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V V V . . . . . . V. TH E513 .v;\ 5". {I . ., ’ ~ _ \, SW3: $22.3 .2? 3.1? ’37; w .t 1 I. '3 . ' '~' 4. g 3 i g afliiitfifififly l K 3—3 This is to certify that the thesis entitled TISSUE DEPOSITION AND CLEARANCE OF AFLATOXINS FROM BROILER CHICKENS presented by CHEN, CHIHOUNG has been accepted towards fulfillment of the requirements for M ° S ' degree in MEIENCE Major professor Date ”orig! ‘3; /7X5 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. [)0 K00” USE ONLY 34%;“ W“ ’EVWJM'E'E Ui (iitfli‘ ‘ ii: TISSUE DEPOSITION AND CLEARANCE OF AFLATOXINS FROM BROILER CHICKENS by Chihoung Chen /%00pr A THESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1983 ABSTRACT TISSUE DEPOSITION AND CLEARANCE OF AFLATOXINS FROM BROILER CHICKENS by Chihoung Chen A trial was conducted to determine the tissue levels of aflatoxins and the amount of time necessary for tissue clearance from broiler chickens fed an aflatoxin—contaminated diet containing 2057 and 1323 ppb of aflatoxins B1 and 32' respectively, for 35 days. The aflatoxins caused depressed growth and enlarged kidneys, livers and gall bladders. Hemorrhagic spots were present on the surfaces of both the muscles and livers of the experimental birds. Livers of some of the experimental chicks were pale and infiltrated with lipid. Analysis of the tissues at 0 day after withdrawal from the aflatoxin-spiked ration showed that aflatoxins were deposited in all tissues, either as the original compounds or as their metabolites. The highest levels were present in the livers and kidneys. Mean values for the combined aflatoxins in all tissues at 0 day were less than 3 ppb. The withdrawal trial showed that broiler chickens rapidly metabolized and removed aflatoxins from their tissues. Four days after placing the chickens on an aflatoxin-free diet, there were no detect- able levels in any of the tissues. To my dear parents Mr. and Mrs. Pao-Shiang and Char-Shun Chen and to my lovely wife Yeong—Tsuey ii ACKNOWLEDGEMENT The author wishes to express his appreciation to his major professor, Dr. A. M. Pearson, for his invaluable support and helpful criticism in preparing this thesis. Special thanks are also given to Dr. T. H. Coleman for his assistance throughout the course of raising and slaughtering the chickens, and serving as a member of the guidence commitee.. The author would like to acknowledge the other members of his advisory commitee, Dr. J. I. Gray and Dr. S. D. Aust for their critical review of the manuscript. The author is also grateful to Dr. R. K. Ringer for his helpful discussions, and to Dr. D. Polin for his providing the formula of the broiler starter diet. Gratitude is also extended to Dr. L. E. Dawson and Dr. J. F. Price for allowing use of their laboratory facilites during this study. Thanks are also expressed to the staff of the MSU Poultry Research Center for their cooperation and assistance in aiding the author during the experimental trial. Lastly, the author is grately indebted to his wife, Yeong-Tsuey, for her continuous understanding, encouragement and helping throughout this study. iii TABLE LIST OF TABLES . . . . . LIST OF FIGURES. . . . . INTRODUCTION . . . . . . REVIEW OF LITERATURE . . OF CONTENTS Discovery of Aflatoxins . . Metabolism of Aflatoxins. . Toxicity of Aflatoxins. . . Chicken Aflatoxicosis . Occurrence of aflatoxins in of Animal Origin. . . EXPERIMENTAL . . . . . . Feeding Trial . . . . Preparation of the diet. Experimental animals . . Slaughtering and collection of samples , . . . Analysis of Aflatoxins. . . Sample preparation Purification of the aflatoxin extract. . . . . . Thin layer chromatography. . . Densitometric analysis of aflatoxins . . . . Analysis of Aflatoxin in the Feed iv 32 43 43 43 45 46 47 w 48 50 52 53 Page Confirmatory Tests for Aflatoxins. . . . . . . 5U Aflatoxin B1. . . . . . . . . . .'. . . . . 5U Aflatoxin M1. . . . . . . . . . . . . . . . 55 General confirmatory test for aflatoxins. . . . . . . . . . . . . . . . . 56 Preparation of Aflatoxin Reference Standards. . . . . . . . . . . . . . . . . . . 56 Fat and Moisture Analysis. . . . . . . . . . . 5? Moisture content. . . . . . . . . . . . . . 57 Fat content . . . . . . . . . . . . . . . . 57 Statistical Analysis . . . . . . . . . . . . 58 Safety Procedures. . . . . . . . . . . . . . . 58 RESULTS AND DISCUSSION. . . . . . . . . . . . . . 60 Feeding Trial. . . . . . . . . . . . . . . . . 60 Gross Observation in the Tissues . . . . . . . 63 Aflatoxin Residues in the Tissues of Chicks Fed a Contaminated Diet. . . . . . . 7O Withdrawal Trial . . . . . . . . . . . . . . . 77 SUMMARYANDCONCLUSIONS............. '86 LIST OF REFERENCES. . . . . . . . . . . . . . . . 88 LIST OF TABLES TABLE Page 1. Ratios of aflatoxin B1 levels in the feed in relation to aflatoxin B1 or M1 levels in edible tissues . . . . . . . . . 41 2. Chicks, broiler starter diet (formula CBS 711). O O I O O I O O O O I O O I I O O O 41+ 3 Performance data showing body weights, body weight gains, average feed intake and average feed efficiency for the 5—week feeding trial. . . . . . . . . . . . . 61 4 Average aflatoxin consumption and daily dosage ratio of the experi— mental group. . . . . . . . . . . . . . . . . 62 5 Summary of the gross lesions observed upon inspection . . . . . . . . . . . . . . . 64 6 Fat and moisture content of livers for both the control and the experimental groups at 0 day after withdrawal from aflatoxin-spiked ration . . . . . . . . . . . 66 7 Internal organ weights in grams of both the control and the experimental groups (0 day and 16 days after withdrawal from aflatoxin-spiked ration). . . . . . . . . . . 67 8 Summary of organ weights expressed as a percentage of live body weight for both the control and the experimental groups (0 day and 16 days after withdrawal from the aflatoxin-spiked ration). . . . . . . . . 68 9 Aflatoxin residues detected in selected chick tissues at 0 day after withdrawal from the aflatoxin-spiked ration tug/kg). . . 71 10 The total amounts of aflatoxins B1 and B2, M1 and M2, and B1, B2, M1 and M2 in the tissues of chicks slaughtered 0 day after withdrawal from the aflatoxin- Spiked ration I O I O I I O O ' I I O I I 0 0 0 72 vi TABLE 11 12 13 14 15 The ratio of aflatoxin B1 to B2 (Bl/B2) in selected chick tissues at 0 day after withdrawal from the aflatoxin-spiked ration . . . . . Ratios of aflatoxins B1 or B2 levels in the feed in relation to aflatoxins M1 or M2 levels in livers and kidneys . . . . . . Aflatoxin residues detected in selected chick tissues at 1 day after withdrawal from the afla- toxin-spiked ration Lug/kg) . . . Aflatoxin residues detected in selected chick tissues at 2 days after withdrawal from the afla- toxin-spiked ration (Hg/kg) . . . Aflatoxin residues detected in selected chick tissues at 4 days after withdrawal from the afla- toxin-Spiked ration (ug/kg) . . . vii Page 73 75 78 80 82. LIST OF FIGURES FIGURE Page 1 Chemical structures of the four principal aflatoxins produced by mold . . . . . . . . . . . . . . 6 2 Structures of the minor mold- derived aflatoxins . . . . . . . . . . . . . 8 3 Metabolism of aflatoxin B1 . . . . . . . . . 15 Metabolism of aflatoxin B2 . . . . . . . . . 16 5 Spotting and scoring pattern for two-dimensional 20 x 20 cm TLC plates I I I I I I I I I I I I I I I I I 51 viii INTRODUCTION Aflatoxins, which are the most significant and hazardous of the mycotoxins of economic and public health cdncern, are a group of secondary metabolites produced by certain strains of As er illi, principally, A. flavus and A, parasiticus. Aflatoxins have been found to oCcur in many agricultural products, particularly those held at high tempe- ratures and high relative humidities. Thus, they become un- avoidable natural contaminants in animal feeds, as well as in some foods used for human consumption. Aflatoxins have been shown to be toxicogenic, carci- nogenic, mutagenic and teratogenic. Epidemiological studies (Shank 23 al., 1972; Peers and Linsell, 1973; Wilson, 1978) have indicated that aflatoxins possess acute toxicity in addition to being hepatocarcinogens to humans. Biologically, the effects of aflatoxins may vary with dose, duration of exposure, species, sex, age and the nutritional status of the animal affected. I vivo, aflatoxins, like many other xenobiotics, may be biotransformed to increase their polarity and their ex- cretability. During the biotransformation process, some of the metabolites of aflatoxins may cause acute or chronic toxicity. There is evidence that the residues of aflatoxins and their biotransformed metabolites that are found in animal tissueslmay become an indirect source of aflatoxin contami- nation in human foods. The present study was designed to investigate: (i) the levels of aflatoxins and their metabolites deposited in the tissues of broiler chickens, and (ii) the length of time required to obtain tissue clearance after removal of aflatoxins from the diet of broiler chickens. REVIEW OF LITERATURE DISCOVERY OF AFLATOXINS Aflatoxins are a group of secondary metabolites produced mostly by certain strains of Aspergillus flavus and Aspergillus parasiticus (Wilson gt,§;., 1968). Raper and Fennell (1965) classified the genus Aspergillus into 18 groups with both Aspergillus flavus and Aspergillus parasiticus being placed into the A. flavus group. Members of this group are broadly identified by the production of yellow-green conidia. A. flavus and A. parasiticus can be. differentiated from each other primarily by the color of their colonies, and the morphology of their conidial struc- tures. The sterigmata of A. flavus are typically biserrate, whereas, those of A. parasiticus are uniserrate (Raper and Fennell, 1965). The recognition in the United Kingdom of an unusual disease of turkeys characterized by striking hepatic lesions was announced by Blount (1961). This condition, which was called "Turkey X" disease, accounted for the loss of more than 100,000 birds. At the same time, a similar incidence occurred in a farm in East Anglia, where over 14,000 ducklings died during a 4 to 5 week period (Asplin and Carnaghan, 1961). A similar condition was soon discovered in pigs (Loosmore and Harding, 1961) and calves (Loosmore and Markson, 1961). Investigations showed that the disease 3 Lt in all these species was caused by a toxic factor contained in imported Brazilian groundnut meal (Blount, 1961; Asplin and Carnaghan, 1961; Loosmore and Harding, 1961; Loosmore and Markson, 1961). Asplin and Carnaghan (1961) fed chickens and ducklings with Brazilian groundnut meal and found that ducklings are highly susceptible to this toxic meal. Sargeant gt él- (1961b) used paper chromatography to separate a crude extract of Brazilian groundnut meal with a mixture of n-butanol and 5 per cent acetic acid as the developing solvent. They found a blue fluorescing spot under UV light with a Rf of 0.7. Later it was shown that ground- nut meal other than that from Brazil contained this component (Wannop, 1961). Tests on groundnuts from Kenya (Carnaghan and Sargeant, 1961), Uganda (Sargeant a: gi., 19610). West Africa (Sargeant gt a;., 1961a) and India (Hornby gt a;., 1962) showed the presence of a toxic component in all samples. Sargeant §t_§;. (19610) isolated a fungus from toxic Ugandan groundnuts and identified it as being A. flavus Link ex Fries. They extracted this fungus with chloroform, and the extract gave positive tests for the toxin when fed to ducklings (Sargeant gt g;., 1961b). In 1962, a British "Interdepartmental Working Party on Groundnut Toxicity Research" proposed the trivial name aflatoxin (A. flavus toxin). Nesbitt gt a;. (1962) showed that the single blue fluorescing spot of the toxin-containing extracts observed by Sargeant gt gi. (1961b) could be split into two components by using alumina chromatoplates developed in a chloroform-methanol (98.5:1.5) 5 mixture. Hartley gt g;. (1963) further separated them into four components by using silica gel G plates developed in a mixture of a chloroform-methanol (98:2). Thus, they discover- ed the four major natural occurring aflatoxins, B1, B2, G1 and G The specific designation was due to their blue (B) and 2. green (G) fluorescence under UV light (Nesbitt gt g;., 1962; Hartley gt_g;., 1963). Their chemical structures are shown in Fig. 1. Allcroft and Carnaghan (1963) reported that extracts of milk from cows fed with toxic groundnut meal contained a toxic substance, which was capable of producing the typical aflatoxin-poisoning in day-old ducklings. DeIongh gt_g;. (1964) found that the toxic substance exhibited blue-violet fluorescence under UV light on a silica gel plate developed with chloroform-methanol (97:3), and had a R value well below f that of aflatoxin B1. The toxic substance was also found in the extract from the dried A. flavus culture grown on crushed groundnuts. At the same time, they fed lactating rats with pure aflatoxin B1, and the rats produced the same "milk toxin" in their milk. Therefore, they concluded that the milk toxin is a biotransformation product instead of coming through direct ingestion (DeIongh gt g;., 1964). Later, Butler and Clifford (1965) found the so called "milk toxin" in the livers of rats given pure aflatoxin B1, either orally or intraperitoneally. Allcroft gt g;. (1966) also isolated the milk toxin from the urine of sheep fed aflatoxin B , and confirmed its chromatographic equivalence. 1 They further proposed the trivial name aflatoxin M for the O 0 ° l AFLATOXIN Bl AFLATOXIN B2 AFLATOXIN G AFLATOXIN G2 Figure 1 -- Chemical structures of the four principal aflatoxins produced by mold. 7 toxin found in milk. Holzapfel gtlgi. (1966) repeated the experiment of Allcroft g:,gl. (1966) and isolated aflatoxin M from the urine of aflatoxin-dosed sheep. They than separat- ed it into two compounds by using paper chromatography. The blue-violet component was named aflatoxin M1, and the violet spot was named aflatoxin M2. Two additional forms of aflatoxin were isolated by Dutton and Heathcote (1966) from cultures of A. flavus. They concluded that the two new aflatoxins were hydroxy- derivatives of aflatoxins B2 and G2, and they named them aflatoxin B28. and G2a’ respectively. The chemical structures of the four minor natually occurring aflatoxins, M1, M2, B2 a and G2a are shown in Fig. 2. METABOLISM OF AFLATOXINS Man and animals are exposed to a great variety of organic chemicals from the environment (Maugh, 1978) which cannot be used for energy production or as building blocks for their own body constituents. These foreign compounds are referred to as xenobiotics (Reeves, 1981). Once these com- pounds have gained access to the body they tend to persist as they distribute themselves in the fat and other tissues, and are difficult to excrete (Klaassen, 1980). They may interact with biologically important molecules and can give rise in this way to a number of pharmacological or toxico- logical reSponses (Reeves, 1981). Nutrients and xenobiotics may be metabolized by identical or analogous routes. While the purpose of nutrient AFLATOXIN M AFLATOXIN M2 AFLATOXIN B2a AFLATOXIN G2a Figure 2 -- Structures of the minor mold-derived aflatoxins. 9 metabolism is the release of caloric energy and incorporation of essential components into vital structures, the purpose of metabolism of xenobiotics is the reduction of their toxicity and/hr the facilitation of their excretion (Reeves, 1981). However, the use of the same metabolic pathway may increase toxicity instead of resulting in detoxification (Hayes, 1975). For this reason, Hayes (1975) referred to all of these meta- bolic reactions as biotransformations instead of by the popular synonym for foreign compound metabolism, detoxication. A number of enzymes in animal organisms are capable of metabolizing lipid-soluble xenobiotics so as to render them more water soluble, and thus make them easier to excrete (Hayes, 1975; Reeves, 1981). Generally, biotransformation of xenobiotics makes them more easily excreted by increasing their polarity and ionization (Reeves, 1981). Williams (1959) proposed that these enzymatic reactions are two types: phase I reactions, whcih involve oxidation, reduction and hydrolysis; and phase II reactions, which consist of conjugation. Phase I reactions generally convert foreign compounds to derivatives, which can then undergo phase II reactions. Microsomal mixed-function oxidases (MFO) are perhaps the most important enzymes involved in phase I reactions. These enzymes are localized in the endoplasmic reticulum, a complex network of membranes within the cell that is continuous with the outer nuclear membrane (Neal, 1980). When a cell is homogenized, the endoplasmic reticulum is degraded to form small vesicles known as microsomes (Palade and Siekevitz, 1956). The enzyme systems, which are sometimes referred to 10 as cytochrome P-450-containing mono-oxygenases, are composed of two enzymes, NADPH-cytochrome P-U5O reductase, and a heme-containing enzyme, cytochrome P-450 (White and Coon, 1980). Cytochrome P-450 was first demonstrated inde- pendently by Klingenberg (1958) and by Garfinkel (1958), and shown to be a carbon monoxide binding pigment, which was located in the microsomal fraction of mammalian liver cells. Its name was derived from the soret peak at 450 nm, which occurs when it is reduced to form complexes with carbon monoxide (Neal, 1980). The general types of reactions catalyzed by cyto- chrome P-45O containing monooxygenases include aromatic hydroxylation, aliphatic hydroxylation, epoxidation, desulfu- ration, sulfoxidation, N-hydroxylation and N-, O- or S- dealkylation (Neal, 1980). The microsomal system contains mutiple forms of cytochrome P-450 (Hodgson, 1979). This system is found predominately in liver. In addition to liver, the cytochrome P-450 dependent activity has been found in vertebrate skin, sebaceous glands, the alveolar epithelium of lung, the intestines, the placenta and the kidney (Ahmad, 1979). Aflatoxin B1 is metabolized by the hepatic micro- somal mixed function oxygenase system to a group of deriva- tives, such as aflatoxin M1, Q1, P1, B2 and aflatoxin B1- a epoxide (Campbell and Hayes, 1976). Aflatoxin B1 can also be reduced by a cytoplasmic reductase to aflatoxicol (Wong and Hsieh, 1978). 11_ In addition to its natural occurrence as a mold metabolite (DeIongh g; g;,, 1964), aflatoxin M1 is also a mixed-function oxidases mediated liver biotransformation product of aflatoxin B1 (Schabort and Steyn, 1969). In the presence of fortified NADPH, crude and isolated micro- somal preparations from liver of many species have been found to transform aflatoxin B1 to the 4—hydroxy derivative, afla— toxin Ml’ which is shown in Fig. 3-pathway 1 (Schabort and Steyn, 1969). Hsieh gt g;. (1976) examined extracts of urine derived from rhesus monkeys, which had been previously dosed with aflatoxin B . They concluded that aflatoxin M 1 1 predominant oxidative metabolite in this species of primate. was the Aflatoxin M2 is a liver metabolite of aflatoxin B2 (Fig. 4-pathway 3) and is, presumably, produced by the same liver fractions that convert aflatoxin B1 to M1 (Roebuck gt a1” 1978). Aflatoxicol (aflatoxin R0) was independently isolat- ed as the degradation product of aflatoxin B1 by various microorganisms by Teunisson and Robertson (1967) and by Detroy and Hesseltine (1968). Teunisson and Robertson (1967) found that a culture of Tetrahymena phriformis W degraded aflatoxin B1 to a bright blue-fluorescing compound with a lower Rf value than B1 on TLC plates. Detroy and Hesseltine (1968) observed that aflatoxin B1 was transformed to a blue- fluorescing hydroxylated compound by Dactylium dendroides. They then named it aflatoxin R0. Later Detroy and Hesseltine (1970) proposed the name aflatoxicol instead of aflatoxin R0. Aflatoxicol in animal tissues was not reported until 12 Patterson and Roberts (1971) produced evidence that aflatoxi- col was a biotransformation product of aflatoxin B1. They found that soluble fractions of liver from chicken, duck, turkey and rabbit were capable of reducing the carbonyl group of the terminal cyclopentenone ring of aflatoxin B1 and B2 to the corresponding aflatoxicol (Fig. 3-pathway 2) and dihydro- ‘ aflatoxicol (Fig. 4-pathway 1). Unlike other metabolites, aflatoxicol biotransformation is catalyzed by a NADP-linked dehydrogenase of the cytosol, which is also known as 17- ketosteroid dehydrogenase (Patterson and Roberts, 1971; 1972). The biotransformation of aflatoxin B1 to aflatoxicol has been shown to be reversible depending on the NADPHZ/NADP ratio in the tissue (Patterson and Roberts, 1972).‘ Aflatoxicol M is a biotransformation product that 1 can be formed either from aflatoxin M1 (Fig. 3-pathway 3) or from aflatoxicol (Fig. 3-pathway 4). The cyclopentenone carbonyl of aflatoxin M1 can undergo reduction by a cytosolic enzyme system, as described for aflatoxicol, to yield afla- toxicol M1 (Salhab gt g;., 1977). Aflatoxicol M1 can also be formed directly by oxidizing aflatoxicol by a mixed function oxidase system (Salhab gt g;., 1977). Aflatoxicol Ml can be reoxidized by liver microsomal fractions to yield aflatoxin M By analogy to the conversion of aflatoxicol to aflatoxin 1I B1, the reoxidation reaction is probably not mediated by the cytochrome P-450 system (Salhab and Edwards, 1977). Aflatoxin P1 is a biotransformation product of 13 aflatoxin B1 resulting from O-demethylation as shown in Fig. 3-pathway 5 (Shank and Wogan, 1965). Shank and Wogan (1965) prepared ring—labeled and O-methyl-labeled aflatoxin B1 by submerging cultures of A. flavus in liquid media containing 1 either E 1- “C J-acetate or methyl-luC-L—methionine. They then administered the labeled toxin orally to Fisher rats. 140 dose was excreted Some 60 per cent of the administered into the feces through the bile. About 20 per cent was excreted into the urine. A further 27 per cent of O—methyl- labeled 140 appeared in the respiratory 002' These results indicated that O-demethylation constitutes a significant metabolic pathway for excretion of aflatoxin B1 by rats. Aflatoxin P1 was subsequently identified as the glucuronide or sulfate conjugate in the urine of monkeys given aflatoxin B1 (Dalezios and Wogan, 1972). Biotransformation of aflatoxin B to P appeared to be a mixed function oxidases dependent 1 1 product of tg_1tttg liver metabolism (Bassir and Emafo, 1970). Aflatoxin Q1 is the product of mixed-function oxidase hydroxylation of aflatoxin B1 at the B-position to the cyclopentenone carbonyl as demonstrated in Fig. 3-pathway. 6 (Masri gt g;., 1974a). Aflatoxin Q1 represents the major metabolite produced from aflatoxin B1 by microsomes of monkey and human liver ta ttttg (Buchi gt g;., 1974; Masri gt gt., 1974a; 1974b). The hydroxylation appears to be carried out by an enzyme system that is different from the one producing aflatoxin M1 (Krieger gt g;., 1975). Salhab and Hsieh (1975) demonstrated that human and monkey liver preparations modified aflatoxin B1 to form a 14 derivative with the ketone carbonyl on the cyclopentenone ring being reduced to a secondary alcohol, and a hydroxyl group introduced onto the B-carbon of the alcohol group (Fig. 3-pathway 7). This newly identified metabolite was named aflatoxicol H1 (Salhab and Hsieh, 1975; Hsieh gt g;., 1977; Salhab and Edwards, 1977). Aflatoxicol H1 may also arise by a direct hydroxylation of aflatoxicol (Fig. 3-path- way 8). Because of the rapid conversion of aflatoxicol to aflatoxin B1, and aflatoxin B1 to aflatoxin Q1 in liver homogenates, it is not clear whether direct hydroxylation of aflatoxicol occurs at an appreciable rate (Salhab and Edwards, 1977). Patterson and Allcroft (1970) suggested that afla- toxin B1 and M1 were reduced by chicken, duckling, guinea pig and mouse liver preparations in vitro to their corresponding reduction products, aflatoxin B2 and M2, with either NADP or NADPH being required as the cofactors (Fig. 4-pathway 2). The active mutagenic form of aflatoxin B1 has been proposed to be the 2,3-epoxide, which is generated metabo- lically from the 2,3-vinyl ether double bond in the terminal furan ring as illustrated in Fig. 3-pathway 9 (Schoental, 1970). Although this labile molecular species has not been isolated, microsomal enzyme activation is an absolute requirement for mutagenicity of aflatoxin B1 (Wong and Hsieh, 1976). Support for the existence of the 2,3-epoxide came from the isolation of 2,3-dihydro-2,3-dihydroxy-aflatoxin B1, a postulated diol detoxification product. The latter was isolated from acid hydrolysates of covalently linked complexes 15 Aflatoxicol M1 Aflatoxicol Aflatoxicol H1 Aflatoxin Q1 5 . Aflatoxin Bl- Aflatoxin P 2.3-epoxide \\\\\\\\\\\ 1 Aflatoxin B 2a 2,3-Dihydro- 2,3-dihydroxy- aflatoxin B1 PROTEIN PROTEIN DNA, RNA t J, l SCHIFF BASE SCHIFF BASE NUCLEIC ACID ADDUCT Figure 3 - Metabolism of aflatoxin B1 2,3-DIHYDRO AFLATOXICOL 16 3 O O I... 0 I. 0 AOL. AFLATOXIN M H OC 3 2 Figure 4 - Metabolism HO'LO’LO O OCH3 AFLATOXIN B2a of aflatoxin B2 17 of aflatoxin B1 and nucleic acids generated ta vitro and in vivo (Swenson gt g;,, 1973; 1974; Neal et g;., 1981). Aflatoxin B2a was first reported as a natUral metabolite in cultures of A. flavus by Dutton and Heathcote (1966; 1968). Using ia.xitre assays of aflatoxin-metabo- lizing activies of different species, Patterson and Roberts (1972) found that the NADPH dependent microsomal enzyme hydrates the vinyl ether at the double bond of aflatoxin B1 to form its 2-hydroxyl derivative, aflatoxin B2a (Fig 3.-- pathway 11). Neal gt g;. (1981) pointed out that previous reports of the production of aflatoxin B2a (Patterson and Roberts, 1972; Gurtoo and Dahms, 1974; Gurtoo and Campbell, 1974) were erroneous. The latter authors identified 2,3- dihydro-Z,3-dihydroxy-aflatoxin B1 as B2a due to the simi- larity of their UV spectral characteristics (Swenson gt g;., 1973; 1977). Thus, there is doubt whether aflatoxin B2a is a significant microsomal metabolite of aflatoxin B1. Aflatoxin B28. was not readily produced from afla- toxin B2 on incubation with rat liver microsomes (Roebuck gt_g;., 1978). However, it does appear to be a major urinary metabolite of aflatoxin B2 in rats, accounting for about 8 per cent of the injected dose (Fig. 4--pathway 4) according to Dann gt a. (1972). TOXICITY OF AFLATOXINS Results from many experiments showed that there are a great many factors that can effect the toxicity of aflatoxins. 18 These include the type of aflatoxin to which one is exposed (Wong and Hsieh, 1976), the dose (Wogan and Newberne, 1967), duration of exposure (Newberne and Butler, 1969), species (Hsieh gt_g;., 1977), age (Newberne and Butler, 1969; Sullman gt g;., 1970), sex (Purchase gt g;., 1973), nutritional status (Hamilton, 1977), pre-xenobiotic exposure (Wong gt_g;,, 1981), environmental factors (Wyatt gt g;., 1977) and the route of administration (Butler, 1964). Aflatoxins B1, B2, C1 and-G2 are the four predomi- nant natural aflatoxins that have been tested for relative toxicity in human embryonic liver cells. Their biological activity was found to decrease in the order of aflatoxin B1 > G1 > G2 > 32 (Zukerman gt g;., 1968; Sullman gt g;., 1970). However, when the toxic effects were tested on day- old ducklings (Carnaghan gt g;., 1963) or chicken embryos \Vesely gt_gt., 1982) and the mutagenetic potency on Salmonella typhimurium (Wong and Hsieh, 1976), the biological effects were decreased in the order of aflatoxin B1 > G1 > B2 > G2. Wong and Hsieh (1976) have studied the relative mutagenicity of aflatoxins and their animal biotransformation products using the Ames test (Ames gt g;., 1973). Wong and Hsieh (1976) found that all isolated biotransformation products of aflatoxin B1 had a decrease in mutagenicity. Aflatoxin M1 had only about 3 per cent of the potency of aflatoxin B1. Wong and Hsieh (1976) also reported that the relative mutagenicity tg_vitro was closely correlated with 1 vivo carcinogenicity data. 19 The potency of aflatoxin M1 in inducing liver tumors in rainbow trout was one-third of that of aflatoxin B1 at a level of 4 ppb in the diet (Sinnhuber et elm 1970). This is in contrast to its ability to cause acute toxicity as demons- trated by Holzapfel gt gt. (1966) and Purchase (1967). They reported that the LD of aflatoxin M1 in ducklings was 50 similar to that of B1, and that similar liver lesions were produced by the two toxins. Recently, Green gt gt. (1982) compared the cyto- toxicity and genotoxicity of aflatoxin B1 and M1 in primary cultures of adult rat hepatocytes. Aflatoxin B1 stimulated the release of lactate dehydrogenase into the culture mediuum, and the loss of viable cells from the monolayer at a lower dose than aflatoxin M1. Genotoxicity, which is determined by an assay for stimulation of DNA repair, demonstrated that aflatoxin B1 is again more potent than aflatoxin M1. Although aflatoxin M1 is less potent than aflatoxin B1 in cytotoxicity and genotoxicity assays using primary cultures of hepatocytes. Green gt gt. (1982)pointed out that aflatoxin M1 is still active at relatively low doses. Thus, they concluded that it is probably a potent hepatotoxin i vivo. The earliest evidence of the carcinogenetic properties of aflatoxins was provided by the experiments of Lancaster gt gt. (1961), who fed rats a diet containing 20 per cent peanut meal that had earlier been identified as a cause of poisoning in poultry flocks. The rats did not show the signs of acute toxicity seen in turkey poults. After a 6 month feeding period, 9 out of 11 rats developed multiple 20 liver tumors and 2 of them had lung tumors. The presence of liver tumors in the absence of cirrhosis, cell necrosis or cellular infiltration suggested that the toxic agent acts directly on the hepatic cells. Schoental (1970) first postulated that the formation of an epoxide intermediate of aflatoxin B1 at its 2,3-double bond might account for its toxicity. Wong and Hsieh (1976) using the Salmonella typhimurium mutant assay (Ames gt g;., 1973) demonstrated that none of the aflatoxins, including the biotransformation metabolites, possessed any mutagenic potency in the absence of an activation factor isolated from rat liver preparations. They concluded that none of these aflatoxins or their biotransformation products are the ultimate mutagenic and/or carcinogenic species. Therefore, Wong and Hsieh (1976) concluded that the 2,3-double bond is involved in both the mutagenetic and carcinogenetic activity of aflatoxins. Garner and Hanson (1971), and Garner gt_g;. (1972) demonstrated that incubation of rat hepatic microsomes with an aflatoxin and a NADPH-generating system produced a metabolite of aflatoxin B1 that was lethal to certain bacteria. None of the aflatoxins were active in the absence of mixed-function oxidases of liver microsomes, and the bacterial toxicity was reduced on the addition of either DNA or RNA. This suggested that the same metabolite was reacting with nucleic acid (Garner e_t. al., 1972). The idea that DNA is probably the critical target of the aflatoxin metabolite was supported by its lethal effect on certain strains of E; coli, which are very sensitive 21 to UV light. They apparently lack the gene product necessary for the repair of DNA that has been damaged by light or other mutagens (Garner and Wright, 1973). Further experimental support for the view that 2,3- epoxide might be an important metabolite of aflatoxin B1 is found in the work of Swenson gt gt. (1973; 1974; 1975). They isolated an 12.11122 product indistinguishable from synthetic 2,3-dihydro-2,3-dihydroxy-aflatoxin B1 from liver microsomes in the presence of RNA. They injected E 3H J-aflatoxin B1 into rats and isolated covalently bound derivatives in hepatic DNA, rRNA and protein. Mild acid hydrolysis of the DNA and rRNA adducts formed a derivative indistinguishable from 2,3-dihydro-2,3-dihydroxy-aflatoxin B1. These observations strongly support the concept that aflatoxin B1 is biotrnas- formed into aflatoxin-2,3-epoxide, which binds covalently through its high electrophilic 0-2 to nucleophilic sites in the DNA and rRNA. Swenson gt gt. (1975) synthesized aflatoxin B1- 2,3-dichloride as a model of the epoxide. The dichloride would be expected to form a resonance stabilized carbonium ion at carbon 2. These workers showed that the dichloride is highly carcinogenic and mutagenic, and reacts tg41ttgg with nucleophiles in the manner expected for the 2,3-epoxide. The compound formed covalent adducts with DNA and RNA, with retention of one half to the chlorine. Polyguanylic acid was the most reactive homopolymer tested towards aflatoxin B1- 2,3-dichloride. This was confirmed by Croy gt gt. (1978), who isolated the 2,3-dihydro-2(7N-guanyl)-3-hydroxy-aflatoxin B1 22 as the principal product after hydrolysis of liver DNA from rats dosed with aflatoxin B1. Wong and Hsieh (1976) had suggested that the 2,3- vinyl ether double bond is not the sole molecular site on the aflatoxin B molecule that determines mutagenic activity. 1 They found that alterations of the cyclopentenone ring, such as its substitution by a terminal lactone ring as in aflatoxin G1, or by 7-hydroxylation as in aflatoxin Q1 or by reduction of the keto group as in aflatoxicol, result in a significant lowering of mutagenic potential, despite the presence of an intact 2,3-double bond. Wong and Hsieh (1978) also suggested that both ifl.XlE£2 and tg tttg formation of aflatoxicol may be an indicator of species sensitivity to aflatoxin induced carci- nogenesis. They found that aflatoxicol was the major metabo- lite in the plasma of Sprague-Dawley rats, dosed orally or intravenously with 1LAC-aflatoxin B1. However, aflatoxicol was not detected in the plasma of similarly dosed mice and monkeys, which are resistant to aflatoxin Bl-induced carcino- genesis. Furthermore, the biotransformation of aflatoxin B1 to aflatoxicol has been shown to be reversible (Patterson and Roberts, 1972; Salhab and Edwards, 1977). The reversibility has been suggested to function as a reservoir for aflatoxin B1 and its metabolites, which prolonges the cellular exposure to the carcinogens, and hence enhances their carcinogenic effects (Hsieh gt gt., 1977). It has been proposed that aflatoxin B1 is converted to aflatoxicol, which is slowly reconverted to aflatoxin B and thus, has the same effect as 1 ’ 23 chronic administration of aflatoxin B1 (Swenson, 1981). Conversion of aflatoxin B2 to aflatoxin B1 (Fig. 4- pathway 2) has been demonstrated directly in duck liver homo- genates (Roebuck gt_gt., 1978) and indirectly in rat liver tg_yttg (Swenson et gt., 1977). It was estimated by Swenson gt gt. (1977) that about 1 per cent of the administered aflatoxin B2 is converted to aflatoxin B1. This agrees with the relative carcinogenic potencies of these toxins (Wogan eta” 1971). At physiological pH, both aflatoxin B28. and 2,3- dihydro-2,3—dihydroxy-aflatoxin B1 may bind to proteins (Patterson and Roberts, 1972; Gurtoo and Campbell, 1974; Ashoor and Chu, 1975; Neal gtgt.,‘1981).‘~ According to Patterson (1977), these two aflatoxins may also bind to various key enzymes and metabolically important liver cell structures, and thus, may be responsible for the acute toxicity of aflatoxin B1. CHICKEN AFLATOXICOSIS Aflatoxicosis is the disease produced when animals ingest aflatoxins, and was first discovered in poultry (Blount, 1961). Asplin and Carnaghan (1961) showed that chickens were more resistant than ducklings to the effects of aflatoxins. Muller gt gt. (1970) used four different levels of aflatoxins B1 and G1, which were equivalent to 500, 1,000, 2,000 and 4,000 ppb of aflatoxin B in the diet, 1 and compared them to a control diet. The diets were fed to chicks, ducklings, goslings, pheasants and turkey poults 24 for four weeks. Results showed that the order of sensitivity from greatest to least was ducklings, turkey poults, goslings, young pheasants and chickens. Arafa gt gt. (1981) determined the relative sensiti- vity of broiler chickens, White Leghorn hens, quail, goslings and turkey poults to increasing dietary levels of aflatoxins from 1 to 21 days of age. Aflatoxins were included in the diet at three concentrations (700, 1,400 and 2,100 ppb of aflatoxin B1 equivalents). Turkey poults and goslings were the most sensitive and quail were intermediate. Broiler chickens and White Leghorn hens were the most tolerant to aflatoxins. These findings agree with the earlier work of Muller gt gt. (1970) and Gumbmann gt gt. (1970) suggesting that different poultry species show varying degrees of tolerace to aflatoxins. In a comparative study, Florentin gt gt. (1969) fed Brahmas, Barred Plymouth Rocks, White Plymouth Rocks, Langshans, Rhode Island Reds, New Hampshires, Single Comb White Leghorns, Delaware x New Hampshires and Cornish x White Plymouth Rock chicks on diets containing 380, 550, 650, 660 and 1,300 ppb of aflatoxin B1 for eight weeks. In compa- rison to the controls, all test groups showed depressed growth, higher mortality, enlarged livers and hearts and an increased fat content in the liver. If the growth and morta- lity effects are considered, the Brahmas were the most susceptible breed and the White Plymouth Rocks the most resis- tant. Mortality rates for New Hampshires and Rhode Island Reds were similar, but the Rhode Island Reds exhibited more growth 25 depression. Performance of the broiler cross chicks was similar to that obtained with White Plymouth Rocks. Smith and Hamilton (1970) determined the sensitivity of 6 different strains of unsexed White Leghorn chicks to aflatoxins by finding the LD5O dose. Results showed that the acute LD varied from 6,500 to 16,500 ppb. Results indicated 50 that there is a considerable variation between different strains of chickens in regard to their susceptibility to aflatoxins. Gumbmann gt gt. (1970) fed an experimental diet with' aflatoxins equivalent to 800 ppb of pure aflatoxin B1 to 18 different strains of cross-bred chickens, turkeys and quail for a period of 2 to 6 weeks. They found that New Hampshire chicks and turkey poults were most adversely affected by aflatoxins. When New Hampshire hens were crossed with Leghorn males or vice versa, the.sensitivity of the chicks to afla— toxins was no longer detectable. They suggested that the susceptibility to aflatoxins is a genetically controlled factor. Asplin and Carnaghan (1961) fed Rhode Island Red chickens a diet containing aflatoxin-contaminated peanut meal. The main effect of feeding the aflatoxins was growth retardation. The gross lesions observable showed that young chickens are more susceptible to the toxin than the older ones. In the chickens dying during the first 3 weeks of the feeding experiment, the livers were pale in color but firm in texture, while the kidneys were pale and slightly enlarged. In the birds which were killed during the following 3 months, 26 the liver and kidney changes were similar but more marked. In those chickens killed during the latter phases of the feeding trial, the organs were normal in texture and color, but discrete white pin point lesions were consistently found in the livers. Smith and Hamilton (1970) also determined the effects of age on the sensitivity of chickens to aflatoxins by finding the LD The birds they used were broiler chickens (Arbor 50' Acre 60 x Peterson). In contrast to the findings of ASplin and Carnaghan (1961), day—old broiler chicks were about as sensitive as birds ready for processing when the sensitivity to aflatoxins was measured in terms of acute LD50' However, when sensitivity was measured in terms of growth rate, young chickens were more sensitive. The growth of day-old chicks was depressed more by aflatoxins than that of 10—day old chicks. Carnaghan gt gt. (1966) fed aflatoxin B1 at the rate of 1,500 ppb to Rhode Island Red chickens. They noted that the livers were enlarged relative to body weight, which was lower than normal. The fat content of the liver was higher than normal. Microscopic changes included periportal fatty infiltration, cell necrosis, progressive biliary proliferation and an increase in connective tissue. Within 3.5 days, there was slight proliferation of the bile duct epithelial cells, and the amount of proliferation became prominent within two through six weeks. A similar pattern of bile duct prolifera- tion was been reported for ducklings (Butler, 1964), turkeys (Wannop, 1961; Siller and Ostler, 1961) and swine (Loosmore and Harding, 1961). 27 The first case of aflatoxicosis in chickens was recognized by Asplin and Carnaghan (1961), who observed that about 30 per cent of a group of 100 Rhode Island Reds were stunted at 2 weeks of age. It was later discovered that the commercial mash being fed contained 6.25 per cent of Brazilian groundnut meal. A number of field cases were also diagnosed in the United Kingdom (Asplin and Carnaghan, 1961), and out- breaks were later reported in Canada (Archibald gt gt., 1962), Australia (Gardiner and Oldroyd, 1965; Hart, 1965) and the United States (Hamilton, 1971). Mortality reached 50 per cent in a flock of laying hens which died within 48 hours after being fed highly toxic maize containing 83,000 and 101,000 ppb of aflatoxin B1. According to Hamilton (1971), a 90 per cent drop in egg production simultaneously occurred. Broiler type chickens are considered to be relatively resistant to aflatoxin toxicity (Archibald gt gt., 1962; Arafa gt gt., 1981). In broiler chickens, aflatoxins limit both the performance and health of the birds. There is an increase in susceptibility to bruising (Tung gt gt., 1971b), impairment of immunity (Thaxton gt gt., 1974), impairment of coagulation (Doerr gt gt., 1976), a decrease in pigmentation (Tung and Hamilton, 1973), an interaction with vitamin deficiencies (Hamilton, 1977). a decrease in bone strength (Huff gt gt., 1977), a decrease in the concentration of the digestive enzymes (Osborne gt gt., 1976) and a decrease in hatchabili— ty of the eggs (Cottier gt gt., 1969). A field case of broiler aflatoxicosis was reported by Smith and Hamilton (1970) that was characterized by a three-fold 28 increase in mortality, a doubling of the normal carcass condemnation rate, an increase in the feed conversion ratio from the normal 2.0 - 2.1 to 2.3 - 2.4 and the final body weight was 0.25 lbs less than normal. Examination of the affected birds revealed a serious infiltration of the peri- cardium, a pale comb, shank and bone marrow, an enlarged and pale liver, an enlarged spleen, an enlarged pancreas, a regressed bursa of Fabricius, and a below normal amount of depot fat. When the feed was fed to young chickens, they showed the same symptoms as birds fed an experimentally contaminated diet. Analysis showed that the ingredients in the suspected feed were contaminated with aflatoxins ranging from 100 to 10,000 ppb. Corn was the primary source of the aflatoxin. When an alternate corn supply free of aflatoxin was fed, there were no symptoms of aflatoxicosis in the chickens. Archibald gt gt. (1962) fed broiler chickens a diet containing peanut meal contaminated with unknown amounts of aflatoxins. They observed no effects for up to two weeks, thereafter uneven growth was shown. On necropsy, pale livers with lesions were observed, but the livers did not appear to be enlarged. Kratzer gt gt. (1969) fed broiler chickens for 8 to 9 weeks with diets containing 0, 400, 800 and 1,600 ppb of mixed aflatoxins. Growth retardation was observed in the groups at the two higher levels. Smith and Hamilton (1970) fed day-old male broiler chickens for three weeks on diets containing aflatoxins at levels of 0, 625, 1,250, 2,500 and 29 10,000 ppb. The effects of different levels of aflatoxins upon body weight of the experimental birds indicated that there was a threshold dOSe of 1,250 ppb, which had to be exceeded before any growth rate depression was observed. Smith and Hamilton (1970) also indicated that the threshold dose became larger with longer periods of administration. At aflatoxins levels of 2,500 ppb and above, enlargement of the spleen and pancreas was apparent, and the size of the bursa of Fabricius was decreased. The lipid content in liver was almost doubled at the higher doses. There was a significant increase in relative liver weight with increasing levels of aflatoxins up to 2,500 ppb. The possibility that aflatoxicosis is a predisposing factor in bruising was investigated by Tung gt gt. (1971b) They determined the minimal amount of energy required to produce bruises on the breast muscle of broiler chickens receiving graded levels of dietary aflatoxins. Doses of aflatoxins as small as 625 ppb, which is insufficient to affect the growth rate of most commercial strains, required 18 per cent less energy to produce bruising when compared to that for control chicks. At levels higher than 2,500 ppb, the amount of energy needed to produce bruising was decreased by about 50 per cent. These authors considered that the bruising effect might be caused by lowered muscle strength or by increas- ed capillary fragility. They found that doses of 625 and 1,250 ppb had no effect on body weight but significantly increased capillary fragility. Tung gt gt. (1971b) fed a diet containing 5,000 ppb 30 of aflatoxins to 2-week old chicks and found that the ca- pillary fragility increased significantly within 24 hours. They further measured the lateral shear strength of the breast muscle and found that the strength of the tissues was decreased at doses as low as 625 ppb of aflatoxin. These findings suggest that dietary aflatoxins produce a generalized loss of tissue strength and integrity, and result in an in- creased susceptibility to bruising. Aflatoxins have been shown to impair blood coagula- tion in a number of Species (Bababunmin and Bassir. 1972). Doerr gt gt. (1976) fed 3-week old broiler chickens with graded levels of dietary aflatoxins (O, 625, 1,250, 2,500, 5,000 and 10,000 ppb) to determine the specific coagulation responses. Tissue thromboplastin was found to posses two active components, one of which was lost during aflatoxicosis. The thromboplastin protein content was unaltered but the lipid content of the blood was depressed at 10,000 ppb of aflatoxins. The activities of coagulation factors VII and V and of fibrinogen were reduced at levels above 2,500 ppb of aflatoxins. The activity of coagulation factor X was depressed at a level of 5,000 ppb of aflatoxins. Prothrombin activity, the most sensitive factor tested, was reduced by feeding aflatoxins at a level of 625 ppb. These data demons- trated that severe impairment of clotting pathway functions occur during aflatoxicosis in chickens, and that prothrombin is primarily affected. The anemia associated with severe aflatoxicosis was initially characterized as aplastic anemia by Forgacs and 31 Carll (1962), and has subsequently been reported as hemolytic anemia by Tung gt gt. (1975). Early studies by Forgacs and Carll (1962) reported that aflatoxins consumed in moldy feeds resulted in a hemorrhagic anemia syndrome characterized by spontaneous hemorrhages in the musculature and internal organs. They concluded that it was caused by aplastic anemia. Wannop (1961) reported that erythrocyte numbers were decreased in a field outbreak of aflatoxicosis in turkeys. Tung gt gt. (1975) found that red blood cell counts, packed red cell volumes and hemoglobin levels were depressed by feeding aflatoxins to chickens. These results are consistent with the presence of aplastic anemia in the birds. However, microscopic examination of the bone marrow revealed a general hyperactivity. A possible explaination for these results may be that aflatoxins result in production of erythrocytes with an impaired integrity. Tung gt gt. (1975) confirmed the occurrence of anemia in chicks during aflatoxicosis and suggested that it was hemolytic in nature. Osborne and Hamilton (1981) fed graded levels of aflatoxins to broiler chicks and found that at 1,250 ppb or above there was a significant decrease in the specific acti- vities of the pancreatic digestive enzymes. The enzymes included pancreatic amylase, trypsin, lipase, RNase and DNase. At 10,000 ppb, the specific activities of amylase, trypsin, lipase, RNase and DNase were reduced to 41, 61, 42, 71 and 74 per cent of their control values, respectively. Osborne gt gt. (1982) observed that feeding aflatoxins at doses as small as 1,250 ppb, which does not inhibit the growth rate 32 of broiler chicks, produced a malabsorption syndrome charac- terized by steatorrhea, hypocarotenoidemia, and a decrease in the concentration of bile salts and of all the pancreatic digestive enzymes. OCCURRENCE OF AFLATOXINSgtN_ FOODS OF ANIMAL ORtgflN According to Jarvis (1975) foods may become conta- minated by mycotoxins through various routes. These routes were summarized as being by direct and indirect contamination. The direct routes of contamination result from mycotoxin production associated with mold growth on food materials of all types. These may include the following: (1) stored natural products intended for direct consumption or for use as food ingredients in manufactured products; (2) mold-ripened foods; and possibly, (3) mold-spoiled manufactured foods. The indirect routes of contamination may result from the use of mycotoxin-contaminated food additives, such as fermentation products of fungal origin, or from mycotoxin residues in animal products resulting from the use of mycotoxin contami- nated animal feedingstuffs (Jarvis, 1975). Allcroft and Carnaghan (1963) first postulated that residues of aflatoxins or their metabolites might be present in the milk, meat or eggs of animals receiving aflatoxin contaminated feeds. They fed ducklings with extracts of milk from cows fed aflatoxin contaminated rations, and found that the ducklings had lesions identical to those produced by administration of aflatoxin B1 directly. The milk toxin was later named aflatoxin M and M2 by Holzapfel gt gt. (1966). 1 33 Several reviews deal with the excretion of aflatoxins in the milk of farm animals (Allcroft, 1969; Kiermeier, 1973; 1977; Patterson, 1977; Rodricks and Stoloff, 1977; Stoloff, 1980). When cattle (Allcroft gt gt., 1968) or sheep (Nabney gt gt,, 1967) were fed rations contaminated with aflatoxin B1, their milk contained aflatoxin M1. Allcroft and Roberts (1968) measured the amount of aflatoxin M1 in milk from cows given diets containing various levels of aflatoxin B1. The daily intake of aflatoxin B ranged from 0.857 to 24 mg. The 1 excretion of aflatoxin M1 was proportional to the intake. There was a linear relationship between the amount of afl- toxin B1 ingested and the level of aflatoxin M1 in the milk. In order to determine the minimum intake of aflatoxin B1 which produces detectable levels of aflatoxin M1 in cow milk, Polan gt gt. (1974) fed graded levels (10, 50, 250 or 1,250 ppb) of aflatoxin B1 to 4 lactating cows for 7 days. On day 8, they substituted E 3H jaflatoxin B1 for a portion of the aflatoxin dose, and fed it an additional 7 days. The milk, blood, urine and feces samples were collected beginning at day 8. Regression analysis indicated that the 15 ppb of aflatoxin B1 was required for aflatoxin M1 to be detectable in the milk. The results generally agreed with those of Allcroft and Roberts (1968), who concluded that the level of aflatoxin B1 fed to milking cows should not exceed 50 ppb in order to insure negligible amounts of aflatoxins in the milk. Patterson gt gt. (1980) fed 6 dairy cows a diet contaminated with 10 ppb of aflatoxin B They found that 1I the amount of aflatoxin M1 in the milk varied from 0.01 to 34 0.33 ug/liter with a mean value of 0.19 ug/liter. Approxi- mately 2.2 per cent of the aflatoxin B1 ingested appeared in the milk as aflatoxin M Rodricks and Stoloff (1977) conclud— 1. ed from literature values that the ratio of aflatoxin B1 in the feed to aflatoxin M1 in the milk of cows was 300. Patterson gt gt. (1980) stated that this ratio varies consi- derably from animal to animal and from one set of data to another, and the overall range for the ratio of aflatoxins in feed to milk varied from 34 to 1,600.. " Allcroft and Carnaghan (1963) on assaying for aflatoxins with day-old ducklings failed to demonstrated toxicity from the livers and eggs of hens fed a ration containing 15 per cent of toxic peanut meal. They also were unable to find any toxic factor in the clotted blood serum and livers from cows fed a concentrate ration containing 20 per cent of toxic peanut meal, or from a pig liver taken from an animal with fatal aflatoxi- cosis. Allcroft and Raymond (1966) using a chemical assay found that the toxic peanut meal was contaminated with 10,000 and 200 ppb of aflatoxin B1 and B2, respectively. Platonow and Beauregard (1965) fed White Leghorn chickens for 2, 4 or 6 weeks on a diet containing 3,100 ppb of aflatoxin B1. The meat and livers from these chickens were fed separately to ferrets for 4 weeks, with each ferret receiving 100 g/day of tissue. No histological or chemical signs of toxicity were observed. A similar study by Allcroft and Lancaster (1966) using tissues from pigs and cattle fed aflatoxin contaminated peanut meal also provided negative results using ferret. Results from these studies were limited 35 by the lack of sensitivity of the ferrets to aflatoxins. Butler (1969) demonstrated that ferrets receiving a diet containing 300 ppb of aflatoxins required 28 months for tumor development. Platonow and Beauregard (1965) showed that ferrets receiving 1,000 ppb of aflatoxins died within 21 days. In addition to feeding the meat and liver from White Leghorn chickens fed aflatoxins to ferrets, Platonow and Beauregard (1965) assayed these tissues chemically by the method of Heusinkveld gt gt. (1965). The technique used was originally developed for determination of aflatoxin levels in peanuts. No detectable amounts of aflatoxins or their fluo- rescent metabolites were observed in the samples using this method. Abram (1965) fed ducklings and chickens a diet containing the meat from birds fed 0.75 ppb of aflatoxins. No histological signs of toxicity were observed. Negative results indicated that the aflatoxin levels in the tissues of the birds were probably below 40 ppb. Similar negative results were obtained for eggs from birds fed the same diet. Butler and Clifford (1965) found that aflatoxin B 1 and M (at that time aflatoxin M and M2 had not been separa— 1 ted) were present in the livers of rats that were given pure aflatoxin B1 by gastric intubation of by intraperitoneal injection. These compounds were also present in both the systemic and the portal blood. The authors suggested that conversion of aflatoxin B1 to M took place in the livers of the rats. Allcroft gt gt. (1966) gave sheep a dose of 1 mg of 36 mixed aflatoxins/kg of body weight in a ratio of 36:52:3:2 of B B2, G and G2, respectively, by either gastric intuba- 1’ 1 tion or intraperitoneal injection. All sheep were killed two hours later. All liver and kidney samples were removed, and chemically analyzed by the method described by DeIongh gt gt. (1964). It was found that the livers of all sheep had afla- toxins Bl’ G1 and M2. The M2 spot was less intense than those of B1 and G1. The kidney extracts showed fluorescent spots similar to those seen in liver extracts, but the inten- sity of aflatoxin M2 was greater than that of B1 and G1. Kratzer gt gt. (1969) fed graded levels of dietary aflatoxins to White Rock chicks and found that at higher levels (800 and 1,600 ppb) adverse biochemical effects were detected in the liver. They analyzed the meat, liver and blood samples by using the method of Wiley (1966). There was no detectable amount of aflatoxin ir the tissues of broilers fed 1,600 ppb of aflatoxins for 60 days prior to slaughter. Similarly, Kratzer gt gt. (1969) observed no aflatoxins in the eggs, meat, liver or blood of hens fed a ration containing 2,700 ppb of aflatoxins for a period of 48 days. According to Kratzer gt gt. (1969) as little as 305 ppb of aflatoxin B could be detected by the method of 1 Wiley (1966). Van Zytveld gt gt. (1970) fed White Rock chicks for 6 weeks with daily dosages ranging from 0.4 to 1.7 g of mixed aflatoxins/kg of body weight. In 15 of 45 test birds, aflatoxins or their metabolites were isolated from the livers and skeletal muscles. Aflatoxins were found mainly 37 in chicks that died or grew poorly during the test and only one bird that reached market weight was shown to have residues of aflatoxins in its tissues. Mabee and Chipley (1973a) intubated 6—week old 1Ll'C-labeled afla- Vantress-Arbor Acre broiler chickens with toxin B1. The daily dose was 0.1 mg/kg of body weight. No toxic effect was observed in broiler chickens during the 14 day experiment. The feces samples were collected every 48 hours during the feeding trial. Five hours after the final dose of aflatoxin was administered, all birds were killed by exsanguination. Samples of leg and breast muscle, gizzard, liver, heart and blood were collected. All the samples were lyophilyzed and extracted with sodium acetate buffer. The supernatant fluid was treated with NaCl and then was further extracted with ethyl acetate. According to the analysis, the 14 broiler chickens excreted 90.64 per cent of the C adminis- tered. or the 1” C retained, 11.04, 9.83, 4.03, 12.52, 31166 and 30.63 per cent was found in the blood, liver, heart, gizzard, breast and leg, respectively. In the combined sample, 81.2 per cent of the radioactivity was confined to the sodium acetate buffer extract. Mabee and Chipley (1973b) further treated the sodium acetate buffer with B-glucuronidase to determine the conjugated aflatoxins, and found that 31.5 per cent of the total radioactivity originally present in the buffered extract was transferred to the chloroform extract. The presence of aflatoxin M1 was confirmed by TLC of the chloroform extract. The authors concluded that broiler if it chickens can metabolize the majority of aflatoxin B1, 38 is administered at relatively low levels. Aflatoxin conju— gates were the predominant forms of the metabolites produced. Aflatoxin B1 was metabolized to aflatoxin Ml' Aflatoxin M1 was shown to be soluble in aqueous extracts and was present mainly as conjugated glucuronides. Mabee and Chipley (1973b) also used the same methods to study aflatoxin retention and excretion with White Leghorn pullets. They found that the average levels of radioactivity detected in the blood, liver, heart, gizzard, breast and leg muscles were 19.5, 16.1, 3.9, 7.2, 26.4 and 26.9 per cent of the total amount retained by the laying hens, respectively. The 14 C detected in liver, heart, gizzard, breast and leg samples accounted for 7.85 per cent of the total dosage. Aflatoxin M1 glucuronides constituted 38.9 per cent of the total conjugates. The authors proposed that in addition to glucuronide conjugates, other metabolites of afltoxin B1 were present, probably as sulfate conjugates. They further treated all the samples with carboxypeptidase A, leucine amino pepti— dase, pepsin or trypsin. Results revealed that an average of 50 per cent of the 140 detected in the ethyl acetate extracts 14 was liberated as the amino acid conjugates of C aflatoxin B (Chipley gt gt., 1974). 2a Mintzlaff gt gt. (1977) divided 120 HNL—hybrid broilers into 8 experimental groups, and fed aflatoxin B1 at levels of 25, 100, 500, 1,000, 2,000, 5,000, 10,000 or 15,000 ppb for 8 weeks. They also fed 5 times as much G1. All birds were killed at the end of the experiment. The livers and muscle tissues were lyophilyzed and analyzed 39 quantitatively for aflatoxin B1 by using the A.O.A.C. method. With this method, about 0.1 ppb of aflatoxin B is detectable 1 in the liver and muscle of broiler chicks. The authors found that on increasing the level of aflatoxin B1 in the feed, the tissue residues in the liver and muscle increased. Liver contained more aflatoxin B1 than muscle, but the amounts in the thigh and breast muscles were not significantly different. The critical aflatoxin B1 concentration which resulted in residues in the tissues was 100 ppb. Results showed that on the average liver contained 0.28 per cent, thigh muscle 0.07 per cent, and breast muscles 0.12 per cent of the total administered aflatoxin B1. Aflatoxin G1 and M1 were also detected in the liver and muscles. Stoloff and Trucksess (1979) analyzed the livers from 4 calves that had been fed with mixed aflatoxins and from 4 pigs that had died after consuming aflatoxin contami- nated feed. There were no statistically significant differences in the levels of aflatoxin B1 or M1 found in different sections of the livers. The levels of aflatoxins in the pig livers were lower than that found in the calf livers, with only one out of the four pig livers containing detectable amounts of aflatoxin B1. Since the pigs died from exposure to aflatoxins, and were not sacrificed as were the calves, these authors suggested that the low level and incidence of aflatoxins in the pig livers may be attributed to poor feed consumption, although postmortem changes could not be ruled out. The withdrawal time required for clearance of afla- toxins from pig tissues was studied by Furtado gt gt. (1982). 40 Two feeding trials involving 20 pigs in each were used. In both trials spiked diets were fed, which contained 551 and 355 ppb of aflatoxins BJ and B2, respectively. After feed- ing 42 days, all pigs were fed the unspiked control diet. There was a significant reduction of aflatoxins in all organs and tissues one day after placing the pigs on an aflatoxin- free ration. After two days, only one pig contained trace amounts of afltoxins in tissues. After four days, there were no detectable levels of aflatoxins in any of the tissues. It was also found that a naturally contaminated diet containing only 20 and 31 ppb of aflatoxins B1 and B2, respectively, resulted in trace amounts of aflatoxins B1, B2, M1 and M2 in the kidneys and livers after a 13-14 hours withdrawal period. Miller gt gt. (1982) also studied the clearance time of aflatoxin residues in swine tissue by performing an acute test. In the acute study, 8 pigs were given a single dose of 1.2 mg of aflatoxins/kg of body weight. Liver, kidney and muscle tissues from these pigs were analyzed for aflatoxin residues. Residue levels were correlated with post-dosage time. Residues were present in the liver and kidney of one pig at 12 hours after dosing, but not in muscle. However, detectable muscle residues were reportedly found in the muscle of pigs killed 24 hours after dosing. Residue levels were 0 nsistently lower at 72 hours than that at 24 hour post-dosing. In a chronic study, Miller gt gt. (1982) fed 30 mixed breed pigs (10 animals/diet) a ration containing 0, 400 or 800 ppb of aflatoxins for 10 weeks. Aflatoxin residues in 41 the liver, kidney and muscle tissues were detected immediate- ly after slaughter using a HPLC method. No detectable resi- dues were found in the muscles from pigs fed 400 ppb of afla- toxins in the diet, but were present in the muscle of pigs fed 800 ppb of aflatoxins. Aflatoxin residues in the kidney and liver were higher than in the muscles of pigs fed 800 ppb. Luthy gt gt. (1980) administered ring-labeled [140]- aflatoxin B1 to 2 pigs over a 9-day period. They found that the major excretory route was feces (accounting for 51 and 65 per cent of the dose) and that less than 20 per cent of the dose was excreted in the urine. Only about 4 per cent of the dose recovered in the feces and urine was in identifiable forms, most of it being aflatoxin M1 and B1 with traces of aflatoxicol. Jacobson and Wiseman (1974) fed laying hens with an aflatoxin-contaminated laying ration. The hens were fed as follows: (1) 10 days—100 ppb, (2) for the following 12 days- 200 ppb, and (3) the next 15 days-400 ppb. The eggs from hens at all three feeding periods contained measurable amounts of aflatoxin B1. The relationship between the amounts of aflatoxin B1 fed and the average amounts found in the eggs appeared to be approximately linear. The average level for aflatoxin B1 in the eggs collected during period one was 0.23 ppb, from period two was 0.78 ppb and from period three was 1.4 ppb. 42 Table 1 -- Ratios of aflatoxin B1 levels in the feed in relation to aflatoxin B1 or M1 levels in edible tissues (Rodricks and Stoloff, 1977). ANIMAL TISSUE AFLATOXIN FEED T0 TISSUE IN TISSUE RATIO Beef Cattle Liver B1 14,000 Dairy Cattle Milk M1 300 Swine Liver B1 800 Layers Eggs B1 2,200 Broiler Liver B1 1,200 A summary of the data from various studies presenting the ratios for the level of aflatoxin B1 in feeds in relation- ship to the level likely to be encountered in selected animal products is presented in Table 1 (Rodricks and Stoloff, 1977). Assuming that the experimental data reflect reality, meat from swine is more likely to be contaminated than poultry meat or beef, since the observed feed/tissue ratios appear to be lower for swine than for broilers or beef cattle. EXPERIMENTAL FEEDING TRIAL Preparation of the Diet During the entire experimental period, the basal diet used was a broiler starter ration, formula CBS 711 prepared at the MSU Swine Research Center. The composi- tion of the diet is given on Table 2. In order to prepare the spiked ration, the crystalline aflatoxin B1 and B2 (Calbiochem-Behring Co. Saw Diego, CA.) were first solubilized with chloroform, and then were diluted to 2 liters in a volumetric flask. The chloroform solution was then divided into two equal fractions and each of the was slurried with 1.5 kg of the pre-dried basal diet that had been held overnight in an oven at 10000. The chloroform was allowed to evaporate in the dark overnight using forced air under a hood. These two fractions of aflatoxin-spiked feed were mixed homogenously together in a 4-speed Reynolds Mixer (Reynolds Electric Co.), and were then divided into three equal fractions by weight. Each of the aflatoxin premixed feed samples was separately transferred to a stainless steel Wenger horizontal mixer (Model No. 61129, Wenger Mixer Manu- facturing Co.), and mixed with additional feed until homogenous to give a final weight of 100 lb. Each 100 lb lot was divided 43 44 Table 2 -- Chick, broiler starter diet (formula CBS 711) Ingredients Amt./1000 Corn, #2 yellow 482.2 Soybean meal, 48% 365 Alfalfa, 17% 68 Corn oil, stabl.a 42 Methionine, dl 0.8 Dicalcium phosphate 22 Limestone 9 Choline cthride, 50% 3.5 Salt _ 3-5 Vitamin mix 3 Mineral mix (c.c.c.) 0.5 Selenium mix (c.c.c.) 0.5 a Ethoxyquin added to corn oil at 125 mg/kg diet. Specifications: Protein, crude 23.4% Metabolizable energy 3.05 kcal/g Fat 6.6% Fiber 3.7% Calcium 1.07% Phosphorus, avail 0.55% Methionine 2% of protein Lysine 5.5% of protein 45 into two 50 lb lots, and was cross mixed with another 50 lb lot during preparation. Finally, the total of 300 lb of aflatoxin-spiked feed was evenly packed into twelve 5-gallon plastic pails, and stored at -20°C. One day before being used, the feed was transferred to a refrigerator and held at approximately 4-8°C until fed. Experimental Animals A total of 48 day-old male Hubbard White Mountain broiler chicks (White Cornish x White Plymouth Rock) ordered from Commercial chicks, Inc. (Thorntown, Inc.). All chickens were housed in a wire floored electrically heated battery (75 x 27 x 7.5 cm) at the MSU Poultry Research Center. All birds were kept in 4 cages under continuous dim illumi- nation with feed and water available gg libitum. After an adjustment period of one week, all chicks were weighed and labeled with a Zip wing band (size 2.75). They were randomly divided into a control and an experimental group. The chicks in the control group were kept in one cage on the upper layer of the battery, while those in the experimental group were randomly placed in three cages on the lower layer of the battery, with 12 chickens per cage. This gave all groups the same floor, feeder and watering Space. The experimental group was fed the basal diet spiked with aflatoxins B1 and B2 at levels of 2057 and 1323 ppb, respectively. The chicks in the control group were fed the 46 unspiked control diet. The feeding period was for 6 weeks. The chicks were weighed weekly, and feed and water consump- tion were recorded daily. At 4 weeks of age, all the chicks were moved into wire floored growing batteries (48 x 31 x 4 inch). At the end of the experimental feeding period, 6 birds from both the control and the experimental groups were slaughtered. The remaining 30 experimental group birds and 6 controls were all fed the uncontaminated basal ration, and held for 1, 2, 4, 8 and 16 days. At the end of each period, 6 experimental chickens were killed. After 16 days, the last 12 birds, 6 from the experimental group and 6 from the con- trol group were killed. Slaughtering and Collection of Samples On the day proceding slaughter at 12 noon the feed and water were removed. Body weights, feed consumption and water consumption data were recorded. The chickens were then taken to the MSU Poultry Science Laboratory. At approximately 3 pm (3 hours after removal of the feed), the chickens were slaughtered. Carcasses of all birds were inspected for gross lesions. Samples of blood, heart, liver, spleen, gizzard, kidney, intestine and of breast and leg muscles were collected and weighed. All samples were frozen and stored at -200C for later analysis. 47 ANALYSIS OF AFLATOXINS Sample Preparation The leg and breast muscle samples were deboned, and the collagenous tissues and excess fat were removed from the lean portions.' Then the muscle samples were passed through a meat grinder (Hobart Manufacturing Co.), using a plate having 3/16-inch diameter holes. The gizzards were opened with scissOrs and any undigested feed was discarded on re- moval of the lining. The tissues were stored at -20°C until analysis. The internal organs (gizzard, heart, liver and spleen) were cut into fine pieces before extraction. Extraction of Aflatoxins from Tissues Extraction and analysis of aflatoxins from raw tissues and the processed samples were carried out according to a 'modification of the procedure of Trucksess and Stoloff (1979). About 25 g of tissue were blended for 2 minutes in a Waring blender at moderate speed with 42 ml of NaCl-citric acid solution (35 g NaCl + 4.8 g citric acid/100 ml H20). Then 200 ml of acetone was added to the homogenate while washing the sides of the blender jar.) The tissue was blended for 3 minutes at moderate speed followed by 2 minutes at high speed. Another 100 ml of acetone were added to wash the blender jar again, and the tissue was blended for an additional 1 minute at high speed. The material was then filtered through fast filtering prefolded filter paper (Whatman 114v), and the filtrate was collected in a 500 ml Erlenmeyer flask. After filtration was completed, the flask was stoppered with 48 an aluminum foil covered cork stopper, and placed in a freezer at —20°C for 5 minutes, which precipitated the excess fat. The precipitated fat was removed by filtration, using the same type filter paper. The filtrate was trans— ferred to another 500 ml Erlenmeyer flask. Then 150 ml of distilled water, 7 g of (NH4)2804 and 20 ml of Pb(0Ac)2 solution (200 g Pb(OAc)2 . 3H20 in 500 ml H20 containing 3 ml of acetic acid and made to volume of one liter with H20) were added. The solution was stirred for 0.5 minute using a magnetic stirring device. Then 10 g of Celite were added to the solution, and stirring was continued for an additional 0.5 minute. The solution was allowed to stand for about 5 minutes before filtering into a 500 ml graduate cylinder using fast filtering prefolded filter paper. Purification of the Aflatoxin Extract Liquid-Liquid Partition The filtrate was transferred to a 500 ml separatory funnel. Then 100 ml of petroleum ether (30-600C b.p.) were added. The separatory funnel was shaken vigorously for about 1 minute. The layers were allowed to separate, and the lower aqueous-acetone layer was drained into a second 500 ml sepa- ratory funnel. The petroleum ether layer was then discarded. Then 50 ml of chloroform were added to the aqueous-acetone solution and the separatory funnel was shaken as before. After the layers separated, the lower chloroform acetone layer was collected in a 500 ml flask. Aflatoxin extraction from 49 the aqueous-acetone layer was repeated one more time using 50 ml of chloroform-acetone (1:1). The aqueous layer was collected in the same 500 ml flask, and the chloroform extract was discarded. The chloroform-acetone extract was then evaporated to dryness in a rotary evaporator (Buchi, Switzerland) using a water bath setting of 37°C. Silica Gel Column Chromatography A 25 x 250 mm chromatographic column (Fisher and Porter) was filled using about 50 ml of chloroform. A slurry of 10 g of silica gel 60 (70-230 mesh ASTM-EM Labo- ratories, Inc.) in 60 ml of chloroform was added to the column. The silica gel was allowed to settle and then the chloroform was drained to about 10 cm above the top of the silica gel. About 2 cm of anhydrous sodium sulfate was layered slowly on top of the silica gel. The excess chloro- form was then drained from the column until it reached the level of the upper layer of sodium sulfate. The aflatoxin extract was dissolved in approximately 5 ml of chloroform-hexane (1:1) and then transferred to the column with a disposable glass pipet. The sides of the flask were washed three more times with 15 ml of chloroform- hexane, and the washings were added to the column. After each addition of the chloroform-hexane extract, the column was drained to the tOp of the packing and the eluate was discarded. Any interfering substances were eluted from the column in 100 ml of ether-hexane (3:1). The eluate was then discarded. The aflatoxins were eluted from the silica gel column 50 with 150 ml of chloroform-methanol (97:3). The eluate was collected in a 250 ml flask, and evaporated to near dryness in a rotary evaporator as described earlier herein. The sample extract was dissolved in acetone and quantitatively transferred to 2 dram vials using a disposable glass pipet. The acetone solution was evaporated to dryness (N-Evap Evapo- rator, Model 106, Organomation Assoc.) under a stream of nitrogen using a water bath setting of 4000. Special care was taken to avoid overheating of the dry extract in order to avoid decomposition. 'The aflatoxin extract was then dissolved in 50 ul of benzene—acetonitrile (9:1), and the vial was sealed with a teflon lined screw cap. The samples were held in the vials at -20°C until removed for analysis. The vial contain- ing the aflatoxins was shaken vigorously for about 1 minute on a vortex shaker before removing the samples for analysis. Thin Layer Chromatography Precoated 20 x 20 cm silica gel plates (Sil-G-HR- 25, Brinkman Instrument Co.) were scored and spotted as shown in Figure 5. A 20,ul sample of the aflatoxin extract was applied to the sample spot with a 25 ul syringe (Sigma Chemi— cal Co.). Standards of 5.0, 1.5, 5.0, 1.5, 2.0 and 2.0 ng of aflatoxins B1' B2, G1, G2, M1 and M2, respectively, were spotted on the plates as standards in both directions of development about 1.5 cm from the edges. The plates were develOped in the first direction with ether-methanol-water (95:4:1) in a sealed and unequili- brated tank. After the development in the first dimension cm 14 cm 51 2nd direction standard spots lst direction sample spot¢ I<-——>I< ate—x 2.5 cm 16 cm 1.5 cm Figure 5 - Spotting and scoring pattern for two- dimensional 20 x 20 cm TLC plates. 52 was completed, the plates were removed from the tank and dried under a hood for about 2 minutes. The drying was finished by placing in a forced-draft oven for 1 minute and the plates were immediately developed in the second dimension with chloroform-acetone (85:15). After development was completed, the plates were removed and dried as before. After drying, the plates were examined visually under UV light and prepared for densitometric analysis. Densitometric Analysis of Aflatoxins Aflatoxins were quantified with a double-beam scanning recording-integrating spectrodesimeter SD 3000-4 (Schoeffel Instruments). The plates were scored prior to spotting as shown in Fig. 5 with a Schoeffel Scoring Device SDA 303, which provides 18 mm strips. The average of three readings of the aflatoxin reference standards (spotted within the three strips parallel to the second direction of development) was used for densitometric comparison in calculating the concen— trations of the sample spots. Scanning was carried out for the analysis of the standard spots. In scanning the sample spots, the plates were viewed under UV light, and each spot was localized within two pencil marks made on the silica gel plates. The marks were about 1 cm apart and located approxi- mately 3 mm ahead of the sample spot along the second dimen- sion of the development. Then the plate was placed on the carrier in such a way as to be driven and scanned parallel to the direction of the imaginary lines. Aflatoxin concentrations were calculated according 53 to the following formula: Jug/kg=(BxYxSxV)/(ZxXxW) where: Area under aflatoxin peak in the sample; Concentration of aflatoxin standard in.ug/ml; .ul of the aflatoxin standard; Dilution of sample extract in,ul; N < U] H: U1 ll Area of aflatoxin standard peak (average of three replications); X =,ul of sample extract spotted on the plate; and W = Grams of sample in the final extract. ANALYSIS OF AFLATOXINS IN THE FEED Analysis for aflatoxins in the feed was carried out according to a modification of the A.O.A.C. method (1965). A 50 g sample of feed was weighed into a 500 ml Erlenmyer flask. Then, 25 ml of water, 25 g of Celite and 250 ml of chloroform were added. The stopper was secured with masking tape and the flask was shaken for 30 minutes on a wrist action shaker (Burrel Corp.). The material was then filtered through prefolded filter paper (Whatman 2v) and the first 150 ml of filtrate were collected, dried and transferred to a 25 x 250 mm chromatographic column as described earlier herein. The aflatoxins were eluted from the column, spotted on 20 x 20 cm TLC plates and quantified as described before for analy- sis of aflatoxins in tissue samples. 54 CONFIRMATORY TESTS FOR AFLATOXINS Aflatoxin B1 A total of 25,ul of sample extract was applied about 1.5 cm from both edges in the lower left corner of a precoated TLC plate (20 x 20 cm Sil-G-HR-25, Brinkman Instruments, Inc.). Approximately 5.0, 1.5, 5.0, 1.5, 2.0 and 2.0 ng of aflatoxins G B1, B G2, M1 and M2 standards, respectively, were spot- 2’ 1’ ted in the same location in both directions of development on the plates. Then the plates were developed in a closed, unlined and unequilibrated tank using ether-methanol-water (95:4:1) in the first dimension and chloroform—acetone (85:15) in the second dimension. After the development was completed, the plates were examined visually under UV light. The spot corresponding to aflatoxin B1 was identified by comparison with the aflatoxin B1 standard. It was marked in the silica gel on the left with a pencil along the second direction of development. Another pencil mark was made about 1 cm apart to the left of the first one. The second mark was used as a guide to apply about 5.0 ng of aflatoxin B1 standard close to the aflatoxin B1 sample Spot. Then 2,ul of trifluoroacetic acid (TFA)-chloroform (1:1) were applied to both of the aflatoxin B1 spots. The plate was allowed to stand in the dark for about 5 minutes at room temperature. Then the plate was dried in a forced-draft oven for 5 minutes. After cooling the plate in the dark at room temperature, another 5.0 ng of aflatoxin B1 standard was applied about 1 cm to the left of the second pencil mark. The plate was 55 developed in the second direction with chloroformaacetone- isopropanol (87:10:3) the same way as described before. After the plate was developed and dried, the chromatogram was examined for the formation of aflatoxin B2a' The chro- matographic equivalence of the sample and the aflatoxin standard spots after treatment with TFA was used as confir- matory test for identifying aflatoxin B1. Afle£2£i2_ml After second dimension was finished, the aflatoxin M1 spot in the sample was marked in the silica gel on the left with a pencil along the direction of the development. Another pencil mark was made about 3 cm apart to the right of the first and close to the aflatoxin M2 spot. The second mark was used as a guide for applying about 2.0 ng of afla— toxin M2 standard. Two ul of TFA-chloroform (1:1) were then applied to both the sample Spot and the aflatoxin M1 standard. The plate was allowed to stand in the dark for about 5 minutes at room temperature. Then the plate was dried in a forced- draft oven for 5 minutes. After cooling the plate in the dark at room temperature, another 2.0 ng of aflatoxin M1 standard was spotted about 1 cm to the right of the second pencil mark. Then the plate was developed perpendicular to the first direction using chloroform-acetone (85:15). After development, the plate was dried and examined under UV light for the formation of the aflatoxin M1 derivative in order to ascertain if the Rf was lower than that of the unreacted aflatoxin M1 standard. The chromatographic equivalence of 56 the sample and the aflatoxin M1 standard spot after treatment with TFA was used as a confirmatory test for the identifying of aflatoxin M1. General Confirmatopy Test for Aftatoxins The technique of Prezybylski (1975) was used for additional confirmation of the presence of aflatoxins at low levels. After development of the TLC plate and identifi- cation of the spots under UV light, the plate was sprayed with 25 per cent sulfuric acid (v/v). Then the plate was dried in a forced-draft oven for 1 minute. Changes in the characteristic fluorescence of aflatoxins B1, B2, M1 and M2 from blue to yellow, and aflatoxins G1 and G2 from green to yellow after the H2804 treatment were used as an additional confirmatory test for the presence of aflatoxins. PREPARATION OF AFLATOXIN REFERENCE STANDARDS Aflatoxins B B G1, G2, M and M2 were obtained 1’ 2’ 1 from Sigma Chemical Company and used as reference standards. Aflatoxins B1 and B2 used for preparation of the spiked diets were purchased from Calbiochem-Behring Corporation. Aflatoxin reference standards were prepared according to the A.O.A.C. method (1975) and contained 0.5, 0.15, 0.5, 0.15, 0.2 and 0.2 ug/ml of aflatoxins B B G G M and 1’ 2’ 1’ 2’ 1 M2, respectively. A solvent mixture of benzene-acetonitrile B G and 1’ 2’ 1 G1, while benzene-acetonitrile (90:10) was utilized as the (98:2) was used as the solvent for aflatoxins B 57 solvent for aflatoxins M1 and M2. The aflatoxin standard solutions were stored at -20°C. FAT AND MOISTURE ANALYSIS Moisture Content The A.O.A.C. (1975) procedure for determining-moisture was used. Four grams of tissue were accurately weighed to four decimal places into a previously dried and tared alumi- num dish (1000C for at least 1 hour). The sample plus the dish were then dried overnight for 18-24 hours in an air con- vection oven at 100°C. The dried sample was cooled in.an desiccator and weighed to four decimal places. The loss in weight was calculated as percentage moisture. Three repli- cates were run for each sample. Fat Content The fat content was determined using the Goldfisch extraction method of the A.O.A.C. (1975). The same sample was used following moisture analysis. The aluminum dish containing the dried meat sample was carefully folded into a porous thimble and clipped into a Goldfisch apparatus. The fat was extracted with anhydrous diethyl ether for appro- ximately 3 hours into a previously dried and tared beaker. The extract was then dried for 1 hour at 10000 in an air convection oven, cooled in a desiccator and weighed as before. The per cent fat was calculated as grams of fat extracted from each one hundred grams of tissue. Three replicates were run for each sample. 58 STATISTICAtTANALYStg Statistical analysis was carried out using the pro- cedures described by Gill (1978). In testing the difference between the means for the control and for the experimental groups, a preliminary F-test was performed to test the difference between two variances. When there was no differ- ence between the two variances at the 80 per cent confidence level, a t-test was used to determine if the difference was significant. If the variances were different at the 80 per cent confidence level, another preliminary test was performed to determine the difference between the two coefficients of variation. When the coefficients of variation were different at the 80 per cent confidence level, a t-like test (Behrens, 1929) was used to determine the significance between the two means. Otherwise, the direct test developed by Lohrding (1969) was applied to determine the significance between the means 0 SAFETY PROCEDURES All glassware and vials in contact with aflatoxins were soaked with 5-6 per cent NaOCl (household bleach) to destroy any residual aflatoxins. Plastic disposable gloves were worn routinely during all work with aflatoxins. Respi- rator masks were worn when mixing and handling the spiked rations. Surface of work areas were routinely scanned with an UV lamp and any contaminated areas were treated by washing thoroughly with 5-6 per cent NaOCl solution. All TLC plates used in aflatoxin analysis were thoroughly soaked in NaOCl 59 solution before discarding. Filter papers and tissue residues resulting from aflatoxin analysis were thoroughly soaked with concentrated ammonium hydroxide solution overnight before discarding. These waste materials, after treatment with ammonia, were collected in plastic bags and placed inside tightly closed containers and labeled properly until removed by the MSU Animal Waste Disposal Unit. The discarded solvents involved in the experiment were removed by the MSU Chemical and Biological Safety Unit. All work involving aflatoxins was done under a hood, this included preparation of silica gel columns, spotting, develOpment and drying of the TLC plates. Similarly, any work involving the use of toxic solvents, such as ammonia, chloroform, benzene, methanol and acetonitrile, was also carried out under a hood. RESULTS AND DISCUSSION FEEDING TRIAL The response of broiler chicks to aflatoxins was determined by growth, feed consumption and feed efficiency data (feed consumed/unit weight gain). Results showing body weights, body weight gains, average feed intake and feed efficiency are presented in Table 3, The amount of aflatoxins consumed and the daily dosage ratio of the experimental group are presented in the Table 4. Final weights and overall mean body weight gains are presented in Table 3 and demonstrate that growth retardation was significant in the experimental group when compared with the control group. There was no significant difference between the feed efficiency for chicks fed aflatoxins and for those in the control group. These results are generally in agreement with those reported by Kratzer gt gt. (1969) and by Smith and Hamilton (1970). The chicks in the experimental group had on average a daily intake of 208 ug of aflatoxin B1 and 134 ug of aflatoxin B2. The daily dosage ratio (DR) has been reported as the best measure of the response of the exposed animals to aflatoxins (Armbrecht gt gt., 1971). The results in Table 4 showed that the DR ratio of aflatoxins B and B2 was high- 1 est in week I and lowest in week V. Thus, the DR ratio 60 61 .Hoo.ovm Pm asonm HMPCoEHMoQNo may smnp anfloa whoa hdpsmoflwflcwflm posflmw macaw Hospsoo oneo .Hoo.ovm pm macaw Hassosflhoaxo one snap nofl>mos zHPCmOHmwsmflm mm; macaw Hompsoo oxen .onwpmH>ob unaccepmo .zHo>Hpooamoh .Qaa MNMH use mmom_mo mao>oa Pm mm was Hm mCaXopmaam wsflcflmpsoo msofipms com 9 .poflc Hohvsoo esp CH manmpoopop ohms msflxopmamm ozm a.aaH_ s.saa m.a o.m.m :.N H.HOH mfiommfifi 65.3H0H B.H3H CNQE QSOMU Dampsosflpomxm m.md N.m: H.: o.Q.m QSOMU N N m sea om.mdma em.HMAa a.ema sees eeoeeeoo va Asasn\eooav ado sea soars sea Amv seen any sense: Ana armada soeoaoaeem coca .mea oasesH soda .w>< sensor Assam weerasem .A9369w ampsoswnomxo map CH mppfin on new macaw Hospsoo one CH mphfln NHV Hmwpp wcfipoom xooslm esp pom monoHOHmwo doom owmno>m cam oxmpcfi pooh ommno>m .mswmw Pcmwos anon .mpnmfloa hoop msflsocm mpmp oocmspowpom II m canoe 62 Table 4 -- Average aflatoxin consumption and daily dosage ratio of the eXperimental group. Avg. Aflatoxin Consum tion Daily Dosa e Per Chick Per Day (ugg Ratioa (DR B1 B2 B1 B2 Week I 129 83 487 313 Week II 138 88 287 185 Week III 213 137 294 189 Week IV 274 176 263 169 Week V 289 186 206 132 Overall 208 134 238 154 . a Amount of aflatoxin calculated from formula DR = Wa/0.5 (Ws + We) t (Armbrecht gt gt., 1971). Where, (Wa);ng of aflatoxin ingested during the interval of time (t) in days, starting weight (Ws) and ending weight (We) in kg. 63 tended to decrease as the chicks grew older. In other words, the young chicks were exposed to a greater amount of aflatoxins per unit body weight when compared to the older ones. This might explain why Asplin and Carnaghan (1961) found young chicks to be more sensitive to aflatoxins than older ones. However, when sensitivity was measured in terms of the LD50, there was no great difference between young chicks and those large enough for processing (Smith and Hamilton, 1970). GROSS OBSERVATIONS ON TISSUES At slaughter, all birds were inspected for gross lesions. On examination of the birds, several distinctive symptoms were observed, which are summarized in Table 5. 'Inspection showed hemorrhagic spots on the surface of the muscles, especially on the breast muscles of the experimental chicks, but not in the controls. Also in the present study hemorrhagic spots were observed on the livers of three chicks in the experimental group. These results agree with those reported by Tung gt gt, (19711», who stated that dietary afla- toxins increase the susceptibility of broiler chickens to bruising. This was because the fragility of the blood vessels increased and the muscle strength decreased. Doerr gt_gt, (1976) reported that aflatoxicosis will impair the coagula- tion of blood. The pale livers observed in the experimental group of the present study were associated with a higher lipid content (Table 6). Tung gt_gt. (1971a; 1972) also indicated that inhibition of lipid transport is a primary lesion during 64 Table 5 -- Summary of the gross lesions observed upon inspection (6 birds in each group). SYMPTOMS Hemorrhagesa Pale liver Gall Bladder Slipped on the Muscle Enlarged Tendon 0 day after withdrawal from aflatoxin-spiked ration Control Group 0 O 0 2 Experimental b Group ' 6 _ 4 4- , O 1 day after withdrawal from aflatoxin-spiked ration Experimental Group 5 0 O 0 2 days after withdrawal from aftgtoxin-spiked ration Experimental Group 6 4 1 0 4 days after withdrawal from aflatoxin—spiked ration Experimental Group 2 2b 0 O 8 dayg after withdrawal from aflatoxin—spiked ration Experimental Group . O 0 0 1 16 days after withdrawal from aflatoxin-spiked ration Control Group 0 O 0 1 Experimental Group 0 O 0 2 a Most of the hemorrhagic spots were on the breast muscle. b On 0 day withdrawal from the aflatoxin-spiked ration, 2 out of 4 livers had hemorrhagic Spots, and on 4 days withdrawal from the aflatoxin-spiked ration 1 out of 2 livers had hemorrhagic spots. 65 chicken aflatoxicosis. They reported that aflatoxins can cause a dose-related increase in the lipid content of the livers of young broilers. This was accompanied by a decrease in total serum lipids, serum phospholipids, serum cholesterol and serum triglycerides. These authors also measured the incorporation of radio-labeled acetate into the long-chain fatty acids of the livers from birds receiving aflatoxins. Results indicated that fatty acid biosynthesis decreased in a dose-related fashion. The authors suggested that aflatoxins inhibit lipid synthesis and transport in broiler chickens. However, hepatic lipid biosynthesis increased in some instan- ces as was reported by Chou and Marth (1975). The weights of the internal organs (hearts, spleens, livers, kidneys, gizzards and intestines) were recorded. The mean for organ weights and a summary of organ weights express— ed as the percentage of the live body weight are presented in Tables 7 and 8. Data from the present experiment (Table 7) showed that the livers and kidneys of the chicks on the experimental diet were significantly heavier at the end of the aflatoxin feeding trial (day 0) than those of the controls (P < 0.01). On the average, the livers of the experimental group were 29 per cent heavier than those of the control group. The difference may in part be due to the higher lipid content of the livers for the experimental birds. The kidneys of the experimental group were 46 per cent heavier than those of the control group. The hearts of the experimental group were slightly 66 Table 6 --Fat and Moisture content of livers for both the control and the experimental groups at 0 day after withdrawal from the aflatoxin-spiked ration. Chick No. Moisture Content (%) Fat Contenta(%) Control Group C1 71.7 1.4 C2 69.9 1.7 C3 69.8 . 0.7 C4 70.2 1.0 C5 73-3 0-3 C6 70.0 1.5 Mean 70.8 1.2 Experimental Group 01 73.1 1.7 02 69.3 2.6b 03 67.4 4.5b 04 62.7 17.7b 05 70.0 3-9 06 65.6 8.8b Mean 68.0 6.5 a The fat content was based on the wet basis. b Gross lesion inspection showed that these birds had pale livers. 67 Table 7 -- Internal organ weights in grams of both the con- trol and the eXperimental groups (0 day and 16 days after withdrawal from aflatoxin-Spiked ration). ORGAN WEIGHTS (grams) Heart Spleen Liver Kidney ‘Gizzard Intestine 0 day after withdrawat from aftatoxtnegptked ration Control Group Mean 9.1 1.6 33.2a 6.5b S.D. 0.8 0.3 6.0 1.9 Experimental Group Mean 10.2 1.9 42.7a 9.4b S.D. 1.0 0.5 4.8 1.4 16 dgys after withdrawal from aflatoxin-spiked- Control Group Mean 12.9 3.4 38.2 9.5 S.D. 2.5 ' 1.2 5.2 2.5 Experimental Group Mean 11.9 2.3 35.6 9.6 S.D. 2.2 0.8 8.1 3.4 28.8 4.8 29.7 203 47.1 5.9 39.5 6.0 67.1 10.2 74.9 12.5 ration 69.9 7.1 66.7 13.0 a The livers from the eXperimental group were significantly heavier than those of the control group (P < 0.01). b Kidneys from the experimental group were significantly heavier than those for the control group (P < 0.01). 68 Table 8 -- Summary of organ weights expressed as a percentage of live body weight for both the control and the experimental groups (0 day and 16 days after with- drawal from aflatoxin-Spiked ration). Heart Spleen ORGANS Liver Kidney Gizzard Intestine 0 day after withdrawal from aflatotin-sniked ration Control Group Mean 0.57 0.10 S.D. 0.05 0.01 Experimental Group Mean 0.62 0.12 S.D. 0.09 0.04 2.03 0.17 2.73a 0.61 0-37 0.15 0.61b 0.16 1.74 0.14 1.82 0.13 4.08 0.51 4.54 0.42 16 days after withdrawal from aflatoxin-spiked ration Control Group Mean 0.47 0.12 1.46 0.37 1.85 2.74 S.D. 0.05 0.04 0.12 0.09 0.28 0.18 Experimental Group Mean 0.54 0.10 1.51 0.39 1.71 3.18 S.D. 0.04 0.02 0.13 0.10 0.28 0.82 a b The livers from the experimental group were significantly heavier than those of the control group (P<0.0005). The kidneys from the experimental group were significantly heavier than those of the control group (P<0.0005). 69 enlarged (Table 7) when compared to those of the control group (P<0.1). Gall bladder enlargement was also noticed in the inspection for gross lesions. Differences in the weights of the livers, kidneys and hearts between the control and the experimental groups were not significant at 16 days after removal of aflatoxins from the diet, but were signifi- cant at all earlier times. In this study, several birds with slipped tendons were observed in both the control and the experimental groups. The incidence of slipped tendons in the control group (3 out of 12) was higher than that in the experimental group (3 out of 36). However, the occurrence of slipped tendons is not believed to be associated with the treatments. These observations are in agreement with Washburn gt gt. (1976), who indicated that the relatively high incidence of leg problems is related to the rapid growth rate of broiler chickens, and that birds growing at comparatively high growth rates are more inclined to develop leg problems. The incidence of all other symptoms of aflatoxicosis gradually decreased with the time elapsing after withdrawal from the aflatoxin-spiked rations. This indicates that the chicks recover from aflatoxicosis after removal of aflatoxins from the ration. Similar results have also been reported by other authors (Blount, 1961; Smith and Hamilton, 1970). 70 AFLAggXIN RESIDUES tN TI-EE: TISSUES 0F CHICKS FED A CONTAMINATED DIET Results of aflatoxin analysis for the tissues of birds slaughtered following removal of the contaminated feed (no withdrawal period) are presented in Table 9. As expected there were no detectable amounts of aflatoxin residues in any of the tissues from the birds in the control group. Measur- able amounts of aflatoxins B1 and B2 were found in all analyz- ed tissues of all the birds fed the aflatoxin-spiked ration. The highest levels were found in the gizzards, and the livers contained the second highest levels. Measurable amounts of aflatoxins M1 and M2, the metabolites of aflatoxins B1 and B2, were also found in the most of the tissues of the chicks in the experimental group. . Although the gizzards had the highest levels of afla- toxins B1 and B2 with mean values of 0.26 and 0.19.ug/kg, respectively, it may have been caused by direct contamination. In the slaughtering procedure, the Opening of the gizzards with scissors may have caused direct contact of the surfaces of the organ with the undigested feed inside the gizzard and may have led to contamination of the tissues. Further support for contamination is found by the ratio of aflatoxin B to 1 B2 in the gizzards (1.32), which is similar to the ratio in the aflatoxin-spiked ration (1.55). The relatively small standard deviation of the ratio (0.24) in the gizzards indi- cated that the individual values were within a rather narrow range. Other tissues did not have B1 to B2 ratios similar 71 Table 9 -- Aflatoxin residues detected in selected chick tissues at 0 day after withdrawal from the aflatoxin-Spiked ration (ug/kg). Levels of Aflatoxinsa Chick No. B M 1 2 1 2 1 2 1 2 ng Muscle Breast Muscle 01 0.13 0.02 tr. 0 0.08 0.02 0.02 tr. 02 0.04 0.02 0.02 0 0.02 0.01 0.03 0 03 0.16 0.01 tr. tr. 0 0.01 O 0 04 0.05 0.02 0.06 0 0.02 0.05 0.05 0 05 0.09 0.02 0.04 0 0.06 0.04 tr. 0.01 06 0.06 0.02 0.03 0 0.08 0.04 0.02 tr. Mean 0.09 0.02 0.02 tr. 0.04 0.03 0.02 tr. Liver Gizzard 01 0.13 0.03 0.05 1.50 0.26 0.20 0 0.31 02 0.29 0.13 0.05 3.00 0.11 0.07 tr. 0 03 0.07 0.06 0.04 6.53 0.50 0.33 tr. 0.05 04 0.22 0.13 0.14 0.64 0.16 0.17 0.03 tr. 05 0.06 0.10 0 1.13 0.35 0.24 0 0.12 06 0.22 0.12 0.06 0.87 0.17 0.15 tr. 0.02 Mean 0.17 0.10 0.06 2.27 0.26 0.19 tr. 0.08 Heartb Kidneyb Mean 0.08 0.06 0 0.19 0.05 0.05 0.10 2.07 a tr = trace amounts, visible but too small of an amount to quantitate (< 0.02.ug/kg). Composite of six samples in one analysis. 72 to that in the feed and such small standard deviations (Table 11). In contrast to aflatoxins B1 and B2, aflatoxins M1 and M2 residues in the gizzard were low (Table 10). This may also indicate that the high levels of aflatoxins B1 and B2 in the gizzards were caused by direct contact with the conta- minated feed. Table 10 -- The total amounts of aflatoxins B1 and B2, M and M2, and B1, B2, M1 and M2 in the tissues of chiCks slaughtered 0 day after withdrawal 1 from the aflatoxin-spiked rationa. AFLATOXINS ( ng/kg) TISSUES B1 + sz M1.+ M20 B1 + B2 + M1 + M2 Leg Muscle 0.11 0.03 0.14 Breast Muscle 0.07 0.02 0.09 Liver 0.27 2.33 2.60 Kidney 0.10 2.08 2.18 Heart 0.14 0.19 0.33 Gizzard 0.45 0.08 0.53 a Data were calculated from the mean values presented in Table 9. The sum of aflatoxins B and B2 represented the carry 1 over of ingested aflatoxins to tissues. The sum of aflatoxins M1 and M2 represented the deposit of aflatoxins metabolites in the tissues. 73 .Coapmfl>op UMMUCMPm O .mm.a 003 000000 eoxeamueaxopsaes 0:0 :0 A0m\fimv 00 or am assessaes to 00000 009 e .m manna SH mosam> Scam popMHSoHMo one; 0900 0 00.0 00.H 00.0 00.0 0.0.0 00. mm. 0m.a 00.H m0.a ma.0 0002 ma.a mmla 00.0 00.0 00 00.H 00.0 00.H 00.: 00 00.0 00.H 00.0 0m.0 00 Hm.a SH.0 0 00.0H m0 sm.e 00.0 00.0 00.0 00 00.H 00.: 00.0 0m.0 H0 hoccflm pmmom 6900000 no>wq odomsz Pmmomm oHomsz mod .02 x0030 mmpmmHe .p.m:oflpmn poxflmmnsfixopmdmm esp Scam stmspnvfls sopmm amp 0 00 0000000 00000 0opooa00 ea A0m\amv 0m or am saxoesfies so 00000 are I- as 00009 7L: The total amount of aflatoxins (B1 + B2 + M1 + M2) was highest in the livers and kidneys (Table 10). These two organs also showed the highest residual levels of aflatoxins M1 and M2 (Table 10). These results agree with those report- ed by Furtado gt gt. (1982), who found the highest amounts of aflatoxins B1, B2, M1 and M2 in the livers and kidneys of pigs fed an aflatoxin-contaminated ration in two different feeding trials. The capacity of the liver and kidneys to concentrate aflatoxins is probably associated with their important role in the metabolism and elimination of xenobiotics (Klaassen, 1980; Neal gt gt., 1981). Except for the livers and kidneys, the levels of afla- toxins M1 and M2 were much lower than the levels of B and B 1 (Table 10). This is probably because of the higher polarity 2 and increased water solubility of aflatoxins M1 and M2, which are more easily eliminated from the tissues than the unmetabo- lized aflatoxins, B1 and B2. These hydroxylated aflatoxins may be excreted either as the original compound or as afla- toxin conjugates after being bound to various endogenous compounds (Mabee and Chipley,1973a; 1973b; Neal gt,gt., 1981). These results are consistent with the findings of Furtado gt gt. (1982) for the tissues of pigs fed aflatoxins. In the present study, the aflatoxin M2 levels in the livers and kidneys were 2.27 and 2.07.0g/kg, respectively. These values were significantly higher than the corresponding aflatoxin M1 level of 0.17 and 0.10 ug/kg for the same organs. The high levels of aflatoxin M2 in the livers and kidneys may be associated with conversion of aflatoxin B1 to B2 and 75 aflatoxin M1 to M2, respectively. Patterson and Allcroft (1970) reported that aflatoxins B1 and M1 were reduced to their corresponding reduction products, Bz'and M2, for tp, vitro liver preparations from chickens, ducklings, guinea pigs and mice. Table 12 -- Ratios of aflatoxins B1 or B2 levels in the feed in relation to aflatoxins M1 or M2 levels in livers and kidneys. FEED.T0 TISSUE AFLATOXIN TISSUE RATIOa’b Liver B1 12,000 M1 34.283 B2 13,228 M2 583 Kidney B1 41,140 M1 20.570 32 26,456 M2 639 a Higher values indicate more efficient tissue clearance. b Values for M1 and M2 were calculated based on the assumption that B1 and B2 were entirely converted to these their respective metabolites. As indicated in the Table 12, the ratio of aflatoxin B1 in the feed in relation to aflatoxin B1 in the livers was 12,100. This value is approximately 10 times higher than that of 1,200 predicted by Rodricks and Stoloff (1977), which was an estimated value taken from the data of several studies (Table 1). The difference may be due to the various parame- ters reported by Rodricks and Stoloff (1977) to effect the 76 pattern of tissue residues. These include: (1) the Species and breed of animals, (2) the level of exposure. (3) the diet and health status of the animals, and (4) the time interval after cessation of aflatoxin exposure before slaughter and collection of samples for analysis. The difference could also be due to the fact that other aflatoxins in the ingested feed may interfere with the metabolism of aflatoxin B1. Swenson (1981) indicated that the biotransformations of afla- toxin B1 to M1, B2 to M2 and G1 to GM1 may be catalyzed by the same enzyme system. Further support for this vieWpoint comes from the study of Mintzlaff gt gt. (1977). in which broilers were fed different levels of aflatoxin B1 and 5 times as much G1 for a period of 8 weeks. The birds were killed at the end of the experiment, and the livers and muscle tissues were ana- lyzed for aflatoxin residues. The authors found that the livers of the chickens fed aflatoxin B1 at a level of 2,000 ppb contained 7.9 ppb of aflatoxin B1. This value is consi- derably higher than the level of aflatoxin B1 found in the livers of the chicks in the present study (0.17 ppb). The difference may be due to the high level of aflatoxin G1 in the feed used in the study by Mintzlaff gt gt. (1977). On calculating the total amount of aflatoxins B1, B2 and G1 in the feed of the present study, the level was equivalent to 563 ppb of aflatoxin B1, which is similar to the level of 500 ppb used in the study of Mintzlaff gt gt. (1977). On comparing these two values, aflatoxin B1 residues for the present study (liver, 0.17; leg muscle, 0.09; breast muscle, 77 0.04 ug/kg of tissue) are fairly comparable to those reported by Mintzlaff gt gt. (1977) for 500 ppb of aflatoxin B1 in the feed (liver, 0.38; leg muscle, 0.06; breast muscle, 0.16 ug/ kg of tissue). According to Wogan (1968) and Smith and Hamilton (1970), the L'D5O of different animals to aflatoxins varies from 500 to 16,5000 .ug/kg of body weight. In the present study, the total amounts of aflatoxins deposited in different tissues ranged from 0.09 to 2.60 ppb at 0 day after withdrawal from the afla— toxin-contaminated diet (Table 10), and the highest level was found in the livers. Thus, it appears that there is little likelihood of acute toxicity to man from eating these afla- toxin-contaminated tissues. However, the levels may be high enough to cause chronic toxic effects during prolonged exposure to the contaminated tissues. WITHDRAWAL TRIAL Results of the withdrawal trial showed that broiler chickens can metabolize and remove aflatoxins from their tissues. One day after withdrawal from the aflatoxin-contaminated feed, measurable amounts of aflatoxins B1 and B2 were still found in the livers, gizzards, leg and breast muscles of most of the chickens (Table 13). Except for one gizzard sample, which had an unusually high level of aflatoxin B1 (0.39 ppb), the levels of aflatoxins B1 and B2 in all the analyzed tissues one day after withdrawal from the contaminated feed were far lower than the levels found in the tissues of the chickens at the time of withdrawal. There were no detectable amounts of 78 Table 13 -- Aflatoxin residues detected in selected chick tissues at 1 day after withdrawal from the aflatoxin-spiked ration (ug/kg). Levels of Aflatoxin a Chick No. B1 B2 M1 M2 B1 B2 M1 M2 Leg Musclg Breast Muscle 11 0.07 0 0 0.03 0 0 0 0 12 0.05 0 0 tr. 0 0.05 0 0 13 0.03 0.06 0 0.08 0.06 0' 0 0 14 0.03 0 0 0 tr. tr. 0 0 15 0.04 0 0 0 0.03 0.05 0 0 16 0.03 0.02 0 0 0.03 0 0 0 Mean 0.04 tr. 0 0.02 0.02 0.02 0 0 tgtgp_ Gizzard 11 0 tr. tr. 0.75 0.39 0.03 tr. 0 12 O 0.05 0.02 0.28 tr. tr. 0 0 13 0.06 0.03 0.08 0.12 0.06 tr. 0.02 0 14 0.19 0.08 0 0.06 0.07 0.06 0 0 15 0.07 0.03 0.13 0.77 tr. 0.02 0 0 16 ' 0.25 0.13 0 0.22 0.07 0.04 tr. tr. Mean 0.10 0.05 0.04 0.38 0.09 0.03 tr. 0 Heartb Kidneyb Mean 0 0 0 0 0 0.03 0 0.16 a tr = trace amounts, visible but too small of an amount to quantitate (< 0.02 ug/kg). b . . . . Compos1te of Six samples ln one analySls. 79 aflatoxins B1 and M1 in the kidneys, but measurable amounts of aflatoxins B2 and M2 were found in the same organ. Hearts were completely free of detectable aflatoxins one day after withdrawal from the contaminated feed. Two days after placing the birds on the aflatoxins free diet, with the exception of the livers, only a few samples contained detectable levels of aflatoxins (Table 14). Measurable amounts of aflatoxin B2 were found in only one liver and one breast muscle sample from two different chickens. However, measurable amounts of aflatoxin M2 were found in the livers (4 out of 6) and breast muscles (2 out of 6) two days after placing the birds on the aflatoxin-free diet. Although the levels of aflatoxins B1, B2 and M1 in the tissues consistently decreased after removal of the birds from the contaminated diet, the levels of aflatoxin M2 in the muscle tissues first tended to increase and then declined during withdrawal. Only trace amounts of aflatoxin M2 were found in the leg muscles of chickens killed without any with- drawal period, but an average of 0.02 ppb of aflatoxin M2 was found one day after withdrawal from the aflatoxin-spiked diet. Only trace amounts of aflatoxin M2 were detected in the breast muscle of chickens killed without any withdrawal period, and no aflatoxin M2 was detected in the same tissue one day after withdrawal from the contaminated feed. However, 2 out of 6 chickens had detectable amounts of aflatoxin M2 in the breast muscles two days after withdrawal from the contaminated feed. This might indicate that the aflatoxin metabolizing enzyme 80 Table 14 -- Aflatoxin residues detected in selected chick tissues at 2 days after withdrawal from the aflatoxin-spiked ration (ug/kg). Level of Aflatoxina Chick No. B1 B2 M1 M2 B1 B2 M1 M2 Leg Muscle Breast Muscle 21 0 0 0 0 0 0 O 0 22 tr. 0 0 0 O 0.03 O O 23 O 0 0 0 O 0 O 0 24 0 0 0 0 0 0 0 0 25 '0 0 O 0 O O 0 0.02 26 0 0 O O O 0 0 0.03 Mean 0 0 0 0 0 tr. 0 tr. Liver Gizzard 21 O 0 0 0.08 0 O O 0 22 0 0 0 0.18 0 0 O 0 23 0 tr. 0 0.04 0 0 0 O 24 0 0.03 O 0.11 0 O 0 O 25 0 tr. 0 O 0 O O O 26 0 0 O 0 O 0 0 0 Mean 0 tr. 0 0.07 O 0 0 O Heartb Kidneyb Mean 0 0 0 0 0 O 0 O a tr = trace amounts, visible but too small of an amount to quantitate (< 0.02.ug/kg). b Composite of six samples in one analysis. 81 systems were altered during aflatoxicosis, but after with- drawal from the aflatoxins that the enzyme systems may have recovered their ability to metabolize the toxic components again. In studying albino rats, Singh and Clausen (1980) found that aflatoxin B1 induced aryl hydrocarbon hydroxylase tem) activity in the liver, but depressed the system in kidney and brain. Results of the withdrawal trial showed that aflatoxins B1 and M chickens than aflatoxins B2 and M2. This may be due to the 1 were more efficiently removed from tissues of broiler fact that aflatoxins B and M1 can be biotransformed into 1 aflatoxins B2 and M2 as mentioned earlier herein (Patterson and Allcroft, 1970). The high excretion rates of aflatoxins B1 and M1 may be also due to the fact that the vinyl ether double bond in the 2,3-position can be easily biotransformed into the 2,3-epoxide and bind to the nucleoside, or be further hydrated into the dihydrodiol compounds of aflatoxins B1 and M1. £0.11322s aflatoxin B1 can be rapidly and almost exclu- sively biotransformed into the dihydrodiol compound of afla- toxin B1 (2,3-dihydro-2,3-dihydroxy-aflatoxin B1) by chicken liver preparations (Neal gt gt., 1981). The vinyl ether double bond is not present in aflatoxins B2 and M2 as shown in Fig. 1 (Brechbuhler gt gt., 1967), which may also help to explain the lower excretion rate of aflatoxins B2 and M2. Four days after putting the birds on an aflatoxin- free diet, there were no detectable levels of aflatoxins in any of the tissues (Table 15). This suggests that placing 82 Table 15 -- Aflatoxin residues detected in selected chick tissues at 4 days after withdrawal from the aflatoxin-spiked ration (Mg/kg). Levels of Aflatoxina Chick No. B B M 1 2 1 2 1 2 1 2 Leg Muscle Breast Muscle 41 0 0 O O 0 0 0 0 42 0 O 0 0 0 0 0 0 43 0 O 0 0 0 0 0 0 44 0 0 0 0 O O O 0 45 O O O 0 O 0 0 0 46 0 0 0 0 0 0 0 0 Mean 0 O 0 0 0 0 O '0 Liver Gizzard 41 0 0 O 0 0 0 0 0 42 O O O O 0 O O O 43 0 0 0 O O O O O 44 O 0 O 0 0 0 O O 45 O 0 0 O 0 0 0 0 46 O O O 0 0 0 0 0 Mean 0 0 0 0 0 0 0 0 b . b Heart Kidney Mean 0 O O 0 0 O 0 0 a tr = trace amounts, visible but too small of an amount to quantitate (< 0.02 ug/kg). b I I I o CompOSlte of Slx samples in one analySls. 83 chickens on an aflatoxin-free diet for 4 days prior to slaugh— ter is adequate to remove detectable levels of free aflatoxins and their metabolites from the tissues of chicks that had pre- viously been fed a highly contaminated diet. This result is consistent with the finding of Furtado gt gt. (1982), who found that pigs cleared aflatoxins from their tissues within 4 days after removal from the diet. The present study suggests that only a small fraction of the aflatoxins consumed was deposited in the tissues of the chickens, and that it was readily removed after withdrawal from the aflatoxin-contaminated feed. Part of the ingested aflatoxins may be excreted into feces as the original afla- toxins, as one of the various metabolites, or as one of their water soluble conjugates. Mabee and Chipley (1973a) found that 90.64 per cent of the inc-activity was excreted in the feces of broiler chickens fed 1[LC-labeled aflatoxin B1. The excretion of aflatoxins or their metabolites into the intestine via the bile also seems to be a major pathway by which absorbed aflatoxins are excreted (Sawhney gt gt., 1973). Mabee and Chipley (1973a) found that aflatoxin M1 glucuronide was one of the major metabolites isolated from 14 the tissues of broiler chickens dosed with C-aflatoxin B1. Aflatoxin P1 has also been found to be a major urinary metabo- lite of aflatoxin B1 in rhesus monkeys according to Dalezios and Wogan (1972). It is excreted predominantly in the form of water soluble glucuronides and sulfate conjugates. Hayes gt gt. (1977) found that upon incubation of 1"LC-aflatoxin B 1 with bovine liver preparations, about 60 per cent of the 84 original activity was transformed to water soluble materials, and approximately 10 per cent was metabolized to aflatoxins M1, Q1 and two unidentified metabolites. Aflatoxin Ql’ however, was not isolated from the tissues of cows fed afla- toxin-contaminated feed. Thus, these authors suggested that aflatoxin Q1 was in conjugated form in vivo. All these con- jugates have enhanced water solubility and reduced solubility in other solvents, such as chloroform; thus they are not ex- tracted by classical methods (Mabee and Chipley, 1973a). T These conjugates are believed to be excreted faster than free aflatoxins because of their higher water solubility (Klaassen, 1980). I < ivo and in vitro studies have provided the evi- dence that 2,3-dihydro-2,3-dihydroxy-aflatoxin B is the 1 major metabolite of aflatoxin B1. Neal gt gt. (1981) pointed out that previous reports on the production of aflatoxin B2a were erroneous (Patterson and Roberts, 1972; Gurtoo and Campbell, 1974; Gurtoo and Dahms, 1974). The error was due to the fact that aflatoxin B and 2,3-dihydro-2,3-dihydroxy- 2a aflatoxin B1 have similar UV Spectral characteristics (Swenson gt gt., 1973; 1977; Neal gt gt., 1981). Some 2,3-dihydro-2,3- dihydroxy—aflatoxin B1 has been found to be covalently bound to the microsomal proteins (Gurtoo and Campbell, 1974). Trans— formation of aflatoxin B1 to 2,3-dihydro-2,3-dihydroxy-afla— toxin B1 was found to be a major pathway when compared to the biotransformation of aflatoxin B to M1 by liver preparations 1 from rabbits, ducklings, guinea pigs and rats (Patterson and 85 Allcroft, 1970). Neal gt gt. (1981) compared the metabolism of aflatoxin B1 in liver preparations from rats, mice, guinea _pigs and chickens. These authors found that chicken liver microsomes metabolized aflatoxin B1 at the highest rate, and it was almost exclusively transformed into 2,3-dihydro-2,3- dihydroxy-aflatoxin B1. If chickens behave the same as in the 12.21322 study (Neal gt_gt., 1981), the formation of the 2,3-dihydro-2,3-dihydroxy-aflatoxin B may be a significant 1 metabolic pathway. Aflatoxin B28. was detected in the urine of rats, which had been injected with aflatoxin B2 (Dann gt gt., 1972). Both aflatoxin B2a and 2,3-dihydro-2,3-dihydroxy-aflatoxin B1 may rearrange themselves to form dialdehydic phenolated reso- nance hydrid ions at physiological and alkaline pH values. In this form, they may react with amino acids, peptites and proteins to form Schiff bases as shown in Fig. 3 (Patterson and Roberts, 1971; 1972; Gurtoo and Campbell, 1974). These aflatoxins will probably remain in the tissues longer than the free or conjugated aflatoxins. This may explain the results of Sawhney gt gt. (1973), who fed White Leghorn hens a single oral dose of ring-labeled aflatoxins, and still re— covered the radio activity in the excrement 7 days after the aflatoxin treatment. Thus, more studies on the toxicity and metabolism of aflatoxin-adducts are needed. SUMMARY AND CONCLUSIONS A feeding trial was conducted to determine the amount of time necessary for tissue clearance of aflatoxins from broiler chickens fed an aflatoxin-contaminated diet. There were 48 chicks in the trial, with 12 being fed the control diet and 36 being used to determine the time necessary for tissue clearance after removal from the contaminated diet. The diet used in the experimental group was spiked with afla- toxins B1 and B2 to give a concentration of 2057 and 1323 11g of aflatoxins B1 and BZ/kg of feed, respectively. The control ration was Shown to be free of aflatoxins. The ini— tial phase of the feeding trial in which the chicks were fed either the control or the spiked diet lasted for 35 days. Results demonstrated that there was a significant difference in the final weight and the rate of weight gain between the controls and the group fed the aflatoxin-spiked ration. The chicks fed aflatoxins exhibited depressed growth and on the average gained 8 per cent less weight than the control chicks over the 35 day feeding trial. There was also a 29 per cent increase in the liver weights and 48 per cent increase in the kidney weights of the chicks fed afla— toxins in comparison to the control group. Tissues and organs from the control group were normal in appearance. On examination for gross lesions in the tissues 86 87 of the experimental chicks, several distinctive symptoms were observed, which included: (1) Hemorrhagic spots were present on the surfaces of both the muscles and the liverS: (2) the livers were pale and exhibited fatty infiltration; and (3) The gall bladders were enlarged. Although slipped tendons were observed, they were present in both the control and the experimental groups and appeared to be related to the rapid growth rates. All symptoms of aflatoxicosis gradually de— creased after withdrawal of the aflatoxin—spiked rations. Analysis of the tissues of chickens at 0 day with- drawal from the aflatoxin-spiked ration showed that only a small fraction of the aflatoxins consumed was deposited in the tissues, either as the original compound or as their metabolites. The aflatoxins were found to be widely distri- buted in all tissues. The highest levels of aflatoxins were present in the liver and kidneys. The mean value for the combined aflatoxins in all tissues was less than 3 ppb. The leg and breast muscles showed the lowest levels of resi— dual aflatoxins with an average of less than 0.2 ppb. Except for the liver and kidneys, the levels of aflatoxins M1 and M2 were much lower than those of B1 and B2. Results from the withdrawal trial showed that the broiler chickens rapidly metabolized and removed aflatoxins from their tissues. Four days after placing the chickens on an aflatoxin-free diet, there were no detectable levels of aflatoxins in any of the tissues. 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