An in vitro comparison of the cytotoxicity of sulphur mustard in melanoma and keratinocyte cell lines

Abstract

<jats:p> 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 H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> 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. </jats:p>

Effect of sulphur mustard on human skin cell lines with differential agent sensitivity

“…These findings are consistent with Smith et al (2001), who also showed that G361 cells were more resistant than SVK14 cells to the effects of H 2 O 2 to a similar extent, alluding to related resistance mechanisms involving an upregulated DNA repair capacity. In the present study, differences in cell population growth rates alone could not account for the greater sensitivity of SVK14 cells to HD, because despite the known deleterious effects of HD on rapidly dividing cells (Maynard, 1987) the G361 cell cultures were found to have a higher growth rate than SVK14 cell cultures.…”

“…The results confirm previous findings by Smith et al (2001), who found that cellular proliferation in G361 cultures could recover following HD exposure, although in the present study this was not evident at HD doses of >60 µM. This is suggestive of a limited repair capability and would explain the markedly greater resistance of G361 cells at 72 h compared with SVK14 cells, significant differences in cell culture viability being found at 20, 30 and 60 µM HD.…”

“…However, other factors cannot be ruled out, because at 30 µM HD the G361 cultures contained a relatively high proportion of cells that remained viable even after BSO pretreatment. In order to probe this further, a comparative analysis on cell cycling activity in SVK14 and G361 cultures was carried out because previous studies have suggested that G361 cells may have a more efficient DNA repair capacity, as indicated by elevated levels of PCNA in this cell line (Smith et al, 2001). The DNA repair involved may be nucleotide excision repair, because PCNA is an essential co-factor in this type of repair (Bootsma, 2001).…”

“…Previous work suggests that increased resistance to HD may be associated with elevated levels of proliferating cell nuclear antigen (PCNA) (Smith et al, 2001), an accessory factor for DNA polymerase-δ and -ε in DNA mismatch repair (Wood and Shivjii, 1997). The PCNA also plays an essential role in nucleotide excision repair (Miura et al, 1996;Tomkinson and Mackey, 1998).…”

Direct binding of sulfur mustard and chloroethyl ethyl sulphide to human cell membrane-associated proteins; implications for sulfur mustard pathology

“…One-dimensional SDS PAGE and Western blotting were carried out as described in reference [14]. The cell fractions were diluted with Laemmli sample buffer (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) to the required protein concentration and then heated at 100 • C for 5 min.…”