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National Peanut Research Laboratory, Dawson, GA 31742. ROBERT A. HILL. Georgia Coastal Plain Experiment Station, Tifton, GA 31793. Aspergillus flavus ...
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14 Mode of Formation of Aflatoxin in Various Nut Fruits and Gross and Histologic Effects of Aflatoxins in Animals RICHARD J. COLE, TIMOTHY H. SANDERS, and PAUL D. BLANKENSHIP National Peanut Research Laboratory, Dawson, GA 31742

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ROBERT A. HILL Georgia Coastal Plain Experiment Station, Tifton, GA 31793 Aspergillus flavus Link and the closely related fungus A. parasiticus Speare are two species of fungi that are capable of invading various food and feed commodities and causing extensive economic losses by contamination of these commodities with highly toxic aflatoxins, thus, posing a threat to animal health. The specific conditions that result in containation of peanut, pecans, almonds, and pistachio nuts with the aflatoxins are discussed in detail. The primary target organ of the aflatoxins in domestic animals is the liver. A discussion of the gross and histological effects of aflatoxin poisoning in rainbow trout and swine is presented. A considerable amount of research e f f o r t has been expended since the discovery of the h i g h l y t o x i c a f l a t o x i n s contaminating B r a z i l i a n peanut meal i n Great B r i t a i n i n the e a r l y I 9 6 0 s . Recently some of t h i s e f f o r t has been expanded to d e f i n e the e t i o l o g y of a f l a t o x i n contamination i n peanuts. The preharvest and storage phases of peanut production are most o f t e n a s s o c i ated with s i g n i f i c a n t a f l a t o x i n contamination. The most extensive a f l a t o x i n contamination of peanuts occurs during preharvest f o l l o w i n g drought s t r e s s . Environmental cond i t i o n s a f f e c t the nature and degree of m i c r o b i a l i n t e r a c t i o n with peanuts. Despite the omnipresence of A s p e r g i l l u s f l a v u s propagules i n the s o i l during peanut f r u i t development, the fungus i s innocuous except during periods of environmental s t r e s s . Recent s t u d i e s have shown that dry s o i l c o n d i t i o n s and an e l e vated s o i l temperature i n the geocarposphere during the l a t t e r 4-6 weeks of the growing season are the major f a c t o r s r e s p o n s i b l e f o r preharvest a f l a t o x i n contamination (1,2)(Table 1). The apparent e f f e c t of dry s o i l c o n d i t i o n s i s to reduce m i c r o b i a l a c t i v i t y and, t h e r e f o r e , antagonism by competitors of A. f l a v u s . The major fungal antagonists of A. f l a v u s are probably A. n i g e r 1

T h i s chapter not subject to U . S . copyright. P u b l i s h e d 1983, A m e r i c a n C h e m i c a l S o c i e t y

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

0.35

23.8

24.0

between rows

o v e r a l l mean under rows

between rows

o v e r a l l mean other ( o i l stock, LSK, and damaged)

o v e r a l l mean e d i b l e s (jumbo, medium, #1, and other e d i b l e )

1.29

o v e r a l l mean under rows

Irrigated Treatment

Drought Treatment

Drought-Cooled Treatment #1

22.5

21.8 16.6

18.0

471

10,516

27.0

25.7 20.5

19.8

2553

19

66

A f l a t o x i n Concentration (ppb)

30.5

30.5

Temperatures During Treatment P e r i o d , °C

12.6

15.4

S o i l Moisture i n Bars Tension

Drought-Heated Treatment

542

22.7

21.3

14.7

17.2

Drought-Cooled Treatment #2

2309

23.6

22.9

15.7

20.4

Drought-Cooled Treatment #3

Table I . Moisture Tension, Mean Geocarposphere Temperatures, and A f l a t o x i n Content During The Treatment P e r i o d f o r the S i x Treatment Regimes

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and Trichoderma sp., which cannot t o l e r a t e low environmental moisture c o n d i t i o n s as can A. f l a v u s . The lower l i m i t of water a c t i v i t y (Aw) capable of s u s t a i n i n g growth i s approximately 0.88 Aw f o r A. niger, whereas 0.78 i s considered the lower l i m i t f o r A. f l a v u s (3). In conjunction with drought s t r e s s , an elevated geocarposphere temperature during the l a t t e r 4-6 weeks of the growing season i s necessary f o r preharvest a f l a t o x i n contamination of peanuts (1,2). Experiments i n which peanuts were grown under drought s t r e s s at s e l e c t e d geocarposphere temperatures, showed that an o v e r a l l mean geocarposphere temperature between 26.732.2°C was r e q u i r e d i n a d d i t i o n to dry s o i l c o n d i t i o n s f o r p r e harvest a f l a t o x i n contamination to develop (Table I ) . Elevated geocarposphere temperatures presumably break down some r e s i s t a n c e mechanism i n the developing peanut f r u i t , enabling A. f l a v u s to grow and produce a f l a t o x i n . Peanut p l a n t s that grow with adequate moisture overlap the row middles during the l a t t e r t h i r d of the growing season i n such a f a s h i o n that the s o i l surface under, and between, the rows i s shaded from d i r e c t s u n l i g h t . I t has been shown that the mean geocarposphere temperature under these c o n d i t i o n s (approximately 24°C) i s not a f f e c t e d g r e a t l y by ambient temperatures (1,2). Peanut p l a n t s grown under severe drought s t r e s s , during the l a t t e r 4-6 weeks of the growing season, recede so as to p a r t i a l l y expose the s o i l surface to d i r e c t s u n l i g h t , which causes an increase i n geocarposphere temperature. Thus, the two major requirements (dry s o i l and elevated geocarposphere temperature) f o r preharvest a f l a t o x i n contamination are achieved. I t has been reported that the a f l a t o x i n content of i n s e c t damaged peanuts i s considerably higher than contaminated undamaged peanuts (1,2.). The l e s s e r c o r n s t a l k borer, which i s the most important s o i l i n s e c t r e s p o n s i b l e f o r peanut k e r n e l damage, favors hot, dry environmental c o n d i t i o n s s i m i l a r to those favored by A. f l a v u s . In a d d i t i o n , i n s e c t i c i d e a p p l i c a t i o n s must be wetted i n t o the s o i l to be e f f e c t i v e . Thus, i n s e c t treatments during drought periods are l a r g e l y i n e f f e c t i v e . The other phase of peanut p r o d u c t i o n i n which a f l a t o x i n contamination may occur i s storage. The primary concern i n maint a i n i n g peanuts a f l a t o x i n - f r e e during storage i s moisture c o n t r o l (4). Required temperatures may be b i o l o g i c a l l y generated i n a mass of peanuts by m i c r o b i a l a c t i o n . The source of moisture may be from a l o t of improperly d r i e d peanuts (stored with greater than 10% m o i s t u r e ) . Condensation of moisture w i t h i n the warehouse, because of an inadequate flow of a i r through the peanuts and the overspace, i s a l s o a cause. T h i s occurs when the moisture condenses before i t i s exhausted from the warehouse. Another source of excessive moisture i s a leaky warehouse r o o f . Peanuts moistened from a leak can become h i g h l y contaminated with a f l a t o x i n and these can, i n t u r n , contaminate a considerable amount of uncontaminated peanuts when they are mixed. Two a d d i t i o n a l

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sources of moisture i n warehouses are high moisture f o r e i g n m a t e r i a l , such as w i l d cucumbers and aqueous i n s e c t i c i d e a p p l i c a t i o n s (4) . In a d d i t i o n to peanuts (which are not b o t a n i c a l l y true n u t s ) , a f l a t o x i n s have been found i n many of the tree nuts i n c l u d i n g p i s t a c h i o s , almonds, pecans, and other tree nuts ( 5 ) . P i s t a c h i o nuts are formed i n c l u s t e r s and enclosed i n a h u l l . They are e i t h e r hand-harvested (imported nuts) or mechanically harvested (domestically produced n u t s ) . Conventional methods of processing harvested nuts are used abroad. T h i s procedure i n v o l v e s sun drying i n the h u l l . The d r i e d nuts are stored e i t h e r i n bulk or i n burlap bags. Before marketing the nuts are dehulled a f t e r soaking them i n water f o r 4-6 h r s . The nuts are then r e washed and d r i e d with sun, or hot a i r d r y e r s . The use of a r t i f i c i a l d r y i n g of p i s t a c h i o s , as compared to sun d r y i n g , i s more d e s i r a b l e and provides l e s s chance f o r mold growth and a f l a t o x i n development (6). Storage of p i s t a c h i o nuts i n the h u l l n e c e s s i t a t e s rewetting before d e h u l l i n g ; t h e r e f o r e , storage of nuts a f t e r d e h u l l i n g would provide l e s s opportunity f o r a f l a t o x i n contamination. A l s o , the presence of the h u l l f a v o r s mold growth under f a v o r a b l e conditions ( 7 ) . Since nuts that are s p l i t are more r e a d i l y invaded by A. f l a v u s than sound nuts, the s p l i t nuts should be separated from i n t a c t nuts a f t e r d e h u l l i n g and should be stored s e p a r a t e l y ( 8 ) . A survey conducted i n 3 areas of the Gajiantep province of Turkey, r e p r e s e n t i n g extremes of geography and c l i m a t e , showed that A. f l a v u s was a common contaminant of harvested stored nuts at a l l stages a f t e r d e h u l l i n g , but there was no evidence of s i g n i f i c a n t a f l a t o x i n contamination of immature or f r e s h l y harvested nuts (2). In c o n t r a s t , i n a study of I r a n i a n p i s t a c h i o nuts, Rahnema (.10). observed contamination of immature nuts (endosperms) while s t i l l on the t r e e . Based on p a r a l l e l observations i n peanuts, the e x p l a n a t i o n may be that A. f l a v u s i s more competitive i n the h o t t e r , more a r i d c l i m a t e . In a d d i t i o n , the higher temperatures may predispose the developing f r u i t to a f l a t o x i n development i n a way s i m i l a r to that observed i n peanuts (1,2). A s p e r g i l l u s f l a v u s i n v a s i o n and a f l a t o x i n contamination i n almonds are u s u a l l y a s s o c i a t e d with damaged k e r n e l s (8,11,12,13). The most s i g n i f i c a n t source of damage to almond k e r n e l s i s caused by the navel orangeworm l a r v a e , Amyelois t r a n s i t e 1 1 a (Walker). The i n s e c t a t t a c k s the almond k e r n e l i n the orchard, a f t e r n a t u r a l s p l i t t i n g of the f r u i t , as i t matures. Apparently, the i n s e c t provides an i n v a s i o n route f o r A. f l a v u s by i n j u r i n g the k e r n e l r a t h e r than s e r v i n g as a v e c t o r of the fungus (14)• The source of A. f l a v u s propagules i s from the environment, where they are disseminated by n a t u r a l f a c t o r s , such as wind and r a i n . Studies have shown that A. f l a v u s i n v a s i o n and a f l a t o x i n development i n almonds occur i n the orchard f o l l o w i n g the n a t u r a l s p l i t t i n g of the h u l l before harvest (12). Normally, the moisture content of

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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the almond k e r n e l at harvest i s i n s u f f i c i e n t to support growth of A. f l a v u s . Although i t i s somewhat unclear at t h i s time whether A. f l a v u s i n v a s i o n and a f l a t o x i n development can occur i n the absence of i n s e c t damage, r e s u l t s suggest that sound almond kernels are r e l a t i v e l y f r e e of a f l a t o x i n contamination. However, surveys over a three-year p e r i o d e s t a b l i s h e d that a f l a t o x i n may occur i n tree nuts, such as almond, throughout the growing area and that only a r e l a t i v e l y few kernels i n a l a r g e population were contaminated (8). The pecan nut may be invaded by numerous fungi i n c l u d i n g the A. f l a v u s group. There i s no c l e a r l y defined evidence that a f l a ­ t o x i n contamination i n pecans i s a preharvest problem s i m i l a r to peanuts. The problem, as c u r r e n t l y understood, occurs when pecans are damaged by i n s e c t s or when they l i e on the ground s e v e r a l days a f t e r normal drop (15). In a d d i t i o n , the b e n e f i t s of timely har­ vest of pecans may he l c s t ^ i f they are not adequately d r i e d and s t o r e d . This i s due to damage i n c u r r e d to the s h e l l s during mechanical h a r v e s t i n g (16). Furthermore, i t was concluded that r e s i s t a n c e due to the s h e l l apparently i s a v a r i e t a l c h a r a c t e r i s ­ t i c that i s r e l a t e d to s h e l l s t r u c t u r e . S e l e c t i o n of v a r i e t i e s on the b a s i s of s h e l l s t r u c t u r e and r e s i s t a n c e to s p l i t t i n g should provide adequate p r o t e c t i o n from A. f l a v u s i n v a s i o n and subsequent a f l a t o x i n contamination. T h i s , i n conjunction with prompt harvesting and good storage p r a c t i c e s , should insure nuts that are a f l a t o x i n ^ f r e e . H i s t o l o g i c e f f e c t s of a f l a t o x i n s i n animals A t y p i c a l gross c l i n i c a l s i g n of chronic a f l a t o x i c o s i s i n animals i s general u n t h r i f t i n e s s r e s u l t i n g from reduced feed con­ sumption and feed conversion e f f i c i e n c y . The major target organ of a f l a t o x i n Βχ i s the l i v e r , with v a r i a b l e adverse e f f e c t s depending on the animal species i n v o l v e d . The potent hepatocarc i n o g e n i c i t y of a f l a t o x i n Βχ i s seen i n the Shasta s t r a i n of r a i n ­ bow t r o u t (17) and i n the F i s c h e r s t r a i n of r a t (Table I I ) ( 1 8 ) . Table I I . Carcinogenic i n Rats A f l a t o x i n Levels (ppb) 0 1 5 15 50 100

E f f e c t s of D i e t a r y Levels of A f l a t o x i n Βχ Number of Animals With Carcinoma 0 2 1 4 20 28

T o t a l Animals Tested 18 22 22 21 25 28

This i s i n c o n t r a s t to the Wister s t r a i n of r a t , which i s not n e a r l y as s u s c e p t i b l e to the carcinogenic e f f e c t s of a f l a t o x i n Βχ (19).

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The rainbow trout i s known to be one of the most s e n s i t i v e animals to the t o x i c e f f e c t s of d i e t a r y a f l a t o x i n , e s p e c i a l l y the hepatocarcinogenic e f f e c t s . The L D ^ Q of pure a f l a t o x i n Βχ i n t r o u t i s 0.5 mg/kg f o r a s i n g l e dose, and 0.3 mg/kg f o r a dose given over a 5-day p e r i o d . Trout fed l e v e l s as low as 0.4 yg/kg i n the d i e t showed a s i g n i f i c a n t i n c i d e n c e of hepatoma a f t e r 12 months exposure. In the l a t t e r case, the t o t a l i n t a k e of a f l a ­ t o x i n Β χ over the one-year p e r i o d would be 0.1-0.2 yg. In t r o u t fed a f l a t o x i n at 20 yg/kg f o r 20 days, i n c i d e n c e of hepatoma was 40% a f t e r 12 months. Recently, i t was reported that when embryonated t r o u t eggs were placed i n an aqueous s o l u t i o n of a f l a t o x i n ΒI (0.5 yg/ml) f o r only 1 hour, the i n c i d e n c e of hepatoma was 40% a f t e r 10 months (20). Table I I shows that the F i s c h e r s t r a i n of r a t i s a l s o very s u s c e p t i b l e to the hepatocarcinogenic e f f e c t s of a f l a t o x i n Βχ (18). Induction of l i v e r carcinoma has been l e s s c o n s i s t e n t i n animals other than the Shasta t r o u t and F i s c h e r r a t . For example, there are few, i f any, d e f i n i t i v e accounts of a f l a t o x i n - i n d u c e d l i v e r cancer i n swine (21). A f l a t o x i c o s i s i n most animals appears to be s i m i l a r to that observed i n swine. The carcass of a p i g s u f f e r i n g from a f l a t o x i c o s i s appears yellow or jaundiced due to impaired l i v e r f u n c t i o n and the r e s u l t a n t accumulation of b i l i ­ r u b i n i n the t i s s u e s . The p e r i t o n e a l c a v i t y and pericardium c o n t a i n excessive f l u i d . The gross appearance of the l i v e r i s o f t e n yellow due to accumulation of f a t . The b i l e i s t y p i c a l l y a straw c o l o r rather than the normal dark c o l o r . On gross obser­ v a t i o n , the l o b u l a r p a t t e r n may be accentuated due to the accumu­ l a t i o n of f a t and an i n c r e a s e of f i b r o u s t i s s u e around the l o b u l e . M i c r o s c o p i c a l l y , there i s f a t t y change, p r o l i f e r a t i o n of b i l e ductules w i t h i n the l o b u l e , karyomegaly (enlarged n u c l e i ) , megaloc y t o c i s (enlarged hepatocytes), nodular r e g e n e r a t i o n (hyperplasia), and p e r i l o b u l a r f i b r o s i s developing u l t i m a t e l y i n t o a d i s s e c t i n g f i b r o s i s reminiscent of the c o n d i t i o n of advanced c i r r h o s i s seen with a l c o h o l i s m i n humans (21). In a f l a t o x i c o s i s , n e c r o s i s of the l i v e r i s c e n t r o l o b u l a r i n swine and t y p i c a l l y p e r i p o r t a l i n other animals, such as the r a t . Although a f l a t o x i n has been shown to be t o x i c to humans (22), the r o l e of these toxins i n human primary l i v e r cancer remains obscure. E p i d e m i o l o g i c a l s t u d i e s i n humans conducted i n p o r t i o n s of A f r i c a show a c o r r e l a t i o n between i n c r e a s e d i n c i d e n c e of human primary l i v e r cancer and i n c r e a s e i n a f l a t o x i n i n t a k e (23). How­ ever, e p i d e m i o l o g i c a l studies i n the U.S. do no i n d i c a t e a c o r r e l a t i o n between i n c i d e n c e of primary human l i v e r cancer and a f l a t o x i n consumption. In the Southeast, the average intake of a f l a t o x i n i s estimated to be nine times greater than the n a t i o n a l average (24). However, according to unpublished data from the U.S. N a t i o n a l Center f o r Health S t a t i s t i c s , the number of deaths from l i v e r cancer i s lower i n each of the eight p o p u l a t i o n groups i n the Southeast as compared to the n a t i o n a l average (24). In summary, the environmental c o n d i t i o n s that appear to be 1

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FEEDS

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c r i t i c a l f o r a f l a t o x i n contamination of nuts i n general are dam­ age, elevated temperature, and i n peanuts, dry s o i l c o n d i t i o n s . The l a t t e r c o n d i t i o n i s r e l a t e d to m i c r o b i a l competition and may be unique to peanuts, since they develop i n a subterranean e n v i r o n ­ ment.

Literature Cited 1. Hill, R. Α.; Blankenship, P. D.; Cole, R. J.; Sanders, T. H. Appl. Environ. Microbiol., in press. 2. Blankenship, P. D.; Cole, R. J.; Sanders, T. H.; Hill, R. Α., in preparation. 3. Hill, R. Α., personal communication. 4. Smith, J. S., Jr.; Davidson, J. I., Jr. Trans. ASAE 1982, 231-236. 5. Stoloff, L. J. Assoc. Off. Anal. Chem. 1978, 63, 1067-1073. 6. Sommer, N. R., personal communication. 7. Denizel, T. Archieves de L'Institut Pasteur Die Tunis 1977, 54, 433-440. 8. Fuller, G.; Spooner, W. W.; King, A. D., Jr. J. Am. Oil Chem. Soc. 1977, 231A-235A. 9. Denizel, T.; Jarvis, B.; Rolfe, E. J. J. Sci. Food Agric. 1976, 27, 1021-1026. 10. Rahnema, R. I.U.P.A.C. Symposium, Göteborg, Sweden, 1972, Abstract. 11. Stoloff, L. Proc. Am. Phytopathol. 1976, 3, 156-172. 12. Phillips, D. J.; Uota, M.; Monticelli, D.; Curtis, C. J . Am. Soc. Hort. Sci. 1976, 100, 19-23. 13. Shade, J. E.; McGreevey, R.; King, A. D., Jr. Appl. Microbiol. 1975, 29, 48-53. 14. Phillips, D. J.; Purcell, S. L.; Stanley, G. I. U. S. Department of Agriculture ARM-W-20, 1980; 12 pp. 15. Schroeder, H. W. HortScience 1976, 11, 53-54. 16. Schroeder, H. W. Archives de L'Institut Pasteur de Tunis 1977, 54, 479-485. 17. Halver, J. W. in "Aflatoxin"; Goldblatt, L. Α., Ed.; Academic Press: New York, 1969; Chap. 10, p. 265. 18. Wogan, G. N.; Palialunga, S.; Newberne, P. M. Fd Cosmet. Toxicol. 1974, 12, 681. 19. Stoloff, L. A. in "Mycotoxins in Human and Animal Health"; Rodricks, J. V.; Hesseltine, C. W.; Mehlman, Μ. Α.; Eds.; Pathotox Publ., Inc.: Park Forest South, Ill., 1977; p. 7. 20. Wales, J. H.; Sinnhuber, R. O.; Hendricks, J. D.; Nixon, J.E.; Eisel, T. A. J. Nat. Cancer Inst. 1978, 60, 1137. 21. Cole, Richard J. Public Health Lab. 1979, 37, 57-68. 22. Van Rensburg, S. J. in "Mycotoxins in Human and Animal Health"; Rodricks, J. V.; Hesseltine, C. W.; Mehlman, Μ. Α., Eds.; Pathotox Publ. Inc.: Park Forest South, Ill., 1977; p. 699. 23. Shank, R. C. in "Mycotoxic Fungi, Mycotoxins, Mycotoxicoses"; Wyllie, T. D.; Morehouse, L. G.; Eds.; Marcel Dekker, Inc.: New York, 1978; Vol. 3; p. 1. 24. "Aflatoxin and Other Mycotoxins: An Agriculture Perspective," Council Agric. Sci. Technol. (CAST) Report No. 80, 1979. RECEIVED May 13, 1983 Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.