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Rubratoxin B and chick embryogenesis: An experimental study
ENVIRONMENTAL
RESEARCH
20, 199-204
Rubratoxin
(1979)
B and Chick Experimental
Embryogenesis: Study
SHAMSHAD H. GILANI,JAMESBANCROFT, Department
of
Anatomy.
AND MURIELREILLY
Nets Jersey Medical New Jerse.v 07103
Received
September
An
School.
Newark.
2 I. 1978
The effects of rubratoxin B :verr studied on the chick embryos of l-3 days of incubation (groups A. B, and C). Rubratoxin B was dissolved in propylene glycol and injected into the air sacs of the eggs at doses ranging from 0.0005 to 0.007 mg per egg. The controls received an equal amount of propylene glycol only (0. I ml per egg). All the embryos including controls were examined at Day 7. In all, 720 eggs were utilized for this investigation. The percentage survival of the embryos on incubation Day 7 ranged from 67 to 40 in group A. 77 to 43 in group B. and 93 to 60 in group C. The LDSUfor the embryos treated on Days I and 2 was 0.001 and 0.005 mg per embryo. respectively. Gross malformations such as short and twisted limbs, short and twisted neck, exencephaly, microphthalmia, everted viscera, reduced body size, and hemorrhage over the body were observed. The frequency and the types of gross abnormalities did not vary much in all the groups. The incidence of malformations in the controls was low. Microscopic examination of Day 1 treated embryos (group A) showed the various types of cardiac anomalies. These include aortic stenosis, ventricular septal defect, thin ventricular wall, atria1 septal defects, and malformations of the valves. The incidence of these cardiac anomalies was low in the experimental embryos. No cardiac anomalies were observed in the control embryos. The results of the present study indicate that rubratoxin B is teratogenic to the overall development of the chick embryos including heart.
INTRODUCTION
Rubratoxin B, a metabolite of Pencillium rubrum, is a mycotoxin that produces hepatitis, nephrosis, and tissue hemorrhage in swine and cattle (Sippel et al.. 1953). In a very comprehensive study, Wogan et al. (1971) noted that chronic treatment of rubratoxin B leads to lesions of liver and spleen, hemorrhagic and congestive lesions of lymph nodes, subendocardial hemorrhage, adrenocortical congestion, and marked edema of the serosal surface of gallbladder in cats and other laboratory animals. There are reports demonstrating that rubratoxin B can induce teratogenic responses in the experimental animals. Koshakji et al. (1973) observed that rubratoxin B was both embryotoxic and teratogenic when administered intraperitoneally to pregnant mice. Hood et al. (1973) found that single intraperitoneal injection of rubratoxin B resulted in significant increases in embryonic mortality. The most common defects noted were exencephaly, malformed pinnae, malformed jaws, umbilical hernias, and open eyes. Evans and Harbison (1977) found rubratoxin B both mutagenic and teratogenic in mice and concluded that the structural requirement for this activity is an c-w-p-unsaturated lactone ring. The present series of experiments was designed to further investigate the teratogenic potential of this mycotoxin on the chick embryogenesis with special emphasis on the heart development. 199 0013-9351/79/050199-06$02.00/O Copyright All rights
@ 1979 by Academic Press. Inc of reproduction in any form reserved.
200
GILANI,
BANCROFT,
MATERIALS
AND REILLY
AND METHODS
Chick embryos of White Leghorn strain were used for all the experiments. The fertile eggs were incubated in a forced draft chick incubator operated at 385°C and a relative humidity of 60-65%. Rubratoxin B was dissolved in propylene glycol and was sterilized prior to injection by passage through a Millipore filter. The injections were made into the air sacs of the eggs at doses ranging from 0.0005 to 0.007 mg per egg on the lst, 2nd, and 3rd days of incubation, respectively (groups A, B, and C). The volume of fluid injected was 0.1 ml per egg. The controls received an equal amount of propylene glycol. Seven batches of eggs (30 eggs per batch) were used each in groups A, B, and C, respectively. In addition, one batch (30 eggs) of controls was utilized for each group. In all, 720 eggs were used. All the embryos were studied on the 7th day of incubation. After noting gross malformations, the embryos were fixed in Bouin’s fluid or in 10% neutral formalin. They were then dehydrated and stored in cedar wood oil. The percentage survival of the embryos indicated that group A (Day 1) was more sensitive to rubratoxin B than the embryos of groups B and C (Days 2 and 3; Table 1). It was therefore considered of interest to examine the embryos of group A for the possible cardiac anomalies. Five embryos from each batch with doses ranging from 0.0007 to 0.005 mg per egg were selected for microscopic sectioning. Histogenetic studies were made with cross and longitudinal sections at 6-8 pm. Tissue sections were stained with hematoxylin and eosin. RESULTS
The percentage survival of the embryos on incubation Day 7 ranged from 67 to 40 in group A, 77 to 43 in group B, and 93 to 60 in group C with similar doses (0.0005-0.007 mg per egg; Table 1). The percentage of embryos that survived decreased as the doses of rubratoxin B were increased in all the groups. Within all the groups, groups A and B appeared to be more sensitive to rubratoxin B than group C. The LD,,for the embryos of groups A and B was 0.001 and 0.0005 mg per egg, respectively (Table 1). TABLE THE PERCENTAGE SURVIVAL THE ADMINISTRATION
1
OF CHICK EMBRYOS ON INCUBATION DAY 7 AFTER OF RUBRATOXIN B ON DAYS 1, 2, AND 3”
Dosage WWegg)
Day 1 (Group A) (%o)
Day 2 (Group B) (%)
Day 3 (Group C) (%,)
0.0005 0.0007 0.0009 0.001 0.003 0.005 0.007
67 63 63 50 40 43 40 87
77 77 73 63 63 50 43 90
93 90 87 77 77 77 60 93
Controls (Propylene glycol)
u Seven batches of eggs (30 eggs per batch) were used each in groups A, B, and C. In addition, one batch (30 eggs) of controls was utilized for each group. In all, 720 eggs were used.
RUBRATOXIN
FREQUENCY OF CONGENITAL TREATED ON DAY 1 (GROUP
I’ Abnormalities abnormality.
were
TABLE 2 ABNORMALITIES IN SURVIVING CHICK A) WITH RUBRATOXIN B AND EXAMINED of abnormalities
EMBRYOS ON DAY 7
t%)” -
Reduced body size
20 19 19 15 12 13 12 26
0.0005 0.0007 0.0009 0.001 0.003 0.005 0.007 Control (Propylene dYCOl)
201
EMBRYOGENESIS
Types
No. of surviving embryos
Dosage hg/egg)
B AND
10 16 21 20 25 23 25 4
noted
Abnormal limbs
Abnormal neck
15 16 21 33 33 31 25 8
in the live embryos.
Exencephaly
15 21 21 33 33 31 25 8
A large
Microphthalmia 0 0 16 20 42 46 33 0
0 0 16 20 42 46 33 0
number
Everted viscera
of embryos
showed
1R 16 0 20 25 ‘3 25 4
more
than one
The teratogenic effects of rubratoxin B were observed all over the body of the embryos in all the groups (Tables 2-4). The gross abnormalities induced by rubratoxin B included microphthalmia (Fig. l), exencephaly (Fig. 2), short and twisted limbs, everted viscera, reduced body size, and hemorrhage all over the body. Within all the groups (A, B, and C), the frequency and types of gross malformations did not vary much. Thetypes of cardiac anomalies observed in embryos in group A included aortic stenosis, ventricular septal defects, thin ventricular walls, atria1 septal defects, and malformations of the valves. The incidence of these cardiac anomalies was
FREQUENCY
Dosage Wkg) 0.0005 0.0@07 0.0009 0.001 0.003 0.005 0.007 Control (Propylene glycol) ’ Abnormalities anomaly.
TABLE 3 OF CONGENITAL ABNORMALITIES IN SURVIVING CHICK (GROUP B) WITH RUBRATOXIN B AND EXAMINED Types
No. of surviving embryos
Reduced body size
24 24 22 19 19 15 13 27
were
13 13 14 21 21 26 23 3
noted
Abnormal limbs 13 17 14 26 21 20 30 3
in the live embryos.
Abnormal neck
of abnormalities
Exencephaly
13 17 14 26 21 20 30 3
A large number
EMBRYOS TREATED ON DAY 7
ON DAY 2
(%)”
Everted viscera
Microphthalmia
0 13 23 21 16 20 23 0
of embryos
0 13 23 21 16 20 23 0
showed
0 0 0 16 16 33 37 0
more
than one
202
GILANI,
BANCROFT, TABLE
FREQUENCY
OF CONGENITAL (GROUP
ABNORMALITIES C) WITH RUBRATOXIN
AND REILLY 4
IN SURVIVING CHICK B AND EXAMINED
EMBRYOS ON DAY
TREATED 7
ON DAY
3
Types of abnormalities (%)”
No. of surviving embryos
body size
limbs
neck
0.0005 0.0007 o.ooo9 0.001 0.003 0.005 0.007 Control (Propylene &!lYCOl)
28 27 26 24 24 24 18 28
7 7 8 13 13 13 17 7
11 15 15 21 21 17 27 7
14 15 15 21 21 17 27
0
0
13 17
0
0
13 17 7
a Abnormalities abnormality.
were noted in the live embryos. A large number of embryos showed more than one
Dosage hih3)
Reduced
Abnormal
Abnormal
Exencephaly 0 0 0 0
Microphthalmia
Everted viscera 0
7 8 13
FIG. 1. Seven-day chick embryos. Embryo at left is from a control group and is normal. Embryo at right is treated with rubratoxin B (0.001 mg per egg) on Day 1 and shows reduced body size and severe microphthalmia. x4.
RUBRATOXIN
B AND
EMBRYOGENESIS
203
low in the rubratoxin B treated embryos. Of 20 surviving embryos, 4 showed cardiac anomalies. No cardiac anomalies were observed in the control embryos. DISCUSSION
The experiments reported indicate that rubratoxin B was both embryoCoxic and teratogenic. It was also dose-dependent. The wide spectrum of gross malformations in the present investigation shows that rubratoxin B has an overall effect on the development of the chick embryos. Like other teratogenic agents this mycotoxin interferes with the early development of the embryonic tissues. Umeda et al. (1970) reported that rubratoxin B increased the mitotic rate of HeLa cells and produced chromosomal damage in postmieotic spermatozoa and described rubratoxin B as a potent mutagen and teratogen. They concluded that the mechanism of rubratoxin B-induced toxicity to the reproductive process was probably electrophilic attack by the lactone moiety to bind to essential mac-
FIG. 2. Seven-day-old embryo showing eye defects, malformations of limbs and neck, cephaly. Embryo was treated with 0.005 mg per egg of rubratoxin on Day 2. x4.
and exen-
204
GILANI,
BANCROFT,
AND REILLY
romolecules irreversibly and inhibit cellular proliferation. In the early stages of heart development, endocardial cushion tissue are rapidly proliferating cellular masses that contribute to the formation of septa and valves (Van Mierop et al., 1962). Administration of rubratoxin B at this stage of cardiac morphogenesis possibly interferes with the growth of cells of endocardial cushion tissue and precipitates their abnormal development. Endocardial cushion defects such as atrial and ventricular defects and malformations of the valves were observed in the present study. The findings in the present study suggest that different doses of rubratoxin B are teratogenic to the overall development of the chick embryos including heart. ACKNOWLEDGMENT This study was supported by The National Foundation-March
of Dimes, White Plains, New York.
REFERENCES Evans, M. A., and Harbison, R. D. (1977). Prenatal toxicity of rubratoxin B and its hydrogenated analog. Toxicol. Appl. Pharmacol. 39,13-22. Hood, R. D., Innes, J. E., and Hayes, A. W. (1973). Effects of rubratoxin B on prenatal development in mice. Bull. Environ. Contam. Toxicol. 10, 200-207. Koshakji, R. P., Wilson, B. J., and Harbison, R. D. (1973). Effect of rubratoxin B on prenatal growth and development in mice. Res. Commun. Chem. Pathol. Pharmacol. 5, 584-592. Sippel, W. L., Bumside, J. E., and Atwood, M. B. (1953). “A Disease of Swine and Cattle Caused by Eating Moldy Corn,” pp. 174-181. Proceedings of the 90th Annual Meeting of the American Veterinary Medical Association. Umeda, M., Saito, A., and Saito, M. (1970). Cytotoxic effects of toxic culture tibrale of Pencillium purpurogenum and its toxic metabohte, rubratoxin B, on HeLa cells. Japan. J. Exp. Med. 40, 490-523. Van Mierop, L. H. S., Alley, R. D., Kausal, H. W., and Stranahan, A. (1962). The anatomy and embryology of endocardial cushion defects. J. Thoruc. Cardiovasc. Surg. 43, 71-83. Wogan, G. N., Edwards, G. S., and Newbern, P. M. (1971). Acute and chronic toxicity of rubratoxin B. Toxicol. Appl. Pharmacol. 19, 712-720.
RESEARCH
20, 199-204
Rubratoxin
(1979)
B and Chick Experimental
Embryogenesis: Study
SHAMSHAD H. GILANI,JAMESBANCROFT, Department
of
Anatomy.
AND MURIELREILLY
Nets Jersey Medical New Jerse.v 07103
Received
September
An
School.
Newark.
2 I. 1978
The effects of rubratoxin B :verr studied on the chick embryos of l-3 days of incubation (groups A. B, and C). Rubratoxin B was dissolved in propylene glycol and injected into the air sacs of the eggs at doses ranging from 0.0005 to 0.007 mg per egg. The controls received an equal amount of propylene glycol only (0. I ml per egg). All the embryos including controls were examined at Day 7. In all, 720 eggs were utilized for this investigation. The percentage survival of the embryos on incubation Day 7 ranged from 67 to 40 in group A. 77 to 43 in group B. and 93 to 60 in group C. The LDSUfor the embryos treated on Days I and 2 was 0.001 and 0.005 mg per embryo. respectively. Gross malformations such as short and twisted limbs, short and twisted neck, exencephaly, microphthalmia, everted viscera, reduced body size, and hemorrhage over the body were observed. The frequency and the types of gross abnormalities did not vary much in all the groups. The incidence of malformations in the controls was low. Microscopic examination of Day 1 treated embryos (group A) showed the various types of cardiac anomalies. These include aortic stenosis, ventricular septal defect, thin ventricular wall, atria1 septal defects, and malformations of the valves. The incidence of these cardiac anomalies was low in the experimental embryos. No cardiac anomalies were observed in the control embryos. The results of the present study indicate that rubratoxin B is teratogenic to the overall development of the chick embryos including heart.
INTRODUCTION
Rubratoxin B, a metabolite of Pencillium rubrum, is a mycotoxin that produces hepatitis, nephrosis, and tissue hemorrhage in swine and cattle (Sippel et al.. 1953). In a very comprehensive study, Wogan et al. (1971) noted that chronic treatment of rubratoxin B leads to lesions of liver and spleen, hemorrhagic and congestive lesions of lymph nodes, subendocardial hemorrhage, adrenocortical congestion, and marked edema of the serosal surface of gallbladder in cats and other laboratory animals. There are reports demonstrating that rubratoxin B can induce teratogenic responses in the experimental animals. Koshakji et al. (1973) observed that rubratoxin B was both embryotoxic and teratogenic when administered intraperitoneally to pregnant mice. Hood et al. (1973) found that single intraperitoneal injection of rubratoxin B resulted in significant increases in embryonic mortality. The most common defects noted were exencephaly, malformed pinnae, malformed jaws, umbilical hernias, and open eyes. Evans and Harbison (1977) found rubratoxin B both mutagenic and teratogenic in mice and concluded that the structural requirement for this activity is an c-w-p-unsaturated lactone ring. The present series of experiments was designed to further investigate the teratogenic potential of this mycotoxin on the chick embryogenesis with special emphasis on the heart development. 199 0013-9351/79/050199-06$02.00/O Copyright All rights
@ 1979 by Academic Press. Inc of reproduction in any form reserved.
200
GILANI,
BANCROFT,
MATERIALS
AND REILLY
AND METHODS
Chick embryos of White Leghorn strain were used for all the experiments. The fertile eggs were incubated in a forced draft chick incubator operated at 385°C and a relative humidity of 60-65%. Rubratoxin B was dissolved in propylene glycol and was sterilized prior to injection by passage through a Millipore filter. The injections were made into the air sacs of the eggs at doses ranging from 0.0005 to 0.007 mg per egg on the lst, 2nd, and 3rd days of incubation, respectively (groups A, B, and C). The volume of fluid injected was 0.1 ml per egg. The controls received an equal amount of propylene glycol. Seven batches of eggs (30 eggs per batch) were used each in groups A, B, and C, respectively. In addition, one batch (30 eggs) of controls was utilized for each group. In all, 720 eggs were used. All the embryos were studied on the 7th day of incubation. After noting gross malformations, the embryos were fixed in Bouin’s fluid or in 10% neutral formalin. They were then dehydrated and stored in cedar wood oil. The percentage survival of the embryos indicated that group A (Day 1) was more sensitive to rubratoxin B than the embryos of groups B and C (Days 2 and 3; Table 1). It was therefore considered of interest to examine the embryos of group A for the possible cardiac anomalies. Five embryos from each batch with doses ranging from 0.0007 to 0.005 mg per egg were selected for microscopic sectioning. Histogenetic studies were made with cross and longitudinal sections at 6-8 pm. Tissue sections were stained with hematoxylin and eosin. RESULTS
The percentage survival of the embryos on incubation Day 7 ranged from 67 to 40 in group A, 77 to 43 in group B, and 93 to 60 in group C with similar doses (0.0005-0.007 mg per egg; Table 1). The percentage of embryos that survived decreased as the doses of rubratoxin B were increased in all the groups. Within all the groups, groups A and B appeared to be more sensitive to rubratoxin B than group C. The LD,,for the embryos of groups A and B was 0.001 and 0.0005 mg per egg, respectively (Table 1). TABLE THE PERCENTAGE SURVIVAL THE ADMINISTRATION
1
OF CHICK EMBRYOS ON INCUBATION DAY 7 AFTER OF RUBRATOXIN B ON DAYS 1, 2, AND 3”
Dosage WWegg)
Day 1 (Group A) (%o)
Day 2 (Group B) (%)
Day 3 (Group C) (%,)
0.0005 0.0007 0.0009 0.001 0.003 0.005 0.007
67 63 63 50 40 43 40 87
77 77 73 63 63 50 43 90
93 90 87 77 77 77 60 93
Controls (Propylene glycol)
u Seven batches of eggs (30 eggs per batch) were used each in groups A, B, and C. In addition, one batch (30 eggs) of controls was utilized for each group. In all, 720 eggs were used.
RUBRATOXIN
FREQUENCY OF CONGENITAL TREATED ON DAY 1 (GROUP
I’ Abnormalities abnormality.
were
TABLE 2 ABNORMALITIES IN SURVIVING CHICK A) WITH RUBRATOXIN B AND EXAMINED of abnormalities
EMBRYOS ON DAY 7
t%)” -
Reduced body size
20 19 19 15 12 13 12 26
0.0005 0.0007 0.0009 0.001 0.003 0.005 0.007 Control (Propylene dYCOl)
201
EMBRYOGENESIS
Types
No. of surviving embryos
Dosage hg/egg)
B AND
10 16 21 20 25 23 25 4
noted
Abnormal limbs
Abnormal neck
15 16 21 33 33 31 25 8
in the live embryos.
Exencephaly
15 21 21 33 33 31 25 8
A large
Microphthalmia 0 0 16 20 42 46 33 0
0 0 16 20 42 46 33 0
number
Everted viscera
of embryos
showed
1R 16 0 20 25 ‘3 25 4
more
than one
The teratogenic effects of rubratoxin B were observed all over the body of the embryos in all the groups (Tables 2-4). The gross abnormalities induced by rubratoxin B included microphthalmia (Fig. l), exencephaly (Fig. 2), short and twisted limbs, everted viscera, reduced body size, and hemorrhage all over the body. Within all the groups (A, B, and C), the frequency and types of gross malformations did not vary much. Thetypes of cardiac anomalies observed in embryos in group A included aortic stenosis, ventricular septal defects, thin ventricular walls, atria1 septal defects, and malformations of the valves. The incidence of these cardiac anomalies was
FREQUENCY
Dosage Wkg) 0.0005 0.0@07 0.0009 0.001 0.003 0.005 0.007 Control (Propylene glycol) ’ Abnormalities anomaly.
TABLE 3 OF CONGENITAL ABNORMALITIES IN SURVIVING CHICK (GROUP B) WITH RUBRATOXIN B AND EXAMINED Types
No. of surviving embryos
Reduced body size
24 24 22 19 19 15 13 27
were
13 13 14 21 21 26 23 3
noted
Abnormal limbs 13 17 14 26 21 20 30 3
in the live embryos.
Abnormal neck
of abnormalities
Exencephaly
13 17 14 26 21 20 30 3
A large number
EMBRYOS TREATED ON DAY 7
ON DAY 2
(%)”
Everted viscera
Microphthalmia
0 13 23 21 16 20 23 0
of embryos
0 13 23 21 16 20 23 0
showed
0 0 0 16 16 33 37 0
more
than one
202
GILANI,
BANCROFT, TABLE
FREQUENCY
OF CONGENITAL (GROUP
ABNORMALITIES C) WITH RUBRATOXIN
AND REILLY 4
IN SURVIVING CHICK B AND EXAMINED
EMBRYOS ON DAY
TREATED 7
ON DAY
3
Types of abnormalities (%)”
No. of surviving embryos
body size
limbs
neck
0.0005 0.0007 o.ooo9 0.001 0.003 0.005 0.007 Control (Propylene &!lYCOl)
28 27 26 24 24 24 18 28
7 7 8 13 13 13 17 7
11 15 15 21 21 17 27 7
14 15 15 21 21 17 27
0
0
13 17
0
0
13 17 7
a Abnormalities abnormality.
were noted in the live embryos. A large number of embryos showed more than one
Dosage hih3)
Reduced
Abnormal
Abnormal
Exencephaly 0 0 0 0
Microphthalmia
Everted viscera 0
7 8 13
FIG. 1. Seven-day chick embryos. Embryo at left is from a control group and is normal. Embryo at right is treated with rubratoxin B (0.001 mg per egg) on Day 1 and shows reduced body size and severe microphthalmia. x4.
RUBRATOXIN
B AND
EMBRYOGENESIS
203
low in the rubratoxin B treated embryos. Of 20 surviving embryos, 4 showed cardiac anomalies. No cardiac anomalies were observed in the control embryos. DISCUSSION
The experiments reported indicate that rubratoxin B was both embryoCoxic and teratogenic. It was also dose-dependent. The wide spectrum of gross malformations in the present investigation shows that rubratoxin B has an overall effect on the development of the chick embryos. Like other teratogenic agents this mycotoxin interferes with the early development of the embryonic tissues. Umeda et al. (1970) reported that rubratoxin B increased the mitotic rate of HeLa cells and produced chromosomal damage in postmieotic spermatozoa and described rubratoxin B as a potent mutagen and teratogen. They concluded that the mechanism of rubratoxin B-induced toxicity to the reproductive process was probably electrophilic attack by the lactone moiety to bind to essential mac-
FIG. 2. Seven-day-old embryo showing eye defects, malformations of limbs and neck, cephaly. Embryo was treated with 0.005 mg per egg of rubratoxin on Day 2. x4.
and exen-
204
GILANI,
BANCROFT,
AND REILLY
romolecules irreversibly and inhibit cellular proliferation. In the early stages of heart development, endocardial cushion tissue are rapidly proliferating cellular masses that contribute to the formation of septa and valves (Van Mierop et al., 1962). Administration of rubratoxin B at this stage of cardiac morphogenesis possibly interferes with the growth of cells of endocardial cushion tissue and precipitates their abnormal development. Endocardial cushion defects such as atrial and ventricular defects and malformations of the valves were observed in the present study. The findings in the present study suggest that different doses of rubratoxin B are teratogenic to the overall development of the chick embryos including heart. ACKNOWLEDGMENT This study was supported by The National Foundation-March
of Dimes, White Plains, New York.
REFERENCES Evans, M. A., and Harbison, R. D. (1977). Prenatal toxicity of rubratoxin B and its hydrogenated analog. Toxicol. Appl. Pharmacol. 39,13-22. Hood, R. D., Innes, J. E., and Hayes, A. W. (1973). Effects of rubratoxin B on prenatal development in mice. Bull. Environ. Contam. Toxicol. 10, 200-207. Koshakji, R. P., Wilson, B. J., and Harbison, R. D. (1973). Effect of rubratoxin B on prenatal growth and development in mice. Res. Commun. Chem. Pathol. Pharmacol. 5, 584-592. Sippel, W. L., Bumside, J. E., and Atwood, M. B. (1953). “A Disease of Swine and Cattle Caused by Eating Moldy Corn,” pp. 174-181. Proceedings of the 90th Annual Meeting of the American Veterinary Medical Association. Umeda, M., Saito, A., and Saito, M. (1970). Cytotoxic effects of toxic culture tibrale of Pencillium purpurogenum and its toxic metabohte, rubratoxin B, on HeLa cells. Japan. J. Exp. Med. 40, 490-523. Van Mierop, L. H. S., Alley, R. D., Kausal, H. W., and Stranahan, A. (1962). The anatomy and embryology of endocardial cushion defects. J. Thoruc. Cardiovasc. Surg. 43, 71-83. Wogan, G. N., Edwards, G. S., and Newbern, P. M. (1971). Acute and chronic toxicity of rubratoxin B. Toxicol. Appl. Pharmacol. 19, 712-720.