No abstract

1 The basal epidermal keratinocytes of the skin are a main target for the vesicating agent, sulphur mustard (SM). A human keratinocyte cell line (SVK-14) has been used to model the effects of SM on the basal epidermal keratinocytes and subsequently to test the efficacy of potential prophylactic compounds in reducing the SM-induced cytotoxicity. 2 The cultures were pretreated with mixtures of methe namine (HMT) and glutathione (GSH) for 1 h prior to exposure to 10 μM SM. The viability of the cultures was then assessed using neutral red (NR) dye uptake and crystal violet DNA staining assays at 24 h intervals post exposure. 3 Pretreatment led to a 1.9 fold increase in culture viability (NR assay) compared to those exposed to SM only, and a 2.3 fold increase in cell number (crystal violet assay). Photomicrography showed that pre treatment preserved the morphology of the cultured cells and maintained their mitotic activity whereas those exposed to SM only show non-proliterative cultures with extensive cellular damage. 4 The results of this study show that it is possible to protect mitotically active cultures from the effects of SM, however the measures must be in place prior to SM exposure.

In vivo, the pigment producing melanocytes are the most susceptible cell type to sulphur mustard (HD) in the epidermal region of pig skin. It has been postulated that this is due to the melanogenic pathway producing a cytotoxic, free radical cascade within the melanocyte following HD poisoning, leading to cellular necrosis and subsequent inflammation. To test this hypothesis, the cytotoxicity of HD was tested in three human melanoma cell lines and compared to SVK-14 human keratinocytes, a cell line in which the response to HD has already been characterised. The results of both neutral red (NR) and gentian violet (GV) assays showed that all three melanoma cell lines, particularly the G361 line, were less susceptible to the toxic effects of HD than the SVK-14 keratinocyte cell line. Preliminary data indicate that the expression level of the DNA repair cofactor, proliferating cell nuclear antigen (PCNA), is up to 13-fold greater in the HD-resistant cell line G361 compared to the HD-sensitive SVK-14 cell line. The data point to the importance of DNA lesions in HD-induced cell death and to potential mechanisms associated with increased resistance to HD. A dose–response study was carried out to confirm the differences between these two cell lines. It was found that the G361 line is 5-fold more resistant to HD and 5.5-fold more resistant to the cytotoxic effects of H2O2 than the SVK-14 line, as determined by the MTT assay. The results suggest that differences in the relative efficiency of DNA repair processes may underlie these responses. Whilst the study indicates the limitations of using melanoma cell lines (in vitro) to model melanocyte responses to HD, analysis of the biochemical basis of the observed differences in sensitivity to HD could assist in the identification of novel therapeutic strategies against HD.

In vivo experiments have shown that melanocytes are more sensitive than keratinocytes to the cytotoxic effects of sulfur mustard when it is applied topically to pig skin.1 It has been hypothesized that this is caused by the uncoupling of the melanogenic pathway by depletion of cellular glutathione, resulting in the uncontrolled production of cytotoxic quinone free-radical species by tyrosinase.2. In the present study, the feasibility of blocking the melanogenic pathway as a means of reducing the cytotoxicity of sulfur mustard was evaluated using kojic acid. Kojic acid is a topically applied depigmenting agent that exerts its effect by acting as a slow-binding, competitive inhibitor of tyrosinase.3 Preincubation of G361 pigmented melanoma cells and mixed cultures of G361 cells and SVK keratinocytes with 2.5 mM kojic acid resulted in significant increases in the viability of these cultures as determined by neutral red (NR) and gentian violet (GV) dye binding assays for up to 48 h following exposure to 50 microM sulfur mustard. The highest levels of protection were seen in the G361 cultures, with a 26.8% increase in culture viability (NR assay) compared with the sulfur-mustard-only controls at 24 h. Preincubation of SVK cells alone with kojic acid resulted in lower increases in viability (2.5% at 24 h by the NR assay). Inhibition of the melanogenic pathway reduces the sensitivity of cultures containing pigment cells to sulfur mustard.

Epidermal melanocytes have a higher sensitivity to sulphur mustard (HD) compared with other skin cell types.1 This may be due to the enzymatic production of melanin precursors exerting an additional cytotoxic effect following HD depletion of the cellular protectant, GSH.2,3 Stimulation of the protein kinase C pathway in melanocytes is known to increase melanin production in melanocytes and melanoma cell lines.4,5 In order to investigate the role of pigment synthesis in HD toxicology, cultures of an unpigmented melanoma cell line (C32) and of a pigmented melanoma line (G361) were treated with the potent diacyl glycerol analogue, oleoyl acetyl glycerol (OAG), in order to determine if protein kinase C-mediated increases in pigment production could increase sensitivity to subsequent HD exposure. Stimulation of C32 cells with OAG exerted a significant protective effect against the cytotoxic effects of HD. However, this was not due to increased melanin synthesis because this cell line cannot synthesize melanin pigments. The protective action observed is postulated to be due to modulation of protein kinase C activity. In contrast, stimulation of G361 melanoma cells with OAG resulted in an increased level of cytotoxicity upon subsequent exposure to HD. Protein kinase C controls several cellular pathways including checkpoints in the cell cycle, stalling the cell in G1 and promoting transition through the G2 /M boundary. Given the genotoxic properties of HD, these two points in the cell cycle are important in determining the overall cytotoxic effect of HD. Control of the cell cycle by protein kinase C modulation and manipulation of melanin synthetic pathways may have therapeutic benefits.