Vol. 37, No. 1
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1979, p. 122-126 0099-2240/79/01-0122/05$02.00/0
Decreased Glycogen Mobilization During Ochratoxicosis in Broiler Chickenst W. E. HUFF,' J. A. DOERR,2 AND P. B. HAMILTON'` Department of Poultry Science' and Department of Microbiology,2 North Carolina State University, Raleigh, North Carolina 27650
Received for publication 30 October 1978
Graded doses of pure ochratoxin A (0, 0.5, 1.0, 2.0, 4.0, and 8.0 ,Ag of toxin per g of feed) were incorporated into a commercial diet which was fed to chickens from hatching to 3 weeks of age, at which time the experiments were terminated. Liver glycogen levels were elevated significantly (P < 0.05) by 4.0 and 8.0 ,ug/g but not lower doses. Glucagon stimulation of glycogen mobilization was inhibited at the same concentrations. Histopathological examination revealed cytoplasmic but not nuclear deposits of glycogen in cells at the periphery of liver lobes. These data demonstrated that ochratoxin inhibited glycogenolysis. Impaired ability to generate glucose from glycogen could account for the increased susceptibility to cold stress previously reported to occur in ochratoxicosis. Based on present and prior observations, it seems possible that ochratoxin induces a syndrome which mimics the glycogen storage disease of type X which is caused by a deficiency in the cyclic AMP-dependent enzyme of the glycogenolytic enzymatic cascade.
Ochratoxin is the term used to designate a group of mycotoxins known by the trivial names of ochratoxin A, B, and C (28, 36) of which ochratoxin A is the most toxic and most abundant. These mycotoxins are produced by Aspergillus ochraceus, from which they receive their name, as well as six additional Aspergillus species (13) and six Penicillium species (4, 25), of which P. viridicatum is the most important synthesizer. Ochratoxin A, based on acute oral 50% lethal dose determinations and minimal growth inhibitory concentration, is the most potent mycotoxin studied in chickens to date (16), and natural outbreaks of ochratoxicosis in poultry have recently been documented (P. B. Hamilton, W. E. Huff, J. R. Harris, and R. D. Wyatt, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, 021, p. 248). These studies, the widespread natural occurrence of fungi producing ochratoxin (27), and the natural occurrence of ochratoxin itself (21, 25, 27) imply that ochratoxin is a threat to both animal and human health. Ochratoxin A, which has multiple toxic effects, has been shown to be a severe nephrotoxin in chickens, swine, dogs, and rats (8, 14, 15, 18, 24, 32). However, the toxicity of ochratoxin is not limited to the kidney alone because it has been shown to cause hepatic degeneration and suppression of hematopoiesis in White Leghorn cockerels (6, 22), impaired blood coagulation (5) and nutrient absorption in broiler chickens (D.
J. Osborne, W. E. Huff, and P. B. Hamilton, Abstr. Poult. Sci. 55:2075, 1976), abortions in cows (30), and teratogenic effects in mice (11). Furthermore, in ducklings ochratoxin has been shown to be a more potent hepatotoxin than a nephrotoxin (34). One of the early descriptions of acute ochratoxicosis (24) reported glycogen accumulation in the livers of rats and, on the basis of histological examination of rat liver tissue, concluded that ochratoxin produced a syndrome similar to either glycogen storage disease VIII or IX (20). However, this was disputed in several reports (3, 21, 31) which demonstrated a reduction in liver glycogen of rats given a single dose of ochratoxin. The documented field outbreaks of ochratoxicosis in poultry (P. B. Hamilton, W. E. Huff, J. R. Harris, and R. D. Wyatt, Abstr. Annu. Meet. Am. Soc. Microbiol., 1977, 021, p. 248) and its emerging importance in swine and human health (9, 18) prompted the present studies of ochratoxin in broiler chickens and its possible role in glycogen metabolism. Chickens would appear to be particularly suitable experimental animals because symptoms of ochratoxicosis in chickens include sensitivity to cold stress (14) which can be explained on the basis of impaired glucose availability (33) resulting from interference with glycogen catabolism.
t Paper no. 5695 of the Journal Series of the North Carolina Agric,ultural Experiment Station, Raleigh, NC 27650.
Husbandry. One-day-old male broiler chicks (Cobb x Cobb) obtained from the university farm
122
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VOL. 37, 1979
GLYCOGEN MOBILIZATION DURING OCHRATOXICOSIS
used in these studies. The birds were housed in electrically heated batteries under continuous illumination with feed and water available ad libitum from 1 day to 3 weeks of age, at which time the experiments were terminated. The feed was a commercial broiler starter ration which was kept free of any medication. Ochratoxicosis was induced by adding known amounts of ochratoxin A dissolved in 95% ethanol into a small portion of the diet which was dried and mixed with the rest of the feed. Toxin production. Ochratoxin A was produced by growing A. ochraceus NRRL 3174 on wheat by the method of Trent et al. (35). Ochratoxin A was extracted from the wheat by the method of Steyn and Van der Merwe (29) and purified by chromatography on thick layers of Silica Gel G (E. M. Laboratories, Elmsford, N. Y., 10523) with benzene-acetic acid (9:1) as the solvent (10). Ochratoxin A was eluted from the silica gel by making a slurry of the silica gel in hot benzene-acetic acid (9:1). The slurry was then filtered, and the procedure was repeated three times until the ochratoxin was removed from the silica gel. The combined filtrates were reduced to approximately 1% of the total volume. Cold benzene was then added to precipitate the ochratoxin A. Ochratoxin A crystals were collected and recrystallized twice from benzene. Analyses. Liver glycogen was measured by the method of either Seifter et al. (26) or Carroll et al. (2). Crystalline glucagon (lot no. AZ 2297) was purchased from Schwarz/Mann, Orangeburg, N. Y. A glucagon solution was prepared by dissolving crystalline glucagon in physiological saline (0.1 mg/ml) immediately before use. The glucagon infusion test was performed by injecting the glucagon solution into the brachial vein at the dose level of 0.2 ml/kg of body weight (12). Blood samples were taken before glucagon administration and 2 min after glucagon administration. The plasma was collected, and plasma glucose values were determined by the method of Dubowski (7). Cloacal temperatures were measured by a thermister temperature probe (model 46, Yellow Springs Instrument Co., Yellow Springs, Ohio). Experimental design. There were four replicates of 10 birds at each of the ochratoxin levels of 0, 0.5, 1.0, 2.0, 4.0, and 8.0 jug of ochratoxin per g of diet. The treatments and birds were completely randomized. Values presented in this paper as percents were subjected to logarithmic transformation for statistical analysis. The replicate means were evaluated statistically by an analysis of variance in which an F ratio was calculated. If the F ratio was ignificant (P < 0.05), the least significant differences between the treatment means were calculated (1). Histopathology. Liver samples including both a peripheral and central segment were fixed in neutral buffered Formalin for 48 h with a change to fresh fixative at 24 h. Thin sections were made from embedded tissues by an independent histological service (Rex Hospital, Raleigh, N.C.) using a duplicate slide technique in which one slide was treated with diastase to remove glycogen before staining both slides with periodic acid-Schiff stain. This approach negated the lack of specificity with the periodic acid-Schiff stain. The slides were evaluated after being presented to the microscopist in a double-blind fashion followed by specific comparisons in a blind fashion. were
123
RESULTS Because liver glycogen was found to be rapidly mobilized in the chickens in preliminary experiments, the best system for killing the animals that gave the highest glycogen levels was determined. Table 1 shows the effect on liver glycogen of killing 2- or 3-week-old chickens via cardiac injection of succinylcholine chloride, which induces immediate cardiac arrest and thus stops circulation to the liver of any hormones that might influence glycogen stability, or via decapitation, which immediately arrests startle responses. Because the excitability and flightiness of chickens is pronounced, procedures were done in both darkened and lighted rooms. The method of killing did not have a significant effect at either age. However, both systems of killing gave significantly (P < 0.05) higher values with 3-week-old birds when conducted in the dark. Consequently, the subsequent experiments utilized 3-week-old birds decapitated in the dark; decapitation was chosen because of its greater ease.
During experimentation on ochratoxicosis, we observed that the experimental birds felt cooler than control birds when handled at death. Also, the intensity and duration of agonal convulsions appeared less in the experimental birds. Therefore, the effects of ochratoxin on cloacal temperatures were measured. The cloacal temperatures were not significantly depressed from the control value of 41.2 ± 0.1°C at any level of ochratoxin, suggesting that the apparently cooler body temperatures of experimental birds were an artifact. However, the cloacal temperature of living birds approximates the core body temperature more closely than it does the surface temperature of a dead bird which is very difficult to measure. The effect of ochratoxin on liver glycogen is presented in Fig. 1. Dietary ochratoxin A significantly (P < 0.05) increased liver glycogen at dose levels of 4.0 and 8.0 jtg/g. The increase in liver glycogen at the highest dose of ochratoxin represented a fourfold increase. This increase in glycogen during ochratoxicosis of broiler chickTABLE 1. Effect of succinykcholine, decapitation, and lighting on liver glycogen % Glycogen at: Killing method Lights 3 weeks 2 weeks On 0.88 + 0.06a 1.00 + 0.07a Succinylcholine On 0.96 ± 0.04a 0.95 ± 0.06a Decapitation Off 0.97 ± 0.06a 1.30 ± 0.13b Succinylcholine Off 0.99 t 0.12a 1.50 ± 0.12b Decapitation a, b Values with different superscripts in a column differ significantly (P < 0.05). Values are the mean ± standard error of the mean of the percent glycogen in liver on a wet weight basis with four groups of 10 birds.
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HUFF, DOERR, AND HAMILTON
could be attributed to either an inhibition of glycogenolysis or stimulation of glycogenesis. The differentiation of whether ochratoxin exhibited its effects on glycogenolysis or glycogenesis was determined by the administration of glucagon. Glucagon is the naturally occurring peptide released by the a-cells of the pancreas and whose normal function is to stimulate glycogenolysis (19). Table 2 shows the effect of ochratoxin on the stimulation of glycogenolysis as measured by an increase in plasma glucose after injection of glucagon. The increased plasma glucose arose, of course, from the glycogen broken down as a result of glucagon activity. Ochratoxin significantly (P < 0.05) inhibited the mobilization of glycogen by glucagon at the dose levels of 4.0 and 8.0 ,ug/g, and at the highest dose of ochratoxin (8.0 ,ug/g) there was even a 4% ens
200
-
z W
100
CD 0
w
OCHRATOXIN ( EXg/g)
FIG. 1. Effect of graded levels of dietary ochratoxin A on liver glycogen of chickens. Data points are mean offourgroups of 10 birds. Vertical bars on data points are standard errors of the mean. TABLE 2. Response ofplasma glucose to glucagon administration during ochratoxicosis Ochratoxin x Ug/g)
4g/g) 0.0 0.5 1.0 2.0 4.0 8.0
Pre-injection
plasma glucose (mg/100 ml) 197 ± 26a 209 ± 20"
219 ± 8a 200 ± 8 195 ± 7a 209 ± lla
Post-injection plasma glucose (mg/100 ml) 243 ± 20b 242 ± 13b 248 ± 1b 219 ± 3b 192 ± 15a 200 ± 8a
a, b Values in a row with different superscripts differ significantly (P < 0.05). Values are the mean ± stan-
dard error of the mean of four groups of 10 chickens. Glucagon was injected at the rate of 20 ,ug/kg of body weight.
APPL. ENVIRON. MICROBIOL.
decrease in plasma glucose after glucagon administration. Histological examination of liver tissue revealed that glycogen distribution in the liver of birds fed 4.0 and 8.0 ,ug/g was affected. Glycogen appeared to be more concentrated in the periphery of the liver lobes than in the center of the lobes. Cells at the periphery of the liver were enlarged, and furthermore the glycogen was seen in the cytoplasm with no nuclear deposition of glycogen.
DISCUSSION Ochratoxin A increased up to fourfold the liver glycogen content of young chickens fed a constant level of ochratoxin for 3 weeks (Fig. 1). This observation agrees closely with those of Purchase and Theron (24) in rats and is contrary to those of other investigators (3, 21, 31) who demonstrated a reduction in the liver glycogen of rats given a single dose of ochratoxin. The kinetic differences between an animal given a single acute dose which results in a spiking concentration in tissues and an animal in a chronic condition approaching a steady state appear obvious. An increase in liver glycogen is characteristic of a family of diseases known as glycogen storage diseases which were well described by McAdams et al. (20) and of which there are 10 types labeled simply with Roman numerals. In seeking detailed information about a glycogen storage disease, it is logical and conventional to classify the disease as 1 of the 10 recognized types for which at least partial mechanisms are known. Purchase and Theron (24) in their pioneering study postulated on the basis of histopathological examination alone that ochratoxin caused a type VIII or IX glycogen storage disease in rats. However, types VIII and IX display an increase of blood sugar in response to glucagon (20) which we did not observe with chickens (Table 2). Glycogen storage disease induced by ochratoxin is type I, VI, or X because they are the only types that show an abnormal (20) response to glucagon stimulation. Type I glycogenosis is further characterized by nuclear inclusions of glycogen, hypoglycemia, and hyperlipemia. Both the present and earlier (16) results demonstrated these symptoms did not occur during ochratoxicosis in chickens, thereby ruling out type I. From the current studies, it cannot be deduced with certainty whether ochratoxin causes type VI or X, although the glycogen deposits being concentrated in cells at the periphery of the lobes of chicken livers are reported to occur in type X and not in type VI (20). Additionally, Pitout (23) described a 70% in-
VOiL. 37, 1979
GLYCOGEN MOBILIZATION DURING OCHRATOXICOSIS
hibition by the in vitro addition of ochratoxin to the system of enzymes which activate phosphorylase, which is the enzyme that actually degrades glycogen to glucose, and furthermore he speculated that ochratoxin exerted its inhibitory effect on the enzymatic cascade which activates phosphorylase by competing with cyclic AMP for the allosteric site of the enzyme protein kinase which activates phosphorylase kinase. Two years later, it was demonstrated that glycogen storage disease of type X is the result of a deficiency in the activity of cyclic AMP-dependent protein kinase (17). Therefore, the findings of Pitout (23) combined with the present data suggest that ochratoxin induces a syndrome which mimics glycogen storage disease of type X. However, an extensive enzymatic analysis is needed before unequivocal statements are made. The inhibition of glycogenolysis by ochratoxin could account for the increased susceptibility to cold stress of birds with ochratoxicosis (14). The inhibition of glucagon stimulation (Table 2) occurred at the same dietary concentrations (4.0 and 8.0 jLg/g) of ochratoxin that made birds more susceptible to cold stress (14). Blood glucose is the immediate energetic reservoir used by the young bird in its attempts to maintain thermal homeostasis in a low ambient temperature (33), and, if ochratoxin inhibits the formation of glucose from glycogen, it stands to reason that birds with ochratoxicosis would be more susceptible to cold stress. The possibility that ochratoxin interferes with hormonal regulation at the second messenger or cyclic AMP level suggests that many other physiological systems are affected during ochratoxicosis. Indeed, mycotoxicologists may now be pressed to explain the specificities of ochratoxicosis. The elaboration of this area would appear rewarding.
7.
8.
9.
10.
11. 12.
13. 14. 15.
16. 17.
18.
19. 20.
ACKNOWLEDGMENTS
21.
We thank Sharon West, Gorum Whitaker, and Nancy Goodwin for their technical assistance.
22.
LITERATURE CITED 1. Bruning, J. L, and B. L. Kintz. 1968. Computational handbook of statistics. Scott Foresman Co., Glenview,
m.
2. Carroll, N. V., R. W. Langley, and J. H. Roe. 1956. The determination of glycogen in liver and muscle by use of anthrone reagent. J. Biol. Chem. 220:583-593. 3. Chu, F. S. 1974. Studies on ochratoxins. Crit. Rev. Toxicol. 3:499-524. 4. Ciegler, A., D. L. Fennell, H. J. Mintzlaff, and L. Leistner. 1972. Ochratoxin synthesis by Penicillium
species. Naturwissenschaften 59:365-366.
5. Doerr, J. A., W. E. Huff, H. T. Tung, R. D. Wyatt, and P. B. Hamilton. 1974. A survey of T-2 toxin, ochratoxin and aflatoxin for their effects on the coagulation of blood in young broiler chickens. Poult. Sci. 53: 1728-1734. 6. Doupnik, B., Jr., and J. C. Peckham. 1970. Mycotox-
23.
125
icity of Aspergillus ochraceus to chicks. Appl. Microbiol. 19:594-597. Dubowski, K. M. 1962. An o-toluidine method for bodyfluid glucose determination. Clin. Chem. 8:215-235. Elling, F., B. Hald, C. Jacobhon, and P. Krogh. 1975. Spontaneous caaes of toxic nephropathy in poultry associated with ochratoxin A. Acta Pathol. Microbiol. Scand. Sect. A 83:739-741. Elling, F., and P. Krogh. 1977. Fungal toxins and Balkan (endemic) nephropathy. Lancet i(8023):1213. Eppley, R. M. 1968. Screening method for zearalenone, aflatoxin and ochratoxin. J. Assoc. Off. Agric. Chem. 51:74-78. Hayes, A. W., R. D. Wood, and H. L Lee. 1973. Teratogenic effects of ochratoxin A in mice. Teratology 9: 93-98. Heald, P. J., P. M. McLachlan, and K. A. Rookledge. 1965. The effects of insulin, glucagon and adrenocorticotropic hormone on the plasma glucose and fatty acids of the domestic fowl. J. Endocrinol. 33:83-95. Hesseltine, C. W., E. E. Vandergraft, K. I. Fennell, M. L Smith, and 0. L Shotwell. 1972. Aspergilli as ochratoxin producers. Mycologia 64:539-550. Huff, W. E., and P. B. Hamilton. 1975. The interaction of ochratoxin A with some environmental extremes. Poult. Sci. 64:1659-1662. Huff, W. E., R. D. Wyatt, and P. B. Hamilton. 1975. Nephrotoxicity of dietary ochratoxin A in broiler chickens. Appl. Microbiol. 30:48-51. Huff, W. E., R. D. Wyatt, T. L. Tucker, and P. B. Hamilton. 1974. Ochratoxicosis in the broiler chicken. Poult. Sci. 53:1585-1591. Hug, G., W. K. Schubert, and G. Chuck. 1970. Loss of cyclic 3', 5' AMP dependent kinase and reduction of phosphorylase kinaae in skeletal muscle of a girl with deactivated phosphorylase and glycogenosis of liver and muscle. Biochem. Biophys. Res. Commun. 40:982-988. Krogh, P., N. H. Axelson, F. Elling, N. Gyrd-Hansen, B. Hald, J. Hyldgaard-Jensen, A. E. Larsen, A. Madsen, H. P. Mortensen, T. Moller, 0. K. Peterson, U. Ravnskov, M. Rostgaard, and 0. Aalund. 1974. Experimental porcine nephropathy. Changes of renal function and structure induced by ochratoxin A contaminated feed. Acta Pathol. Microbiol. Scand. 246: 1-20. Lehninger, 0. L 1975. Biochemistry. Worth Publishers, Inc., New York. McAdams, J. A., G. Hug, and K. E. Bove. 1974. Glycogen storage diseases, types I to X. Criteria for morphologic diagnosis. Human Pathol. 5:463-487. Munro, I. C., P. M. Scott, C. A. Moodie, and R. F. Willes. 1973. Ochratoxin A occurrence and toxicity. J. Am. Vet. Med. Assoc. 163:1269-1273. Peckham, J. C., B. Doupnik, and 0. H. Jones, Jr. 1971. Acute toxicity of ochratoxin A and B in chicks. Appl. Microbiol. 21:492-494. Pitout, M. J. 1968. The effect of ochratoxin A on glycogen storage in the rat liver. Toxicol. Appl. Pharmacol. 13:
299-306. 24. Purchase, L. F. H., and J. J. Theron. 1968. The acute toxicity of ochratoxin A to rats. Food Cosmet. Toxicol. 6:479-483. 25. Scott, P. M., W. Van Walbeek, B. Kennedy, and D. Anyeti. 1970. Occurrence of a mycotoxin, ochratoxin A, in wheat and isolation of ochratoxin A and citrinin producing strains of Penicillium viridicatum. Can. J. Plant Sci. 50:583-585. 26. Seifter, S., S. Dayton, B. Novic, and E. Muntwyler. 1950. The estimation of glycogen with the anthrone reagent. Arch. Biochem. 25:191-200. 27. Shotwell, 0. L, C. W. Hesseltine, and M. L. Goulden. 1969. Ochratoxin A: occurrence as natural contaminant of a corn sample. Appl. Microbiol. 17:765-766.
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28. Steyn, P. S., and C. W. Holzapfel. 1967. The synthesis of ochratoxin A. and B, metabolites of Aspergillus ochraceus Wilh. Tetrahedron 23:4449-4461. 29. Steyn, P. S., and K. J. Van der Merwe. 1966. Detection and estimation of ochratoxin A. Nature (London) 211: 418. 30. Still, P. E., A. W. Mackiln, W. E. Ribelin, and E. B. Smalley. 1971. Relationship of ochratoxin A to foetal death in laboratory and domestic animals. Nature (Lor.don) 234:563-564. 31. Suzuki, S., and T. Satoh. 1973. Effects of ochratouin A on tisue glycogen levels in rats. Jpn. J. Pharmacol. 23: 415419. 32. Szczech, G. M., W. W. Carlton, and J. Tuite. 1973. Ochratoxicosis in beagle dogs. Vet. Pathol. 10:135-154.
APPL. ENVIRON. MICROBIOL. 33. Thaxton, P., R. D. Wyatt, and P. B. Hamilton. 1974. The effect of environmental temperature on paratyphoid infection in the neonatal chicken. Poult. Sci. 53: 88-94. 34. Theron, J. J., K. K. Van der Merwe, N. Leibenberg, H. J. B. Joubert, and W. NeL 1966. Acute liver injury in ducklings and rats as a result of ochratoxin poisoning. J. Pathol. Bacteriol. 91:521-629. 35. Trent, H. L., M. E. Butz, and F. S. Chu. 1971. Production of ochratoxin in different cereal products by Aspergillus ochraceus. Appl. Microbiol. 21:1032-1035. 36. Van der Merwe, K. J., P. S. Steyn, and L Fourie. 1965. Mycotoxins. II. The constitution of ochratoxin A, and C, metabolites of Aspergilus ochraceus Wilh. J. Chem. Soc. 1965, p. 7083-7088.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1979, p. 122-126 0099-2240/79/01-0122/05$02.00/0
Decreased Glycogen Mobilization During Ochratoxicosis in Broiler Chickenst W. E. HUFF,' J. A. DOERR,2 AND P. B. HAMILTON'` Department of Poultry Science' and Department of Microbiology,2 North Carolina State University, Raleigh, North Carolina 27650
Received for publication 30 October 1978
Graded doses of pure ochratoxin A (0, 0.5, 1.0, 2.0, 4.0, and 8.0 ,Ag of toxin per g of feed) were incorporated into a commercial diet which was fed to chickens from hatching to 3 weeks of age, at which time the experiments were terminated. Liver glycogen levels were elevated significantly (P < 0.05) by 4.0 and 8.0 ,ug/g but not lower doses. Glucagon stimulation of glycogen mobilization was inhibited at the same concentrations. Histopathological examination revealed cytoplasmic but not nuclear deposits of glycogen in cells at the periphery of liver lobes. These data demonstrated that ochratoxin inhibited glycogenolysis. Impaired ability to generate glucose from glycogen could account for the increased susceptibility to cold stress previously reported to occur in ochratoxicosis. Based on present and prior observations, it seems possible that ochratoxin induces a syndrome which mimics the glycogen storage disease of type X which is caused by a deficiency in the cyclic AMP-dependent enzyme of the glycogenolytic enzymatic cascade.
Ochratoxin is the term used to designate a group of mycotoxins known by the trivial names of ochratoxin A, B, and C (28, 36) of which ochratoxin A is the most toxic and most abundant. These mycotoxins are produced by Aspergillus ochraceus, from which they receive their name, as well as six additional Aspergillus species (13) and six Penicillium species (4, 25), of which P. viridicatum is the most important synthesizer. Ochratoxin A, based on acute oral 50% lethal dose determinations and minimal growth inhibitory concentration, is the most potent mycotoxin studied in chickens to date (16), and natural outbreaks of ochratoxicosis in poultry have recently been documented (P. B. Hamilton, W. E. Huff, J. R. Harris, and R. D. Wyatt, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, 021, p. 248). These studies, the widespread natural occurrence of fungi producing ochratoxin (27), and the natural occurrence of ochratoxin itself (21, 25, 27) imply that ochratoxin is a threat to both animal and human health. Ochratoxin A, which has multiple toxic effects, has been shown to be a severe nephrotoxin in chickens, swine, dogs, and rats (8, 14, 15, 18, 24, 32). However, the toxicity of ochratoxin is not limited to the kidney alone because it has been shown to cause hepatic degeneration and suppression of hematopoiesis in White Leghorn cockerels (6, 22), impaired blood coagulation (5) and nutrient absorption in broiler chickens (D.
J. Osborne, W. E. Huff, and P. B. Hamilton, Abstr. Poult. Sci. 55:2075, 1976), abortions in cows (30), and teratogenic effects in mice (11). Furthermore, in ducklings ochratoxin has been shown to be a more potent hepatotoxin than a nephrotoxin (34). One of the early descriptions of acute ochratoxicosis (24) reported glycogen accumulation in the livers of rats and, on the basis of histological examination of rat liver tissue, concluded that ochratoxin produced a syndrome similar to either glycogen storage disease VIII or IX (20). However, this was disputed in several reports (3, 21, 31) which demonstrated a reduction in liver glycogen of rats given a single dose of ochratoxin. The documented field outbreaks of ochratoxicosis in poultry (P. B. Hamilton, W. E. Huff, J. R. Harris, and R. D. Wyatt, Abstr. Annu. Meet. Am. Soc. Microbiol., 1977, 021, p. 248) and its emerging importance in swine and human health (9, 18) prompted the present studies of ochratoxin in broiler chickens and its possible role in glycogen metabolism. Chickens would appear to be particularly suitable experimental animals because symptoms of ochratoxicosis in chickens include sensitivity to cold stress (14) which can be explained on the basis of impaired glucose availability (33) resulting from interference with glycogen catabolism.
t Paper no. 5695 of the Journal Series of the North Carolina Agric,ultural Experiment Station, Raleigh, NC 27650.
Husbandry. One-day-old male broiler chicks (Cobb x Cobb) obtained from the university farm
122
MATERLA1S AND METHODS
VOL. 37, 1979
GLYCOGEN MOBILIZATION DURING OCHRATOXICOSIS
used in these studies. The birds were housed in electrically heated batteries under continuous illumination with feed and water available ad libitum from 1 day to 3 weeks of age, at which time the experiments were terminated. The feed was a commercial broiler starter ration which was kept free of any medication. Ochratoxicosis was induced by adding known amounts of ochratoxin A dissolved in 95% ethanol into a small portion of the diet which was dried and mixed with the rest of the feed. Toxin production. Ochratoxin A was produced by growing A. ochraceus NRRL 3174 on wheat by the method of Trent et al. (35). Ochratoxin A was extracted from the wheat by the method of Steyn and Van der Merwe (29) and purified by chromatography on thick layers of Silica Gel G (E. M. Laboratories, Elmsford, N. Y., 10523) with benzene-acetic acid (9:1) as the solvent (10). Ochratoxin A was eluted from the silica gel by making a slurry of the silica gel in hot benzene-acetic acid (9:1). The slurry was then filtered, and the procedure was repeated three times until the ochratoxin was removed from the silica gel. The combined filtrates were reduced to approximately 1% of the total volume. Cold benzene was then added to precipitate the ochratoxin A. Ochratoxin A crystals were collected and recrystallized twice from benzene. Analyses. Liver glycogen was measured by the method of either Seifter et al. (26) or Carroll et al. (2). Crystalline glucagon (lot no. AZ 2297) was purchased from Schwarz/Mann, Orangeburg, N. Y. A glucagon solution was prepared by dissolving crystalline glucagon in physiological saline (0.1 mg/ml) immediately before use. The glucagon infusion test was performed by injecting the glucagon solution into the brachial vein at the dose level of 0.2 ml/kg of body weight (12). Blood samples were taken before glucagon administration and 2 min after glucagon administration. The plasma was collected, and plasma glucose values were determined by the method of Dubowski (7). Cloacal temperatures were measured by a thermister temperature probe (model 46, Yellow Springs Instrument Co., Yellow Springs, Ohio). Experimental design. There were four replicates of 10 birds at each of the ochratoxin levels of 0, 0.5, 1.0, 2.0, 4.0, and 8.0 jug of ochratoxin per g of diet. The treatments and birds were completely randomized. Values presented in this paper as percents were subjected to logarithmic transformation for statistical analysis. The replicate means were evaluated statistically by an analysis of variance in which an F ratio was calculated. If the F ratio was ignificant (P < 0.05), the least significant differences between the treatment means were calculated (1). Histopathology. Liver samples including both a peripheral and central segment were fixed in neutral buffered Formalin for 48 h with a change to fresh fixative at 24 h. Thin sections were made from embedded tissues by an independent histological service (Rex Hospital, Raleigh, N.C.) using a duplicate slide technique in which one slide was treated with diastase to remove glycogen before staining both slides with periodic acid-Schiff stain. This approach negated the lack of specificity with the periodic acid-Schiff stain. The slides were evaluated after being presented to the microscopist in a double-blind fashion followed by specific comparisons in a blind fashion. were
123
RESULTS Because liver glycogen was found to be rapidly mobilized in the chickens in preliminary experiments, the best system for killing the animals that gave the highest glycogen levels was determined. Table 1 shows the effect on liver glycogen of killing 2- or 3-week-old chickens via cardiac injection of succinylcholine chloride, which induces immediate cardiac arrest and thus stops circulation to the liver of any hormones that might influence glycogen stability, or via decapitation, which immediately arrests startle responses. Because the excitability and flightiness of chickens is pronounced, procedures were done in both darkened and lighted rooms. The method of killing did not have a significant effect at either age. However, both systems of killing gave significantly (P < 0.05) higher values with 3-week-old birds when conducted in the dark. Consequently, the subsequent experiments utilized 3-week-old birds decapitated in the dark; decapitation was chosen because of its greater ease.
During experimentation on ochratoxicosis, we observed that the experimental birds felt cooler than control birds when handled at death. Also, the intensity and duration of agonal convulsions appeared less in the experimental birds. Therefore, the effects of ochratoxin on cloacal temperatures were measured. The cloacal temperatures were not significantly depressed from the control value of 41.2 ± 0.1°C at any level of ochratoxin, suggesting that the apparently cooler body temperatures of experimental birds were an artifact. However, the cloacal temperature of living birds approximates the core body temperature more closely than it does the surface temperature of a dead bird which is very difficult to measure. The effect of ochratoxin on liver glycogen is presented in Fig. 1. Dietary ochratoxin A significantly (P < 0.05) increased liver glycogen at dose levels of 4.0 and 8.0 jtg/g. The increase in liver glycogen at the highest dose of ochratoxin represented a fourfold increase. This increase in glycogen during ochratoxicosis of broiler chickTABLE 1. Effect of succinykcholine, decapitation, and lighting on liver glycogen % Glycogen at: Killing method Lights 3 weeks 2 weeks On 0.88 + 0.06a 1.00 + 0.07a Succinylcholine On 0.96 ± 0.04a 0.95 ± 0.06a Decapitation Off 0.97 ± 0.06a 1.30 ± 0.13b Succinylcholine Off 0.99 t 0.12a 1.50 ± 0.12b Decapitation a, b Values with different superscripts in a column differ significantly (P < 0.05). Values are the mean ± standard error of the mean of the percent glycogen in liver on a wet weight basis with four groups of 10 birds.
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HUFF, DOERR, AND HAMILTON
could be attributed to either an inhibition of glycogenolysis or stimulation of glycogenesis. The differentiation of whether ochratoxin exhibited its effects on glycogenolysis or glycogenesis was determined by the administration of glucagon. Glucagon is the naturally occurring peptide released by the a-cells of the pancreas and whose normal function is to stimulate glycogenolysis (19). Table 2 shows the effect of ochratoxin on the stimulation of glycogenolysis as measured by an increase in plasma glucose after injection of glucagon. The increased plasma glucose arose, of course, from the glycogen broken down as a result of glucagon activity. Ochratoxin significantly (P < 0.05) inhibited the mobilization of glycogen by glucagon at the dose levels of 4.0 and 8.0 ,ug/g, and at the highest dose of ochratoxin (8.0 ,ug/g) there was even a 4% ens
200
-
z W
100
CD 0
w
OCHRATOXIN ( EXg/g)
FIG. 1. Effect of graded levels of dietary ochratoxin A on liver glycogen of chickens. Data points are mean offourgroups of 10 birds. Vertical bars on data points are standard errors of the mean. TABLE 2. Response ofplasma glucose to glucagon administration during ochratoxicosis Ochratoxin x Ug/g)
4g/g) 0.0 0.5 1.0 2.0 4.0 8.0
Pre-injection
plasma glucose (mg/100 ml) 197 ± 26a 209 ± 20"
219 ± 8a 200 ± 8 195 ± 7a 209 ± lla
Post-injection plasma glucose (mg/100 ml) 243 ± 20b 242 ± 13b 248 ± 1b 219 ± 3b 192 ± 15a 200 ± 8a
a, b Values in a row with different superscripts differ significantly (P < 0.05). Values are the mean ± stan-
dard error of the mean of four groups of 10 chickens. Glucagon was injected at the rate of 20 ,ug/kg of body weight.
APPL. ENVIRON. MICROBIOL.
decrease in plasma glucose after glucagon administration. Histological examination of liver tissue revealed that glycogen distribution in the liver of birds fed 4.0 and 8.0 ,ug/g was affected. Glycogen appeared to be more concentrated in the periphery of the liver lobes than in the center of the lobes. Cells at the periphery of the liver were enlarged, and furthermore the glycogen was seen in the cytoplasm with no nuclear deposition of glycogen.
DISCUSSION Ochratoxin A increased up to fourfold the liver glycogen content of young chickens fed a constant level of ochratoxin for 3 weeks (Fig. 1). This observation agrees closely with those of Purchase and Theron (24) in rats and is contrary to those of other investigators (3, 21, 31) who demonstrated a reduction in the liver glycogen of rats given a single dose of ochratoxin. The kinetic differences between an animal given a single acute dose which results in a spiking concentration in tissues and an animal in a chronic condition approaching a steady state appear obvious. An increase in liver glycogen is characteristic of a family of diseases known as glycogen storage diseases which were well described by McAdams et al. (20) and of which there are 10 types labeled simply with Roman numerals. In seeking detailed information about a glycogen storage disease, it is logical and conventional to classify the disease as 1 of the 10 recognized types for which at least partial mechanisms are known. Purchase and Theron (24) in their pioneering study postulated on the basis of histopathological examination alone that ochratoxin caused a type VIII or IX glycogen storage disease in rats. However, types VIII and IX display an increase of blood sugar in response to glucagon (20) which we did not observe with chickens (Table 2). Glycogen storage disease induced by ochratoxin is type I, VI, or X because they are the only types that show an abnormal (20) response to glucagon stimulation. Type I glycogenosis is further characterized by nuclear inclusions of glycogen, hypoglycemia, and hyperlipemia. Both the present and earlier (16) results demonstrated these symptoms did not occur during ochratoxicosis in chickens, thereby ruling out type I. From the current studies, it cannot be deduced with certainty whether ochratoxin causes type VI or X, although the glycogen deposits being concentrated in cells at the periphery of the lobes of chicken livers are reported to occur in type X and not in type VI (20). Additionally, Pitout (23) described a 70% in-
VOiL. 37, 1979
GLYCOGEN MOBILIZATION DURING OCHRATOXICOSIS
hibition by the in vitro addition of ochratoxin to the system of enzymes which activate phosphorylase, which is the enzyme that actually degrades glycogen to glucose, and furthermore he speculated that ochratoxin exerted its inhibitory effect on the enzymatic cascade which activates phosphorylase by competing with cyclic AMP for the allosteric site of the enzyme protein kinase which activates phosphorylase kinase. Two years later, it was demonstrated that glycogen storage disease of type X is the result of a deficiency in the activity of cyclic AMP-dependent protein kinase (17). Therefore, the findings of Pitout (23) combined with the present data suggest that ochratoxin induces a syndrome which mimics glycogen storage disease of type X. However, an extensive enzymatic analysis is needed before unequivocal statements are made. The inhibition of glycogenolysis by ochratoxin could account for the increased susceptibility to cold stress of birds with ochratoxicosis (14). The inhibition of glucagon stimulation (Table 2) occurred at the same dietary concentrations (4.0 and 8.0 jLg/g) of ochratoxin that made birds more susceptible to cold stress (14). Blood glucose is the immediate energetic reservoir used by the young bird in its attempts to maintain thermal homeostasis in a low ambient temperature (33), and, if ochratoxin inhibits the formation of glucose from glycogen, it stands to reason that birds with ochratoxicosis would be more susceptible to cold stress. The possibility that ochratoxin interferes with hormonal regulation at the second messenger or cyclic AMP level suggests that many other physiological systems are affected during ochratoxicosis. Indeed, mycotoxicologists may now be pressed to explain the specificities of ochratoxicosis. The elaboration of this area would appear rewarding.
7.
8.
9.
10.
11. 12.
13. 14. 15.
16. 17.
18.
19. 20.
ACKNOWLEDGMENTS
21.
We thank Sharon West, Gorum Whitaker, and Nancy Goodwin for their technical assistance.
22.
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