Akira Yoshitake

Hepatic UDP-glucuronyltransferase(s) activity toward thyroid hormones in rats: Induction and effects on serum thyroid hormone levels following treatment with various enzyme inducers

Induction of hepatic UDP-glucuronyltransferase(s) (hUDP-GT(s] activity toward thyroid hormones and the relationship between the activity and the serum thyroid hormones or the thyroid stimulating hormone (TSH) level were examined in male Sprague-Dawley rats after four consecutive ip doses of various hepatic enzyme inducers at 75-150 mg/kg/day. hUDP-GT activity toward thyroxine (T4; hUDP-GT-T4) was induced by treatment with beta-naphthoflavone, 3-methylcholanthrene (3-MC), polychlorinated biphenyls, or pregnenolone-16 alpha-carbonitrile. However, no significant induction was observed for isosafrole administration and in the cases of phenobarbital and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane slight decreases were found. The induction profile of hUDP-GT-T4 for these inducers was approximately the same as that of hUDP-GT activity toward triiodothyronine (T3; hUDP-GT-T3), indicating that these two thyroid hormones (T4 and T3) are glucuronidated by the same hUDP-GT(s). Moreover, the induction profile of both hUDP-GT-T4 and hUDP-GT-T3 was similar to that of hUDP-GT toward 1-naphthol, but not chloramphenicol, suggesting that T4 and T3 belong to the so-called group-1 substrates which are preferentially glucuronidated by hUDP-GT(s) inducible by treatment with 3-MC. Decreases in serum T4 levels clearly correlated with an increase in hUDP-GT-T4 activity, indicating that serum T4 levels are directly affected by hUDP-GT-T4 activity. However, no direct correlation between decrease in thyroid hormone levels and compensatory increase in TSH levels was found.

Behavior of α2u-globulin accumulating in kidneys of male rats treated with d-limonene: Kidney-type α2u-globulin in the urine as a marker of d-limonene nephropathy

Abstract Effects of d -limonene on α 2 u -globulin in the kidneys, urine and serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male rats with d -limonene by gavage for 14 consecutive days (300 mg/kg/day) caused accumulation of hyaline droplets in renal proximal tubule cells, and a marked intensification of a protein band corresponding to the kidney-type α 2 u -globulin, with a molecular weight calculated to be approximately 16 kDa. However, no significant changes in the serum α 2 u -globulin (native-type) band, of approximately 19 kDa, were observed between treated rats and controls, suggesting that circulating α 2 u -globulin levels were not affected by the d -limonene administration. While the molecular weight of the major α 2 u -globulin in the urine from control rats was the same as that in the serum (native-type), marked increase in the protein band corresponding to kidney-type- α 2 u -globulin was observed in the urine from treated rats. The results were indicative of elimination of α 2 u -globulin from the kidney to urine, the appearance of kidney-type- α 2 u -globulin in urine implying disruption or exfoliation of proximal tubule cells. Therefore, it is suggested that the presence of the α 2 u -globulin (kidney-type) in the urine might be used as an indicator of chemically induced α 2 u -globulin nephropathy.

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole (2- and 3-BHA) in the rat. (II): Metabolism in forestomach and covalent binding to tissue macromolecules.

The mechanism of action of 2(3)-tert-butyl-4-hydroxyanisole (2-BHA or 3-BHA) on rat forestomach epithelium was studied by examining the metabolites of BHA in the stomach and the covalent binding of BHA to macromolecules in the forestomach epithelium. Male F344 rats 6 weeks old were given a single intragastric injection of 1 g/kg body wt of [tert-14C]-3-BHA (Bu-3-BHA) or [methyl-14C]-3-BHA (Me-3-BHA), and 6 h later BHA metabolites in the forestomach, glandular stomach and stomach contents were examined by thin-layer chromatography. No significant amounts of metabolites were detected in the forestomach or glandular stomach epithelium and almost all the radioactivity in these tissues was extracted with organic solvents. In in vitro experiments also, no significant amounts of metabolites were detected when the 9000 g supernatant of the forestomach or glandular stomach epithelium, or gastric juice was incubated with Bu-3-BHA in the absence or presence of NADPH. In binding studies, rats were given Bu-3-BHA, [tert-14C]-2-BHA (Bu-2-BHA), Me-3-BHA or [methyl-14C] butylated hydroxytoluene (Me-BHT) intragastrically at a dose of 1 g/kg body wt with or without pretreatment with unlabelled 1% 3-BHA or BHT in the diet for 6 days. Six hours after treatment with a labelled compound, the rats were sacrificed and the DNA, RNA and protein of their forestomach, glandular stomach, liver and kidney were isolated. Bu-3-BHA, Bu-2-BHA and Me-3-BHA did not bind covalently to forestomach DNA or RNA, and the amounts of radioactivity of these compounds bound to proteins in the 4 tissues were similar. These findings suggest that BHA acts on the forestomach epithelium directly without metabolic activation, and that its action is not related to its binding to DNA or RNA.

EFFECTS OF PYRETHROID INSECTICIDES ON GAP JUNCTIONAL INTERCELLULAR COMMUNICATIONS IN Balb/c3T3 CELLS BY DYE-TRANSFER ASSAY

The effects of fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin, p-chlorophenylisovaleric acid (CPIA, major metabolite of fenvalerate) and DDT, a liver tumor promoter, on gap junctional intercellular communication (GJIC) were examined in Balb/c3T3 cells by dye-transfer assay. Separate groups of Balb/c3T3 cells were exposed to the chemicals for 1 day. On the following day, GJIC was measured by counting the number of dye-transferring cells per injection of Lucifer Yellow under a fluorescent microscope. Fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin and DDT inhibited GJIC at noncytotoxic concentrations, while CPIA did not inhibit GJIC even, at a cytotoxic concentration. It is concluded that the examined pyrethroid insecticides, but not a metabolite, have inhibitory effects on GJIC in Balb/c3T3 cells.

Substrate specificity for formation of cholesterol ester conjugates from fenvalerate analogues and for granuloma formation

1. The substrate specificity of microsomal carboxyesterase(s) responsible for the formation of cholesteryl [2R]-2-(4-chlorophenyl) isovalerate from fenvalerate was investigated by incubating mouse kidney microsomes with 14C-cholesterol and the following substrates: fenvalerate isomers, fenvalerate analogues, other pyrethroids, methoprene and cycloprate analogues. Among the four isomers of fenvalerate, only the [2R, alpha S]-isomer yielded a cholesterol ester, being identical with the result obtained in the in vivo study. Some fenvalerate analogues produced cholesterol ester conjugates, but no other pyrethroids nor methoprene produced such conjugates. Some cycloprate analogues gave the corresponding cholesterol ester, the yields of which were dependent on their carbon-chain lengths. 2. Cholesterol ester formation in vitro from these fenvalerate analogues was well correlated with granuloma formation observed when the analogues were given to mice at 3000 ppm for a month. 3. Steroids other than cholesterol were also investigated as acceptors of the acid moiety of the [2R, alpha S]-isomer by incubating solubilized carboxyesterase(s) with the [2R, alpha S]-isomer in the presence of egg lecithin and several steroids. Dehydroisoandrosterone and pregnenolone were found to give the corresponding ester conjugates.

Effect of Phenobarbital on Hepatic Gap Junctional Intercellular Communication in Rats

The effects of in vivo exposure to phenobarbital (PB) on hepatic gap junctional intercellular communication (GJIC) and conncxin protein expression in Sprague-Dawley rats were examined by in vivo/in vitro dye-transfer assay, immunohistochemical staining, and by Western blot analysis. PB (50 mg/kg) was administered orally once a day for up to 6 wk. The average size of the dye spread after injection of Lucifer Yellow decreased at week 1 and remained at the same level until week 6. The area and number of connexin 32 (Cx32) spots per hepatocyte in the central zone of liver lobules decreased from week 1 to week 6, but no change of Cx32 spots in the peripheral zone was observed. The average area and number of connexin 26 (Cx26) spots per hepatocytes showed no clear change through the experimental periods. The decreased level of Cx32 protein in plasma membranes was observed in the PB group. These results suggest that PB, a liver tumor-promoting agent, inhibits hepatic GJIC in vivo in rats and that aberrant Cx32 protein expression and/or localization may be responsible for this effect.

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole (2- and 3-BHA) in the rat (I): Excretion of BHA in urine, feces and expired air and distribution of BHA in the main organs.

The mechanism of the carcinogenic or toxic action of BHA on rat forestomach was examined by studies on the excretion and tissue distribution of radioactivity in F344 male rats given tert-butyl- or methoxy-labelled 3-BHA orally. Within 2 days after a single oral dose of labelled BHA at 1 g/kg body wt, 87-96% of the 14C was excreted, mainly in the urine with smaller amounts in the feces and expired air. More 14C was found in the tissues of rats given the methoxy-labelled compounds. The distributions of 14C in the forestomach and the glandular stomach were similar. At 168 h after treatment, more 14C was found in the forestomach of rats given 2-BHA than in that of rats given 3-BHA. These results indicate that excretion of BHA is rapid, that 4-O-methyl demethylation may take place readily and that demethylated methyl group may become distributed non-specifically in tissues. The carcinogenic or toxic action of BHA on the forestomach does not seem to be due accumulation of BHA in the forestomach.

In vivo and in vitro pharmacokinetics and metabolism studies of 26,26,26,27,27,27-F6-1,25(OH)2 vitamin D3 (Falecalcitriol) in rat: induction of vitamin D3-24-hydroxylase (CYP24) responsible for 23S-hydroxylation in target tissues and the drop in serum levels.

1. 26,26,26,27,27,27-F6,-1,25(OH)2 vitamin D3, Falecalcitriol, the hexafluorinated analogue of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biological activity appears related to F6-1,25(OH)2 vitamin D3 metabolism to F6-1,23S,25(OH)3 vitamin D3, a bioactive 23S-hydroxylated form which is resistant to further metabolism. 2. In the present in vivo studies, the repeated oral administration of [3H]F6-1,25(OH)2 vitamin D3 to rat resulted in a significant reduction of the radioactivity and the F6-1,25(OH)2 vitamin D3 concentrations in serum, especially at the 2 h maximum point after each dosing. Additionally, F6-1,23S,25(OH)3 vitamin D3 in the serum and small intestine was increased by the prior administration of F6-1,25(OH)2 vitamin D3. 3. Further in vitro investigation showed [3H]F6-1,25(OH)2 vitamin D3 to be metabolized to F6-1,23S,25(OH)3 vitamin D3 by kidney and small intestine homogenates of rat, the reaction being increased by the prior administration of F6-1,25(OH)2 vitamin D3. Moreover, this latter treatment was associated with a marked increase of CYP24 mRNA in the small intestine within 4 h after dosing. 4. The results indicate that in vivo metabolism of F6-1,25(OH)2 vitamin D3 to F6-1,23S,25(OH)3 vitamin D3 is catalysed by CYP24, the enzyme being induced by prior substrate exposure.

Metabolism of N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide (S-23121) in the rat: I. Identification of a new, sulphonic acid type of conjugate

1. Several metabolites of 14C-labelled N-[4-chloro-2-fluoro-5-[(1-methyl- 2-propynyl)oxy]phenyl]-3,4,5,6-tetra-hydrophthalimide (S-23121) were identified. 2. The major urinary metabolites were found to be 4-chloro-2-fluoro-5-hydroxyaniline, its sulphate and glucuronide by t.l.c. cochromatography with authentic standards. 3. The major faecal metabolites in addition to the parent compound were six sulphonic acid conjugates having a sulphonic acid group incorporated into the double bond of the 3,4,5,6-tetrahydrophthalimide moiety. These sulphonic acid conjugates have never been reported previously for this type of compound. 4. To confirm the mechanism of biosynthesis of the sulphonic acid conjugates, sodium sulphate, cysteine and glutathione labelled with 35S were administered to the male rat together with unlabelled S-23121. The same faecal metabolites as those detected in faeces of the rat dosed with 14C-labelled S-23121 were similarly found after dosing with any of the 35S-labelled chemicals. Their biosynthesis was most pronounced with 35S-labelled sodium sulphate, implying that the sulphonic acid is incorporated into the double bond after reduction of sulphate to sulphite.

Distribution and metabolism of F6-1,25(OH)2 Vitamin D3 and 1,25(OH)2 Vitamin D3 in the bones of rats dosed with tritium-labeled compounds

26,26,26,27,27,27-Hexafluo-1,25(OH)2 vitamin D3, the hexafluorinated analog of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biologic activity in the kidneys and small intestine appears to be related to F6-1,25(OH)2 vitamin D3 metabolism to ST-232, 26,26,26,27,27,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3, a bioactive 23S-hydroxylated form that is resistant to further metabolism. Since F6-1,25(OH)2 vitamin D3 is considered to prevent osteoporotic decrease in bone mass by suppressing bone turnover, we here compared the distribution and metabolism of [1 beta-3H]F6-1,25(OH)2 vitamin D3 and [1 beta-3H]1,25(OH)2 vitamin D3 in bones of rats by autoradiography and radio-HPLC. In the dosed groups, radioactivity was detected locally in the metaphysis, the modeling site in bones. As compared with the [1 beta-3H]1,25(OH)2 vitamin D3 case, [1 beta-3H]F6-1,25(OH)2 vitamin D3 was significantly retained in this site, and moreover, it mainly persisted as unchanged compound and ST-232. These findings indicate that the reason for the higher potency of F6-1,25(OH)2 vitamin D3 than 1,25(OH)2 vitamin D3 in bones are linked with increased distribution and reduced metabolism.