Toxicity Studies of Metabolites of Some Fungal Isolates in

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1983, p Vol. 46, No /83/ $02.00/0 Copyright 1983, American Society for Microbiology Toxicity Studies of Metabolites of Some Fungal Isolates in Albino Micet BHARATI PATHAK,' N. SETHI,2 JOYOTI GUPTA,' AND V. C. VORA'* Division of Fermentation Technology1 and Division of Experimental Medicine,2 Central Drug Research Institute, Lucknow, India Received 20 June 1983/Accepted 23 June 1983 Crude metabolites of 21 of 60 fungal cultures isolated from some of the common cereals collected from different parts of India were found to be toxic. Of these toxin-producing fungi, 79% caused hepatic pathology of varying severity in mice. Serum glutamate pyruvate transaminase values and blood urea nitrogen were found to be high in such experimental animals. Mycotoxins may cause morbidity due to positive organ pathology, even without lethal effects on test animals during experimentation. Such histopathological findings have been reported by Gupta et al. (3) and Itakura and Kinosita (7). Hematological parameters in a variety of experimental animals have been reported from the testing of pure toxins (2, 4, 6, 11). The present communication deals with the effects of the crude fungal toxins on tissue pathology, hematopoietic system, and blood chemistry. Collection of samples, identification of fungi, isolation of crude toxins, and biological assay on mice were carried out as reported earlier (3, 7). The samples, from the farms and grain markets of two northern states of India, were collected in polythene bags (ca. 200 g each) and were stored under laboratory conditions. The fungal cultures were grown on natural substrates. The chloroform extract of these fungal metabolites was dried in vacuo. The crude metabolites thus obtained were tested for toxicity. The crude extract, suspended in propylene glycol at a concentration of 100 mg/ml (wt/vol), was administered intraperitoneally to groups of five mice (average weight, 18 to 20 g) for 4 consecutive days at a dose level of 5 mg per mouse. On the fifth and final day, a dose of 10 mg per mouse was administered. Control sets were administered propylene glycol and an extract of the sterile, uninoculated medium. The experiment was terminated on day 7. Toxicity was graded as follows: 4+ (high toxicity: three of five mice died after the first dose); 3+ (medium toxicity: three of five mice died after the second and third doses); 2+ (moderate toxicity: three of t Communication no five mice died after the fourth and fifth doses); and 1+ (mild toxicity: lesions were observed without mortality). A daily record of the behavior (appetite, activity, and response to external stimuli) and general health of all of the animals was maintained. The body weights and hemograms (hemoglobin; total and differential leukocyte count) of the animals were determined initially and at the time of necropsy. At the end of the experimental period, the surviving animals were observed for 48 h and then were sacrificed. Blood samples were collected for biochemical and hematological estimations. At necropsy, different organs of the sacrificed animals were weighed, and gross pathology was recorded. The organs were fixed in 10% neutral buffered Formalin for histological examination. Paraffin sections of these organs were prepared and stained by routine hematoxylin and eosin stains. Total and differential leukocyte counts were carried out by using standard leukocyte pipettes and an improved Neubauer counting chamber (10). Blood smears were stained with Leishman stain. Hemoglobin was determined by the acid hematin method of Sahli and expressed as gram percent (10). Blood urea nitrogen (BUN) and serum glutamate pyruvate transaminase (SGPT) were determined by the methods of Archer and Robb (1) and Bergmeyer and Bernt (2a), respectively. The toxicity of 60 crude extracts (of fungal cultures) was studied. Of the 60 fungal cultures, 38.7% (22 cultures) were found to be toxic, and 61.3% (38 cultures) were nontoxic. Among the toxic cultures, two (9.1%) showed toxicity of 2+ grade, and 90.9% were of 1+ grade. Enzyme values and microscopic pathological data were tabulated (Table 1). Animals with high enzyme values and microscopic pathology showed gains Downloaded from on March 15, 2019 by guest 944

2 VOL. 46, 1983 NOTES 945 I A Photomicrograph of liver, showing cytoplasmic vacuolization (x750). in individual liver weight. Spleens and kidneys as compared with organs from healthy animals, did not show any apparent change. In addition, the animals showed loss of weight, gross and microscopic evidence of hepatic pathology in the form of focal cell necrosis, and fatty degeneration of various severity. Because hematological parameters were found to be essentially within the normal range (comparable to the control animals), it is evident that the crude metabolites had no effect on the hematopoietic system. In 77.8% of the animals, there was an increase in SGPT and BUN values (Table 1). Though BUN values were high (compared with those of the control group) in the majority of the test animals, no gross or microscopic pathology could be recorded in kidneys, except in the case of Syncephalastrum sp. and Mycelia sterilia. Cloudy swelling in the first case and degeneration of tubular epithelium in the second case were observed. However, SGPT values were FIG. 2. Photomicrograph of liver, showing focal necrosis (x 190). FIG. 3. Liver section, showing fatty midzonal infiltration (x 190).

3 946 NOTES APPL. ENVIRON. MICROBIOL. TABLE 1. Histopathological and biochemical observations in mice surviving the administration of crude toxin" Relative liver wt Biochemical parameters' Organismanulueo and culture no. gpob (g per 100 g of Pertinent microscopic observations SGPT BUN body wt ± SD) (W units ± SD) (mg% ± SD) Alternaria sp. (Pl-W-1203) 5.18 ± 1.01 Cytoplasmic vacuolization (+) 22.6 ± ± 2.07 (Fig. 1) Aspergillus sp. (P1-G-342) 7.51 ± 0.73 Focal necrosis (++) 37.4 ± ± 2.97 A. flavus (Pl-G-358) 6.42 ± 1.32 Cytoplasmic vacuolization 19.8 ± ± 3.27 periportal (+) A. fumigatus (Pl-G-166) 4.35 ± 0.59 Normal 11.2 ± ± 2.51 A. japonicus (Pl-W-953) 7.4 ± 1.06 Necrosis 32 ± ± 1.82 A. nidulans (PI-G-331) 6.31 ± 0.68 Focal necrosis (+) (Fig. 2) 31.8 ± ± 2.41 A. niger (Pl-G-141) 6.17 ± 1.55 Fatty midzonal infiltration 34.8 ± ± 2.17 (+) (Fig. 3) A. terreus (PI-G-206) 7.36 ± 0.56 Focal necrosis (+++) 51 ± ± 2.07 A. ustus (P1-G-144) 4.0 ± 0.58 Normal 11 ± ± 2.07 Chaetomium sp. (P1-G-182) 3.8 ± 0.9 Normal 10.8 ± ± 2.39 Cladosporium sp. (P1-G-183) 6.25 ± 1.19 Focal necrosis (+) 31.4 ± ± 3.54 Curvularia sp. (Pl-W-1607) 6.58 ± 0.72 Early necrosis 27 ± ± 2.39 Fusarium sp. (PI-R-297) 5.2 ± 0.53 Focal necrosis (++) 38 ± ± 2.3 Mycelia sterilia (P1-G-152) 5.87 ± 0.91 Focal necrosis (+++) 42.8 ± ± 3.83 Paecilomyces sp. (P1-G-2) 5.94 ± 0.76 Focal necrosis (++) 41.5 ± ± 3.7 Penicillium sp. (P1-G-148) 5.72 ± 0.72 Focal necrosis (++) 31.2 ± ± 2.7 Syncephalastrum sp. (P1-G ± 0.47 Fatty midzonal infiltration 37.4 ± ± ) (+) Trichoderma sp. (Pl-W-286) 3.24 ± 0.81 Normal 17.1 ± ± 2.39 Control Gram (Cicer arietinum) 3.87 ± 0.43 Normal 16.8 ± ± 2.12 Rice (Oryza sativa) 3.86 ± 0.62 Normal 11.2 ± ± 2.59 Wheat (Triticum aestivum) 4.0 ± 1.21 Normal 17.2 ± ± 1.64 a Five animals were used in the experiment. b G, Gram; R, rice; W, wheat. c W units, Wroblewski units; mg%, milligram percent. significantly high in most of the animals treated with metabolites of Aspergillus japonicus, A. nidulans, A. niger, Aspergillus sp., A. terreus, Cladosporium sp., Fusarium sp., Mycelia sterilia, Paecilomyces sp., Penicillium sp., and Syncephalastrum sp. The relative increase in the SGPT values was found to be proportional to the severity of hepatic lesions. Financial support for the investigation from the Department of Science and Technology (grant no. HCS/DSTI421/77), Government of India, is gratefully acknowledged. LITERATURE CITED 1. Archer and Robb Tests of renal efficiency. II. Urease-nesslerization method, p. 96. In G. A. Harrison (ed.), Chemical methods in clinical medicine, 3rd ed. J. & A. Churchill Ltd., London. 2. Avram, N Haematological aspects of mycotoxicosis in intensively reared piglets. Pasteur 14: a.Bergmeyer, H. U., and E. Bernt Colorimetric assay of Reitman and Frankel, p In H. U. Bergmeyer (ed.), Methods of Enzymatic Analysis. Academic Press, Inc., London. 3. Gupta, J., B. Pathak, N. Sethi, and V. C. Vora Histopathology of mycotoxicosis produced in Swiss albino mice by metabolites of some fungal isolates. Appl. Environ. Microbiol. 41: Gupta, M., S. Bandopadhyay, B. Paul, and S. K. Majumdar Haematological changes produced in mice by ochratoxin A. Toxicology 14: Hansen, In H. U. Bergmeyer (ed.), Methods of enzymatic analysis, p Academic Press, Inc., London. 6. Hughes, B. L., and J. E. Jones Haematology of single comb white Leghorn pullets fed aflatoxin contaminated and ammonia treated corn. Poult. Sci. 58: Itakura, H., and R. Kinosita Toxic fungi isolated from Uganda foodstuffs. A histopathological study of acute toxicity of fungal culture filtrates. Trop. Med. (Nagasaki) 17: Lillehoj, E. B., and A. Ceigler Mycotoxin synergism, p In D. Schlessinger (ed.), Microbiology American Society for Microbiology, Washington, D.C. 9. Mall, 0. P., P. Mehta, S. C. Agarwal, and V. C. Vora A survey offungal contaminants offield crops with a view to evaluate their ability to produce mycotoxins. I. Fungal contaminants of wheat and barley harvested during spring, Ind. J. Microbiol. 19: Newberne, P. M Mycotoxins: toxicity, carcinogenicity and the influence of various nutritional conditions. Environ. Health Perspect. 9: Sato, N., T. Ito., H. Kumada, Y. Ueno, K. Asano, M. Saito, K. Ohtsubo, I. Ueno, and Y. Hatanaka Toxicological approaches to the metabolites of Fusaria. XIII. Hematological changes in mice by a single and repeated administration of trichothecenes. J. Toxicol. Sci. 3:

4 VOL. 46, 1983 NOTES Thacker, H. L., W. W. Carlton, and G. A. Sansing Citrinin mycotoxicosis in the guinea pigs. Food Cosmet. Toxicol. 15: Wintrobe, M. M Clinical haematology, 5th ed., p Lea & Febiger, Philadelphia, Pa. 14. Wogan, G. N Dietary factors and special epidermiological situations of liver cancer in Thailand and Africa. Cancer Res. 35: Zimmermann, J. L., W. W. Carlton, and J. Tuits Mycotoxicosis produced in rats by cultural products of an isolate of Aspergillis ochraceits. Food Cosmet. Toxicol. 16:

5 ERRATA Toxicity Studies of Metabolites of Some Fungal Isolates in Albino Mice BHARATI PATHAK, N. SETHI, JOYOTI GUPTA, AND V. C. VORA Division of Fermentation Technology and Division of Experimental Medicine, Central Drug Research Institute, Lucknow, India Volume 46, no. 4, page 944, column 1, paragraph 1, line 5: "Gupta et al. (3) and Itakura and Kinosita (7)" should read "Gupta et al. (3), Itakura and Kinosita (7), Thacker et al. (12), and Zimmerman et al. (15)." Page 944, column 1, paragraph 3, line 3: "... as reported earlier (3, 7)" should read... as reported earlier (3, 9)." Page 944, column 2, paragraph 1, lines 4 and 7: Reference 10 should be reference 13. Pages , Literature Cited: The following references should be deleted: numbers 5, 8, 10, and 11. Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae A. K. KUNDU AND S. MANNA Department of Microbiology, East India Pharmaceutical Works Ltd., Calcutta-60, India Volume 30, no. 4, p : Proteolytic activities throughout should be multiplied by 3.2 to express correct units. 1453