* Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris VII Denis-Diderot, Tour 5354, E3 case 7073, 2 place Jussieu, 75251 Paris cedex 05, France; and
Centre d'Etudes du Bouchet (Defense Medical Research Center), 91710 Vert Le Petit, France
Received May 2, 2000; accepted August 5, 2000
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ABSTRACT |
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Key Words: sulfur mustard; nitrogen mustard; comparative protection; respiratory epithelium in culture.
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INTRODUCTION |
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The respiratory tract is one of the main targets of SM, and is the site of the most disabling lesions for exposed subjects (Balali, 1984). However, few studies have been performed on in vitro models of respiratory epithelial cells (Chevillard et al., 1992
; Guiliani et al., 1994). Furthermore, although the toxicity of the two mustards has been compared (Calvet et al., 1999a
; Smith et al., 1998
), protection studies against these agents have rarely been performed on the same biological model (Gray et al., 1994
; Vojvodic et al., 1985
; Wormser et al., 1997
), and never on respiratory epithelium.
We have previously investigated the protection afforded by several classes of molecules against HN2 using the human bronchial epithelial cell line 16HBE14o- as a model of human respiratory cells (Rappeneau et al., 2000). We found that the sulfhydryl-containing molecule, N-acetylcysteine (NAC), and the radioprotector, WR-1065, classically described for their scavenging properties (Griggs, 1998
; Zandwijk, 1995), reduced HN2 toxicity mainly by direct extracellular interaction with HN2. These agents prevented metabolic disruption and sulfhydryl group depletion in both immediate and delayed cotreatment (up to 3 h). Several antioxidants also proved to be ineffective against HN2 toxicity and PARP inhibitors only afforded transient protection against HN2-induced metabolic disruption during the first hours of exposure (Rappeneau et al., 2000
).
In the present study, we systematically tested the efficacy of these molecules against either SM or HN2 toxicity on the 16HBE14o- cell model as well as several other molecules reported to have a protective effect against mustards (Lindsay and Hambrook, 1997; Sawyer and Risk, 2000
). As several drugs, administered individually, demonstrated a moderate protective effect against SM, we also studied the possibility of improving protection by using combinations of several drugs. The efficacy against SM and HN2, of all the molecules investigated in this study, was determined by measuring the cleavage of the highly soluble tetrazolium salt 4-[3-4(iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) by mitochondrial succinate dehydrogenases as an indicator of cytotoxicity (Miyasaki et al., 1999
).
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MATERIALS AND METHODS |
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Cell cultures.
16HBE14o- cell line was obtained from Dr. Gruenert (San Francisco, USA) and was originally isolated from human bronchial epithelial cells transformed by origin-defective SV40 (Cozens et al., 1994). Cells were routinely plated at a density of 28,000 cells/well in 96-well titre plates coated with type I collagen at 4 µg/cm2. Cells were grown in DMEM/Ham F12 (pH 7.4) supplemented with 10% FCS and 0.25 µg/ml fungizone and antibiotics (7 U/ml penicillin, 100 µg/ml streptomycin) and were cultured at 37°C in 95% air5% CO2.
Chemical treatment.
Confluent monolayers were used in each assay. Before each treatment, the medium was removed and cells were rinsed with DMEM/Ham F12 not containing either phenol red or HEPES. All treatments were carried out in DMEM/Ham F12 not containing either phenol red or HEPES.
HN2 stock solution (50 mM) was prepared in sterile distilled water and stored at 20°C for up to one month. SM was stored in ethanol (1 M) at 20°C. Further mustard dilutions were performed in serum-free culture medium immediately before use. For SM exposure, the final ethanol concentration was less than 0.1% (v/v).
Comparison of SM and HN2 cytotoxic effects.
Two protocols were used to compare the toxicity of SM and HN2 on this cell culture model:
Two treatment strategies were used to test the efficacy of candidate molecules against SM or HN2:
WST-1 reduction assay.
WST-1 reduction assay is a colorimetric assay for quantification of cytotoxicity, based on cleavage of the WST-1 tetrazolium salt by mitochondrial dehydrogenases in viable cells (Ishiyama et al., 1995; Miyasaki et al., 1999
). In contrast with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which is cleaved to water-insoluble formazan crystals and therefore has to be solubilized after cleavage, WST-1 yields water-soluble products that can be measured without an additional solubilization step (Ishiyama et al., 1995
). Solubilization can be a source of problems when studying epithelial cells 24 h after intoxication by mustards, as careful removal of the dye-containing medium prior to dissolution of the reduced dye can result in aspiration of living cell aggregates (Guiliani et al., 1994; Sawyer, 1999a
). MTT and WST-1 reduction assays were compared after 4 h of cell exposure to SM or HN2, i.e., before cell detachment is observed, and similar results were obtained. Microscopic examinations were systematically performed during all experiments and confirmed the results obtained with WST-1 assay (data not shown).
After mustard exposure, cells were rinsed twice with DMEM/Ham F12 not containing either phenol red or HEPES, to avoid any interference by phenol red or test substances used for protection with WST-1 reduction. Cells were then incubated with DMEM/Ham F12 not containing either phenol red or HEPES. At 4 or 24 h after initial mustard exposure, 20 µl/well of WST-1 reagent (diluted 1:1 in DMEM/Ham F12 not containing either phenol red or HEPES), was added to each sample. After a 2-h incubation in a humidified atmosphere (37°C, 5% CO2), the optical density of the wells was determined spectrophotometrically at a wavelength of 450 nm with a reference wavelength of 630 nm, using a DYNEX MRX microplate reader. A background control (absorbance of culture medium plus WST-1 in the absence of cells) was used as blank. Results were expressed as a percentage of the control WST-1 cleavage exposed to medium only (the reference 100% viable cell control). These data are expressed as mean values ± SD (n = 8). The protective ratio was calculated according to the following: viability of cells treated with mustard + candidate molecule/viability of cells treated with HN2 alone.
Statistical analysis.
Data were analysed using 1-way analysis of variance. Subsequent to this, groups were compared with each other using the Dunnett's test. Significant differences between groups were assumed if the p value was < 0.05.
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RESULTS |
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Kinetics of Protection
Of the various individual or combination treatments found to be effective against SM, NAC and NAC + DOX appeared to be the most relevant for potential treatment due to their therapeutic tolerance, which excluded the choice of L-TC. As both compounds were effective when added simultaneously with SM, we tested the protection afforded by NAC and NAC + DOX as delayed cotreatment, i.e, added at different times after initial exposure to SM (Fig. 5).
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DISCUSSION |
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Our first strategy consisted of decreasing the bioavailable amount of mustard in order to reduce the immediate lesions induced. For this purpose, scavengers such as the sulfhydryl-containing molecules, NAC and WR-1065 (Rappeneau et al., 2000), or the nucleophile HMT (Lindsay and Hambrook, 1997
) were used to trap mustards. NAC and HMT were found to prevent both SM and HN2 toxicity on 16HBE14o- cells, whereas WR-1065 was only effective against HN2 (Figure 3
). Although the protection provided by HMT against SM or HN2 toxicity was relatively weak, our study confirms its protective effect on the 16HBE14o- cell model, as reported on other upper respiratory cell lines (Andrew and Lindsay, 1998
). The protection provided by scavengers such as NAC or WR-1065 was better against HN2 than SM, emphasizing the difference of reactivity between SM and HN2, as the efficacy of NAC addition against SM cytotoxicity declined extremely rapidly and no statistically significant protection was observed 15 min after the initial SM exposure. In view of the short half-life of SM in aqueous solution (about 6 min under incubation conditions) (Meier and Johnson, 1992
), this could have been indicative of a chemical interaction, in which NAC effectively binds SM and prevent its toxicity, as previously described for HN2 (Rappeneau et al., 2000
).
Our second strategy was to prevent the secondary biochemical consequences of alkylation induced by mustards such as metabolic disruption, by using NC, a PARP inhibitor and NAD+ precursor (Meier et al., 1987; Mol et al., 1989
) and DMTU, as free oxygen radical scavenger, to reduce oxidative stress (Fig. 3
). As previously shown on 16HBE14o- cells for HN2 (Rappeneau et al., 2000
), NC provided a transient protection against SM (data not shown), but was ineffective at 24 h. DMTU afforded relatively good protection against HN2 toxicity, but was ineffective against SM. Although DMTU is classically described as a free radical scavenger (Fox, 1984
), its antioxidant properties may not be related to its protective mechanism against HN2, as several other antioxidants, such as iron chelators or lipid peroxidation inhibitors, failed to modulate HN2 toxicity in 16HBE14o- cells (Rappeneau et al., 2000
). It is therefore tempting to speculate on the way in which DMTU exerts its protective effect against HN2 toxicity, as very few data concerning a particular mechanism of action are available at the present time.
Based on the report that the L-arginine analogues, L-TC and L-NAME, have a potent protective activity against SM toxicity on chick embryo neurons (Sawyer et al., 1998), these compounds were also investigated in our study. Our results showed that L-TC was also effective against SM and HN2 on a model of human respiratory epithelium. However, L-NAME was only slightly effective against HN2. Although the protective mechanism of these agents remains unclear, L-TC is supposed to act while SM is still outside the cell, by reducing its bioavailability (Sawyer et al., 1999b). Moreover, Sawyer et al. (1998) demonstrated that the protective activity of L-TC is not associated with its interaction with SM, nor with its NOS (nitric oxide synthase)-inhibiting potency, because other potent NOS inhibitors such as L-C and MeTC were ineffective on 16HBE14o- cells, as observed on the chick embryo neuron model (Sawyer et al., 1998
). The tetracycline, doxycycline (DOX), was investigated for its anti-gelatinase properties (Petrinec et al. 1996
), as a previous study by Calvet et al. (1999b) evidenced the potential involvement of gelatinases in SM-induced respiratory lesions in vivo and opened up new therapeutic possibilities. However, our results showed that DOX is weakly effective against SM and ineffective against HN2 on 16HBE14o- used as individual treatment (Fig. 3
).
Overall, the level of protection afforded by agents used individually, such as NAC and L-TC, against SM is relatively weak compared to the levels obtained against HN2. Moreover, as the various molecules used individually induced low protection against SM, and taking into account the potential mechanisms of action of SM with multiple targets in the cell, it appeared necessary to test drug combinations in order to improve the level of protection against SM. We therefore combined several compounds demonstrating a certain degree of efficacy in individual treatment against SM. These combinations consisted of agents reducing the bioavailability of the mustard and compounds possibly acting on the consequences of alkylation, as it can be hypothesized that scavenging of the mustard reduces the available quantity of mustard and can potentiate the efficacy of a drug acting on the consequences of alkylation, which would be ineffective against higher doses of mustards. In our study, this hypothesis was revealed to be accurate. Thus, the combinations L-TC + DOX, NAC + DOX, NAC + DMTU, and NAC + HMT were found to be more effective than each of these compounds used individually (Fig. 4). The most therapeutically relevant of these effective combinations appears to be the NAC + DOX combination. Although the other compounds can be useful tools to study the mechanism of the toxic action of mustards, L-TC is of no practical value as an antidote, due to its NOS characteristic of action, and DMTU is potentially toxic in vivo (Beehler et al., 1994
; Dooms-Gossens et al., 1979; Lewis et al, 1994
). NAC and DOX are both interesting molecules that are already used in the treatment of numerous diseases (Cotgreave, 1997
; Lokeshwar, 1999
) and as they have multiple beneficial properties that can be useful against the multiple effects of mustards. Indeed, NAC exhibits scavenging properties, antioxidant and anti-gelatinase properties (Cotgreave, 1997
), and while DOX exhibited antioxidant (Wasil et al., 1988
) and anti-gelatinase properties (Lokeshwar, 1999
). Further investigations will have to determine the importance of proteases in SM-induced injury, testing several anti-proteases. Furthermore, NAC and DOX are known to be well tolerated in humans and the combination of NAC + DOX was found to be protective when administered during the first 90 min after onset of SM exposure (Fig. 5
), a time interval compatible with the therapeutic use of this combination. However, this efficacy, evidenced on an in vitro model of respiratory cells, needs to be confirmed on other cell culture models, i.e., other targets, such as keratinocytes, and then by in vivo studies.
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ACKNOWLEDGMENTS |
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NOTES |
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