Oxidative cell damage in Kat-sod assay of oxyhalides as inorganic disinfection by-products and their occurrence by ozonation.

Nine oxyhalides as possible inorganic disinfection by-products were tested for oxidative cell damage by Kat-sod assay with E. coli mutant strains deficient in the active oxygen-scavenging enzymes. Chlorine dioxide, chlorite, and iodate were highly cytotoxic, whereas in the presence of cysteine, bromate (BrO3-) and metaperiodate (IO4-) showed more growth inhibition toward the superoxide dismutase-deficient strains than the wild strain. BrO3- also showed oxidative mutagenicity with cysteine or glutathione ethyl ester in S. typhimurium TA 100. To identify oxyhalides formed by ozonation of raw water containing sea water, the occurrence of ozonation by-products of bromide and iodide was investigated. The results indicate that BrO3- is toxicologically one of the most remarkable oxyhalides detectable in drinking water because IO4- was not detected in the ozonated solution of iodide, and the ozonation condition to lower BrO3- is to keep it neutral in the presence of ammonium ion.

[Selenium methylation and toxicity mechanism of selenocystine].

Selenium is an essential trace element and a toxicant for animals. Selenocystine, a selenium-containing amino acid, is one of the chemical forms in which selenium exists in food. This review summarized recent studies on the toxicity mechanism of selenocystine in experimental animals. Hepatotoxicity is caused by repeated oral administration of selenocystine. Selenocystine is metabolized by reduced glutathione and/or glutathione reductase to hydrogen selenide via selenocysteine-glutathione selenenyl sulfide. The hydrogen selenide is a key intermediate in the selenium methylation metabolism of inorganic and organic selenium compounds. Accumulation of the hydrogen selenide resulting from inhibition of the selenium methylation metabolism, detoxification metabolic pathway of selenium, is found in animals following repeated administration of a toxic dose of selenocystine. The excess of the hydrogen selenide produced by inhibition of the selenium methylation metabolism contributes to the hepatotoxicity caused by selenocystine.

Disinfection by-products in the chlorination of organic nitrogen compounds: by-products from kynurenine.

Volatile by-products in the chlorination of 3 humic acids as naturally-occurring substances and 37 nitrogen compounds normally found in excrement were analyzed, and as result kynurenine, a urinary metabolite of tryptophan was found a suitable model compound for dichloroacetonitrile-forming precursors. Possible pathways for the formation of chlorination by-products from kynurenine were also proposed by identification and kinetic properties of by-products decomposed further from each product.

Mechanisms of selenium methylation and toxicity in mice treated with selenocystine.

Mechanisms 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 50 mg/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 20 mg/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 (100 mumol/kg), a known potent inhibitor of the SAM-dependent methyltransferase, at 30 min before oral treatment of Se-Cys (10, 20, of 50 mg/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.

This 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 (50 mg/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 beta-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 glutathione reductase yielding CySeH, which is decomposed by selenocysteine beta-lyase to hydrogen selenide. CySeSG may be a stable precursor of hydrogen selenide in animals.

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.

Distribution and chemical form of selenium in mice after administration of selenocystine.

To elucidate the relationship between chemical forms of selenium in tissues and subacute liver damage induced by selenocystine (T. Hasegawa et al., Arch. Toxicol., 68, 91 (1994)), the distribution and chemical form of selenium were investigated in ICR male mice treated with the chemical orally (50 mg/kg) and intravenously (5 mg/kg). The time-distribution of selenium in plasma, erythrocytes and liver after separate administration varied. However, Sephadex G-150 chromatograms of plasma, and stroma-free hemolysate from mice treated orally or intravenously with selenocystine, revealed that selenium exists mainly in the albumin and hemoglobin fractions, respectively, and is neither route- or time-dependent. Sephadex G-150 chromatograms of liver cytosol of the animals 1 h after oral administration or 1 and 6 h after intravenous administration showed two selenium-containing fractions, void volume and a low-molecular fraction (Kav = 0.85); 6 h after oral treatment, however, animals had an additional high-molecular fraction (Kav = 0.45). Levels of acid-volatile selenium and dialyzable selenium in the fraction with a Kav value of 0.45 were similar, being 31.2% and 30.3%, respectively. No acid-volatile selenium was recognized in the non-dialyzable high-molecular fraction. The present study demonstrated that when selenocystine is administered orally to mice, the selenium which produces acid-volatile selenium by acidification may bind to protein sulfhydryl groups in the liver cytosol; this was not seen in the case of intravenous administration.

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.

Identification of polycyclic aromatic hydrocarbons in mutagenic adsorbates to a copper-phthalocyanine derivative recovered from municipal river water.

A study was made to identify polycyclic aromatic hydrocarbons (PAHs) in the mutagenic adsorbate to blue cotton recovered from the water of the Katsura River which is a tributary of the Yodo River, a typical municipal river. As blue cotton bears a covalently bound copper-phthalocyanine derivative which can adsorb PAHs over 3 rings, PAHs in the adsorbate were separated into 4 fractions (I-IV) by Sephadex LH-20 gel chromatography. Fractions III and IV showed high direct and indirect frameshift mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98, especially in YG1024 with S9 mix, whereas these fractions showed less mutagenicity in TA98NR or TA98/1,8-DNP6. These results suggest that mutagenic nitroarenes and aminoarenes are present in both fractions. The retention times of some peaks separated from both fractions using high performance liquid chromatography (HPLC) with a fluorescence detector were identical with those of authentic PAHs. Gas chromatography-mass spectrometry of some HPLC fractions demonstrated that anthraquinone, azulene derivative, quinoline derivative, chrysene and benzo[b]fluoranthene are probably contained in these fractions.

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen.

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.

DNA lesion in rat hepatocytes induced by in vitro and in vivo exposure to glyoxal.

The alkaline elution technique was applied to measure the damage of rat hepatic DNA following exposure to glyoxal. DNA single-strand breaks were induced after exposure of primary-cultured hepatocytes to 0.1-0.6 mg/ml glyoxal for 60 min, while no DNA cross-link was observed. Single-strand breaks were also detected in livers of rats within 2 h following a single oral exposure at 200-1000 mg/kg body weight, and the frequency of the breaks reached a maximum around 9 h after exposure. The breaks were almost fully repaired 24 h after exposure to any dose. However, hardly any DNA lesions were detected in other tissues following exposure to 1000 mg/kg glyoxal. Thus, the present results indicate that glyoxal causes DNA single-strand breaks in rat hepatocytes following in vitro and in vivo exposure.

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.

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water.

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.

Mutagenicity on chlorination of products formed by ozonation of naphthoresorcinol in water.

The mutagenicity of products formed by chlorination after ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated and subsequently chlorinated naphthoresorcinol was directly mutagenic, as was ozonated naphthoresorcinol, in both strains tested. The mutagenic activity at chlorination with 8 equivalents of chlorine per mole of naphthoresorcinol after ozonation was markedly higher than that at only ozonation. Of the identified ozonation products of naphthoresorcinol, muconic acid, after chlorination with 2 or 4 equivalents of chlorine per mole of the compound, induced direct mutagenicity against TA98 and TA100. The chlorination of glyoxal with 0.5 and 1 chlorine equivalents per mole of the compound was shown to produce direct mutagenicity toward TA98. The identification of the chlorination products of these compounds is also discussed.

Generation of hypophosphatemia in rats by continuous oral administration of cadmium.

To obtain further information on the negative calcium balance caused by cadmium (Cd), the factors associated with serum calcium and phosphorus homeostasis other than inhibition of intestinal calcium absorption were studied by using parathyroid hormone (PTH) and 1 alpha-hydroxycholecalciferol (1 alpha-OH-D3). In rats exposed to Cd for 30 or 90 days, the concentrations of serum calcium after treatment with PTH, parathyroidectomy (PTX) or 1 alpha-OH-D3 showed almost the same patterns as those of control animals. It was considered that the mechanism of regulation of calcium in Cd-exposed rats was normal. The continuous oral administration of Cd generated hypophosphatemia in rats. On the other hand, in 30- and 90-day-treated rats, the low concentration of serum phosphorus caused by Cd was further decreased by administration of PTH, whereas it was increased by PTX. The hypophosphatemia found in rats exposed to Cd for 30 days, but not for 90 days, was reversed by treatment with 1 alpha-OH-D3. From these results, it was concluded that the hypophosphatemia caused by long term oral administration of Cd resulted from secondary hyperparathyroidism, due to inhibited calcium absorption from the intestine, as was demonstrated previously.

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions.

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.

Influence of cadmium on the metabolism of vitamin D3 in rats.

In order to obtain further information on the effects of cadmium (Cd) on the mechanism of activation of vitamin D3 in the kidney, the serum concentrations of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] were determined by radioimmunoassay techniques. The serum concentration of 1,25(OH)2D3 in Cd-exposed rats was always higher than that in control rats. The concentrations of serum 1,25(OH)2D3 in parathyroidectomized rats (PTX), both in the control and in the Cd-exposed groups, were markedly lower than those in non-PTX rats. On the other hand, the concentration of serum 24,25(OH)2D3 in Cd-exposed rats was less than that in control rats. In other words, the pathway from secretion of parathyroid hormone (PTH) to synthesis of 1,25(OH)2D3 or 24,25(OH)2D3 was normal, but the pathway from stimulation of PTH to secretion of PTH was abnormal. These results suggest that rats exposed to Cd for 90 days were in a state of either deficiency of 1,25(OH)2D3 or excess secretion of parathyroid hormone. Although hypophosphatemia occurred in the Cd-exposed rats, the 1,25(OH)2D3 serum level in rats was not increased by PTX. On the basis of these results, it is suggested that hypophosphatemia occurring after the exposure of rats to Cd is a secondary hypophosphatemia.

Comparative studies of chromosomal aberration induced by trivalent and pentavalent arsenic.

The comparative cytogenetic effects on the synthesis of DNA, RNA and protein in cultured mammalian cells of trivalent and pentavalent arsenic were investigated. The chromosome-breaking activity in cultured leukocytes was significantly higher for the compounds with trivalent (NaAsO2, AsCl3 and As2O3) than with pentavalent arsenic (Na2HAsO4, H3AsO4 and As2O5). The activity in cultured human skin fibroblasts was similar to that in leukocyte cultures. The colony-forming capacity after exposure to arsenicals indicated that trivalent was more toxic than pentavalent arsenic. In the response of DNA, RNA and protein synthesis, both trivalent and pentavalent arsenic inhibited DNA and protein synthesis in leukocytes.

Metabolic fate of chromium compounds. I. Comparative behavior of chromium in rat administered with Na251CrO4 and 51CrCl3.

Comparative metabolic fate of labelled chromium chloride and sodium chromate and interaction of these compounds in the rat liver and blood were investigated after their oral and intravenous administration. Gastrointestinal absorption of both compounds was below 1% of the oral dose, but trivalent chromium showed higher radioactivity than the hexavalent form in rats (biological half-life: CrCl3 91.79 days, Na2CrO4 22.24 days). The higher residual activity of the trivalent chromium was also observed after intravenous administration. Both forms of chromium were excreted more in the urine via the kidney than in the intestinal tract after intravenous administration. When 51CrCl3 and Na251CrO4 were injected into rats, in the time-distribution patterns of 51Cr in the organs, a significant difference was shown between oxidation states of the two compounds, especially in subcellular fractions of the liver and blood constituents. This significant difference mainly observed in the rat blood came from the fact that trivalent chromium possessed a high binding activity for transferrin in plasma, while hexavalent chromium was permeable into red cells and bound with hemoglobin.