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Metabolism and acute toxicity of aflatoxin B1 in rats
Chem.-Biol. Interactfans, 7 (1973) 283-287 0 Elsevier Scientific Publishing Company, Amslerdam-Printed
in The Nethcrluncis
METABOLlSM AND ACUTE TOXICITY OF AFLATOXIN
1. F. H. PURCHASE*,
M. STEYN”
AND
283
B, IN RATS
T. C. GILFILLAN***
*National Research institate$or Nutritional Diseases, So& African Medical Rtuarch (tnncil. Private Bag X380: **Division of Toxicology, National Research Institute for Nutritional Diseases. Suurh African Medical Research Council. Private kg X380; aad ***Statistics Division. Natioyl Research Institute for Mathematical Scknces, Council for Scientific and Industrial Research, P.0. Box 395. Pretoria (South Africa) (Received September 14th. 1972) (Revision received April 2nd. 1973) (Accepted July 30th. 1973)
SUMMARY
Female rats are more resistant to the acute oral toxicity of af!atoxin B, and have higher tissue levels of aflatoxin M ,, a fluorescent hydroxylated mctabolite, than males. These differences are reduced by castration, as castrated animals of either sex are more resistant and have higher tissue levels than entire males and females. ‘The toxicity of a single oral dose of aflatoxin B, appears to be invcrscly related to levels of aflatoxin M, in the tissues.
INTROUUC I’ION
There are considerable differences in the susceptibility of various species to the acute toxic effects of aflatoxiu B,, LDSo values ranging from 0.40 mg/kg in l-day-old ducklings to 17.9 mg/kg in 150-g female rats’. The range of LD5,, values reported in rats is almost as great, with the neonate as the most susceptible (0.56 mg/kg) and the susceptibility decreasing with age (weanling 6.0 mg/kg, 150-g female 17.9 mg/kp)’ . The significance of various metabolic pathways has been sought by studying interspecies differences. PA-I JERSON AND ALLCROFT~ found no correlation between the susceptibility of 9 avian and mammalian species and either levels of hepatic microsomal enzymes or the ability of liver to metabolise aflatoxin B, k hxx There was also no correlation between the excretion of aflatoxin B, and M, in urine and faeces and acute toxicity in the 5 species. PURCHASE AND STEYN~ studied the levels * Present address: Imperial Chemical Industries Ltd., Industrial Hygiene Research Laboratories, Alderley Park, Macclesfield, Cheshire, SKI0 4TJ (Grext Britain). Abbreviation: DMSO. dimethylsulphoxide.
I. F. H.
284
PURCHASE et d.
of aflatoxin MI in the tissues of rats dosed with &toxin B,. The levels of aflatoxin M, were higher in females and, as females are reported* to be more resistant than males to the acute effects of &toxin B,, it was concluded that high levels of aflatoxin MI indicated a more rapid breakdown and hence a higher resistance to a&toxin B1. Castration eliminated the difference in levels of aflatoxin MI and administration of steroid hormones to castrated animals had no effect on these levels5. A major problem of interpreting these results is that, although the metabolic studies reported in a single paper were carried out in the same laboratory, the measurement of susceptibility (LDs, values) were obtained from the literature. Comparison of LDS, values obtained by various authors under varying conditions may be difficult as is well illustrated by the differences in the LDs, of aflatoxin B1 in sheep reported by WOGAN” ( > 500 mg/kg) and by ARMBRECHT et ai.’ (2.0 mg/kg). This difference is large enough to change the classification of the sheep from a resistant to a susceptible species in PATTERSONAND ALLCROFT’S work2 and therefore affect the conclusions. This report correlates LDs, values with the aflatoxin MI levels reported previously3*3, both observations having been made on the same strain of rats. METHODS
Metabolic
strcdies
The methods were described previously”.“. Male and female rats were castrated at 4 weeks (:t: 60 g) and kept for 6 weeks by which time their average weights were 223 g and 211 g, respective!y. The castrated rats, and a separate group of entire rats (male 359 g and female 220 g), were housed in individual cages and dosed with aflatoxin B, (IO mg/kg) dissolved in dimethylsulphoxide (IO mg/ml). Groups of rats were killed at intervals and their livers, kidneys, stomachs and intestines assayed3 for aflatoxin B, and M , . L DSO deternrinatiorl
Male and female rats were castrated at 4 weeks of age (& 60 g) and kept for approximately 12 weeks when their average weights were 343 g and 291 g, respectively. Groups of 30 adult entire rats of each sex (male 30.; g and female 186 g) and the castrated rats (30 of each sex) were used for determination of LDS, values. Each group of 30 ra!* was divided into 6 subgroups of 5 r&s. The 6 subgroups of rats received dose3 of aflatoxin B, dissolved in dimethyl sutphoxide (DMSO) of 0, I .O, 2.34, 5.49, 12.85 and 30.27 r-g/kg body weigh.:, respectively. The total dose of DMSO was I ml/kg body weight except in the group receiving 30.27 mg/kg where a larger quantity of DMSO (2 ml/kg) was used. The rats were observed for 14 days. The LD,, values were determined according to WEIL’S technique*, Rats were from our outbred Wistar-deritled colony, the experiments being carried out between January 1969 p.nd May 197I. Aflatoxin B, was crystalline and contained more than 98 % aflatoxin B, on assay by UV absorption spectrophotometry9.
TOXICITY
285
BI
OF AFLATOXIN
Metabolic studies
A detailed comparison of the results of the metabolic studies has been published previously 3*s.There was a significantly higher level of atlatoxin M in the liver, kidney and intestine of female rats than male rats. There was also fess aflatoxin Br in the stomacns of female than of male rats. The differences were virtually abolished by castration (Table 1). TABLE
I
RESULTS OBTAINED FROM ANALYSIS OF VARIANCE IN MALE AND FEMALE, ENTIRE AND CASTRATED RATS . -. . __ Orgun
Difference between the sexes
Liver (MI) Kidney (M,) Intestines (M,) Stomach (B,)
Entire
Castrated
Male
Female
-I- -t4-f
i -
-I- + +
++ -
i--k
-
-
+
_- , no significant difference;
Difference between entire and castrated rats
f,
significant at the 5 % level;
-t-ii- +. significant
-t-+
-t-tat the I Y. Icvel.
A further facet of this work which is now presented is that there were significantly lower levels of aflatoxin M, in the tissues of the entire rats than in the tissues of castrated rats of the same sex. There was also a significantly smaller amount of aflatoxin B, in the stomach of castrated rats (Table I). As there was a difference between the body weight of the entire and castrated rats, an analysis of covariance, with body weight as covariate, was performed in all cases where an analysis of variance had been done. As no significant regression coefficients appeared, the original analysis of variance results were retained. LD,,
values
The LDSOvalues (and confidence limits) are: entire males, entire females, cast rated males, castrated females,
1.53 (I.OO- 2.34) mg/kg; 5.96 (3.93- 9.05) mg/kg; 8.38 (5.49-12.85) mg/kg; 9.93 (7.06-13.36) mg/kg.
In the case of entire and castrated females the figures in brackets are 95 ‘$4confidence limits. In the case of entire and castrated males, because the calculated/valuer’ is zero, the figures in brackets are the next lowest and highest dose respectively, which correspond to approximately 95 ‘A confidence limits. DlSCUSSION
The LD,, values of aftatoxin B, in entire rats, which are similar to those
I. P. H.
286
PURCHASE et Ui.
reported in the literature le4, show that the female is more resistant to the afiatoxin B1 than the male, Female rats also absorb4 more &toxin from their stomachs and had higher levels of ahtoxin M 1 in their livers, kidneys and intestines than did male rats, indicating that the metabolism of aflatoxin B1 is more efficient in females than in males. Aflatoxin MI is, however, a relatively minor metabolic product of aflatoxin B1. Radiotracer studies in rats indicate that 25% of a&toxin BI is metabolised by demethylation and the rest is excreted in the urine and faeces’O. The identity of the labelled urinary and faecal metabolites was not established but studies in other species, where the urinary and faecal metabolites were identified by their fluorescence, indicate that aflatoxin M, was a minor excretion product2*11-14. The total quantity of aflatoxin MI present at any one timr was less than 3.1% of the initial dose of aflatoxin 8, in entire rats and somewhat higher in castrated rats. Thus, although aflatoxin MI does not appear to account for a major part of aflatoxin B, metabolism, tissue levels correlate with toxicity in entire and castrated rats, indicating that it represents an important pathway of aflatoxin BI metabolism. Although the acute toxicity of aflatoxin M, is equal to that of aflatoxin B1 in ducklingsIS, and their toxic effects are quantitatively similar in rats16, aflatoxin MI does not contribute to the overall toxic effects of aflatoxin B, because only small qunntitics of aflatoxin MI arc present at any one time, and there is an inverse relationship between aflatoxin 8, toxicity and aflatoxin M, tissue levels. Other metabolites, such as the 2,3-epoxide which is presumed to be the metabolite that binds covalently to nucleic acids”, may contribute more towards toxicity. Rapid formation of aflacSx\in M,, and possibly larger quantities of c ther metabolites such as desmethyl allatoxin B, (ref. 15). would reduce the amount of aflatoxin Bt available for conversion to more toxic metabolites. Castration removed most of the sex differences in terms of both metabolism and susceptibility - there was no statistically significant difference in the levels of &toxin in kidney, intestine and stomach of llale and female castrated rats, and LD,, values (8.38 and 9.93 mg/kg respectively) were similar. As the sex difference in metabolism is not due to specific interaction b:tween aflatoxin B, metabolism and steroid hormones5, it is surprising that both metabolism and LDS, values were significantly greater in castrated than entire ani nals.
REFERENCES I 2 3 4 5 6
P. M. NW~ERF;E ANV W. H. BUTLER, Acute and chronic cffccts of aflatoxin on the liver of domestic and laboratory animals; a review, Cancer Res.. 29 (1969) 236-250. D. S. P. PATTERSONAND R. A. ALLCROFT, Metabolism of aflatoxin in susceptible and resistant animal species. Fd. Cost~ef. Tosieol.. 8 (1970) 43-53. I. F. H. PURCHASFAND M. S~EYN, The metabolism of aflatoxin B, in rats, Brit. J. Cmcer, 23 ( 1969) 800-805. W. H. BLJ~LER.Acute toxicity of aflatoxin 9, in rats, Brit. J. Cancer, 18 (1964) 756-762. M. STEYNAND I. F. H. PURCHASE.Atiatoxin metabolism, in 1. F. H. PURCHASE(Ed.), Mycoroxins irr Human Health, Macmillan, London, 1971. p. 47. G. N. W~GAN. Aflntoxin risks and control measures, Fed. Proc., 27 (1968) 932-938.
TOXICITYOF APLATOXINB1 7
8 9
10 11 12 13 14 15 16 17
287
B. H. ARMBRECHT,W.T. SHALKOP, L. D. ROLLINS,A. E. POHLAND MD L. STOLOFF.Acute toxicity of atbttoxin Br in whethers, Nutwe, 225 (1970) 1062. C. S. Welt,, Tables for the convenient calculation of median+ffective dose (LDse or ED& and instructions for their use, Biometrics, 8 (1952) 249-263. 3. V. RODRICK~,L. STOLOFF, W. A. PONS, H. A. ROBERTKINAND L. A. GOLDIILA~,Molar absorptivity values for affatoxins and justitication for their use as criteria of purity of analytical standards, J. .4ss. 08. .rlfl&t. Chem., 53 (1970) 96-101. G. N. WOGAP!, S. S. E.w/?vs AND R. C. SHANK, Excretion and tissue distribution of radioactivity from afla:osin B b-lAC in rats, Cunce? Res., 27 (1967) 1729-1736. J. DALEZI(W, G. i-L ‘+FC.GAN AND S. Wtux~te, Aflatorin P, anew afiatoxin metabolite in monkeys, Science, 171 (1971) %4-585. J. NABNEY, M. B. B~JRB~~~:, R. ALLCRO~ A!G~0. LEH~S,Metabolism of aflatoxin in sheep; Excretion pattern in the lactating ewe, Fd, Cosmet. Toxicol., 5 (1967) 11-17. R. ALLCROFT, A. ROOERS, G. Lxwrs, J. NUNEY AND P. E. BEST,Metabolism of aflatoxin in sheep, Excretion of the “milk toxin”, Nurure, 209 (1966) 154-155. R. ALL~ROFT,B. A. ROBERTSAND M. K. LLOYD,Excretion of aflatoxin in the lactating cow, Fd. Cosmet. Toxicol., 6 (1968) 619-625. 1. F. H. PURCHASE, Acute toxicity of aflatoxins MI and M2 in one-day-old ducklings, Fd. Cosmer. Toxicol., 5 (1967) 739-342. R. S. PONG AND G. N. WOGAN,Toxicity and biochemical and fine structural effects of synthetic aflatoxins M t and B, in rat liver, J. Nod. Cancer Inst., 47 (1971) 585-592. R. C. GARNER, Microsomal-dependent binding of agatoxin Bt to DNA, RNA, polyribonuhotides and protein in v&o, C&m.-Biol. fnrerac~ionr, 6 (1973) 125-129.
in The Nethcrluncis
METABOLlSM AND ACUTE TOXICITY OF AFLATOXIN
1. F. H. PURCHASE*,
M. STEYN”
AND
283
B, IN RATS
T. C. GILFILLAN***
*National Research institate$or Nutritional Diseases, So& African Medical Rtuarch (tnncil. Private Bag X380: **Division of Toxicology, National Research Institute for Nutritional Diseases. Suurh African Medical Research Council. Private kg X380; aad ***Statistics Division. Natioyl Research Institute for Mathematical Scknces, Council for Scientific and Industrial Research, P.0. Box 395. Pretoria (South Africa) (Received September 14th. 1972) (Revision received April 2nd. 1973) (Accepted July 30th. 1973)
SUMMARY
Female rats are more resistant to the acute oral toxicity of af!atoxin B, and have higher tissue levels of aflatoxin M ,, a fluorescent hydroxylated mctabolite, than males. These differences are reduced by castration, as castrated animals of either sex are more resistant and have higher tissue levels than entire males and females. ‘The toxicity of a single oral dose of aflatoxin B, appears to be invcrscly related to levels of aflatoxin M, in the tissues.
INTROUUC I’ION
There are considerable differences in the susceptibility of various species to the acute toxic effects of aflatoxiu B,, LDSo values ranging from 0.40 mg/kg in l-day-old ducklings to 17.9 mg/kg in 150-g female rats’. The range of LD5,, values reported in rats is almost as great, with the neonate as the most susceptible (0.56 mg/kg) and the susceptibility decreasing with age (weanling 6.0 mg/kg, 150-g female 17.9 mg/kp)’ . The significance of various metabolic pathways has been sought by studying interspecies differences. PA-I JERSON AND ALLCROFT~ found no correlation between the susceptibility of 9 avian and mammalian species and either levels of hepatic microsomal enzymes or the ability of liver to metabolise aflatoxin B, k hxx There was also no correlation between the excretion of aflatoxin B, and M, in urine and faeces and acute toxicity in the 5 species. PURCHASE AND STEYN~ studied the levels * Present address: Imperial Chemical Industries Ltd., Industrial Hygiene Research Laboratories, Alderley Park, Macclesfield, Cheshire, SKI0 4TJ (Grext Britain). Abbreviation: DMSO. dimethylsulphoxide.
I. F. H.
284
PURCHASE et d.
of aflatoxin MI in the tissues of rats dosed with &toxin B,. The levels of aflatoxin M, were higher in females and, as females are reported* to be more resistant than males to the acute effects of &toxin B,, it was concluded that high levels of aflatoxin MI indicated a more rapid breakdown and hence a higher resistance to a&toxin B1. Castration eliminated the difference in levels of aflatoxin MI and administration of steroid hormones to castrated animals had no effect on these levels5. A major problem of interpreting these results is that, although the metabolic studies reported in a single paper were carried out in the same laboratory, the measurement of susceptibility (LDs, values) were obtained from the literature. Comparison of LDS, values obtained by various authors under varying conditions may be difficult as is well illustrated by the differences in the LDs, of aflatoxin B1 in sheep reported by WOGAN” ( > 500 mg/kg) and by ARMBRECHT et ai.’ (2.0 mg/kg). This difference is large enough to change the classification of the sheep from a resistant to a susceptible species in PATTERSONAND ALLCROFT’S work2 and therefore affect the conclusions. This report correlates LDs, values with the aflatoxin MI levels reported previously3*3, both observations having been made on the same strain of rats. METHODS
Metabolic
strcdies
The methods were described previously”.“. Male and female rats were castrated at 4 weeks (:t: 60 g) and kept for 6 weeks by which time their average weights were 223 g and 211 g, respective!y. The castrated rats, and a separate group of entire rats (male 359 g and female 220 g), were housed in individual cages and dosed with aflatoxin B, (IO mg/kg) dissolved in dimethylsulphoxide (IO mg/ml). Groups of rats were killed at intervals and their livers, kidneys, stomachs and intestines assayed3 for aflatoxin B, and M , . L DSO deternrinatiorl
Male and female rats were castrated at 4 weeks of age (& 60 g) and kept for approximately 12 weeks when their average weights were 343 g and 291 g, respectively. Groups of 30 adult entire rats of each sex (male 30.; g and female 186 g) and the castrated rats (30 of each sex) were used for determination of LDS, values. Each group of 30 ra!* was divided into 6 subgroups of 5 r&s. The 6 subgroups of rats received dose3 of aflatoxin B, dissolved in dimethyl sutphoxide (DMSO) of 0, I .O, 2.34, 5.49, 12.85 and 30.27 r-g/kg body weigh.:, respectively. The total dose of DMSO was I ml/kg body weight except in the group receiving 30.27 mg/kg where a larger quantity of DMSO (2 ml/kg) was used. The rats were observed for 14 days. The LD,, values were determined according to WEIL’S technique*, Rats were from our outbred Wistar-deritled colony, the experiments being carried out between January 1969 p.nd May 197I. Aflatoxin B, was crystalline and contained more than 98 % aflatoxin B, on assay by UV absorption spectrophotometry9.
TOXICITY
285
BI
OF AFLATOXIN
Metabolic studies
A detailed comparison of the results of the metabolic studies has been published previously 3*s.There was a significantly higher level of atlatoxin M in the liver, kidney and intestine of female rats than male rats. There was also fess aflatoxin Br in the stomacns of female than of male rats. The differences were virtually abolished by castration (Table 1). TABLE
I
RESULTS OBTAINED FROM ANALYSIS OF VARIANCE IN MALE AND FEMALE, ENTIRE AND CASTRATED RATS . -. . __ Orgun
Difference between the sexes
Liver (MI) Kidney (M,) Intestines (M,) Stomach (B,)
Entire
Castrated
Male
Female
-I- -t4-f
i -
-I- + +
++ -
i--k
-
-
+
_- , no significant difference;
Difference between entire and castrated rats
f,
significant at the 5 % level;
-t-ii- +. significant
-t-+
-t-tat the I Y. Icvel.
A further facet of this work which is now presented is that there were significantly lower levels of aflatoxin M, in the tissues of the entire rats than in the tissues of castrated rats of the same sex. There was also a significantly smaller amount of aflatoxin B, in the stomach of castrated rats (Table I). As there was a difference between the body weight of the entire and castrated rats, an analysis of covariance, with body weight as covariate, was performed in all cases where an analysis of variance had been done. As no significant regression coefficients appeared, the original analysis of variance results were retained. LD,,
values
The LDSOvalues (and confidence limits) are: entire males, entire females, cast rated males, castrated females,
1.53 (I.OO- 2.34) mg/kg; 5.96 (3.93- 9.05) mg/kg; 8.38 (5.49-12.85) mg/kg; 9.93 (7.06-13.36) mg/kg.
In the case of entire and castrated females the figures in brackets are 95 ‘$4confidence limits. In the case of entire and castrated males, because the calculated/valuer’ is zero, the figures in brackets are the next lowest and highest dose respectively, which correspond to approximately 95 ‘A confidence limits. DlSCUSSION
The LD,, values of aftatoxin B, in entire rats, which are similar to those
I. P. H.
286
PURCHASE et Ui.
reported in the literature le4, show that the female is more resistant to the afiatoxin B1 than the male, Female rats also absorb4 more &toxin from their stomachs and had higher levels of ahtoxin M 1 in their livers, kidneys and intestines than did male rats, indicating that the metabolism of aflatoxin B1 is more efficient in females than in males. Aflatoxin MI is, however, a relatively minor metabolic product of aflatoxin B1. Radiotracer studies in rats indicate that 25% of a&toxin BI is metabolised by demethylation and the rest is excreted in the urine and faeces’O. The identity of the labelled urinary and faecal metabolites was not established but studies in other species, where the urinary and faecal metabolites were identified by their fluorescence, indicate that aflatoxin M, was a minor excretion product2*11-14. The total quantity of aflatoxin MI present at any one timr was less than 3.1% of the initial dose of aflatoxin 8, in entire rats and somewhat higher in castrated rats. Thus, although aflatoxin MI does not appear to account for a major part of aflatoxin B, metabolism, tissue levels correlate with toxicity in entire and castrated rats, indicating that it represents an important pathway of aflatoxin BI metabolism. Although the acute toxicity of aflatoxin M, is equal to that of aflatoxin B1 in ducklingsIS, and their toxic effects are quantitatively similar in rats16, aflatoxin MI does not contribute to the overall toxic effects of aflatoxin B, because only small qunntitics of aflatoxin MI arc present at any one time, and there is an inverse relationship between aflatoxin 8, toxicity and aflatoxin M, tissue levels. Other metabolites, such as the 2,3-epoxide which is presumed to be the metabolite that binds covalently to nucleic acids”, may contribute more towards toxicity. Rapid formation of aflacSx\in M,, and possibly larger quantities of c ther metabolites such as desmethyl allatoxin B, (ref. 15). would reduce the amount of aflatoxin Bt available for conversion to more toxic metabolites. Castration removed most of the sex differences in terms of both metabolism and susceptibility - there was no statistically significant difference in the levels of &toxin in kidney, intestine and stomach of llale and female castrated rats, and LD,, values (8.38 and 9.93 mg/kg respectively) were similar. As the sex difference in metabolism is not due to specific interaction b:tween aflatoxin B, metabolism and steroid hormones5, it is surprising that both metabolism and LDS, values were significantly greater in castrated than entire ani nals.
REFERENCES I 2 3 4 5 6
P. M. NW~ERF;E ANV W. H. BUTLER, Acute and chronic cffccts of aflatoxin on the liver of domestic and laboratory animals; a review, Cancer Res.. 29 (1969) 236-250. D. S. P. PATTERSONAND R. A. ALLCROFT, Metabolism of aflatoxin in susceptible and resistant animal species. Fd. Cost~ef. Tosieol.. 8 (1970) 43-53. I. F. H. PURCHASFAND M. S~EYN, The metabolism of aflatoxin B, in rats, Brit. J. Cmcer, 23 ( 1969) 800-805. W. H. BLJ~LER.Acute toxicity of aflatoxin 9, in rats, Brit. J. Cancer, 18 (1964) 756-762. M. STEYNAND I. F. H. PURCHASE.Atiatoxin metabolism, in 1. F. H. PURCHASE(Ed.), Mycoroxins irr Human Health, Macmillan, London, 1971. p. 47. G. N. W~GAN. Aflntoxin risks and control measures, Fed. Proc., 27 (1968) 932-938.
TOXICITYOF APLATOXINB1 7
8 9
10 11 12 13 14 15 16 17
287
B. H. ARMBRECHT,W.T. SHALKOP, L. D. ROLLINS,A. E. POHLAND MD L. STOLOFF.Acute toxicity of atbttoxin Br in whethers, Nutwe, 225 (1970) 1062. C. S. Welt,, Tables for the convenient calculation of median+ffective dose (LDse or ED& and instructions for their use, Biometrics, 8 (1952) 249-263. 3. V. RODRICK~,L. STOLOFF, W. A. PONS, H. A. ROBERTKINAND L. A. GOLDIILA~,Molar absorptivity values for affatoxins and justitication for their use as criteria of purity of analytical standards, J. .4ss. 08. .rlfl&t. Chem., 53 (1970) 96-101. G. N. WOGAP!, S. S. E.w/?vs AND R. C. SHANK, Excretion and tissue distribution of radioactivity from afla:osin B b-lAC in rats, Cunce? Res., 27 (1967) 1729-1736. J. DALEZI(W, G. i-L ‘+FC.GAN AND S. Wtux~te, Aflatorin P, anew afiatoxin metabolite in monkeys, Science, 171 (1971) %4-585. J. NABNEY, M. B. B~JRB~~~:, R. ALLCRO~ A!G~0. LEH~S,Metabolism of aflatoxin in sheep; Excretion pattern in the lactating ewe, Fd, Cosmet. Toxicol., 5 (1967) 11-17. R. ALLCROFT, A. ROOERS, G. Lxwrs, J. NUNEY AND P. E. BEST,Metabolism of aflatoxin in sheep, Excretion of the “milk toxin”, Nurure, 209 (1966) 154-155. R. ALL~ROFT,B. A. ROBERTSAND M. K. LLOYD,Excretion of aflatoxin in the lactating cow, Fd. Cosmet. Toxicol., 6 (1968) 619-625. 1. F. H. PURCHASE, Acute toxicity of aflatoxins MI and M2 in one-day-old ducklings, Fd. Cosmer. Toxicol., 5 (1967) 739-342. R. S. PONG AND G. N. WOGAN,Toxicity and biochemical and fine structural effects of synthetic aflatoxins M t and B, in rat liver, J. Nod. Cancer Inst., 47 (1971) 585-592. R. C. GARNER, Microsomal-dependent binding of agatoxin Bt to DNA, RNA, polyribonuhotides and protein in v&o, C&m.-Biol. fnrerac~ionr, 6 (1973) 125-129.