Naohiko Isobe

Biotransformation and enzymatic reactions of synthetic pyrethroids in mammals.

Synthetic pyrethroids, a major insecticide group, are used worldwide for controlling indoor and agricultural pests. Extensive mammalian metabolism studies have been carried out since the late 1960s, and major metabolic reactions have been found to be oxidation of the acid or alcohol moiety, ester cleavage, and conjugation reactions. In addition, various conjugation reactions occur in mammals, forming hydrophilic and lipophilic conjugates. Pyrethroids are generally rapidly metabolized in mammals and completely excreted from the body in a short period. Human and laboratory animals share similar metabolic reactions for pyrethroids. Oxidation reactions in humans are mediated by several CYP isoforms. On the other hand, ester bonds of pyrethroids are hydrolyzed mainly by carboxylesterase(s).

Evaluation of in vitro methods for detecting the effects of various chemicals on the human progesterone receptor, with a focus on pyrethroid insecticides

The progesterone receptor (PR) is associated with physiological events such as implantation and the maintenance of pregnancy. Recently, it has become a social concern that chemicals may exert agonistic or antagonistic effects on hormone receptors. Therefore, we examined the effects of various chemicals on the human PR, with a focus on pyrethroid insecticides, using three in vitro methods. Eight pyrethroid insecticides (fenvalerate, d-allethrin, d-phenothrin, prallethrin, empenthrin, permethrin, cypermethrin and imiprothrin), examples of environmental pollutants and positive control chemicals were subjected to a reporter gene assay (luciferase assay) using human breast cancer T-47D cells, a two-hybrid assay and a binding assay using the same whole cells or receptors (cell-free). In none of these did the eight pyrethroid insecticides show any binding to the PR, agonistic or antagonistic effects.

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.

Mechanism of prolongation of pentobarbital-induced sleeping time by empenthrin in mice

The effect of empenthrin, a synthetic pyrethroid, on pentobarbital (PTB)-induced sleeping time was examined in mice and rats. In mice, pretreatment with empenthrin prolonged PTB-induced sleeping time in a dose-dependent manner. The maximum effect on PTB-sleeping time was noted when mice were pretreated orally with empenthrin 2-4 h before PTB injection. However, empenthrin did not change the sleeping time induced by diethyl ether which is hardly metabolized in liver. Empenthrin inhibited the clearance of serum PTB in mic, but did not change the PTB concentration in serum at which animals recovered from sleeping. To examine the effect of PTB on metabolic enzymes in mouse liver, PTB was incubated aerobically with a hepatic microsomal fraction in the presence of NADPH at 37 degrees C. Empenthrin inhibited the vitro metabolism of PTB dose-dependently. In rats, empenthrin neither changed the PTB sleeping time, nor inhibited the clearance of serum PTB. No inhibitory effect of empenthrin was observed on the in vitro metabolism of PTB using rat hepatic microsomal fraction. These findings indicate that empenthrin prolongs PTB-sleeping time in mice through an inhibition of the PTB-metabolizing enzyme(s) in the liver , an effect that does not occur in rats. Also, there is a clear species-specificity in the inhibitory effect of empenthrin on the PTB-metabolizing enzyme(s).

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole in the rat (III): Metabolites in the urine and feces.

The urinary and fecal metabolites of orally administered 2-tert-butyl-4-hydroxyanisole (2-BHA) and 3-tert-butyl-4-hydroxyanisole (3-BHA) in rats were identified. Samples of 2-day pooled urine and feces of rats given a single intragastric dose of 1 g/kg body wt of tert[butyl-14C]3-BHA (*Bu-3-BHA). tert[butyl-14C]2-BHA (*Bu-2-BHA), [methyl-14C]3-BHA (*Me-3-BHA) or [methyl-14C]-2-BHA (*Me-2-BHA) were analyzed by comparing thin-layer chromatography (TLC) retentions with authentic standards. Conjugated metabolites were identified after enzymatic hydrolysis. Proton magnetic resonance spectroscopy and electron impact mass spectrometry were used for confirmation of the authentic standards. In rats given 3-BHA, a major metabolite in the urine was 3-BHA-glucuronide with a smaller amount of tert-butylhydroquinone (TBHQ)-sulfate, while unchanged 3-BHA and 3-BHA-glucuronide were detected in the feces. In rats given 2-BHA, the main metabolites were the sulfate conjugates of 2-BHA, 4-tert-butyl-5-methoxy-1,2-benzoquinone (2-TBOQ) and the glucuronide of 2-BHA in the urine, while unchanged 2-BHA was found in the feces.

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.

Differences in α2u-globulins increased in male rat kidneys following treatment with several α2u-globulin accumulating agents: cystein protease(s) play(s) an important role in production of kidney-type-α2u-globulin

Abstract Effects of α 2u -globulin accumulating agents on α 2u -globulins in rat kidneys were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male animals with decalin (150 mg/kg), 2,2,4-trimethylpentane (50 mg/kg), isophorone (150 mg/kg), d -limonene (150 mg/kg) or 1,4-dichlorobenzene (150 mg/kg) by gavage for 14 consecutive days in each case resulted in a marked intensification of a protein band corresonding to kidney-type- α 2u -globulin with a molecular mass calculated to be ∼ 16 kDa. However, intraperitoneal treatment with leupeptin and E-64 (two times 0.07 mmol/kg, for each), well known cystein protease inhibitors, while only slightly increasing this kidney-type- α 2u -globulin band, caused the intensification of a ∼ 19-kDa molecular mass protein band which was revealed to be a native-type- α 2u -globulin by SDS-PAGE and immunoblotting. These results indicated that at least two types of α 2u -globulin can be increased in male rat kidney by chemical treatment. Moreover, cystein protease(s) appear(s) to play an important role in the degradation of α 2u -globulin and particularly in the conversion of native-type- α 2u -globulin to kidney-type- α 2u -globulin in rat kidneys.

Initial induction and subsequent reduction of α2u-globulin in urine and serum of mature male rats after repeated intraperitoneal injections of (anti)estrogen

The influence of sex (anti)hormones on expression of alpha(2u)-globulin (a2uG) is complex and has not been sufficiently detailed. In order to assess the specificity of sex (anti)hormone action on a2uG expression and the utility of this approach as a sensitive screening method, mature male rats were given daily intraperitoneal injections of 17beta-estradiol (E2), dihydrotestosterone (DHT), tamoxifen (TX) and flutamide (FL) for 5 consecutive days. They were employed as representatives of estrogen, androgen, antiestrogen and antiandrogen categories, respectively. Urinary a2uG was specifically altered with E2 (1 microg/kg/day) and TX (50 mg/kg/day), but not by DHT (1 mg/kg/day) or FL (50 mg/kg/day). E2 and TX temporarily increased urinary a2uG on days 1 or 2, and days 2-4, respectively, followed by a return to the control level, and then a decrease with E2. The reduction in urinary a2uG on day 6 was more pronounced than the drop in serum a2uG. Serum hormone levels, and liver and testis weights were not remarkably altered with any treatment. Another strong xenoestrogen, diethylstilbestrol, also significantly reduced urinary and serum a2uG at 1 mg/kg/day on day 6. However, the other xenoestrogens (100 mg/kg/day of bisphenol A, nonylphenol, and dichlorodiphenyltrichloroethane, and 10 mg/kg/day of dieldrin) and phytoestrogens (10 mg/kg/day of genistein and daidzein) were without any appreciable influence. The results indicate that urinary a2uG is a sensitive indicator of estrogen action in mature male rats, with two different responses, initial induction and subsequent reduction.

Research to develop a predicting system of mammalian subacute toxicity, (3). Construction of a predictive toxicokinetics model.

A new predictive toxicokinetics model was developed to estimate subacute toxicity (target organs, severity, etc.) of non-congeneric industrial chemicals, where the chemical structures and physico-chemical properties are only available. Thus, a physiological pharmacokinetics model, which consists of blood, liver, kidney (these were experimentally found as major toxicological targets), muscle and fat compartments, was established to simulate the chemical concentrations in organs/tissues with pharmacokinetic parameters by means of Runge-Kutta-Gill algorithm. The pharmacokinetic parameters, i.e. absorption rate, absorption ratio, hepatic extraction ratio of metabolism and renal clearance were calculated by using separately established Quantitative Structure-Pharmacokinetics Relationship equations. The developed predictive model was then applied to simulations of 43 non-congeneric industrial chemicals. The chemical concentrations in organs/tissues after single oral administration were simulated, and their maximum concentrations (Cmaxs) and area under the concentration-time curves (AUCs) were calculated. Fast Inverse Laplace Transform was newly applied for the purpose of simulation of 28-day repeated dose toxicity. Simulated concentrations of 28 days repeated dose were, however, found to be the same as those of simple repetitions of a single administration per day because of the short half-lives of non-congeneric industrial chemicals. A comparison of subacute toxicity data with Cmaxs and AUCs in a single dose scenario suggested that the organs/tissues with relatively high concentrations of tested chemical substances were the most sensitive targets within a chemical. Chemical concentrations in liver, for instance, were correlated with the severity of hepatotoxicity among the chemicals. It was also suggested that to improve and widen the present approach, data of metabolite and reactivity of non-congeneric industrial chemicals to organs/tissues, receptors, etc. should be incorporated into the model.

Metabolism of (Z)-(1R,3R)-Profluthrin in Rats.

When [benzyl-α-(14)C]-labeled (Z)-(1R,3R)-profluthrin (2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2-dimethyl-3-(prop-1-enyl) cyclopropanecarboxylate, a newly developed pyrethroid) was administered orally to rats at 1 mg/kg, around 70% was absorbed, metabolized, and mainly excreted into urine within 48 h. Radioactivity in plasma reached Cmax at 6-8 h, and decreased (half-life; 37-52 h). A similar tendency was observed also in tissues. Absorption rate was slightly lower at high dose, while kinetics and distribution did not change. Eight metabolites were detected in urine and one in feces. Most of the (14)C in feces was unabsorbed (Z)-(1R,3R)-profluthrin. The main metabolic reactions were ester cleavage, hydroxylation of the methyl group on the C4-position of the benzene ring, and its glucuronidation or oxidation to carboxylic acid. Oxidation of the geminal dimethyl on the cyclopropane-C2 to carboxylic acid, oxidation followed by hydration of the propenyl double bond, and ω-oxidation to carboxylic acid and mercapturic acid conjugation of the benzyl alcohol were observed as minor reactions.