Experimental Studies On Sulfur Mustard Produced Genotoxicity And Their Molecular Mechanisms

Sulfur mustard, a blister agent, is one of the most important chemical warfare agents. It is also a typical biofunctional alkylating agent that has the capacity to cross-link biological molecules such as DNA, resulting in alteration of structure and information of DNA. Sulfur mustard not only developed cytotoxicity, but also developed genotoxicity and reproduction toxicity. The hazardous manner and extent need further investigation. Although the cytotoxicity of sulfur mustard has been well studied, the genotoxicity and reproduction toxicity have been poorly investigated. In this study, several techniques including Ames test, chromosomal aberration analysis, micronucleus test and single cell gel electrophoresis assay were employed to study the mutagenicity both in vivo and in vitro. The genetic endpoints tested in this study include chromosomal aberration and gene mutation. The mutation frequency and mutational spectra of tk locus were analyzed with the methods of mouse lymphoma assay, microscopic examination, RT-PCR, electrophoresis analysis and DNA sequencing. At the same time, sulfur mustard-induced reproductive system damage were determined by analysis of maternal toxicity and embryo toxicity of mice, counting the sperm deformity and pathomorphology of testis as well. The basic genetic toxicology character of sulfur mustard was further explained. The effect of sulfur mustard on the glutathione S-transferase (GSTs) activity was illustrated with Wistar rat spleen lymphocytes and mouse lymphoma L5178Y cells. In addition, the protective effects of glutathione (GSH) on CHL cells were also studied. The main results are as follows:1. Sulfur mustard was demonstrated a marked mutagen. The results from Ames test, chromosomal aberration analysis, micronucleus test and single cell gel electrophoresis assay were both positive.2. Sulfur mustard produced severe spermatogenic structural injury in mice testis. After sulfur mustard injection, the body weight decreased, the activities ability weakened and the percentage of abnormal sperm increased significantly(P<0.01). It caused scarceness and deletion of mature sperm in seminiferous tubule, chromatin condensed, vacuolation, edema,necrosis and shedding of spermatogenic cells.3. Sulfur mustard displayed maternal toxicity and fetal abnormalities after ip injections in KM mice. At the range of 0.2-5.0mg, sulfur mustard displayed no influence on reproductive ability of pregnant mice and the weight of placent, but the body weight increase of pregnant mice reduced and hematom was the most common effect of fetal appearance. The main influence on fetal skeleton included absent centrum, bipartite ossification of sternebra, sternebra remains, split cervical arch and cervical rib.4. Sulfur mustard displayed considerable cytotoxicity and induced tk locus mutation with mutation frequency 2-15 times higher than that of spontaneous mutation frequency of L5178Y3.7.2c-tk+/- cells at the range of 10-30 μmol/L. The mutation frequency (MF) increased with the concentration increases of sulfur mustard. There were two different phenotypes of mutation colonies: large and small colonies. But most of them were small colonies. The molecular mutagensis mechamism of sulfur mustard was studied by cDNA sequencing of the tk gene mutants. There was 34 point mutation. Most of them were base substitution (67.6%, 23/34); others were frameshift mutation (32.4%, 11/34). 64.7% (22/34) base substitution belongs to trans version. Most of the point mutation was G→T(434), T →G(629) transversion and the frame shift of -C(912). The results were reported at first time, which need to be verified in future.5. GSH partly against sulfur mustard -induced DNA impairment in CHL cells. GSTs may act in the mechanism of carcinogenic and anticarcinogenic capability. The activities of GST were induced by lower doses of sulfur mustard whereas inhibited by higher doses both in lymphocytes separated from Wistar rat spleen and L5178Y cells. The lasting time of the activities of GST in lymphocytes was relatively short, on the contrary, relatively long in L5178Y cells.