)VIESI.} RETURNING MATERIALS: Place in book drop to LIBRARJES remove this checkout from __. your record. FINES wi‘H ~ be charged if book is returned after the date stamped below. DEPOSITION AND CLEARANCE OF AFLATOXINS IN THE EGGS AND TISSUES OF LAYING HENS FED A CONTAMINATED DIET By Arlene Wolzak-Kappes A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 198& ABSTRACT DEPOSITION AND CLEARANCE OF AFLATOXINS IN THE EGGS AND TISSUES OF LAYING HENS FED A CONTAMINATED DIET By Arlene Wolzak-Kappes A trial was conducted to determine the levels of afla- toxins deposited in the eggs and tissues of laying hens fed an aflatoxin-spiked diet and the time necessary to achieve clearance upon removal of the contaminated diet. Sixty-four hens were fed a ration containing 3.310 and 1,680 ug/kg of aflatoxins B1 and B2. respectively, for h weeks, while 16 control hens received an aflatoxin free ration. At the end of aflatoxin feeding. 16 treated and 8 control hens were sac- rificed. The remaining treated and control hens were fed the unspiked ration for 32 days. Eight treated hens were sacri- ficed at 2, h, 8, 16 and 2h days after withdrawal. All remaining hens were slaughtered at 32 days. Aflatoxins caused a significant decrease in egg produc- tion and egg weights by the third and fourth weeks of feed- ing, respectively. Both returned to normal by the second week of withdrawal. At the end of aflatoxin feeding, livers. kidneys and spleens of treated hens were significantly larger, while ovaries and hearts were significantly smaller than controls. Livers were pale and hemorrhagic and ovaries had Arlene Wolzak-Kappes only small ova (( 25 mm). After 32 days withdrawal all organ weights, except hearts, were not different from controls. Transfer of aflatoxins to the eggs occurred rapidly with levels reaching a maximum after A-5 days and remaining rela- tively constant throughout aflatoxin feeding. Combined resi- due levels in whole eggs were (0.5 ug/kg. Levels of afla- toxins B2, M1 and M2 were similar in yolk and albumen, while levels of B1 and B2a were higher in the yolk. Aflatoxin residues were widely distributed in tissues, but comprised only a small fraction of total consumption. Aflatoxin B2a was tentatively identified as the most abun- dant residue. Highest residue levels were detected in giz- zard, kidneys and liver and amounted to combined levels of. (.3 ug/kg in each. Upon removal of the spiked diet, residues in eggs and tissues decreased rapidly. There were differences between tissues and between hens in the time required for clearance. AFB2 was still detected in the liver of one hen 32 days after withdrawal. However, no detectable aflatoxins were present in eggs and most tissues after A and 8 days on the unspiked diet, respectively. ACKNOWLEDGEMENTS I would like to express appreciation to my major professor, Dr. A.M. Pearson for for his help and guidance during completion of this project. Appreciation is also extended to Dr. S.D. Aust, Dr. M. Bennink, Dr. J.R. Brunner and Dr. J.I. Gray for serving as members of the Guidance Committee and for reviewing this thesis. Special thanks are expressed to Dr. T.H. Coleman and to the staff of the Poultry Research Center for their help during the experimental trial and to Dr. W.T. Magee for his advice in the statistical analysis of the data. I would like to thank my parents for their love and encouragement at all times. and my husband, Ewald, for his loving support during these last difficult years. ii TABLE OF CONTENTS Page LIST OF TABLES ............................... vi LIST OF FIGURES .............................. Viii INTRODUCTION ................................. 1 REVIEW OF LITERATURE ......................... A Discovery of Aflatoxins .................... 4 Characterization and Structure Elucidation .. 5 Chemistry of Aflatoxins .................... 7 Occurrence of Aflatoxins ................... 10 Biological Activity of Aflatoxins .......... 11 Aflatoxicosis in Man ....................... 15 Metabolism and Toxicity of Aflatoxins ...... 19 specific Effects of Aflatoxin on Laying Hens. 3h Occurrence of Aflatoxins in Foods of Animal Origin ..................................... #5 Stability of Aflatoxins in Foods ........... 61 EXPERIMENTAL ................................. 68 Feeding Trial .............................. 68 Preparation of the Diet .................. 68 Experimental Birds ....................... 71 Analysis of Aflatoxins ..................... 72 Analysis of Aflatoxins in Eggs ........... 72 Sample Preparation ..................... 72 Extraction of Aflatoxins from Eggs ..... Purification of Aflatoxins ............. LiquidéLiquid Partition .............. Cleanup by Silica Gel Column Chromato- graphy ..IOIOOOOOOOOIIAOOOOOO.00......O Thin Layer Chromatography ............... Qualitative Thin Layer Chromatography . Quantitative Thin Layer Chromato- graphy 0.00.00.........IOIOOOOOOOIOOO. Densitometric Analysis ................ Analysis of Aflatoxins in Tissues ........ Sample Preparation ..................... Extraction of Aflatoxins from Tissues ... Purification of the Aflatoxin Extract .. Liquid-Liquid Partition .............. Cleanup by Silica Gel Column Chromato- graphy 0........OIOOOOOOOIOOOOOOIOOOOO Quantitative Thin Layer Chromatography .. Densitometric Analysis ................. Analysis of Aflatoxins in the Feed ....... Confirmatory Tests for Aflatoxins ......... General Test for Aflatoxins ............ Aflatoxin B1 ............................ Aflatoxin M1 ........................... Three Dimensional Chromatography ....... Preparation of Reference Standards ........ MOiSture AnalbrSiS ...-00000000000000000000000 iv 7A 75 '75 76 77 77 79 82 83 83 8h 85 85 86 86 87 87 88 88 88 89 9o 91 92 COOking Of Egg samples ....IOOOOOOOOOOCOOCIOO Safety Procedures . Statistical Analysis RESULTS AND DISCUSSION FEEDING TRIAL ..... Feed and Aflatoxin Consumption ........... Effect of Aflatoxins on Egg Production .... Effect of Aflatoxins on Egg Weight ....... Effects of Aflatoxins on Body Weights ..... Gross Observations on Tissues ............ Effects of Aflatoxins on Organ Weights .... AFLATOXIN RESIDUES Tentative Identification of Unknown Aflato- xin Metabolite in Eggs and Tissues of Laying Hens ..... Aflatoxin Residues Fed a Contaminated Aflatoxin Residues Egg Yolks ....... Aflatoxin Residues Withdrawal ...... in the Eggs of Hens Diet IDIOIIOOOOOOOIOOOO in Egg Albumen and in Eggs During Whale Eggs ......OIOOOOO......OOOOOOOOOO Egg Albumen and Egg Yolks ............... Effects of Cooking Aflatoxins in Eggs Aflatoxin Residues in the Tissues and Organs on the Levels of of Hens Fed an Aflatoxin-Contaminated Diet. Aflatoxins Residues in Tissues and Organs of Hens During Withdrawal ................ SUWARY ......OIOOOOOOOOOOI.0.0.0....0...... LIST OF REFERENCES V 92 9A 95 96 96 96 98 10A 109 113 116 122 122 12A 130 135 135 137 139 1&5 159 172 176 Table 10 11 12 13 1h LIST OF TABLES Ratios of Aflatoxin B1 Levels in the Feed in Relation to Aflatoxin B1 or M1 Levels in Edible Tissues (Rodricks and Stoloff. 1977) ooocoo0.000000000000000000.00.000.00 Nutrient Specifications of the Layer Hen maSh ....00......00............OIOOOCOCOOO Vitamin and Trace Mineral Mix for Layer maSh .0.0.....IOOOOOOOOOOOOOOOOOO0.0.0.... Aflatoxins Consumed by Laying Hens ....... Effect of Aflatoxins on Egg Production ... Effect of Aflatoxins on Egg Production and Egg Weights During the Feeding Trial ..... Effect of Aflatoxins on Egg Weight ....... Average Liver Weights of Aflatoxin-Fed Hens During Clearance .................... Effect of Aflatoxins on the Liver of Hens. Effect of Aflatoxins on Ova Development .. Effect of Aflatoxins on Organ Weights Ex— pressed as Percent of Body Weight ........ Average Organ Weights as Percent of Body Weight of Laying Hens During Withdrawal .. Comparative Thin Layer Chromatography of the Unknown Aflatoxin Metabolite in Dif- ferent Solvent Systems ................... Aflatoxin Residues Detected in Eggs During the First Week of Aflatoxin Feeding ...... vi Page 47 69 70 99 100 101 105 112 11A 115 117 121 123 125 Table 15 16 17 18 19 20 21 22 23 2h 25 26 27 Aflatoxin Residues Detected in Eggs During the Aflatoxin Feeding Period ............... Aflatoxin Residues Detected in the Albumen and Yolks of Eggs Laid During Aflatoxin Feeding I00.0.00........IIOOOOOOOOOOOOO...O. Aflatoxin Residues in Eggs After Withdrawal from the AflatOXin-Spiked Diet 0 o o I o o o o o o o O 0 Aflatoxin Residues Detected in the Albumen and Yolk of Eggs After Withdrawal from the AflatOXin-Spiked Ration coo-000.000.000.000 Effect of Cooking on Aflatoxin Levels in Eggs From Hens Fed an Aflatoxin—Spiked Diet ......I..........OOCOOOCOOOCOIOOOI..... Percent Recoveries of Spiked Aflatoxins During COOking Of Eggs 00.000000000000000... Aflatoxin Residues Detected in the Tissues of Aflatoxin-Fed Hens at the End of the Aflatoxin Feeding Period .................-- Average Total Amounts and Ratios Between Aflatoxins B1 and 82 and Their Metabolites M1, M2 and 32a Deposited in Tissues of Hens Fed an Aflatoxin-Spiked Diet ............-- Average Calculated Values for Aflatoxins Found in the Tissues of Hens Sacrificed at the End Of AflatOXin Feeding coo-0000000000 Ratios of Aflatoxins B1 and B2 in the Feed in Relation to Aflatoxins B1, B2, M1, M2 and B2a in the Tissues of Hens ............ Aflatoxin Residues Detected in the Tissues of Hens After 2 Days of Withdrawal From the AflatOXin-Spiked Diet 0 O O O O O O O O O I O O O O O O O O O I Aflatoxin Residues Detected in the Tissues of Hens After A Days of Withdrawal From the AflatoxinaSpiked Diet ............... Aflatoxin Residues Detected in the Tissues of Hens After 8 Days of Withdrawal From the AflatOXin-Spiked Diet OOOOOOIOOOOOOOOOOIOO vii 131 136 138 1&0 1A3 146 152 155 157 161 164 167 Figure 10 11 12 13 1h LIST OF FIGURES Structures of the major aflatoxins produced by Aspergillus fungi Structures of minor aflatoxins produced by Aspergillus fungi OOIOOOOOOOOOIOOCIOOIOI... ... Transformation of Aflatoxin B1 to various metabOlites ......OOOOOOOOOOIOOCO0.0.0.0... Metabolic transformations of Aflatoxin B2 .. Experimental Design of the Feeding Trial ... Spotting and Scoring Pattern for Two-Dimen- sion Double 10x20 cm TLC (Screening) ...... Spotting and Scoring Pattern for Two-Dimen- sional 20x20 cm TLC plate ................. Feed consumption by Laying Hens ........... Effect of Aflatoxins Effect of Aflatoxins Effect of Aflatoxins Egg Weight Expressed on Egg Production on Egg Weight ........ on Egg Production and as Percent of Control . Body Weight of Hens During the Feeding Trial .........0......IIIOOOOOCIOOOIIIOI... Changes in Body Weight of Hens During the Withdrawal Trial ......IOICCOCIOOOUOCOO.... Organ Weights as Percent of Body Weight of Hens During the Withdrawal Trial .......... viii Page 22 32 73 78 8O 97 102 106 108 110 111 119 INTRODUCTION Aflatoxins are secondary metabolites of certain strains of Aspergillus flavus and A. parasiticus. A. flavus, a common food spoilage fungus, can grow in a variety of foods and feeds either before harvest or during storage. The envi- ronmental requirements for aflatoxin production are relati- vely non—specific and almost any natural substrate sustaining the growth of this mold is also suitable for aflatoxin pro- duction. Experimentally. aflatoxins have been shown to be potent hepatocarcinogens in different animal species (Lancaster gt al., 1961; Wogan, 1973). as well as mutagens (Lilly, 1965) and teratogens (Di Paolo ££.§l-: 1967). Epidemiological studies in Africa and Southeast Asia have shown a strong correlation between aflatoxin ingestion and primary liver cell cancer (Peers gt a;., 1976; Shank gt_gi., 1972). The biological effects of aflatoxins vary with dose, du- ration of exposure, species, age and nutritional status of the affected animal. Aflatoxins are metabolized by cytoplas- mic or microsomal enzymes in the liver to more polar deriva- tives which can undergo conjugation with endogenous compounds. The increased polarity and water solubility of the conjuga- ted aflatoxin metabolites facilitates the excretion of the ingested nonpolar aflatoxins. 1 Consumption of aflatoxin contaminated feeds is responsi- ble for.aflatoxicosis in farm animals. Exposure to dietary levels which fail to induce overt aflatoxicosis in food producing animals may constitute a special hazard to humans (Armbrecht. 1971). It has been demonstrated that part of the ingested aflatoxins may be retained as the original afla- toxin or as one or several metabolites, some of which pos- sess toxic properties (Rodricks and Stoloff. 1977; Furtado ‘gg‘ai., 1982). The levels of aflatoxin residues found in products of animals consuming contaminated feed are very low compared to those present in directly contaminated feed and foods. Nevertheless. the potential risk to humans of prolonged exposure to low levels of aflatoxins in animal products can not be overlooked considering that under such conditions several animal species develop liver tumors (Wogan ‘gt‘ai., 197A; Sinnhuber gt a;.. 1970). The determination of aflatoxin residues in animal tissues is important in the evaluation of direct exposure of humans to these extremely toxic compounds. The assessment of low levels of aflatoxin residues deposited in eggs and tissues of hens consuming aflatoxin contaminated feed has been limi- ted by the complex lipid-protein matrix of animal tissues and by the presence of many fluorescent compounds. These factors have also limited the determination of the length of time required to achieve egg and tissue clearance of afla- toxin residues transferred from the feed. The present study was designed to: 1) Investigate the levels of aflatoxins and their metabolites carried over to eggs and those deposited in the tissues of laying hens fed an aflatoxin contaminated diet; 2) To determine the length of time required to achieve egg and tissue clearance after removal of aflatoxins from the diet of the laying hens; and 3) Finally to evaluate the effects of cooking on the aflato- xin levels in eggs. REVIEW OF LITERATURE Discovery of Aflatoxins Toxic metabolites of fungal origin have affected the health of both man and animals for many centuries (Hesseltine. 1979). During the 1950s, "moldy feed toxicosis" was described as being a serious livestock problem (Forgacs and Carll, 1962). Scientific interest on mycotoxins and the discovery of aflatoxins occurred in the early 1960s as a result of an acute outbreak of a lethal disease in turkey poults that caused an estimated loss of at least 100,000 birds in England. The disease, called Turkey X disease by Blount (1961), was characterized by anorexia, lethargy and muscular weakness. Postmortem examination indicated that the liver of the affect- ed birds was pale, fatty and showed extensive necrosis, as well as bile duct proliferation. At the same time British farmers suffered losses of partridge and pheasant poults (Asplin and Carnaghan. 1961). In addition several early re- ports suggested that cattle (Loosmore and Markson. 1961), pigs (Loosmore and Harding. 1961), chickens and ducklings (Asplin and Carnaghan. 1961), and sheep were also affected. In every case the outbreak occurred when animals had been fed rations containing groundnut meal imported from 5 Brazil. Although the Brazilian meal was the first implicated (Allcroft gplal., 1961), certain batches from other countries including Nigeria, West Africa, Gambia. East Africa and India also showed similar toxic properties (Sargeant ggwal.. 1961a). This distribution pattern suggested that moldy feed was in- volved. Sargeant ggwal. (1961b) identified the toxin produc- ing mold in the feed as Aspergillus flavus. The name aflatoé xin (from Aspergillus flavus tgxin) was given to the toxic chemicals of A, flavus before it was recognized that it com- prised a complex mixture of compounds (Nesbitt 2£.§l°9 1962). Characterization and Structure Elucidation Sargeant 23 El! (1961c) isolated a toxic material that had a blue fluorescence under UV light from groundnut meal. and later described the original bioassay method using 1-day old ducklings (Sargeant 22.El°v 1961b). Nesbitt §t_ai. (1962) resolved the toxic material into two fluorescent spots under UV light by thin layer chromatography (TLC) using alu- mina plates. These spots differed in the color of their fluorescence. The faster moving spot fluoresced blue, while the slower one exhibited a green fluorescence. For conve- nience these authors referred to them as aflatoxin B1 (blue fluorescence). and aflatoxin G1 (green fluorescence). The isolation and characterization of the four main afla- toxins was reported by Hartley 33 El- (1963). A crude mixture of the aflatoxins was extracted from a sterilized groundnut meal, which had been inoculated with a toxigenic strain of ‘A. flavus, and resolved into several fluorescent spots. The four main aflatoxins were separated on silica gel chromato- plates and named B1. B2..G1 and G2. based on the color of their fluorescence and on their relative chromatographic mobility. Further research into the chemical nature of these com- pounds led to structure elucidation of aflatoxins B1 and G1 by Asao 33.3i. (1965), and of B2 by Chang 23 al. (1963). Van der Merwe gg‘al. (1963) had shown that 82 and G2 were the dihydro-derivatives of B1 and G1. respectively. The la- boratory synthesis of aflatoxin B1 (AFBl) (Bfichi 23 al., 1967) and of B2 (Roberts gtflal., 1968) confirmed the struc- tures. Allcroft and Carnaghan (1962, 1963) found that cows fed "cattle cake" rich in aflatoxins produced milk that was toxic to 1-day old ducklings. Analysis of the milk (de Iongh gt. a;., 1964) showed only traces of AFB1, but demonstrated the presence of a blue violet fluorescent component which had an R1. value considerably smaller than pure AFBl. They called this new aflatoxin, "milk toxin". The "milk toxin" was also found in rat milk (de Iongh 33 al., 1964) and in the liver of rats (Butler and Clifford, 1965) fed AFBl. indicating that at least some of the ingested aflatoxin is converted in the liver into the "milk toxin". Allcroft 23 El- (1966) found "milk toxin" in the urine of non-lactating sheep given a single dose of mixed aflatoxins (B1. 32, G1 and G2). As a result of the observation that the "milk toxin” was present in other tissues and fluids of ani— mals ingesting aflatoxins, these authors suggested the trivial name of aflatoxin M for this new compound. Holzapfel 33_§i. (1966) isolated aflatoxin M from the urine of adult sheep given an i.p. dose of mixed aflatoxins, and were able to re- solve it into two components, which they designated as afla- toxins M1 and M2 on the basis of chromatographic mobility. Chemical and spectroscopic analysis led to structure eluci- dation of aflatoxin M1 (AFMI) as the 4-hydroxy-derivative of AFB1 and of M2 as the dihydro-derivative of AFM1 (Holzapfel 23 31-. 1966). Dutton and Heathcote (1966) isolated two additional afla- toxins from cultures of A, flavus, one of which fluoresced blue and the other green. These compounds were identified by spectroscopic analysis and selected chemical reactions as the 2-hydroxy-derivatives of aflatoxins B2 and G2, and were named aflatoxins B2a and G2a' respectively. These two aflato- xins are relatively non toxic to ducklings (Dutton and Heathcote, 1968). Chemistry of Aflatoxins The chemical structures of the above mentioned aflatoxins are shown in Figures 1 and 2. Aflatoxins are highly substitu- ted coumarins containing a fused dihydrofuran moiety. In addition this basic nucleus is attached to a pentenone ring as in aflatoxins B1, B2, M1 and M2, or to a 6-membered lactone as in the G series (Jones, 1978). In addition. the presence of other substituents,particularly hydroxyl groups, the re- duction of the ketone function in the cyclopentanone ring and the reduction of the 2,3-double bond. give rise to the different forms of aflatoxin. As many as 17 different com- pounds, all designated as aflatoxins have been isolated (WHO, 1979). These are mainly products of aflatoxin metab- olism by animals consuming contaminated feed and will be dis- cussed later herein. kolo O ..3 Loi. o Aflatoxin B1 Aflatoxin B2 3 I o lo’LO O OCH Aflatoxin G Aflatoxin G 1 2 Figure 1 - Structures of the major aflatoxins produced by Aspergillus fungi. 9 Aflatoxins are soluble in moderately polar solvents like methanol and chloroform, but only slightly soluble in water (WHO, 1979). The toxins absorb UV light (362-363 nm) with extinction coefficients varying from 16,100 for aflatoxins B2 and G1 to 21,800 for AFBI. Fluorescence emission is at 425 nm for aflatoxins B1 and B2, and at 450 nm for aflatoxins G1 and G2 (Wilson and Hayes, 1973). 0 0 ”.1... ° I. "cl 0 3 Aflatoxin M1 0 0 0 O | o I O * L o ,L ,L o HO/LO ooH3 H . O 0 OCH3 Aflatoxin B2a Aflatoxin G2a Figure 2 - Structures of minor aflatoxins produced by Aspergillus fungi. 10 Occurrence of Aflatoxins The presence of mycotoxins in foodstuffs can result from either direct or indirect contamination (Jarvis, 1975). Di- rect contamination occurs from the synthesis of mycotoxins by specific strains of fungi and is influenced by environmen- tal factors such as humidity and temperature. Consequently, mycotoxin contamination of foodstuffs varies with geographi- cal location. production and storage practices, as well as with the type of food since some food commodities are more suitable Substrates for fungal growth than others (Ciegler, 1978; Frazier and Westhoff, 1978). .Indirect routes of con- tamination are the use of aflatoxin contaminated food addi- tives or the transfer of mycotoxin residues to animal prod- ucts resulting from the use ofmycotoxin contaminated feed- stuffs (Jarvis, 1975). Aflatoxins are produced by some strains of Aspergillus flavus and A, parasiticus on many feed or food ingredients whenever the moisture content reaches 14 to 25% in a tempera- ture range of 25 to 45°C, if an adequate level of zinc is present (Edds. 1979). The water content of the substrate influences aflatoxin production mainly by its effect on the growth of A. flavus (Davis and Diener, 1970). Davis and Diener (1970) have shown that the lower moisture limit for the growth and production of aflatoxins by A, flavus is such that the moisture content is in equilibrium with a relative humidity of 85%. 11 Most of the toxigenic strains of A. flavus. which contam- inate plant products. mainly synthesize AFBl, followed by AFGl, while AFB2 and AFG2 occur in lower concentrations (Diener and Davis, 1966). The ratio of AFB1 to AFG1 varies directly with the storage temperature and with the strain (Schroeder and Ashworth, 1966). Other aflatoxins may be produced in minor amounts by cultures of A. flavus and A, parasiticus, includ- ing aflatoxins M1, M2, BZa' G2a' GM1 and aflatoxicol. Stoloff (1977) has reviewed the occurrence of aflatoxins in foods and feedstuffs. On the basis of the surveys carried out, he concluded that corn is the dietary staple in which aflatoxins are most likely to be encountered. Groundnuts and other oilseeds, such as cottonseed, Brazil nuts and pis- tachio nuts, bear a considerable risk of contamination. In the United States, the Southeast has the greatest risk of aflatoxin contamination of corn, while in some underdeveloped countries a major exposure can occur through unrefrigerated prepared foods at the household level (Stoloff, 1977)- Biological Activity of Aflatoxins Hayes (1978) has classified the effects of aflatoxins into two general groups: 1) short term effects including acute toxicity, and 2) long term effects, which include chronic toxicity. The suscpetibility of animals to both acute and chronic toxicity from aflatoxins depends on a number of factors. These include host factors, such as species (Ciegler, 1975), 12 sex (Purchase 23 filo: 1973), age (Newberne and Butler, 1969), nutritional status (Hamilton, 1977), and a number of external factors including dose (Wogan and Newberne, 1967), pre-xeno- biotic exposure (Wong 22.2i-v 1981; McGrew 2E §;., 1982), duration of exposure (Newberne and Butler, 1969), route of administration (Butler, 1964) and environmental factors (Wyatt 2£,§l-. 1977)- Up to the present, no animal species has been found to be resistant to the lethal effects of aflatoxins (Wogan, 1977). Busby and Wogan (1981) have stated that acute toxicity of AFB1 to mammals and birds may vary over two orders of magnitude between a senSitive species like the rabbit (LD5O = 0.3 mg/kg) and an insensitive species like the mouse (LDSO’ approximately 40 mg/kg). ‘ AFB1 is the most potent of the 12 to 15 analogs produced by A, flavus and A, parasiticus (Hayes, 1980). It is also the most commonly occurring member of the group, thus most studies dealing with aflatoxins have concentrated on AFB1 (Applebaum EELEL-' 1982). Small changes in chemical structure of aflatoxins markedly modify their biological activity. Thus, the LD5O for l-day-old ducklings of aflatoxins B2 and G2 (the 2,3-dihydro-derivatives of aflatoxins B1 and G1, re- spectively) were 4 to 5 times higher than that of aflatoxins B1 and G1 (Carnaghan 2£.§l-v 1963). The liver is the main target organ for aflatoxin poison- ing. Hepatic lesions caused by aflatoxins include prolifer- ation of the bile duct (Carnaghan 2£.§l-v 1966; Krogh E£.El-' 13 1973), central lobular necrosis, fatty infiltration (Carnaghan £2.2l-v 1966) and primary hepatocarcinoma (Lancaster 2£.El'v 1961). Some studies, however, have shown that aflatoxin B1, and especially AFGl, can cause necrosis of the kidney tubules and hemorrhagic lesions in other organs such as the lungs and adrenal glands (Butler and Lijinsky, 1970; Epstein gglgl., 19693 Wogan, 1973)- Experiments by Lancaster 2£.§l' (1961) provided the earliest evidence of the carcinogenic properties of aflato- xins. These researchers fed rats a diet containing 20% peanut meal that had been identified as the cause of poisoning in poultry flocks. No signs of acute toxicity were seen in the rats, but after 6 months 9 out of 11 rats developed multiple liver tumors and 2 of them had lung tumors. Early studies of tumor induction in rats using approximately 10 ug of AFB1 per day led Butler and Clifford (1965) to conclude that AFB1 is the most potent natural carcinogen. According to Apple- baum £E.§l° (1982), this conclusion still holds true. The other aflatoxins are also considered to be carcino- genic, although they are far less potent (Butler and Clifford, 1965). The variation in potency appears to be related to differences in chemical structure (Wogan §£.§l-I 1971). Wong and Hsieh (1976) have concluded that the 2,3—double bond present in aflatoxins B1 and G1, but not in aflatoxins B2 and G2, is involved in the carcinogenicity and mutagenicity of aflatoxins. The potency of AFM1 (the 4-hydroxy-derivative of AFB1) in inducing liver tumors in the rainbow trout was 14 only one-third of that of AFB1 (Sinnhuber g£_AA., 1970). This is in contrast to its acute toxicity to l-day-old ducklings, which was qualitatively and quantitatively similar to that of AFB1 (Holzapfel 23.2l-v 1966; Purchase, 1967). The mutagenicity of aflatoxins was recognized much later following the development of reliable methods for such tests (Ames 2£.§l-c 1973). Mc Cann 2£.§l- (1975) noted that on a molar basis, AFB1 was the second most potent mutagen of 300 compounds tested by the Ames test. Later, Wong and Hsieh (1976) screened aflatoxins and their animal biotransformation products for carcinogenic potential. They found that all the metabolites tested were less active than AFBl. The most mutagenic metabolite tested was aflatoxicol, which was only 22.8% as mutagenic as AFBl, and it was followed by aflatoxins 1. These workers concluded that the relative in vitro mutageni- G1 and M1, which had only about 3% of the potency of AFB city of aflatoxins correlated qualitatively with in vivo carcinogenic data. Various studies on the metabolism and mode of action of aflatoxins have provided evidence that they require metabolic activation to elicit mutagenesis (Ames E£.§l°t 1973; Ong, 1975). Wong and Hsieh (1976) demonstrated that neither afla- toxicol nor aflatoxins M1, Q1 or B2a possess mutagenic activ- ity in the absence of the activation factor from rat liver preparations. This indicated that none of these metabolites are the ultimate mutagenic/carcinogenic species. 15 The cytotoxicity and genotoxicity of aflatoxins B1 and M1 in primary cultures of adult rat hepatocytes were compared by Green 33 El- (1982). AFB1 was a more potent genotoxic and cytotoxic agent then AFMI, although these authors pointed out that AFM1 is still active at relatively low doses. Thus, they concluded that AFM1 is probably a potent hepatocarcinogen in vivo. AFB1 has been found to be a potent teratogen in hamsters (Elis and Di Paolo, 1967), chickens (Bassir and Adekunle, 1970) and Japanese killifish (Llewellyn :1; sin 1977). AFB1 has also been shown to have marked suppressive effects on the development of acquired immunity through its action on the cell mediated immune system (Pier, 1981). Aflatoxicosis in Man Human exposure to aflatoxins can occur directly by in- gestion of aflatoxin contaminated foods like corn and nuts, or indirectly by consuming foods of animal origin (milk, eggs or meat), which contain aflatoxins derived from animals that consume contaminated feed (Hayes, 1980). A few cases of suspected aflatoxin poisoning in Asia and Africa have been reported (Krishnamachari g£,§A., 1975; Campbell and Stoloff, 1974). Information available is limited since contamination is most frequent in areas where medical services are poor and many cases go unnoticed. In 1974, aflatoxins were implicated in an outbreak of hepatitis in 16 India (Krishnamachari g£_A;., 1975) during which 106 out of 397 patients died. Corn samples were found to contain between 6.25 and 15.6 mg/kg of aflatoxins. It has also been suggested that the condition known as Indian childhood cirrhosis might be due in part to aflatoxin poisoning (Amla 22.2l-v 1971). Liver biopsies of Indian children, who accidentally ingested large quantities of protein rich peanut meal contaminated with aflatoxins, showed the characteristic bile duct proliferation caused by aflato- xins. The urine of some of these children also contained aflatoxins. . It has been suggested that other human diseases could be related to aflatoxin consumption, including: 1) encephalopathy and fatty degeneration of the viscera (EFDV) also known as Reye's syndrome (Ryan EE.§l-v 1979), and 2) primary hepatic carcinoma (Oettle, 1964). EFDV has been recognized as a major cause of morbidity and mortality among infants since it was first described (Reye 2£.§$-v 1963). It may affect children from only a few months of age up to adolescence. Symptoms progress from a mild viral illness with vomiting and abdominal pain to cerebral involvement with coma (Hayes, 1980). Reye's syndrome is endemic in northeast Thailand, an area of high rice and low protein consumption (Olson 23 file. 1970). A study of aflatoxin contamination of Thai foods indicates seasonal and geographical patterns coincident with EFDV (Bourgeouis E£.§£-: 1969). Traces of AFB1 have been. found in the body tissues of many Thai children, but in a 17 series of EFDV patients, 22 had high concentrations in their tissues or gastrointestinal contents (Shank 22.2l-' 1971). This suggests that there is probably a chronic, low level of ingestion of the toxin throughout the population in this region. Cases of Reye's syndrome in association with aflatoxin contamination have also been reported in the United States (Chaves-Carballo §£_AA., 1976; Ryan 23_AA., 1979), Czechoslovakia (Dvorackova 22.2i-v 1977) and New Zealand (Becroft-and Webster, 1972). A The probable involvement of mycotoxins in the genesis of human tumors was first suggested by Oettle (1964) in an extensive review of cancer in Africa. He noted that in South Africa hepatoma was concentrated among the Negroid races and that cirrhosis was a common precursor. A high incidence of human liver cancer occurred in areas of high humidity and temperature and in relatively primitive populations. Fungi require high temperatures and humidity to grow, and will grow in food stored under such primitive conditions (Hayes, 1980). Therefore, the ingestion of food contaminated with mycotoxins could provide a reasonable explanation for the high incidence of liver cancer in certain areas of Africa, the Far East and South America. A number of studies in various regions of Africa and Asia have related the ingestion of aflatoxins to the incidence of hepatoma in man. Evidence for this association has been suggested in Uganda (Alpert g£‘§;., 1971), Taiwan (Tung and Ling, 1968), Kenya (Peers and Linsell, 1973), Swaziland 18 (Peers 2£.él-v 1976), Mozambique (Van Rensburg EE.§l-v 1974), the Philippines (Campbell and Salamat, 1971), Southeast Asia (Shank EEHEl-v 1972), Senegal (Payet EEHEi-v 1966) and India (Amla 2£.éi-v 1971). Van Rensburg 22.3l- (1974) summarized some epidemiological evidence on the incidence of liver carci- noma and probable aflatoxin contamination of the food supply, which is presented in the following table: Summary of the Available Data on Aflatoxin Ingestion Levels and the Incidence of Liver Cancer Country - Area Estimated afla- Incidence of liver toxin intake, cancer cases, ne/ks/day /105/yr(a Kenya - high altitude 3.5 0.7 Thailand - Songkhla 5.0 2.0 Kenya—middle altitude 5.8 2.9 Kenya-low altitude 10.0 4.2 Thailand-Ratburi 45.0 6.0 Mozambique-Inhambane 222.4 - 25.4 a) Cancer rate per 100,000 inhabitants per year No such studies exist for any United States population, however, livers from 3 of 6 patients who died of primary liver cancer contained detectable levels of AFB1 (Hayes, 1980). The validity of the apparent link between consumption of aflatoxin contaminated foods and primary liver cancer in humans has 19 been recently questioned by Stoloff (Anonymous, 1984). He noted that the small excess of liver cancers in white males from the Southeast area of the United States (6-10%), where contamination of foods and feeds with aflatoxins is more common, over white males from the North and West is far from the big difference that would be expected based on the prob- able greater consumption of aflatoxin contaminated foods by those living in the Southeast. With respect to the evidence gathered in Africa and Asia, Stoloff (Anonymous, 1984) favors the hypothesis that chronic infection by hepatitis B virus is etiologically related to primary liver cancer. It will probably be necessary to look at the data again and continue studies to establish if a real link exists between aflatoxins and human liver carcinoma. _ Metabolism and Toxicity of Aflatoxins Animals and man are exposed to a variety of naturally occurring foreign compounds or xenobiotics (Linsell, 1977). Most of these compounds are nonpolar and difficult to excrete (Klaassen, 1980). and thus require metabolic transformation. The essential effects of xenobiotic metabolism are detoxifi- cation and elimination, involving the transformation of li- pophilic compounds into more polar, water soluble metabolites that can be more easily excreted via bile or urine (Park, 1982). However, in some cases, metabolic transformation of a foreign compound enhances its reactivity toward important 20 biological molecules and results in increased toxicity (Swick, 1984). Thus, Hayes (1975) proposed the use of the term bio- transformation for all of the metabolic reactions of xeno- biotics. The rate of metabolism of a xenobiotic strongly influ- ences the intensity of its effect (Swick, 1984). Although many tissues possess xenobiotic metabolizing capacity (Blum- berg, 1978; Guengerich, 1977), the liver is the most active site for most xenobiotics (Park, 1982). The reactions in- volved in the metabolism of xenobiotics are catalyzed by rel- atively non-specific enzymes and lead to a variety of differ- ent metabolites (Gillette, 1979). Williams (1959) pointed out that these reactions can be classified into two general groups. Phase I biotransformations include those reactions that convert one functional group into another (e.g., oxida- tion), those that split neutral compounds to form a frag- ment having polar groups (e.g., hydrolysis of esters or am- ides), and those which introduce polar groups into nonpolar compounds (e.g., hydroxylation). Phase II biotransformations involve the conjugation of polar groups of foreign compounds with glucuronate, sulfate, glycine, glutathione, methyl groups and water. The most important enzymes involved in Phase I reactions are localized in the hepatic endoplasmic reticulum, a network of intracellular tubules which on homogenization disrupts to form particles called microsomes (Gillette, 1979). The micro- somal enzymes require both NADPH and oxygen (Gillette, 1966), 21 thus are named mixed-function oxidases (MFO). The enzyme systems, which are sometimes referred to as cytochrome P-450 monooxygenases, are composed of two enzymes: NADPH-cytochrome P-450 reductase and a heme-containing enzyme, cytochrome P-450 (White and Coon, 1980). There are multiple forms of the terminal enzyme cytochrome P-450 both in animals and man, which have selective but not specific substrate requirements (Park, 1982). The evidence for this has been recently reviewed by Lu and West (1980). AFB1 is metabolized by the hepatic microsomal mixed- function oxidase system to form a group of hydroxylated derivatives (Campbell and Hayes, 1976), which possess distinct- ly different toxic properties (Wong and Hsieh, 1976). AFB1 can also be reduced by a cytoplasmic reductase to aflatoxicol (Wong and Hsieh, 1978). The qualitative and quantitative com- position of the aflatoxin metabolites is apparently species SPBCifiC (Masri §£,§l-. 1974a) and can be‘correlated with differences in susceptibility among animals (Hsieh 23 §;., 1977). The pathways for formation of the major metabolites of AFB1 are shown in Figure 3. Aflatoxin M1, the 4-hydroxy-derivative of AFBl, was the first metabolite identified, and was so named because of its presence in milk where it occurred in the protein fraction (Allcroft and Carnaghan, 1963). Secretion of AFM1 into milk as a percentage of aflatoxin in the diet varies from less than 1% to 3% (Polan EE.§l-n 1974). Microsomal enzymes have been suggested as being responsible for AFM1 production via 22 .mmpmmswCOO mammasm no opflsohsosHm HafipCTpom so saccx n * mmcfiHOQMme msowhm> on Hm :wXOpmHm< mo Soapmsnowmzmne I m mesmwm ... 28:33 .. ...—.3232“ . L-.. 1. © . . sen”... .. s.~e~¢.u< su_ _n”u.II. n—uaoa< .. ..nopcaaa ¢.u.e.. \\\n 9.. o- up¢usocoo —1_ use.:»«_.sa...ae .. ..uapa..<..uogasa.a . .. -n.~.eca.=_a-n.~ ._ _ .. L. ~ ... a.“ ”bu-aa¢ o—uc u_uau.¢ law“... 22...: :22: o; .1 23 4-hydroxylation of AFB (Portman 22.2l-v 1968) (Figure 3, path- 1 way 1). AFM1 is produced in vitro from AFB1 by liver microso- mes from a variety of species including human beings (Masri g; l§;,,.1974a;‘ Campbell gt §l°v 1970). AFM1 is excreted into the urine of AFB -treated sheep (Allcroft EE.§l°v 1966) and 1 monkeys (Dalezios and Wogan, 1972). The glucuronide of AFM1 was reported in the tissue and excreta of chickens (Mabee and Chipley, 1973a). Several reports suggest that AFM1 is less toxic (Garner 23,3A., 1972) and less carcinogenic (Sinnhuber g£_AA., 1970) than AFBl, although on administration to duck- lings it induces liver lesions identical to those caused by AFB1 (Allcroft and Carnaghan, 1963). Aflatoxin P1 is produced by O-demethylation of AFB1 (Figure 3, pathway 2). The formation of this metabolite was suspected by Shank and Wogan (1965) when a significant propor- 11+C-methoxy-labeled tion of the radioactivity administered as AFB1 to rats appeared in the respired 002. Later, Dalezios £2.§l- (1971) found that AFP1 is the major excretory product in the urine of AFBl-treated monkeys where it was present as glucuronide or sulfate conjugates. The overall role of this metabolite in aflatoxin toxicity has not been fully elucidated (Swick, 1984). Hydroxylation of the molecule allows conjuga- tion reactions to occur, thus rendering it excretable. However, the molecule retains both the 2,3-unsaturated furan and the lactone portion of the coumarin, generally assumed to be 1 required for aflatoxin toxicity (Wong and Hsieh, 1976). Wong and Hsieh (1980) have found that species, which are relatively 24 resistant to the carcinogenic effect of AFB1, are more reactive in the in vivo conversion of AFB1 to AFP1 and water soluble conjugates. Aflatoxin Q1 is produced by hydroxylation of the carbon atom at the /€-position relative to the carbonyl group of the pentenone ring (Masri ££.§l-: 1974b), AFQ1 represents one- third to one-half of the metabolites produced in vivo from AFB1 by human (Bdchi 22.3l'! 1974) or monkey liver (Masri At §l°1 1974b). AFQ1 appears to be much less toxic than AFB1 (Hsieh 2E.El-v 1974) suggesting that its formation is a mecha- nism of detoxification in primates (Figure 3, pathway 3). Aflatoxicol (AFL) is the secondary alcohol formed by the reduction of the carbonyl group in the cyclopentenone moiety of AFB1 (Figure 3, pathway 4). This reaction is catalyzed by NADPH-dependent cytoplasmic enzymes, which also reduce the ketone function of aflatoxin B2 to form the corresponding di- hydro-aflatoxicol (Patterson and Roberts, 1971). Patterson and Roberts (1972) observed that rabbit and bird liver homo- genates were active aflatoxicol producers, whereas, rodent and sheep preparations were inactive. The susceptibility of different species to the carcinogenic effects of AFB1 has been associated with their ability to produce aflatoxicol (Hsieh 2313A., 1977). Aflatoxicol is less toxic and less mutagenic than AFB1 (Wong and Hsieh, 1976; Campbell and Hayes, 1976), although it is the most mutagenic of the AFB metabo- 1 lites (Wong and Hsieh, 1976). Aflatoxicol is readily converted to AFB1 by a microsomal 25 enzyme system that does not appear to involve cytochrome P-450 (Salhab and Edwards, 1977). The reversibility of this enzy- matic reaction led to the theory that aflatoxicol may serve as an intracellular reservoir of AFBl, prolonging cellular exposure to AFB1 and thereby enhancing its carcinogenic effect (Wong and Hsieh, 1978). Recently, Kumagai §£.El- (1983) found that the interconversion of AFB1 and aflatoxicol occurred in red blood cell suspensions, with resistant species having the highest rate of conversion. This finding needs further clarification. Aflatoxicol M1 can be formed through reduction of the carbonyl group in the pentenone ring of AFM1 by a cytosolic enzyme (Figure 3, pathway 5). or by the microsomal mixed- function oxidation of aflatoxicol (Figure 3, pathway 6) (Salhab g£_AA., 1977). Aflatoxicol H1 is a dihydroxyl-derivative of AFB1 with substitutions at the cyclopentenone carbonyl and at the/9- carbon (Figure 3, pathway 7). SaHmab and Hsieh (1975) report- ed this compound was formed from AFB1 when both the microsomes and a soluble enzyme preparation of either human or monkey liver were used. This compound may arise from the reduction of AFQ1 (Figure 3, pathway 8), or by oxidation of aflatoxi- col (Figure 3, pathway 7). However, the exact mechanism operating in the formation of aflatoxicol H1 is not known (Salhab and Edwards, 1977). Aflatoxin B 2a ure 3, pathway 9). Pohland 22.§l- (1968) reported formation is the hemiacetal derivative of AFB1 (Fig- 26 of this compound by the acid catalyzed addition of water to the vinyl double bond in the terminal furan ring of AFB1. AFB2a is considerably less toxic (Pohland At A;., 1968) and less mutagenic (Wong and Hsieh, 1976) than AFBl. Patterson and Roberts (1970) found that liver microsomal preparations of several mammalian and avian species metabolized AFB1 into a metabolite with spectral characteristics similar to those of AFBZa' This compound was found to be unstable under phys- iological conditions and was assumed to bind to protein (Fig- ure 3, pathway 12b). These conditions favor the dialdehyde- phenolate conversion. The nature of the reaction was presumed to be Schiff base formation between the aldehyde groups of ring-opened AFB2a and the free amino groups of protein and amino acids (Gurtoo and Dahms, 1974). Considerable controversy has arisen recently regarding AFBZa' Lin EE.§l° (1978) were not able to demonstrate the formation of AFB2a after incubation of AFB1 with rat or hamster liver microsomes. They suggested that formation of the AFBl- dihydrodiol (Figure 3, pathways 10 and 11), which has identi- cal spectral properties to AFB2a and reacts similarly with proteins, may be the same protein binding metabolite previously reported. Furthermore, Neal 2£.§l- (1981) pointed out that earlier reports on the production of AFBZa (Patterson and Roberts, 1970; Gurtoo and Dahms, 1974) were erroneous, being based on the wrong identification of AFBl-dihydrodiol as AFB2a due to the similarity of their ultraviolet spectral data (Swenson Eiuél'v 1973). The dihydrodiol of AFB1 27 (2,3-dihydro-2,3-dihydroxy-AFB1) showed little or no toxic, carcinogenic or mutagenic activity (Swenson 33 §;., 1975: Coles £3.2l-v 1980). Bioactivation of AFB to the highly reactive and labile 1 intermediate, aflatoxin B1-2,3-epoxide (Figure 3, pathway 10) has been proposed as the ultimate mechanism by which mutagen- esis and carcinogenesis are manifested (Garner £3.Ai., 1972). Schoental (1970) first postulated that formation of an epoxide intermediate at the 2,3-double bond might account for the toxicity of AFB1. Later, Garner and Hanson (1971) and Garner E£.§l- (1972) demonstrated that incubation of rat liver micro- somes with AFB1 and a NADPH-generating system produced a me- tabolite that was lethal to certain bacteria. No lethal effect was observed in the absence of mixed-function oxidases and toxicity was reduced on addition of either DNA or RNA. This suggested that the same toxic metabolite reacted with nucleic acids. Strong evidence for the formation of aflatoxin B1-2,3- epoxide was presented by Swenson gt‘gl. (1973), who isolated the dihydrodiol after mild hydrolysis of the RNA-aflatoxin B1 adduct formed by microsomal oxidation of AFB1 in the presence of RNA. Later, Swenson 2£.§l- (1974) found that acid hydrol- ysis of the DNA- and RNA-bound derivatives of AFB1 formed in vivo in rat liver, released a major share of bound aflatoxin as the dihydrodiol (Figure 3, pathway 12a). Although binding of AFB1 to liver proteins was observed, the level was only 4 to 7% of that observed~with nucleic acids. 28 Swenson 2£.§$-_(1975) suggested that aflatoxin B1-2,3- epoxide formed an electrophilic carbonium ion at carbon 2, which could react with nucleophilic nitrogen and oxygen atoms in the nucleic acids to yield glycoside-like linkages suscep- tible to acid hydrolysis. Since the extreme reactivity of the AFB1-2,3-epoxide precluded its isolation and synthesis, Swenson 2E.§l- (1975) synthesized the more stable AFBl-2,3-dichloride as a model of the epoxide. These workers showed that the di- chloride was a more potent mutagen and carcinogen than AFBI, and that it reacted in vitro with nucleophiles in the same manner as was expected for the 2,3-epoxide. The model com- pound formed covalent adducts with DNA, RNA, protein and amino acids by reaction with their nucleophilic centers. It reacted more readily with polynucleotides than with mononucleotides and especially with polyguanylic acid. Further evidence of the greater reactivity of the epoxide toward the guanine resi- dues in nucleic acids was reported by Essigman 22 El. (1977), who isolated and identified 2,3-dihydro-2-(N7-guanyl)-3-hydroxy- aflatoxin B1 as the major adduct formed from AFB1 in vitro, accounting for approximately 90% of the aflatoxin bound to DNA (Figure 3, pathway 13). Lin Agigl. (1977) and Croy g§_gl, (1978) have established that this compound is also the major adduct formed from AFB1 by rat liver in vivo. The relative biological hazard posed by the formation of aflatoxin adducts with different classes of cellular macro- .molecules is unknown (Busby and Wogan, 1981). However, in studies comparing in vivo macromolecular binding of the toxic 29 AFB1 and the relatively nontoxic AFB2 to rat liver DNA, rRNA and protein, Swenson.§£,gi. (1977) noted that AFB2 bound to nucleic acids at approximately 1% the level of AFB 0n the 1. other hand, protein binding of AFB2 was 35 to 70% of that ob- served with AFBl. This suggested that aflatoxin-protein adducts are relatively unimportant in the manifestation of toxic and carcinogenic effects. ' Raj 2£.§i- (1975) detected a polar fluorescent ninhydrin- positive compound on in vitro incubation of AFB1 with rat liver microsomes and reduced glutathione (GSH). Later, Degen and Neumann (1978) isolated the glutathione conjugate as the major component in the bile of rats dosed with 1“ C-AFBl, and identi- fied it as 2,3-dihydro—2-(S-glutathionyl)-3-hydroxy-aflatoxin B1. They also reported its formation in vitro on incubation of rat liver postmitochondrial supernatant with AFB1 and 3H- GSH. These results provided further evidence for the existence of AFB1-2,3-epoxide and also suggested an important metabolic role for GSH in protection from aflatoxin toxicity. Earlier, Mgbodile 2£.§l- (1975) had shown that depletion of the GSH levels in the liver of rats made them more susceptible to the toxic effects of AFB1. Similarly, Allen-Hoffmann and Campbell (1977) demonstrated that binding of AFB metabolites to DNA 1 is inversely related to the hepatic GSH levels. Metabolism plays a prominent role in determining the toxicity of AFB1 (Campbell and Hayes, 1976).‘ Various path- ways are known to occur in the hepatocyte. Hsieh EESél- (1977) indicated that the ultimate or net toxicity is determined by _1 30 the partitioning of the toxin among the various pathways. Us- ing hepatic enzyme preparations in the study of in vitro metabo- lism of AFBI, they revealed correlations between: 1) the sus- ceptibility of a species to acute effects of AFB1 and both their ability to reduce‘AFB1 to aflatoxicol and their overall rate of metabolism, and, 2) the susceptibility of a species to the carcinogenic effects of AFB1 and their relative activi- ties in forming aflatoxicol, AFQ1 and conjugated metabolites. .'Later, Wong and Hsieh (1980) compared in vivo metabolism and toxicokinetics of AFB1 in monkey, rat and mouse. They noted that species susceptibility to the acute and carcinogenic ef- fects of AFB1 is closely related to the rate and extent of tissue penetration, distribution, metabolism and elimination. Species relatively sensitive to the acute effects of AFB1 showed a higher volume of distribution, a higher equilibri- um transfer rate constant, higher levels of total aflatoxins in liver and plasma, and a longer plasma biological half-life of AFB1. Species relatively resistant to the carcinogenic effect of AFB1 were more active in the in vivo conversion of AFB1 to AFP1 and water soluble metabolites. Wei g§_AA, (1978) compared the mutagenic activities of water soluble AFB1 conjugates before and after/B-glucuronidase and sulfatase hydrolysis. The conjugates were obtained from urine of rhesus monkeys and primary hepatocyte cultures pre- pared from adult rats and mice. The water soluble conjugates were found to have no or very low mutagenic activity as measur- ed by the Ames test. After enzymatic hydrolysis, however, the 31 activity increased several fold, It was also suggested that the intestinal microflora, which can cleave these conjugates, may play an important role determining the toxic effect of aflato- xins. The genotoxicity of various aflatoxin residue fractions. in the livers of rats fed lac-AFBI was evaluated by Jaggi 33 AA. (1980). They isolated the macromolecules and water soluble conjugates from the liver, administered these fractions orally to other rats and determined the amount of radioactivity incorporated into liver DNA of the second group of rats. They concluded that macromolecule-bound AFB1 derivatives are at least 4,000 times less active than AFB1 in terms of covalent binding to rat liver DNA, and that the water soluble conjugates are at least 100 times less potent than AFB1 itself. Aflatoxin B2, the dihydro-derivative of AFBl, occurs nat- urally as a mold metabolite (Hartley, 3 §;., 1963). Much less is known about the metabolism of AFB2, although some me- tabolites have been identified (Figure 4). Roebuck gglgg. (1978) compared the in vitro metabolism of AFB2 by postmito- chondrial supernatant fractions of duck, rat, mouse and human livers. Duck liver had the highest activity and produced AFBI, aflatoxicol 1, aflatoxicol 2, AFM1 and AFMZ. Rat, mouse and human hepatic preparations produced no detectable AFBl, and only produced small amounts of compounds thought to be afla- toxins Q2 and P2 (the 2,3-dihydro-derivatives of aflatoxins Q1 and P1, respectively). Aflatoxin B28. was not produced readily from AFB2 on incubation with rat liver microsomes (Schabort and Steyn, 1969). However, it appears that AFB2a 32 mm :onamah< Ho mcowpmsuoemcmmp owHODMme I a shaman ~a =.xeh'25 mm) at Death 0 Treated 0/16 0 Control 8/8 2 5/ 8 4 4/8 8 8/8 16 7/8 24 8/8 32 Treated 5/8 32 Control 6/8 Three of the 16 hens killed at 0 days had reabsorbed ova. As shown in Table 10 none of the aflatoxin-fed hens had ova larger than 25 mm in diameter at the end of the afla- toxin feeding period. Control hens showed an average of 6 large ova (Q1) 25 mm) at this time. Eight days after aflato- xin withdrawal, all hens sacrificed had large ova, thus in- 116 dicating that their laying potential had returned to normal. These results are in agreement with those of Trucksess At 3;. (1983), who reported that the livers and ovaries of laying hens were affected most during aflatoxin feeding. Effects of Aflatoxins on Organ Weights The average organ weights as a percentage of body weight at the end of the aflatoxin feeding period (0 days) and at the end of withdrawal (32 days) for the aflatoxin-fed and control hens are shown in Table 11. These results demon- strate that at the end of the aflatoxin feeding period the size of the livers, kidneys, ovaries, spleens and hearts from the aflatoxin-fed hens differed significantly from those of the control. Livers, kidneys and spleens were enlarged by approximately 61, 14 and 20% over control values, respectively. Ovaries and hearts of treated hens were smaller than those of controls by 27 and 7.5%, respectively. At the end of the withdrawal period, the weights of all organs, except for the hearts, were not significantly different from the controls, indicating that recovery had occurred. Although the liver is the main target organ, AFB1 is known to cause damage to other tissues and organs (Hayes, 1980: Wogan, 1973). Van Zytveld 2£.§i- (1970) reported pale, swollen and enlarged kidney in poultry fed aflatoxin contam- inated rations. Chen (1983) reported that the kidneys of aflatoxin-fed chicks were 46% heavier than those of the 117 E cannon secs soehm n .m.m .2802 0:0 kc honed osmosmpm AN Atv mo.ovm .Ho AI; Ho.ovm .zaucm0ngsmwm howmwe mpmwhomhomsm usopoewfio new; oswa meow esp sw.mosam> AH * H0.0 H00.0 0 «0.0 s00.0 s mm * H0.0 nos.0 0 H0.0 HR0 0H 0 names 00.0 0a.0 0 00.0 ma.0 s mm * no.0 s0H.0 0 00.0 mma.0 0a 0 sooaam 00.0 Hm.m 0 No.0 sfi.m s mm a 00.0 csm.m 0 mm.0 omm.m 0A 0 moamoso 00.0 sm.0 0 00.0 00.0 s mm * no.0 ome.0 0 a0.o 00s.0 0H 0 msosoae 00.0 ms.s 0 00.0 mm.a s mm no.0 mm.a 0 s0.0 om.a 0H 0 ososoao ma.0 0H.m 0 ss.0 00.H s mm as as.0 nma.m_ 0 NH.0 oas.m. 0a 0 mo>aq mv.m.m scenes 0 N0.m.m ”Samoa s- Hosoaosofis Hoepsoo UOHISHXOpmHm< 909mm when cameo pswfloz zoom we psooeom mm oommonaxm mpgwwos cameo so mSHROpme< mo poogmm I “a manna 118 control group. Smith and Hamilton (1970) observed enlargement of the spleen and pancreas in young broiler chickens fed afla- toxins. Enlargement of the spleen and kidneys in laying hens after aflatoxin feeding was reported by Howarth and Wyatt (1976), while Hamilton and Garlich (1971) did not detect any changes in the same organs in laying hens. These differ- ences are probably due to variation in the strains of birds and in the levels and/or purity of the aflatoxins fed as well as to duration of feeding. The significantly smaller weight of the hearts from the aflatoxin-fed hens at the end of aflatoxin feeding and also at the end of the withdrawal trial was unexpected. In con- trast, Chen (1983) reported that the hearts of aflatoxin-fed chickens were slightly enlarged over those of controls (P(0.10). Butler (1966) reported that the only change seen in the hearts of guinea pigs dosed with AFB1 was an occasion- al area of fatty degeneration of the myocardium. Although aflatoxin residues were detected in the hearts of the hens in the present study, it is not clear if this effect was 1 caused by aflatoxins. Mabee and Chipley (1973b) and Sawhney 22.20- (1973) also detected aflatoxin residues in the heart of aflatoxin-fed hens, but reported that there were no evi- dent lesions in any of the tissues of the test hens. Average organ weights as percent of body weight for the aflatoxin-fed hens during the withdrawal period are pre- sented in Figure 14. Control values at 0 and 32 days of withdrawal are also indicated. The graph indicates that 1fl19 1T. .5. S S 2 3 =3 ... § 3 i L0" KIDNEY K" A I" l f \ ' "- um: ‘K. 8.! . ' E" . res. In in ‘35—‘10“ (lumMNETue(mwflI Figure 14 - Organ Weights as Percent of Body Weight of Hens During the Withdrawal Trial Control values are indicated by anlg at 0 and 32 days, 0, L, G, H, K and S, representing ovaries, liver, gizzard, heart, kidneys and spleen, respectively. 120 the size of the spleen. heart, kidneys and gizzard showed little variation, whereas, the livers and ovaries changed markedly during withdrawal, but were similar to control values at 32 days. Present results are in general agreement with previous reports indicating a relatively fast recovery in organ weights upon removal of aflatoxins from the feed (Blount, 1961: Smith and Hamilton, 1970: Hamilton and Garlich, 1972, Chen, 1983). Organ weights during withdrawal were analyzed by one- way analysis of variance over time (Snedecor and Cochran, 1967). Significant F ratios (P<0.05) were obtained for liver, ovaries and kidney, while heart, spleen and gizzard showed no significant change during withdrawal. Values having a significant F ratio were compared against the 0 days value (Dunnett, 1955, 1964) to establish when the difference was significant. Average organ weights as percent of body weight for liver, kidneys and ovaries during withdrawal are shown in Table 12. The data indicate that liver and kidney size were signifi-” cantly smaller (P'(0.01) at 16 and 32 days of withdrawal, respectively, than at 0 days. Ovary size, however, was sig- nificantly larger at 8 and 24 days of withdrawal than the 0 day value, but not at 16 and 32 days. When compared against control values, no statistical difference in ovary size was found at 32 days. This apparent discrepancy may be associ- ated with a decrease in the laying potential of the hens as they became older (North, 1978), which could affect both the 121 C ohmsvm :mos hoesm .m.m .:008 cap ho scene unaccepm 000.0 mowem>o .noo.o u hocowx .mmm.o u no>wa_.mem moumzwm some nouum AN 8.0 valet; 03 0002000020 one 0300, 0000 o 0:» Sop.“ msweowhflo mosam> .03H0> name 0 on» pmcfimmm noMMQSOO who; mesa» unonowmflo pm mosam> A“ 00.0 s. 00.0 as 00.0 0 0000 00 0* 00.0 00.0 ** 00.0 0 mass 00 00.0 00.0 as 00.0 0 name 00 0* 00.0 00.0 00.0 0 0000 0 00.0 00.0 00.0 0 name 0 00.0 00.0 00.0 0 0000 0 00.0 00.0 00.0 00 name 0 mofipm>o hoseflx no>fiq_ : HmsmnoSpwz psmflos zoom mo pcooeom mm mpsmwma cameo owmeo>< hopmm mama N.Hv 0030000003 000000 0:0: mSHzmq mo pnwfloa zoom mo psoopom mm munwfioa cameo ommuo>< I N“ manna 122 aflatoxin-fed and control hens regardless of aflatoxin feed- ing. In addition, the statistical tests used in these com- parisons were different and could affect sensitivity. AFLATOXIN RESIDUES Tentative Identification.of"Unknown Aflatoxin Metabolite in Eggs and Tissues of Laying Hens - In addition to aflatoxins B1, B , M1 and M2, a blue vio- 2 let fluorescent spot, which showed a lower mobility (Rf) than. AFM2 in both directions of development, was detected in eggs and tissue samples of hens fed the aflatoxin contaminated diet. The spot was not present in control eggs and showed a similar pattern of appearance and clearance to other aflato- xin residues detected in eggs and tissue samples. The un- known spot turned yellow under UV light on spraying the plate with 25% H280“, indicating that this compound may be an afla- toxin metabolite (A.O.A.C., 1975a). Since the unknown spot had a lower Rf than aflatoxins B1 and B2, and aflatoxins M1 and M2 (Figures 1 and 2), a me- tabolite of higher polarity was suspected. The unknown spot was then extracted from the TLC plate and chromatographed in three solvent systems with other hydroxylated aflatoxin me- M M and 1' 2' Q1 2a’ 2a aflatoxin Bl-dihydrodiol. The hemiacetal derivatives of tabolites including aflatoxins M , B 123 aflatoxins B1 and M1, namely aflatoxins B28‘ and MZa' were prepared in situ by additiOn of TFA/CHCl3 (1:1) as described earlier herein. Aflatoxin Bl-dihydrodiol was prepared from AFB1 as described by Swenson 22.10- (1974). Results of the comparative chromatograms are presented in Table 13. The data indicate that the mobility of the un- known compound was very similar to that of AFBZa* Table 13 - Comparative Thin Layer Chromatography of the ‘Unknown Aflatoxin Metabolite in Different Solvent Systems Compound . Rf(1 in: . CHCl3-Acetone CHClB-Acetone- Ether-MeOH- Isopropanol Water (83:17) (85:10:5) (90:8:2) UNKNOWN 0.14 0.40 0.32 AFM1 0.22 0.48 0.41 AFM2 0.19 0.45 0.35 AFBZa 0.19 0.41 0.32 AFM2a 0.03 0.20 0.23 1) Rf (Relative mobility) = Distance of center of spot from origin/ Distance of solvent front from origin (Ganshirt, 1965) 124 Attempts were made to identify the unknown metabolite by mass spectrometry techniques (MS), including negative ion chemical ionization and electron impact. Relatively small amounts recovered from the TLC plates along with contamination by plasticizers precluded positive identification. However, since the mobility and fluorescence of the unknown spot were similar to those of AFB2a' it was tentatively identified as AFBZa' Thus standards for AFB23 were prepared from AFB1 treated with TPA/OHCI3 (1.1) on each plate and the unknown spot was quantified as AFBZa' These values are included in the tables for aflatoxin residues in eggs and tissues as an indication of relative amounts of this compound. Aflatoxin Residues in the Eggs of Hens Fed a Contaminated Diet Aflatoxin levels for the eggs of hens during the first week of feeding the aflatoxin contaminated diet are presented in Table 14. Each value presents the level of aflatoxins in a composite sample of two eggs that were randomly selected and pooled together for analysis. The levels of aflatoxins in the eggs on days 10, 14, 18, 22, 25 and 28 of the aflato- xin feeding trial are shown in Table 15. Mean values of aflatoxin residues are also shown in both tables. The data presented indicate that the transfer of afla- toxins from the feed into eggs occurred rapidly after initial B feeding of the contaminated ration. Aflatoxins B M 1’ 2’ 2 1255 Table 14 - Aflatoxin Residues Detected in Eggs During the First Week of Aflatoxin Feeding 1 Aflatoxin (ug/k )( Day Sample '3 B M $I'JE— B 1 2 1 2 2a 1 1 0 0 0 tr tr ' tr 0.01 0 0.01 0.02 3 tr tr 0 0.01 0.03 Mean ' tr tr 0 tr 0.02 2 1 tr 0.03 0 0.01 0.05 _ tr 0.02 0 0.03 0.07 3 0.02 0.01 0 0.01 0 Mean tr 0.02 0 0.02 0.04 3 1 0.03 0.03 0 0.03 0.07 ~ 0.07 0.03 0 0.02 0.05 3 0.02' '0.03 0 0.03 0.06 Mean . 0.04 0.03 o 0.03 0.06 4 ’ 1 0.05 0.04 0.01 0.05 0.07 0.06 '0.04 0.01 0.03 0.08 3 0.01 0.02 O 0.03 0.05 Mean 0.04 0.03 tr 0.04 0.07 5 1 0.06 0.03 tr 0.05 0.09 ‘ 0.05 0.03 tr 0.03 0.05 3 0.05 0.02 O 0.03 0.05 Mean 0.05 0.03 tr 0.04 0.06 6 1 0.01 0.03 O 0.02 0.07 0.02 0.03 0 0.05 0.11 3 0.02 0.03 O 0.02 0.05 Mean 0.02 0.03 0 0.03 0.06 7 1 0.04 0.03 tr 0.05 0.05 2 0.05 0.03 0.01 0.02 0.06 3 0.03 0.04 0 0.02 0.07 Mean 0.04 0.03 tr 0.03 0.06 .1 tr 3 trace amounts, visible but too small to quantitats ((0.01 ug/kg) 126 Table 15 - Aflatoxin Residues Detected in Eggs During the Aflatoxin Feeding Period Aflatoxin (ug/kg)<1 Day Sample 31 B2 M1 M2 B2a 10 1 0.07 0.03 0.02 0.03 0.10 2 0.05 0.04 tr 0.04 0.11 3 0.05 0.02 0.01 0.01 0.07 Mean 0.06 0.03 0.01 0.03 0.09 14 1 0.06 0.05 0.01 0.02 0.07 2 0.03 0.03 0.03 0.04 0.07 3 0.05 0.03 0.01 0.01 0.07 Mean 0.04 0.04 0.02 0.02 0.07 18 1 0.10 0.06 tr 0.02 0.07 2 0.03 0.03 tr 0.02 0.05 3 0.03 0.03 0.01 0.02 0.07 Mean 0.05 0.03 tr 0.02 0.05 22 1 0.03 0.04 0.01 0.04 0.08 2 0.01 0.04 tr 0.03 0.08 3 tr 0.01 0 0.02 0.05 Mean 0.01 0.03 tr 0.03 0.07” 25 1 0.09 0.07 0.02 0.07 0.11 2 0.04 0.05 0.03 0.04 0.07 3 0.03 0.02 0 0.02 0.06 Mean 0.05 0.05 0.02 0.04 0.08 28 1 0.05 0.04 0.01 0.02 0.08 2 0.06 0.06 tr 0.03 0.06 3 0.03 0.01 0 0.01 0.02 Mean 0.05 0.04 tr 0.02 0.05 1) tr = trace amounts, visible but too small to quantitate (( 0.01 ug/kg) 127 and B2a appeared in trace or low but measurable amounts in the eggs laid only one day after consumption of the aflato— xin-spiked ration. The mean values in Tables 14 and 15 show that the levels of aflatoxin residues rapidly increased and reached a maximum value, which remained relatively con- stant as long as the hens continued to consume the aflatoxin- spiked diet. Results agree with the findings of Trucksess gt El- (1983). In a longer feeding trial, Ldtzsch and Leistner (1976) found that the maximum levels of aflatoxin residues were found after 8-10 days of aflatoxin feeding and decreased af- terwards, even before removal from the contaminated diet. After feeding hens at a level of approximately 3,000 ug of AFBl/kg of diet, they reported that the average level of AFB1' in the eggs was 0.03 ug/kg, which is similar to the levels found in the present study. Jacobson and Wiseman (1974), however, reported average amounts of 2.2 and 3.6 ug of AFB1/ kg in the albumen and yolks of eggs laid by Arbor Arce hens fed with only 100 ug of AFBl/kg diet. Trucksess gt EL- (1983) fed hens a ration containing 8,000 ug of AFBl/kg, a level about 2.4 times higher than that used in the present study. Unlike Trucksess 23.2l- (1983), who did not find AFM1 in any of the eggs analyzed, AFM1 was found in some samples in the present study although the levels were very low. The failure of Trucksess 21.2l- (1983) to detect AFM1 could be due in part to less sensitive analytical methodology and/or to the naturally low levels of free AFMl. 128 Extraction of AFM1 from eggs is difficult and requires preheating according to Gregory and Manley (1981). They con- cluded that no AFM1 could be recovered using the official A.0.A.C. method for AFB1 in eggs (Trucksess 33111;” 1977) which uses no heating, no acidulant or protein precipitants other than a saturated solution of sodium chloride. The method used by Trucksess 22.20- (1983) has no heating step and uses only a saturated sodium chloride solution to precip- itate the protein (Trucksess and Stoloff, 1984). The method used in the present study for the analysis of eggs included a two—stage sequential heating step prior to extraction, and is a modification of the method for the anal- ysis of aflatoxins in animal tissues (Trucksess and Stoloff, 1979). 'The need for the preheating step was seen by the fact that either poor or no recovery of added AFM1 and AFM2 occurred in spiked samples. Phase separation was also noted in samples heated in a boiling water bath for 20 min as recommended by Gregory and Manley (1981). This problem was solved by heating the samples in a 60°C water bath for 30 min, and then gradual- ly increasing the temperature to 72°C over a 15 min period. After heating, recovery of AFM1 and AFM2 improved, but a slight decrease in recovery of AFB1 and AFB2 was noted. As shown in Tables 14 and 15, the levels of AFB2 in eggs were in general slighlty lower than those of AFB1. Since the ratio of AFB1 to AFB2 in the feed was approximately 2.3, it appears that the metabolism of AFB2 is less efficient. A similar observation was made by Furtado 2£.E$- (1982) in 129 relation to the levels of AFB1 and AFB2 in the liver and kid- ney of pigs fed rations contaminated with AFB1 and AFBZ, and by Chen gt EA. (1984) in the tissues of broiler chickens. The levels of AFMZ, the hydroxylated metabolite of AFB2, were also higher than those of AFMl, the hydroxylated metab- olite of AFB1. This again is in agreement with observations of Furtado gt AA. (1982) and of Chen gt 3;. (1984), who con- cluded that the removal of aflatoxins B2 and M2 from the kid- neys and livers of pigs and chickens, respectively is some- what impaired in relation to that of aflatoxins B1 and M1. The relatively high levels of AFB2 and AFM2 may also be asso- ciated with the conversion of AFB1 to AFBZ, and of AFM1 to AFMZ. Patterson and Allcroft (1970) reported that AFB1 and AFM1 were reduced in vitro to the corresponding reduction products, AFB2 and AFMZ, by liver preparations from chickens, ducklings, guinea pigs and mice. Trucksess 23.20- (1983) reported the presence of aflato- xicol in eggs laid by hens fed an AFBl-spiked diet. Aflato- xicol was determined after separation on a 018 reverse phaée high performance liquid chromatographic system, because it could not be resolved from interferences by TLC. In the pre- sent study, the presence of aflatoxicol in the eggs could not be established, since extracts were resolved only by TLC. A strongly fluorescent compound normally present in eggs showed the same mobility than pure aflatoxicol in the chloro- form/acetone (83:17) developing system, but differenct Rf in the ether/methanol/water (95:4:1) system. In addition to 130 being present in control eggs, this fluorescent compound showed no change in the color of its fluorescence when the plate was sprayed with 25% H280“, thus eliminating the possi- bility of it being an aflatoxin residue (A.0.A.C., 1975a). Aflatoxin Residues in Egg Albumen and Egg Yolks Egg albumen and egg yolk were analyzed separately during aflatoxin feeding to establish the distribution of aflatoxin residues in the liquid egg. Five to six eggs were separated and the combined yolks and albumen (about 90-100g) were ana- lyzed separately by the same procedure followed in the analy- sis of whole eggs. Levels of aflatoxins in albumen and yolks are presented in Table 16. It can be seen that the residue levels in both the albumen and yolks showed similar patterns to those ob- served in whole eggs. ‘After two days of feeding the aflato- xin-spiked diet, trace to measurable amounts of all aflato- xins, except AFMI, could be detected in both the albumen and yolks. The levels increased rapidly showing a maximum value around the 8th day of aflatoxin feeding. Rapid growth of an ovum begins about 10 days before the yolk is released from the ovary (Nesheim 22.20:: 1979). Thus, it may be expected that maximum levels in the yolk would be reached after this period of time and they would remain rel- atively constant as long as aflatoxin feeding continues. However, a slight decrease in aflatoxin levels in both the 131 Table 16 - Aflatoxin Residues Detected in the Albumen and Yolks of Eggs Laid During Aflatoxin Feeding 2 Day Sample‘1 Aflatoxin-(“E/kg)( B1 B2 "1 "2 B2a 502282! 2 1 0.02 0.02 o 0.03 0.05 2 tr 0.01 0 0.02 0.03 5 1 0.02 0.02 o 0.02 0.06 2 0.01 0.02 o 0.01 0.04 8 1 0.06 0.03 tr 0.02 0.07 2 0.06 0.03 0.02 0.02 0.05 15 1 0.04 0.02 tr 0.03 0.04 2 0.02 0.02 tr 0.03 0.05 22 1 0.02 0.03 tr 0.02 0.08 2 0.06 0.04 0.01 0.04 0.13 28 1 0.04 0.03 0.01 0.02 0.06 2 0.03 0.02 0.01 0.02 0.04 10Lx 2 1 0.01 0.03 o 0.01 0.03 2 0.02 ‘o.02 0 0.02 0.03 5 1 -0.02 0.02 0 0.02 0.08 2 0.03 0.02 0 0.02 0.09 8 1 ‘0005 000“ 0002 0002 0008 2 0.05 0.02 0.01 0.03 0.09 15 1 0.04 0.02 0.01 0.01 0.07 2 0.05 0.02 0.02 0.04 0.09 22 1 0.04 0.03 tr 0.03 0.08 2 0.06 0.04 0.02 0.04 0.11 . 28 1 0.04 0.02- 0.01 0.03 0.08 2 0.04 0.02 0.02 0.01 0.06 1’ Each sample was a composite of 5-6 eggs for every day tr I trace amounts, visible but too small to quantitato ((0.01 was/ks) 132 albumen and yolks occurred toward the end of the feeding pe- riod. Similar results were reported by Ldtzsch and Leistner (1976) who suggested that hens fed aflatoxins for relatively I long periods of time may become adapted, which may result in lower residue levels in the eggs. Comparison of the levels of the various aflatoxins in separated eggs indicates that similar amounts of aflatoxins B2, M1 and M2 were recovered from albumen and yolks, while slightly higher amounts of AFB1 were recovered from the yolks. Similar observations for the deposition of AFB1 in eggs were reported by Ldtzsch and Leistner (1976). Jacobson and Wise- man (1974) reported that the level of AFB1 recovered from the yolk was 1.6 times higher than that in the albumen. Using 1“ C-AFBI, Sawhney gt QA. (1973) detected increasing levels of aflatoxins in egg components after administration of a single oral dose. After oviposition, the highest level of radioactivity was detected in shell membranes, followed by the yolks and albumen, respectively. No attempt was made to further characterize which metabolites were deposited. ” Trucksess gt 3;. (1983) found the same levels of aflatoxins in the ova and eggs at the end of aflatoxin feeding. They proposed that transmission appeared to be relatively constant throughout the proceSs of egg formation, although actual anal- ysis of egg albumen and yolks was not carried out separately. In the present study, the levels of AFM2 were higher than those of AFM1 in both the albumen and yolks. The pres- ence of the 2.3-double bond in the AFM1 molecule makes it 133 more reactive than AFM2 toward biotransformation by microsomal enzymes in the liver (Swenson, 1981) and could account for the higher levels of free AFM2 recovered. In addition, Mabee and Chipley (1973b) showed that most of the AFM1 formed upon feeding AFB1 to laying hens was found in the aqueous buffer extract and was bound to glucuronate and perhaps to other water soluble molecules. AFB2a was recovered in considerably higher amounts from the yolk than from the albumen. AFB2a is known to bind very readily with proteins, amino acids and peptides at physiolog- ical and alkaline pH values. AFBZa rearranges itself into dialdehydic phenolate resonance hybrid ions, which can then react with amino acids, peptides and proteins to form Schiff ‘ bases (Gurtoo and Campbell, 1974; Ashoor and Chu, 1975). The lower amounts of AFB2a recovered from the albumen could be the result of real differences in deposition or from the higher amount bound to the protein in the albumen, since the protein in the albumen accounts for about 92% of the dry matter and fonly for about 50% in the yolk (Nesheim At A;., 1979). As indicated by Brackett and Marth (1982) for cheese and by Murthy 2£.El: (1975) for beef, the interactions be- tween aflatoxins and proteins in animal products are not well understood. Thus, conclusive statements on the causes for the differences can not be made at this time. I As indicated earlier, the hens were exposed to a DR of 213 and 108 ug for aflatoxins B1 and B2, respectively. 134 Assuming an average weight of 1.6 kg/hen, daily consumption of AFB1 and AFB2 would be about 340 ug and 170 ug per hen, respectively. Comparison of these values to the levels re- covered from eggs (Tables 14, 15 and 16) suggests that only ‘small amounts of aflatoxins are transferred to the eggs either as the original aflatoxins or their unbound or free hydroxylated metabolites. The level of total aflatoxins added to the diet in the present study was about 5,000 ug/kg, which is 50 times higher than the action level of 100 ug/kg of total aflatoxins offi- cially set by the FDA for the rations of beef cattle, swine and poultry (Anonymous, 1982). Although contamination at the levels used in the present study is not encountered normally, it may occur under some circumstances. For example, a naturally occurring case of feed contamination at levels of about 83 and 110 mg of aflatoxins/kg of ration was reported by Hamilton (1971) in a commercial laying flock. 0n the basis of present results, it can be concluded that if aflatoxin residues in the feed are in compliance with actibn levels, the transmission of aflatoxins into the eggs of laying hens poses little or no potential public health hazard. This conclusion is supported by the results in previous reports (Sims gt A;., 1970: Ldtzsch and Leistner, 1976, 1977). 135 Aflatoxin Residues in Eggs During Withdrawal Whole Eggs Aflatoxin levels in whole eggs during withdrawal from the aflatoxin-spiked diet are presented in Table 17. The data indicate that upon withdrawal aflatoxin residues disap- pear from the eggs as rapidly as they appear during aflatoxin feeding. Low levels of aflatoxins were detected at one and two days of clearance. After three days, one egg sample had traces of AFB1 and AFBZ, and two samples had trace to low but measurable amounts of AFM2 and AFBZa' By the fourth day of withdrawal no detectable residues were found in any of the samples. Trucksess 22.20- (1983) reported that aflatoxin residues in eggs decreased rapidly after feeding of the contaminated ration was discontinued. They detected no AFB1 in eggs laid six days after withdrawal, although low levels of aflatoxicol (0.01 ug/kg) were present after seven days. Differences in the time required to achieve clearance between their study and the present experiment are probably due to variation in the levels and duration of feeding. In the present study, a lower level of aflatoxins combined with a longer feeding period. may have resulted in adaptation of the hens to the toxin, and could be responsible for lower residue levels and faster clearance as reported by Ldtzsch and Leistner (1977). 136 Table 17 - Aflatoxin Residues in Eggs After Withdrawal from the Aflatoxin-spiked Diet ' Days after Sample Aflatoxin (U57 kg) Withdrawal B1 32 M1 M2 B2a 1 1 0.03 0.02 tr tr 0.03 2 0.02 0.01 0 0.01 0.04 3 0.03 0.02 0 tr ' 0.03 Mean 0.03 0.02 0 tr 0.03 2 1 tr tr tr 0 0.02 2 0.02 tr 0 tr 0.04 3 0.01 tr 0 tr 0.03 Mean 0.01 tr 0 0 - 0.03 3 1 tr tr 0 0.01 0.02 2 0 0 0 tr tr 3 0 0 0 O 0 Mean 0 0 0 tr 0.01 4 1 0 0 ' 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 Mean 0 0 0 0 0 M 1) tr = trace amounts, visible but too small to quantitate (< 0.01 ug/kg) 137 Egg Albumen and Egg Yolks Egg albumen and egg yolks were analyzed separately dur- ing withdrawal to establish if there were differences in clearance patterns between the egg components. Since egg production by the aflatoxin-fed hens was very low at the end of aflatoxin feeding and some hens had been sacrificed, only a few eggs were available for analysis. Therefore the albu- men and yolks at three and four days of withdrawal could not be analyzed. 1 Levels of aflatoxins in albumen and yolks after withdraw- al are shown in Table 18. The data indicate that clearance of aflatoxin residues from the albumen occurred faster than from the yolks. Only trace amounts were detected in the al~ bumen at two days, and no residues were present at five days. In contrast, measurable levels were present in the yolks at two days and traces were still detected in one sample up to six days after removal from the contaminated ration. The presence of residues in the yolks of eggs laid five to six days after withdrawal may be due to deposition during the initial days of ova maturation (Nesheim 23.20:: 1979). Maturation of the yolks of eggs laid five to six days after withdrawal would have begun four to five days before withdraw- al and thus could have accumulated some aflatoxin residues. Sawhney 22.30: (1973) reported that large ova (> 10 mm) 14 retained C activity four days after administration of a 138 Table 18 - Aflatoxin Residues Detected in the Albumen and Yolk of Eggs After Withdrawal from the Aflato- xin-Spiked Ration Aflatoxin (uglkg)(1 Days after Withdrawal Sample B1 32 M1 M2 32a ALBUMEN 1 1 0.02 0.01 O 0.01 0.02 2 tr tr 0 tr 0 2 1 tr tr 0 tr tr 2 tr tr 0 tr 3.4(2 5 1 0 0 0 0 0 2 0 0 0 0 0 6 1 O 0 0 0 0 2 0 0 0 0 0 IOLK 1 1 0.03 0.01 tr 0.01 0.05 2 0.02 0.01 0.01 tr 0.05 2 1 0.01 .tr 0 0.01 0.05 2 0.02 0.01 0 0.01 0.04 2 3.4‘ 5 1 0 0 0 0 0 2 tr tr 0 tr tr 6 1 tr tr 0 0 tr . 2 0 0 0 0 tr 7 0 0 0 0 O 2 0 0 0 0 0 1) tr - trace amounts, visible but too small to quantitate ( (0.01 ug/kg) Not enough eggs available for separate analysis 139 single oral dose of 1“ C-AFBI, and that the activity was pre- sent in small ova ((.10 mm) up to seven days after dosing. Trucksess gt El- (1983) reported that aflatoxin residues were present in the ova of hens sacrificed at the end of aflatoxin feeding, but had disappeared after 7 days of clearance. Sim- ilar results were obtained in the present study (Table 27). Growth of the ovum and addition of the albumen free of aflatoxins to the whole egg have a dilution effect, which may account for faster clearance of aflatoxin residues. In a normal egg, the yolk constitutes only about 36% of the liquid portion (Powrie, 1976). Furthermore, eggs laid by hens fed aflatoxins are smaller as a result of a decrease in yolk size (Huff gt al., 1975). Thus, the contribution of the yolk to the aflatoxin content of the whole egg during' clearance was probably below detection limits. Results support the conclusion that aflatoxin residues in eggs decrease rapidly after withdrawal of the contami- nated diet. Clearance of aflatoxin residues can be achieved within three to four days from either the whole egg or the" egg white and within six to seven days from the egg yolk. Effects of Cooking on the Levels of Aflatoxins in Eggs The effects of cooking on the levels of aflatoxins in the eggs of hens fed an aflatoxin contaminated diet are pre- sented in Table 19. Eggs from day 15 of aflatoxin feeding were selected for analysis since this was an intermediate 140 Table 19 - Effect of Cooking on Aflatoxin Levels in Eggs From Hens Fed an Aflatoxin-Spiked Diet Aflatoxin Level (ug/kg) SAMPLE B1 B2 M1 M2 32a Replication 1 Raw 0.03 0.01 tr 0.01 0.0h Steamed 0.03 0.01 tr 0.04 0.06 Fried 0.03 0.02 tr 0.03 0.04 Replication 2 Raw 0.02 0.03 tr 0.02 0.07 - Steamed 0.02 0.02 tr 0.02 0.05 Fried 0.02 0.02 tr 0.03 0.03 Replication 3 Raw 0.51 0.01 tr 0.01 0.05 Steamed 0.02 0.02 tr 0.01 0.03 Fried 0.02 0.02 0.01 0.02 0.03“ 1&1 time in the feeding trial and adequate numbers of eggs were available. As indicated by the data, no major changes in the afla- toxin levels of the eggs were observed upon cooking. Most levels remained the same, others showed a slight decrease and some increased with cooking, even after correcting for moisture losses. Problems were encountered in establishing the effects of processing on eggsnaturally contaminated by aflatoxins since the amounts present were very low and the method was not sufficiently sensitive and/or precise to detect small differences at the low levels. Variability in the recovery of AFM1 during processing of dairy products has been reported to be a problem in ex- periments designed to study the effects of processing on AFM1 levels (Van Egmond gt_§l., 1977; Wiseman and Marth, 1983a; Brackett and Marth, 1982). Wiseman and Marth (1983a) reported inconsistent recoveries between trials in processing of cul- tured dairy products when working at levels as high as 3 to 6 ug og AFMl/kg. They concluded that a better understanding of the interactions between AFM1 and casein will be neces- sary in order to explain the results of processing. Simi- larly, interactions between aflatoxins and the egg proteins are not understood and may explain the variability in results from the present study. In a second experiment, control eggs were spiked with B 0.5, o.15,~o.u and 0.2 ug/kg of aflatoxins B 2, M1 and 1’ M2, respectively, and cooked as described for the naturally 142 contaminated eggs. Although the levels of aflatoxins were higher than for the naturally contaminated eggs, they were considerably lower than those used by Wiseman and Marth (1983a) in the study of cultured dairy products. Results for the spiked eggs are presented in Table 20 as percent recoveries. Values ranged from #0 to 108% indicating considerable vari- ability. Similar to the naturally contaminated eggs, there was no consistent trend in the effects of cooking. With two different methods of cooking (steaming and frying), AFBl remained constant for both in the first replication and in- creased in the second. In the third replication, AFB1 recov- ery decreased with steaming but increased with frying. The differences on comparing these results were not statistically significant due to small number of samples and the large degree of variability. Higher recoveries of aflatoxins from processed or aged dairy products than from the corresponding raw milk or unaged products have been reported in several studies (Van Egmond 33 al., 1977; Wiseman and Marth, 1983a; Brackett and Marth, 1982). Furtado gt al. (1981) also noted a slight increase in the average residual levels of aflatoxins upon frying of sliced pork bellies. In the present study, temperatures reached at the center of the steamed and fried eggs were 78 and 86-8700, respec- tively. Total cooking times were 5 min for steamed eggs and 2 min for the fried eggs. Results of the present study indicate that aflatoxins were quite heat stable during 143 Table 20 - Percent Recoveries of Spiked Aflatoxins During Cooking of Eggs % Recovery of Aflatoxins(1’2 SAMPLE B1 32 M1 M2 Replication 1 Raw 46 62 101 69 Steamed 41 66 92, 64 Fried 43 48 47 72 Replication 2 Raw 62 54 94 73 Steamed 81 60 94 88 Fried 81 62 94 7O Replication 3 Raw 52 46 86 108 Steamed 4O 63 81 71 Fried 62 52 91 76 Mean of Replications Raw 53 54 94 83 Steamed 54 , 63 89 7h Fried 62 54 77 73 1) Spiking levels of raw egg were 0.5, 0.15, 0.4 and 0.2 ug/ kg of aflatoxins B1, B2, M1 and M2, respectively. Correc- tions were made to account for moisture losses during cooking. 2) % Recovery = Amount recovered from sample x 100 Amount added to sample 144 cooking. Little or no reduction would be expected during cooking of eggs under these conditions due to the heat sta- bility of aflatoxins (Goldblatt, 1971). In support of these reults,Peers and Linsell (1975) have shown that AFB1 is not degraded in peanut and corn oils until the temperature reaches 250°C, which is close to its decomposition tempera- ture. Likewise, Allcroft and Cranaghan (1963) found that pasteurization of 'toxic' milk at 80°C for 45 sec or at 70°C for 30 min, or roller drying did not reduce its toxicity to 1-day old ducklings. Wiseman and Marth (1983b) also found no change in the AFM1 content of naturally contaminated milk after heating for 2 hours at 100°C. Furtado gt a; (1981) reported that cooking and/or pro- cessing of pork had some effect in lowering the levels of aflatoxins B1 and B2. Although inactivation was in the range of 15 to 30%, it was not statistically significant. The authors concluded that aflatoxins are quite stable during cooking and/or processing and that the procedures used were not effective in reducing the levels in contaminated meat, which is in essential agreement with results of the current study. Present results suggest that steaming or frying had no major effect on the levels of aflatoxins present in the raw eggs. This was probably a result of the stability of the aflatoxins to heatiJLcombination with low cooking tempera- tures and short cooking times. At the low levels of afla- toxins present in the raw eggs, the sensitivity and precision 145 of the method were not high enough to detect consistent dif- ferences. The action level officially set by the FDA in raw and processed foods for human consumption is 20 ug/kg. At the levels of aflatoxins found in the eggs of hens consuming a spiked ration ((.0.5 ug/kg), the significance of the small changes on cooking are of doubtful importance. This provides further support for concluding that the potential hazard for human exposure to aflatoxins by consumption of raw or cooked eggs is negligible. Aflatoxin Residues in the Tissues and Organs of Hens Fed an Aflatoxin-Contaminated Diet Aflatoxin levels for the tissues and organs of hens sacrificed at the end of the aflatoxin feeding period are presented in Table 21. Although 16 treated hens were slaugh- tered at this time, analysis were carried out on the tissues of only eight randomly selected hens. Extra hens had been included in the aflatoxin feeding experiment to have enough muscle samples for processing experiments. As shown in Table 21, aflatoxin levels in breast and leg muscles were very low. Thus, processing was not carried out, since it was evident that at these low levels the method used is not sensitive and/or precise. enough to detect the small changes occurring during processing. As shown in Table 21, measurable amounts of both AFB1 and AFB2 were carried over to all tissues and organs of the 1.1L63 fable 2! - Aflatoxin Residuen Detected in the fleeces 0t Aflatoxin-red Ilene ct the End _ cf the Aflatoxin Feeding Period hen l or Supie Aflatoxin bevelu (up/kg) '1 ’2 "1 '2 '20 I’1 '2 |‘1 I'2 '2. mm m 202 0 0 0 0 tr 0.05 0.05 0 tr 0.02 205 0 0 0 0 tr 0.00 0.05 0 0.01 0.00 207 0 tr 0 0.02 0.01 0.01 0.22 0 0.01 0.00 200 0 tr 0 0 tr 0.02 0.02 0 tr 0.00 210 0.05 0.05 0: 0.02 0.05 0.11 0.07 0.01 0.02 0.05 211 tr 0.01 0 tr 0 0.02 0.00 tr 0.01 0.02 216 0 0 0 0 0.01 0.02 0.02 0 tr 0.02 255 0 0 0 tr 0.01 1: 0.02 0 tr 0.05 ecun" 0.01 0.01 0 0.01 0.02 0.05 0.06 0 0.01 0.05 m m 202 0.00 0.05 tr 0.01 2.21 0.55 0.25 0 0 tr 205 0.22 0.09 0 0.01 0.50 1.09 0.75 0.01 0.02 0.07 207 0.00 0.25 0.05 0.10 5.07 0.95 0.52 0 0.02 0.10 200 0.07 0.00 0 tr 0.55 0.56 0.22 0 0 0.00 210 0.26 0.26 0 0.01 0.55 1.29 0.60 0 0 0 211 0.12 0.07 an 00 1.16 0.00 0.10 tr tr 0.07 216 0.20 0.25 0.00 0.00 1.55 0.50 0.50 tr 0: 0.06 2,, 002° 00°, 00°, 00°, 2032 . 00,. 0086 02' 00°! 00°? soan‘z 0.20 0.15 0.02 0.05 1.52 - 0.67 0.50 0 0.01 0.05 ms? m“ 202 0.07 0.05 0.01 0.02 0.00 _ 2050210 0.15 0.00 0.02 0.01 0.02 200 0.00 0.02 0.01 0.02 0.05 211 0.00 0.05 tr 0.01 0.02 216 . 0.00 0.02 tr tr 0.0) 255 0.05 0.02 0 tr 0.00 0000‘: 0.07 0.05 0.01 0.01 0.06 0.25 0.10 tr 0.01 0.10 um: am 1‘5 (207.200. 205.216) 0.00 0.05 0 0 0.06 0.27 0.16 0.07 0.05 1.90 11" (255. 211. 205.210) '0.10 0.05 0: 0.02 0.05 0.07 0.50 0.11 0.00 2.29 (5 ii! 20 .212 ‘203.2093 IA 0.55 0.20 0.10 0.07 1.95 (3 IV 21 .201 (202.2103 an 0.27 0.12 0.11 0.00 2.50 0000‘: 0.15 0.05 0 0.01 0.05 0.09 0.20 0.10 0.07 2.12 mm mm P001 ‘(6 0006 0007 Sr 0e06 0001 0002 0005. O ‘r 0002 2001 0“ 0.12 0.10 0: 0.01 0.00 0.05 0.00 0 tr 0.01 lesn‘z 0.09 0.00 tr 0.01 0.05 0.02 0.00 0 tr 0.01 1) tr I trace ascents - visible but tee call to quntitcte (( 0.01 Mg) Ill-chrcsetccrunetweli received. u-nctunlyeed 2’ leans calculated ass-inc trauma”, which is the largest 0.01-It which would not he assemble ” Ovaries ct hens 205 end 210 were ccshined fer minis. The every 0! hen 207 was not mined because it wee too can 4) he spleens tree ell 16 hens were contained for analysis (30.6 3) 5) Scspie represents 0 ccepceite 0! heart and kidney eagles tor the hens indicated in parenthesis 6) Peel A - Ccsbined blood 0! hens 201. 203, 205. 207. 209. 210. 211 end 216 P00]. I I Mined bleed of hens 202, 200. 206, 200. 2!). 210, 212. “‘23) 1117 hens fed the contaminated diet. The lowest levels were de- tected in breast and leg muscles and in blood serum, followed by the ovaries and the blood clot. Higher levels were detected in all other excised organs. The highest levels of both AFB1 and AFB2 were detected in the gizzard, followed by the kidneys, liver, spleen and heart, respectively. The high levels of AFB1 and AFB2 in the gizzard may have been caused in part by direct contamination from the feed during slaughtering as reported by Chen gt 3;. (1984). Additional evidence for this in the present study is the high ratio between the amounts of aflatoxins B1 and B2 in comparison to the total aflatoxins levels detected in the gizzard and, conversely, the low metabolite to parent or ingested residues ratio (Table 22). Sawhney 23 El- (1973) 14 also reported a high concentration of 0 activity in the luC-AFBI. Howev- er, they concluded that this suggested either slow absorption crop and gizzard of hens dosed orally with or passage, or both, from this segment of the alimentary canal. The levels of aflatoxins B1 and 32 in the blood clot were considerably higher than those in the serum. This is in agreement with results of Kumagai gt.al. (1983), who showed that upon incubation of blood from different animal species with 14 C-AFBI, high levels of radioactivity were associated with blood cells. They suggested that AFB1 may bind some component of the blood cells. Since the radioactiv- ity detected in the plasma was present in the plasma protein fraction, they proposed that in vivo AFB1 may be carried by 148 both the plasma proteins and the blood cells. Aflatoxins M1 and M2, the metabolites of AFB1 and AFB2, respectively, were detected in low amounts in all organs and tissues. The highest levels of these metabolites were found in the kidneys and liver. The compound tentatively identified as AFBZa was the most abundant metabolite detected in all tissues of aflatoxin-fed hens. The highest levels were found in kidneys and liver, while the lowest amounts occurred in breast and leg muscles and in the blood serum and clot. Results of the present study are in general agreement with reports by Sawhney 33 al. (1973) and Mabee and Chipley 14 (1973b), who detected 0 activity in all tissues and fluids 14c-AFB analyzed from laying hens dosed or fed with 1. Sim- ilarly, Trucksess gt al. (1983) reported the presence of AFB1, AFM1 and aflatoxicol in various tissues of hens fed a diet contaminated with AFBI. Chen gt El' (1984) also reported that aflatoxins were deposited, either as the original com- pound fed (AFB1 and AFBZ) or as their metabolites (AFM1 and AFMZ), in all tissues of chickens fed an aflatoxin-spiked diet. Aflatoxin B2a' the hemiacetal derivative of AFB1, is considered to be‘a metabolite of AFB1 (Patterson and Roberts, 1970; Gurtoo and Campbell, 1974), although it was also a major urinaty metabolite in rats injected with AFB2 (Dann gt al., 1972). Patterson and Roberts (1970) reported that the liver of chicks, guinea pigs and mice metabolized AFB1 into small amounts of AFM1 (5-10%). whereas, the major metabolite 149 (90%) was AFB Later, Patterson and Roberts (1972) stated 2a' that the yields of the hemiacetal were difficult to assess because of binding with free amino groups in proteins and amino acids to form Schiff base compounds. In the present study, the use of strong protein and nucleic acid precipitants, such as ammonium sulfate and lead acetate, in combination with with acidulating agents and competitive reactants, such as citric acid and saturated sodium chloride solution, may account for the detection of AFBZa' These reagents may cause dissociation of the bonds formed with cellular components and thus release AFBZa' This possibility is supported by find- ings of Ashoor and Chu (1975), who have shown that the inter- actions of AFB2a with amino acids, proteins and liver micro- somes in vitro are reversible and pH dependent. Neal 33 El. (1981) have pointed out that earlier reports on the production of AFB2a (Patterson and Roberts, 1970; Gurtoo and Dahms, 1974) were erroneous being based on iden- tification of AFBl-dihydrodiol as AFB2a due to the similarity of their ultraviolet spectra (Swenson 33 al., 1973). In the present study, however, the unknown metabolite was tenta- tively identified as AFBZa’ since upon comparison of its chromatographic mobility in different solvent systems, it had an Rf similar to AFB2a and not to AFBl-dihydrodiol. Further evidence indicating the formation of AFB2a by chickens and laying hens dosed with 1L‘LC-AFBI was presented by Chipley 33 al. (1974). They reported that upon enzymatic treatment ethyl acetate extracts of tissues and organs of dosed birds, 150 14 an average of 50% of the C detected was a liberated peptide or amino acid conjugate of 14C-AFBZa. Identification was based on comparison of the Rf values and absorbance maxima of the isolated metabolite, which were identical to those of a prepared standard of AFBZa' Gregory 33 El! (1983) detected compounds having the chromatographic behavior and fluorescence properties of AFB2a and AFM2a in tissues of turkey poults fed a diet containing 500 ug of AFBl/kg for 21 days. Dashek gt El. (1983) also reported an unidentified fluorescent compound with an Rf similar to AFB2a which was present in the blood, liver and excreta of quail dosed with mixed aflatoxins. They reported, however, that the UV absorption spectra of the presumed AFB2a failed to yield the expected absorption maxima. Furtado 33 al. (1979) tentatively identified AFB2a in the tissues of pigs fed aflatoxins. No positive identifica- tion was made due to the lack of standards. However, in a later study, Furtado 23.2i- (1982) concluded that the spot previously identified as AFB2a was really AFM2 on comparison of its chromatographic mobility with authentic AFB2a and AFM2 standards. Aflatoxins M2 and B2a are very similar chem- ically and structurally (Figure 2), differing only by the position of the hydroxy group in the molecule. Thus, they show similar chromatographic characteristics and exhibit similar blue-violet fluorescence under UV light. Positive identification of this metabolite by mass spectra will be required to definitely establish whether the compound iso- lated in the present study is AFBZa' 151 Total amounts of ingested aflatoxins and their metabolites deposited in the tissues and organs, as well as some relation- ships between them are presented in Table 22. As shown, the highest concentration of aflatoxin residues was found in the liver and kidneys. These results agree with those reported by Furtado gt El- (1982) and by Chen gt al. (1984), who found that upon feeding aflatoxin-spiked diets to pigs and chickens, respectively, the highest amounts of aflatoxins were detected in the liver and kidneys. The high capacity of the liver and kidneys to concentrate toxic compounds in comparison to other organs is probably related to the important role they play in elimination of xenobiotics (Klaassen, 1980). Both the kidney and the liver have the capacity to excrete and metabolize many chemicals, although the main metabolizing organ is the liver (Klaassen, 1980). In the present study, the higher proportion of free metabolites (M1 + M2 + 32a) over the total or the parent (B1 + B2) aflatoxins found in the liver and kidneys in compar- ison to other organs (Table 22) indicates their importance-‘ in aflatoxin metabolism by the laying hen. Thus, the liver and kidney had on average 4.8 and 3.3 times more metabolites than parent aflatoxins, respectively. This probably reflects the transformation of AFB1 and AFB2 to metabolites by these organs. As indicated in Table 22, the breast and ovaries con- tained approximately the same amounts of both the parent afla- toxins and their metabolites, while the other tissues had 152 + vacuum u Hmpoe mmpeaonmpms .MSHuomh EHxOpmahm we use on» pm_mmsmmwu 059 :w m~mm< was sz< .Hzm< on mmuHHOQMQoE .mmm< can “mms Mo pszosm on» on Uzommohhoo mcfixOpmem pneumm am manna CH mozam> cams Scum pmpmHZOHMo who; Mama AH 00.0 00.0 00.0 HH.H 00.0 00.“ eemueeu 50.0 00.0 00.0 50.0 “0.0 00.0 sseom 000Hm 05.0 00.0 00.0 H5.0 00.0 5H.0 00H0 cooam Hm.0 05.0 05.0 00.0 ”a.0 00.0 cmmaem 00.0 05.0 05.0 05.0 00.0 0a.0 0050: 00.0 20.0 00.0 mi.0 00.0 00.0 504 00.0 00.0 00.0 50.0 50.0 0H.0 mmeem>0 00.H 00.0 00.0 00.0 50.0 50.0 000005 50.0 55.0 05.0 50.5 05.5 00.0 msmzeex 05.: 00.0 5H.0 00.H 5m.a 00.0 00>eq gcmpnm Hapoa Hmmoe mopfiHOQMpoE mopwaonmpmz pamgmm Hence mopHHoQMpms psopmm msmmwe owumm wa\wzv mafixopmaw< 50 ivpmem 0mxeemscexoamse< :5 com mCmm 5o mosmmwa CH pmpwmommm mmm 625 NS . S mmpHHOQMpmS pflmze 0:5 mm was Hm mafixoumam< smozpmm mowpmm can mussos< H5309 ow5hm>< 1 mm manna 153 higher parent molecule residues. This is probably due to the higher polarity and increased water solubility of the hydrox- ylated metabolites, which are more easily removed from the tissues than unmetabolized aflatoxins B1 and B2. Excretion of the hydroxylated metabolites may be either as the original compound, or more likely as aflatoxin conjugates after bind- ing to various endogenous compounds (Mabee and Chipley, 1973b; Neal gt al., 1981). The low proportionof hydroxylated.metabolites in the blood components in the present study may indicate that if they are present, they are more likely bound to protein or other components and remain in the water soluble portion of the extract (Mabee and Chipley, 1973b). The gizzard had the lowest metabolite concentration, which as discussed earlier may indicate contamination from the feed during slaughter (Chen gt al., 1984). Results of the present study demonstrate that only a small fraction of the aflatoxins consumed was deposited in the tissues of hens, either as the original aflatoxins orr as their metabolites. These results are in agreement with those of Mabee and Chipley (1973b) and of Sawhney 2£.§l- (1973), who indicated that laying hens can metabolize the majority of AFB1 when administered at relatively low doses. Sawhney 33 al. (1973) reported that 7 days after administra- trion of a single oral dose of 1“ C-AFBl, 70.61% had been recovered in the excrement. Similarly, Mabee and Chipley (1973b) reported recovery of only 7.85% of the total 154 radioactivity administered in the tissues of the laying hens while the rest was excreted. They also noted that only 10% of the radioactivity present in the sample extracts was chloroform soluble and concluded that aflatoxin conjugates are the predominant metabolites. They suggested that forma- tion and deposition of conjugates may explain conflicting reports dealing with the levels of aflatoxins from animal tissues. In the present study efforts were concentrated on the determination of free aflatoxins becauSe the mutagenici- ty, genotoxicity and acute toxicity of the water soluble con- jugates is very low in comparison to that of free residues (Wei 23 al., 1978; Jaggi 32 31., 1980). The importance of water soluble conjugates: however, can not be disregarded, since conjugated aflatoxins can be liberated from the tissues in the presence of the appropriate enzyme(s) (Mabee and Chipley, 1973a: Chipley _e_t_ gin 19711, Wei at. _a_1_1_.,1978) and may contribute to the risk of chronic toxicity during long term ingestion of contaminated tissues. The total amounts of aflatoxin residues in each tissue were calculated from multiplying the total concentration in each tissue (Table 22) by the average weight of the corre- sponding tissue. These values and the contribution of each tissue as a percentage of the total aflatoxins recovered are presented in Table 23. As shown, the highest absolute amount of aflatoxins was found in the liver followed by the gizzard, leg and kidneys. The small size of the kidneys accounts for 155 Table 23 - Average Calculated Values for Aflatoxins Found in the Tissues of Hens Sacrificed at the End of Aflatoxin Feeding Tissue Average Total Aflatoxin Percent of Total weight, g(1 Content, ug(2 Recovered<3 Liver 53.8 0.101 43.3 Kidneys 7.9 0.023 9.9 Breast 185.2 0.007 3.0 Ovaries 49.0 0.009 3.9 Leg 195.5 0.025 10.7 Heart 6.5 0.016 6.9 Spleen 1.8 0.009 3.9 Blood“L 101.8 0.014 6.0 Gizzard 25.9 0.029 12.4 Total 0.233 100.0 1) Average weight of each tissue from 8 aflatoxin-fed hens sacrificed at 0 days. 2) Total Aflatoxin Content (ug)= Total aflatoxin concentra- tion (ug/kg from Table 22) x Organ weight (g)/1,000 % of total recovered;= Igtal afllatgxins in a tissue x 100 Total aflatoxin content of all tissues 3) 4) Blood volume was calculated under the assumption that it represents about 6% of the weight of a mature hen (Nesheim 23 31., 1979) 156 their low net contribution in spite of having the highest total concentration (Table 22). In contrast, leg muscle, which contained only about 4% of the concentration present in the kidneys, contributed a larger percentage of the total because leg weight was about 25 times that of the kidneys. Results demonstrate that in absolute terms very small amounts of aflatoxins were actually present in tissues of hens fed an aflatoxin-contaminated diet. Rodricks and Stoloff (1977) summarized a large number of studies on carry over of aflatoxin residues from feed to edi- ble tissues of food producing animals. They tabulated the data in terms of the ratio between the level of aflatoxins in the feed and the level in the tissue or animal product. This was intended to provide regulatory and public health officials with the information necessary to control human exposure to aflatoxins by placing restrictions on the afla- toxin levels in feeds. Table 24 presents the calculated ratios derived from the present study for kidneys, liver and leg muscle. The lowest ratios between the parent aflaé toxin in feed and parent aflatoxin in tissue occurred in the kidneys and liver, respectively. This reflects the ability of these organs to concentrate the ingested aflatoxins as discussed earlier. When the metabolites present in the tis- sues were included, the ratio decreased markedly, particularly for AFBI. This suggests that deposition of the parent afla- toxins is low while higher amounts of metabolites are present, probably as a result of the ability of laying hens to 157 Table 24 - Ratios of Aflatoxins B1 and B2 in the Feed in Relation to Aflatoxins B1, B in the Tissues of Hens 2, M1, M2 and B2a (1 Tissue Aflatoxin Aflatoxin Ratio of Level in in Feed in Tissue(2 Feed to Level in Tissue Liver B1 B1 16,550 B1 B1+M1+B2a 1,902 Liver B2 B2 12,923 B2 B2+M2 10,500 Kidney B1 B1 6,755 B1 B1+M1+B2a 1,221 Kidney B2 B2 8,400 B2 B2+M2 6,222 Leg B1 B1 110.333 B1 Bl+M1+B23 55,167 Leg 32 B2 28,000 B2 B2+M2 24,000 1) Higher values indicate less residues from a given amount of aflatoxins in the feed It is assumed that AFM1 and AFB2a are only metabolites of 2) AFB the tissues 1, and that AFM2 is the only metabolite of AFB2 in 158 metabolize and excrete these compounds on administration at relatively low doses (Mabee and Chipley, 1973b; Sawhney gt gin 1973). Three studies summarized by Rodricks and Stoloff (1977) give values of 59, 537 and ) 1,860 for the ratio between AFB1 in the feed and AFB1 in the liver of hens. These values are all smaller than the 16,550 derived in the present study, but point out the great amount of variability that can be encountered on estimating this parameter. The variability that can occur is emphasized by Patterson gt 3;. (1980), who reexamined the ratios between AFB1 in the feed and AFM1 in cow's milk estimated by Rodricks and Stoloff (1977), and found an overall range of 34 to 1,600. They also reported a similar degree of variation between 6 cows that they - studied in a single experiment (Patterson 33 31., 1980). As indicated by Rodricks and Stoloff (1977), the dif- ferences in the feed/tissue ratios may be due to a number of factors which are known to quantitatively and in some cases even qualitatively affect tissue deposition of xenobiotics. These include: 1) the species and breed of animals; 2) dose, mode and duration of exposure; 3) nutritional and health status of the animal; and 4) length of time between cessation of exposure and collection of samples. Ldtzsch and Leistner (1977) suggested that long term feeding of aflatoxins to lay- ing hens resulted in adaptation to aflatoxins with lower levels being deposited in their eggs. This type of response would probably also have an effect on feed/tissue ratios. 159 Total aflatoxin residues in the tissues analyzed in the present study were below 3 ug/kg (Table 22), and thus were all less than the action level of 20 ug/kg officially set by the FDA for aflatoxin levels allowable in foods and food in- gredients intended for human consumption. Furthermore, the total aflatoxin levels found in the breast and leg muscles, which would be more commonly consumed than organ meat, had even lower values and averaged less than 0.2 ug/kg. 0n the basis of these results it can be concluded that there is little risk for humans of acute toxicity from exposure to aflatoxins in the tissues of aflatoxin-fed hens. Neverthe- less, the possibility of long term or chronic exposure to free aflatoxins and those which could be released from the water soluble conjugates can not be disregarded. This is in agreement with the conclusion that aflatoxin residues depos- ited in the tissues of pigs (Jaggi 23 31., 1980; Furtado 23 31., 1982) and Of broiler chickens (Chen gt 31., 1984) constitute little risk to humans consuming them. Aflatoxin Residues in Tissues and Organs of Hens During Withdrawal Upon removal of the contaminated feed, aflatoxin resi- dues in the tissues of hens decreased considerably in a short period of time. Two days after withdrawal from the contami- nated feed, no aflatoxins were detected in the spleens and 160 hearts of the laying hens (Table 25). All other tissues analyzed had trace to low but measurable amounts of one or more of the aflatoxin residues. The levels, however, were far lower than those found in the corresponding tissues at the end of the aflatoxin-feeding period, particularly in the gizzard, kidneys and liver. which were the organs with the highest levels at 0 days. Thus, the average level of AFB1 in kidneys decreased from 0.49 ug/kg at 0 days to 0.01 ug/kg after two days on the aflatoxin-free diet. The marked decrease in the content of aflatoxins B1 and B2 in the liver and kidneys is probably due to the important role these organs play in xenobiotic metabolism and excretion (Klaassen, 1980). Williams gt 2.1. (1965)and Millburn e}; a_]_... (1967) tentatively concluded that compounds with high molecu- lar weight () 300) and containing two or more aromatic rings tended to be excreted into the bile. Several studies using ring labeled AFB have indicated that aflatoxins are prefer- I entially excreted through the bile (Sawhney 33 31., 1973; Harland and Cardeilhac, 19753 Falk gt 31., 1965). The metabolic activity of the liver and kidneys may also be responsible for the higher levels of AFB2a present 2 days after withdrawal, since AFB2a is a known metabolite of both AFB1 (Patterson and Roberts, 1970; Gurtoo and Campbell, 1974) and AFB2 (Dann 33 31., 1972). AFB2a has been tenta- tively identified in the tissues and excreta of laying hens 14 dosed with C-AFBl, either in its free form (Harland and Cardeilhac, 1975) or conjugated to peptides or amino acids 161 211010 25 - Aflatoxin leeidees Detected in the tissues 0: Ilene After 2 We 01' Iithdrewal tree the Aflatoxin-Spiked Diet Aflatoxin bevelu (W llen 000,10 “w ‘1 '2 '1 I2 '2. ’1 '2. I1 II2 '20 m m 217 0 tr 0 0 tr tr 0.0) 0 0 tr 210 0 0 0 0 01- 0 0 0 e 0 219 0 01- 0 0 01- 01» 0.01 0 0 0.02 220 01- 0.02 0 0 01- 0 01- 0 0 01- 221 0 0 0 0 01- 01- 0.01 0 0 01- 222 0.01 0.02 0 In 101 0.01 0.02 0 0 01- 225 01- 0.02 0 0 tr 0: 0.02 0 tr 01- 220' 01- 01- 0 0 e 0 0 0 01- 0 11.011" 0 0.01 0 0 0 0 0.01 0 0 0.01 4 217 0 0 0 0 1a 1a 0.01 0 0 0 210 0 0 0 0 0.09 0 01- 0 0 0 219 0 0 c 0 0.01 01- 0.01 0 0 0 220 0 0 0 0 0.00 0 0 0 0 0 221 00- 0.02 0 u- 0.05 0.05 0.05 0 0 0 222 0 0 0 0 02- 0.00 0.05 0 0 01- 22) 0 00°! I II. .0“ 0 .I’ 0 0 ‘1' 220 0 0 0 0 0.02 0 0 112 00 110 seen“ 0 '0 0 0 0.05 0.01 0.01 0 0 0 mm m" 217 01- 01- 0 tr 0.01 210 0.05 0.02 0.01 0.02 0.05 219 0.05 0.01 0 0 01- 220 0.01 0.01 e 0 - tr - ' 221 0.05 0.02 c 0.01 0.00 222 0.02 0.05 0 0 0 225 0.02 0.02 0 tr 0.02 220 0.00 0.02 0 0 0.02 00011“ 0.05 0.02 0 tr 0.02 0 0 0 0 0 In: 1" 1210.220. ‘ (0 .225 0 c e 0 0 0.01 c 0 0.05 n (31:33 0 0 0.02 0.01 0 c 0.05 a...“ c 0 c 0 0 0.01 0.01 0 0 0.05 mm" mm" mm 0.05 0.05 0 0 01- 0.01 0.01 0 0 01- " tr - trace ucents - visible bet tee —ll te qfiatitate (£0.01 W 3 - ehrceatcgres nct well received ” leans calculated ass-ins tr - 0.0050. which is the largest ascent which would not he aeseerahle 3’ the spleens (renaiiOhenewere ccshined rcrmlyeie 0) Sawple represents a ccapeeite 0! heart and kidney sa-ples ter the hens indicated in parenthesis Pre- ceatised bleed et all I hens 162 where it accounted for as much as 50% of the 140 label upon enzymatic tratment of the sodium acetate extracts of tissues and excreta (Chipley gt al., 1974). Although the metabolite detected in the present study was tentatively identified as AFBZa’ controversy over its identity has arisen recently (Neal gt al., 1981). It appears, however, that the metabolite in the present study is the same compound reported and iden- tified as AFB2a by other researchers working with laying hens (Harland and Cardeilhac, 1975; Mabee and Chipley, 1973b: Chipley gt 31., 1974). As discussed previously, contamination of the gizzard during slaughter with aflatoxins B and B2 from the feed may 1 have been responsible for the high levels of AFB1 and AFB2 present on day 0. Chen gt éi- (1984) also reported contami- nation of chicken gizzards from the feed during slaughter. As shown in Table 25, the levels of aflatoxins B1 and B2 in the gizzard decreased markedly after two days on the aflato- xin-free diet. This may be due to the fact that contamination from the feed was no longer possible. 7 After two days on the aflatoxin-free diet, the highest levels of both AFB1 and AFB2 were detected in the blood clot. The presence of aflatoxins in the blood may be expected, since the blood functions in the transport of nutrients, me- tabolites, hormones and waste products in the hen (Nesheim 33 al., 1979). The higher levels in the clot are in agreement with the results of Kumagai gt a; (1983) who reported that blood cells appear to carry more AFB1 than plasma proteins. 163 Aflatoxins B1 and B2 were still detected in the breast, leg, gizzard and ovaries of almost all hens sacrificed 2 days after withdrawal. The ovaries had relatively high levels of aflatoxins B1, B2 and B2a' Similarly, Sawhney gt a1. (1973) noted an increase with time after dosing on the specific ac- tivity of the small ova from hens administered 14C-AFBl. In 'the present study, aflatoxins M1 and M2 were detected in only a few samples. The low levels of these metabolites in the tissues are probably due to their high polarity and water solubility, which contribute to their excretion. Mabee and Chipley (1973b) reported that AFM1 in the tissues of laying hens fed aflatoxins is present mainly in the form of a con- jugated glucuronide and perhaps also bound tosulfate, both of which are soluble in aqueous extracts and are not recover- ed by traditional chloroform extraction. Four days after withdrawal from the aflatoxin contami- nated diet, trace to measurable amounts of aflatoxins were detected in only a few tissues of some hens (Table 26). One hen, 227, had trace to measurable amounts of aflatoxins B1” and B2 in the breast, leg and liver. Upon reviewing some other performance parameters of this hen, it was noted that she had stepped laying with no evident signs of recovery. In fact, the ovaries weighed only 7.2 g, and thus were to small to be analyzed. Her liver was still significantly en- larged at slaughter, comprising about 6.75% of her body weight compared to a control value of approximately 2.1%. The large size of the liver may have had a dilution effect 1.631; table 26 - Aflatoxin Residues Detected in the fleeces 01‘ Ilene After 0 Days 0: lithdreeai Pres the Aflatoxin-Spiked Diet Aflatoxin leveln (cg/kg) llen I cr Sample '1 02 111 02 02. 0, '2 a, 112 02'| m m 225 0 0 0 0 0 0 0 o 0 0 226 o 0 0 0 0 0 0 0 0 0 227 tr tr 0 II h! 0.01 0.0! 0 0 0 220 0 0 0- 0 0 0 0 0 0 0 229 o 0 ' 0 0 0 0 0 0 0 0 250 0 0 0 0 0 0 0 0 0 0 251 0 0 . 0 0 0 0 0 0 0 0 252 0 0 0 0 0 0 0 0 0 0 Ian“ 0 0 0 0 0 0 0 0 e 0 m m 225 0 0 0 0 0 ' 0 0 0 0 0 226 0 0 0 0 0 0 0 0 0 0 227 tr 01- 0 0 21- 0 0 0 0 0 220 0 0 0 0 0 0 0 0 0 0 229 0 tr , 0 0 0.00 0 tr 0 0 III 250 0 0 0 0 0.07 0 0 0 0 0 251 0 0 0 0 01- 0 0 0 0 0 252 0 0 0 0 0.00 0 0 0 0 0 110011“ 0 0 0 0 0.02 0 0 0 0 0 9101102 2:10:0" 225 0 0 0 0 0 226 0 0 0 0 227“ u _ 220 0 tr 0 0 tr 229 0 0 0 0 o 250 0 0 0 0 tr 251 0 0 0 0 0 252 0 0 0 0 0.01 110011“ 0 0 0 0 0 0 0 0 0 0 1:10: llulllll 1‘5 (226.220 , . ) 0 0 0 0 0 0 0 0 0 0 11" (225.227 251.252) 0 0 0 0.01 0 0 01- 00.1" 0 0 0 0 0 0 0 0 21222.912:)‘ ‘. 11222.211103‘ 1111 hens 0 0 0 0 0 01- 0.01 0 0 0 ‘1?" - trace acute . visible bet tee 0.11 to quantitate ((0.01 Wu) III - ehrc-tcgras net well received, M - net analysed 2) Isms calculated ass—ing tr 0 0.0050. which is the largest escut which wcaid not be assemble 3’ The spleens free all hens were ccabined tcr analysis fhecnrycrhenMwaemtmlyud_beaau itwastceull 5’ Semis representsaceapcaite ci‘heartallihidney-apieetcrthetcarhenechcwn in prentheeie " nee 00.011100 01000 or 011 0011- 165 on the residue levels and could account for the fact that only trace amounts of aflatoxins were detected in this organ. Hen 227 also lost 225 g of body weight during the 4 days of clear- ance. From these observations it is evident that this hen appeared to be more sensitive to aflatoxins and had not re- covered before slaughter. No aflatoxin residues were detected in the heart or spleen of hens sacrificed four days after withdrawal from the spiked ration. However, blood serum contained some AF32 and one kidney sample contained traces of AFB2a and AFBZ. AFB2a was detected in 5 liver and 3 ovary samples on day 4. Both AFB2a and AFBl-dihydrodiol may react with proteins, peptides and amino acids at physiological and alkaline pH values to form Schiff bases (Gurtoo and Dahms, 1974; Neal 33 al., 1981). Aflatoxin residues bound to tissue components probably remain in the tissues longer than the free or con- jugated aflatoxins. This may explain the persistence of AFB2a residues in the present study and may account for the fact that radioactivity was still detected in the excreta and tis- sues of hens dosed with 1“ C-AFB1 at 7 days after dosing (Sawhney 23 91., 1973). Under the extraction conditions used in the present study, dissociation of the bonds of AFB2a to cellular components, which have been reported to be revers- ible and pH dependent (Ashoor and Chu, 1975), could result in the release and detection of AFB2a over a longer period of time. No aflatoxin residues were detected in the breast, leg, 166 gizzard and ovaries of hens sacrificed after 8 days on the aflatoxin-free diet (Table 27). Four liver samples had trace to measurable, but low levels of either aflatoxins B1, B2 or B2a' One of the composite kidney samples contained some AFB and AFB , and the blood clot had AFBl, AFB2 and 2 2a traces of AFB However, by day 16, no aflatoxins were re- 2a' covered from the kidneys, blood clot or serum. Performance data for hens showing aflatoxin residues af- ter 8 days on the aflatoxin-free diet were examined. Hen 240, which had residues of aflatoxins B1, B2 and B28. in the liver, had stopped laying during the third week of aflatoxin feeding. The ovary size of this hen at death was normal, however, and there were large and medium ova present, which would indicate that recovery had begun. Liver size appeared to be normal , although it was still slightly pale and showed some petechial hemorrhages, which may account for decreased ability of this hen to metabolize aflatoxins that were found in the liver. Results of the present study indicate that differences exist between individual hens in the amount of time required to achieve tissue clearance upon removal of aflatoxins from the feed. Parameters like egg production and the size and condition of the liver and ovaries serve as indicators of recovery from aflatoxin exposure and appear to be related to the likelihood of detecting aflatoxin residues in the tissues of hens. Similarly, Van Zytveld 23 El- (1970) detected afla- toxins in the tissues of chickens that grew poorly or died 1677 Table 27 - Aflatoxin Residues Detected in the Tissues 0f Hens After 8 Days of Withdrawal from the Aflatoxin-Spiked Diet Aflatoxin I.cvei(l (us/ks) Hen I or Sanple B1 B2 '1 '2 320 B1 B2 '1 '2 32. a 2152 5&9 235 0 0 0 0 0 0 O 0 O 0 236 0 O 0 0 O O 0 0 0 O 237 0 0 0 O 0 O O 0 0 0 238 O O O O 0 0 O O 0 0 239 0 0 0 0 0 O 0 0 0 O 2&0 O O 0 0 0 0 O 0 0 O 2h1 0 0 O 0 0 0 0 O O 0 242 0 0 O 0 O 0 0 0 O O I0an‘2 0 o 0 0 0 0 0 0 o o m M 2 235 0 0 0 0 0 O 0 0 O 0 236 ‘ 0 0 0 0 tr 0 0 0 0 0 237 tr tr 0 O O 0 O 0 NR NR 238 0 0 0 0 0.02 0 0 O O 0 239 O 0 O 0 0 O O O O O 240 tr 0.01 0 0 0.01 0 0 O 0 0 241 0 0 0 0 0 0 O 0 0 0 242 0 0 0 0 0 0 O 0 0 O Isan‘z 0 o 0 0 0 0 0 0 0 o 2!A§£§§ 235 0 0 0 o 0 235 O 0 . 0 0 0 237 o o 0 0 0 238 O 0 0 O 0 239 O 0 0 O O 2&0 0 0 0 0 0 251 O 0 0 O 0 202 0 0 0 O 0 lean‘z 0 o 0 0 0 51.21.22 1‘3 (235.239. - 237.2 2) 0 0 0 O 0 11(3 (236.2 3 2 0,2 1 0 0.01 O 0 tr 0.0012 0 tr 1 0 0 0 01000 0102‘“ 01000 szauu‘h All hens 0.02 0.03 0 0 tr 0 O 0 0 O 1) tr I trace aneunts - visible but too .0011 to quantititatc ((0.01 ug/hg) NR I 0hr0aat0graa not well resolved 2) Means calculated accusing tr I 0.0058. the largest amount that would not be measurable 2; Sample represents a coaposite of kidney saspice ter the hens shown in parenthesis Pros cenbined blood of all 8 hens 168 during aflatoxin feeding, but residues were found in only one chicken reaching market weight. Since it was noted that egg production and liver size appeared to be related to the likelihood of finding aflatoxin residues in the tissues of layinghens, the livers of hens 214 (slaughtered on day 16), hen 257 (slaughtered on day 2M) and hen 261 (slaughtered on day 32) were analyzed. Liver size at death had returned to normal for these hens, although only hen 244 had resumed normal egg production. No residues were detected in the livers of hens. 244 or 257, but 0.011- 11ng of AFB2 were detected in the liver of hen 261. After finding AFB2 residues in the liver of hen 261, the livers of all hens slaughtered on the last day of the experi- ment (32 days) were analyzed. No aflatoxin residues were detected in any of the livers of the other hens sacrificed at this time. The fact that hen 261 still had AFB2 residues in the liver further indicates the existence of individual vari- ation in response to aflatoxins, and was also manifested by gross observations at slaughter including an enlarged spleen, reabsorbed ova and weight loss over the entire clearance pe- riod. Present results demonstrate that residue levels decrease rapidly, but complete tissue clearance may require longer periods or time in some hens which may be more sensitive to the effects of aflatoxins. Pier (1981) concluded that when animals of the same age and breeding are fed aflatoxins exper- imentally, there is often a marked variation in the effects 169 on different individuals in the same experimental group. Recently, Marks and Wyatt (1979) indicated that at least part of the within-breed resistance to the effects of aflatoxins may be hereditary. They reported that the offspring of quail selected for resistance to the effects of aflatoxin had significantly lower mortality on diets containing aflatoxins than those produced by unselected parents. In a study of genetic resiStance of chickens to aflatoxins, Williams gt 3;. (1980) also reported that individual responses of the birds were quite variable, suggesting that selection for resistant progeny could reduce aflatoxin sensitivity in poultry. It would be interesting to know, however, if resistant birds also deposit less tissue residues and achieve faster clear- ance. Species differences in response to aflatoxins have been reported by several workers (Ciegler, 1975; Pier, 1981). Comparison of the present study with the results of Furtado gt al. (1982) and of Chen gt 3;. (1984) on the time required for aflatoxin clearance from tissues of pigs and chickens,[ respectively, further demonstrates the ekistence of species differences in the response to aflatoxins. Both studies re- ported clearance of aflatoxin residues from all tissues after 1 days on an aflatoxin-free diet. In the present study, how- ever, clearance of aflatoxin residues in the breast, leg, gizzard and ovaries required 8 days of withdrawal, and for the kidneys and blood 16 days. One liver sample still had measurable levels of AFB2 32 days after withdrawal. 170 Slow clearance of aflatoxin residues from the tissues of hens dosed with 1AC-AFBI has also been reported by Sawhney gt éi- (1973). These authors found that approximately 29% of the administered dose still remained in the tissues of laying hens 7 days after treatment. Trucksess 33 El- (1983) de- tected aflatoxicol in the muscle of most hens and AFB1 in the liver_of one hen 7 days after withdrawal of aflatoxin from the diet, but residues were not detectable in the kidneys, blood and ovaries at this time. Studies using labeled aflatoxins have shown that most of the administered dose appears in the tissues and excreta of the dosed animals as water soluble conjugated aflatoxin metab- olites (Bassir and Osiyemi, 1967; Mabee and Chipley, 1973a, 1973b). Harland and Cardeilhac (1975) reported that .nly 25% of the 1“ 1LLC-AFBI could be extracted with chloroform, while most (75%) In C excreted in the bile and urine of hens dosed with of the C excreted remained in the aqueous phase. Similarly, Mabee and Chipley (1973b) reported that on average 80% of the radioactivity in the excreta and tissues of layer hens was.- confined to the sodium acetate portion of the extracts. They concluded that metabolism of aflatoxins by animals, which results in the formation of conjugates, may account for the conflicting reports and failure to isolate aflatoxins in animal products. Due to their high polarity and water solubility, aflato- xin conjugates would not be extracted by the solvents used in the present study. However, the possibility that aflatoxin 171 conjugates may remain in the tissues of hens following with- drawal from the contaminated feed is more unlikely than is the case for the free aflatoxins. Gregory gt El! (1983) re- ported that both free and conjugated aflatoxins were cleared at rapid and similar rates from the tissues of turkey poults that had been fed a diet spiked with AFBl. Previous studies have not followed the clearance of afla- toxins from laying hens for such a long period of time. This allowed us to observe different response patterns to aflato- xins in terms of egg production, gross lesions and aflatoxin residues in different tissues. There were differences in the length of time required to achieve clearance of aflatoxin res- idues from different tissues of the laying hens. In fact, 'one liver still had measurable amounts of AFB2 32 days after withdrawal from the contaminated feed. In general, however, no residues were detected in most tissues after 8 days on the aflatoxin-free diet. The importance of water soluble conjugates and covalent- ly bound aflatoxins can not be disregarded, since Wei gt 3;. (1978) have shown that the intestinal microflora of the rat has the ability to hydrolyze aflatoxin conjugates and to release free toxins. This may contribute to the risk of chronic toxicity during long term exposure to contaminated tissues. More information on the presence, toxicity and me- tabolism of aflatoxin conjugates and adducts is needed to fully determine the importance of these residues in tissues of animals destined for human consumption. SUMMARY A feeding trial was conducted to determine the levels of aflatoxins deposited in the eggs and tissues of hens fed an aflatoxin-spiked diet for 4 weeks. The aflatoxin-spiked diet contained 3,310 ug of AFB and 1,680 ug of AFB2 per kg. 1 The amount of time necessary to achieve egg and tissue clear- ance upon removal of the spiked diet was also ascertained over a period of 32 days. Aflatoxin feeding resulted in a significant decrease in both egg production and egg weights by the third and fourth weeks of feeding, respectively. At the end of aflatoxin feeding, however, there was no significant difference in body weights for the treated and control hens. After feeding aflatoxins for h weeks, 16 treated and 8 control hens were sacrificed, examined for gross lesions and selected tissues were analyzed for residues. The livers appeared slightly to very pale with some petechial hemor- rhages or with large hemorrhages close to the edges. None of the ovaries of the treated hens had ova larger than 25 mm in diameter. The livers, kidneys and spleens of the aflato- xin fed hens were 61, 1h and 20% larger than those of con- trols, respectively. In contrast, the ovaries and hearts were 27 and 7.5% smaller than those of the controls. 172 173 Egg production and egg weights of the treated hens re- turned to control values by the end of the second week of clearance. After 32 days on the aflatoxin-free ration, only the hearts of the treated hens were different from the con- trols, remaining significantly smaller. All other organs had returned to control values. Transfer of aflatoxins from the feed into eggs occurred rapidly. Residue levels in whole eggs increased to a maxi- mum by 445 days and remained relatively constant throughout aflatoxin feeding. Aflatoxins B2, M1 and M2 levels were sim- ilar in the yolk and albumen, while higher levels of AFB 1 and AFB were recovered from the yolk. The mean value for 2a the combined aflatoxins in the whole egg was less than 0.5 ug/kg. Clearance of aflatoxin residues from the albumen occurred faster than from the yolk. Due to an insufficient number of eggs, analysis of separated eggs could not be carried out on days 3 and 4 of withdrawal. However, no res- idues were detected in the albumen and the yolk after 5 and 7 days of withdrawal, respectively. No aflatoxin residues” could be recovered from whole eggs after feeding the aflato- xin- free diet for four days, probably as a result of dilution. The effects of cooking on the levels of aflatoxins in naturally and artificially contaminated eggs were studied. At the low levels of aflatoxins present, the sensitivity and precision of the method were not high enough to detect con- sistent differences. In general, however, no major effects on aflatoxins were observed with cooking. 174 Only a small fraction of the aflatoxins consumed was deposited in the tissues, either as the original compounds or as their metabolites. The aflatoxins were found widely distributed in all tissues. The compound tentatively identi-' fied as AFB2a was the most abundant residue. The highest levels of aflatoxins were detected in the gizzard, kidneys and liver, with average combined concentrations of less than 3 ug/kg each. The lowest residue levels were detected in the breast, blood serum and leg. The breast muscle had a total concentration of less than 0.1 ug/kg. Upon withdrawal from the contaminated feed, residue levels in the tissues decreased rapidly. However, complete tissue clearance required longer for some hens than for others, which could result from greater absorption or‘ slower clearance. By two days after removal of the contaminated feed, aflato- xin residues in all tissues had decreased markedly, with no aflatoxins being detected in the heart or spleen. No afla- toxin residues were detected in the breast, leg, gizzard and ovaries of hens sacrificed 8 days after withdrawal, or in . the kidneys and blood at 16 days. However, one hen out of seven still had measurable amounts of AFB2 in the liver at 32 days after withdrawal. Results of the present study indicated that differences exist between tissues and between individual hens in the amount of time required to achieve tissue clearance. However, few residues were detected in the eggs and in most tissues 175 after h and 8 days on the aflatoxin-free diet, respectively. Thus, there appears to be little or no risk to humans on consuming eggs or meat from hens 8 days after removal of aflatoxins from the feed. LIST OF REFERENCES Abbott, W.W. and Couch, J.R. 1971. Fatty liver syndrome. Proc. Distillers Feed Conf. 26:27. Abrams, L. 1965. Mycotoxicosis. J. South Africa Vet. Med. Ass. 36:5. Allcroft, R. and Carnaghan, R.B.A. 1962. Groundnut toxicity: Aspergillus flavus toxin (aflatoxin) in animal products: preliminary communication. Vet. Rec. 7h:863. Allcroft, R. and Carnaghan, R.B.A. 1963. Groundnut toxicity: An examination for toxin in human food products from animals fed toxic groundnut meal. Vet. Rec. 75:259. Allcroft, R., Carnaghan, R.B.A., Sargeant, K. and O'Kelly, J. 1961. A toxic factor in Brazilian groundnut meal. Vet. Rec. 73:h28. Allcroft, R. and Raymond, W.D. 1966. Toxic groundnut meal: Biological and chemical assays of a large batch of a 'reference' meal used for experimental work. Vet. Rec. 79:122. ' Allcroft, R. and Roberts, B.A. 1968. Toxic groundnut meal: The relationship between aflatoxin B1 intake by cows and excretion of aflatoxin M1 in milk. Vet. Rec. 82:116. Allcroft, R., Rogers, R., Lewis, G., Nabney, J. and Best, E. 1966.. Metabolism of aflatoxin in sheep: Excretion of' the milk toxin. Nature (London) 209:15h. Allen-Hoffmann, B.L. and Campbell, T.C. 1977. The relationship between hepatic glutathione levels and the formation of aflatoxin B -DNA adducts as influenced by dietary protein intake. Fed. Proc. 36.1116. Alpert, M.E., Hutt, M.S.R., Wogan, G.N. and Davidson, 0.8. 1971. Assoc1ation between aflatoxin content and hepa- _toma frequency in Uganda. Cancer 28:253. Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. 1973. CarCinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for de- tection. Proc. Natl. Acad. Sci. U.S.A. 70:2281. 176 177 Amla, I., Kamala, G.S., Goplakrishna, G.S., Jayaraj, A.P., Sreenivasamurthy, V. and Parpia, H.A.B. 1971. Cirrhosis in children from a peanutmeal contaminated with aflato- xin. Am. J. Clin. Nutr. 2h:609. Anonymous. 1982. FDA raises aflatoxin action level for cot- tonseed meal. Food Chem. News Aug. 2, pp. uo-ua. Anonymous. 198a. Validity of aflatoxin link to human cancer questioned. Food Chem. News Jan. 30, pp. 32-33. Applebaum, R.S., Bracket, R.E., Wiseman, D.W. and Marth, E.H. 1982. Aflatoxin: Toxicity to dairy cattle and occur- rence in milk and milk products - A review. J. Food Protection 45:752. Applebaum, R. S. and Marth, E. H. 1982. Fate of aflatoxin M1 in cottage cheese. J. Food Protection. h5:903. Armbrecht, B. H. 1971. Aflatoxin residues in food and feed fierived from plant and animal sources. Residue Rev. 1 13 Armbrecht, B.H., Wiseman, H.G., Shalkip, W.P. and Geleta, J. N. 1971. Swine aflatoxicosis. I. An assessment of growth efficiency and other response in growing pigs fed aflatoxin. Environm. Physiol. 1: 198. Asao, T., Buchi, G. , Abdel- Kader, M. M., Chang, S. B., Wick, E. L. and Wogan, G. N. 1965. The structures of aflato- xins B1 and G1. J. Am. Chem. Soc. 87:882. Ashoor, S.H. and Chu, F.S. 1975. Interaction of aflatoxin B2a with amino acids and proteins. Biochem. Pharmacol. 2h:1799. Asplin, F.D. and Carnaghan, R.B.A. 1961. The toxicity of certain groundnut meals for poultry with special refer- ence to their effect on ducklings and chickens. Vet. Rec. 73:1215. Association of Official Analytical Chemists. 1975a. Changes in Official Methods of Analysis made at the 88th Annual Meeting, Oct. 1h-17, 1971. J. Ass. Off. Anal. Chem. 58:393- Association of Official Analytical Chemists. 1975b. Official Methods of Analysis. 12th ed. Washington, D.C. Association of Official Analytical Chemists. 1980. Official Methods of Analysis. 13th ed. Washington, D.C. Association of Official Analytical Chemists. 1965. Official Methods of Analysis. 10th ed. Wash1ngton, D.C. 178 Babubunmin, E.A. and Bassir, O. 1972. Species differences in the anticoagulation activities of aflatoxin B1 and h-hy- droxy coumarin. Afr. J. Med. Sci. 3:97. Bassir, O. and Adekunle, A. 1970. Teratogenic action of afla- toxin Bo , palmotoxin B and palmotoxin Go on the chick embryo. J. Pathol. 102:49. Bassir, O. and Osiyemi, F. 1967. Biliary excretion of afla- toxin in the rat after a single dose. Nature (London) 215:882. Becroft, D.M. and Webster, D.R. 1972. Aflatoxins and Reye's disease. Br. Med. J. 4:117. Blount, W.P. 1961. Turkey "X” disease.- J. Br. Turkey Fed. 9:52. Blumberg, W.E. 1978. Enzymatic modification of environmental intoxicants: The role of cytochrome P-450. Quarterly Rev. Biophys. 11:h81. Bossard, E.H. and Combs, G.F. 1970. Lipotropic agents in broiler breeder rations. Poultry Sci. h9:599. Bourgeois, C.H., Keschamras, N., Comer, D.S., Harikul, S., Evans, R., Olson, C., Smith, T. and Beck, M.R. 1969. Udon encephalopathy: Fatal cerebral edema and fatty degeneration of the viscera in Thai children. J. Med. Ass. Thailand 52:553. Brackett, R.E. and Marth, E.H. 1982. Fate of aflatoxin M in Parmesan and Mozzarella cheese. J. Food Protection “5:597. Brekke, O.L., Sinnhuber, R.O., Peplinski, A.J., Wales, J.H., Putnam, G.B., Lee, D.L. and Ciegler, A. 1977. Aflato- xin in corn: Ammonia inactivation and bioassay with rainbow trout. J. Appl. Environ. Microbiol. 3h:3h. Brown, J.M.M. and Abrams, L. 1965. Biochemical studies on aflatoxicosis. Onderstepoort J. Vet. Res. 32:119. Buchi, G., Foulkes, D.M., Kurono, M., Mitchell, G.F. and Schneider, R.S. 1967. Total synthesis of racemic afla- toxin 31' J. Am. Chem. Soc. 89:6745. Buchi, G. , Muller, P.M., Roebuck, B.D. and Wogan, G.N. 197k. Aflatoxin Q1: A major metabolite of aflatoxin B1 produced by human liver. Res. Commun, Chem. Pathol. Pharmacol. 8:585. 179 Busby, W.E.Jr. and Wogan, G.N. 1981. Aflatoxins. In: Myco- toxins and N-nitroso Compounds: Environmental Risks Volume II. R. Shank (Ed.) CRC Press Inc. Boca Raton, Florida pp. 4-27. Butler, W.H. 1964. Acute toxicity of aflatoxin Blin rats. Brit. J. Cancer. 18:756. Butler, W.H. 1966. Acute toxicity of aflatoxin B1 in guinea p1gs. J. Pathol. Bacterial. 91:277. Butler, W.H. and Clifford, J.I. 1965. Extraction of afla- toxin from rat liver. Nature (London) 206:1045. Butler, W.H. and Lijinsky, W. 1970. Acute toxicity of afla- toxin G1 to the rat. J. Pathol. 102:209. Campbell, T.C., Caedo, J.P., Bulatao-Jayme, J., Salamat, L. and Eugel, R.W. 1970. Aflatoxin M1 in human urine. Nature (London) 227:403. Campbell, T.C. and Hayes, J.R. 1976. The role of aflatoxin _ metabolism in its toxic lesion. Toxicol. Appl. Pharmacol. 35:199. Campbell, T.C. and Salamat, L. 1971. Aflatoxin ingestion and excretion by humans. In: Mycotoxins in Human Health. I.F.H. Purchase (Ed.) Macmillan Press, London ppe 271-280 e Campbell, T.C. and Stoloff, L. 1974. Implications of myco- toxins for human health. J. Agric. Food Chem. 22:1006. Carnaghan, R.B.A., Hartley, R.D. andlD‘Kelly, J. 1963. Toxicity and fluorescence properties of the aflatoxins. Nature (London) 200:1101. Carnaghan, R.B.A., Lewis, G., Patterson, D.S.P. and Allcroft, R. 1966. Biochemical and pathological aspects of groundnut poisoning in chickens. Pathol. Vet. 3:601. Chang, S.B., Abdel-Kader, M.M., Wick, E.L. and Wogan, G.N. 1963. Aflatoxin B : Chemical identity and biological activity. Science 142:1191. Chaves-Carballo, E., Elifson, R.D. and Gomez, M.R. 1976. An aflatoxin in the liver of a patient with Reye-John- son.syndrome. Mayo Clin. Proc. 51:48. Chen, C. 1983. Tissue deposition and clearance of aflato- xins from broiler chickes. Master Thesis. Michigan State University. East Lansing, Michigan. 180 Chen, 0., Pearson, A.M., Coleman, T.H., Gray, J.I., Pestka, J.J. and Aust, S.D. 1984. Tissue deposition and clearance of aflatoxins from broiler chickens fed a contaminated diet. Food Chem. Toxicol. 22:447. Chipley, J.R., Mabee, M.S., Applegate, K.L. and Dreyfuss, M. S. 1974. Further charact rization of tissue distri- bution and metabolism of 1 C-aflatoxin B1 in chickens. Appl. Microbiol. 28:1027. Ciegler, A. 1975. Mycotoxins: Occurrence, chemistry, bio- logical activity. Lloydia 38:21. Ciegler, A. 1978. Fungi that produce mycotoxins: conditions and occurrence. Mycopathologia 65:5. Coles, B.F., Welch, A.M., Herzog, P.J., Smith, J.R.L. and Garner, R.C. 1980. Biological and chemical studies on 8,9-dihydroxy-8,9-dihydro-aflatoxin B1 and some of its esters. Carcinogenesis 1:79. Conney, A.H., Lu, A.Y.H., Levin, W. Somogyi, A., West, 8., Jacobson, M., Ryan, D. and Kuntzman, R. 1973. Effect of enzyme inducers on substrate specificity of the cytochrome P-450's. Drug Metab. Dispos. 1:199. Conway, H.F., Anderson, R.A. and Bagley, E.B. 1978. Detox- ification of aflatoxin-contami ted corn by roasting. Cereal Chem. 55:115. Cottier, G.J., Moore, C.H., Diener, U.L. and Davis, N.D. 1968. Aflatoxin studies with White Rock chickens. Poultry Sci. 47:1663. Cottier, G.J., Moore, C.H., Diener, U.L. and Davis, N.D. 1969. The effect of feeding four levels of aflatoxin on hatchability and subsequent performance of broilers. Poultry Sci. 8:1797. Croy, R.G., Essigman, J.M., Reinhold, U.N. and Wogan, G.N. 1978. Identification of the principal aflatoxin B1- DNA adduct formed in vivo in rat liver. Proc. Nat l. Acad. Sci. U.S.A. 75:1745. Dalezios, J.I. and Wogan, G.N. 1972. Metabolism of aflato- xin B1 in rhesus monkeys. Cancer Res. 32:2297. Dalezios, J.I., Wogan, G.N. and Weinbeb, S.M. 1971. Afla- toxin P : A new aflatoxin metabolite in monkeys. Science 171:584. 181 Dann, R.E., Mutscher L.A. and Couri, D. 1972. The in vivo metabolism of l["C-labeled aflatoxins B1, B and G1 in rats. Res. Commun, Chem. Pathol. Pharmaco . 3: 667. Dashek, W.V., Barker, S.M., Statkiewicz, W.H., Shanks, E.T. and Llewellyn, G.C. 1983. Histochemical analysis of liver cells from short term aflatoxin-dosed and non- dosed Coturnix coturnix japonica. 1. Aflatoxin-sensi- tive quail. Poultry Sci. 62:2347. Davis, N.D. and Diener, U.L. 1970. Environmental factors affecting the production of aflatoxin. In: Proceedings of the First US-Japan Conference on "Toxic Microorga- nisms" M. Herzberg (Ed.) US Government Printing Office Washington, D.C. pp. 43-47. Degen, G.H. and Neumann, H.G. 1978. The major metabolite of aflatoxin B1 in the rat is a glutathione conjugate. Chem. Biol. Interactions 22:239. De Iongh, H., Vles, R.O. and Van Pelt, J.G. 1964. Milk of mammals fed an aflatoxin-containing diet. Nature (Lon- don) 202:466. Detroy, R.W. and Hesseltine, C.W. 1968. Isolation and bio- logical activity of a microbial conversion product of aflatoxin Bl“ Nature (London) 219:967. Diener, U.L. and Davis, N.D. 1966. Aflatoxin production by isolates of Aspergillus flavus. Phytopathology 5 :1390. Di Paolo, J.A., Elis, J. and Erwin, H. 1967. Teratogenic response by hamsters, rats and mice to aflatoxin B1. Nature (London) 215:638. Doerr, J.A., Wyatt, R.D. and Hamilton, P.B. 1976. Impair? ment of coagulation function during aflatoxicosis in young chickens. Toxicol. Appl. Pharmacol. 35:437. Donaldson, W.E., Tung, H.T. and Hamilton, P.B. 1972. De- pression of fatty acid synthesis in chick liver (Gallus domesticus) by aflatoxin. Comp. Biochem. Physiol. 41Bx843. Doyle, M.P., Applebaum, R.S., Brackett, R.E. and Marth, E.H. 1982. Physical, chemical and biological degradation of mycotoxins in foods and agricultural commodities. J. Food Protection 45:964. Dunnett, C.W. 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Ass. 50:1096. 182 Dunnett, C.W. 1964. New tables for multiple comparisons with a control. Biometrics 20:482. Dutton, M.F. and Heathcote, J.G. 1966. Two new hydroxyafla- toxins. Biochem J. 101:21. Dutton, M. F. and Heathcote, J. G. 1968. Structure, biochemi- cal properties and origins of aflatoxins 32a and G2a' Chem. Ind. 7: 418. Dvorackova, I., Kusak, V., Veseoi, D., Vesela, J. and Nes- nidal, P. 1977. Aflatoxin and encephalopathy with , fatty degeneration of viscera. Ann. Nutr. Alim. 31: 977- Edds, G.T. 1979. Aflatoxins. Overview. In: Conference on Mycotoxins in Animal Feeds and Grains Related to Animal Health. Bureau of Veterinary Medicine, FDA. Rockville, Maryland pp. 80-84. Elis, J. and Di Paolo, J.A. 1967. Aflatoxin B1: induction of malformations. Arch. Pathol. 83:53. Epstein, S. M., Bartus, B. and Farber, E. 1969. Renal epi- thelial neoplasms induced in male Wistar rats by oral aflatoxin B1. Cancer Res. 29:1045. Essigman, J. M., Croy, R. G., Nadzan, A. M., Busby, W.P. Jr. Reninhold, U. N., Buchi, G. and Wogan, G. N. 1977. Structural identification of the major DNA adduct formed b aflatoxin B in vitro. Proc. Natl. Acad. Sci. U.S.A. 74:1870. 1 Exarchos, 0.0. and Gentry , R.E. 1982 . Effect of aflatoxin B1 on egg production. Avian Diseases 26:191. Falk, H. L., Thompson. S. J. and Kotin, P. 1965. Metabolism 2f Sflatoxin1 B in the rat. Proc. Am. Ass. Cancer Res. :1 Forgacs, J. and Carll, W.T. 1962. Mycotoxicoses. Adv. Vet. Sci. 7:273. Frazier, W.C. and Westhoff, D.C. 1978. Food Microbiology 3rd. ed. McGraw Hill Book Co. New York, N.Y. pp. 3-16. Furtado, R. M. 1980. The effects of withdrawal and process- ing upon the levels of aflatoxins in the tissues of pigs fed a contaminated ration. Ph. D. Dissertation. Michi- gan State University, East Lansing, MI. 183 Furtado, R.M., Pearson, A.M., Gray, J.I., Hogberg, M.G. and Miller, E.R. 1981. Effects of cooking and/or processing upon levels of aflatoxins in meat from pigs fed a contam- inated diet. J. Food Sci. 46:1306. Furtado, R.M., Pearson, A.M., Hogberg, M.G. and Miller, E.R. 1979. Aflatoxin residues in the tissues of pigs fed a contaminated diet. J. Agric. Food Chem. 27:1351. Furtado, R.M., Pearson, A.M., Hogberg, M.G., Miller, E.R., Aust, S.D. 1982. Withdrawal time required for clear- ance of aflatoxins from pig tissues. J. Agric. Food Chem. 30:101. Ganshirt, H. 1965. Documentation of thin layer chromato- grams. In: Thin Layer Chromatography. A Laboratory HanngoEa E. Stahl (Ed.) Springer-Verlag, Berlin PP- - - Garlich, J.D., Tung, H.T. and Hamilton, P.B. 1973. The effects of short term feeding of aflatoxin on egg pro- duction and some plasma constituents of the laying hen. Poultry Sci. 52:2206. Garner, R.C. and Hanson, R.S. 1971. Formation of a factor lethal for S. typhimurium TA1530 and TA1531 on incuba- tion of aflatox1n B w1th rat liver microsomes. Biochem. Biophys. Res. Comm . 45:774. Garner, R.G., Miller, E.C. and Miller, J.A. 1972. Liver microsomal metabolism of aflatoxin B to a reactive derivative toxic to Salmonella typhimurium TA1530. Cancer Res. 32:205 . Gillette,J3R. 1966. Biochemistry of drug oxidation and seduction by enzymes in hepatic endoplasmic reticulum. Advan. Pharmacol. 4:219. Gillette, J.R. 1979. Effects of induction of cytochrome P-450 enzymes on the concentration of foreign compounds and their metabolites and on the toxicological effects of these compounds. Drug Metab. Rev. 10:59. Goldblatt, L.A. 1971. Control and removal of aflatoxin. J. Amer. Oil Chem. Soc. 48:605. Green, C.E., Rice, D.W., Hsieh, D.P.H. and Byard, J.L. 1982. The comparative metabolism and toxic potency of aflato- xin B and aflatoxin M1 in primary cultures of adult- rat hepatocytes. Food Chem. Toxicol. 20:53. 184 Gregory, J.F. III, Golstein, S.L. and Edds, G.T. 1983. Me- tabolite distribution and rate of tissue clearance in turkeys fed a diet containing aflatoxin 31' Food Chem. Toxicol. 21:463. Gregory, J.F. III, and Manley, D. 1981. High performance liquid chromatographic determination of aflatoxins in animal tissues and products. J. Ass. Off. Anal. Chem. 64:144. Guengerich, F.P. 1977. Separation and purification of mul- tiple forms of microsomal P-450. J. Biol. Chem. 252: 3970- . Gumbmann, M.R., Williams, S.N., Booth, A.N.,Vohra, P., Ernst, R.A., Bethard, M. 1970. Aflatoxin susceptibility in various breeds of poultry. Proc. Soc. Exptl. Biol. Med. 134:683. Gurtoo, H.L. and Campbell, T.C. 1974. Metabolism of aflato- xin B1 and its metabolism-dependent and independent bind- ing to rat hepatic microsomes. Molec. Pharmacol. 10:776. Gurtoo, H.L. and Dahms, R. 1974. On the nature of the binding of aflatoxin 32a to rat hepatic microsomes. Res. Commun. Chem. Pathol. Pharmacol. 9:107. Hamilton, P.B. 1971. A natural and extremely severe occur- rencgaof aflatoxicosis in laying hens. Poultry Sci. 50:1 0. Hamilton, P.B. 1977. Interrelationships of mycotoxins with nutrition. Fed. Proc. 36:1899. Hamilton, P.B. and Garlich, J.D. 1971. Aflatoxin as a pos- sible cause of fatty liver syndrome in laying hens. Poultry Sci. 50:800. Hamilton, P.B. and Garlich, J.D. 1972. Failure of vitamin supplementation to alter the fatty liver syndrome caused by aflatoxin. Poultry Sci. 51:688. Harland, E.C. and Cardeilhac, P.T. 1975. Excretion of car- bon-14-labeled aflatoxin B in bile, urine and intesti- nal contents of the chicken. Am. J. Vet. Res. 36:909. Hartley, R.D., Nesbitt, B.F. and O'Kelly, J. 1963. Toxic metabolites of Aspergillus flavus. Nature (London) 198:1056. ‘ ' Hayes, W.J. 1975. Toxicology of Pesticides. Williams and Wilkins, Co. Baltimore, Maryland. 185 Hayes, A. W. -1978. Biological activities of mycotoxins. Mycopathologia. 65:29. Hayes, A.W. 1980. Mycotoxins: A review of biological ef- fecgs and their role in human diseases. Clin. Toxicol. 17: 5. Hayes, J.H., Polan, C.E. and Campbell, T.C. 1977. Bovine liver metabolism and tissue distribution of aflatoxin B1. J. Agric. Food Chem. 25:1189. Hesseltine, C.W. 1979. Introduction, definition and history of mycotoxins of importance to animal production. In: Interactions of Mycotoxins in Animal Production. Pro- ceedings of a Symposium. July 13, 1978. National Aca- demy of Sciences, Washington, D.C. pp. 3-18. Holzapfel, C.W., Steyn, P.S. and Purchase, I.F.H.. 1966. Isolation and structure of aflatoxins M1 and M2. Tetra- hedron Lett. 25:2799. Howarth, B. Jr. and Wyatt, R.D. 1976. Effect of dietary aflatoxin on fertility, hatchability and progeny per- formance of nroiler breeder hens. Appl. Environ. Micro- biol. 31:680. . Hsieh, D.P.H., Salhab, A.S., Wong, J.J. and Yang, S.L. 1974. Toxicity of aflatoxin Q as evaluated with the chicken embryo and bacterial auxotrophs. Toxicol. Appl. Phar- macol. 30:237. HSiehg DePeHe, wong' ZeAe’ wong’ JeJe, MiChaS, Ce and Rueb" ° ber, B.H. 1977. Comparative metabolism of aflatoxin. In: Mycotoxins in Human and Animal Health. J.V. Ro- dricks, C.W. Hesseltine and Mahlman, M.A. (Eds.) Pa- thotox Publishers, Inc. Park Forest South, Illinois PP- 37-50. Huff, W.E., Wyatt, R.D. .and Hamilton, P.B. 1975. Effects of dietary aflatoxin on certain egg yolk parameters. Poultry Sci. 54:2014. Hughes, B.L. Barnett, B D , Jones J E and Dick J W p . e e e e g e e 1 e Safety of feeding aflatoxin-inactivated corn to White 979 Leghorn-Layer breeders. Poultry Sci. 5811202. Hughes, B.L. and Jones J E 1979 Hematolo ' . , . . . gy of Sin le comb White Leghorn pullets fed aflatoxin-contaminaged and ammon1a-treated corn. Poultry Sci. 58:981. 186 Jacobson, W.C., Harmeyer, W.C. and Wiseman, H.G. 1971. De- termination of aflatoxins 31 and M1 in milk. J. Dairy. Sci. 54:21. Jacobson, W.C. and Wiseman, H.G. 1974. The transmission of aflatoxin B1 into eggs. Poultry Sci. 53:1743. Jaggi, W., Lutz, W.H., Luthy, J., Zweifel, U. and Schlatter, Ch. 1980. In vivo covalent binding of aflatoxin meta- bolites isolated from animal tissue to rat-liver DNA. Food Cosmet. Toxicol. 18:257. Jarvis, B. 1975. Mycotoxins in foods - Their occurrence and significance. Intern. J. Environ. Studies. 8:187. Jensen, A.H., Brekke, O.L., Frank, R. and Peplinski, A.J. 1977. Acceptance and utilization by swine of aflatoxin- contaminated corn treated with aqueous or gaseous ammonia. J. Anim. Sci. 45:8. . Jones, B.D. 1978. Chemistry of aflatoxin and related com- pounds. In: Mycotoxic Fungi, Mycotoxins, M cotoxicoses. Vol. I. T.D. Wylie and L.G. Morehouse (Edsg. Marcel Dekker, Inc. New York, N.Y. pp. 136-143. Keyl, A.C. and Booth, A.N. 1971. Aflatoxin effects in live- stock. J. Amer. Oil Chem. Soc. 48:599. Kiermeier, F. 1977. Significance of aflatoxins in the dairy industry. Annual Bulletin International Dairy Federation. Document 98. Kiermeier, F. and Mashaley, R. 1977. Einfluss der molkerei- technischen Behandlung der Rohmilch auf den Aflatoxin-M1 Gehalt der daraus hergestellten Produkte. Z. Lebensm. Unters. Forsch. 164:183. Kiermeier, F., Weiss, G., Behringer, G. and Miller, M. 1977a. Ueber das Vorkommen und den Gehalt von Aflatox1n Mg in Kasen des Handels. Z. Lebensm. Unters. Forsch. 1 3:268. Kiermeier, F.G., Weiss, G., Behringer, G., Miller, M. and Ranfft, K. 1977b. Vorkommen und Gehalt an Aflatox1n M1 in molkereianlieferungs Milch. Z. Lebensm. Unters. Forsch. 163:171. Klaassen, C.D. 1980. Absorption, distribution and excretion of toxicants. In: Toxicology. 2nd. ed. Cha ter 3.. Doul, J., Klaassen, C.D. and Amdur, M.O. (Eds.. Macm1- llan Publishing Co. Inc. London. pp. 28-55. 187 Kratzer, F.H., Bandy, D., Wiley, M. and Booth, A.N. 1969. Aflatoxin effects in poultry. Proc. Soc. Exptl. Biol. Med. 131:1281. Krishnamachari, V.R., Nagarjan, R.V., Bhat, R.V. and Telak, T.B.G. 1975. Hepatitis due to aflatoxicosis: An out- break in western India. Lancet (May 10, 1975):1061. Krogh, P., Hald, B., Hasselager, E., Madsen, A., Montensen, H.P., Larsen, A.E. and Campbell A.D. 1973. Aflatoxin residues in bacon pigs. Pure Appl. Chem. 35:275. Kumagai, S., Nakano, N. and Aibara, K. 1983. Interactions of aflatoxin B and blood components of various species in vitro: Interconversion of aflatoxin B1 and aflatoxi- col in blood. Toxicol. Appl. Pharmacol. 67:292. Lancaster, C.M., Jenkins, P.B. and Philip, J.M. 1961. To- xicity associated with certain samples of groundnuts. Nature (London) 192:1095. Lee, L.S., Cucullu, A.E., Franz, A.D. Jr. and Pons, N.A. Jr. 1969. Destruction of aflatoxins in peanuts during dry and oil roasting. J. Agric. Food Chem. 17:451. Lilly, L.J. 1965. Induction of chromosome aberrations by aflatoxin. Nature (London) 207:433. . Lin, J.-K., Kennan, K.A., Miller, E.C. and Miller, J.A. 1978. Reduced nicotinamide adenine dinucleotide phosphate-de- pendent formation of 2,3-dihydro-2,3-dihydroxy-aflatoxin B from aflatoxin B by hepatic microsomes. Cancer Res. 138:2424. 1 Lin, J.-K., Miller, J.A. and Miller, E.C. 1977. 2.3-dihy- dro-2,3-dihydroxy-aflatoxin B1, a major hydrolysis pro- duct of aflatoxin B -DNA or ribosomal RNA adducts formed in hepatic microsome-mediated reactions and in rat liver in vivo. Cancer Res. 37:4430. Linsell, C.A. 1977. Aflatoxins. In: Environment and Man Volume Six. The Chemical Environment. J. Lenihan and W.W. Fletcher (Eds.) Academic Press, New York, N.Y. pp. 121-137. Llewellyn, G.C., Stephenson, C.A. and Hofman, J.W. 1977. Aflatoxin B induced toxicity and teratogenicity in Ja- panese medaka eggs (Oryzias latipes). Toxicon 15:582. 188 Loosmore, R.M. and Harding, J.D. 1961., A toxic factor in Brazilian groundnut causing liver damage in pigs. Vet. Rec. 73:1362. . Loosmore, R.M. and Markson, L.M. 1961. Poisoning of cattle by Brazilian groundnut meal. Vet. Rec. 73:813. Ldtzsch, R. and Leistner, L. 1976. Aflatoxin-Ruckstande in Hahnereiern und Eiprodukten. Die Fleischwirtschaft 1221777. Ldtzsch, R. and Leistner, L. 1977. Transmission of aflato- xins into eggs and egg products. Ann. Nutr. Alim. 31: 499. Ldtzsch, R, Leistner, L. and Ghosh, M.K. 1976. Carry-over Effekt van Aflatoxinen bei Eiern japanischer Zwegwachteln (Coturnix coturnix japonica). -Die Fleischwirtschaft 1.231773e Lu, A.Y. and West, S.B. 1980. Multiplicity of mammalian cytochromes P-450. Pharmacol. Rev. 31:277. Luter, L., Wyslouzil, W. and Kashyap, S.C. 1982. The des- truction of aflatoxins in peanuts by microwave roasting. Can. Inst. Food Technol. J. 15:236. Lynch, G.P. 1972. Mycotoxins in feedstuffs and their effect on dairy cattle. J. Dairy Sci. 55:1243. Mabee, M.S. and Chipley, J.R. 1973a!L Tissue distribution and metabolism of aflatoxinB1 -1 C in broiler chickens. Appl. Microbial. 25:763. Mabee, M.S. and Chipley, J.R. 1973b. Tissue distribution and metabolism of aflatoxin B1-140 in layer chickes.” J. Food Sci. 38:566. Mann, C.E., Codifer, L.P., Jr. and Dallear, F.G. 1967. Effects of heat on aflatoxins in oilseed meals. J. Agric. Food Chem. 15:1090. Marks, H.L. and Wyatt, R.D. 1979. Genetic resistance to aflatox1n 1n japanese quail. Science 206:1329. 189 Marth, E.H. and Doyle, M.P. 1979. Update on molds: degra- dation of aflatoxins. Food Technol. 33:81. Masri, M.S., Booth, A.N., Hsieh, D.P.H. 1974a. Comparative metabolic conversion of aflatoxin B to M and Q1 by monkey, rat, and chicken liver. Life Sci} 15: 203. Masri, M.S., Garcia, V.S. and Page, J.R. 1969. Aflatoxin M content of milk from cows fed known amounts of aflato- xin. Vet. Rec. 84:146. Masri, M.S., Haddan, W.P., Lund, R.E. and Hsieh, D.P. 1974b. Aflatoxin Ql' A newly identified major metabolite of aflatoxin B1 in monkey liver. J. Agric. Food Chem. 22:512. Mc Cann, J., Choi, E., Yamasaki, E. and Ames, B.N. 1975. Detection of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72:5135. McGrew, P.B., Barnhart, R.M., Mertens, D.R. and Wyatt, R.D. 1982. Some effects of phenobarbital dosing of dairy cattle on aflatoxin M1 and fat in milk. J. Dairy Sci. 65:1227. McKinney, J.D., Cavanagh, G.C., Bell, J.T., Hoverslund, A.S. Nelson, D.M., Pearson, J. and Selkirk, R.J. 1973. Ef- fects of ammoniation on aflatoxins in rations fed to lactating cows. J. Amer. Oil Chem. Soc. 50:79. Mgbodile, M.U.K., Holscher, M. and Neal, R.A. 1975. A possible protective role for reduced glutathione in afla- toxin B toxicity: Effect of pretreatment of rats with phenobarbital and 3-methyl chalantrene on aflatoxin B1 toxicity. Toxicol. Appl. Pharmacol. 34:1281. Millburn, P., Smith, R.L. ans Williams, R.T. 1967. Biliary excretion of foreign compounds, biphenyl, stilbestrol * and phenolphtalein in rat: Molecular weight, polarity and metabolism as factors in biliary excretion. Bia- chem. J. 104:1275. Miller, D.M., Wilson, D.M., Wyatt, R.D., McKinney, J.K., Crawell, W.A. and Stuart, B.P. 1982. Mycotoxins: High performance liquid chromatographic determination and clearance time of aflatoxin residues in swine tissues. J. Ass. Off. Anal. Chem. 65:1. 190 Murthy, T.R.K., Jemmali, M. and Rosset, R. 1975. Studies of recovery of aflatoxin B1 injected in frozen beef at different intervals of storage. Z. Lebensm. Unters. Forsch. 158:5. Neal, G.B., Judah, D.J., Stirpe, F. and Patterson, D.S.P. 1981. The formation of 2,3-dihydro-2,3-dihydroxy-afla- toxin B by the metabolism of aflatoxin B by liver microsomes isolated from certain avian and mammalian species and the possible role of this metabolite in the acute toxicity of aflatoxin B1' Toxicol. Appl. Phar- macol. 58:431. Nesbitt, B.P., O'Kelly, J., Sargeant, K. and Sheridan, A. 1962. Toxic metabolites of Aspergillus flavus. Nature (London) 195:1062. Nesheim, M.C., Austic, R.E. and Card, L.E. 1979. Poultry Production. 12th. ed. Lea & Febiger, Philadelphia, Pennsylvania. pp. 16-57. Newberne, P.M. and Butler, W.H. 1969. Acute and chronic effects of aflatoxin in the liver of domestic and laboratory animals: A review. Cancer Res. 29:236. Norred, W.P. -4982. Use of ammonia treatment to destroy . aflatoxins in corn. J. Food Protection 45:972. North, M.G. 1978. .Commercial Chicken Production Manual. 2nd ed. Avi Publishing Co. Westpart, Connecticut. Obidoa, 0.and Siddiqui, H.T. 1978. Aflatoxin inhibition of avian hepatic mitochondria. Biochem. Pharmacol. 27: 547 - Oettle, A.G. 1964. Cancer in Africa, especially in regions south of the Sahara. J. Nat. Cancer Inst. 33:383. ' Olson, L.C., Bourgeois, C.H., Jr., Kreshamras, N., Harikul, S., Sanyakorn, C.K. and Grassman, R.A. 1970. Ence- phalopathy and fatty degeneration of the viscera in Thai children. Amer. J. Dis. Child. 120:1. Ong, T.M. 1975. Aflatoxin mutagenesis. Mutation Res. 32: Park, B.H. 1982. Assessment of the drug metabolizing capa- ' city of the liver. Br. J. Clin. Pharmacol. 14: 31. Patterson, D.S.P. 1977. Metabolism of aflatoxin and other mycotoxins in relation to their toxicity and the accumu- lation of residues in animal tissues. Pure Appl. Chem. 49:1723. 191 Patterson, D.S.P. and Allcroft, R. 1970. Metabolism of afla- toxin in susceptible and resistant animal species. Food Cosmet. Toxicol. 8:43. Patterson, D.S.P., Glancy, E.M. and Roberts, B.A. 1980. The carry-over of aflatoxin M into the milk of cows fed rations containing a low dancentratian of aflatoxin B1. Food Cosmet. Toxicol. 18:35. Patterson, D.S.P. and Roberts, B.A. 1970. The formation of aflatoxins B and G a and their degradation products in vitro detgiification of aflatoxin by levers of certain gvian and mammalian species. Food Cosmet. Toxicol. :527. Patterson, D.S.P. and Roberts, B.A. ~1971. The in vitro re- duction of aflatoxins B and B2 by soluble avian liver enzymes. Food Cosmet. Toxicol. 9:829. Patterson, D.S.P. and Roberts, B.A.’ 1972. .Aflatoxin metabo- lism in duck-liver homogenates. The relative importance of reversible cyclopentenone reduction and hemiacetal formation. Food Cosmet. Toxicol. 10:501. Payet, M., Cross, J., Quenum,C., Sankale, M. and Maulanier, M. 1966. Deus observations d'enfants ayart consumme de facon pralongee des farines sauillees par Aspergillus flavus. Presse Med. 74:649. Peers, P.G., Gilman, C.A. and Linsell, C.A. 1976. Dietary aflatoxins and human liver cancer, a study in Swaziland. Int. J. Cancer 17:167. Peers, P.G. and Linsell, C.A. 1973. Dietary aflatoxins and liver cancer- A population based study in Kenya. Br. J. Cancer 27:473 Peers, P.G. and Linsell, C.A. 1975. Aflatoxin contamination and its heat stability in Indian caok1ng oils. Trap. Sci. 17:229. Pier A.C. 1981. Mycotoxins and animal health. Adv. Vet. Sci. Comp. Med. 25:185. Pohland, A.E., Cushmac, M.E. and Andrellos, P.J. 1968. Afla- toxin Bl. J. Ass. Off. Anal. Chem. 51:907. Polan, C.E., Hayes, J.R. and Campbell, T.C. 1974. Consumption and fate of aflatoxin 31 by lactating cows. J. Agric. Food Chem. 22:635. Polzhofer, K. 1977. Aflatoxinbestimmung in Milch und Milch- produkten. Z. Lebensm. Unters. Forsch. 163:175. 192 Pang, R.S. and Wogan, G.N. 1971. Toxicity and biochemical and fine structural effects of synthetic aflatoxins M1 and B1 in rat liver. J. Natl. Cancer Inst. 47:485. Partman, R.S., Plowman, K.M., and Campbell, T.C. 1968. Afla- toxin metabolism by liver microsomal preparations by two different species. Biochem. Biophys. Res. Commun. 33: 711. Powrie, W.D. 1976. Characteristics of edible fluids of ani- mal origin: eggs. In: Principles of Food Science Pt. I Food Chemistry. 0. R. Fennema (Ed.) Marcel Dekker, Inc. New York, N.Y. pp. 659-675. Przybylski, W. 1975. Formation of aflatoxin derivatives on thin layer plates. J. Ass. Off. Anal. Chem. 58:163. Purchase, I.F.H. 1967. Acute toxicity of aflatoxins M1 and Ma in one-day old ducklings. Food Cosmet. Toxicol. 5: 3 9- Purchase, I.F.H. 1972. Aflatoxin residues in food of animal origin. Food Cosmet. Toxicol. 10:531. Purchase, I.F.H., Steyn, M. and Gifillan, T.C. 1973. Meta- bolism and acute toxicity of aflatoxin B1 in rats. Chem. Biol. Interactions 7:283. Purchase, I.F.H., Steyn, M., Rinsma, R. and Tustin, R.C. 1972. Reduction of the aflatoxin M1 content of milk by process- ing. Food Cosmet. Toxicol. 10:383. Raj, A.J., Santhanam, K., Gupta, R.P. and Venkitasubramanian, T.A. 1975. Oxidative metabolism of aflatoxin B : Obser- vations on the formation of epoxide-glutathine conjugate. Chem. Biol. Interactions 11:301. Reed, J.R., Deacon, L.E., Farr, F. and Couch, J.R. 1968. Inositol and fatty liver 3 drome. Proc. Texas N t . Conf. 23:204. yn , u r Reye, R.D., Morgan, G. and Baral, J. 1963. Encephalopathy and fatty degeneration of the viscera. A d' ' of childhood. Lancet 2:749. lsease ent1ty .Roberts, J.C., Sheppard, A.H., Knight, J.A. and Ruffey, P. 1968. Studies 1n mycological chemistry 22. Total syn- thesis of (+-)-aflataxin 32. J. Chem. Soc. (C):22. 193 Rodricks, J.V. and Stoloff, L. 1977. Aflatoxin residues from contaminated feed in edible tissues of food producing animals. In: Mycotoxins in.Human and Animal Health. J.V. Rodricks, C.W. Hesseltine and M.A. Mehlman (Eds.) Pathggo; Publishers Inc., Park Forest South, Illinois. PP- - 9- Roebuck, B.D., Siegel, W.C. and Wagan, G.N. 1978. In vitro metabolism of aflatoxin B2 by animal and human liver. Cancer Res. 38:999. Ryan, N.G., Hogan, G.R., Hayes, A.W., Unger, P.D. and Siraj, M.Y. 1979. Aflatoxin B : its role in the etiology of Reye's syndrome. Pediat ics 64:71. Salhab, A.S., Abramson, F.P., Gelhoed, G.W. and Edwards, 0.8. 1977. Aflatoxicol M1, a new metabolite of aflatoxicol. Xenobiotica 7:401. Salhab, A.S. and Edwards, 0.8. 1977. Comparative in vitro metabolism of aflatoxicol by liver preparations from animals and humans. Cancer Res. 37:1016. Salhab, A.S. and Hsieh, D.P.H. 1975. Aflatoxin H : a major metabolite of aflatoxin B1 produced by human And Rhesus monkeys livers in vitro. Res. Commun. Pathol. Pharmacol. 10: 19. Sargeant, K., Allcroft, R. and Carnaghan, R.B.A. 1961a. Ground- nut toxicity. Vet. Rec. 73:865. Sargeant, K., O'Kelly, J., Carnaghan, R.B.A. and Allcroft, R. 1961b. The assay of a toxic principle in certain ground- nut meals. Vet. Rec. 73:1219. Sargeant, K., O'Kelly, J., Carnaghan, R.B.A. and Allcroft, R. 1961c. Toxicity associated with certain samples of ground- nuts. Nature (Londan) 192:1096. Sawhney, D.S., Vadehra, D.V. and Baker, R.C. 1973. The meta- bolism of 1 C-aflatoxins in laying hens. Poultry Sci. 52:1302. Schabort, J.C. and Steyn, M. 1969. Substrate and phenobarbital inducible aflatoxin 4-hydroxylation and aflatoxin metab- olism by rat liver microsomes. Biochem. Pharmacol. 18: 22 1. Schoental, R. 1970. Hepatotoxic activity of retrorsine, sen- kirkine and hydroxysenkirkine in newborn rats, and the role of epoxides in carcinogenesis by p rrolizidine al- kaloids and aflatoxins. Nature (London 227:401. 194 Schroeder, R.W. and Ashworth, L.J. 1966. Aflatoxins: some factors affecting production and location of toxins in Aspergillus flavus-oryzae. J. Stored Prod. Res. I:267. Schuller, P.L., Verhulsdonk, C.A. and Paulsch, W.E. 1977. Aflatoxin M in liquid and powdered milk. Zesz. probl. Postep. Nau . roln. 189:255. Shank, R.O., Bourgeois, C.H., Keschamras, N. and Chandauimol, P. 1971. Aflatoxins in autopsy specimens from Thai children with acute disease of unknown etiology. Food Cosmet. Toxicol. 9:501. Shank, R.C. and Wagan, G.N. 1965. Distribution and excretion oi éuc-labeled aflatoxin B1 in the rat. Fed. Proc. 2 : 27. ‘ Shank, R.O., Wogan, G.N., Gibson, J.B. and Nondasuta, A. 1972. Dietary aflatoxins and human liver cancer. II. Aflatoxins in market foods and foodstuffs of Thailand and Hong Kong. Food Cosmet. Toxicol. 10:61. Sims, W.M., Kelly, D.C. and Sanford, P.E. 1970. A study of aflatoxins in laying hens. Poultry Sci. 49:1082. Sinnhuber, R.O., Lee, D.J., Wales, J.H., Landers, M.K. and Keyl, A.C. 1970. Aflatoxin M1- a potent liver carcino- gen for rainbow trout. Fed. Proc. 29:568. Smith, J.W. and Hamilton, P.B. 1970. Aflatoxicosis in the broiler chicken. Poultry Sco. 49:207. Snedecor G.W. and Cochran, W.C. 1967. Statistical Methods. 6th, ed. Iowa State University Press, Ames, Iowa. Southern, L.L. and Clawson, A.J. 1980. Ammoniation of corn contaminated with aflatoxin and its effects on growing J. Animal Sci. 50:459. Stoloff, L. 1977. Aflatoxins-An Overview. In: Mycotoxins in Human and Animal Health. J.V. Rodricks, C.W. Hessel- tine and M.A. Mehlman (Eds.) Pathotox Publishers, Inc. Park Forest South, Illinois. pp. 7-27. Stoloff, L. 1980. Aflatoxin control: Past and present. J. Ass. Off. Anal. Chem. 63:1067. Stoloff, L. and Trucksess, M.W. 1978. Survey for aflatoxin 31 in chicken eggs. J. Ass. Off. Anal. Chem. 61:995. 195 Stoloff, L., Trucksess, M.W., Hardin, N., Francis, O.J., Hayes, J.R., Polan, C.E., Cambell, T.C. 1975. Stability of aflatoxin M1 in milk. J. Dairy Sci. 58:1789. Strzelecki, E.L. 1973. Behavior of aflatoxins in some meat products. Acta Microbiol. Pol. Ser. B. 5:171. Swenson, D.H. 1981. Metabolic activation and detoxication of aflatoxins. In: Reviews in Biochemical Toxicology. E. Hodgson, J.R. Bend and R.M. Philpot (Eds.) Elsevier North Holland, Inc. New York, N.Y. Vol. 3 p. 155. Swenson, D.H., Lin, J.H., Miller, E.C. and Miller, J.A. 1977. Aflatoxin B1-2,3-oxide as a probable intermediate in the covalent binding of aflatoxins B and B to rat liver DNA and ribosomal RNA in viva. Cancer es. 32:172. Swenson, D.H., Miller, E.C. and Miller, J.A. 1974. Aflato- xin B1-2,3-oxide: Evidence of its formation in rat liver in vivo and by human liver microsomes in vitro. Biochem. Biophys. Res. Commun. 60:1036. Swenson, B.H., Miller, J.A. and Miller, E.C. 1973. 2.3-di- hydro-2,3-dihydroxy-aflatoxin B1: An acid hydrolysis product of an RNA-aflatoxin B1 adduct formed by hamster and rat liver microsomes in vitro. Biochem. Biophys. Res. Commun. 53:1260. Swenson, D.H., Miller, J.A. and Miller, E.C. 1975. The reac- tivity ahd carcinogenicity of aflatoxin B1-2,3-dichlori- de, a model for the putative 2.3-oxide metabolite of aflatoxin B1. Cancer Res. 35:3811. Swick, R.A. 1984. Hepatic metabolism and bioactivatian of mycotoxins and plant toxins. J. Animal Sci. 58:1017. Trucksess, M.W. and Stoloff, L. 1979. Extraction, cleanup and quantitative determination of aflatoxin B1 and M1 in beef liver. J. Ass. Off. Anal. Chem. 62:1080. Trucksess, M.W. and Stoloff, L. 1984. Determination of afla- toxicol and aflatoxins B1 and M1 in eggs. J. Ass. Off. Anal. Chem. 67:317. Trucksess, M.W.,.Stoloff, L., Pans; W.A., Cucullu, A.E., Lee, L.S. and Franz, A.O. 1977. Thin layer chromatographic determination of aflatoxin B1 in eggs. J Ass. Off. Anal. Chem. 60:795. . Trucksess, M.W., Stoloff, L., Young, K., Wyatt, R.D. and Mi- ller, B.L. 1983. Aflatoxicol and aflatoxins B1 and M1 in eggs and tlssues of hens consuming aflatoxin-conta- m1nated feed. J. Poultry Sci. 62:2176. 196 Tung, H.T., Donaldson, W.E. and Hamilton, P.B. 1972. Alter- ed lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22:97. Tung, T.C. and Ling, H.G. 1968. Study on aflatoxin of foodstuffs in Taiwan. J. Vitaminol. 14:48. Ulloa-Sosa, M. and Schroeder, H.W. 1969. Note on aflatoxin decomposition in the rocess of making tortillas from corn. Cereal Chem. 6:397. Van Egmond, R.P. 1983. Mycotoxins in dairy products. Food Chem. 11:289. Van Egmond, H.P., Paulsch, W.E., Veriga, H.A. and Schuller, P.L. 1977. The effects of processing on aflatoxin M content of milk and milk products. Arch. Inst. Pasteur (Tunis) 54:381. Van der Linde, J.A., Van der Frens, A.M. and Van Esch, G.J. 1965. Experiments with cows fed groundnut meal contain- ing aflatoxin. In: Mycotoxins in Foodstuffs. G.N. Wogan (Ed.) MIT Press, Cambridge, Massachusetts p. 247. Van der Merwe, K.J., Fourie, L. and Scott, de B. 1963. The structures of aflatoxins. Chem Ind. p. 1660. Van Rensburg, S.J., Van der Watt, J.J., Purchase, I.F.H., Pe- reira Coutinho, L. and Markahm, R. 1974. Primary can- cer rate and aflatoxin intake in a high cancer area. S. Afr. Med. J. 48:2508a. Van Zytveld, W.A., Kelley, D.C. and Dennis, S.M. 1970. Afla- toxins or their metabolites in livers and skeletal muscles of chicken. Poultry Sci. 49:1351. ‘ Waltking, A.E. 1971. Fate of aflatoxin during roasting and storage of contaminated peanut products. J. Ass. Off. Anal. Chem. 54:533. Washburn, K.W. and Wyatt, R.D. 1978. Effects and mechanism of action of aflatoxin on shell quality. Poultry Sci. 57:111. Wei, C.I., Decad, C.M., Wong, Z.A., Byard, J.L. and Hsieh, D. P.H. 1978. Characterization and mutagenicity of water- soluble conjugates of aflatoxin B1. Toxicol. Appl. Pharmacol. 45:274. White, R.E. and Coon, M.J. 1980. Oxygen activation by cyto- chrome P-450. Ann. Rev. Biochem. 49:315. 197 Williams, C.M., Colwell, W.M. and Rose, L.P. 1980.. Genetic resistance of chickens to aflatoxin assessed w1th organ culture techniques. Avian Diseases 24:415. Williams, R.T. 1959. Detoxicatian Mechanisms. John Wiley, New York, N.Y. Williams, R.T., Millburn, P. and Smith, R.L. 1965. The in- fluence of enterohepatic circulation on toxicity of drugs. Ann. New York Acad. Sci. 123:110. Wilson, B.J. and Hayes, A.W. 1973. Microbial toxins. In: Toxicants occurring naturally in foods. Comittee on Food Protection, Food and Nutrition Board. Natl. Res. Council/Natl. Acad. Sci. Washington, D.C. Wiseman, D.W. and Marth, E.H. 1983a. Behavior of aflato- xin M1 in yogurt, buttermilk and kefir, J. Food Pro- tection 46:115. - Wiseman, D.W. and Marth, E.H. 1983b. Heat and acid stability of aflatoxin M1 in naturally and artificially contami- nated milk. Milchwissenschaft 38:464. Wogan, G.N. 1964. Experimental toxicity and carcinogenicity of aflatoxins. In: Mycotoxins in Foodstuffs. G.N. Wogan (Ed.) MIT Press, Cambridge, Massachusetts. p. 163. Wogan, G.N. 1968. Biochemical responses to aflatoxins. Cancer Res. 28:2282. Wogan, G.N. 1973. Aflatoxin carcinogenesis. In: Methods in Cancer Research H. Busch (Ed.) Academic Press, New York. NeYe Ppe 309-31111». Wogan, G.N. 1977. Made of action of aflatoxins. In: Myco- toxins in Human and Animal Health. J.V. Rodricks, C.W. Hesseltine and M.A. Mehlman (Eds.) Pathotox Publishers Inc., Park Forest South, Illinois. p. 29. Wogan, G.N., Edwards, 0.8. and Newberne, P.M. 1971. Struc- ture activity relationship in toxicity and carcinogeni- city af aflatoxins and analogs. Cancer Res. 31:1936. Wagan, G.N. and Newberne, P.M. 1967. Dose-response charac- teristics of aflatoxin B1 carcinogenesis in the rat. Cancer Res. 27:2370. Wogan, G.N. and Paglialunga, S. 1974. Carcinogenicity of synthetic aflatoxin M1 in rats. Food Cosmet. Toxicol. 12:381. 198 Wogan, G. N., Paglialunga S., Newberne, P. M. 1974. Carcino- genic effects of low dietary levels of aflatoxin B1 in rats. Food Cosmet. Toxicol. 12:381. Wong, J.J. and Hsieh, D.P.H. 1976. Mutagenicity of aflato- xins related to their metabolism and carcinogenic poten- tial. Proc. Natl. Acad. Sci. U.S.A. 73:2241. Wong, Z.A. and Hsieh, D.P.H. 1978. Aflatoxicol: major afla- toxin B1 metabolite in rat plasma. Science 200:325. Wong, Z.A. and Hsieh, D.P.H. 1980. The comparative metabo- lism and toxicokinetics of aflatoxin B in the monkey, rat and mouse. Toxicol. Appl. Pharmacol. 55:115. Wong, Z.A., Wei, C.J., Rice, D.W. and Hsieh, D.P.H. 1981. Effects of phenobarbital pretreatment on the metabolism and toxicokinetics of aflatoxin B1 in the Rhesus monkey. Toxicol. Appl. Pharmacol. 60:387. World Health Organization (United Nations Environmental Pro- gramme). 1979. Mycotoxins. Environmental Health Criteria II. W.H.O. Geneva, Switzerland. Wyatt, R.D., Lockhart, W.C. and Huston, T.M. 1977. Increased resistance of chickens to acute aflatoxicosis by acclimation to low environmental temperatures. Poultry