Tatsuya Hasegawa

Mechanisms of selenium methylation and toxicity in mice treated with selenocystine

Abstractu2002Mechanisms of selenium methylation and toxicity were investigated in the liver of ICR male mice treated with selenocystine. To elucidate the selenium methylation mechanism, animals received a single oral administration of selenocystine (Se-Cys; 5, 10, 20, 30, 40, or 50u2005mg/kg). In the liver, both accumulation of total selenium and production of trimethylselenonium (TMSe) as the end-product of methylation were increased by the dose of Se-Cys. A negative correlation was found between production of TMSe and level of S-adenosylmethionine (SAM) as methyl donor. The relationship between Se-Cys toxicity and selenium methylation was determined by giving mice repeated oral administration of Se-Cys (10 or 20u2005mg/kg) for 10 days. The animals exposed only to the high dose showed a significant rise of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in plasma. Urinary total selenium increased with Se-Cys dose. TMSe content in urine represented 85% of total selenium at the low dose and 25% at the high dose. The potential of Se-methylation and activity of methionine adenosyltransferase, the enzyme responsible for SAM synthesis, and the level of SAM in the liver were determined. The high dose resulted in inactivation of Se-methylation and decrease in SAM level due to the inhibition of methionine adenosyltransferase activity. To learn whether hepatic toxicity is induced by depressing selenium methylation ability, mice were injected intraperitoneally with periodate-oxidized adenosine (100u2005μmol/kg), a known potent inhibitor of the SAM-dependent methyltransferase, at 30u2005min before oral treatment of Se-Cys (10, 20, or 50u2005mg/kg). Liver toxicity induced by selenocystine was enhanced by inhibition of selenium methylation. These results suggest that TMSe was produced by SAM-dependent methyltransferases, which are identical with those involved in the methylation of inorganic selenium compounds such as selenite, in the liver of mice orally administered Se-Cys. Depression of selenium methylation ability resulting from inactivation of methionine adenosyltransferase and Se-methylation via enzymic reaction was also found in mice following repeated oral administration of a toxic dose of Se-Cys. The excess selenides accumulating during the depression of selenium methylation ability may be involved in the liver toxicity caused by Se-Cys.

Identification and metabolism of selenocysteine-glutathione selenenyl sulfide (CySeSG) in small intestine of mice orally exposed to selenocystine

Abstractu2002This investigation was carried out to elucidate the chemical form of selenium-containing metabolite in small intestine of ICR male mice orally administered selenocystine (CySeSeCy). The metabolite in intestinal cytosol of mice treated with CySeSeCy (50u2004mg/kg) was identified as selenocysteine-glutathione selenenyl sulfide (CySeSG) by high performance liquid chromatography using a gel filtration and reversed phase column. Hydrogen selenide formation was caused as a result of the anaerobic reaction between the CySeSG and liver cytosol containing selenocysteine β-lyase, which specifically acts on selenocysteine (CySeH). Effects of GSH or glutathione reductase on hydrogen selenide formation from CySeSG reacted with the liver cytosol were examined. The CySeSG was nonenzymatically reduced to CySeH by excess GSH in the liver cytosol. It was also recognized that CySeSG was enzymatically reduced to CySeH by glutathione reductase in the presence of NADPH. These results indicate that the chemical form of this metabolite is CySeSG, which has a molecular weight of 473, the CySeSG is then reduced by excess GSH and/or gluta- thione reductase yielding CySeH, which is decomposed by selenocysteine β-lyase to hydrogen selenide. CySeSG may be a stable precursor of hydrogen selenide in animals.

Subchronic oral toxicity of glyoxal via drinking water in rats

The subchronic oral toxicity of glyoxal via drinking water and the effect on in vivo protein synthesis in tissues following a single treatment with this substance were assessed in Sprague-Dawley male rats. Animals received drinking water containing glyoxal levels of 2000, 4000, and 6000 mg/liter ad libitum for 30, 60, and 90 days in Phase I. In Phase II, the high-dose and control-1 groups fed the diet ad libitum, and a diet-limited control-2 group given the same amount of diet as consumed by the high-dose group were maintained for 90 and 180 days. The study designs included observations of clinical signs, body weights, major organ weights, gross and histopathological examinations, serum clinical chemistry, and biochemical examinations such as glyoxalase activity and glutathione content in selected tissues. Body weight gain and organ weights significantly decreased with dosage. Although consumption of food and water was also depressed in the exposed group, the reduction of body weight gain was greater in the high-dose group than in the diet-limited control 2 group. Histopathological examinations revealed only a slight papillary change in the kidneys from the high-dose group at both 90 and 180 days terminations in Phase II. The induction of both glyoxalase I and II was observed in liver and erythrocytes at 30-day termination of the exposed groups. Serum enzyme and protein levels were significantly reduced by the mid- and/or high-dose exposures. With a single oral high-dose treatment of glyoxal, a great decline in the incorporation of L-[3H]leucine was shown particularly in the liver, and this probably led in part to a reduction in the serum protein levels in rats following subchronic exposure to glyoxal. These data indicated an overall low degree of systemic toxicity to rats exposed subchronically to glyoxal via drinking water.

Toxicity and chemical form of selenium in the liver of mice orally administered selenocystine for 90 days

The subacute oral toxicity of selenocystine and chemical form of selenium in the liver following exposure to this compound were assessed in ICR male mice. Animals were dosed 6 days/week for 30, 60 or 90 days with 0, 5, 10 or 15 mg/kg per day. Body weight gain decreased with dosage. The activities of aspartate aminotransferase and alanine aminotransferase in plasma were significantly elevated at the highest dose level after 60 days and at the two higher dose levels after 90 days of exposure. However, the level of selenium content in the liver was the same at the two higher dosages at both 60 and 90 days of exposure. The subcellular distribution of selenium in the liver from mice treated with selenocystine showed that the major part of the total selenium content, 68.3–72.1%, existed in the cytosolic fraction. Sephadex G-150 chromatograms of liver cytosol of the animals administered selenocystine revealed three selenium-containing fractions which involve glutathione peroxidase (molecular weight 80 000) high molecular (molecular weight 55 000–60 000) and low molecular (molecular weight <10 000) substances. Selenium content and acid-volatile selenium content in the high molecular weight fraction increased with exposure time to selenocystine. Thus, in a subacute toxicity study selenocystine given for 90 days caused hepatic damage in mice, depending on the acid-volatile selenium content in the liver cytosol.

Chemical form of selenium-containing metabolite in small intestine and liver of mice following orally administered selenocystine.

The chemical form of a selenium-containing metabolite in the small intestine following a single oral administration of selenocystine was investigated with ICR male mice. Selenium content in the small intestine of animals treated with 50 mg/kg selenocystine significantly increased 15 min, 1 h and 6 h after treatment. In contrast, selenocystine significantly depressed the intestinal reduced glutathione (GSH) level at 1 h after administration. A significant negative correlation between the selenium level and the level of GSH in the small intestine was observed (r=−0.83, p<0.001). Analysis of the intestinal metabolite of selenocystine showed that selenium-containing metabolites elute in two fractions from a Sephadex G-25 column: the low-molecular fraction (peak I) contained the selenocystine, while the high-molecular fraction (peak II) contained selenocysteine-containing metabolite. An in vitro experiment was performed to gain insight into the mechanism for selenocysteine-containing metabolite production in the intestinal cytosol. When selenocystine or selenocysteine reacted with excess GSH in the presence of intestinal homogenate, the peak II fraction which involved the selenocysteine-containing metabolite was recognized in the Sephadex G-25 chromatogram. From an examination of the distribution of the selenocysteine-containing metabolite, it was recognized that this metabolite exists in plasma and liver cytosol of mice after oral administration of selenocystine. These results suggested that the mice treated with selenocystine produce selenocysteine-containing metabolite by reaction of selenocystine with excess GSH in the small intestine, and the metabolite is then transported to the liver through blood plasma.