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

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.

Diisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard.

The A549 cell line was used to assess the ability of diisopropylglutathione (DIPE) to protect against a 100 microM challenge dose of sulphur mustard (HD) using gentian violet (GV), thiazolyl blue (MTT) and neutral red (NR) assays as indicators of cell culture viability. As part of a continuing study of the efficacy of protective nucleophiles as candidate treatments for HD poisoning, several different combinations of protectant and HD were used to determine the optimal means of protecting A549 cells from the effects of HD. It was found that DIPE (4 mM) could protect cells against the effects of HD though for optimal effect, DIPE had to be present at the time of HD challenge. Cultures protected with DIPE were up to 2.9-fold more viable than HD exposed cells 48 h after HD challenge when using the GV, MTT and NR assays to assess viability. Observations by phase contrast microscopy of GV stained cultures confirmed these findings. Pretreating A549 cultures with DIPE for 1 h followed by its removal prior to HD challenge did maintain cell viability, though at a relatively low level (only up to 1.4-fold more viable than HD only exposed cells). DIPE was also able to protect HD exposed A549 cultures when added to cell cultures at intervals of up to 12 to 15 min after the initial HD exposure, though viability tended to decrease over this period, so that at 1 h, addition of DIPE did not maintain the viability of the cultures. This is the first such report of the anti-HD protectant properties of DIPE in A549 cells. It is concluded that the protection observed against HD is probably largely due to extracellular inactivation of HD by DIPE.

Local and systemic responses against ricin toxin promoted by toxoid or peptide vaccines alone or in liposomal formulations.

The objective of this study was to develop a vaccine which would ultimately protect man from the lethal effects of inhaled ricin toxin. Porton rats have previously been protected from lethal quantities of inhaled ricin by subcutaneous (s.c.) ricin toxoid vaccine, but not without lung damage. This situation might be improved by an alternative vaccine such as the A chain of ricin, already known to protect against inhaled ricin. Another option would be to improve respiratory tract immunity by local vaccination in conjunction with liposomal formulation with a view to enhancing lung secretion of immune IgA. While boosted s.c. doses of ricin toxoid or A chain produced indistinguishable systemic immune responses 3 weeks later, when delivered by the intratracheal (i.t.) route, the A chain failed to elicit a specific immune response, unlike ricin toxoid. This situation was overcome by liposomal formulations and although ricin toxoid was readily encapsulated in liposomes, A chain was not. However, by simply mixing A chain and liposomes in the same weight ratio determined for liposomal toxoid, systemic immune responses for each formulation were indistinguishable 1 week after boosting. Ricin antibody responses in lung fluid monitored 1, 3, 7 and 14 days after i.t. challenge with ricin were statistically indistinguishable, but the group vaccinated with liposomal toxoid secreted 28.7% IgA compared with 0.9-14.9% for the A chain liposomal group. From this, it might be anticipated that the lungs would be better protected by liposomally-encapsulated ricin toxoid than by the A chain-liposome mixture.

Liposomally-encapsulated ricin toxoid vaccine delivered intratracheally elicits a good immune response and protects against a lethal pulmonary dose of ricin toxin.

A small study was performed to examine whether the instillation of ricin toxoid vaccine into the lungs of Porton rats offered protection from lethal effects of subsequent intratracheal challenge with ricin toxin. Further the immune response to liposomally-encapsulated vaccine and the protection offered was compared with vaccine either adsorbed to Alhydrogel adjuvant or as a simple aqueous solution. The formaldehyde-treated ricin toxin vaccine (RTV) was administered at two dose levels, 500 and 100 micrograms kg-1 body weight to groups of rats, on two occasions by intratracheal instillation. Liposomally-encapsulated vaccine (LRTV) produced a higher titre of ricin-specific antibodies than Alhydrogel-vaccine (ARTV) and vaccine solution. When challenged with 3 LD50 of ricin by intratracheal instillation 7 weeks after the second vaccine instillation, all rats in both LRTV dose groups survived with minimal signs of incapacitation. Analysis of antibody secretion by spleen cells, 14 days post challenge, showed that the IgG isotype in the LRTV group was significantly higher than that in the ARTV and RTV groups and also that the proportion of specific IgA in lung fluid was higher in the LRTV group than in the ARTV and RTV groups. The results of this study indicate that effective vaccinations against inhaled ricin could be achieved with liposomally-encapsulated ricin toxoid, via the lung and should be investigated further.

Protection of A549 cells against the toxic effects of sulphur mustard by hexamethylenetetramine.

The A549 cell line was used as a model of the deep lung to study the toxicity and mechanism of action of sulphur mustard (HD), using the neutral red (NR) dye retention and gentian violet (GV) assays as indices of cell viability. It was found that exposure to concentrations in excess of 40 microM HD resulted in a rapid onset of toxicity. Exposure to 1000 microM HD reduced viability in A549 cell cultures to 61% after 2 h (control cultures = 100%), whereas exposure to 40 microM HD did not result in deleterious effects until 26 h at which point viability fell to only 84% (NR assay). Agarose gel electrophoresis of cell cultures exposed to 40 and 1000 microM HD and harvested at 4.5, 19 and 43 h after exposure to HD, indicated that cell death was due to necrosis, despite the observation that at the higher concentration of HD cells displayed many of the features common to cells undergoing apoptotic death. The ability of hexamethylenetetramine (HMT) to protect A549 cells against the effects of an LC50 challenge dose of HD was assessed using the GV and NR assays. It was found that HMT (15 mM) could protect cells against the effects of HD though HMT had to be present at the time of HD challenge. Cultures treated with HD only were 49% viable at 48 h after HD challenge, compared to 101% for protected cultures (NR assay) and 58% and 91% for unprotected and protected cultures respectively using the GV assay. Morphological observations of GV and NR stained cultures confirmed these findings. HMT concentrations of 2.5 to 25 mM were used. Maximal protection against the toxic effects of HD (LC50) was found at 10 to 25 mM HMT. Over this concentration range, HMT did not exert any toxic effects on A549 cells. Pretreatment of A549 cultures with HMT followed by its removal prior to HD challenge had no protective effect. Similarly, treating cultures with HD followed by addition of HMT did not increase the viability of the cultures, even if the HMT was added immediately after HD exposure. HMT was found to protect against the toxic effects of HD, though it must be present at the time of HD challenge. A549 cells were found to be a valuable experimental model for studying the toxicology of HD and other lung damaging agents, and for screening other compounds for potential therapeutic efficacy as a prelude to studies with non-transformed cell culture systems and in vivo models.

Monoisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard.

1. The A549 cell line was used to assess the toxicity of sulphur mustard (HD), using gentian violet (GV) and neutral red (NR) dyes as indicators of cell viability. It was found that exposure to concentrations in excess of 40 microM HD resulted in a rapid onset of toxicity. 2. The ability of monoisopropylglutathione ester (MIPE) to protect A549 cells against the effects of a 100 microM challenge dose of HD was determined using the NR and GV assays. It was found that MIPE (8 mM) could protect cells against the effects of HD though MIPE had to be present at the time of HD challenge. Cultures protected with MIPE were two times more viable than HD exposed cells 48 h after HD challenge when using the GV and NR assays to assess viability. Observations by phase contrast microscopy of NR and GV stained cultures confirmed these findings. Addition of MIPE after previously exposing the A549 cultures to HD (for up to 5 min) maintained cell viability at 72% compared to 37% for unprotected cultures, after which time viability fell significantly so that at 10 min there was no difference in viability between the MIPE treated and untreated cultures. 3. Pretreating A549 cultures with MIPE for 1 h followed by its removal prior to HD challenge did not maintain cell viability. Treatment of cultures with HD for 1 h followed by addition of MIPE did not maintain the viability of the cultures, thus the window within which it was possible for MIPE to rescue cell cultures from the effects of HD was of short duration. 4. High performance liquid chromatography was used to determine the biochemical basis of the actions of MIPE. It was found that whilst intracellular levels of cysteine were increased up to 40-fold following treatment of A549 cell cultures with MIPE, levels of reduced glutathione did not rise. The lack of protection seen in cultures pretreated with MIPE for 1 h prior to HD exposure suggests that raising intracellular cysteine levels was not an effective strategy for protecting cells from the effects of HD. The protection observed is probably due to extracellular inactivation of HD by MIPE.

Examination of toxicity of Clostridium perfringens -toxin in the MDCK cell line.

The epithelial Madin Darby canine kidney (MDCK) cell line was examined as a model to study the toxicity of Clostridium perfringens -toxin. The MDCK cell line was used because it is a monolayer cell line sensitive to -toxin. Using the neutral red (NR) retention assay (an indicator of lysosomal integrity), the concentration of toxin causing death in 50% of the cell population (LC(50)) was 900 pM, although this was found to vary between production batches. -Toxin was found to act rapidly but with a lag phase of 1 hr (NR assay). Pulsing the cultures with toxin (up to 4800 pM) indicated that the duration of exposure required to exert an effect was potentially very short (2.5 min). Increasing the duration of exposure beyond 3 hr did not decrease cell viability any further. Experiments with protease inhibitors failed to inactivate the toxin. Ethylenediaminetetraacetic acid (EDTA) was found to potentiate the lethality of the toxin by 90% This may be due to non-specific chaotropic effects such as membrane destabilization. By exposing cultures of MDCK cells to -toxin for a second time, resistance to the effects of the toxin was increased by 43%. The factor(s) controlling resistance to the toxin may have a heritable component.

Metabolism of thiodiglycol (2,2'-thiobis-ethanol): isolation and identification of urinary metabolites following intraperitoneal administration to rat.

1. The metabolism of thiodiglycol, 2,2'-thiobis-ethanol, was investigated following i.p. administration to rat. 2. Approximately 90% of the administered dose was excreted in the 0-24-h urine. Four metabolites were isolated by h.p.l.c. and identified by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards. 3. Thiodiglycol sulphoxide was the major metabolite accounting for approximately > or = 90% of the excreted radioactivity following i.p. injection of 13C4, 35S-thiodiglycol. Thiodiglycol sulphone, S-(2-hydroxyethylthio)acetic acid and S-(2-hydroxy-ethylsulphinyl)acetic acid were identified as minor metabolites. 4. Analysis for thiodiglycol by GC-MS indicated approximately 0.5-1% of the applied dose was excreted unmetabolized.

Biological fate of sulphur mustard (1,1'-thiobis(2-chloroethane)): uptake, distribution and retention of 35S in skin and in blood after cutaneous application of 35S-sulphur mustard in rat and comparison with human blood in vitro.

1. During the 6-h occluded cutaneous application of 35S-sulphur mustard vapour to rat, most of the dose, approximately 75%, passed through the skin and was systemically-distributed. Up to 25% of the 35S was retained in the skin, up to 30% was excreted in the urine and 5-8% was present in the blood, by the end of the application. 2. 35S initially declined rapidly in skin and then more slowly with a half-life of approximately 7.4 days. Some of the early loss was as sulphur mustard vapour from a possible depot of this compound which was larger with increase in dose. There was some apparent continuing uptake from such a depot into the systemic circulation. 3. The decline of 35S in blood was much slower than that from skin. About one-fifth of the original post-exposure level in the blood still present 6 weeks later. 4. The 35S in blood was mainly in the red cell contents as reaction products of haemoglobin with sulphur mustard. Its persistence in the systemic circulation, after an initial rapid decline due to its removal from the plasma, reflected the 65 day life span of these cells. Significant levels of 35S were found in the plasma only for a few days (t1/2 = 2.4 days). 5. Uptake and subsequent distribution of 35S in rat and human blood in vitro during gradual exposure to 35S-sulphur mustard using a novel method were similar. 35S in the red cells was associated mainly with the haemoglobin protein, but slightly greater binding with human, rather than with rat, plasma components was indicated.

Biological fate of sulphur mustard, 1,1'-thiobis(2-chloroethane): isolation and identification of urinary metabolites following intraperitoneal administration to rat.

1. The metabolism of sulphur mustard, 1,1'-thiobis(2-chloroethane), in vivo was investigated following i.p. administration to rat. 2. Approx. 60% of dose was excreted in the 24 h urine. Many metabolites were present; nine have been isolated by h.p.l.c. and characterized by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards. 3. Some metabolites result from initial hydrolysis of the sulphur mustard, but the majority are formed by conjugation with glutathione. These are further metabolized to N-acetylcysteine conjugates, or to methylthio/methylysulphinyl derivatives by a pathway probably involving beta-lyase, accompanied by oxidation of the mustard sulphur atom to sulphoxide or sulphone. 4. Thiodiglycol sulphoxide, 1,1'-sulphonylbis[2-S(N-acetylcysteinyl)ethane] and 1,1'-sulphonylbis[2-methylsulphinyl)ethane] or 1-methylsulphinyl-2-[2-(methylthio ethylsulphonyl]ethane were the most prevalent metabolites resulting from the three major pathways. Metabolic pathways for the formation of the excretion products are proposed.

Biological fate of sulfur mustard, 1,1'-thiobis(2-chloroethane). Urinary excretion profiles of hydrolysis products and beta-lyase metabolites of sulfur mustard after cutaneous application in rats.

The urinary excretion profiles of some metabolites of sulfur mustard were determined by gas chromatography/mass spectrometry after cutaneous application of sulfur mustard in rats. Excretion profiles of the individual metabolites thiodiglycol and thiodiglycol sulfoxide, derived from the hydrolysis of sulfur mustard, were determined in different groups of three rats. Concentrations of thiodiglycol detected increased up to 10 fold after treatment of the urine with hydrochloric acid, presumably because of the excretion of acid-labile esters of thiodiglycol. Free thiodiglycol, free plus esterified thiodiglycol, and thiodiglycol sulfoxide excreted over 8 days accounted for less than 0.3%, 1-1.5%, and 3.4-4.3%, respectively, of the applied dose of sulfur mustard. In a further study, a modified analytical method was applied to determine these hydrolysis products and their acid-labile esters as the single analyte thiodiglycol, after treatment with acidic titanium trichloride. The excretion profile of the combined hydrolysis products was compared with the excretion profile of a different group of metabolites of sulfur mustard derived from the glutathione/beta-lyase pathway. These were also reduced to a common analyte, 1,1'-sulfonylbis-[2-(methylthio)ethane], after similar treatment with titanium trichloride. Urinary excretion of hydrolysis products determined in 4 rats over 8 days accounted for 3.7-13.6% of an applied cutaneous dose of sulfur mustard. Urinary excretion of beta-lyase metabolites accounted for 2.5-5.3% of the applied dose in the same group of rats. The excretion of beta-lyase products showed a much sharper decline than was observed for the hydrolysis products of sulfur mustard.

Biological fate of sulphur mustard (1,1'-thio-bis(2-chloroethane)): urinary and faecal excretion of 35S by rat after injection or cutaneous application of 35S-labelled sulphur mustard.

1. A technique for the quantitative cutaneous application of sulphur mustard vapour is described. The maximum rate of uptake of the agent vapour by shaved rat skin in vivo was estimated as 7 micrograms/cm2 per min. 2. The major route of excretion was through the kidneys: greater than 70% of the radioactivity of all doses was excreted in 3 days after either i.v. or i.p. injections of 35S-labelled sulphur mustard, but less (50-70% depending on dose) was excreted in the same time following cutaneous application. 3. This urinary excretion of 35S had a half-life of 1.4 days, and the decreased proportion of the applied dose excreted following cutaneous application may be accounted for by retention of radioactivity in the skin. 4. The contribution of faecal excretion was greater following cutaneous application.