Nobuyuki Susa

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.

Protective effect of vitamin E on chromium (VI)-induced cytotoxicity and lipid peroxidation in primary cultures of rat hepatocytes

Abstract Pretreatment of primary cultures of rat hepatocytes with α-tocopherol succinate (vitamin E) for 20 h prior to exposure to K2Cr2O7 resulted in a marked decrease of chromium (VI)-induced cytotoxicity, as evaluated by the leakage of lactate dehydrogenase, without affecting cellular uptake and subcellular distribution of chromium. The levels of chromium (VI)-induced lipid peroxidation, as monitored by malondialdehyde formation, were also inhibited by pretreatment with the vitamin. Pretreatment with vitamin E normalized the levels of nonenzymatic antioxidants such as glutathione and vitamin C suppressed by dichromate, and caused a distinct accumulation of vitamin E in hepatocytes. However, vitamin E pretreatment did not affect the activities of enzymatic antioxidants including glutathione reductase, superoxide dismutase, and catalase suppressed by dichromate. These results indicate that the protective effect of vitamin E against chromium (VI)-induced cytotoxicity as well as lipid peroxidation, may be associated more with the level of nonenzymatic antioxidants than the activity of enzymatic antioxidants.

The comet assay in eight mouse organs: results with 24 azo compounds

The genotoxicity of 24 azo compounds selected from IARC (International Agency for Research on Cancer) groups 2A, 2B, and 3 were determined by the comet (alkaline single cell gel electrophoresis, SCG) assay in eight mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8, and 24 h after treatment. For the 17 azo compounds, the assay was positive in at least one organ; (1) 14 and 12 azo compounds induced DNA damage in the colon and liver, respectively, (2) the genotoxic effect of most of them was greatest in the colon, and (3) there were high positive responses in the gastrointestinal organs, but those organs are not targets for carcinogenesis. One possible explanation for this discrepancy is that the assay detects DNA damage induced shortly after administration of a relatively high dose, while carcinogenicity is detected after long treatment with relatively low doses. The metabolic enzymes may become saturated following high doses and the rates and pathways of metabolic activation and detoxification may differ following high single doses vs. low long-term doses. Furthermore, considering that spontaneous colon tumors are very rare in rats and mice, the ability to detect tumorigenic effects in the colon of those animals might be lower than the ability to detect genotoxic events in the comet assay. The in vivo comet assay, which has advantage of reflecting test chemical absorption, distribution, and excretion as well as metabolism, should be effective for estimating the risk posed by azo dyes to humans in spite of the difference in dosage regimen.

Butyltin and phenyltin compounds in some marine fishery products on the Japanese market.

The degree of butyltin and phenyltin contamination was determined in samples of 11 species of fish products that were representative of the Japanese fish market. We observed high contamination levels of these organotin compounds in cultured marine products. Mean butyltin contamination of natural (nonfarmed) marine products, however, were relatively low, compared with the cultured products. Phenyltin concentrations in the described samples were generally much lower than levels of butyltin compounds. Levels of organotin compounds in these marine products have decreased drastically as a result of legal controls instituted by the Japanese government in 1990. On the basis of calculated tolerable daily intake levels of tributyltin and triphenyltin, we concluded that the levels of organotin compounds in the marine products on the Japanese market were not sufficiently high to have any effect on human health. However, we also determined that some of the cultured marine products were contaminated with undesirable levels of tributyltin.

Vibrio vulnificus induces macrophage apoptosis in vitro and in vivo.

ABSTRACT In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of V. vulnificus.

Comparison of hepatotoxicity caused by mono-, di- and tributyltin compounds in mice

The in vivo induction of hepatotoxicity, as evaluated by the activity of ornithine carbamyl transferase in serum, was investigated in mice administered orally with the following three butyltin compounds: tributyltin chloride (TBTC), dibutyltin dichloride (DBTC) and monobutyltin trichloride (MBTC). The minimal concentrations of TBTC and DBTC that caused hepatotoxicity at 24 h after oral administration were 180 μmol and 60 μmol/kg, respectively, while MBTC did not induce liver injury even at 7000 μmol/kg. Additionally, when the administered doses were equivalent (180 μmol/kg), a time course (3–96 h) study revealed that the hepatotoxicity of TBTC and DBTC appeared at 24 and 12 h, respectively, but that MBTC showed no hepatotoxicity even at 96 h. The amounts of Sn excreted into urine for 4 days were 1.5 fold greater with TBTC than with DBTC treatment and were lowest in MBTC group. Similarly, the total liver Sn content was 2- to 5-fold greater in the TBTC group than in the DBTC group whereas the liver Sn content in the MBTC treatment showed the lowest value throughout the 3- to 96-h period. Thus, the non-hepatotoxicity of MBTC may be due either to low absorption through the digestive tract of mice or to the low levels of Sn in liver; however, the level of Sn in liver was not associated with the induction of hepatotoxicity by TBTC and DBTC. The analysis of metabolites of TBTC (180 μmol/kg) and DBTC (60 μmol/kg) at equivalent hepatotoxicity showed that the main tin compounds in the liver after the administration of TBTC were dibutyltin and monobutyltin as well as inorganic tin compounds, while most (>78%) of the total tin compounds in the liver of mice treated with DBTC was in the form of dibutyltin. In addition, the levels of monobutyltin and inorganic tin compounds in the livers of mice treated with TBTC were greater than those with DBTC, but the levels of dibutyltin did not differ significantly between TBTC and DBTC. The levels of lipid peroxidation (LPO) and hepatic glutathione (GSH) content in the liver showed a transitory increase after the administration of MBTC and TBTC, respectively. These results suggest that DBTC is more hepatotoxic than TBTC, and that dibutyltin inside the cells may be the main form of tin which is responsible for induction of hepatotoxicity following in vivo administration of TBTC and DBTC. The generation of free radical species, as evaluated by LPO and GSH levels, may not be associated with the hepatotoxicity caused by butyltin compounds.

Organ-specific genotoxicity of the potent rodent bladder carcinogens o-anisidine and p-cresidine

We used a modification of the alkaline single-cell gel electrophoresis (SCG) (Comet) assay to evaluate the in vivo genotoxicity of two potent rodent bladder carcinogens, o-anisidine and p-cresidine, in mouse liver, lung, kidney, brain, and bone marrow, and in the mucosa of stomach, colon, and bladder. Male CD-1 mice (8 weeks old) were sacrificed 3 and 24 h after oral administration of o-anisidine at 690 mg/kg or p-cresidine at 595 mg/kg. Both chemicals were dissolved in olive oil. Both chemicals yielded statistically significant DNA damage in bladder mucosa 3 and 24 h after treatment. o-Anisidine yielded DNA damage in the colon at 3 h, but not at 24 h. No significant effects were observed in any other organs. Our results suggest the importance of the urinary bladder as a sentinel organ for evaluating chemical genotoxicity in rodents.

Vanadate and chromate reduction by saccharides and L-ascorbic acid: effect of the isolated V(IV) and Cr(III) products on DNA nicking, lipid peroxidation, cytotoxicity and on enzymatic and non-enzymatic antioxidants

Vanadate and chromate reduction in the presence of biocomponents like saccharides and L-ascorbic acid is demonstrated to generate V(IV) and Cr(III) species, respectively. The incipiently formed (V(IV) and Cr(III) species are demonstrated to cause single strand and plasmid DNA nicking, lipid peroxidation and cytotoxicity in isolated rat hepatocytes. The V(IV) complexes depleted the levels of non-enzymatic antioxidants like L-ascorbic acid and glutathione and parallelly increased levels of enzymatic antioxidants like glutathione reductase, glutathione S-transferase, glutathione peroxidase, superoxide dismutase and catalases. However, the Cr(III) species did not have any significant effect on the antioxidant enzymes. Thus the reducing and complexing abilities of biogenic ligands and the corresponding effects of the V(IV) and Cr(III) species generated from vanadate and chromate reduction, respectively, may lead to a better understanding of the toxic effects of the corresponding metal ions.

Protective effect of deferoxamine on chromium (VI)-induced DNA single-strand breaks, cytotoxicity, and lipid peroxidation in primary cultures of rat hepatocytes.

Abstract Incubation of primary cultures of rat hepatocytes with K2Cr2O7 and deferoxamine (DFO), an iron chelator, resulted in a marked decrease in cellular levels of DNA single-strand breaks caused by K2Cr2O7. Cellular treatment with DFO also suppressed both dichromate-induced cytotoxicity – evaluated by the leakage of lactate dehydrogenase, and lipid peroxidation – as monitored by malondialdehyde formation. In addition, treatment with DFO attenuated the suppression of the levels of vitamin E and C as well as the inhibition of alkaline phosphatase and glutathione peroxidase activity attributed to K2Cr2O7. However, DFO had no influence on the cellular level of glutathione or the activity of glutathione reductase and superoxide dismutase suppressed by dichromate. Under the same experimental conditions, cellular uptake and distribution of chromium were not affected by DFO. These results indicate that DFO protects cells from chromium(VI)-induced DNA strand breaks, cytotoxicity, lipid peroxidation, vitamin E and C depression, and glutathione peroxidase inhibition. The role of antioxidants in chromium(VI)-induced cytotoxicity, DNA breaks, and lipid peroxidation is discussed.

Role of cytochrome P450 in hepatotoxicity induced by di- and tributyltin compounds in mice

The role of cytochrome P450 in the induction of hepatotoxicity by butyltin compounds such as tributyltin chloride (TBTC) and dibutyltin dichloride (DBTC) was investigated in vivo. The pretreatment of mice with SKF-525A, which decreased hepatic levels of cytochrome P450, suppressed TBTC-induced hepatotoxicity, as estimated by serum ornithine carbamyl transferase activity, whereas pretreatment with phenobarbital (PB), which increased the levels of cytochrome P450, enhanced the hepatotoxicity of TBTC. In the case of DBTC, PB pretreatment enhanced hepatotoxicity, while SKF-525A had no effect. Under these experimental conditions only PB pretreatment was found to increase hepatic levels of tin in mice treated with TBTC. These results suggest that hepatic metabolism of butyltin compounds by cytochrome P450 is more closely related to the induction of hepatotoxicity by TBTC than by DBTC. The active tin compounds formed during hepatic metabolism, which are responsible for induction of hepatotoxicity, will be discussed