Metabolism of Cyanox in rat. II. Sex-related differences in oxidative dearylation and desulphuration.

1. To examine the metabolites of Cyanox (O-4-cyanophenyl O,O-dimethyl phosphorothioate, cyanophos, CYAP) in brain, liver, blood cells and plasma during the early toxic period, the male and female rat was administered a single oral dose of [phenyl-14C]Cyanox at dose levels of 50 mg/kg and killed 5, 10 and 20 min thereafter. 2. Sex-related differences in the concentrations of metabolites were observed. Cyanoxon, produced by oxidative desulphuration, was observed in the brains of both sexes at all time points, but the concentrations were 2-6 times higher in the male. The same metabolite was detected in the liver, blood cells and plasma of the male but not the female. The total concentrations of oxidative dearylation metabolites (4-cyanophenol + 4-cyanophenylsulphate + glucuronide of 4-cyanophenol) in plasma, blood cell, brain and liver were larger in the male at all time points than those in the female, whereas the reverse was the case for demethylated metabolites (desmethylcyanox + desmethylcyanoxon) in all tissues except for the brain. 3. Studies of the in vitro metabolism of Cyanox revealed no sex-related difference for hepatic cytosolic fractions in terms of the major in vitro metabolic reaction, demethylation. On the other hand, the major reactions in microsomal fractions, oxidative desulphuration and oxidative dearylation, were significantly (2-3 times) greater in the male than in the female. 4. Oxidative desulphuration and oxidative dearylation, involving cytochrome P450 enzymes, were inhibited by male-specific rat CYP2C11 antiserum. The degree of inhibition was more pronounced in the male case. Thus, the results strongly suggest that the 2C family of cytochrome P450 (male, CYP2C11 and CYP2C13; female, CYP2C12) contributes to oxidative desulphuration and dearylation of cyanox in the rat and that the activity of male-specific CYP2C11 (and CYP2C13) is greater than that of female-specific CYP2C12. The consequent greater formation of cyanoxon in the male is consistent with the higher toxicity in this sex.

Metabolism of 7-fluoro-6-(3,4,5,6-tetrahydrophthalimido)-4- (2-propynyl)-2H-1,4-benzoxazin-3(4H)-one (S-53482, flumioxazin) in the rat: II. Identification of reduced metabolites.

On single oral administration of (14)C-S-53482 [7-fluoro-6-(3,4,5, 6-tetrahydrophthalimido)-4-(2-propynyl)-2H-1,4-benzoxazin-3( 4H)-one, Flumioxazin] labeled at the 1- and 2-positions of tetrahydrophthaloyl group to rats at 1 (low dose) or 100 (high dose) mg/kg, the radiocarbon was almost completely eliminated within 7 days after administration in both groups with generally very low residual (14)C tissue levels. The predominant excretion route was via the feces. The major fecal and urinary metabolites involved reduction or sulfonic acid addition reactions at the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety and hydroxylation of the cyclohexene or cyclohexane ring. One urinary and four fecal metabolites were identified using chromatographic techniques and spectroanalyses (NMR and MS). Three of five identified metabolites were unique forms, reduced at the 1,2-double bond of the 3,4,5, 6-tetrahydrophthalimide moiety. On the basis of the metabolites identified in this study, the metabolic pathways of S-53482 in rats are proposed. To specify tissues forming reduced metabolites, an in vitro study was conducted. Reduction was found to take place in red blood cells.

Metabolism of tetramethrin isomers in rat: IV. Tissues responsible for formation of reduced and hydrated metabolites.

1. To identify the sites of formation of the reduced metabolites, 3-hydroxy-cyclohexane-1,2-dicarboximide (3-OH-HPI-1 and -2), 1,2-cyclohexanedicarboxylic acid (TCDA) and 1-hydroxy-1,2-cyclohexanedicarboxylic acid (1-OH-HPA), in rat treated with 14C-labelled (1RS, trans)-tetramethrin, [3,4,5,6-tetrahydrophthalimidomethyl (1RS, trans)-chrysanthemate], bile-duct cannulated animals were orally or intravenously administered 14C-labelled 3,4,5,6-tetrahydrophthalimide (TPI) or 3,4,5,6-tetrahydrophthalic acid (THPA), precursors of these metabolites, and bile, urine and faeces were collected for analysis. 2. 3-OH-HPI-1 and 3-OH-HPI-2, which are cis-form reduced metabolites, and 1-OH-HPA were detected in bile and urine samples of the bile-cannulated rat treated intravenously and orally with 14C-labelled TPI, indicating their formation in tissues or blood. TCDA, a trans-form reduced metabolite, was not detected in bile, urine or faeces of the bile-cannulated rat treated intravenously with 14C-THPA, but was found in the faeces after oral application, indicating formation in the gastrointestinal tract. 3. To clarify whether 1-OH-HPA is produced from THPA via TCDA (hydroxylation via reduction) or by direct addition of H2O to its double bond (hydration), rats were orally administered 14C-labelled TCDA, and metabolites in urine and faeces were analysed. The observed lack of 1-OH-HPA indicated formation by direct addition of H2O to the double-bond of THPA. 4. To specify which tissues form reduced and hydrated metabolites, in vitro metabolism studies were carried out. Reduction to the cis-form was found to take place in blood cells, reduction to the trans-form took place in the gastrointestinal tract contents, and hydration took place in the liver and the intestinal tract contents.

Metabolism of tetramethrin isomers in rat. III. Stereochemistry of reduced metabolites.

1. Three main urinary metabolites, two isomers of 3-hydroxycyclohexane-1,2-dicarboximide (3-OH-HPI-1 and 2) and 1,2-tetrahydrodicarboxylic acid (TCDA) were purified from rat treated with (1RS, trans)-tetramethrin [3,4,5,6-tetrahydrophthalimidomethyl (1RS, trans)-chrysanthemate]. 2. To elucidate the mechanism of formation of these reduced metabolites, the stereochemistry of 3-OH-HPI-1, 3-OH-HPI-2 and TCDA was clarified by chemical reactions, spectroanalysis (nmr) and X-ray analysis. 3. The sole difference in configuration between 3-OH-HPI-1 and 3-OH-HPI-2 was found to be the orientation of the hydroxyl group to the cyclohexane ring, and both of these reduced metabolites showed cis-addition of two hydrogens. In contrast, reduction resulted in the trans form with TCDA. 4. These findings indicate the existence of two different reduction reaction mechanisms in the rat.

Metabolism of Cyanox in the rat. I. Absorption, disposition, excretion and biotransformation.

1. To examine the metabolic fate of Cyanox [O-4-cyanophenyl O, O-dimethyl phosphorothioate, cyanophos, CYAP], rats of both sexes were administered [phenyl-4C]Cyanox as a single oral dose at levels of 0.5 mg/kg (low-dose group) or 25 mg/kg (high-dose group), or as multiple doses at 50 mg/kg/day once daily for 7 days (repeat-dose group). 2. The radiocarbon was almost completely eliminated from rats within 7 days after administration in both low- and high-dose groups. 14C-recoveries (expressed as % relative to the dosed 14C) in faeces and urine were 2-3 and 95-96% respectively for the low-dose and 13-14 and 86% respectively for the high-dose. 3. 14C-tissue residues on the seventh day after a single administration were generally low. Peak 14C-concentrations in blood and kidney occurred at 0.5 h (high-dose) and decreased rapidly thereafter. 4. Sex-related differences in the amounts of metabolites were observed in both groups. With the low-dose, the major metabolite was 4-cyanophenylsulphate in both sexes. However, in the high-dose, the major metabolites were 4-cyanophenyl sulphate and desmethylcyanoxon in males, but 4-cyanophenyl sulphate and desmethylcyanox in females. These findings indicate that the amounts or the types of enzymes responsible for oxidative desulphuration or oxidative dearylation in males are different from those in females. In the male rat given repeat doses significant differences in the amounts of metabolites in excreta were observed between early and final dosing. 5. The greater formation of desmethylcyanoxon in the male rat in the high-dose case is consistent with the higher incidence of toxicity in this sex.

[Clinical usefulness of a trypsin radioimmunoassay kit].

From the clinical use of RIA-gnost trypsin kit, the following results were obtained. 1. Standard curve showed a steep and good curve was shown. 2. Incubation: The condition for the first incubation was set at the room temperature for 10-24 hours and that for the second incubation at the room temperature for 3-5 hours. With these settings, satisfactory results were obtained. 3. Reproducibility and recovery: The C.V. of the reproducibility and the recovery were considered superior, and the values were below 10% and +/- 3%, respectively. 4. Correlation between trypsin and serum elestase-1: An excellent positive correlation (coefficient of correlation r = 0.889) was shown. 5. Serum trypsin concentration of normal and pancreatic diseases: The normal range was from 100 to 500 ng/ml. Acute pancreatitis rose obviously. Diabetes mellitus and chronic pancreatitis was below 500 ng/ml and the pancreatic cancer showed a tendency to scatter in the range of 50-1,250 ng/ml. The above results indicated that serum trypsin can be easily measured with high precision by using this method. Thus the method is considered useful for the diagnosis of pancreatic diseases.