Sulfur Mustard Induced Toxicological Effects On Skin And Its Possible Countermeasures: An Experimental Study Focused On GST And TFPI-2

Sulfur mustard (SM),"the king of the chemical warfare agents,"is a typical vesicant agent. Skin is the major target for SM., the most prominent toxic effects of which are extremely slow healing lesions and blisters resulting in ulcer and vesicle of the skin, and promoting secondary infections.Although the toxicity of SM has been investigated since it was used in the World War I. Presently, the mechanisms underlying sulfur mustard-induced skin damage are not fully elucidated and no antidote exists, apart from its characteristics of multiple stratification and multiple targets. Current treatment strategy is designed to relieve symptoms. Critical questions are proposed to increase our understanding of SM toxicity and accelerate the development of vesicant therapeutics.We focus on Glutathione S-transferase (GST) and Tissue factor pathway inhibitor-2 (TFPI-2) which closely related with SM, based on the accumulation of our work previously and the research in progress of SM. The interest of this paper is further to discover the role of GST in apoptosis and to acquire TFPI-2 in vitro, and to know about the potent pharmacology of TFPI-2 in SM. The results are as follows:1. Relation between the variation of GST activity and apoptosis induced by SM.The variation of GST activity is dose- and time-dependent of SM in mouse ear model. GST activity decreased significantly at 48h post exposure, and also decreased at the dose 160mg/ml SM. It suggested that the skin can't scavenge free radicals and sulfur mustard at these conditions. The result is also in accordance with the proteomic analysis, confirmed with the decrease of GST after exposure of SM.We explored the condition of primary fibroblast apoptotic induced by SM.A large number of fibroblast apoptotic were observed in a study of exposure to 100μM SM at 16h, GST activity decreased at the same condition. This result is consistent with our previous findings that the decrease in the expression of GST might relate to epidermal basal cells apoptosis (not published).2. Effects of increased GST on apoptosis induced by SM.We obtain GST gene by PCR from a mouse liver, directly insert GST gene into plasmid pCMV, and successfully transfect GST gene into HaCaT by lipofectamine. The cell expressed exogenous GST gene was named as HaCaT/GST.The percentage of apoptosis cell decreased in HaCaT/GST compared with HaCaT at 16h post exposure to 100μM SM. Such investigation may confirm that GST participates in the apoptosis in SM, and can alleviate the toxicity of SM.The activity of SOD, the GSH and MDA level were measured at 6h, 12h and 24h after SM administration. The results show that both SOD activity and GSH level were significantly decreased after post-exposure, while the MDA level was enhanced in HaCaT. These results suggested that endogenous antioxidant system was interrupted by the excess free radical production and lipid per-oxidation. So SM induced apoptosis may be associated with the antioxidant defense systems interruption, and the GSH may also have relation with this progress.3. Molecular cloning, prokaryotic expression and purification of TFPI-2 and the first Kunitz-type domain of TFPI-2.The TFPI-2, obtained by PCR from a women placenta, was directly inserted into expression vector. And the first Kunitz-type domain of TFPI-2 (TFPI-2/KD1), screened in TFPI-2, was also cloned into expression vector. After cell expression, TFPI-2 expressed as inclusion bodies, while TFPI-2/KD1 exist in supernatant. These results implied that TFPI-2/KD1 in E coli has biological activity. Purification of recombinant TFPI-2/KD1 was performed using His-tag.4. Biological function of TFPI-2/KD1.4.1 Biological activity assay of TFPI-2/KD1.Subsequently TFPI-2/KD1 inhibits the activity of MMPs. Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of purified KD1 on trypsin and MMPs individually.The result suggested that the protein can inhibit MMP-2 and MMP-9 at 20μM of TFPI-2/KD1, and dose-dependently inhibit trypsin. This result implied that TFPI-2/KD1 can inhibit serine protease.4.2 Effects of TFPI-2/KD1 on inflammatory cytokines release after SM exposure.The release of inflammatory cytokines including interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) significantly increased at 8h after exposure of 100μM SM in HaCaT. At the dose of 1mg/mL and 2mg/mL TFPI-2/KD1 can significantly suppress SM-increased IL-8 and IL-6 can also be inhibited when administrate 1mg/ml TFPI-2/KD1. But TFPI-2/KD1 has no effect on TNF-α. The fact that TFPI-2/KD1 can inhibit the production of inflammatory cytokines suggested it can at least partly alleviate the toxicity of SM.4.3 Changes of skin blood perfusion and histopathology induced by SM.To assessment the lesion depth of different dose SM-exposure (1μl, 2.5μl, 5μl, 10μl); the dermal blood flow was studied by laser Doppler perfusion imaging (LDPI) for three days. And after 72h post exposure, the skins were processed for routine H & E histological evaluation to determine the lesion.Blood flow of the damage areas became sufficient in a short time. By the time going on the blood flow of damaged skin reduced, and around it circulation increased. This phenomenon was dose- and time- dependent. The result suggested the subcutaneous'vascular were damaged.The skin biopsies showed that the normal rabbit skin composed with epidermis, dermis, and subcutaneous tissue. A biopsy from 1μl SM exposed skin showed epidermal erosion, ulceration, sub-epidermal bullous (>5mm), hair follicles and sebaceous glands hyperplasia. Along with the increased poison dose, part of the epidermis disappear, necrosis, exudative; vasodilator and congestive in dermis; inflammatory cell infiltration into adnexa, covered the acute inflammatory response. At the dose of 5μl SM, biopsies showed epidermis focal necrosis and obvious dermal hemorrhage.4.4 Intervention roles of TFPI-2/KD1 on skin inflammation induced by SM. TFPI-2/KD1 has no effect on the blood flow, which can reflect the degree of SM induced inflammatory. H & E histological evaluation suggested that TFPI-2/KD1 can partly alleviate the inflammation response to SM, such as the reduction of neutrophil infiltrating. This indicated that single drug can't effectively alleviate the SM toxicity on skin.In summary, GST and TFPI-2/KD1 may play intervention roles in SM skin damage by different pathways. The former is correlated with the apoptosis and the later can partly against with the acute inflammation. Whereas SM has multiple stratification and multiple targets, the mitigation of single drug is limited, we must seek for the key mediator and take the multiple drug/agent medical countermeasures.