Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 38, Num. 1, 2006, pp. 38-42

Indian Journal of Pharmacology, Vol. 38, No. 1, January-February, 2006, pp. 38-42

Research Paper

Effect of trolox and quercetin on sulfur mustard-induced cytotoxicity in human peripheral blood lymphocytes

Bhattacharya R, Tulsawani R K, Vijayaraghavan R

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior-474002, Madhya Pradesh, India
Correspondence Address: R. Bhattacharya. E-mail: rbhattacharya41@rediffmail.com

Code Number: ph06007

Abstract

Keywords: Alkylating agent, antioxidant, cytotprotective, WBC

Introduction

Although India is a signatory to the Chemical Weapons Convention, threat from unscrupulous use of sulfur mustard (SM; bis [2-chloroethyl] sulfide), a potential chemical warfare agent still persists.[1],[2] SM is a highly reactive bifunctional alkylating agent that causes blisters and several other injuries on exposure.[3],[4],[5] SM forms sulfonium ion in the body and alkylates DNA, leading to strand breaks and cell death.[3],[5] Owing to the high electrophilic property of the sulfonium ion, SM binds to several cell components and causes various toxic effects on different cell types or tissues.[3], [4], [6] A variety of substances such as free radical scavengers, inhibitors of cell death, promoters of cell survival, radioprotectors, and numerous other pharmacological agents of diverse action have been shown to attenuate SM toxicity in vivo and in vitro . [7],[8],[9],[10],[11],[12] Protective effects of various antioxidants, viz. vitamin E, glutathione, and thiol reagents such as dithiothreitol and N -acetylcysteine (NAC) have also been reported against toxicity of SM or its analogs.[13], [14] However, none of the agents have been approved for human use so far.

The best protection against SM poisoning is avoidance of contact and in the event of contact, rapid decontamination or detoxification of the contaminated area should be done. Only a few chemical decontaminants for human use have shown very good efficacy.[15], [16] The possible strategies to counter SM poisoning include (i) prevention of SM-induced alkylation of macromolecules, (ii) reversal of alkylation, and (iii) prevention or reversal of cascade of biochemical events following alkylation. Clinical manifestation of SM exposure is delayed and often has an extended latent phase of injury.[6] In view of this, many researchers have proposed prophylaxis as a more pragmatic approach for SM poisoning,[7], [10], [12], [14] more so because of the risks involved in the destruction of SM or during inspection by the Organization for Prohibition of Chemical Weapons. In the present study, the effect of trolox (a water-soluble analog of α-tocopherol; vitamin E) and quercetin (a polyphenolic natural bioflavonoid) was evaluated against SM-induced cytotoxicity in human peripheral blood lymphocytes (PBL). Protective efficacy of trolox and quercetin was studied by pretreatment (-0.5 h), simultaneous treatment (0 h), or post-treatment (+0.5 h) to SM exposure.

Materials and Methods

Chemicals
Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman 2-carboxylic acid), quercetin dihydrate (3-3′,4′, 5, 7-pentahydroxy flavone), MTT (3-4,5-dimethyl thiazol- Z -yl)-2,5-diphenyltetrazolium bromide), HEPES (histopaque-1077), fetal bovine serum (FBS), phosphate buffered saline (PBS), and Trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). DCFH-DA (2′,7′-dichlorofluoroscin diacetate) was from Molecular Probes, USA. Hank′s balanced salt solution (HBSS), penicillin, and streptomycin were from Hi Media India Ltd., Mumbai, and other chemicals of highest purity were from BDH or Qualigen India Ltd., Mumbai. Lactate dehydrogenase (LDH) diagnostic kit was purchased from Merck India Ltd., Mumbai. SM was synthesized in the Synthetic Chemistry Division of Defence Research and Development Establishment (DRDE), Gwalior, and was found to be> 99% pure by gas-chromatography analysis.

Lymphocyte preparation
Human PBLs were isolated from fresh heparinized venous blood obtained from normal volunteers.[17] Blood was diluted with equal volume of HBSS (pH 7.4) plus 15 mM HEPES. The sample was then layered on HEPES and centrifuged at 400 g for 30 min at 18°C. Lymphocytes were removed from the aqueous Ficoll interface and washed three times with HBSS. Viability of the cells was found to be more than 95%, as determined by Trypan blue dye exclusion (TBDE).[18] Cells were washed in HBSS and treated with equal volumes of TB (6.2 mM) and NaCl (0.8M) and gently mixed. After 2 min, cells were counted under inverted microscope (Olympus, Japan) using hemocytometer. The percentage of cells excluding dye was calculated immediately after washing. The cell suspension was diluted to a final density of 1 X 10[6] cells/ml of HBSS, containing 15 mM HEPES, 5% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Aseptic techniques were used throughout the preparation of the lymphocytes. Cells were incubated (2 ml/well) in 24 well plates (Nunc, Denmark) maintained at 37°C in humidified atmosphere rich in 5% CO2 + 95% air.

Treatments
Preparation of the solutions and concentrations of SM or the antidotes were based on our previous studies.[12], [19] Cytotoxicity of various concentrations (20-640 mM) of SM, in the presence or absence of 10 mM trolox or quercetin (-0.5, 0, or +0.5 h), was determined in the cells after 6-h exposure. The control cells and SM-treated cells received equal amounts of PBS at the time points corresponding to the antidote treatments. The cell viability was measured by TBDE. In a separate experiment, a cytotoxic concentration of SM (80 mM) was challenged with pretreatment (-0.5 h) of 10 mM trolox or quercetin alone and the cell viability was measured after 6 h by TBDE and leakage of LDH. At this point of time, mitochondrial integrity and peroxide levels were also measured by MTT and DCFH-DA assay, respectively.

Leakage of LDH
After termination of the experiment, intracellular and extracellular (medium) LDH was estimated by commercial diagnostic kit at a wavelength of 340 nm using a plate reader (Tecan SPECTRAFluor Plus, Austria). Leakage of LDH was expressed as percentage of the total.[20]

MTT assay
Mitochondrial integrity of the cells was measured by reduction of mitochondrial enzyme succinate dehydrogenase by MTT. The MTT reduction was carried out by the method of Mosman.[21] Briefly, MTT was dissolved in culture medium at a concentration of 0.5 mg/ml and filtered to remove the small amount of insoluble residue. MTT-containing medium was added to each well in a volume of 0.5 ml and incubated for 3 h. Thereafter, the supernatant was removed and 1.25 ml of a solution of 0.4N HCl-isopropanol (1 : 24 v/v) was added to extract and solubilize the formazan. After 0.5 h at room temperature, the absorbance of the formazan was read at a wavelength of 570 nm and expressed as (_ΔOD⁵⁷⁰).

Measurement of peroxides
The assay is based on the principle that DCFH-DA, a nonpolar and nonfluorescent compound, can diffuse through the cell membrane and deacetylated by cytosolic esterases to yield polar, nonfluorescent DCFH (2′, 7′-dichlorofluoroscin). DCFH is trapped within the cytoplasm, where it reacts with peroxides to form DCF (2′, 7′-dichlorofluorescein), which can be measured by fluorometer at excitation and emission wavelengths of 500 and 520 nm, respectively.[22] Cells were centrifuged at 50 g for 1 min, resuspended in HBSS containing 1.6 µM DCFH-DA, and incubated at 37°C for 2 min. At the end of the incubation, the fluorescence intensity was measured and results expressed as percent control.

Photomicrography
In a separate experiment, cells were exposed to 320 mM SM for 6 h, in the presence or absence of 10 mM trolox or quercetin (-0.5 h). Cells were stained with Papanicolaou solution 3b and observed under light microscope for morphological changes.

Statistical analysis
The results are expressed as mean±SEM ( n =4). The data were analyzed by one-way ANOVA followed by Student-Newman-Keuls test for comparison. Statistical significance was considered at P< 0.05.

Results

Discussion

Various in vitro models have been used to delineate SM toxicity or its antagonism, but human lymphocyte culture is a model with many advantages.[23] Previous study from this laboratory revealed that pretreatment of trolox, but not quercetin, was cytoprotective against cyanide poisoning in vitro .[19] In view of the oxidative injury reported with SM toxicity, we evaluated the effects of pretreatment, simultaneous treatment, or post-treatment of trolox or quercetin against the same in human lymphocytes in vitro. SM causes cytotoxicity through lipid peroxidation, which is mediated by depletion of reduced glutathione (GSH).[3] Flavonoids (gossypin and hydroxyethyl rutaside) and vitamin E have been shown to decrease SM-induced lipid peroxidation in mice, and this protection is attributed to their possible antioxidant and free-radical scavenging properties.[7]

In the present study, the dose-response of SM was similar to that observed previously in liver slice culture, but the severity of toxicity was more.[12] Pretreatment with trolox generally protected against the cell membrane-damaging effects (TBDE, LDH release) of SM, but did not protect the mitochondrial activity, which is more sensitive to free radicals. Perhaps, the diminished mitochondrial activity was sufficient to sustain the cell viability and would have restored to normalcy at a later stage. Trolox, however, protects cyanide-induced DNA fragmentation and mitochondrial and nuclear dysfunction by attenuating the peroxide levels in thymocytes in vitro.[19] Reactive oxygen species mediated cytotoxicity and DNA damage is usually accompanied by depletion of intracellular GSH.[24] Trolox is known to penetrate biomembranes and protect mammalian cells from oxidative damage and DNA fragmentation, and it is considered to be more potent than vitamin E.[25] Generation of free radicals is considered to be one of the earliest events preceding cell death.[24] Because of this reason cells pretreated with the antioxidants could only show significant protection as compared with simultaneous or post-treatment. Lymphocytes are known to lack the ability to synthesize cysteine, a primary component for GSH, and its intracellular glutathione level depends largely on the extracellular cysteine. Therefore, GSH-mediated protection by trolox cannot be anticipated as observed in case of NAC, a precursor to glutathione.[26] Antioxidant properties of quercetin is ascribed to its ability to interrupt membrane lipid peroxidation rather than scavenging the free radicals, which is excessively generated in SM toxicity. Perhaps for this reason we did not observe appreciable protection by quercetin. Also, in our previous study we did not observe notable protection by quercetin.[19] The present study also indicates that trolox was better than quercetin in terms of reducing the levels of peroxide generated by SM. This also suggests strong antioxidant property of trolox, which has been widely recognized for many pathological conditions.[25] In view of the present findings, prophylactic implications of trolox alone or with other agents cannot be overlooked against SM poisoning. Detailed animal studies would further validate its scope.

Acknowledgment

References

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