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Susceptibility of vitamin A-deficient rats to aflatoxin
Fd Cosmet. Toxicol. Vol. 11, pp. 467-470. Pergamon Press 1973. Printed in Great Britain
Susceptibility of Vitamin A-deficient Rats to Aflatoxin G. SRIRANGA REDDY,T. B. G. TILAK and D. KRISHNAMURTHI National Institute of Nutrition, hutian Council of Medical Research, Hyderabad 500007, A.P., India (Received 28 November 1972)
Summary--Weanling albino rats were divided into two groups each of six males and six females. They were maintained on a vitamin A-free diet, one group being fed ad lib. and the second being pair-fed with the first. Rats in this second group were given an oral supplement of 100 IU vitamin A/animal/day. At the end of a feeding period of 9 wk, a crystalline preparation of aflatoxin was administered in a single ip dose of 3"5 mg/kg body weight. Male rats in the first group showed an increased susceptibility to the toxin as evidenced by rapid mortality and severe liver damage, midzonal in distribution. Their hepatic stores of vitamin A were found to have been severely depleted. Vitamin A-supplemented male rats showed only mild hepatic damage of the periportal parenchyma. Liver damage was minimal in female rats of both groups and was unaffected by the amount of vitamin A stored in the liver.
Introduction The toxic effects of aflatoxins in laboratory animals and birds have been shown to be affected by dietary protein (Madhavan & Gopalan, 1965; Madhavan, Rao & Tulpule, 1967), fat (Hamilton, Hsi-Tang Tung, Harris, Gainer & Donaldson, 1972; Smith, Hill & Hamilton, 1971) and lipotrophic factors (Rogers & Newberne, 1971). Compared with ariatoxin-treated rats on a normal diet, a decreased incidence of hepatomas and an increase in the occurrence of colonic carcinomas were reported in vitamin A-deficient rats given aflatoxin (Newberne & Rogers, 1972). It was reported earlier that aflatoxicosis in animals (Allcroft & Lewis, 1963; Keyl, Booth, Masri, G u m b m a n n & Gagne, 1970) and in birds (Carnaghan, Lewis, Patterson & Allcroft, 1966) reduced hepatic and serum levels of vitamin A. The present communication deals with the response of vitamin A-deficient rats to a single toxic dose of aflatoxin.
Experimental Animals and diet. Weanling albino rats of both sexes weighing between 28 and 50 g were selected from a limited number of litters from the stock colony of the National Institute of Nutrition. They were divided into two groups matched in respect of litter mates and body weights and consisting in each case of six males and six females. The rats of both the groups were maintained on a vitamin A-free diet consisting of vitamin A-free casein (20 7oo), maize starch (60 7oo), cottonseed oil (5 ~), vitamin mixture (1 ~ ) , salt mixture (470) and sucrose (10 ~o). The casein was refluxed with ethanol for 6 hr, extracted with ether and ovendried at 110°C for 48 hr. Each gram of vitamin mixture contained vitamin K (0.5 rag), thiamine hydroehloride (0:5 rag), riboflavin (1.0 nag), pyridoxine hydroehloride (0-4 rag), pantothenic acid (4-0 rag), niacin (4-0 rag), inositol (25-0 mg), p-aminobenzoic acid (10.0 rag), vitamin B12 (2"0/zg), biotin (0.02 rag), folic acid (0.2 rag) and vitamin E (10 IU). In addition choline chloride (100 mg/100 g diet) and vitamin D (12-5/zg/100 g diet) were added. 467
468
G. SRIRANGAREDDY, T. B. G. TILAK and D. KRISHNAMURTHI
Animals in group 1 received the diet ad lib. and each animal of this group had its pair-fed control rat of the corresponding sex in group 2. The latter group of animals received in addition an oral supplement of vitamin A at the rate of 100 iU/rat/day throughout the experiment. The rats were housed individually and were given water ad lib. Treatment. Each rat was weighed weekly. After 9 wk on the test diets, each animal was given ip a mixture of crystalline aflatoxins dissolved in propylene glycol in a concentration of 1 mg/ml. The crystalline aflatoxin preparation, given in a dose of 3"5 mg/kg body weight, included aflatoxins B1 (44 ~o), G1 (44 700) and B2 and G2 (2 70). Some 40 hr after the toxin was administered, all the male rats in group 1 died. Although no adverse effects were noted in any other animals, the experiment was terminated at this point, the rest of the animals in both groups being killed. The liver from each of the animals that died or was killed was removed and weighed immediately. Determination o f liver levels o f vitamin A. Part of the liver from each animal was immediately frozen and preserved for not more than 1 wk at --20°C for the estimation of vitamin A by the Carr-Price method (Embree, Ames, Lehman & Harris, 1957). Histology. The rest of each liver was fixed in 10 ~ neutral buffered formol-saline. Paraffin blocks of the tissue were sectioned at 5/z and stained with haematoxylin and eosin, with periodic acid-Schiff (PAS) reagent before and after salivary digestion, and with Masson's trichrome stain. Results
Rats in both groups continued to gain weight until the toxin was administered at the end of 9 wk. The body weights of the male and female rats of group 1 were comparable with those of the corresponding pair-fed controls of group 2 (Table 1). Hepatic stores of vitamin A in the rats of group 1 were significantly lower (P < 0.001) than those of the animals of group 2, but within each group, the hepatic vitamin A levels were comparable in males and females (Table 1). As already mentioned, vitamin A-deficient male rats (group 1) died about 40 hr after aflatoxin administration. The vitamin A-deficient females (group 1) and all the vitamin Asupplemented animals (group 2) showed no adverse reactions. Table 1. Mean body weights and hepatic vitamin A levels in rats fed diets deficient in or supplemented with vitamin A for 9 wk
Group
Dietary feeding regime
Vitamin A intake (IU/rat/day)
1
Ad lib.
2
Pair-fed
0 100
1
Ad lib.
2
Pair-fed
0 100
Mean body weight (g) Initial Males 45.5 4- 1.94 45-7 ± 2.42
Terminal
Vitamin A (IU/whole liver)
226.7 4- 8.26 36.5 4- 6-16"t 201.04- 10.77 2128-14- 153"56
Females
40"0 -4- 2"34 37-5 ± 2.14
136-8-4-2.40 130.04- 4.11
18-6 -4- 3"42* 2299-14- 111-49
tMean values for five rats, since in one rat, liver vitamin A could not be estimated. Values are means -t- SEM for determinations in groups of six animals except where indicated otherwise and those marked with an asterisk differ significantly (paired t test) from those for the corresponding vitamin A-supplemented, pair-fed group: *P < 0-001.
AFLATOXIN EFFECTS IN VITAMIN A DEFICIENCY
469
Liver histology
The livers of the vitamin A-deficient male rats (group I) were grossly congested, but those of the females in this group and both sexes of the other group appeared normal. Histologically, the livers of the male rats of group 1 showed midzonal necrosis which extended into the centritobular and periportal areas, leaving a narrow ring of parenchymal cells surrounding the central veins and portal tracts. The surviving cells were severely vacuolated, less basophilic than normal and PAS negative and often exhibited cytoplasmic eosinophilic globular hyaline. Knpffer cells in general were prominent and more numerous. Bile-duct proliferation was absent, but the epithelial cells were plump and prominent. Male rats given vitamin A supplements (group 2) also showed liver damage. This was principally periportal in distribution, but often spread irregularly into adjacent midzones. Spotty and focal cell necrosis and fatty change were observed in the areas of surviving parenchymal cells, which showed a loss of cytoplasmic basophilia and eosinophilic degeneration. In three animals, glycogen was found only in the centrilobutar areas, while in the remaining three it had disappeared from entire lobules. Prominence of the bile-duct epithelium and a variable degree of Kupffer-cell proliferation were also found in these animals, as in the rats of the other group. The histopathological changes found in the male rats were thus similar in type in the two groups, but differed considerably in magnitude and distribution. Liver damage found in the female rats was minimal and almost identical between the groups. The only changes observed were a mild loss of cytoplasmic basophilia, the presence of cytoplasmic eosinophilic globular hyaline in a few periportal hepatocytes and plumpness of the bile-duct epithelium. Liver glycogen was depleted, however, in the liver cells of the periportal zones. Discussion
The nature of the interaction between aflatoxin and vitamin A is not yet clear, but observations made in the present study are important in the context of the wide prevalence of vitamin A deficiency in children in developing countries (Arroyave, Wilson, Mendez, Behar & Scrimshaw, 1961; Reddy & Srikantia, 1966). Experimentally induced proteincalorie malnutrition has been reported to increase the susceptibility of animals to aflatoxins (Madhavan & Gopalan, 1965; Madhavan et al. 1967), and vitamin A deficiency is yet another situation to be taken into account before safe limits are prescribed for this toxin in the human diet. The aflatoxin-induced liver damage in male rats given the vitamin supplement (group 2) involved the periportal parenchyma, as was reported earlier (Butler, 1970). On the other hand, vitamin A-deficient male rats (group 1) mainly exhibited midzonal lesions, which extended, however, towards the peripheral and centrilobular zones. Thus a chronic deficiency of vitamin A apparently alters the distribution of demonstrable liver damage. The manner in which this is brought about requires elucidation. Although hepatic stores of vitamin A were depleted to a similar extent in the males and females of group 1, hardly any liver injury was observed in the female rats. A relative resistance to aflatoxin in female rats has already been reported (Butler, 1970). In relation to the LDso reported for female rats (Butler, 1970), the dose of aflatoxin used in the present study was very small. Acknowledgements--The authors wish to express their thanks to Mr. A. Nadamuni Naidu for his help with
470
G. SRIRANGA REDDY, T. B. G. TILAK a n d D. KRISHNAMURTHI
the statistical analysis of the data and Dr. K. Seetharam Bhat for his help with vitamin A determinations. They also acknowledge the technical assistance of Mr. N. K. Sreedharan and Mr. M. Purushotham. REFERENCES Allcroft, Ruth & Lewis, Gwyneth (1963). Groundnut toxicity in cattle: Experimental poisoning of calves and a report on clincial effects on older cattle. Vet. Rec. 75, 487. Arroyave, G., Wilson, D., Mendez, B. M. & Scrimshaw, N. S. (1961). Serum and liver vitamin A and lipids in children with severe protein malnutrition. Am. J. clin. Nutr. 9, 180. Butler, W. H. (1970). Liver injury induced by aflatoxin. In Progress in Liver Diseases. Vol. 3. Edited by H. Popper and F. Schaffner. p. 408. Grune and Stratton, New York. Carnaghan, R. B. A., Lewis, G., Patterson, D. S. P. & Allcroft, Ruth (1966). Biochemical and pathological aspects of groundnut poisoning in chickens. Path. vet. 3, 601. Embree, N. D., Ames, S. R., Lehman, R. W. & Harris, P. L. (1957). Determination of vitamin A. In Methods o f Biochemical Analysis. Vol. 4. Edited by D. Glick. p. 92. Interscience Publishers, Inc., New York. Hamilton, P. B., Hsi-Tang Tung, Harris, J. R., Gainer, J. H. & Donaldson, W. E. (1972). The effect of dietary fat on aflatoxicosis in turkeys. Poult. Sci. 51, 165. Keyl, A. C., Booth, A. N., Masri, M. S., Gumbmann, M. R. & Gagne, W. E. (1970). Chronic effects of aflatoxin in farm animal feeding studies. In Proceedings of the First U.S.-Japan Conference on Toxic Micro-organisms, Honolulu, Hawaii. Edited by M. Herzberg. p. 72. US Department of the Interior and U J N R Panels on Toxic Micro-organisms, Washington, D.C. Madhavan, T. V. & Gopalan, C. (1965). Effect of dietary protein on aflatoxin liver injury in weanling rats. Archs Path. 80, 123. Madhavan, T. V., Ran, K. S. & Tulpule, P. G. (1967). Effect of dietary protein level on susceptibility of monkeys to aflatoxin liver injury. Indian J. med. Res. 53, 984. Newberne, P. M. & Rogers, A. R. (1972). Vitamin A, liver and colon carcinoma in rats fed low levels of aflatoxin. Toxic. appl. Pharmac. 22, 280. Reddy, V. & Srikantia, S. G. (1966). Serum vitamin A in kwashiorkor. Am. J. clin. Nutr. 18, 105. Rogers, Adrianne E. & Newberne, P. M. (1971). Diet and aflatoxin B1 toxicity in rats. Toxic. appl. Pharmac. 20, 113. Smith, J. W., Hill, C. H. & Hamilton, P. B. (1971). The effect of dietary modifications on aflatoxicosis in the broiler chicken. Poult. Sci. 50, 768.
Susceptibility of Vitamin A-deficient Rats to Aflatoxin G. SRIRANGA REDDY,T. B. G. TILAK and D. KRISHNAMURTHI National Institute of Nutrition, hutian Council of Medical Research, Hyderabad 500007, A.P., India (Received 28 November 1972)
Summary--Weanling albino rats were divided into two groups each of six males and six females. They were maintained on a vitamin A-free diet, one group being fed ad lib. and the second being pair-fed with the first. Rats in this second group were given an oral supplement of 100 IU vitamin A/animal/day. At the end of a feeding period of 9 wk, a crystalline preparation of aflatoxin was administered in a single ip dose of 3"5 mg/kg body weight. Male rats in the first group showed an increased susceptibility to the toxin as evidenced by rapid mortality and severe liver damage, midzonal in distribution. Their hepatic stores of vitamin A were found to have been severely depleted. Vitamin A-supplemented male rats showed only mild hepatic damage of the periportal parenchyma. Liver damage was minimal in female rats of both groups and was unaffected by the amount of vitamin A stored in the liver.
Introduction The toxic effects of aflatoxins in laboratory animals and birds have been shown to be affected by dietary protein (Madhavan & Gopalan, 1965; Madhavan, Rao & Tulpule, 1967), fat (Hamilton, Hsi-Tang Tung, Harris, Gainer & Donaldson, 1972; Smith, Hill & Hamilton, 1971) and lipotrophic factors (Rogers & Newberne, 1971). Compared with ariatoxin-treated rats on a normal diet, a decreased incidence of hepatomas and an increase in the occurrence of colonic carcinomas were reported in vitamin A-deficient rats given aflatoxin (Newberne & Rogers, 1972). It was reported earlier that aflatoxicosis in animals (Allcroft & Lewis, 1963; Keyl, Booth, Masri, G u m b m a n n & Gagne, 1970) and in birds (Carnaghan, Lewis, Patterson & Allcroft, 1966) reduced hepatic and serum levels of vitamin A. The present communication deals with the response of vitamin A-deficient rats to a single toxic dose of aflatoxin.
Experimental Animals and diet. Weanling albino rats of both sexes weighing between 28 and 50 g were selected from a limited number of litters from the stock colony of the National Institute of Nutrition. They were divided into two groups matched in respect of litter mates and body weights and consisting in each case of six males and six females. The rats of both the groups were maintained on a vitamin A-free diet consisting of vitamin A-free casein (20 7oo), maize starch (60 7oo), cottonseed oil (5 ~), vitamin mixture (1 ~ ) , salt mixture (470) and sucrose (10 ~o). The casein was refluxed with ethanol for 6 hr, extracted with ether and ovendried at 110°C for 48 hr. Each gram of vitamin mixture contained vitamin K (0.5 rag), thiamine hydroehloride (0:5 rag), riboflavin (1.0 nag), pyridoxine hydroehloride (0-4 rag), pantothenic acid (4-0 rag), niacin (4-0 rag), inositol (25-0 mg), p-aminobenzoic acid (10.0 rag), vitamin B12 (2"0/zg), biotin (0.02 rag), folic acid (0.2 rag) and vitamin E (10 IU). In addition choline chloride (100 mg/100 g diet) and vitamin D (12-5/zg/100 g diet) were added. 467
468
G. SRIRANGAREDDY, T. B. G. TILAK and D. KRISHNAMURTHI
Animals in group 1 received the diet ad lib. and each animal of this group had its pair-fed control rat of the corresponding sex in group 2. The latter group of animals received in addition an oral supplement of vitamin A at the rate of 100 iU/rat/day throughout the experiment. The rats were housed individually and were given water ad lib. Treatment. Each rat was weighed weekly. After 9 wk on the test diets, each animal was given ip a mixture of crystalline aflatoxins dissolved in propylene glycol in a concentration of 1 mg/ml. The crystalline aflatoxin preparation, given in a dose of 3"5 mg/kg body weight, included aflatoxins B1 (44 ~o), G1 (44 700) and B2 and G2 (2 70). Some 40 hr after the toxin was administered, all the male rats in group 1 died. Although no adverse effects were noted in any other animals, the experiment was terminated at this point, the rest of the animals in both groups being killed. The liver from each of the animals that died or was killed was removed and weighed immediately. Determination o f liver levels o f vitamin A. Part of the liver from each animal was immediately frozen and preserved for not more than 1 wk at --20°C for the estimation of vitamin A by the Carr-Price method (Embree, Ames, Lehman & Harris, 1957). Histology. The rest of each liver was fixed in 10 ~ neutral buffered formol-saline. Paraffin blocks of the tissue were sectioned at 5/z and stained with haematoxylin and eosin, with periodic acid-Schiff (PAS) reagent before and after salivary digestion, and with Masson's trichrome stain. Results
Rats in both groups continued to gain weight until the toxin was administered at the end of 9 wk. The body weights of the male and female rats of group 1 were comparable with those of the corresponding pair-fed controls of group 2 (Table 1). Hepatic stores of vitamin A in the rats of group 1 were significantly lower (P < 0.001) than those of the animals of group 2, but within each group, the hepatic vitamin A levels were comparable in males and females (Table 1). As already mentioned, vitamin A-deficient male rats (group 1) died about 40 hr after aflatoxin administration. The vitamin A-deficient females (group 1) and all the vitamin Asupplemented animals (group 2) showed no adverse reactions. Table 1. Mean body weights and hepatic vitamin A levels in rats fed diets deficient in or supplemented with vitamin A for 9 wk
Group
Dietary feeding regime
Vitamin A intake (IU/rat/day)
1
Ad lib.
2
Pair-fed
0 100
1
Ad lib.
2
Pair-fed
0 100
Mean body weight (g) Initial Males 45.5 4- 1.94 45-7 ± 2.42
Terminal
Vitamin A (IU/whole liver)
226.7 4- 8.26 36.5 4- 6-16"t 201.04- 10.77 2128-14- 153"56
Females
40"0 -4- 2"34 37-5 ± 2.14
136-8-4-2.40 130.04- 4.11
18-6 -4- 3"42* 2299-14- 111-49
tMean values for five rats, since in one rat, liver vitamin A could not be estimated. Values are means -t- SEM for determinations in groups of six animals except where indicated otherwise and those marked with an asterisk differ significantly (paired t test) from those for the corresponding vitamin A-supplemented, pair-fed group: *P < 0-001.
AFLATOXIN EFFECTS IN VITAMIN A DEFICIENCY
469
Liver histology
The livers of the vitamin A-deficient male rats (group I) were grossly congested, but those of the females in this group and both sexes of the other group appeared normal. Histologically, the livers of the male rats of group 1 showed midzonal necrosis which extended into the centritobular and periportal areas, leaving a narrow ring of parenchymal cells surrounding the central veins and portal tracts. The surviving cells were severely vacuolated, less basophilic than normal and PAS negative and often exhibited cytoplasmic eosinophilic globular hyaline. Knpffer cells in general were prominent and more numerous. Bile-duct proliferation was absent, but the epithelial cells were plump and prominent. Male rats given vitamin A supplements (group 2) also showed liver damage. This was principally periportal in distribution, but often spread irregularly into adjacent midzones. Spotty and focal cell necrosis and fatty change were observed in the areas of surviving parenchymal cells, which showed a loss of cytoplasmic basophilia and eosinophilic degeneration. In three animals, glycogen was found only in the centrilobutar areas, while in the remaining three it had disappeared from entire lobules. Prominence of the bile-duct epithelium and a variable degree of Kupffer-cell proliferation were also found in these animals, as in the rats of the other group. The histopathological changes found in the male rats were thus similar in type in the two groups, but differed considerably in magnitude and distribution. Liver damage found in the female rats was minimal and almost identical between the groups. The only changes observed were a mild loss of cytoplasmic basophilia, the presence of cytoplasmic eosinophilic globular hyaline in a few periportal hepatocytes and plumpness of the bile-duct epithelium. Liver glycogen was depleted, however, in the liver cells of the periportal zones. Discussion
The nature of the interaction between aflatoxin and vitamin A is not yet clear, but observations made in the present study are important in the context of the wide prevalence of vitamin A deficiency in children in developing countries (Arroyave, Wilson, Mendez, Behar & Scrimshaw, 1961; Reddy & Srikantia, 1966). Experimentally induced proteincalorie malnutrition has been reported to increase the susceptibility of animals to aflatoxins (Madhavan & Gopalan, 1965; Madhavan et al. 1967), and vitamin A deficiency is yet another situation to be taken into account before safe limits are prescribed for this toxin in the human diet. The aflatoxin-induced liver damage in male rats given the vitamin supplement (group 2) involved the periportal parenchyma, as was reported earlier (Butler, 1970). On the other hand, vitamin A-deficient male rats (group 1) mainly exhibited midzonal lesions, which extended, however, towards the peripheral and centrilobular zones. Thus a chronic deficiency of vitamin A apparently alters the distribution of demonstrable liver damage. The manner in which this is brought about requires elucidation. Although hepatic stores of vitamin A were depleted to a similar extent in the males and females of group 1, hardly any liver injury was observed in the female rats. A relative resistance to aflatoxin in female rats has already been reported (Butler, 1970). In relation to the LDso reported for female rats (Butler, 1970), the dose of aflatoxin used in the present study was very small. Acknowledgements--The authors wish to express their thanks to Mr. A. Nadamuni Naidu for his help with
470
G. SRIRANGA REDDY, T. B. G. TILAK a n d D. KRISHNAMURTHI
the statistical analysis of the data and Dr. K. Seetharam Bhat for his help with vitamin A determinations. They also acknowledge the technical assistance of Mr. N. K. Sreedharan and Mr. M. Purushotham. REFERENCES Allcroft, Ruth & Lewis, Gwyneth (1963). Groundnut toxicity in cattle: Experimental poisoning of calves and a report on clincial effects on older cattle. Vet. Rec. 75, 487. Arroyave, G., Wilson, D., Mendez, B. M. & Scrimshaw, N. S. (1961). Serum and liver vitamin A and lipids in children with severe protein malnutrition. Am. J. clin. Nutr. 9, 180. Butler, W. H. (1970). Liver injury induced by aflatoxin. In Progress in Liver Diseases. Vol. 3. Edited by H. Popper and F. Schaffner. p. 408. Grune and Stratton, New York. Carnaghan, R. B. A., Lewis, G., Patterson, D. S. P. & Allcroft, Ruth (1966). Biochemical and pathological aspects of groundnut poisoning in chickens. Path. vet. 3, 601. Embree, N. D., Ames, S. R., Lehman, R. W. & Harris, P. L. (1957). Determination of vitamin A. In Methods o f Biochemical Analysis. Vol. 4. Edited by D. Glick. p. 92. Interscience Publishers, Inc., New York. Hamilton, P. B., Hsi-Tang Tung, Harris, J. R., Gainer, J. H. & Donaldson, W. E. (1972). The effect of dietary fat on aflatoxicosis in turkeys. Poult. Sci. 51, 165. Keyl, A. C., Booth, A. N., Masri, M. S., Gumbmann, M. R. & Gagne, W. E. (1970). Chronic effects of aflatoxin in farm animal feeding studies. In Proceedings of the First U.S.-Japan Conference on Toxic Micro-organisms, Honolulu, Hawaii. Edited by M. Herzberg. p. 72. US Department of the Interior and U J N R Panels on Toxic Micro-organisms, Washington, D.C. Madhavan, T. V. & Gopalan, C. (1965). Effect of dietary protein on aflatoxin liver injury in weanling rats. Archs Path. 80, 123. Madhavan, T. V., Ran, K. S. & Tulpule, P. G. (1967). Effect of dietary protein level on susceptibility of monkeys to aflatoxin liver injury. Indian J. med. Res. 53, 984. Newberne, P. M. & Rogers, A. R. (1972). Vitamin A, liver and colon carcinoma in rats fed low levels of aflatoxin. Toxic. appl. Pharmac. 22, 280. Reddy, V. & Srikantia, S. G. (1966). Serum vitamin A in kwashiorkor. Am. J. clin. Nutr. 18, 105. Rogers, Adrianne E. & Newberne, P. M. (1971). Diet and aflatoxin B1 toxicity in rats. Toxic. appl. Pharmac. 20, 113. Smith, J. W., Hill, C. H. & Hamilton, P. B. (1971). The effect of dietary modifications on aflatoxicosis in the broiler chicken. Poult. Sci. 50, 768.