JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol.18, 257–259 (1998)

Effect of Sulphur Mustard Inhalation Exposureon some Urinary Variables in Mice

Om Kumar* and R. VijayaraghavanDivision of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior-474002, India.

Key words: sulphur mustard; inhalation exposure; uric acid; creatine; creatinine; mice

The effect of sulphur mustard (2,29-dichlorodiethyl sulphide) exposure through inhalation at 0.5. 1.0and 2.0 lc50 (21.2, 42.3 and 84.6 mg m23 for 1 h) on some urinary variables was studied in femalemice at 6, 24 and 48 h and 7 days post-exposure. The urinary excretion and circulatory bloodaccumulation of uric acid increased significantly. The level of creatine was also elevated significantly ascompared to the control at 2lc50. It is concluded that sulphur mustard alkylates DNA and triggerscatabolism of apurinated purine bases in a very short time. The increase in uric acid excretion in urinecan be detected only when the exposure concentration is high. 1998 John Wiley & Sons, Ltd.

INTRODUCTION

Sulphur mustard (SM, 2,29-dichlorodiethyl sulphide) isan alkylating agent and reports are available of itsrecent use during the Iran–Iraq war.1,2 Sulphur mustardhas mutagenic and carcinogenic properties and alkyl-ates DNA, leading to a series of reactions.3,4 It notonly alkylates DNA but also reacts with membranes,RNA and proteins.5 Alkylation of DNA leads to DNAbreaks, which activates the chromosomal enzyme poly-(ADP-ribose) polymerase, resulting in the depletion ofcellular NAD1 and inhibition of glycolysis, leading tocell death.3,6 Apoptosis, a form of programmed celldeath due to DNA fragmentation, is also reported inSM intoxication.7 We reported earlier that topicallyapplied SM increased the excretion of uric acid in adose-dependent manner due to purine catabolism.8

Because SM can also enter the system by inhalation,in the present study we have investigated the effect ofSM inhalation on some urinary variables in connectionwith its role in purine catabolism.

EXPERIMENTAL

Chemicals

Sulphur mustard was synthesized in the chemistrydivision of this Institute and the purity was.95% asanalysed by gas chromatography. All other chemicalsused were of analytical grade.

Animals

Female albino mice bred in this Institute and weighing24–28 g were used for this study. The animals were

* Correspondence to: Dr Om Kumar, Division of Pharmacologyand Toxicology, Defence Research and Development Establishment,Gwalior-474002, India.

CCC 0260–437X/98/040257–03$17.50 Received 4 November 1997 1998 John Wiley & Sons, Ltd. Revised 20 February 1998

Accepted 21 February 1998

maintained on a bedding of rice husk in polypropylenecages and fed on a standard pellet diet (Amrut Feed).Food and water were givenad libitum.

Inhalation exposure

The mice were exposed to the vapours of SM asdescribed by Vijayaraghavan.9 Briefly, the inhalationexposure chamber was made of PTFE, positioned hori-zontally for exposing ten mice at a time for head-onlyexposures (DRDE exposure chamber, Model 2A). Aknown quantity of SM was diluted in 10 ml of acetoneand the solution was pumped into the liquid pick-upcapillary of a compressed-air nebulizer at a rate of8 ml h21. The vapours were directed into the exposurechamber, which was maintained at a constant airflowof 20 l min21. The outgoing air from the exposurechamber was passed through a series of sodiumhydroxide solution and a trap containing activated car-bon and then exhausted out in a fume hood. Thechamber air was sampled and analysed using a gaschromatograph (Aimil-Nucon, India). The mice wererestrained in individual body plethysmographs made ofglass and exposed head only to 0.5, 1.0 and 2.0lc50

(21.2, 42.3 and 84.6 mg m23, respectively; lc50 542.3 mg m23 for the 1-h exposure and 14-day obser-vation period) of SM vapours for a duration of 1 h.For each concentration 30 mice were exposed. Onlyten mice were exposed at a time and three exposureswere done for each concentration. After exposure tothe same concentration the animals were mixed andsacrificed randomly at 6, 24 and 48 h and 7 dayspost-exposure.

Biochemical analysis

The blood and urine samples were collected fromanimals exposed to all three concentrations (0.5, 1.0and 2.0 lc50) at 6, 24 and 48 h and 7 days post-exposure. The mice were lightly anaesthetized withether and blood was collected from the orbital sinusin heparinized vials. Then the animals were killed by

258 O. KUMAR AND R. VIJAYARAGHAVAN

cervical dislocation, which empties the bladder; theurine is collected on a piece of aluminium foil. Theblood was analysed for uric acid and urine was ana-lysed for uric acid, creatine and creatinine. Uric acidwas determined by the phosphotungstic acid method,10

and creatine and creatinine were analysed by the pic-rate method.11

Statistical analysis

The data were analysed by one-way ANOVA, andDunnett’s method was used for comparison of controland SM-exposed groups. For statistical analysis, Sigma-Stat (Jandel Scientific Corporation San Rafael, CA,USA) was used.

RESULTS

There was no mortality during exposure. The miceshowed sensory irritation characterized by a pausebetween inspiration and expiration, and the respiratoryfrequency decreased (data not shown). There was nomortality after exposure in the 0.5lc50 group. How-ever, in the 1.0lc50 and 2.0lc50 groups the micestarted to die after 5 days. Five mice died within 7days in the 1.0lc50 group and eight mice died withinthe 2.0lc50 group.

The effect of SM inhalation on blood uric acid andurinary uric acid at various time intervals is presentedin Table 1. There was a significant time- and dose-dependent increase in the excretion of urinary uric acidfollowing SM exposure. Blood uric acid also increasedin a dose- and time-dependent manner.

Urinary excretion of creatine and creatinine follow-ing SM inhalation is shown in Table 2. The creatineand creatinine levels increased significantly in a time-dependent manner in the 2.0lc50 group. However, atlower doses of SM inhalation, i.e. 0.5 and 1.0lc50,the creatine and creatinine levels were not altered.

Table 1. Effect of sulphur mustard inhalation on blood and urinary uric acida

Blood uric acid (mg dl21) Urinary uric acid (mg dl21)

6 h 24 h 48 h 7 days 6 h 24 h 48 h 7 days

Control 1.31 6 0.12 35.15 6 3.68

0.5 lc50 SM 1.98 2.67* 2.85* 2.46* 56.35 88.32* 97.85* 79.15*60.20 60.21 60.23 60.17 68.18 610.59 66.07 66.18

1.0 lc50 SM 2.39 3.41* 3.34* 3.44* 55.68* 76.89* 109.52* 102.99*60.08 60.24 60.31 60.23 66.20 66.42 67.93 66.15

2.0 lc50 SM 3.09* 4.98* 7.86* 5.43* 67.80* 92.03* 125.46* 170.52*60.24 60.34 60.32 60.33 65.29 66.98 68.24 610.37

F 35.60 21.30

P ,0.001 ,0.001

aValues are means 6 SEM (n 5 6; except 2 lc50/7 days, n 5 4); *significantly different from control by Dunnett’s method.

1998 John Wiley & Sons, Ltd. J. Appl. Toxicol.18, 257–259 (1998)

DISCUSSION

The results of this study show that SM inhalationexposure increases uric acid accumulation in the blood.Inhaled SM enters the systemic circulation and alkyl-ates the DNA, leading to DNA breaks and apurination.It is known that SM can cause monofunctional adductsat N-7 in guanine, N-3 in adenine and bifunctionaladducts at N-7 in two guanines adjacent to one anotheron the same strand, or in two guanines of oppositesite strands, with trace amounts of monofunctionaladducts on other purines and pyrimidines.12 This resultsin cleavage of DNA at apurinic sites by specific apur-inic endonucleases.13 Apurinated bases are catabolizedto hypoxanthine and xanthine, and finally to uric acid,resulting in the increased level of blood uric acid.Although the food consumption is reduced after SMinhalation, the increase in uric acid level was notassociated with starvation because starvation decreasesthe uric acid level.14 Sulphur mustard injury is similarto thermal injury. An increased uric acid level is alsoreported in thermal injury due to an increase in xan-thine oxidase activity.15

Excretion of urinary uric acid was also increasedsignificantly following sublethal and lethal exposure toSM. Similar results were observed by us when SMwas applied topically at lethal and sublethal doses inrats.8 The increase in urinary excretion may also beassociated with increased apurination by SM.

Sulphur mustard exposure at the higher dose(2.0lc50) significantly elevated the urinary excretionof creatine as early as 6 h post-exposure. The DNAstrand breaks due to alkylation cause activation of thechromosomal enzyme poly(ADP-ribose) polymerase,which utilizes NAD1 as its substrate and causesdepletion of cellular NAD1 and interferes in the energymetabolism of the cell by inhibiting glycolysis. Tofulfil the required energy demand of cells, storedenergy in the form of phosphorylcreatine is used. Phos-phorylcreatine is formed by ATP and creatine inmuscles during the resting stage. This reaction isreversed during energy demand, to maintain the supply

259SULPHUR MUSTARD INHALATION AND URINARY VARIABLES

Table 2. Effect of sulphur mustard inhalation on urinary excretion of creatine and creatininea

Creatine (mg dl21) Creatinine (mg dl21)

6 h 24 h 48 h 7 days 6 h 24 h 48 h 7 days

Control 0.34 6 0.08 0.25 6 0.06

0.5 lc50 SM 0.31 0.33 0.39 0.45 0.29 0.28 0.36 0.3560.04 60.06 60.15 60.09 60.04 60.06 60.02 60.04

1.0 lc50 SM 0.36 0.39 0.42 0.46 0.32 0.40 0.32 0.3460.04 60.08 60.12 60.08 60.04 60.02 60.02 60.06

2.0 lc50 SM 1.13* 2.78* 2.85* 2.92* 0.43 0.43 0.66* 0.72*60.22 60.42 60.20 60.34 60.05 60.06 60.12 60.14

F 39.60 5.34

P ,0.001 ,0.001

aValues are means 6 SEM (n 5 6; except 2 lc50/7 days, n 5 4); *significantly different from control by Dunnett’s method.

of ATP, which is the most immediate source of energyfor muscle contraction. Phosphorylcreatine breaks downinto ATP and creatine and the excess amount ofcreatine is excreted in the urine following SM toxicitydue to severe muscular breakdown. Creatinuria is alsoreported during starvation and extensive muscularbreakdown.14

A significant increase in the level of creatinine at48 h and 7 days after exposure to the higher doses ofSM may be due to the conversion of some phosphor-ylcreatine into creatinine. This study shows that alky-lation of DNA by SM triggers catabolism of purinebases in a very short time. The exploitation of purine

REFERENCES

1. U. Wormser, Toxicology of mustard gas. Trends Pharma-col. Sci. 120, 164–167 (1991).

2.. M. Balali-Mood and A. Navaein, Clinical and paraclinicalfindings in 233 patients with sulphur mustard poisoning.Proc. 2nd World Congress on Biological and ChemicalWarfare, Ghent, Belgium (1986).

3. B. Papirmeister, C. L. Gross, H. L. Meier, J. P. Petrali,and J. B. Johnson, Molecular basis for mustard-inducedvesication. Fundam. Appl. Toxicol. 5, S134–S149 (1985).

4. P. D. Lawley and P. Brooks, Molecular mechanism of thecytotoxic action of difunctional alkylating agents and ofresistance to the action. Nature (London) 206, 480–483(1965).

5. S. M. Somani, and S. R. Babu, Therapeutic review: toxico-dynamics of sulphur mustard. Int. J. Clin. Pharmacol. Ther.Toxicol. 27, 419–435 (1989).

6. C. L. Gross, H. L. Meier, B. Papirmeister, F. B. Brinkly andJ. B. Johnson, Sulphur mustard lowers NAD concen-trations in human skin grafted to achymic nude mice.Toxicol. Appl. Pharmacol. 81, 85–90 (1985).

7. M. I. Dabrowsk, L. L. Becks, J. L. Lelli, Jr., M. G. Levesand D. B. Hinshaw, Sulfur mustard induces apoptosis andnecrosis in endothelial cells. Toxicol. Appl. Pharmacol.142, 568–583 (1996).

8. O. Kumar and R. Vijayaraghavan, Effect on physiologicalvariables and urinary metabolites following a single der-

1998 John Wiley & Sons, Ltd. J. Appl. Toxicol.18, 257–259 (1998)

catabolism may be a better indicator for SM toxicity.The increase in uric acid excretion in urine can bedetected only when the exposure concentration is high.For low-level exposures when there is no clinicalmanifestation, there may not be an appreciable increasein uric acid excretion. A more suitable marker may berequired for these exposures.

Acknowledgement

Authors are thankful to Dr R. V. Swamy, Director of the DefenceResearch and Development Establishment, Gwalior, for his keeninterest in this study.

mal application of sulphur mustard in rats. Def. Sci. J. 47,389–394 (1997).

9. R. Vijayaraghavan, Modification of breathing patterninduced by inhaled sulphur mustard in mice. Arch. Toxicol.71, 157–164 (1997).

10. W. T. Caraway, Uric acid, In Standard Methods of ClinicalChemistry, ed. by D. Seligson, 4, 239–247. Academic Press,New York (1963).

11. N. W. Tietz, Fundamentals of Clinical Chemistry, 2nd Edn,pp. 47–51. W. B. Saunders, Philadelphia, PA (1976).

12. P. Brookes and P. D. Lawley, The reaction of mono anddi-functional alkylating agents with nucleic acid. Biochim.J. 80, 496–503 (1961).

13. N. A. Berger, G. W. Sikorski, S. J. Petzold and K. K.Kurohara, Association of poly(adenosine diphosphoribose)syntheses with DNA damage and repair in normal humanlymphocytes. J. Chem Invest. 63, 1164–1171 (1979).

14. W. E. Ganong, Energy balance, metabolism and nutrition.In Review of Medical Physiology, 13th Edn, pp. 243–247.Prentice-Hall International Editions, Appleton and Lang,CA (1987).

15. H. P. Friedl, G. O. Till, O. Trentz and P. A. Ward, Role ofhistamine, complement and xanthine oxidase in thermalinjury of skin. Am J. Pathol. 135, 203–217 (1989).