Toxicology, 14 (1979) 95--98 © Elsevier/North-Holland Scientific Publishers Ltd.

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HEMATOLOGICAL CHANGES PRODUCED IN MICE BY OCHRATOXIN A

MALAYA GUPTA, SUMITRA BANDOPADHYAY, B. PAUL and S.K. MAJUMDER

Division of Pharmacology, Department of Pharmacy, Jadaupur University, Calcutta 700032 (India)

(Received June 1st, 1979) (Accepted September 7th, 1979)

SUMMARY

The effects of ochratoxin A (5 mg/kg, intraperitoneal) on clotting time, hemoglobin, total count of red blood cells and white blood cells and differ° ential count of white blood cells were studied in mice. This mycotoxin caused a significant decrease in the hemoglobin level and in the total count of red blood ceils. Among white blood cells there was a significant increase in small lymphocytes and a significant fall in the count of neutrophils, basophils and monocytes in treated mice. The administration of ochratoxin A also prolonged the clotting time by more than 6-fold.

INTRODUCTION

Ochratoxin A is one of the most potent fungal toxins known. It has been identified as the major causative agent of the toxic effects of Aspergillus ochraceus, contaminated agricultural products including wheat, corn and peanuts [1--3]. The toxin produces structural aberration in duckling hepa- tocytes mitochondria, fat ty vacuolation in liver [4] and inhibits mitochon- drial respiration in rat liver [5]. Ochratoxin A is also a potent nephrotoxin in rat and other animals [4,6]. It is capable of blocking cell division in tissue culture [7]. In this paper we report that the administration of ochra- toxin A to mice caused extensive changes in hematologic parameters.

Abbreviations: RBC, red blood corpuscles; WBC, white blood corpuscles.

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MATERIALS AND METHODS

Toxin Ochratoxin A used in these studies was isolated and purified from Asper-

gillus wentii by the method of Van der Merwe et al [3]. Moistened rice (500 g) in a 500 ml Erlenmeyer flask was sterilized at 15 lbs. steam pressure for 30 min. The rice sample was inoculated with a spore suspension of A. wentii, and incubated at 28°C for 7 days. Crude toxin was extracted with 500 ml of chloroform. The chloroform extract was evaporated to 30 ml and 20 ml hexane were added. A brownish white precipitate was obtained. The precipitate was dissolved in 5 ml CHC13. Toxin was extracted from CHC13 solution with aqueous NaHCO3 (0.5 M). The aqueous layer was acidified and the white precipitate was dissolved in 5 ml CHC13. The process was repeated and the resulting chloroform solution was added to hexane and kept for 24 h at 5°C; the white crystalline material which formed was then collected by filtration. Ochratoxin A was identified and tested for puri ty by thin-layer chromatography [8]. The isolated material was chemically pure.

Animals Male Albino mice weighing 15--20 g were used. The animals were fed a

standard pellet diet and given tap water ad libitum. They were divided into 3 groups, each group containing 25 male mice. Ochratoxin A dissolved in propylene glycol (1 mg/ml) was injected intraperitoneally in the first group at a dose of 5 mg/kg. The second group, (vehicle controls) was given propyl- ene glycol at a dose of 5 ml/kg intraperitoneaUy. A third group received, 0.9% NaC1 (w/v) at a dose of 5 ml/kg intraperitoneally. This group served as the control group. The injections were given once weekly for 6 weeks. For

TABLE I

CLOTTING TIME, HEMOGLOBIN CONTENT, TOTAL COUNT OF RBC AND WBC OF THE WHOLE BLOOD OF P R O P Y L E N E GLYCOL AND OCHRATOXIN A T R E A T E D MICE a

P ropy lene Ochra tox in A glycol (14) (15)

Clot t ing t ime (s) hemoglob in c o n t e n t in g 100 ml whole b lood Total c o u n t of R B C / m m 3 Total coun t o f WBC/mm 3

31.60 ± 1.84 12.5 ± 0.34

1 1 . 1 3 x 106 ± 6.03

4606.25 +- 204.3

196.67 -+ 2.99* 9.29 ± 0.32*

4 . 0 5 6 x 106.± 1 .82"

8979.75 t 231 .14"*

aNumbers in parentheses deno te the n u m b e r of animals in each case. Values shown above are mean results ± S.E. 'P ' was calculated by S tuden t ' s t-test. The same no ta t ions are fo l lowed in Table II. *P < 0 . 0 1 ; * * P < 0.05.

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TABLE II

DIFFERENTIAL COUNT OF WBC IN THE WHOLE BLOOD OF PROPYLENE GLYCOL AND OCHRATOXIN A TREATED MICE a Results expressed in percent WBC

Propylene Ochratoxin A glycol (14) (15)

Lymphocytes (a) Small 54.74 ± 1.06 70.08 ± 1.05" (b) Large 19.57 -+ 0.78 15.16 ± 0.47*

Monocytes 5.00 -+ 0.37 2.66 ± 0.36* Basophils 1.00 ~ 0.22 0.25 + 0.11"* Neutrophils 19.57 ± 0.92 11.91 ± 0.61"

aNumber in parentheses denotes the number of animals. *P < 0.01 ; **P < 0.02.

the hematological studies, the tips of the tails of the mice were cut of f and blood was collected for hematological measurement.

Measurement of hematologic parameters The total counts of red blood corpuscles (RBC) and white blood corpuscles

(WBC) were estimated with a hem ocy t ome te r [9]. Clotting t ime was measured by the capillary glass tube me thod [9]. Hemoglobin con ten t of whole blood was determined by the Hellige me thod [9]. Differential count of leucocytes was measured by the Leishman staining method [10]. The data presented in Table I and II were calculated as average values + S.E. of blood samples of 14 propylene glycol t reated and 15 ochratoxin A treated mice.

RESULTS AND DISCUSSION

Following intraperi toneal injection of ochratoxin A in mice, the clotting t ime of blood was increased by more than 600% when compared with the controls (Table I). Simultaneously WBC count increased nearly 2-fold. In contrast, bo th the hemoglobin conten t and RBC count decreased; the con- centrat ion of red cells in the blood of toxin t reated mice was less than half that in propylene glycol or saline t reated animals.

With the except ion of small lymphocytes , whose number was increased significantly in the ochratoxin t reated mice, the numbers of o ther cells (monocytes , basophils and neutrophils) were decreased. (Table II).

Although the exact mechanism for the product ion of anaemia in these animals is no t known, it has been repor ted that the pylorus and duodenum are altered resulting in defective absorption of nutr ients in ochratoxin A treated rat; thus anaemia could result from malabsorption [11]. However, the prolongation of clotting time by ochratoxin A may be due to liver

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disorder. Since mos t o f the clot t ing factors are synthes ized in the liver [12] , it is possible tha t the increased c lot t ing time is a consequence o f the defici- ency o f the c lo t t ing factor(s) due to liver injury caused by the tox in [3,4] . As discussed above ochra tox in A increased the n u m b e r of small l ympho- cytes in b lood while m o n o c y t e s , basophils and neut rophi ls were found to be decreased (Table II). The mechanism of this d iscr iminatory ef fec t o f the tox in on di f ferent subpopula t ion of white cells is n o t known. It may be possible tha t the decrease o f neutrophi ls by ochra tox in A was a di rect ac t ion o f the latter on bone mar row o f the animal [13] . On the o ther hand the increased small l y m p h o c y t e c o u n t might be related to the hepa- t o tox ic e f fec t o f the m y c o t o x i n [4] .

ACKNOWLEDGEMENTS

The authors are grateful to the Indian Council of Agricultural Research (New Delhi, India) for financial support. Thanks are due to Dr. C.W. Hesseitine, Northern Regional Research Laboratory, U.S. Department of Agriculture, U.S.A. for the supply of standard samples of mycotoxins. They also wish to thank Mr. J.K. Dey, Department of Pharmacy (Jadavpur University, Calcutta).

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