Chemico-Biological Interactions 134 (2001) 1–12

Protective effect of various antioxidants on thetoxicity of sulphur mustard administered tomice by inhalation or percutaneous routes

Om Kumar *, K. Sugendran, R. VijayaraghavanDi6ision of Pharmacology and Toxicology, Defence Research and De6elopment Establishment,

Gwalior 474002, India

Received 7 June 2000; accepted 9 September 2000

Abstract

Protective effect of various antioxidants, trolox (water soluble analogue of vitamin E),quercetin (bioflavonoid) and glutathione reduced (GSH), was studied following sulphurmustard (SM) intoxication. SM, a blistering agent was administered to Swiss albino femalemice through inhalation (1 LC50=42.3 mg/m3 for 1 h duration; 14 days observation formortality) and percutaneous (1 LD50=154.7 mg/kg; 7 days observation for mortality)routes. The antioxidants were administered three times at the dose of trolox, 500 mg/kg;quercetin, 5 mg/kg and GSH, 400 mg/kg body weight by intraperitoneal injection, oneimmediately following SM exposure, then once each day for 2 days after SM treatment. Theeffect of antioxidants on survival, markers of oxidative damage and purine metabolites wasinvestigated. Survival study animals were observed for 14 days. Oxidative markers (in blood,liver and lung) and purine metabolites (in blood and urine) were investigated 72 h after SMtreatment. Survival time increased significantly following trolox and quercetin treatmentsthrough the inhalation route. Significant decrease in GSH and increase in the level ofmalondialdehyde (MDA) indicated oxidative damage to liver and lung tissues following SMinhalation and percutaneous exposure. Blood and urinary uric acid, end product of purinemetabolism showed an increased following both routes of exposures. The antioxidants,trolox and quercetin protected the liver and lung tissues from oxidative damage caused bySM exposure through inhalation and percutaneous routes. This study showed that antioxi-dants could enhance survival time, protect liver and lung from oxidative damage and reduce

www.elsevier.com/locate/chembiont

Abbre6iations: GSH, glutathione reduced; MDA, malondialdehyde; SM, sulphur mustard.* Corresponding author. Fax: +91-751-341148.E-mail address: drde@gwr1.dot.net.in (O. Kumar).

0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0009 -2797 (00 )00209 -X

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–122

accumulation of purine metabolites in blood following SM intoxication. © 2001 ElsevierScience Ireland Ltd. All rights reserved.

Keywords: Sulphur mustard; Inhalation; Percutaneous; Protection; Antioxidant; Trolox; Quercetin;Glutathione; Malondialdehyde; Uric acid

1. Introduction

Sulphur mustard (SM, 2,2%-dichloro diethyl sulphide) is a potent alkylating andblistering agent [1]. It has been stockpiled in a number of countries as a chemicalwarfare agent [2]. The distribution, metabolism and elimination of SM has beeninvestigated and reported [3–5]. SM reacts in aqueous phase with compoundscontaining nucleophilic functional groups like amino, sulfhydryl, carboxylic andhydroxyl in proteins and nucleic acid. The toxicity of SM is due to interaction withone or more cell constituents.

SM has mutagenic and carcinogenic properties, and alkylates DNA leading to aseries of reactions [6,7]. SM not only alkylates DNA but also reacts with mem-branes, RNA and proteins [1]. Alkylation of DNA leads to DNA strand breakswhich activates the chromosomal enzyme poly (ADP-ribose) polymerase, resultingin the depletion of cellular NAD+ and inhibition of glycolysis leading to cell deathdue to disturbance in the intracellular energy balance [6,8]. The cytotoxic mecha-nism of alkylating agents has been explained by the induction of degradationenzymes. Effect of SM on stability of lysosomal membrane, and release of hy-drolytic enzymes like cystine protease and phospholipase in cytoplasm has beenreported [9]. Enhanced activity of phospholipase-A2 degrades membranephospolipids which causes release of archidonic acid from the cell membrane [10].SM induced DNA damage in various visceral organs is reported in an animalmodel after dermal (percutaneous) and inhalation exposure [11]. Earlier, it had beenreported that dermal and SM inhalation caused accumulation of uric acid in bloodand increased excretion of urinary uric acid in a dose dependent manner due topurine catabolism [12,13].

A separate speculative mechanism of SM cytotoxicity based on SM-inducedglutathione reduced (GSH) depletion, but distinct from the thiol-Ca2+ hypothesiswas proposed. According to the lipid peroxidation hypothesis, the principal toxicconsequence of GSH depletion is the formation of toxic lipid peroxides [14].Respiring cells continuously generate partially reduced oxygen species from endoge-nous sources by spontaneous autoxidation of electron transport carriers in mito-chondria or as a consequence of the action of cytoplasmic oxidases. These partiallyreduced oxygen species are normally detoxified by GSH dependent mechanisms[15]. But in the absence of GSH, they are transformed by iron-requiring reactionsinto highly toxic oxidants via H2O2-dependent reaction [16]. Generated oxidisingagents reacts with membrane phospholipids to form lipid peroxides, initiating achain reaction of lipid peroxidation which can lead to alterations in membranefluidity, loss of membrane protein function, loss of membrane integrity and celldeath [15].

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–12 3

SM can cause monofunctional adducts at N-7 in guanine, N-3 in adenine andbifunctional adducts at N-7 in two guanines adjacent to one another on thesame strand, or in two guanines of opposite site strands, with trace amounts ofmonofunctional adducts on other purine and pyrimidines [17]. This results incleavage of DNA at apurinic sites by specific apurinic endonucleases [18].Thepurine bases are the product of alkylated DNA following SM intoxication andthese apurinated purine bases leaves uric acid as the end product.

When free radicals and other reactive species are generated in living systems, awide variety of antioxidants come into play to combat the situation. SM andvarious other toxic chemicals have been reported to deplete the GSH contentand the reduction of GSH has been shown to have an inverse relationship withlipid peroxidation [16,19]. Vitamin E, is an antioxidant and as a scavenger offree radicals inhibits lipid peroxidation [20]. Augmentation of intracellular levelsof GSH provide partial protection against cytotoxicity of SM [21]. Vitamin E,gossypin and hydroxyethyl rutaside reduced the malondialdehyde (MDA) levelsignificantly following dermal application of SM. However, these treatments didnot alter depletion of GSH following SM exposure [22]. Allopurinol an inhibitorof xanthine oxidase was shown to protect pancreatic b cells in vitro againstcytotoxic action of streptozotocin, an alkylating agent, by preventing enzymaticgeneration of superoxide anion O−

2 [16,23]. A number of other scavengers of freeradicals and other oxidant species have been reported to protect against lipidperoxidation and cytotoxicity in a variety of in vitro and in vivo systems [24–26]. In the present study the protective effect of various antioxidants trolox,quercetin and GSH on the SM induced toxicity by percutaneous and inhalationroutes has been investigated. Percutaneous and inhalation are the most commonroute of human exposure to SM.

2. Materials and methods

2.1. Chemicals

SM was synthesised in the Synthetic Chemistry Division of Defence Researchand Development Establishment, (DRDE), Gwalior and its purity was \95%,as analysed by gas chromatography. Trolox, quercetin and GSH were purchasedfrom Sigma (St. Louis, MO). All other chemicals used were of analytical grade.

2.2. Animals

Swiss albino female mice, bred in the authors’ establishment, weighing between24 and 28 g were used for this study. The animals were maintained on a dustfree rice husk bedding in polypropylene cages. Animals were fed a standardpellet diet (Amrut Laboratory Feeds Pvt., India). Food and water were given ad

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–124

libitum. Animals were exposed to SM either through inhalation or percutaneousroutes. This study has the approval of the Institute’s Ethical Committee.

2.3. Percutaneous exposure

For percutaneous exposure SM was dissolved in polyethylene glycol (PEG) 300and about 0.1 ml was applied and smeared on the skin on a circular area of about1.5 cm diameter (the hair on the application site was closely clipped 24 h before SMapplication). The animals were held for 1–2 min and then left in the cage. The SMwas applied as a single dose of 154.7 mg/kg body weight equivalent to 1.0 LD50 (7days observation for mortality) [22].

2.4. Inhalation exposure

The mice were exposed to the vapours of SM as described by Vijayaraghavan[27]. Briefly, the inhalation exposure chamber made of PTFE, was positionedhorizontally for exposing ten mice at a time for head only exposures (DRDEexposure chamber, Model 2A). A known quantity of SM was diluted in 10 ml ofacetone and the solution was pumped into the liquid pick-up capillary of acompressed air nebuliser at the rate of 8 ml/h. The vapours were directed into theexposure chamber which was maintained at a constant airflow of 20 l/min. Theoutgoing air from the exposure chamber was passed through a series of sodiumhydroxide solutions and a trap containing activated carbon and then exhausted outthrough a fume hood. The chamber air was sampled and analysed using a gaschromatograph (Aimil-Nucon, India). The mice were restrained in individual bodyplethysmographs made of glass and exposed head only to 1.0 LC50 (42.3 mg/m3 for1 h exposure) SM vapour.

2.5. Sur6i6al study

For the survival study, SM was administered percutaneously (1.0 LD50) and byinhalation (1.0 LC50). Animals of both the exposure routes were divided into fourgroup of five animals each and one of the following antioxidant treatments wasadministered three times by intraperitoneal injection, once immediately followingSM exposure, then once each day for 2 days after SM treatment. Trolox and GSHwere dissolved in distilled water and quercetin in PEG-300. The mortality of theanimals was recorded up to 14 days of post exposure.

TreatmentsGroupsSMGroup-ISM+Trolox (500 mg/kg, i.p.)Group-IISM+Quercetin (5 mg/kg, i.p.)Group-III

Group-IV SM+GSH (400 mg/kg, i.p.)

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–12 5

2.6. Biochemical study

For biochemical studies SM was administered percutaneously (1.0 LD50) andby inhalation (1.0 LC50). Animals of both the exposure routes were divided intofour groups of five animals each, separately, and one of the following treatmentswas administered three times by intraperitoneal injection, once immediately fol-lowing SM exposure, then once each day for two days after SM treatment. Acontrol group (group-I) without any treatment was maintained for both percuta-neous and inhalation groups.

TreatmentsGroupsControlGroup-ISulphur mustardGroup-IISM+Trolox (500 mg/kg, i.p.)Group-IIISM+Quercetin (5 mg/kg, i.p.)Group-IVSM+GSH (400 mg/kg, i.p.)Group-V

The body weights of the animals were recorded daily. The food and waterintake were also monitored. The animals were sacrificed for biochemical studies72 h after SM application. The animals were lightly anaesthetised with etherand blood was collected from orbital sinus in heparinised vials. Then theanimals were sacrificed by cervical dislocation, that emptied the bladder. Theurine was collected on a piece of aluminium foil. Lung and liver tissues werealso collected.

The level of GSH in blood, lung and hepatic tissue was analysed by colorimet-ric assay of non-protein sulfhydryl content using the standard procedure [28].Hepatic and lung lipid peroxidation was determined by measuring the level ofMDA according to the method of Buege and Aust [29]. One hundred milligramsof tissue sample was directly homogenised in 5 ml of thiobarbituric acid reagentand boiled for 30 min. The contents of the tube were cooled, centrifuged andabsorbance of the clear supernatant was measured at 535 nm. The amount ofMDA formed was calculated using a molar extinction coefficient of 1.56×105/Mper cm. The level of blood uric acid and urinary uric acid was analyzed byphosphotungstic method [30].

2.7. Statistical analysis

The survival study data was analysed by Friedman repeated measuresANOVA on ranks with Dunnett’s multiple comparison. The biochemical datawas analysed by one way ANOVA with Dunnett’s multiple comparison. A prob-ability of less than 0.05 was taken as significant. SigmaStat (Jandel ScientificCorporation, San Rafael, CA) was used for statistical analysis.

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–126

3. Results

There was no mortality during inhalation exposure. The mice showed sensoryirritation characterised by a pause between inspiration and expiration, and therespiratory frequency decreased during SM inhalation (data not shown).

Exposure to SM through both the routes caused a decrease in body weight (Fig.1). SM exposed animals showed less feed and water intake in comparison to controlanimals. The body weight started decreasing after 24 h post exposure and decreasewas significant on the 3rd day post exposure in both routes. The reduction in body

Fig. 1. Effect of antioxidants on percent change in body weight following sulphur mustard (SM)exposure through percutaneous and inhalation routes in mice. (A) Control; (B) SM; (C) Trolox; (D)Quercetin; (E) GSH. a PB0.05 compared to control group. b PB0.05 compared to SM group.

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–12 7

Table 1Effect of antioxidants on percent survival time following sulphur mustard (SM, 1 LC50) inhalation inmicea

Treatments 75%Median 25%

100.00.00100.0SM12.5Trolox 12.5* 0.0021.90.00Quercetin 12.5*

GSH 0.00 62.525.0*

a Chi-square=25.7 with 3 degree of freedom; n=5. Friedman repeated measures ANOVA on rankswith Dunnett’s multiple comparison. Median, percent of mice dying within 7 days of post exposure. The25th and 75th percentile are calculated by SigmaStat.

* PB0.001 compared to SM group.

weight was more prominent in percutaneous exposure in comparison to inhalationexposure. Antioxidants, trolox and quercetin were able to partially protect bodyweight loss. However, GSH did not show any effect on the body weight (Fig. 1).

The effect of antioxidants on survival time following SM inhalation is presentedin Table 1. Survival time was calculated on the basis of 14 days observation period.Median represents percent of mice dying at 7 days of post exposure. The median(50th percentile) and 25th and 75th percentile were obtained from SigmaStat.Trolox, quercetin, GSH and SM showed 12.5, 12.5, 25.0 and 100.0% mortality on7th day, respectively. A significant increase in survival time was recorded in troloxand quercetin treated groups over the SM group. The percent mortality in percuta-neously exposed SM groups following antioxidant treatments are presented inTable 2. Trolox, quercetin, and GSH treatments did not show significantprotection.

Table 3 summarises the effect of trolox, quercetin and GSH on lipid peroxidationand GSH levels in blood, lung and hepatic tissue following 1 LC50 SM exposure. Asignificant decrease was observed in the level of hepatic GSH in the SM treatedgroup over control. A similar decrease was observed in lung and blood GSH levels.The hepatic GSH depletion was protected by all the three antioxidants. The level of

Table 2Effect of antioxidants on percent survival time following sulphur mustard (SM, 1 LD50) administrationthrough percutaneous route in micea

25%Treatments 75%Median

60.0SM 0.00 100.0100.00.000.00Trolox

20.00Quercetin 5.00 75.0GSH 0.0080.00 100.0

a Chi-square=10.3 with 3 degree of freedom. n=5. Friedman repeated measures ANOVA on rankswith Dunnett’s multiple comparison. Median, percent of mice dying within 7 days of post exposure. The25th and 75th percentile are calculated by SigmaStat.

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–128

Table 3Effect of various antioxidants on reduced glutathione (GSH) and malondialdehyde (MDA) levelfollowing inhalation exposure of sulphur mustard (SM, 1 LC50) in micea

Lung GSH Liver MDA Lung MDALiver GSHTreatments Blood GSH

5.4990.5835.4090.98 4.7190.24 6.1890.56 6.3690.77Control2.9190.18 12.3891.06*3.1790.29* 11.2790.60*29.0096.68SM

5.5490.34**29.5094.43 3.9690.83 8.7490.57*,** 8.1290.57**TroloxQuercetin 5.8390.39** 3.9790.27 9.5690.72* 7.4791.12**29.5094.30

4.9290.20** 9.9690.80*5.9190.51** 7.7690.80**25.2095.21GSH

a Values are mean9S.E.M.; n=5. GSH, mmol/l of blood and mmol/g of tissue. MDA, malondialde-hyde as nmol/g of tissue.

* PB0.05 compared to control group not exposed to SM.** PB0.05 compared to SM group.

MDA was significantly increased in hepatic as well as in lung tissues, in the SMgroup compared to control. Only trolox was able to decrease hepatic lipid peroxida-tion while all the three antioxidants trolox, quercetin and GSH were able todecrease lipid peroxidation in the lung as well as liver (Table 3). The GSH depletionwas more in hepatic tissue while lipid peroxidation was more prominent in lungfollowing inhalation of SM.

The effect of antioxidants on GSH and MDA following percutaneous adminis-tration of SM are shown in Table 4. The blood and hepatic GSH decreased whilethe level of hepatic MDA was increased significantly 72 h after exposure to SM. Allthese effects were protected by trolox, quercetin and GSH. However, the level ofGSH and MDA were not altered in lung tissue following percutaneous exposure ofSM.

The level of uric acid in the blood and urine following inhalation exposure areshown in Table 5. The level of uric acid in the blood increased significantly and anincrease in urinary excretion of uric acid was also observed. Among the antioxi-dants only trolox has shown beneficial effect on restoring blood uric acid level.

Table 4Effect of antioxidants on reduced glutathione (GSH) and malondialdehyde (MDA) level followingpercutaneous administration of sulphur mustard (SM, 1 LD50) in micea

Liver MDA Lung MDALiver GSHTreatments Lung GSHBlood GSH

35.4090.98 5.4990.58 4.7190.24 6.1890.56 6.3690.77Control26.5090.95* 3.8890.21*SM 3.3990.22 11.0690.88* 7.9390.87

4.0890.48Trolox 5.3290.23**31.8090.99** 5.3890.266.9690.38**5.0090.19** 6.2490.2930.1091.62* 6.4390.49**Quercetin 4.6590.36

GSH 6.4590.558.9590.74**3.5790.185.1990.10**34.9091.59**

a Values are mean9S.E.M.; n=5. GSH, mmol/l of blood and mmol/g of tissue. MDA, malondialde-hyde as nmol/g of tissue.

* PB0.05 compared to control group not exposed to SM.** PB0.05 compared to SM group.

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–12 9

Table 5Effect of antioxidants on purine metabolites following inhalation exposure of sulphur mustard (SM, 1LC50) in micea

Treatments Blood uric acid (mg/dl) Urinary uric acid (mg/dl)

2.6490.22Control 54.7498.00SM 5.1590.29* 119.3197.21*

116.1294.50*4.1690.12*,**TroloxQuercetin 5.4990.41* 104.6696.66*

107.8994.83*3.7090.6**GSH

a Values are mean9S.E.M.; n=5.* PB0.05 compared to control group not exposed to SM.** PB0.05 compared to SM group.

However, it has no effect on excretion of urinary uric acid following inhalationexposure. Effect of antioxidants on purine catabolism following SM administrationthrough the percutaneous route in mice is presented in Table 6. The level of blooduric acid and urinary uric acid are increased significantly after 72 h of postexposure. Similar to inhalation exposure only trolox had a beneficial effect onrestoring the blood uric acid and urinary uric acid following percutaneousexposure.

4. Discussion

The mechanism of SM induced injury is not fully understood and no effectivedrug is known against the local and systemic injuries caused by SM. The bestprotection remains contact avoidance and in the event of contact, rapid decontam-ination or detoxification of the contaminated site. Other than the decontaminationthere are three possible strategies for prevention of injuries caused by SM. The firstis to attempt to prevent SM from alkylating critical target molecules; second,reverse the alkylation reaction after it has occurred; and third, to prevent or reverse

Table 6Effect of antioxidants on purine metabolites following percutaneous administration of sulphur mustard(SM, 1 LD50) in micea

Urinary uric acid (mg/dl)Blood uric acid (mg/dl)Treatments

2.4290.28 62.4098.00Control3.9890.32*SM 91.2997.42*

Trolox 2.9790.22** 63.4596.02**Quercetin 7.1091.54* 110.33911.87*GSH 5.5191.13* 67.4799.70

a Values are mean9S.E.M.; n=5.* PB0.05 compared to control group not exposed to SM.** PB0.05 compared to SM group.

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–1210

the secondary biochemical consequences of alkylation that occur during the latentphase of injury, before the onset of irreversible cell and tissue destruction. Serioustechnical and practical barriers are associated with each of these approaches. Thepresent study is based on the hypothesis suggested for SM induced cytotoxicitythrough lipid peroxidation occurring as a result of the formation of reactive oxygenintermediates as a consequence of GSH depletion [15,31]. The protective effect oftrolox, a water soluble analogue of a-tocopherol; quercetin, a bioflavonoid andGSH after percutaneous and inhalation exposure of SM were studied.

A progressive loss in body weight of the animals was a consistent observationafter percutaneous administration of SM that is similar with other reports [32].Dose dependent decrease in body weight following SM administration in rats andmice have been reported [12,13]. In the present study trolox and quercetin protectedthe body weight reduction. While trolox and quercetin both increased survival timesignificantly following inhalation exposure, they did not show any significantprotection following percutaneous administration of SM. An earlier study showedthat flavonoids (gossypin and hydroxyethyl rutaside) and vitamin E increasedsurvival time, protected loss of body weight and decreased lipid peroxidationfollowing percutaneous exposure of SM [22]. The protection may be due to theantioxidant and free radical scavenging properties of these agents [33–35].

Inhalation exposure of SM depleted GSH in blood, lung and hepatic tissue andinduced hepatic and lung lipid peroxidation as evidenced by an enhanced level ofMDA. Single subcutaneous injection of a mono functional SM, butyl 2-chloroethylsulphide (BCS) also induced lipid peroxidation and depleted GSH content of thebrain [22,31]. Only trolox reduced hepatic lipid peroxidation, while trolox,quercetin and GSH were able to reduce lung lipid peroxidation.

The results of this study show that SM inhalation and percutaneous exposureincrease uric acid accumulation in the blood. Inhaled SM enters the systemiccirculation and alkylates the DNA leading to DNA strand breaks and apurination.Apurinated bases are catabolised to hypoxanthine, xanthine, and finally to uricacid, resulting in the increased level of blood uric acid. This effect was antagonisedby antioxidant trolox. Protection of blood uric acid may be due to the inhibition ofADP degradation which is the end product of ATP loss after DNA alkylation.Trolox did not significantly increase survival time of animals after percutaneousexposure but it restored blood and urinary uric acid level. This indicates that thesetwo parameters were not interrelated. Decrease uric acid level is normally associ-ated with starvation, but in present study though there was a decline in food intakeafter SM administration, an increased uric acid level was observed [36]. SM injuryis similar to thermal injury. Increased uric acid level is also reported in thermalinjury due to increased xanthine oxidase activity [37]. Increased excretion of uricacid has been reported following percutaneous administration of SM [12]. Theincrease in urinary excretion may be associated with increased apurination by SM.

In the present study various antioxidants have been used as protective agentsagainst SM toxicity through percutaneous and inhalation routes. SM depleted GSHand increased lipid peroxidation in a route specific manner. Trolox ability to reduceSM induced toxicity may be due to its antioxidants properties. The protection given

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–12 11

by the antioxidants were dependent on the route of exposure of SM. More study isrequired on the dose response of the antioxidants for various doses of SM and ata different time course.

Acknowledgements

Authors are thankful to Dr R.V. Swamy, Director, Defence research andDevelopment Establishment, Gwalior, for his keen interest and providing necessaryfacilities for this study.

References

[1] S.M. Somani, S.R. Babu, Toxicodynamics of sulphur mustard, Int. J. Clin. Pharmacol. Ther.Toxicol. 27 (1989) 419–435.

[2] United Nations Security Council, 1988, Report of the mission dispatched by the Security-Generalto investigate allegation of the use of chemical weapons in the conflict between the Islamic states ofIran and Iraq. Report S-20134 (UNSC: New York).

[3] J.L. Hambrook, J.M. Harrison, D.J. Howells, C. Schock, Biological fate of sulphur mustard,1,1%-thiobis (2-chloroethane). Urinary and faecal excretion of 35S by rat after injection or cutaneousapplication of 35S sulphur mustard, Xenobiotica 22 (1992) 65–67.

[4] R.M. Black, J.L. Hambrook, D.J. Howells, R.W. Read, Biological fate of sulphur mustard,1,-1%-thiobis (2-chloroethane). Urinary excretion profiles of hydrolysis products and B-lyase metab-olized of sulphur mustard after cutaneous application in rat, J. Anal. Toxicol. 16 (1992) 79–84.

[5] R.M. Black, K. Brewster, R.J. Clarke, J.L. Hambrook, J.M. Harrson, D.J. Howells, Metabolism ofthiodiglycol (2,2%-thiobis-ethanol): isolation and identification of urinary metabolites followingintraperitoneal administration to rat, Xenobiotica 23 (1993) 473–481.

[6] B. Papirmeister, C.L. Gross, H.L. Meier, J.P. Petrali, J.B. Johnson, Molecular basis for mustard-in-duced vesication, Fundam. Appl. Toxicol. 5 (1985) S134–S149.

[7] P.D. Lawley, P. Brooks, Molecular mechanism of the cytotoxic action of difunctional alkylatingagents and of resistance to the action, Nature 206 (1965) 480–483.

[8] C.L. Gross, H.L. Meier, B. Papirmeister, F.B. Brinkly, J.B. Johnson, Sulphur mustard lowers NADconcentrations in human skin grafted to achymic nude mice, Toxicol. Appl. Pharmacol. 81 (1985)85–90.

[9] S. Shin, D.S. Choi, Y.B. Kim, S.H. Cha, D.E. Sok, The release of lysosomal arylsulfatase from liverlysosomes exposed to 2-chloroethylethyl sulfide, Chem. Biol. Interact. 97 (1995) 229–238.

[10] R. Ray, R.H. Legere, B.J. Majerus, J.P. Petrali, Sulfur mustard induced increase in intracellular freecalcium level and arachidonic acid release from cell membrane, Toxicol. Appl. Pharmacol. 131(1995) 44–52.

[11] P.V. Lakshman Rao, R. Vijayaraghavan, A.S.B. Bhaskar, Sulphur mustard induced damage in miceafter dermal and inhalation exposure, Toxicology 139 (1999) 39–51.

[12] O. Kumar, R. Vijayaraghavan, Effect on physiological variables and urinary metabolites followinga single dermal application of sulphur mustard in rats, Def. Sci. J. 47 (1997) 389–394.

[13] O. Kumar, R. Vijayaraghavan, Effect of sulphur mustard inhalation exposure on some urinaryvariables in mice, J. Appl. Toxicol. 18 (1998) 257–259.

[14] J.M.C. Gutteridge, B. Halliwell, The measurement and mechanism of lipid peroxidation inbiological systems, Trends Biochem. Sci. 15 (1990) 129–135.

[15] B. Papirmeister, A.J. Feister, S.I. Robinson, R.D. Ford, Medical Defence Against Mustard Gas:Molecular Mechanisms of Cytotoxicity, CRC Press, Boca Raton, FL, 1991, pp. 155–209.

[16] B. Halliwel, Oxidants and human disease: some new concepts, FASEB J. 1 (1987) 358–364.

O. Kumar et al. / Chemico-Biological Interactions 134 (2001) 1–1212

[17] P. Brookes, P.D. Lawley, The reaction of mono and di-functional alkylating agents with nucleicacid, Biochim. J. 80 (1961) 496–503.

[18] N.A. Berger, G.W. Sikorski, S.J. Petzold, K.K. Kurohara, Association of poly (adenosine diphos-phoribose) syntheses with DNA damage and repair in normal human lymphocytes, J. Chem. Invest.63 (1979) 1164–1171.

[19] V.T. Maddaiah, Glutathione correlates with lipid peroxidation in liver mitochondria of triiodothy-ronine injected hypophysectomised rats, FASEB J. 4 (1990) 1513–1519.

[20] T. Nakayma, M. Kodama, C. Nagata, Free radical formation in DNA by lipid peroxidation, Agric.Biol. Chem. 48 (1984) 571–578.

[21] C.L. Gross, J.K. Innace, R.C. Hovatter, H.L. Meier, W.J. Smith, Biochemical manipulation ofintracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulphurmustard, Cell Biol. Toxicol. 9 (1993) 259–267.

[22] R. Vijayaraghavan, K. Sugendran, S.C. Pant, K. Husain, R.C. Malhotra, Dermal intoxication ofmice with bis(2-chloroethyl) sulphide and the protective effect of flavonoids, Toxicology 69 (1991)35–42.

[23] M. Nukatsuka, Y. Yoshimura, M. Nishida, J. Kawada, Allopurinol protects pancreatic b cells fromthe cytotoxic effect of streptozotocin: in vitro study, J. Pharmacobio-Dyn. 13 (1990) 259–262.

[24] H.G. Dhertzer, M. Sainsbury, Protection against carbon tetrachloride hepatotoxicity by 5,10-dihy-droindeno (1,2-b) indol, a potent inhibitor of lipid peroxidation, Food Chem. Toxicol. 26 (1988)517–522.

[25] I.T. Numan, M.Q. Hassan, S.J. Stohs, Protective effects of antioxidants against endrin-inducedlipid peroxidation, glutathione depletion, and lethality in rats, Arch. Environ. Contamin. Toxicol.19 (1990) 302–306.

[26] R.J. Ruch, S.J. Cheng, J.E. Klaunig, Prevention of cytotoxicity and inhibition of intercellularcommunication by antioxidant catechins isolated from chinese green tea, Carcinogenesis 10 (1989)1003–1008.

[27] R. Vijayaraghavan, Modification of breathing pattern induced by inhaled sulphur mustard in mice,Arch. Toxicol. 71 (1997) 157–164.

[28] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77.[29] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302–306.[30] W.T. Caraway, in: D. Seligson (Ed.), Standard Methods of Clinical Chemistry, Academic Press,

New York, 1963, p. 239.[31] N.M. Elsayed, S.T. Omaye, G.J. Klain, G.L. Inase, E.T. Dahlberg, C.R. Wheeler, D.W. Korte, Jr,

Response to mouse brain to a single subcutaneous injection of the monofunctional sulphurmustard, butyl 2-chloroethyl sulphide (BCS), Toxicology 58 (1989) 11–17.

[32] K.S. Venkataswaran, V. Neeraja, K. Sugendran, N. Gopalan, R. Vijayaraghavan, S.C. Pant, A.O.Prakash, R.C. Malhotra, Dose dependent effect on lymphoid organs following a single dermalapplication of sulphur mustard in mice, Hum. Exp. Toxicol. 13 (1994) 147–151.

[33] W. Bors, M. Sara, Radical scavenging by flavonoid antioxidants, Free Radic. Res. Commun. 2(1998) 289–296.

[34] J. Robak, R.J. Gryglewski, Flavonoids are scavengers of superoxide anions, Biochem. Pharmacol.37 (1998) 837–843.

[35] A.K. Ratty, J. Sunamoto, N.P. Das, Interaction of flavonoids with 1,1-diphenyl-2-picrylhydrazylfree radical, liposomal and soybean lipooxigenase-1, Biochem. Pharmacol. 37 (1998) 989–994.

[36] W.F. Ganong, Energy balance, metabolism and nutrition, in: Review of Medical Physiology, 13thedition, Prentice-Hall International Editions, Appleton and Lang, California, 1987, pp. 243–247.

[37] H.P. Friedl, G.O. Till, O. Trentz, P.A. Ward, Role of histamine, complement and xanthine oxidasein thermal injury of skin, Am. J. Pathol. 135 (1989) 203–217.

.