Kunio Shiba

α2u-Globulins in the urine of male rats: a reliable indicator for α2u-globulin accumulation in the kidney

Abstract Increases in kidney-type- α 2 u -globulin (αG-K, molecular weight approximately 16 kDa) were detected in the urine of male adult rats treated with d -limonene by immunoblotting analysis using an antiserum which distinguishes native-type- α 2 u -globulin (αG-N, molecular weight approximately 19 kDa) from aG-K. When male adult rats received d -limonene by gavage (0–300 mg/kg/day) for 14 consecutive days, dose-dependent increases in urinary excretion of αG-K were observed at a dosage level of more than 30 mg/kg/day. This was found to be directly correlated with alterations in the concentration of renal αG-K as well as the accumulation of hyaline droplets in proximal convoluted tubule (PCT) epithelial cells in the kidneys. Marked elevation of urinary αG-K was also noted following oral treatment of adult male rats with 2,2,4-trimethylpentane (TMP), 1,4-dichlorobenzene (DCB), decalin and isophorone (ISP) by gavage (1.5 mmol/kg/day) for 7 consecutive days, again in association with increased concentrations of renal αG-K and hyaline droplet accumulation in renal PCT epithelial cells. However, no such increases in urinary αG-K were observed for male adult rats treated with nephrotoxic chemicals such as puromycin aminonucleoside (PAN) (15 mg/kg/day, s.c., 14 consecutive days) or hexachloro-1,3-butadiene (HCBD) (100 mg/kg/day, p.o., 5 consecutive days), lacking the ability to cause kidney accumulation of the hyaline droplets and αG-K. The findings in this study thus indicate that measurement of urinary αG-K can give a reliable estimates not only of the potential to cause renal accumulation of α 2 u -globulin but also of its magnitude.

Metabolism of tetramethrin isomers in rat. I. Identification of a sulphonic acid type of conjugate and reduced metabolites

1. Urinary and faecal metabolites in rat treated with 14C-labelled (1RS, trans)-tetramethrin [3,4,5,6-tetrahydrophthalimidomethyl (1RS, trans)-chrysanthemate] were identified using chromatographic techniques and spectroanalyses (nmr and ms). 2. 3-Hydroxy-cyclohexane-1,2-dicarboximide was found to be a major and unique urinary metabolite, reduced at the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety. 3. The major faecal metabolites were sulphonic acid conjugates, having a sulphonic acid group incorporated into the double bond of the 3,4,5,6-tetrahydrophthalimide moiety. 4. On the basis of the metabolites identified here, the major biotransformation reactions of trans-tetramethrin in rats are: (1) cleavage of the ester linkage; (2) cleavage of the imide linkage; (3) hydroxylation of the cyclohexene or cyclohexane ring of the 3,4,5,6-tetrahydrophthalimide moiety; (4) oxidation at the methyl group of the isobutenyl moiety; (5) reduction at the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety; and (6) incorporation of a sulphonic acid group into the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety.

Identification of two new types of S-linked conjugates of Etoc in rat.

1. Two major metabolites of 14C-labelled (4S,1R)-trans-Etoc[(S)-2-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1R)-trans-chrysanthemate] were purified using a combination of chromatographic techniques and identified by spectroanalysis (nmr(HMBC) and FAB-, TSP-MS). These were established as new types of S-linked conjugates (sulphonic acid and mercapturic acid types). 2. To examine the mechanism of formation of the sulphonic acid and mercapturic acid conjugates, sodium sulphate or glutathione labelled with 35S were administered to rat along with unlabelled trans-Etoc. Both sulphonic acid and mercapturic acid conjugates were found in the excreta, more of the former being yielded with 35S-sodium sulphate than with 35S-glutathione, implying that a sulphonic acid was incorporated into the double bond of a possible intermediate after reduction of sulphate to sulphite. The mercapturic acid conjugate was produced only with 35S-glutathione, implying incorporation of glutathione into the triple bond before subsequent generation of mercapturic acid from the glutathione conjugate. 3. Additional investigation of whether or not the mercapturic acid conjugate was produced by mixing the alcohol moiety of Etoc, PGL (4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one) and N-acetyl-L-cysteine under alkaline conditions. However, spectral data for the synthesized compound were not the same as those of the metabolite generated in vivo. That is, the addition reaction appeared to proceed by anti-Markownikovs rule, whereas the in vivo metabolite was apparently formed according to Markownikovs rule. Addition of glutathione at a triple bond has not been reported to our knowledge for any other foreign compounds in mammalian species.

Metabolism of tetramethrin isomers in rat: II. Identification and quantitation of metabolites

1. To examine the metabolic fate of (1RS, trans)- or (1RS, cis)-tetramethrin [3, 4, 5, 6-tetrahydrophthalimidomethyl (1RS, trans)- or (1RS, cis)-chrysanthemate], rat was administered a single oral dose of trans- or cis-[alcohol-14C]tetramethrin at dose levels of 2 or 250 mg/kg. 2. The radiocarbon was almost completely eliminated from rat within 7 days after administration in all groups. 14C-recoveries (expressed as percentages relative to the dosed 14C) in faeces and urine were 38-58 and 42-58% respectively in rat administrated trans-[alcohol-14C]tetramethrin, and in faeces and urine were 66-91 and 9-31% respectively in rat administered cis-[alcohol-14C]tetramethrin. 3. Fourteen metabolites found in excreta were purified by using several chromatographic techniques and identified by spectroanalyses (nmr and MS). Five sulphonate derivatives and three dicarboxylic acid derivatives were found. 4. The main metabolites were sulphonate derivatives in the faeces, and in the urine, alcohols, dicarboxylic acid and reduced metabolites derived from the 3,4,5,6-tetrahydrophthalimide moiety.

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.

Radioactive benzodiazepine derivatives

The present invention relates to a novel radioactive benzodiazepine derivative represented by the formula: ##STR1## wherein R is hydrogen or a lower alkyl; X* is radioactive iodine or bromine; Y is hydrogen or a halogen; and Z is a halogen or nitro group; and the salt thereof. The compound of the present invention enables to non-invasively detect the presence of benzodiazepine receptors in human or animal brains, other organs or tissues. Additionally, it enables to dynamically trace the change in receptor concentration either. It is very useful as a radioactive diagnostic agent for nuclear medicine in vitro or in vivo or as a radio-pharmaceutical.

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.