Efficient Protection of Human Bronchial Epithelial Cells against Sulfur and Nitrogen Mustard Cytotoxicity Using Drug Combinations

S. Rappeneau*,1, A. Baeza-Squiban*, F. Marano* and J.-H. Calvet{dagger}

* Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris VII Denis-Diderot, Tour 53–54, E3 case 7073, 2 place Jussieu, 75251 Paris cedex 05, France; and {dagger} Centre d'Etudes du Bouchet (Defense Medical Research Center), 91710 Vert Le Petit, France

Received May 2, 2000; accepted August 5, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to test the efficacy of several candidate molecules against sulfur mustard (SM) and nitrogen mustard (HN2) using a human bronchial-epithelial cell line (16HBE14o-). Candidate molecules were chosen on the basis of the known cytotoxicity mechanisms of mustards or their efficacy previously observed on other cellular models. It included the sulfhydryl-containing molecules N-acetyl-cysteine (NAC) and WR-1065, the nucleophile hexamethylenetetramine (HMT), the energy-level stabilizer niacinamide (NC), the antioxidant dimethylthiourea (DMTU), L-arginine analogues such as L-thiocitrulline (L-TC) and L-nitroarginine methyl ester (L-NAME), and the anti-gelatinase doxycycline (DOX). Their efficacy was determined using 2-(4-[3-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium (WST-1) reduction by viable cells 24 h after initial exposure to 100 µM HN2 or SM. On individual immediate cotreatment, some molecules exhibited selective protection against only one mustard, such as DMTU and WR-1065 against HN2 and DOX against SM, whereas NAC and L-TC were effective against both SM and HN2 cytotoxicity. However, as the level of protection against SM was always weak compared to HN2, several combinations were investigated against SM to improve the protection. The effective combinations (L-TC + DOX, NAC + DOX, NAC + DMTU, NAC + HMT, NC + DOX) combined agents, reducing the bioavailability of the mustard with compounds possibly acting on the consequences of alkylation. One of these combinations, NAC + DOX, appeared to be the most interesting, as these agents are already used in human therapy. It exhibited good efficacy in delayed cotreatment (up to 90 min) against SM.

Key Words: sulfur mustard; nitrogen mustard; comparative protection; respiratory epithelium in culture.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfur mustard (2,2`-dichlorodiethyl sulfide, SM) and nitrogen mustard (2,2`-dichlorodiethylamine, mechlorethamine, HN2) are alkylating agents that have been used for many years as chemical weapons and therapeutic drugs, respectively (Papirmeister et al., 1991Go). SM is a highly toxic chemical warfare agent and still remains a threat to both civilians and military personnel (Eisenmenger et al., 1991Go). Although some beneficial effects have been observed with some drugs in tissue culture systems, the antidote activity of the test compounds was always too weak to be used as protectants against SM (Dacre and Goldman, 1996Go; Smith, 1999Go). HN2, which has a similar molecular structure to SM, is commonly used as an anticancer drug and remains an important therapeutic agent in the treatment of early-stage mycosis fungoides (Breneman et al., 1991Go). However, this agent is also extremely toxic and its use is accompanied by severe side effects (Colvin, 1982Go). Despite many years of research into these agents, the cytotoxic mechanisms induced by mustards and the initial events leading to cell death have not been fully elucidated. The mechanism of the mustard injury is linked to alkylation of cellular targets. DNA alkylation provokes the activation of poly(ADP-ribose)-polymerase (PARP) resulting in a rapid depletion of the NAD+/ATP metabolites leading to cell death (Papirmeister et al., 1991Go). Moreover, oxidative stress is likely involved in mustard-induced acute toxicity following glutathion depletion (Giuliani et al., 1997Go; Rappeneau et al., 1999Go).

The respiratory tract is one of the main targets of SM, and is the site of the most disabling lesions for exposed subjects (Balali, 1984Go). However, few studies have been performed on in vitro models of respiratory epithelial cells (Chevillard et al., 1992Go; Guiliani et al., 1994). Furthermore, although the toxicity of the two mustards has been compared (Calvet et al., 1999aGo; Smith et al., 1998Go), protection studies against these agents have rarely been performed on the same biological model (Gray et al., 1994Go; Vojvodic et al., 1985Go; Wormser et al., 1997Go), 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., 2000Go). We found that the sulfhydryl-containing molecule, N-acetylcysteine (NAC), and the radioprotector, WR-1065, classically described for their scavenging properties (Griggs, 1998Go; 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., 2000Go).

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, 1997Go; Sawyer and Risk, 2000Go). 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., 1999Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemical and reagents.
Mechlorethamine hydrochloride (HN2), N-acetylcysteine (NAC), N,N`-dimethylthiourea (DMTU), niacinamide (NC), L-citrulline (L-C), S-methyl-L-thiocitrulline (MeTC), L-thiocitrulline (L-TC), L-nitroarginine methyl ester (L-NAME), doxycycline (DOX), hexamethylenetetramine (HMT), and type I collagen were purchased from Sigma Chemical Co. (St Louis, MO, USA). S-2-[3-aminopropylamino]ethanethiol (WR-1065) was obtained from D.R.E.T (Direction Recherches et Etudes Techniques). SM (purity>98%) was obtained from the Chemistry Department of Centre d'Etudes du Bouchet (Defense Medical Center, France). Cell culture reagents were purchased from Life Technologies (Gibco, Cergy-Pontoise, France): Dulbecco's modified Eagle's medium (DMEM / Ham F12), phenol red and HEPES-free DMEM/Ham F12, fungizone, fetal calf serum (FCS), antibiotics (penicillin, streptomycin). WST-1 reagent (2-(4-[3-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) was purchased from Boehringer Mannheim (Indianapolis, IN).

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., 1994Go). 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% air–5% 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:

  1. In order to determine the concentration effect, cell cultures were exposed to various SM or HN2 concentrations for an exposure time of 4 h. Cell cultures were then rinsed and either immediately assayed for cytotoxicity or incubated in fresh culture medium for a further 20 h in order to determine delayed toxicity.
  2. In order to determine the time-dependence effect, cell cultures were exposed to 500 µM SM or HN2 for exposure times ranging from 5 to 60 min. The dose of 500 µM was chosen in order to obtain a significant cytotoxic effect after 4 h of exposure. Cell cultures were then rinsed and incubated in fresh medium prior to determining cytotoxicity at 4 or 24 h after initial exposure. A 4-h exposure time was chosen because previous experiments showed that longer exposure times to the mustard did not alter the cytotoxicity at 24 h (data not shown).

Two treatment strategies were used to test the efficacy of candidate molecules against SM or HN2:

  1. The candidate molecule was added to the culture medium simultaneously with the mustards, i.e., immediate cotreatment.
  2. The candidate molecule was added to the culture medium as delayed treatment, 2.5 to 90 min after the initial mustard exposure. In both cases, the incubation mixture was removed 4 h after the initial mustard exposure. Cells were then rinsed and incubated in fresh medium for a further 20 h at 37°C. Analyses were performed at 24 h from the initial exposure. Drug combinations were tested according to the same procedures.

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., 1995Go; Miyasaki et al., 1999Go). 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., 1995Go). 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, 1999aGo). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Comparison of SM and HN2 Toxicity on 16HBE14o- Cells
Figure 1Go shows the typical concentration-response curves obtained when 16HBE14o- cell cultures were exposed to SM or HN2. After 4 h of exposure to mustards, the cultures were either assayed for immediate cytotoxicity (Fig. 1AGo) or were incubated for a further 20 h in fresh culture medium prior to determining delayed cytotoxicity (Fig. 1BGo), using the WST-1 assay. Under these conditions, the toxicity of HN2 was greater than that of SM by 4 h of exposure (Fig. 1AGo), and increased at 24 h (Fig. 1BGo). The IC50 at 24 h was about 12 µM and 30 µM for HN2 and SM, respectively. The dose of 100 µM was chosen as suitable to test and compare the protective effect, at 24 h, of candidate molecules against the acute toxicity induced by SM and HN2.



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FIG. 1. Cytotoxic effect of sulphur mustard (SM) (black) and nitrogen mustard (HN2) (grey) concentrations in 16HBE14o- cells. After 4 h of exposure to mustards, the cultures were assayed for cytotoxicity (A), or were incubated for a further 20 h in fresh culture medium prior to determining cytotoxicity (B) using the WST-1 assay. Data shown represent a typical experiment using 8 wells per data point. At 4 h, significant cytotoxic effects were observed with 50 µM and 100 µM for HN2 and SM, respectively. *A significant difference (p < 0.05) was observed between SM- and HN2-treated cells.

 
However, the pattern of toxicity was quite different when SM and HN2 were applied for a short exposure time, as SM was more toxic than HN2 under these conditions (Fig. 2Go). Cell cultures were exposed to mustard (500 µM) for times ranging from 5 to 60 min. A higher dose of mustard (i.e., 500 µM) was used in order to obtain a significant toxic effect for short exposure times. Cell cultures were then rinsed and incubated in fresh medium prior to determining cytotoxicity at 4 h (Fig. 2AGo) or 24 h (Fig. 2BGo) after initial exposure, using the WST-1 assay. Significantly higher cytotoxicity was observed with SM compared to HN2 for exposure times ranging from 15 to 60 min at 4 h (Fig. 2AGo) and with exposure times ranging from 5 to 60 min at 24 h (Fig. 2BGo). Furthermore, at 24 h, maximum cytotoxicity was observed after 15 min of exposure to SM, while the toxicity of HN2 increased dramatically as a function of exposure time during the first hour of exposure (Fig. 2BGo).



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FIG. 2. Effect of short-time exposure to SM (black) or HN2 (grey) on 16HBE14o-cells. Cell cultures were exposed to 500 µM SM or HN2 for exposure times ranging from 5 to 60 min, and were then rinsed and incubated in fresh culture medium prior to determining cytotoxicity at 4 h (A) or 24 h (B) after initial exposure, using the WST-1 assay. Data shown represent a typical experiment using 8 wells per data point. *A significant difference (p < 0.05) was observed between SM- and HN2-treated cells.

 
Protection Afforded by Individual Compounds
All candidate molecules investigated in this study were chosen according to their reported efficacy against mustards. These included the sulfhydryl-containing molecules NAC and WR-1065, the PARP inhibitor NC (Rappeneau et al., 2000Go), HMT (Lindsay and Hambrook, 1997Go), several L-arginine analogues such as L-TC and L-NAME (Sawyer et al., 1996Go, 1998Go), and tetracycline DOX (J. H. Calvet, personal communications). All these molecules were tested at non-cytotoxic concentrations. Cultured cells were exposed to 100 µM of SM or HN2 for 4 h with or without candidate molecules added as immediate cotreatment. Treated cells were then rinsed with medium and incubated for a further 20 h in fresh culture medium prior to determining cytotoxicity, using the WST-1 assay (Fig. 3Go).



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FIG. 3. Comparison of the protective effects provided by several molecules against SM (A) and HN2 (B) toxicity in 16HBE14o- cells. Cell cultures were exposed to 100 µM of SM or HN2 for 4 h, with or without immediate cotreatment with candidate molecules. Cells were then incubated for a further 20 h in fresh culture medium, prior to determining cytotoxicity using the WST-1 assay. The molecules used at the indicated concentrations were added as immediate cotreatment. Data shown represent a typical experiment using 8 wells per data point. *A significant protective effect (p < 0.05) was observed compared to SM or HN2 treatment only.

 
Among the candidate molecules, only NAC (10 mM), HMT (10 mM) and L-TC (10 mM) significantly reduced both HN2- and SM-induced cytotoxicity at 24 h (Figs. 3A and 3BGoGo). However, these compounds were more effective against HN2 than against SM on 16HBE14o- cells, as the protective ratio was 4.9 and 1.6 with NAC (10 mM), 5.2 and 1.6 with L-TC (10 mM), and 1.7 and 1.2 with HMT (10 mM) for HN2- and SM-treated cultures, respectively (Table 1Go). Furthermore, DOX (50 µg/ml) and NC (10 mM) provided relatively weak protection against SM only, with protective ratios of 1.3 and 1.2, respectively, while DMTU (10 mM), WR-1065 (1 mM) and L-NAME (10 mM) were found to be effective against HN2 only, with protective ratios of 3.9, 3.1, and 1.7, respectively (Table 1Go).


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TABLE 1 Protective Ratios following Treatment with Several Molecules against HN2 or SM
 
Protection Afforded by Drug Combinations against SM
To improve the low protection against SM evidenced with molecules used individually, we tested several drug combinations. No significant cytotoxic effects were observed with the drug combinations tested on 16HBE14o- cells. Cultured cells were exposed to 100 µM of SM for 4 h with or without drug combinations added as immediate cotreatment. Treated cells were then rinsed with medium and incubated for a further 20 h in fresh culture medium prior to determining cytotoxicity using the WST-1 assay (Fig. 4Go).



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FIG. 4. Effect of several combinations of molecules against SM toxicity on 16HBE14o- cells. Cell cultures were exposed to 100 µM SM for 4 h with or without a combination of molecules and were then incubated for a further 20 h in fresh culture medium, prior to determining cytotoxicity using the WST-1 assay. The molecules used at the indicated concentrations were added as immediate cotreatment. Data shown represent a typical experiment using 8 wells per data point. *Values significantly different (p < 0.05) from corresponding SM treatment only. +Values significantly different (p < 0.05) from each molecule used alone.

 
Five drug combinations improved the protection afforded by individual drugs (Fig. 4Go): L-TC + DOX, NAC + DOX, NAC + DMTU, NAC + HMT, NC + DOX. Three of the combinations, including NAC, presented similar degrees of efficacy: NAC + DOX, NAC + DMTU, NAC + HMT, with protective ratios of 2.0, 2.0, and 1.9 (Table 1Go). Two other combinations, including DOX, were also effective: L-TC + DOX and DOX + NC, with protective ratios of 2.3 and 1.7. Drug combinations did not clearly improve the high level of protection against HN2 already demonstrated with the molecules used individually (Table 1Go).

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. 5Go).



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FIG. 5. Effect of delayed addition of NAC (A) or NAC in combination with DOX (B) to 16HBE14o- cells exposed to SM. Cell cultures were exposed to 100 µM SM for 4 h. During this incubation period, NAC (10 mM) or NAC in combination with DOX (50 µg/ml) were added to the medium at the indicated time after the initial SM exposure. Treated cells were then incubated for a further 20 h in fresh culture medium prior to determining cytotoxicity, using the WST-1 assay. Data shown represent a typical experiment using 8 wells per data point. *Values significantly different (p < 0.05) from corresponding SM treatment only.

 
The protection afforded by NAC (10 mM) decreased as a function of the time interval before addition and remained significant when NAC was added during the first 15 min of SM exposure (Fig. 5AGo). The protection afforded by NAC in combination with DOX (50 µg/ml) also decreased as a function of the time interval before addition, but remained significant when the combination was added during the first 90 min after initial exposure to SM (Fig. 5BGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we tested the protective action of several agents against SM- and HN2-induced cytotoxicity on human bronchial epithelial cells. The toxicity of SM and HN2 on the 16HBE14o- cell line, used as a model of airway epithelial cells, was characterized and compared using the WST-1 assay, at 4 and 24 h after initial exposure. HN2 exhibited greater toxicity than SM after a long exposure time (4 h) (Fig. 1Go). However, the toxicity of SM was observed extremely rapidly compared to HN2 and SM was more toxic than HN2 for short exposure times (less than 1 h) (Fig. 2Go), as maximum toxicity was reached after 15 min of exposure to SM, while HN2 toxicity increased with exposure time. The different behavior of the two mustards is in accordance with the results of a previous study on a model of rabbit tracheal epithelial cells in primary culture (Calvet et al., 1999aGo). The greater toxicity of HN2 for longer contact times could be explained by a greater stability of the product or its metabolites in the culture medium (Cummings et al., 1993Go; Kirk, 1985Go). The greater toxicity of SM for short contact times could be explained by a higher reactivity, due to the presence of sulfhydryl groups or better penetration into the cell (Smith et al., 1998Go), as SM is highly fat-soluble, which facilitates its passage across the cell membrane (Papirmeister et al., 1991Go), whereas HN2 needs a choline transport carrier to penetrate into the cell (Goldenberg et al., 1971Go). The differences observed in our model of respiratory epithelial cells confirm the fact that the two mustards are not completely interchangeable in terms of toxicity and emphasize the need to act rapidly to prevent SM-induced lesions. In this context, we performed immediate cotreatment with several candidate molecules reported to be effective against SM or HN2 and tested their efficacy at 24 h after the initial mustard exposure, conditions where HN2 is more toxic than SM.

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., 2000Go), or the nucleophile HMT (Lindsay and Hambrook, 1997Go) 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 3Go). 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, 1998Go). 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, 1992Go), 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., 2000Go).

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., 1987Go; Mol et al., 1989Go) and DMTU, as free oxygen radical scavenger, to reduce oxidative stress (Fig. 3Go). As previously shown on 16HBE14o- cells for HN2 (Rappeneau et al., 2000Go), 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, 1984Go), 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., 2000Go). 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., 1998Go), 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., 1998Go). The tetracycline, doxycycline (DOX), was investigated for its anti-gelatinase properties (Petrinec et al. 1996Go), 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. 3Go).

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. 4Go). 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., 1994Go; Dooms-Gossens et al., 1979; Lewis et al, 1994Go). NAC and DOX are both interesting molecules that are already used in the treatment of numerous diseases (Cotgreave, 1997Go; Lokeshwar, 1999Go) 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, 1997Go), and while DOX exhibited antioxidant (Wasil et al., 1988Go) and anti-gelatinase properties (Lokeshwar, 1999Go). 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. 5Go), 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.


    ACKNOWLEDGMENTS
 
We acknowledge Dr. D. C. Gruenert for providing us with the human bronchial cell line. This work was supported by the Délégation Générale pour l'Armement (D.G.A/D.S.P N°95–151) and by a grant from Caisse d'Assurance Maladie des Professions Libérales Provinces.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 33–1–44–27–69–99. E-mail: rappeneau{at}paris7.jussieu.fr. Back


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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