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Teratogenicity of ochratoxin A in chick embryos
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
SHORT Teratogenicity
46,543-546
(1978)
COMMUNICATION
of Ochratoxin
A in Chick Embryos
Teratogenicity of Ochratoxin A in Chick Embryos. GILANI, S. H., BANCROFT, J., AND REILY, (1978). Toxicol. Appl. Pharmacol. 46,543-546. Experiments were conducted to evaluate the teratogenic potential of ochratoxin A given to developing chick embryos. Ochratoxin A was dissolved in propylene glycol and injected into embryonating chicken eggs at doses ranging from 0.0005 to 0.007 mg/egg. The injections were made into the air sacs of eggs after 48,72, and 96 hr of incubation (Groups A, B, and C). Control eggs were injected with an equivalent volume of propylene glycol (0.1 ml/egg). In all, 720 chicken eggs were used for this study. All embryos were examined at Day 8. The LD,, for embryos in Groups A, B, and C was 0.0007,0.003, and 0.007 mg/egg, respectively. The following malformations were observed: short and twisted limbs, short and twisted neck, microphthalmia, exencephaly, everted viscera, and reduced body size. Microscopic examination of whole embryos treated at 48 hr (Group A) showed ventricular septal defects, aortic stenosis, and malformations of the valves. The results of the present study indicate that ochratoxin A is teratogenic. M.
Ochratoxin A is a mycotoxin produced by several fungal species included in the genera Aspergillus and Penicillium. This mycotoxin can contaminate agricultural commodities
and thus food for both animals and humans and has been associated with toxic nephropathy in pigs and poultry under natural conditions (Krogh et al., 1973; Elling et al., 1975). The characteristic changes are tubular degeneration and atrophy accompanied by interstitial fibrosis. Other experimental effects in several laboratory species include: mucohemorrhagic enteritis of the cecum, colon, and rectum; tonsillitis; enlargement of lymph nodes (Szczech et al., 1973); liver damage (Suzuki et al., 1975); growth retardation; and reduced food consumption (Munro et al., 1974). Ochratoxin A is teratogenic in several species of animals. Still et al. (197 1) presented evidence for a possible relationship between ochratoxin A contamination of feedstuff and bovine abortion. This mycotoxin was reported to be teratogenic in mice by Hayes et al. (1974), in rats by More and Galtier (1974) and Brown et al. (1976), and in hamsters by Hood et al. (1976). The purpose of these experiments is to analyze the effects of ochratoxin A on chick embryos and to further define its teratogenic potential. METHODS
Eggs from White Leghorn chickens were incubated at 38.5“C for 48 (Group A), 72 (Group B), and 96 hr (Group C). Seven batches of eggs (30 eggs/batch) were used for each group. Each batch of eggs represented a dose level, hence seven dose levels of ochratoxin A were tested in each test group. In addition there was one control batch of 30 eggs for each group. In all, 720 eggs were used to gather data for the results 543
0041~8X/78/0462-0543S02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
544
SHORT
COMMUNICATION
reported. Ochratoxin A obtained in crystalline form from Makor Chemicals, Israel, was dissolved in propylene glycol and injected by asceptic technique into the air sac at dose levels ranging from 0.0005 to 0.0007 mg/egg. The volume of each injection was 0.1 ml/egg. All solutions were prepared fresh and were sterilized prior to injection by passage through a Millipore filter (0.45 pm). Control eggs were injected with an equivalent volume of sterile propylene glycol. All the eggs were injected only once. On Day 8, the live embryos were removed from the eggs and examined for the presence of gross malformations. All the live embryos (normal and deformed) were fixed in 10% neutral formalin or in Bouin’s fluid. They were then dehydrated and stored in cedar wood oil. Survival data indicated that embryos in Group A were more sensitive to ochratoxin A than embryos in Groups B and C. The embryos in Group A were therefore examined for cardiac anomalies. Five embryos from each trial (at dose levels ranging from 0.0005 to 0.005 mg/egg) were selected for examination by light microscopy. Whole embryos were fixed in Bouin’s fluid, dehydrated in alcohol, embedded in paraffin, and sectioned serially in transverse and sagittal planes at 6 to 8 ,um. DelatIeld’s or Harris’ hematoxylin and eosin were used for staining of the sections. RESULTS
Data for survival of the embryos are given in Table 1. There was a prominent doseresponse relationship regarding the embryolethal effects of ochratoxin A in all three test groups. Embryos treated at 48 hr of incubation (Group A) were more sensitive to ochratoxin A than were embryos treated at 72 or 96 hr. The LD,, for embryos was 0.0007,0.003, and 0.007 mg/egg, respectively, in Groups A, B, and C (Table 1). TABLE SURVIVAL OF CHICK
1
EMBRYOS FROM EGGS TREATED OCHRATOXIN A
WITH
Viable embryos on Day 8 of incubation Ochratoxin bde88)
A
Group A
0.0005 0.0007” 0.0009 0.00 1 0.003b 0.005 o.007c Control (propylene glycol) for embryos in Group A. b LDSO for embryos in Group B. c LD50 for embryos in Group C. a LDSO
(48 hr)
Group B (72 hr)
Group C (96 hr)
63 50 43 43 17 17 7 90
87 60 77 60 50 43 43 87
100 93 93 87 90 60 50 87
SHORT
COMMUNICATION
545
Malformations in treated embryos consisted of microphthalmia, exencephaly, short and twisted limbs, short and twisted neck, everted viscera, and reduced body size. The malformations found in treated embryos were similar regardless of when the embryos were exposed. The types of cardiac anomalies observed in embryos in Group A included ventricular septal defects, aortic stenosis, thin ventricular walls, atrial septal defects and malformations of the valves. The overall incidence of cardiac anomalies was low. Six of thirty surviving embryos (at dose levels ranging from 0.0005 to 0.005 mg/egg) showed cardiac anomalies. No dose-response relationship was observed. DISCUSSION
Several malformations found in this study were similar to those reported previously. Reduced body size, microphthahnia, short and twisted limbs, everted viscera, and short and twisted necks have been reported in mice (Hayes et al., 1974), in rats (Still et al., 1971; Brown et al., 1974; More and Galtier, 1974), and in hamsters (Hood et al., 1976). Meisner and Chan (1974) reported that ochratoxin A is an inhibitor of mitochondrial transport systems. Steyn et al. (1975) demonstrated that in monkey kidney epithelial cells the predominant toxic effect of ochratoxin A was on cells in the metaphase. They concluded that abnormal mitosis results in the formation of multinucleated cells or cell death. In the early stages of cardiogenesis, the endocardial cushions are rapidly proliferating cellular masses that contribute to the formation of valves and septa (Van Mierop et al., 1962). Administration of ochratoxin A at this stage of heart development could interfere with the normal development of the endocardial cushions and result in the kinds of cardiac anomalies we observed. The results of the present study indicate that ochratoxin A is teratogenic in chickens. ACKNOWLEDGMENT This study wassupportedby the National Foundation-March of Dimes,White Plains,New York. REFERENCES BROWN,M. H., SZCZECH, G. M., ANDPURMALIS, B. P. (1976).Teratogenicand toxic effectsof ochratoxinA in rats. Toxicol. Appl. Pharmacol. 37, 33 l-338. ELLING,F., HOLD, B., JACOBSEN, C., AND KROGH, P. (1975). Spontaneouscasesof toxic nephropathyin poultry associated with ochratoxin A. Acta Pathol. Microbial. Stand. Sect. A 83,739-741.
HAYES,A. W., HOOD,R. D., ANDLEE, H. L. (1974). Teratogeniceffectsof ochratoxin A in mice.Teratology 9, 93-97. HOOD, R. D., NAUGHTON, M. J., ANDHAYES,A. W. (1976).Prenataleffectsof ochratoxin A in hamsters.Teratology 13, 11-14. KROGH,P., HOLD,B., ANDPEDERSON, E. J. (1973).Occurrenceof ochratoxin A and citrinin in cerealsassociated with mycotoxic porcinenephropathy.Acta Pathol. Microbial. &and. Sect. B 81,689-695.
MEISNER,H., AND CHAN, S. (1974). Ochratoxin A, an inhibitor of mitochondrialtransport system.Biochemistry 13,2795-3000.
546
SHORT
COMMUNICATION
J., AND GALTIER, P. (1974). Toxicite de l’ochratoxine A: 1, Effet embryotoxique et teratogene chez le rat. Ann. Rech. Vet. 5, 167-178. MIJNRO, I. C., MOODIE, C. A., GOODMAN, T. IS., SCOTT,P. M., AND GRICE, H. C. (1974). Toxicologic changesin rats fed graded dietary levels of ochratoxin A. Toxicol. Appl. MoRB,
Pharmacol.
28,180-188.
P. S., VLEGGAAR, R., Du PREEZ, N. P., BLYTH, A. A., AND SEEGERS, J. C. (1975).The in vitro toxicity of analogsof ochratoxin A in monkey kidney epithelialcells.Toxicol. Appl. Pharmacol. 32, 198-203. STILL, P. E., MACKLIN, A. W., RIBELIN, W. E., AND SMALLEY, E. B. (1971). Relationshipof ochratoxin A to fetal deathin laboratory anddomesticanimals.Nature (London) 234, 563STEYN,
564. S., SATOH, T., AND YAMAZAKI, M. (1975). Effect of ochratoxin A on carbohydrate metabolismin rat liver. Toxicol. Appl. Pharmacol. 32, 116-122. SZCZECH, G. M., CARLTON, W. W., AND TUITE, J. (1973).Ochratoxicosisin Beagledogs: 11, Pathology. Vet. Pathol. 10,219-231. VAN MIEROP, L. H. S., ALLEY, R. D., KAUSAL, H. W., AND STRANAHAN, A. (1962). The anatomy and embryology of endocardialcushiondefects.J. Thorac. Cardiovasc. Surg. 43, 71-83. SHAMSHAD H. GILANI SUZUKI,
JAMES BANCROFT MURIEL REILY
Department of Anatomy New Jersey Medical School Newark, New Jersey 07103 Received March 22,1978;
accepted July lo,1978
AND
APPLIED
PHARMACOLOGY
SHORT Teratogenicity
46,543-546
(1978)
COMMUNICATION
of Ochratoxin
A in Chick Embryos
Teratogenicity of Ochratoxin A in Chick Embryos. GILANI, S. H., BANCROFT, J., AND REILY, (1978). Toxicol. Appl. Pharmacol. 46,543-546. Experiments were conducted to evaluate the teratogenic potential of ochratoxin A given to developing chick embryos. Ochratoxin A was dissolved in propylene glycol and injected into embryonating chicken eggs at doses ranging from 0.0005 to 0.007 mg/egg. The injections were made into the air sacs of eggs after 48,72, and 96 hr of incubation (Groups A, B, and C). Control eggs were injected with an equivalent volume of propylene glycol (0.1 ml/egg). In all, 720 chicken eggs were used for this study. All embryos were examined at Day 8. The LD,, for embryos in Groups A, B, and C was 0.0007,0.003, and 0.007 mg/egg, respectively. The following malformations were observed: short and twisted limbs, short and twisted neck, microphthalmia, exencephaly, everted viscera, and reduced body size. Microscopic examination of whole embryos treated at 48 hr (Group A) showed ventricular septal defects, aortic stenosis, and malformations of the valves. The results of the present study indicate that ochratoxin A is teratogenic. M.
Ochratoxin A is a mycotoxin produced by several fungal species included in the genera Aspergillus and Penicillium. This mycotoxin can contaminate agricultural commodities
and thus food for both animals and humans and has been associated with toxic nephropathy in pigs and poultry under natural conditions (Krogh et al., 1973; Elling et al., 1975). The characteristic changes are tubular degeneration and atrophy accompanied by interstitial fibrosis. Other experimental effects in several laboratory species include: mucohemorrhagic enteritis of the cecum, colon, and rectum; tonsillitis; enlargement of lymph nodes (Szczech et al., 1973); liver damage (Suzuki et al., 1975); growth retardation; and reduced food consumption (Munro et al., 1974). Ochratoxin A is teratogenic in several species of animals. Still et al. (197 1) presented evidence for a possible relationship between ochratoxin A contamination of feedstuff and bovine abortion. This mycotoxin was reported to be teratogenic in mice by Hayes et al. (1974), in rats by More and Galtier (1974) and Brown et al. (1976), and in hamsters by Hood et al. (1976). The purpose of these experiments is to analyze the effects of ochratoxin A on chick embryos and to further define its teratogenic potential. METHODS
Eggs from White Leghorn chickens were incubated at 38.5“C for 48 (Group A), 72 (Group B), and 96 hr (Group C). Seven batches of eggs (30 eggs/batch) were used for each group. Each batch of eggs represented a dose level, hence seven dose levels of ochratoxin A were tested in each test group. In addition there was one control batch of 30 eggs for each group. In all, 720 eggs were used to gather data for the results 543
0041~8X/78/0462-0543S02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
544
SHORT
COMMUNICATION
reported. Ochratoxin A obtained in crystalline form from Makor Chemicals, Israel, was dissolved in propylene glycol and injected by asceptic technique into the air sac at dose levels ranging from 0.0005 to 0.0007 mg/egg. The volume of each injection was 0.1 ml/egg. All solutions were prepared fresh and were sterilized prior to injection by passage through a Millipore filter (0.45 pm). Control eggs were injected with an equivalent volume of sterile propylene glycol. All the eggs were injected only once. On Day 8, the live embryos were removed from the eggs and examined for the presence of gross malformations. All the live embryos (normal and deformed) were fixed in 10% neutral formalin or in Bouin’s fluid. They were then dehydrated and stored in cedar wood oil. Survival data indicated that embryos in Group A were more sensitive to ochratoxin A than embryos in Groups B and C. The embryos in Group A were therefore examined for cardiac anomalies. Five embryos from each trial (at dose levels ranging from 0.0005 to 0.005 mg/egg) were selected for examination by light microscopy. Whole embryos were fixed in Bouin’s fluid, dehydrated in alcohol, embedded in paraffin, and sectioned serially in transverse and sagittal planes at 6 to 8 ,um. DelatIeld’s or Harris’ hematoxylin and eosin were used for staining of the sections. RESULTS
Data for survival of the embryos are given in Table 1. There was a prominent doseresponse relationship regarding the embryolethal effects of ochratoxin A in all three test groups. Embryos treated at 48 hr of incubation (Group A) were more sensitive to ochratoxin A than were embryos treated at 72 or 96 hr. The LD,, for embryos was 0.0007,0.003, and 0.007 mg/egg, respectively, in Groups A, B, and C (Table 1). TABLE SURVIVAL OF CHICK
1
EMBRYOS FROM EGGS TREATED OCHRATOXIN A
WITH
Viable embryos on Day 8 of incubation Ochratoxin bde88)
A
Group A
0.0005 0.0007” 0.0009 0.00 1 0.003b 0.005 o.007c Control (propylene glycol) for embryos in Group A. b LDSO for embryos in Group B. c LD50 for embryos in Group C. a LDSO
(48 hr)
Group B (72 hr)
Group C (96 hr)
63 50 43 43 17 17 7 90
87 60 77 60 50 43 43 87
100 93 93 87 90 60 50 87
SHORT
COMMUNICATION
545
Malformations in treated embryos consisted of microphthalmia, exencephaly, short and twisted limbs, short and twisted neck, everted viscera, and reduced body size. The malformations found in treated embryos were similar regardless of when the embryos were exposed. The types of cardiac anomalies observed in embryos in Group A included ventricular septal defects, aortic stenosis, thin ventricular walls, atrial septal defects and malformations of the valves. The overall incidence of cardiac anomalies was low. Six of thirty surviving embryos (at dose levels ranging from 0.0005 to 0.005 mg/egg) showed cardiac anomalies. No dose-response relationship was observed. DISCUSSION
Several malformations found in this study were similar to those reported previously. Reduced body size, microphthahnia, short and twisted limbs, everted viscera, and short and twisted necks have been reported in mice (Hayes et al., 1974), in rats (Still et al., 1971; Brown et al., 1974; More and Galtier, 1974), and in hamsters (Hood et al., 1976). Meisner and Chan (1974) reported that ochratoxin A is an inhibitor of mitochondrial transport systems. Steyn et al. (1975) demonstrated that in monkey kidney epithelial cells the predominant toxic effect of ochratoxin A was on cells in the metaphase. They concluded that abnormal mitosis results in the formation of multinucleated cells or cell death. In the early stages of cardiogenesis, the endocardial cushions are rapidly proliferating cellular masses that contribute to the formation of valves and septa (Van Mierop et al., 1962). Administration of ochratoxin A at this stage of heart development could interfere with the normal development of the endocardial cushions and result in the kinds of cardiac anomalies we observed. The results of the present study indicate that ochratoxin A is teratogenic in chickens. ACKNOWLEDGMENT This study wassupportedby the National Foundation-March of Dimes,White Plains,New York. REFERENCES BROWN,M. H., SZCZECH, G. M., ANDPURMALIS, B. P. (1976).Teratogenicand toxic effectsof ochratoxinA in rats. Toxicol. Appl. Pharmacol. 37, 33 l-338. ELLING,F., HOLD, B., JACOBSEN, C., AND KROGH, P. (1975). Spontaneouscasesof toxic nephropathyin poultry associated with ochratoxin A. Acta Pathol. Microbial. Stand. Sect. A 83,739-741.
HAYES,A. W., HOOD,R. D., ANDLEE, H. L. (1974). Teratogeniceffectsof ochratoxin A in mice.Teratology 9, 93-97. HOOD, R. D., NAUGHTON, M. J., ANDHAYES,A. W. (1976).Prenataleffectsof ochratoxin A in hamsters.Teratology 13, 11-14. KROGH,P., HOLD,B., ANDPEDERSON, E. J. (1973).Occurrenceof ochratoxin A and citrinin in cerealsassociated with mycotoxic porcinenephropathy.Acta Pathol. Microbial. &and. Sect. B 81,689-695.
MEISNER,H., AND CHAN, S. (1974). Ochratoxin A, an inhibitor of mitochondrialtransport system.Biochemistry 13,2795-3000.
546
SHORT
COMMUNICATION
J., AND GALTIER, P. (1974). Toxicite de l’ochratoxine A: 1, Effet embryotoxique et teratogene chez le rat. Ann. Rech. Vet. 5, 167-178. MIJNRO, I. C., MOODIE, C. A., GOODMAN, T. IS., SCOTT,P. M., AND GRICE, H. C. (1974). Toxicologic changesin rats fed graded dietary levels of ochratoxin A. Toxicol. Appl. MoRB,
Pharmacol.
28,180-188.
P. S., VLEGGAAR, R., Du PREEZ, N. P., BLYTH, A. A., AND SEEGERS, J. C. (1975).The in vitro toxicity of analogsof ochratoxin A in monkey kidney epithelialcells.Toxicol. Appl. Pharmacol. 32, 198-203. STILL, P. E., MACKLIN, A. W., RIBELIN, W. E., AND SMALLEY, E. B. (1971). Relationshipof ochratoxin A to fetal deathin laboratory anddomesticanimals.Nature (London) 234, 563STEYN,
564. S., SATOH, T., AND YAMAZAKI, M. (1975). Effect of ochratoxin A on carbohydrate metabolismin rat liver. Toxicol. Appl. Pharmacol. 32, 116-122. SZCZECH, G. M., CARLTON, W. W., AND TUITE, J. (1973).Ochratoxicosisin Beagledogs: 11, Pathology. Vet. Pathol. 10,219-231. VAN MIEROP, L. H. S., ALLEY, R. D., KAUSAL, H. W., AND STRANAHAN, A. (1962). The anatomy and embryology of endocardialcushiondefects.J. Thorac. Cardiovasc. Surg. 43, 71-83. SHAMSHAD H. GILANI SUZUKI,
JAMES BANCROFT MURIEL REILY
Department of Anatomy New Jersey Medical School Newark, New Jersey 07103 Received March 22,1978;
accepted July lo,1978