The Metabolism of 14 C Aflatoxins in Laying Hens

The Metabolism of 14C Aflatoxins in Laying Hens D. S. SAWHNEY, D. V. VADEHEA AND R. C. BAKER Department of Poultry Science, Cornell University, Ithaca, New York 14850 (Received for publication October 13, 1972) ABSTRACT Sodium acetate-l- 14 C labeled aflatoxins were produced by growing Aspergillus flavus strain NRRL-2999 on rice. A single 0.29 /tCi. oral dose of aflatoxins was administered to laying White Leghorn hens. The radioactivity distribution and its equivalents in various tissues, at one, four and seven days after the administration of the dose were determined. Seven days after treatment, 70.61% of the dose was recovered in the excrement. The excretion of aflatoxins or their metabolites into the intestine via the bile seemed to be the major pathway by which absorbed aflatoxins were excreted. All the components of eggs laid at various intervals showed " C activity. Edible parts of the carcass showed varied amounts of 14C aflatoxins and/or their metabolites at all the periods studied. The time necessary to eliminate one-half of the radioactive aflatoxins from the body was found to be 66.82 hours. The liver, crop, gizzard and fecal material when fed in the diets were toxic to the duckling. POULTRY SCIENCE 52: 1302-1309, 1973

appeared in carbon dioxide but 70-80% OLLOWING the discovery of ana- of the activity was excreted in feces and toxins which are produced by certain urine. In these animals, the liver retained strains of Aspergillus flavus (Sargeant 6 to 9% of radioactivity. The public health hazard of fungal et al., 1961) and their carcinogencity in various species (Allcroft and Carnaghan, metabolites related to the consumption of 1963) an increasing interest has developed food of animal origin intended for human in mycotoxicological investigations. At consumption is reported by Purchase et al. present, there is little known concerning (1967) although Allcroft and Carnaghan the distribution and potential health haz- (1963), using the duckling for assay were ard of these toxins or their metabolites in inable to show toxicity in livers from various foods of animal origin destined for chicken fed toxic rations, clotted blood human consumption. It was observed by serum from cows on toxic feed, livers from delongh et al. (1964) that lactating cattle pigs dying of aflatoxicosis and in eggs from fed peanut meal containing aflatoxins had hens fed a toxic ration. Van Zytveld et al. a compound in the milk which was toxic (1970) reported that anatoxins and/or to the duckling. They also reported that their metabolites were found in skeletal lactating rats converted aflatoxin Bi into muscle and livers of chickens fed diets the toxic metabolite. Metabolism of ana- containing high doses of toxins. At prestoxin occurs primarily in the liver (Sporn ent, there is a growing epidemiological et al., 1966) but metabolic pathways are evidence showing that aflatoxins are hepanot fully understood. Wogan el al. (1967) toxic in humans (Robinson, 1967). The injected 14C ring labeled aflatoxin intra- evaluation of the potential hazard of the peritoneally in rats and during the follow- aflatoxins or their metabolite in meat and ing 24 hour period, virtually no activity eggs could be significant. Because of the lack of sensitive analytical methods capable of detecting all possible products, This study was supported by the Health, Educa- radiochemical analysis may be used as an tion and Welfare Public Service Grant. alternative. INTRODUCTION

F

A preliminary report of part of this paper was presented at the 61st Annual Meeting of Poultry Science, Columbus, Ohio, 1972.

The purpose of the present investigation was: (a) to determine the modes of

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AFLATOXINS METABOLISM

1303

absorption, distribution and elimination of dietary aflatoxins; (b) to quantitate aflatoxins or their metabolites in various edible portions avoiding the interference of substances normally present in the tissues; (c) to determine the toxicity of the excreted aflatoxins or their metabolites to sensitive species; (d) to determine the time required to eliminate the toxins.

/uCi.) of the radioactive aflatoxins dissolved in 3 ml. of dimethyl sulfoxide (DMSO) was administered via a stomach tube and washed with 2 to 4 ml. of propylene glycol to insure that the complete dose entered through the esophagus into the crop. The hens were returned to their cages and given free access to feed and water. Droppings from each hen were collected in preweighed pans made from MATERIALS AND METHODS heavy duty aluminum foil. The eggs were Preparation and purification of aflatoxin. collected either from the uterus or after The radioactive aflatoxins were prepared they were laid. The eggs were cooked in by growing the culture of Aspergillus Cryovac bags by placing the bags in boilflavus strain NRRL-2999 on rice contain- ing water for five minutes. The egg shell ing sodium acetate-l-14C at pH 5.7 (Adye membranes, egg white and yolk were sepaand Mateles, 1964). The aflatoxins were rated, lyophilized and stored at 2°C. until produced on rice by the method of Shot- analyzed. Three hens were sacrificed at well et al. (1966). The inoculated rice was one, four or seven days following the treatincubated at 25°C. in a New Brunswick ment by administering 5 to 7 ml. of chloroshaker set at 120 revolutions per minute. form orally. Blood samples (15 ml.) were The radioactive aflatoxins were purified taken by cardiac puncture using a hepariby column chromatography procedure of nized syringe while the bird was under Shotwell et al. (1966). The fractionated anaesthesia. Immediately after each sacriaflatoxins were further purified by thin fice, various tissues and the digestive layer chromatography (Hsieh and Ma- tract along with its contents and other teles, 1971). The concentration of the organs were excised under conditions aflatoxins in chloroform was calculated which prevent cross contamination by from the optical density of the chloroform blood, body fluid or dissecting instrusolutions by using appropriate molar ex- ments. Highly vascular organs were retinction coefficients (Bi, 21,800; B2, moved last. All the samples were freeze 20,800; Gi, 16,100; G2, 19,300) at 360 nm. dried and stored at 2°C. until analyzed. by the method of Asao et al. (1963). The composition of aflatoxins produced under Preparation of samples. Duplicate subthe conditions described was Bi, 78%; B2, samples of 100 mg. of freeze dried material 2%; Gi, 17%; G2, 3 % . The specific ac- were transferred to 20 ml. glass counting tivity was 8.03 juCi./m. mole expressed vials with foil lined caps provided with a on the basis of aflatoxin Bi. 0.3 inch teflon lining (The Chemical Test birds, treatment and sampling. White Leghorn hens from Cornell strain K, 20-22 weeks of age, weighing 1,600 to 1,800 gm. were selected. The hens were allowed to adjust to their environment for one week and their clutches were determined. A single dose (11.26 mg., 0.29

Rubber Company, Cleveland, Ohio). Water (0.5 ml.) was added and the vial was covered with a cap. The mixture was allowed to stand at room temperature for four hours, followed by the addition of 2 ml. of NCS solubilizer (Amersham/ Searle, Des Plaines, Illinois). The preparation was incubated at 43 to 45°C. for 20

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D . S. S A W H N E Y , D . V. VADEHRA AND R. C. B A K E R

to 24 hours with occasional shaking. A complete digestion of the material was evident when a clear solution was obtained. In a few cases, an additional 0.5 ml. of the reagent followed by incubation for eight to ten hours was necessary to achieve complete digestion of the sample. At the end of digestion, 0.25 ml. of hydrogen peroxide (30%) was added as a decolorizing agent. After cooling to room temperature, 10 ml. of the liquid scintillation mixture was added and the samples were allowed to remain a t room temperature in the dark for one day before counting. Determination of 14C activity. Radioactivity was determined by the use of a Nuclear Chicago well counter model 6804. The scintillation medium was a toluene solution containing 18.05 gm. of 2,5 diphenyloxadole (PPO) and 378.90 mg. of 1,40 bis[2 (4-methyl-5-phenyloxazolyl)]-benzene (dimethyl POPOP) obtained from Packard Instrument Company, Chicago, Illinois. The extent of quench in each case was determined b y using an internal 14C standard. Counting efficiency of 5 0 % was considered as the minimum acceptable level for quench corrections. Background samples were prepared by processing samples from control hens using procedures identical to those used for samples from treated chickens. All the samples and backgrounds were prepared in duplicate and the average of the two determinations was taken as the activity for the individual sample. For determination of the recovery, samples from untreated birds were fortified with known counts of 14 C aflatoxins and processed in the same manner as described. Mean recoveries of added material were 96.0 + 3 % for various samples. Quantitation

of aflatoxins.

T h e quantita-

tion of aflatoxins or their metabolites was done on the basis t h a t 14C in the various samples was of equivalent nature to t h a t of the parent compound or their toxic metabolites. T h e estimation limit of this procedure for various samples was 1.74 p.p.b. of 14C aflatoxins or their metabolites equivalents. Duckling bioassay. The toxicity of the labeled aflatoxins was conducted on three day old Pekin ducklings. The ducklings were fed crop, gizzard, liver and fecal material which possessed high radioactivity. The test materials were mixed with 50 percent of the normal diet by weight and fed to groups of three ducklings for a period of five days or less depending on time of survival. The control group was fed a diet containing normal fecal material. Mortality, reduction in weight, and abnormal liver changes were used as the criteria for toxicity as studied by Allcroft and Carnaghan (1963). RESULTS At the end of each experiment, the chickens were sacrificed and the tissues used for examination were excised. The hens on the aflatoxins diet, weighed 5.5% less at the end of seven days than their weight on the first day of the study. No toxicological lesions were observed in any of the tissues of the test hens. Combined urinary-fecal excretions of 14C activity. Figure 1 and Table 1 show the rate of combined urinary-fecal excretion of 14C activity from the hens. In the first 24 hours, 28.06% of the dose was eliminated followed by a decline. A sharp increase in the accumulated total urinaryfecal excretions was observed on the first day after the administration of anatoxin followed by a slow increase. The data in Table 1 show the elimination of 14C ac-

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AFLATOXINS METABOLISM

ACCUMULATED DAILY

> o z u

0

1

2

3

4

DAYS, FOLLOWING SINGLE DOSE OF

5 ,4

6

7

C AFIATOXIN

FIG. 1. Daily rate and accumulated total urinary-fecal excretion of 14C activity from hens following a single oral dose of acetate-labeled aflatoxins.

tivity recovered from the feces at various time intervals. A total of 70.61% of the activity was recovered seven days after the administration of aflatoxins. U

C activity in the egg. Table 2 shows the C activity in various components of the eggs collected at several intervals after ovulation. All the components of the eggs laid at various periods had higher 14C activity when compared to those at ten hours after ovulation. The activity de-

14

TABLE 1.—Urinary-fecal excretion ofuC activity and amount of aflatoxins or their metabolites during various periods following a single dose of labeled aflatoxins {11.26 mg., 0.29 pCi.)

Period m

0- 24 24- 48 48- 96 96-168 Total

creased in egg white 14 hours after oviposition. The yolk and shell membranes showed an increase in activity from ten hours onwards. Table 3 shows the calculated equivalent of aflatoxins or their metabolites in the various components of the eggs at the end of various periods. Tissue deposit of UC activity. The data obtained by radiochemical analysis of the tissues from the hens fed acetate-14Clabelled aflatoxins are summarized in Tables 4, 5 and Figure 2. TABLE 2.—The distribution of radioactivity in the white, yolk, and membranes of eggs at li intervals after a single oral dose of C labeled aflatoxins

Mg. of Total dose of aflatoxins DPM/gm. aflatoxins or their metabolites eliminated per gm. (%) 9,040 5,924 5,651 2,161

0.158 0.103 0.099 0.038

28.06 18.30 17.54 6.71

22,776

0.3976

70.61

DPM/gm.i Component

1

Dry weight basis.

1

Ovi position time

10 hr.

14 hr.*

24 hr.

48 hr.

286 293 186

444 369 510

313 370

302 420

Egg white Yolk Shell membrane 2

1

Time after ovulatio l

Freeze dried samples.

Average counts for two eggs. Others are the average of three

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D. S. SAWHNEY, D. V. VADEHRA AND R. C. BAKER

TABLE 3.—The amount1 of aflatoxins or their metabolites in the egg white, yolk and membranes of the eggs at different intervals after a single dose of UC labeled aflatoxins (11.26 mg., 0.29 nd.)

Component

Egg white Yolk Shell membranesi 1

Time after ovulation -

Oviposition time

10 hr. Mg./gm.

Hhr.

24 hr , ng./gm.

4.98 5.10 3.23

7.72 6.42 8.87

5.25 6.43 8.26

48 hr.

7.31 12.47

Calculated equivalents of aflatoxins or their metabolites.

Table 4 shows the specific activity in various tissues of the hens. At day one, the highest activity was in the bile, followed by the liver and reproductive organs. The gizzard, wing muscle, breast muscle, leg muscle and heart had nearly half as much activity as did the liver. The adipose tissue and skin had approximately TABLE 4.—Distribution of radioactivity in various tissues of hens 1, 4 and 7 days after a single dose of UC labeled aflatoxins DPM/gm. of tissue 1 day 4 days 7 days 4951 Gizzard Reproductive organs 771 Large ova ( > 10 mm.) 386 Small ova ( < 10 mm.) 423 Spleen 158 Kidney 320 Liver 929 Bile 2,306 Wing muscle 468 Breast muscle 402 Leg muscle 345 Adipose tissue 237 Skin 269 Pancreas 292 Heart 431 Lungs and trachea 184 Adrenal and thyroid 230 Total DPM 8,227 % of total dose/gm. 1.280 Gastrointestinal tract Contents Crop and gizzard 3,175 Digestive tract 5,050 Total DPM 8,225 % of total dose 1.27

256 493 282 388 474 N.A.2 405 785 120 200 290 530 1,068 609 1,211 467 380 360 370 567 451 326 351 277 186 246 465 721 404 948 482 466 462 397 6,565 6,900 1.02 1.07 5,931 1,801 6,400 2,428 12,331 4,229 1.91 0.655

1 Each value is an average of six determinations from three hens. 2 N. A. = not available.

one third the specific activity as the liver. The spleen had the lowest activity. Four days after the administration of toxins, the specific activity increased in the large ova, leg muscle, adipose tissue and pancreas. The bile, liver, gizzard, reproductive organs, small ova, wing muscle, breast muscle, skin and heart showed a decrease in radioactivity for the same period. Seven days after the oral dose, all the tissues still possessed the activity and there was an increase in activity of the small ova, spleen, kidney, breast muscle, pancreas and heart. Toxicity of 14C aflatoxins or their metabolites. Ducklings fed the diet containing feces from normal hens containing no aflatoxins or their metabolites grew normally and showed no abnormal liver changes. Deaths (3/3), (2/3) occurred within 72 hours in ducklings when the diet TABLE 5.—The amount1 of aflatoxins, or their metabolites in the tissues of hens 1, 4 and 7 days after a single oral dose of 14C labeled aflatoxins

Sample

Aflatoxins or their metabolites /jg./gm. 1 day 4 days 7 days

Gizzard 8.65 4.48 8.63 Reproductive organs 13.48 4.94 6.79 Large ova ( > 10 mm.) 6.75 8.29 N.A.2 Small ova ( < 10 mm.) 7.40 7.09 13.72 Spleen and kidney 8.29 7.14 12.47 Liver . 16.27 18.69 10.65 Bile 40.33 21.19 8.17 Wing muscle 8.18 6.65 6.29 Breast muscle 7.04 6.46 9.9 Leg muscle 6.04 7.89 5.70 Adipose tissue 4.14 6.14 4.84 Skin 4.71 3.25 4.31 Pancreas 5.10 8.13 12.54 Heart 7.53 7.07 16.59 Lungs and trachea 3.20 7.80 8.10 Adrenal and thyroid 4.00 8.05 6.90 Gastrointestinal tract Contents Crop and gizzard 55.24 103.10 31.33 Digestive tract 87.87 111.36 42.26 1 Calculated equivalents of aflatoxins on their metabolites. 2 N. A. = not available.

AFLATOXINS METABOLISM

1307

o

<

DAYS FOLLOWING SINGLE DOSE OF M C AFLATOXIN

FIG. 2. Change in the level of radioactivity in the hen tissues following a single oral dose of acetate-labeled aflatoxins. containing radioactive contents of crop and gizzard (pooled) and fecal material was fed. There was a 50% reduction in body weight when a diet containing toxic liver was fed. The livers of sacrificed ducklings showed marked liver lesions similar to those fed unmetabolized aflatoxins. The calculated amount of aflatoxins or their metabolites in various samples which showed toxicity in a duckling were 80 jug., 410 ng., and 2,500 ng. in crop and gizzard, liver and fecal contents, respectively. DISCUSSION Aflatoxins or their metabolites were eliminated fairly rapidly through the combined urinary-fecal excretion. Twentyeight percent of the dose was eliminated in the excrement in 24 hours, while the total of only 70.61% of the dose administered was recovered from the excrements after seven days. At both one and four days, the specific activity of the bile was greater than that of any other tissue, indi-

cating that the excretion of toxins into the intestine is via the bile by which absorbed aflatoxins are removed from the body. The fact that specific activity of the post bile duct digestive tract contents was higher than that of the crop and gizzard contents at day one also supports the findings that the aflatoxins were concentratively excreted into the bile. Wogan (1967) also reported that the major excretory route of the ring labeled material was through the biliary excretion into feces. Falk et al. (1965) also reached similar conclusions when biliary excretion of aflatoxins was studied by fluorescence techniques. The high biliary excretion of aflatoxins is in accordance with the tentative conclusions of Williams et al. (1965) and Milburn et al. (1967) that the compounds of high molecular weight (more than 300) and containing two or more aromatic rings tended to be excreted into the bile. Aflatoxins or metabolites were detected

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D . S. S A W H N E Y , D . V. V A D E H R A AND R. C. B A K E R

in all the components of the eggs as early as ten hours after ovulation and 14 hours after oviposition. Aflatoxins could have reached the various components of the ovary and oviduct or its secretion through the blood. The ovary and oviduct had considerable activity on day one. The 14C aflatoxins equivalent declined in the egg white at 48 hours while in the yolk and shell membranes it increased. These differences could be due to the formation, the structural, and the compositional differences of the various egg components (Sturkie, 1965). Dissipation of total 14 Caflatoxins in various tissues showed the presence of radioactivity in all the tissues at one day and seven days indicating rapid absorption but slow elimination. At day one, the liver, reproductive organs and kidneys had a higher concentration of 14 C activity which could be due to involvement of these organs with the elimination of the aflatoxins. Twenty-nine percent of the administered activity was retained at the end of seven days, showing poorer efficiency of elimination of aflatoxins in chickens as compared to rats which excreted 7 0 - 8 0 % 24 hours after the intraperitoneal administered dose (Wogan et al., 1967). T h e differences in results could be due to the mode of administration or species a n d / o r both. T h e high concentration of 14C aflatoxins in the crop and gizzard contents suggested a slow absorption or passage or both from this segment of the alimentary canal. The rest of the digestive tract also had a high concentration of 14C activity. This may be attributable to the constant elimination of toxins or metabolites via the bile. The rate of depletion of aflatoxins from the body followed the first order reaction kinetics. I n Figure 2, the half-life of total aflatoxins equaled 66.82 hours. Earlier investigations of the excretion or detection of aflatoxins have necessarily

been based upon the detection of the compound or its metabolites by the fluorescence technique, or, in some cases, toxicity measurement. Thus, Allcroft and Carnaghan (1963) found t h a t milk from cows fed diets contaminated with aflatoxins were toxic to ducklings but contained no detectable aflatoxin Bi. Platnow (1965) also was unable to extract aflatoxins or their fluorescent metabolites from breast meat, leg meat or liver from a broiler fed a ration of 3.1 p.p.m. aflatoxin for six weeks. Sims et al. (1970) also could not detect any fluorescent metabolites in the eggs or liver of hens fed dietary aflatoxins. Van Zytveld (1970), however, could detect fluorescent aflatoxin a n d / o r metabolites in the muscles and livers of only 15 out of 45 broilers when 256.6 mg. to 513.0 mg. of aflatoxins were administered daily for six weeks. Wiseman (1968) found aflatoxins in the hen eggs when the hens were fed ad libitum aflatoxin-contaminated feed at 0.4 p.p.m. aflatoxin. These positive results were obtained by using column and thin layer chromatography for the estimation of aflatoxins. The results of this study show t h a t all the components of the egg and edible parts of the carcass had different concentrations of the aflatoxins a n d / o r their metabolites at all periods studied. Eighty fig. of the toxins in the pooled contents of the crop and gizzard were as toxic as 2,500 ng. in feces to a duckling, whereas 410 jug. in liver contents caused reduction in body weight and liver lesions. The duckling bioassay in this study showed t h a t aflatoxins or their metabolites present in the crop and gizzard contents and those eliminated in feces were still as toxic as native aflatoxins, although the amount required in each case to cause mortality was different. These differences in toxicity of various materials could be due to one or a combination of the following: alteration of solubility, binding to various cellu-

AFLATOXINS METABOLISM

lar constituents, availability, structural alterations or the extent of breakdown of aflatoxins, as suggested by Wogan el al. (1967). The results of the study suggest that potential health and environmental hazards related to the consumption of parts of carcass having high 14C activity and from the dispersal of feces of chickens that have ingested anatoxins should not be disregarded. REFERENCES Adye, J., and R. I. Mateles, 1964. Incorporation of labeled compounds into aflatoxins. Biochem. Biophys. Acta, 86: 418-420. Asao, T., G. Buchi, M. M. Abdel-Kadar, S. B. Chang, E. L. Wick and G. N. Wogan, 1963. Aflatoxins B and G. J. Am. Chem. Soc. 85: 17061707. Allcroft, R., and R. B. A. Carnaghan, 1963. Groundnut toxicity: An examination for toxin in human food products from animals fed toxic groundnut meal. Vet. Rec. 75: 259-263. Carnaghan, R. B. A., R. D. Hartley and J. O'Kelley, 1963. Toxicity and fluorescence properties of the aflatoxins. Nature, 200: 1101. delongh, H., R. O. Vies and J. G. van Pelt, 1964. Milk of mammals fed an aflatoxin-containing diet. Nature, 202: 466-467. Falk, H. L., S. J. Thompson and P. Kotin, 1965. Metabolism of aflatoxin Bi in the rat. Proc. Am. Assoc. Cancer Res. 6: 18. Hsieh, D. P. H., and R. I. Mateles, 1971. Preparation of labeled aflatoxins with high specific activities. Appl. Micro. 13: 208-211. Millburn, P., P. L. Smiyh and R. T. Williams, 1967. Biliary excretion of foreign compounds, biphenyl, stilbestrol and phenolpthalein in rat: Molecular weight, polarity and metabolism as factors in biliary excretion. Biochem. J. 104:

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1275-1281. Plantonow, N., 1965. Investigation of the possibility of the presence of aflatoxin in meat and liver of chickens fed toxic groundnut meal. Vet. Rec. 77: 1028. Purchase, I. F. M., 1967. Fungal metabolites as potential carcinogens, with particular reference to their role in aetiology of hepatoma. S. African Med. J. 41:406-413. Robinson, P., 1967. Infantile cirrhosis of the liver in India. Clin. Pediatrics, 6: 57-62. Sargeant, K., A. Sheridan, J. O'Kelley and R. B. A. Carnaghan, 1961. Toxicity associated with certain samples of groundnuts. Nature, 192: 10961097. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425-428. Sims, W. M., Jr:, D. C. Kelley and P. E. Sanford, 1970. A study of aflatoxicosis in laying hens. Poultry Sci. 49: 1082-1084. Sporn, M. B., C. W. Dingman and H. L. Phelps, 1966. Aflatoxin Bi: Binding to DNA in vitro and alteration of RNA metabolism in vivo. Science, 49: 1351-1356. Sturkie, P. D., 1965. Avian Physiology. 2nd ed., p. 474, Comstock Publ., Ithaca, New York 14850. Van Zytveld, W. A., D. C. Kelley and S. M. Dennis, 1970. Aflatoxins or their metabolites in livers and skeletal muscles of chicken. Poultry Sci. 49: 1351-1356. Williams, R. T., P. Millburn and R. L. Smith, 1965. The influence of enterohepatic circulation on toxicity of drugs. Ann. New York Acad. Sci. 123: 110-124. Wiseman, H. G., 1968. Personal communications. Div. Vet. Research, Bureau Vet. Med. Dept. Health, Education and Welfare, Agricultural Research Center, Beltsville, Maryland 20705. Wogan, G. N., G. S. Edwards and R. C. Shank, 1967. Excretion and tissue distribution of radioactivity from aflatoxin Bi-14C in rats. Cancer Res. 27: 1729-1736.

OCTOBER 29-30. SOUTHEASTERN POULTRY AND EGG ASSOCIATION POULTRY HEALTH SEMINAR, EXECUTIVE PARK MOTOR HOTEL, ATLANTA, GEORGIA. DECEMBER 3-4. SOUTHEASTERN POULTRY AND EGG ASSOCIATION WOMEN EXECUTIVE SECRETARIES' SEMINAR, RAMADA INN (AIRPORT), ATLANTA, GEORGIA.