Reduction in peripheral lymphocytes and thymus atrophy induced by organotin compounds in vivo.

To clarify the involvement of apoptosis in the immunotoxicity of organotin compounds, we examined the induction of apoptosis in the peripheral lymphocytes and thymus of mice treated with triphenyltin (TPT), tributyltin (TBT) or dexamethasone (Dex). Application of TPT or TBT and Dex resulted in a transient reduction in peripheral lymphocytes at 3 to 6 hr, and thymus atrophy was observed at 6 and 24 hr after administration. Lymphocyte subpopulation analysis showed that TPT and TBT induced a greater reduction in B cells than in T cells. The maximum levels of organotin in the blood were about 450 ng TPT/ml in the TPT-treated mice, and 170 ng TBT/ml in the TBT-treated mice. When the isolated peripheral lymphocytes were incubated with the organotins at 500 ng/ml, TPT and TBT induced necrosis in over 70% of cells, while both organotins caused lower percentages of apoptosis as well as necrosis after 3 hr at 100 ng/ml. In the thymus, although in vivo treatment of mice with Dex caused apoptosis, neither apoptotic nor necrotic thymocytes were observed in the TPT- and TBT-treated mice, indicating that the thymus atrophy might be caused by the antiproliferative effects of these organotin compounds. Thus, our results did not support the idea that apoptosis played a decisive part in the immunotoxicity of the organotin compounds in vivo.

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate.

Hydroxyl radical (*OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO3) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the *OH generation after injection of Fe-NTA or KBrO3 in the kidneys. A significant increase in 8-hydroxy-2'-deoxyguanosine in nuclei of the kidney was detected only in the KBrO3 treated mice, while the comet assay showed that the Fe-NTA and KBrO3 treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by *OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of *OH generation in cellular injury induced by chemicals in vivo.

Assessment of DNA damage in multiple organs of mice after whole body X-irradiation using the comet assay.

The comet assay was performed to elucidate the linearity of calibration curves and detection limits for DNA damage in multiple organs of whole body X-irradiated mice, and rates of reduction in DNA damage by DNA repair during the irradiation period were estimated in the respective organs by comparing the rates of increase in DNA damage at different absorbed dose rates of X-rays. Of the assay parameters, tail length and the percentage DNA in the tail showed a higher sensitivity to DNA damage in most organs than Olive tail moment. Data at the higher absorbed dose rates (2.22 or 1.44 Gy/min) showed good correlations between absorbed doses and these two parameters, with correlation coefficients of more than 0.7 in many organs. However, this assay had difficulty detecting DNA damage at the lower absorption dose rate (0.72 Gy/min). The estimated rates of increase in DNA damage and those of DNA repair during the irradiation period in the respective organs suggested differences in the radiosensitivity of nuclear DNA and DNA repair capacity among organs. Our results indicated that absorbed dose rates of 1.0-1.3 Gy/min or greater were needed to induce detectable DNA damages by the comet assay in many organs.

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation.

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.