Cisplatin-DNA adduct formation in rat spermatozoa and its effect on fetal development.

Exposure of males to some genotoxic chemicals causes DNA damage in spermatozoa resulting in embryotoxicity and developmental defects in their offspring. This study demonstrates that cisplatin-DNA adducts could be measured in spermatozoa following treatment with the antineoplastic drug, cisplatin. The formation of spermatozoa cisplatin-DNA adducts showed dose and time-dependent increases both in vitro, and in vivo up to 168 h (7 days) after dosing. Treatment of rats with 10 mg cisplatin/kg resulted in spermatozoa Pt-GG adduct levels of approximately 1.0 fmol/microg DNA. When cisplatin-treated male rats were bred to untreated females 6-24 h after cisplatin administration, no adverse developmental effects or decreases in body weight were seen in the offspring although there was a trend towards increased early embryo mortality.

Skin sensitization testing: the relevance of rechallenge and pretreatment with sodium lauryl sulfate in the guinea pig maximization test.

The guinea pig maximization test is one of the preferred test methods for the identification of skin sensitizers. The OECD/EC test guidelines allow for the conduct of a rechallenge in case doubtful reactions are obtained after challenge. The relevance of rechallenging was investigated by performing multiple challenges (up to four) in the maximization test with four well-known sensitizers of varying strength: nickel sulfate, sulfathiazole, benzocaine, and 1-chloro-2,4-dinitrobenzene. In addition, the effect of sodium lauryl sulfate (SLS)-pretreatment during topical induction with weak sensitizers on rechallenging was investigated. In contrast to what has frequently been hypothesized, rechallenge did not result in an increase of skin reaction as compared with the reactions observed after the first treatment. SLS pretreatment was very effective in increasing the initial challenge response to weak sensitizers. Subsequent rechallenging in these cases however again showed a decrease in sensitivity of the animals.

The thymus atrophy-inducing organotin compound DBTC inhibits the binding of thymocytes to thymic epithelial cells.

In this study, it is examined whether the organotin compound di-n-butyltindichloride (DBTC), which has been shown to inhibit immature thymocyte proliferation, is able to disturb the binding between thymocytes and thymic epithelial cells (TEC). To that end, an enzyme-linked binding assay was developed in which the amount of binding of Thy-1+ (mAb ER4)-thymocytes to the rat-derived TEC-line IT45R1 (IT45R1-TEC) could be detected. It was found that preincubation of thymocytes with 3-5 microM DBTC for 30 min inhibited the binding by 50-60% during a 1 h adhesion period. By extending the preincubation period to 1 h and the adhesion period to 22 h, 0.1 microM DBTC was already sufficient to reduce the binding with 60-80%. Further characterization of the binding revealed that splenic lymphocytes were unable to bind to the MHC class II-negative IT45R1-TEC. Since dextran sulfate inhibited the binding as well, sulfated polysaccharide-binding molecules such as Thy-1 and CD2 are likely to be involved in the binding. Electron microscopy showed filament-containing microvilli at the site of interaction. The results are discussed in relation to the mechanism of DBTC-induced thymus atrophy.

Comparative toxicity of organotin compounds to rainbow trout (Oncorhynchus mykiss) yolk sac fry.

The comparative toxicity of various organotin compounds was investigated in early life stages of the rainbow trout. Beginning with yolk sac fry, trout were continuously exposed for 110 days to tributyl- (TBTC), triphenyl- (TPhTC) or tricyclohexyltin chloride (TCHTC) at concentrations of 0.12-15 nM, to trimethyltin chloride (TMTC) at concentrations of 3-75 nM or to dibutyl- (DBTC) or diphenyltin chloride (DPhTC) at 160-4000 nM. The diorganotin compounds DBTC and DPhTC were about three orders of magnitude less toxic than the triorganotin homologs TBTC and TPhTC. Both for DBTC and DPhTC, a no-observable-effect concentration (NOEC) of 160 nM was established, corresponding to 40 and 60 ppb, respectively. Of the triorganotin compounds, TCHTC appeared to be the most toxic, inducing 100% mortality within 1 week at a concentration of 3 nM. Only a few trout survived exposure to 0.6 nM TCHTC for 110 days. TBTC and TPhTC caused acute mortality at a concentration of 15 nM. For both TBTC and TPhTC a NOEC of 0.12 nM was established, corresponding to water concentrations of 40 and 50 ppt, respectively. Histopathological examination revealed depletion of glycogen in liver cells of both di- and triorganotin exposed fish, except in the case of TMTC. No signs of toxicity were observed in fish exposed to up to 75 nM TMTC, the highest concentration tested. Atrophy of the thymus, the most prominent sign of toxicity of di- and tributyltin compounds in mammalian species, was not observed in early life stages of rainbow trout. Tail melanization was observed in the groups exposed to 3 nM TPhTC, 3 nM TBTC, 800 nM DBTC and 800 nM DPhTC. At the end of the exposure period, resistance to infection was examined by an intraperitoneal challenge with Aeromonas hydrophila, a secondary pathogenic bacterium to fish. Resistance of bacterial challenge was found to be decreased even at the lowest-effect concentration of both di- and triorganotin compounds.

Dibutyltin and tributyltin compounds induce thymus atrophy in rats due to a selective action on thymic lymphoblasts.

Di-n-butyltin dichloride (DBTC) or tri-n-butyltin chloride (TBTC) given in the diets of rats have previously been shown to cause atrophy of the thymus and subsequently suppression of the T-cell-dependent immune responses. To study the mechanism of the immunotoxic effects, the dose-effect relationships and the kinetics of the thymus atrophy caused by DBTC and TBTC were investigated in detail. A single oral dose of DBTC or TBTC to rats induced a dose-related reduction of relative thymus weight, which was maximal 4 days after intubation. The log dose-effect relationships for both compounds were linear and ran parallel over a dose range of 5-60 mg/kg. Dose levels calculated to cause 50% reduction of relative thymus weight were 18 mg DBTC and 29 mg TBTC per kg body wt. A single oral dose of mono-n-butyltin trichloride (MBTC), however, did not cause thymus atrophy at dose levels up to 180 mg/kg. The kinetics of the dibutyltin- and tributyltin-induced thymus atrophy in rats were investigated by measuring thymus weight, total thymic cell count, number of small, intermediate and large cells and the incorporation of DNA, RNA and protein precursors into isolated thymocytes during a period of 9 days after a single oral dose. DBTC and TBTC caused atrophy of the thymus due to a selective reduction in the number of rapidly proliferating lymphoblasts in the first 2 days after dosing. As a consequence the large pool of small lymphocytes declined in the following 2 days. On the fourth day, when atrophy was most pronounced, the frequency of the lymphoblasts increased above the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of tri-n-butyltin chloride on macromolecular synthesis and ATP levels of rat thymocyte subpopulations obtained by centrifugal elutriation.

Using centrifugal elutration, rat thymocytes were separated into three fractions by size. Fraction 1 (F1) consisted of a large population (greater than 88% of all cells) of small, nonproliferating thymocytes. Fraction 2 (F2; 7% of all cells) was enriched in medium-sized thymocytes and showed an increased macromolecular synthesis. In the small fraction 3 (F3; less than 5% of all cells), large thymocytes, monocytes, granulocytes and cells in mitosis were concentrated. F3 demonstrated the highest proliferative activity. Fractions were characterized by size, cell morphology, DNA, RNA and protein synthesis and steady state ATP levels. The effects of the inhibitor of oxidative phosphorylation, tri-n-butyltin chloride (TBTC) on ATP levels and the incorporation of DNA, RNA and protein precursors were investigated for each thymocyte fraction. Although ATP levels increased with increasing thymocyte volume, TBTC reduced ATP levels in each subfraction proportionally. The incorporation of thymidine and leucine was markedly reduced in all fractions by TBTC, but not to the same extend. Dependent on the TBTC concentration, the rapidly proliferating cells of F2 and F3 were less affected compared to the noncycling cells of F1. The incorporation of uridine in the unfractionated cells and in F2 and F3 was also decreased by TBTC. However, at concentrations between 0.1 and 1 microM, TBTC stimulated uridine incorporation in the small thymocytes of F1. Therefore, fractionation of thymocyte suspensions was necessary to detect differential effects of TBTC on subpopulations. Centrifugal elutriation was found to be a useful tool with which to obtain subfractions of isolated rat thymocytes.

Biological activity of organotin compounds--an overview.

As a consequence of the rapid expansion of the uses and applications of the organotin compounds, the concern about their environmental and health effects is increasing. The main subject of this overview is the current understanding of the mammalian toxicity of the organotin compounds. Four different types of target organ toxicity, namely neurotoxicity, hepatoxicity, immunotoxicity, and cutaneous toxicity, are discussed in more detail. The effects of the organotin compounds on the mitochondrial and cellular level are summarized and discussed in relation to the mode of action of these compounds on the central nervous system, the liver and bile duct, the immune system, and the skin.

Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes.

The inhibitor of oxidative phosphorylation tri-n-butyltin chloride (TBTC) causes membrane damage and disintegration of isolated rat thymocytes at concentrations higher than 1 microM. From a concentration of 0.1 microM, TBTC disturbs energy metabolism as indicated by an increase in methylglucose uptake, glucose consumption and lactate production and by a decrease in cellular ATP levels. Over the same TBTC concentration range, the incorporation of DNA, RNA and protein precursors are markedly reduced. Moreover the production of cyclic AMP upon stimulation of the cells with prostaglandin E1 is effectively inhibited. These effects cannot be explained by an inhibition of nucleoside kinase activity, amino acid uptake or adenylate cyclase activity. The effects of TBTC on macromolecular synthesis and cyclic AMP production are possibly due to a disturbance of the cellular energy state.

Effects of various inhibitors of oxidative phosphorylation on energy metabolism, macromolecular synthesis and cyclic AMP production in isolated rat thymocytes. A regulating role for the cellular energy state in macromolecular synthesis and cyclic AMP production.

Inhibitors of oxidative phosphorylation such as several triorganotin compounds, oligomycin, 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone suppress energy metabolism of isolated rat thymocytes as indicated by a reduction of ATP levels, an increase in glucose consumption and by a marked accumulation of lactate. Also these compounds effectively inhibit the incorporation of DNA, RNA and protein precursors into acid-precipitable material of thymocytes. Moreover, the prostaglandin E1-induced elevation of cAMP is markedly reduced by these inhibitors. A correlation is observed between the effects on energy metabolism, macromolecular synthesis and cAMP production, since from a series of trialkyltin chlorides, tri-n-propyltin, tri-n-butyltin and tri-n-hexyltin are very effective inhibitors of these functions, while trimethyltin and tri-n-octyltin affect neither of them; other inhibitors of oxidative phosphorylation, each of them with quite different mechanisms of action, also inhibit macromolecular synthesis and cAMP production. The finding that a rise in intracellular ATP concentrations leads to a reversion of the tri-n-butyltin-induced inhibition of cAMP production and uridine incorporation, indicates a regulating role for the cellular energy state in these aspects of cellular function.

Triorganotin-induced cytotoxicity to rat thymus, bone marrow and red blood cells as determined by several in vitro assays.

To further investigate the immunotoxic effects of tri-n-propyltin chloride (TPTC), tri-n-butyltin chloride (TBTC) and triphenyltin chloride (TPhTC) several cytotoxicity tests with a series of trialkyltin chlorides and TPhTC were carried out, using isolated rat thymocytes as target cells. Thymocytes, cultured in a serum-supplemented medium, were exposed to organotin concentrations ranging from 0.01 to 10 microM for periods up to 30 h. Parameters such as cell count, trypan blue exclusion, chromium release, thymidine incorporation and cyclic AMP production were used to evaluate the cytotoxicity of these compounds. The more lipophilic compounds TPTC, TBTC, tri-n-hexyltin chloride (THTC) and TPhTC appeared most cytotoxic, reducing thymidine incorporation at concentrations as low as 0.05-1 microM. Membrane damage as determined by trypan blue exclusion and chromium release occurred at higher levels (1-10 microM). The water soluble homologue trimethyltin chloride (TMTC) was least effective in all test models. When phosphate-buffered saline supplemented with glucose was used as incubation medium, TBTC appeared more cytotoxic to thymocytes. Using this medium in 5-h incubations the cytotoxicity of TBTC to thymus, bone marrow and red blood cells was compared. Bone marrow cells were slightly less sensitive than thymocytes, while red cells were relatively resistant. In conclusion, of the triorganotin compounds especially the lipophilic homologues are cytotoxic in vitro.

Toxicity of triorganotin compounds: comparative in vivo studies with a series of trialkyltin compounds and triphenyltin chloride in male rats.

In 2-week feeding studies, a series of trialkyltin chlorides and triphenyltin chloride were fed to male weanling rats at different dietary concentrations to evaluate their toxic effects, especially on the brains and the lymphoid organs, thymus and spleen. The lower trialkyltin homologs, trimethyltin chloride (TMTC) and triethyltin chloride (TETC), were neurotoxic, causing neuronal degradation and cerebral edema, respectively, at dietary concentrations of 15 ppm. The intermediate homologs, tri-n-propyltin chloride (TPTC) and tri-n-butyltin chloride (TBTC), and the aromatic compound, triphenyltin chloride (TPhTC), caused a dose-related reduction of thymus weight. At a dietary concentration of 150 ppm decreases in thymus weight to 53, 39, and 81% of controls were found following treatment with TPTC, TBTC, and TPhTC, respectively. Microscopically, thymus atrophy was associated with a lymphocyte depletion in the thymic cortex. Only 16% of the total number of nucleated thymocytes could be isolated from rats fed 150 ppm TBTC. These effects were completely reversed within 2 weeks. Slight thymus atrophy was observed after feeding a relatively high dose of 150 mg tri-n-hexyltin chloride (THTC)/kg diet, whereas tri-n-octyltin chloride (TOTC) was ineffective. A dose-related decrease in spleen weight was noticed after 2 weeks feeding of TPTC, TBTC, and TPhTC. Liver weights were increased in rats fed TBTC, THTC, and TPhTC for 2 weeks. Nevertheless, no enlarged livers and normal spleen weights were found upon feeding 100 ppm TPTC or TBTC for 4 weeks, whereas thymus weight was severely decreased. Therefore, atrophy of the thymus was considered to be the predominant effect of the intermediate trialkyltins (TPTC, TBTC). From this study it is concluded that the lower trialkyltins (TMTC, TETC) are essentially neurotoxic, the intermediate trialkytins (TPTC, TBTC) and triphenyltin are primarily immunotoxic, and the higher homologs (THTC, TOTC) are only slightly toxic or not toxic at all.