Hisao Matsui

Comparative study of the metabolism of triphenyltin in hamsters and rats after a single oral treatment with triphenyltin chloride

Our previous work has shown that triphenyltin compound induces the diabetogenic effects, such as hyperglycemia and hypertriglyceridemia, on hamsters, but not on rats. In the present study, it is examined whether the species differences in the metabolic fate of triphenyltin exist for susceptibility between hamsters and rats. Triphenyltin chloride was orally dosed to hamsters and rats, and triphenyltin and its metabolites, mono- and diphenyltin, and inorganic tin, in liver, kidney, pancreas, brain, and blood were determined by gas chromatography periodically for 96 h after the treatment. Triphenyltin levels in the tissues of both species were almost maxima within 24 h after treatment. Although there were relatively high levels of triphenyltin in the tissues of hamsters dosed with triphenyltin chloride, compared with those in the rats, the proportion of metabolites to triphenyltin were lower than those in the rats. In particular, hamsters were more susceptible than rats to the pancreatic accumulations of triphenyltin and good correlation exists between the tin concentrations in the pancreas and the plasma glucose levels in triphenyltin-treated hamsters. These findings suggest that the dearylation of absorbed triphenyltin in hamsters is slower than that in rats and that triphenyltin-induced hyperglycemic action depends upon the amount of tin compounds absorbed into the pancreas. Furthermore, most of the tin compounds in the brains of both species were triphenyltin. This result shows that the metabolism of triphenyltin in the brains of both species was different from that in other tissues.

Glucose Intolerance Induced by a High-Fat/ Low-Carbohydrate Diet in Rats

We examined the time course of effects of a high-fat/low-carbohydrate (HF/LC) diet on the impairment of glucose tolerance in rats, clarified whether insulin secretion and sensitivity were impaired by the HF/LC diet, and investigated the relationship between the increased nonesterified fatty acids (NEFA) after HF/LC diet feeding and insulin secretion and sensitivity. We found that glucose tolerance and the postglucose-loading insulin secretion were impaired after 3 and 7 d on the HF/LC diet. The glucose intolerance was accompanied by a rise in the fasting plasma NEFA level. When stimulated with 15 mmol/L of glucose, the insulin secretion was impaired in pancreatic islets from rats fed the HF/LC diet. Rats fed the HF/LC diet showed insulin resistance in vivo. The glucose-stimulated insulin secretion was inhibited in the islets following 24-h culture with palmitic acid. The 24-h infusion of palmitic acid decreased whole-body insulin sensitivity. In summary, at least 3 d on a HF/LC diet is needed to induce glucose intolerance in rats, and the impairment may be induced by decreased insulin secretion and sensitivity, which is related to the increase in the plasma NEFA level.

An ethanol-extract of Ampelopsis brevipedunculata (Vitaceae) berries decreases ferrous iron-stimulated hepatocyte injury in culture.

We characterized the effects of an ethanol-extract of the berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae), a plant used in folk medicine to treat liver disease, on rat hepatocyte injury occurring spontaneously, stimulated with ferrous iron and with xanthine oxidase in combination with hypoxanthine or stimulated with ethanol in serum-free culture. Total intracellular and extracellular activities of lactate dehydrogenase (LDH) accumulating during incubation and the percentage of intracellular LDH activity released into culture medium were routinely measured, to evaluate the degree of the injury. The extract decreased a high level of LDH release spontaneously occurring and an elevated level of LDH release stimulated with ferrous iron to approximately the level caused by antioxidants, such as superoxide dismutase, pyruvate and dimethyl sulfoxide. Xanthine oxidase-stimulated LDH release was not decreased by the extract. Ethanol-stimulated LDH release was decreased by the extract when the spontaneous release level was comparatively high. These results indicate that the extract inhibits intact hepatocytes from degrading, by the toxic effect of iron released from primary injured hepatocytes through the generation of reactive oxygen species. The major antitoxic activity of the extract was found in an undialyzable fraction. Sugars were necessary to exert the activity as estimated by periodate oxidation of the extract.

Effects of pretreatment with cytochrome P-450 inducers, especially phenobarbital on triphenyltin metabolism and toxicity in hamsters.

The effects of cytochrome P-450 (CYP) induction by phenobarbital (PB), CYP 2B, 2C, and 3A inducer in mammalians, on triphenyltin metabolism and toxicity in hamsters were studied. A single dose of 50 mg/kg of triphenyltin chloride was given by gavage to hamsters after pretreatment with or without PB for 3 days continuously at a daily dose of 80 mg/kg intraperitoneally (i.p.). Although the triphenyltin produced marked but reversible hyperglycemia and hypertriglyceridemia in PB-untreated hamsters, the pretreatment of hamsters with PB, which increased levels of CYP, suppressed the diabetogenic effects compared with PB-untreated hamsters. Furthermore, we investigated whether the mitigation of triphenyltin-induced diabetogenic toxicity by PB pretreatment is due to an alteration of triphenyltin metabolism. Triphenyltin and its metabolites in liver, kidneys, pancreas and brain were determined by gas chromatography periodically for 96 h after triphenyltin administration in both groups of hamsters. The initial triphenyltin levels in the tissues of PB-pretreated hamsters were about half of those in the tissues of PB-untreated hamsters and PB pretreatment accelerated metabolism of triphenyltin at early stage in hamsters. We also examined the other CYP 1A and 2A inducers, beta-naphthoflavone (B-NF) and 3-methylcholanthrene (MC). The PB pretreatment showed the strongest suppression of the toxicity at 24 h after the triphenyltin intubation, compared with the effects of B-NF and MC. In addition, the maximum proportion of diphenyltin to parent triphenyltin in pancreas was observed in PB-treated hamsters. These findings suggest that the induction of CYP system enzymes affects the metabolism and toxicity of triphenyltin in hamsters. Especially, based on effects of PB and other CYP inducers, PB induction has a key role in suppressing the diabetogenic action of triphenyltin, i.e. by decreasing triphenyltin accumulation in the hamsters.

Impaired Cytosolic Ca2+ Response to Glucose and Gastric Inhibitory Polypeptide in Pancreatic β-Cells from Triphenyltin-Induced Diabetic Hamster

Oral administration of a single dose of triphenyltin compounds induces diabetes with decreased insulin secretion in rabbits and hamsters after 2–3 days without any morphological changes in pancreatic islets. In the present study, to test the possibility that the impaired insulin secretion induced by triphenyltin compounds could result from an impaired Ca2+ response in pancreatic β-cells, we investigated the effect of triphenyltin-chloride (TPTCl) administration on the changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by secretagogues, such as glucose, high K+, gastric inhibitory polypeptide (GIP), and acetylcholine (ACh) in hamster pancreatic β-cells. TPTCl administration caused partial suppression in 10 mm K+-induced rise in [Ca2+]i without suppressing the increase in [Ca2+]i evoked by 20–50 mm K+. Administration of TPTCl strongly inhibited the rises in [Ca2+]i induced by 27.8 mm glucose, 100 μm ACh in the presence of 5.5 mm glucose, and by 100 nm GIP in the presence of 5.5 mm glucose. In t...

Effects of pretreatment with SKF-525A on triphenyltin metabolism and toxicity in mice

The effects of cytochrome P-450 inhibition by alpha-phenyl-alpha-propylbenzeneacetic acid 2-[diethylamino]-ethyl ester hydrochloride (SKF-525A), which inhibits the activity of a number of cytochrome P-450s, on triphenyltin metabolism and toxicity in mice were studied. At 24 h after triphenyltin administration, the triphenyltin levels in the tissues of SKF-525A-pretreated mice were about three times of those in the tissues of SKF-525A-untreated mice and the ratio of metabolites to parent triphenyltin in the tissues of SKF-525A-pretreated mice was lower than those in the tissues of SKF-525A-untreated mice. These data indicate that the pretreatment of SKF-525A decelerated the triphenyltin metabolism and increased triphenyltin accumulation in the tissues of mice. Although triphenyltin did not affect plasma glucose levels of in the SKF-525A-untreated mice, the triphenyltin produced marked hyperglycemia in SKF-525A-pretreated mice. These results suggest that the inhibition of cytochrome P-450 system enzymes by SKF-525A affects the metabolism and toxicity of triphenyltin and has a key role in inducing the hyperglycemic action of triphenyltin, i.e. by increasing triphenyltin accumulation in the mice.

Gas chromatographic determination of inorganic tin in rat urine after a single oral administration of stannous chloride and mono-, di-, and triphenyltin chloride

A method is described for the determination of inorganic tin by gas chromatography with flame photometric detection. The inorganic tins, stannous and stannic, were extracted with hydrochloric acid and n-hexane-benzene in the presence of 0.05% tropolone, and both inorganic tins were pentylated to tetrapentyltin with a Grignard reagent prior to gas chromatography. The absolute limit of detection for tetrapentyltin was 3 pg as tin. The recovery of stannous chloride added to rat urine samples was 80.2 +/- 2.4% (mean +/- S.D., n = 8). The application of this method to the study of urinary excretion of inorganic tin and organotin compounds in rats following oral administration of tin compounds is presented. The urinary excretion of tin compounds was observed over a period of 96 h following administration of stannous chloride or phenyltin compounds. Most of the inorganic tin was excreted into urine within 24 h after administration of stannous chloride. In the experiments on organotin administration, the level of the excretion as total tin for monophenyltin reached a maximum ca. 0-24 h after administration, whereas the maxima for di- and triphenyltin were found after 24-48 h and 48-72 h, respectively. The predominant excretion product of these tin compounds found in urine was monophenyltin.

Effects of Ampelopsis brevipedunculata (Vitaceae) extract on hepatic M cell culture: function in collagen biosynthesis

A spirits-extracted fraction of berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae) is used in Japanese folk medicine to treat liver disease. Since such an extract has been shown to inhibit formation of collagen fibers by rat hepatic M cells, it was felt that the extract acts as an inhibitor of hepatic fibrosis. Amino acid analysis of fibrous substances developing on M cell layers and of a cell lysate fraction indicated that an A. brevipedunculata extract inhibited collagen formation. Biosynthesis of non-collagenous proteins and collagen was evaluated by measuring the extent of [3H]tryptophan incorporation into a protein fraction and the rate of [3H]proline incorporation into a collagenase-digestible fraction, respectively. In contrast to the results of the analysis of the fibrous substances, the A. brevipedunculata extract failed to decrease synthesis of non-collagenous proteins and collagen unless cell proliferation was inhibited. There was no detectable level of collagenolytic activity in the M cell culture with the A. brevipedunculata extract. The decrease in accumulation of collagen, therefore, appeared to be a consequence of the proliferation-inhibitory effect of the A. brevipedunculata extract. Such inhibitory activity was found in a macromolecular fraction that contained abundant sugars but lacked proteins.

Ampelopsis brevipedunculata (Vitaceae) extract stimulates collagen synthesis through superoxide generation in the serum-free cultures of rat dermal fibroblasts and Ito cells

We describe the effects of an ethanol-extracted fraction of berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae), a plant used in folk medicine to treat liver disease, on the synthesis of non-collagenous proteins and collagen by rat collagen-producible cells such as dermal fibroblasts and liver non-parenchymal Ito cells. The generation of superoxide and hydroxyl radical was assessed by measuring the reduction of cytochrome c and the formation of thiobarbituric acid-reactive substances from deoxyribose, respectively. The synthesis of non-collagenous proteins and collagen as evaluated by measuring the extent of [3H]tryptophan incorporation into a total protein fraction of culture products and the [3H]proline-incorporating rate into a collagenase-digestible protein fraction, respectively. Both types of cells promptly synthesized only collagen in response to a dialyzable fraction of the extract. Major activity to generate oxygen free radicals accumulated in the dialyzable fraction whereas activity to decrease ferrous iron-mediated generation of the radicals accumulated in an undialyzable fraction of the extract. Stimulation of collagen synthesis was caused by superoxide because addition of superoxide dismutase but not pyruvate, an antioxidant of hydrogen peroxide, or dimethyl sulfoxide, an antioxidant of the hydroxyl radical, abrogated the stimulatory effect. The extract may arrest the progress of liver injury mediated by oxygen free radicals generated in the presence of ferrous iron.

Inhibitory effects of phenyltin compounds on stimulus-induced changes in cytosolic free calcium and plasma membrane potential of human neutrophils

To clarify the inhibitory mechanisms by triphenyltin chloride (TPTCl) on Superoxide anion formation in neutrophils, the effects of phenyltin compounds [TPTCl, diphenyltin dichloride (DPTCl2) and phenyltin trichloride (MPTCl3)] on the increase of cytosolic free calcium and the changes in membrane potential in neutrophils stimulated byn-formyl-methionyl-leucyl-phenylalanine (FMLP) were examined. TPTCl and DPTCh concentration dependently inhibited the increase of fluorescence intensity of the dye 3,3′-dipropyl-thiodicarbocyanine iodide [diS-C3-(5)] (membrane potential probe) in neutrophils induced by 0.1ΜM FMLP in the presence or absence of extracellular calcium (1.26 mM). TPTCl had a greater inhibitory effect on FMLP-mediated membrane potential change than that of DPTCl2. In the presence of extracellular calcium, TPTCl and DPTCl2 increased intracellular free calcium ([Ca2+]i) of unstimulated fura-2-loaded neutrophils at concentrations from 1.0 to 10 ΜM TPTCl and from 2.5 to 10 ΜM DPTCl2. TPTCl and DPTCl2 also increased slightly, in the absence of extracellular calcium, [Ca2+]i without stimulation of FMLP in neutrophils. However, TPTCl and DPTCl2 significantly inhibited the rise of [Ca2+]i in neutrophils stimulated by FMLP at concentrations from 2.5 ΜM to 10 ΜM TPTCl and at a concentration of 10 ΜM DPTCl2 in the absence of extracellular calcium. TPTCl and DPTCl2 significantly inhibited the Superoxide anion production by FMLP at concentrations over 2.5 ΜM in the presence of extracellular calcium. In the absence of extracellular calcium, TPTCl and DPTCl2 also inhibited the Superoxide anion production by FMLP at concentrations over 1.5 ΜM TPTCl and over 5.0 ΜM DPTCl2. Our results suggest that TPTCl and DPTCl2 are potent inhibitors of membrane potential change and intracellular Ca2+ signal transduction in FMLP stimulation, in association with superoxide production in neutrophils.