine mainly as the isomeric mercapturic acids. 3. An acetylated glutathione conjugate was isolated as a major metabolite in bile (8% dose). The acetyl group was shown by mass spectrometry to be on the glutamyl moiety. 4. A glutamylcysteine conjugate of trichlorobenzene was also isolated from bile as a major metabolite (8% dose). 5. Trichlorothiophenols were deduced not to be intermediates or end-products of enzymic metabolism of trichlorobenzene in rats because 14C-2,4,5-trichlorothiophenol dosed i.p. to rats was excreted as the S-glucuronide (17% dose) and as S-(methylsulphonyl-dichlorophenyl)-mercapturic acid (36% dose).

Glutathione-S-transferase-mediated methylthio displacement and turnover in the metabolism of pentachlorothioanisole by rats.

1. The metabolism of pentachlorothioanisole to bis-(methylthio)tetrachlorobenzene was shown to involve turnover of about 50% of the original methylthio group. 2. Metabolites of the displaced methylthio group were methanesulphinic acid and sulphate in the urine, carbon dioxide in the expired air, and unidentified sulphur-containing compounds in the bile.

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.

Evidence for the absence of cysteine S-conjugate N-acetyltransferase activity in the metabolism of propachlor, naphthalene, and dichlobanil in calves.

1. The glutathione conjugate of 2-chloro-N-isopropyl[1-14C]acetanilide (14C-propachlor) was perfused through a calf kidney in situ; 23% of the dose was excreted in the perfused kidney urine as the cysteine conjugate, no mercapturic acid was detected. 2. A 5-day-old calf dosed orally with 14C-propachlor excreted 70% dose in the urine as the cysteine conjugate; no mercapturic acid was detected. Rumen microflora were established in the calf (5 weeks older) and the experiment was repeated with the same results. 3. When the same calf was dosed 1 week later with 14C-naphthalene, 99% dose was excreted in the urine, mostly as the dihydrodiol-glucuronide (34%) and the dihydrohydroxy-cysteine conjugate (47%); no mercapturate was detected. 4. A 9-day-old calf dosed orally with 2,6,-dichlorobenzo[14C]nitrile (14C-dichlobanil) excreted 67% dose in the urine as cysteine conjugates (34%), and products of cysteine conjugate beta-lyase cleavage of cysteine conjugates (30%); no mercapturates were detected. 5. Cysteine S-conjugate N-acetyltransferase activity in calf kidney and liver was about 10% of that in the corresponding rat tissues.

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.

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 Bowman's 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 Bowman's 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 Bowman's 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 the glutathione conjugate of propachlor by in situ perfused kidneys and livers of rats.

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.

Methylsulphone metabolites of m-dichlorobenzene as ligands for alpha 2u-globulin in rat kidney and urine.

1. Kidneys from rats given oral doses of either 3,5- or 2,4- dichlorophenyl[14C]methylsulphide contained the corresponding sulphones that were associated with a protein which has been isolated and characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis to be alpha 2u-globulin. 2. The same methylsulphone-alpha 2u-globulin complex was isolated from the urine from rats doses with 3,5-dichlorophenyl[14C]methylsulphide (0.3-1.0% of dose, 8.5% of 14C in urine. 3. The stoichiometry of binding of the isolated methylsulphone-alpha 2u-globulin complex was shown to be 0.54-0.78 nmol/nmol protein. This binding was not covalent.