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Effects of aflatoxin on some marker enzymes of lysosomes
665
SHORT COMMUNICATIONS
BBA 23 638
Effects of aflatoxin on some m a r k e r enzymes of lysosomes Aflatoxins are a group of metabolic products of Aspergillus/tavus which have been studied intensively because of their carcinogenic properties in several speciesl.We have observed that the carcasses of chickens that died as a result of acute aflatoxicosis decomposed more rapidly than those of control animals. It is well established that post-mortem decomposition of tissues is a result of the activity of lysosomal enzymes 2. The present communication describes the effects of aflatoxicosis in chicken on the activities of acid phosphatase (EC 3.1.3.2) and fl-glueuronidase (EC 3.2.I.3i), two marker enzymes for lysosomes. Experimental aflatoxicosis was induced in chickens by adding known amounts of aflatoxin to a commercial diet. Aflatoxin was produced on rice by growing A. flavus N R R L 2999 according to the method of SHOTWELLet al. a. The moldy rice was steamed, dried, and ground to a fine powder. It was analyzed for aflatoxin content by the method of NABNEY AND lXTESBITTa with the modification of WISEMAN5. The commercial feed was analyzed to ensure freedom from aflatoxin before use. Each experimental treatment was given to four groups of ten male chicks each. The experimental designs were completely randomized. Capillary fragility of the birds was determined by the method of BACKARACH et al. 8. At the end of the feeding interval, the chickens were killed and sections of the liver and breast muscle were removed, blotted, weighed, and chilled. The tissues were homogenized in 0.25 M sucrose by 5 strokes of a P o t t e r Elvehjem homogenizer equipped with a Teflon pestle. Triton X-Ioo was added to the homogenates at a concentration of o.i %. Acid phosphatase and fl-glucuronidase were measured by the methods of GIANETTO AND DE DUVE7. The protein content of the homogenates was measured as described by LAYNEs using bovine serum albumin as the standard. The effect of graded doses of dietary aflatoxin on the specific activity of acid phosphatase in muscle homogenates is shown in Fig. I. Similar data on the specific activity of acid phosphatase and fl-glucuronidase in liver homogenates are shown in Fig. 2. It can be seen that the activities of these enzymes were increased by doses of aflatoxin as low as 0.625/~g/g of diet. A similar dose response was noted previously for an increase in capillary fragility, a decrease in lateral shear strength of muscle, and a decrease in minimal energy required to bruise (unpublished work). Doses of 2.5/*g/g are required before the growth rate is depressed in this experimental system g. Because the above data were obtained with 3-week-old chickens which had been fed a diet containing aflatoxin since hatching, it seemed desirable to determine the time required for the increase of "lysosomal" enzymes. Fig. 3 shows results obtained when aflatoxin at a dose of 5/zg/g was introduced into the diet of 2-week-old chickens. A significant increase in liver acid phosphatase occurred within 48 h which coincided with an increase in capillary fragility. The present finding that "lysosomal" enzymes are increased during aflatoxicosis in the chicken is contrary to those of BALASUBRAMANIAM et al. lo who reported that acid phosphatase did not increase in rat liver following an intraperitoneal injection of aflatoxin. These contrary findings m a y be the result of species difference, or the result of aflatoxin being administered as a single dose which can be metabolized and excreted rapidly as compared with continual dosing through the diet. Biochim. Biophys. Acta, 222 (197 o) 665-667
666
SHORT COMMUNICATIONS
3.0 W .J
8.0
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~
ACID PHOSPHATASE
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AFLATOXIN (IJg/g)
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AFLATOXI N (Pg/g)
Fig. I. Effect of dietary aflatoxin on the specific activity of acid p h o s p h a t a s e in muscle homogenates. The chickens were fed graded doses of aflatoxin from h a t c h i n g until the h o m o g e n a t e s were m a d e at three weeks of age. D e t e r m i n a t i o n of enzyme activities was as described in the text. E a c h point is the m e a n of four groups of ten chickens and the vertical bars are the s t a n d a r d errors of the mean. Fig. 2. Effect of dietary aflatoxin on the specific activity of acid p h o s p h a t a s e and fl-glucuronidase in liver homogenates. The chickens were fed graded doses of aflatoxin from h a t c h i n g until the h o m o g e n a t e s were m a d e at three weeks of age. E n z y m e s were m e a s u r e d as described in the text. E a c h p o i n t is the m e a n of four groups of ten chickens and the vertical bars are the s t a n d a r d errors of the mean.
80,
z
60
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u_
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20
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Fig. 3. Response of capillary fragility and acid p h o s p h a t a s e to the incorporation of 5/~g/g of aflatoxin into the diet of z-week-old chickens. The m e a s u r e m e n t s were made as described in the text. E a c h p o i n t is the difference between the m e a n of four groups of ten, treated chickens and the m e a n of four groups of ten, u n t r e a t e d chickens of the same age. The vertical bars on the d a t a points are the s t a n d a r d errors of the difference.
Biochim. Biophys. Acta, zz2 (197o) 665-667
SHORT COMMUNICATIONS
667
The induction of a rapid loss of tissue strength and integrity by doses of aflatoxin that do not affect the body growth rate represents a new biochemical reaction for aflatoxin. Interference with growth is presumably caused by the general inhibition of protein and nucleic acid synthesis1whereas the rapid loss of tissue strength suggests a degradation of preformed structural elements. The concomitant increase of "]ysosomal" enzyme activity and capillary fragility in dose-related fashions during aflatoxicosis suggests a possible cause and effect relationship. Further work will be required to establish such a relationship conclusively. The degradation or digestion of both cellular and extracellular elements by lysosomal enzymes is well established2 and such digestion may occur in aflatoxicosis. The finding that aflatoxin can cause a generalized loss of tissue strength and integrity without affecting body weight emphasizes again the importance of mycotoxins in public and animal health. These findings suggest that mycotoxins can have an insidious acute toxicity aside from their documented carcinogenic properties.
Department of Poultry Science, North Carolina State University, Raleigh, N.C. 276o 7 (U.S.A.)
HsI~TANG TUNG W . E . DONALDSON P . B . HAMILTON
I A. CIEGLER AND E. B. LILLEHOJ, Advan. Appl. Microbiol., IO (1968) 155. 2 C. DE D o v e AND R. WATTIAUX, Ann. Rev. Physiol., 28 (1966) 435. 3 0 . L. SHOTWELL, C. W. HESSELTINE, R. D. STUBBLEFIELD AND W. G. SORENSON, Appl. Microbiol., 14 (1966) 425 . 4 J. NABNEY AND B. F. NESBITT, Analyst, 90 (I965) 155. H. G. WISEMAN, W. C. JACOBSON AND W. C. HARDMEY'ER, J. ASSOg. O~C. Agr. Chemists, 50 (1967) 982. 6 A. L. BACKARACH, M. G. COATES AND T. R. MIDDLETON, Biochem. J., 36 (1942) 4o7 . 7 IR. GIANETTO AND C. DE DUVE, Biochem. J., 59 (I955) 433. 8 E. LAYNE, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w York, 1957, p. 448. 9 J. W. SMITH AND P. B. HAMILTON, Poultry Sci., 49 (197 o) 207. IO K. BALASOBRAMANIAN, S. WIJESUNDERA, S. iNI. ARSECULERATNE AND G. E. TENNEKOON, Toxicon, 7 (1969) 159.
Received August I3th, 197° Biochim. Biophys. Acta, 222 (197 o) 665--667
SHORT COMMUNICATIONS
BBA 23 638
Effects of aflatoxin on some m a r k e r enzymes of lysosomes Aflatoxins are a group of metabolic products of Aspergillus/tavus which have been studied intensively because of their carcinogenic properties in several speciesl.We have observed that the carcasses of chickens that died as a result of acute aflatoxicosis decomposed more rapidly than those of control animals. It is well established that post-mortem decomposition of tissues is a result of the activity of lysosomal enzymes 2. The present communication describes the effects of aflatoxicosis in chicken on the activities of acid phosphatase (EC 3.1.3.2) and fl-glueuronidase (EC 3.2.I.3i), two marker enzymes for lysosomes. Experimental aflatoxicosis was induced in chickens by adding known amounts of aflatoxin to a commercial diet. Aflatoxin was produced on rice by growing A. flavus N R R L 2999 according to the method of SHOTWELLet al. a. The moldy rice was steamed, dried, and ground to a fine powder. It was analyzed for aflatoxin content by the method of NABNEY AND lXTESBITTa with the modification of WISEMAN5. The commercial feed was analyzed to ensure freedom from aflatoxin before use. Each experimental treatment was given to four groups of ten male chicks each. The experimental designs were completely randomized. Capillary fragility of the birds was determined by the method of BACKARACH et al. 8. At the end of the feeding interval, the chickens were killed and sections of the liver and breast muscle were removed, blotted, weighed, and chilled. The tissues were homogenized in 0.25 M sucrose by 5 strokes of a P o t t e r Elvehjem homogenizer equipped with a Teflon pestle. Triton X-Ioo was added to the homogenates at a concentration of o.i %. Acid phosphatase and fl-glucuronidase were measured by the methods of GIANETTO AND DE DUVE7. The protein content of the homogenates was measured as described by LAYNEs using bovine serum albumin as the standard. The effect of graded doses of dietary aflatoxin on the specific activity of acid phosphatase in muscle homogenates is shown in Fig. I. Similar data on the specific activity of acid phosphatase and fl-glucuronidase in liver homogenates are shown in Fig. 2. It can be seen that the activities of these enzymes were increased by doses of aflatoxin as low as 0.625/~g/g of diet. A similar dose response was noted previously for an increase in capillary fragility, a decrease in lateral shear strength of muscle, and a decrease in minimal energy required to bruise (unpublished work). Doses of 2.5/*g/g are required before the growth rate is depressed in this experimental system g. Because the above data were obtained with 3-week-old chickens which had been fed a diet containing aflatoxin since hatching, it seemed desirable to determine the time required for the increase of "lysosomal" enzymes. Fig. 3 shows results obtained when aflatoxin at a dose of 5/zg/g was introduced into the diet of 2-week-old chickens. A significant increase in liver acid phosphatase occurred within 48 h which coincided with an increase in capillary fragility. The present finding that "lysosomal" enzymes are increased during aflatoxicosis in the chicken is contrary to those of BALASUBRAMANIAM et al. lo who reported that acid phosphatase did not increase in rat liver following an intraperitoneal injection of aflatoxin. These contrary findings m a y be the result of species difference, or the result of aflatoxin being administered as a single dose which can be metabolized and excreted rapidly as compared with continual dosing through the diet. Biochim. Biophys. Acta, 222 (197 o) 665-667
666
SHORT COMMUNICATIONS
3.0 W .J
8.0
m
~
ACID PHOSPHATASE
L~
c
z~
z~6.0 -.o
-~2.o
>-
a.
> E 410 ~-
~~_
glucuronidase
~2.0 u2'~_
~2 0
0
I
I
2.5
5.0
AFLATOXIN (IJg/g)
O_
O~
t
I
O
2.5
5.0
AFLATOXI N (Pg/g)
Fig. I. Effect of dietary aflatoxin on the specific activity of acid p h o s p h a t a s e in muscle homogenates. The chickens were fed graded doses of aflatoxin from h a t c h i n g until the h o m o g e n a t e s were m a d e at three weeks of age. D e t e r m i n a t i o n of enzyme activities was as described in the text. E a c h point is the m e a n of four groups of ten chickens and the vertical bars are the s t a n d a r d errors of the mean. Fig. 2. Effect of dietary aflatoxin on the specific activity of acid p h o s p h a t a s e and fl-glucuronidase in liver homogenates. The chickens were fed graded doses of aflatoxin from h a t c h i n g until the h o m o g e n a t e s were m a d e at three weeks of age. E n z y m e s were m e a s u r e d as described in the text. E a c h p o i n t is the m e a n of four groups of ten chickens and the vertical bars are the s t a n d a r d errors of the mean.
80,
z
60
X 0 I-
A
u_
40
o
E
Z ~ 1
'~
20
hi W b-
"'
I-"- ~ b- ¢
0
~,o p
w
E
.°%o5 x m
g ~
o 0
I
2 3 T I M E (doys)
4
Fig. 3. Response of capillary fragility and acid p h o s p h a t a s e to the incorporation of 5/~g/g of aflatoxin into the diet of z-week-old chickens. The m e a s u r e m e n t s were made as described in the text. E a c h p o i n t is the difference between the m e a n of four groups of ten, treated chickens and the m e a n of four groups of ten, u n t r e a t e d chickens of the same age. The vertical bars on the d a t a points are the s t a n d a r d errors of the difference.
Biochim. Biophys. Acta, zz2 (197o) 665-667
SHORT COMMUNICATIONS
667
The induction of a rapid loss of tissue strength and integrity by doses of aflatoxin that do not affect the body growth rate represents a new biochemical reaction for aflatoxin. Interference with growth is presumably caused by the general inhibition of protein and nucleic acid synthesis1whereas the rapid loss of tissue strength suggests a degradation of preformed structural elements. The concomitant increase of "]ysosomal" enzyme activity and capillary fragility in dose-related fashions during aflatoxicosis suggests a possible cause and effect relationship. Further work will be required to establish such a relationship conclusively. The degradation or digestion of both cellular and extracellular elements by lysosomal enzymes is well established2 and such digestion may occur in aflatoxicosis. The finding that aflatoxin can cause a generalized loss of tissue strength and integrity without affecting body weight emphasizes again the importance of mycotoxins in public and animal health. These findings suggest that mycotoxins can have an insidious acute toxicity aside from their documented carcinogenic properties.
Department of Poultry Science, North Carolina State University, Raleigh, N.C. 276o 7 (U.S.A.)
HsI~TANG TUNG W . E . DONALDSON P . B . HAMILTON
I A. CIEGLER AND E. B. LILLEHOJ, Advan. Appl. Microbiol., IO (1968) 155. 2 C. DE D o v e AND R. WATTIAUX, Ann. Rev. Physiol., 28 (1966) 435. 3 0 . L. SHOTWELL, C. W. HESSELTINE, R. D. STUBBLEFIELD AND W. G. SORENSON, Appl. Microbiol., 14 (1966) 425 . 4 J. NABNEY AND B. F. NESBITT, Analyst, 90 (I965) 155. H. G. WISEMAN, W. C. JACOBSON AND W. C. HARDMEY'ER, J. ASSOg. O~C. Agr. Chemists, 50 (1967) 982. 6 A. L. BACKARACH, M. G. COATES AND T. R. MIDDLETON, Biochem. J., 36 (1942) 4o7 . 7 IR. GIANETTO AND C. DE DUVE, Biochem. J., 59 (I955) 433. 8 E. LAYNE, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w York, 1957, p. 448. 9 J. W. SMITH AND P. B. HAMILTON, Poultry Sci., 49 (197 o) 207. IO K. BALASOBRAMANIAN, S. WIJESUNDERA, S. iNI. ARSECULERATNE AND G. E. TENNEKOON, Toxicon, 7 (1969) 159.
Received August I3th, 197° Biochim. Biophys. Acta, 222 (197 o) 665--667