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
Received June 4, 1999; accepted October 11, 1999
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ABSTRACT |
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Key Words: mechlorethamine; protection; respiratory epithelium; interaction; PARP; oxidative stress.
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INTRODUCTION |
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There is still no useful treatment or therapeutic antidote available to combat therapeutic nitrogen-mustard cellular damage, and the recent reintroduction of SM into the modern chemical weapons repertoire would cause particular problems during conflict (Eisenmenger et al., 1991), since there is no effective and safe universal decontamination procedure (Dacre and Goldman, 1996
).
Despite many years of research into these agents, cytotoxic mechanisms induced by mustards and the initial events leading to cell death are still not fully understood. Papirmeister et al. (1985) have proposed a biochemical mechanism for mustard-induced toxicity involving the process of PARP following DNA alkylation, resulting in rapid depletion of the NAD+/ATP metabolite leading to cell death (Meier et al., 1987, 1996).
Beside the alkylation of DNA, considered to be the most significant injury to cells from mustards, the oxidative stress is likely involved in alkylating agentsacute toxicity (Chen and Stevens, 1991; Giuliani et al.,1997
; Khan et al.,1992
; Liu et al., 1996
; Rappeneau et al., 1999
). Indeed, alkylating agents are known to induce glutathione (GSH) depletion (Gamcsik et al., 1990
; Gross et al., 1993
) which likely contributes to lipid peroxidation and cell death.
Based on these observed cytotoxic mechanisms, the present study was performed to find potent molecules capable of protecting the respiratory epithelium, which represents one of the main targets of mustards, using the prototype nitrogen mustard HN2 as a model. The discovery of potent molecules against HN2 toxicity and the understanding of these toxic mechanisms on respiratory epithelium are important for the development of specific therapy and may serve for further investigation into SM toxicity. Indeed, HN2 and SM produce similar lesions on rabbit tracheal epithelium (Calvet et al., 1999) and on human skin (Gentilhomme et al., 1992
; Smith et al., 1998
) in vitro.
A number of PARP inhibitors, sulfhydryl group donors, and antioxidants were evaluated against HN2, using the 16HBE14o- cell line as an in vitro model of human respiratory epithelium, and considering metabolic disruption, sulfhydryl depletion, oxidative stress induction, and loss of cell viability as endpoints.
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MATERIALS AND METHODS |
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Cell Cultures
The 16HBE14o- cell line was obtained from Dr. Gruenert (San Francisco, CA) and was originally isolated from human bronchial epithelial cells transformed by origin-defective SV40 (Cozens et al., 1994). Cells were plated onto dishes coated with type I collagen at 4 µg/cm2. Cells were grown in DMEM/Ham F12 supplemented with 2% Ultroser G and 0.25µg/ml fungizone and antibiotics (7 U/ml penicillin, 100 µg/ml streptomycin) and were cultured at 37°C in a 95% air-5% CO2.
MTT reduction assays were carried out in 96-well microplates, whereas ATP level and flow cytometric assays were carried out in 12-well plates. Confluent monolayers (125,000 cells per cm2) were used in each protective test.
Chemical Treatment
Before each treatment, the medium was removed and cells were washed with DMEM/Ham F12 without serum. All treatments were carried out in DMEM/Ham F12 without serum. Three treatment strategies were used to test the efficiency of candidate molecules against HN2: (1) the agent was added to the culture medium in immediate treatment with HN2, (2) the agent was added to the culture medium in delayed treatment after the beginning of HN2 exposure (from 1 to 3 h), or (3) the cultures were preincubated for 24 h with the agent prior to HN2 treatment. All the tested molecules were used at non cytotoxic concentration.
N2 stock solution (50 mM) was prepared in sterile distilled water and stored at 20°C for up to one month. BHT and -tocopherol were dissolved in ethanol whereas silymarin and PHN were dissolved in DMSO. NAC, OTC, WR-2721, and WR-1065 were dissolved in DMEM/Ham F12. NAC and OTC stock solutions were buffered with NaOH 9N.
MTT Reduction Assay
The method is based on the ability of living cells to reduce MTT tetrazolium salt into MTT formazan by the mitochondrial enzyme succinate-deshydrogenase (Mosmann, 1983). Following the 4-h exposure to HN2, cells were washed with DMEM/Ham F12 without phenol red or HEPES to ensure that test substances do not interfere with the MTT reduction. The cells were then incubated for 2 h with MTT solution (0.5 mg/ml PBS) diluted in DMEM/Ham F12 without phenol red or HEPES. MTT formazan was extracted in DMSO. Measurement of optical density was performed at 560nm with a DYNEX MRX microplate reader. DMSO solution was used as blank reference. The optical density of the control (cell culture without any treatment) corresponds to 100% MTT reduction. Results were expressed as a percentage of the control exposed to DMEM/Ham F12 only, and data were presented as mean values ± SD (n = 16).
ATP Level Assay
The ATP level of each sample was assayed using a LUMAC Luminometer Model M2010A, according to the manufacturer's procedure (Perstorp S.A., Division Lumac, Bezons, France). After the toxic treatment, cells were rinsed twice with PBS and were resuspended in trypsin-EDTA, which was inhibited by 10% of FCS. An aliquot of each suspension was lysed by a dilution 1:100, into boiling 25 mM HEPES buffer, at pH 7.75 containing 10 mM magnesium acetate, 2 mM EDTA, and 3 mM sodium azide. The cell extracts were kept in plastic tubes at 20°C. The reaction mixture for the luminescent ATP determination consisted of 100 µl of firefly luciferin-luciferase freshly diluted in buffer medium, according to the manufacturer's recommendation, and 100 µl of cell extract. The ATP level of each sample was calculated using the internal standard method by the addition of a known ATP quantity (Boehringer, Mannheim, Germany). Another aliquot (80 µl) was used to count living cells with 20 µl at 0.2 % of trypan blue. Results were expressed as a percentage of the control ATP level in living cells and data were presented as mean values ± SD (n = 3).
Flow Cytometric Quantification of Cell Viability
After a 24-h exposure to HN2, cells were resuspended in trypsin-EDTA, which was inhibited by the addition of 10% FCS. DNA of dead cells was counterstained with 5 µM PI prior to analysis. Ten thousand cells were analyzed from each assay. Viability was related to that of control cells exposed to DMEM/Ham F12 only (the reference 100% viable cell control). Data were expressed as mean values ± SD (n = 3).
Intracellular sulfhydryl and peroxide levels determinations
Sulfhydryl and peroxide levels were assessed using fluorescent probes monobromobimane (mBBr) and dichlorofluorescein-diacetate (H2DCF-DA), respectively. mBBr forms a fluorescent adduct with sulfhydryl groups. After toxic treatment, cells were labeled with 40 µM mBBr for 10 min at room temperature in HBSS in dim light and held on ice until analysis. Attached cells were then trypsinized and the trypsin action stopped by the addition of 10% FCS. H2DCF-DA is deacetylated by intracellular esterases forming H2DCFH, which, in the presence of a variety of intracellular peroxides, is oxidized to the highly fluorescent compound, 2',7'-dichlorofluorescein (DCF). Cells were loaded for 30 min with 200 µM H2DCF-DA in HBSS prior to HN2 or TBHP treatment. TBHP is a lipoperoxide, and was used as a positive control of oxidative stress. Prior to analysis, PI (3 µg/ml) was added to cell preparations to ascertain viability.
Cytometric Measurements
The analysis was performed using an EPICS-Elite-ESP flow cytometer (Coultronics, France). A 15 mW air-cooled argon-ion laser tuned at 488 nm was used for DCF fluorescence and an innova 905 argon-ion laser (Coherent, France) running at 100mW output in multiline 351363 nm mode was used for mBBr fluorescence. DCF, mBBr and PI fluorescence were respectively collected through 525, 470, and 620 nm band pass-filter. Forward and right-angle scattered light was used to select cells. Fluorescence of 10,000 PI-negative cells was analyzed for each assay.
Statistical Analysis
All results are representative of at least 2 separate experiments. The Student's t-test was used to determine if any increase in protection obtained by treating cultures with protective candidate molecule was significant compared to HN2-only treated cultures. The minimal level of significance chosen was p < 0.05.
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RESULTS |
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Effects of PARP Inhibitors against HN2
The PARP inhibitors niacinamide (NC), 3-aminobenzamide (3-AB), and 6(5H)phenanthridinone (PHN) were investigated. These agents were added either in immediate treatment with HN2 (Table 1, Fig. 4A
) or their addition was delayed until after the beginning of HN2 exposure (Fig. 3A
).
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After a 24- h HN2 exposure.
While PARP inhibitors were efficient protectors against HN2-induced metabolic dysfunction over a 4-h exposure period, NC (5 mM) and 3-AB (1 mM) did not prevent cell death determined 24 h after the beginning of HN2 exposure, if one were to consider the cellular PI-dye exclusion assay (Fig. 4A).
Effects of Sulfhydryl Group Donors against HN2
N-acetylcysteine (NAC), L-oxothiazolidine 4-carboxylate (OTC), amifostine (WR-2721), and its active metabolite WR-1065 were investigated. These agents were either added in immediate treatment with HN2 (Table 1, Figs. 4B, 5, 6A
), or the addition was delayed until after the beginning of HN2 exposure (Figs. 3B, 4C, and 5
), or used in a 24-h pretreatment prior to HN2 exposure (Fig. 5
).
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The flow cytometric analysis following the 3-h exposure to HN2 revealed that NAC (10 mM) treatment significantly lowered intracellular sulfhydryl depletion (Fig. 5). Moreover, NAC (1 mM) was found to prevent the increase of intracellular peroxides induced by HN2 or TBHP treatments (Fig. 6A
).
Upon delayed addition (up to 3 h after the beginning of HN2 exposure), NAC (10 mM) and WR-1065 (1 mM) still prevented the decrease of MTT reduction (Fig. 3B). The best protection, however, is obtained when NAC or WR-1065 is added in immediate treatment with HN2, and protection diminishes at 1-h intervals in terms of MTT reduction (Fig. 3B
) and sulfhydryl levels (Fig. 5
).
Pretreatments (24 h) with the intracellular precursors of sulfhydryl groups NAC (10 mM), OTC (10 mM) and WR-2721 (1 mM) prior to HN2 treatment did not provide any significant protection (data not shown). Furthermore, flow cytometric analysis showed that there was no detectable increase in the intracellular sulfhydryl level following 24-h pretreatment with 10 mM NAC (Fig. 5) or 10 mM OTC (data not shown), compared to control or HN2-treated cells.
After a 24-h HN2 exposure.
NAC (10 mM) used in immediate treatment was found to be efficient in the prevention of the HN2-induced cell death contrary to WR-2721 (1mM), WR-1065 (1mM) and OTC (10mM) considering the cellular PI-dye exclusion assay (fig.4B). However, after 1 h HN2 exposure, NAC addition was ineffective and cell viability in NAC treated cultures fell to levels comparable to that of HN2 only exposed cells (fig.4C
).
Effect of Lipid Peroxidation Inhibitors and Iron Chelators against HN2
Iron chelators (OP, DFO) and inhibitors of lipid peroxidation (silymarin, BHT, -tocopherol) were assessed on HN2 cytotoxicity upon immediate treatment with HN2.
The immediate addition of -tocopherol (up to 0.1 mM) is effective at preventing the HN2-induced decrease of MTT reduction following a 4-h exposure, contrary to silymarin (0.1 mM), BHT (0.1 mM), OP (0.25 mM), and DFO (0.5 mM) (Table 1
). However,
-tocopherol (0.1 mM) only provides 15% of protection measured by the MTT assay. Furthermore, the flow cytometric analysis following a 3-h exposure to HN2 demonstrated that
-tocopherol (0.1 mM) was ineffective to prevent both intracellular sulfhydryl depletion (data not shown) and peroxide production (Fig. 6B
). On the contrary,
-tocopherol (0.1 mM) markedly reduced the TBHP-induced peroxide production (Fig. 6B
).
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DISCUSSION |
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One possible strategy to overcome the adverse effect of HN2 on cells is to diminish alkylation of vital cellular components with sulfhydryl-containing nucleophiles (Barbin and Bartsh, 1989). For this purpose, the cellular cysteine precursors NAC and OTC (Meister, 1994) as well as the organic thiophosphate amifostine (WR-2721) and its active sulfhydryl metabolite, the WR-1065, which are chemo- and radioprotective (Foster-Nora and Siden, 1997
; Griggs, 1998
; Yudas, 1979
), were investigated as chemoprotectors in 16HBE14o- cells. In the present study, the sulfhydryl-containing nucleophiles NAC and WR-1065 were found to prevent HN2-induced metabolic injury on 16HBE14o- cells. However, only NAC was found capable of preventing loss of cell viability induced by HN2 following 24 h of exposure, on the condition that NAC was added immediately. Our results suggest that the efficiency of NAC and WR-1065 was mainly due to their direct chemical interaction with HN2 in the culture medium, leading to HN2 inactivation.
First, pretreatments (up to 24 h) with the intracellular precursors of sulfhydryl groups NAC, OTC, or WR-2721 prior to HN2 exposure, or treatments with OTC and WR-2721 during HN2 exposure are ineffective to protect 16HBE14o- cells. On the contrary, the direct addition of sulfhydryl groups immediately available in the medium using NAC or WR-1065 is beneficial against HN2 during treatment, mainly preventing the cellular sulfhydryl depletion and cell injury. Pretreatment inefficiency with agents needing a metabolic step to release their active thiol compound may be due to the relative ineffectiveness of these agents to increase sulfhydryl levels on 16HBE14o- cells, or it may be due to the relatively high HN2 dose challenge used in our investigations. Indeed, we have observed that pre-treatment with NAC, OTC or WR-2721 did not significantly increase the intracellular sulfhydryl levels detected by flow cytometry, as compared to the controls. Moreover, while it was observed that pretreatments with NAC or OTC prior to SM challenge led to an increase of intracellular levels of GSH within human lymphocytes, only a slight decrease in cytotoxicity after treatment with relatively low SM concentrations was observed (Gross et al., 1993, 1997
). Thus, pre-treatments with sulfhydryl group donors appear to be an ineffective or insufficient strategy for cell protection against the mustards.
Secondly, addition of NAC or WR-1065 is effective against HN2 during the first few h of exposure on both metabolic injury and sulfhydryl-level depletion. Elsewhere, these agents are all the more beneficial since they are added early in the cells` exposure to HN2. Some reactive forms of still HN2 remain available in the incubation medium for up to 3 h of exposure. Indeed, it was observed that following a 3-h exposure the incubation medium induced significant intracellular sulfhydryl depletion on freshly cultured cells. Thus, these results suggest that NAC and WR-1065 interact with HN2 in the incubation medium before HN2 reacts with cellular targets. Similar results were obtained with hexamethylenetetramine, a nucleophilic compound, which was found to protect A549 cells against SM, though it was necessary to be present at the time of SM exposure (Lindsay and Hambrook, 1997). Elsewhere, an increase in viability of A549 cells was found to occur when treating A549 cells with the monoisopropylglutathione ester at the time of SM challenge compared to cells only pretreated with this agent, suggesting an additional extracellular inactivation of SM by monoisopropylglutathione ester (Lindsay et al., 1997
).
Another strategy to overcome the adverse effects of HN2 on the cell, is to diminish the toxic consequences of cellular target alkylation, such as metabolic disruption and oxidative stress, due to PARP activation and GSH depletion, respectively. Used in immediate treatment with HN2, the PARP inhibitors NC, 3-AB, and PHN (Griffin et al., 1995) were greatly effective in preventing HN2-induced metabolic dysfunction during the first few hours of exposure in 16HBE14o- cells. These agents act as inhibitors of the cascade responsible for generating the HN2 lesions, and not as HN2 scavengers. Indeed, it was observed that NC did not prevent HN2-induced sulfhydryl depletion in 16HBE14o- cells (unpublished data). Moreover, it has been demonstrated that PARP inhibitors prevented SM-induced cytotoxicity on human lymphocytes, even when added to the medium after a period of time sufficient for complete removal of SM though hydrolysis or reaction with cellular components (Meier et al., 1996). Nevertheless, restriction of HN2-induced metabolic dysfunction with PARP inhibitors is not sufficient to provide long-term survival following 24 h of exposure to HN2, suggesting that mechanisms other than NAD+/ATP depletion may play an important role in HN2-induced cell death in 16HBE14o- cells. Results obtained on 16HBE14o- cells are concomitant with those obtained on cultured human epidermal cells, on which NC was effective at preventing a drop in NAD+ content following a 4-h exposure to SM, but cell viability was not preserved over a 24-h period (Mol et al., 1989
). In contrast, it was observed that PARP inhibitors were effective in protection of human lymphocytes from SM-induced cell death over a 24-h exposure (Meier and Johnson, 1992
). The reason for this discrepancy is likely due to lymphocytes not dividing in culture, whereas both the epidermal and bronchial epithelial cells actively replicate in culture, and thus are more sensitive to DNA damage.
Beside DNA alkylation and the subsequent PARP activation, oxidative stress may contribute to HN2-induced injury. In this study, we have shown that the antioxidant NAC was able to reduce the peroxide augmentation induced by HN2 in 16HBE14o- cells. The antioxidant property of NAC may intervene in this effect since it was observed that NAC prevents peroxide augmentation following TBHP treatment (Ochi and Miyaura, 1989). Moreover, reduction of sulfhydryl alkylation, in particular GSH, using NAC, must be also strongly implicated in oxidative stress induced by HN2. Among the other antioxidant molecules tested, only the lipid peroxidation inhibitor,
-tocopherol, slightly reduced the effect of HN2 on MTT reduction, in a dose-dependent manner. However, while
-tocopherol markedly prevented oxidative stress induced by the lipoperoxide, TBHP, no modifications of intracellular peroxide and sulfhydryl levels were observed with this agent in HN2-treated cells. Moreover, the lipid peroxidation inhibitors, silymarin (Morazzoni and Bombardelli, 1995
) and BHT, and the iron chelators DFO and OP, which have shown protective effects against HN2-toxicity in hepatocyte and skin models (Khan et al., 1992
; Wormser and Nyska, 1991
), were all ineffective on 16HBE14o- cells. On the other hand, all the tested antioxidants were effective at preventing the oxidative stress induced by the lipoperoxide TBHP (unpublished data). The oxidative stress induced by HN2 does not appear as fundamental in the HN2-induced injury on 16HBE14o- cell, as metabolic injury is not prevented by several antioxidants.
In this study, the kinetics of protection by PARP and sulfhydryl-containing nucleophiles clearly showed that the protective treatment must be initiated during the first hour after HN2-exposure on 16HBE14o- cells. It only reduces, however, the amount of injuries induced by HN2, since PARP inhibitors and WR-1065 only provide a transient protection and do not affect the fatal outcome following 24 h of exposure on 16HBE14o-cells. In fact, HN2 rapidly induces irreversible cellular damage on 16HBE14o- cells, where NAC addition was ineffective on HN2-induced cell death following 1 h of exposure.
In order to be really effective, a protective treatment against mustards must take all molecular mechanisms of cytotoxicity into account. Therefore, it would be interesting to combine several individual potent agents, each blocking one of the toxic mechanisms induced by mustards. Therefore, a combination of potent scavengers, stabilizers of energetic metabolites, and antioxidants must be investigated. Finding a potent combination may have further possible implications for in vivo conditions.
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ACKNOWLEDGMENTS |
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NOTES |
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