FOLLOWING the discovery of anatoxins

Transcription

1 The Metabolism of 14 C Aflatoxins in Laying Hens D. S. SAWHNEY, D. V. VADEHEA AND R. C. BAKER Department of Poultry Science, Cornell University, Ithaca, New York (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 14 C 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 hours. The liver, crop, gizzard and fecal material when fed in the diets were toxic to the duckling. INTRODUCTION FOLLOWING the discovery of anatoxins which are produced by certain strains of Aspergillus flavus (Sargeant et al., 1961) and their carcinogencity in various species (Allcroft and Carnaghan, 1963) an increasing interest has developed in mycotoxicological investigations. At present, there is little known concerning the distribution and potential health hazard of these toxins or their metabolites in various foods of animal origin destined for human consumption. It was observed by delongh et al. (1964) that lactating cattle fed peanut meal containing aflatoxins had a compound in the milk which was toxic to the duckling. They also reported that lactating rats converted aflatoxin Bi into the toxic metabolite. Metabolism of anatoxin occurs primarily in the liver (Sporn et al., 1966) but metabolic pathways are not fully understood. Wogan el al. (1967) injected 14 C ring labeled aflatoxin intraperitoneally in rats and during the following 24 hour period, virtually no activity This study was supported by the Health, Education and Welfare Public Service Grant. A preliminary report of part of this paper was presented at the 61st Annual Meeting of Poultry Science, Columbus, Ohio, POULTRY SCIENCE 52: , 1973 appeared in carbon dioxide but 70-80% of the activity was excreted in feces and urine. In these animals, the liver retained 6 to 9% of radioactivity. The public health hazard of fungal metabolites related to the consumption of food of animal origin intended for human consumption is reported by Purchase et al. (1967) although Allcroft and Carnaghan (1963), using the duckling for assay were inable to show toxicity in livers from chicken fed toxic rations, clotted blood serum from cows on toxic feed, livers from pigs dying of aflatoxicosis and in eggs from hens fed a toxic ration. Van Zytveld et al. (1970) reported that anatoxins and/or their metabolites were found in skeletal muscle and livers of chickens fed diets containing high doses of toxins. At present, there is a growing epidemiological evidence showing that aflatoxins are hepatoxic in humans (Robinson, 1967). The evaluation of the potential hazard of the aflatoxins or their metabolite in meat and eggs could be significant. Because of the lack of sensitive analytical methods capable of detecting all possible products, radiochemical analysis may be used as an alternative. The purpose of the present investigation was: (a) to determine the modes of

2 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. MATERIALS AND METHODS Preparation and purification of aflatoxin. The radioactive aflatoxins were prepared by growing the culture of Aspergillus flavus strain NRRL-2999 on rice containing sodium acetate-l- 14 C at ph 5.7 (Adye and Mateles, 1964). The aflatoxins were produced on rice by the method of Shotwell et al. (1966). The inoculated rice was incubated at 25 C. in a New Brunswick shaker set at 120 revolutions per minute. The radioactive aflatoxins were purified by column chromatography procedure of Shotwell et al. (1966). The fractionated aflatoxins were further purified by thin layer chromatography (Hsieh and Mateles, 1971). The concentration of the aflatoxins in chloroform was calculated from the optical density of the chloroform solutions by using appropriate molar extinction coefficients (Bi, 21,800; B2, 20,800; Gi, 16,100; G 2, 19,300) at 360 nm. by the method of Asao et al. (1963). The composition of aflatoxins produced under the conditions described was Bi, 78%; B2, 2%; Gi, 17%; G 2, 3%. The specific activity was 8.03 juci./m. mole expressed on the basis of aflatoxin Bi. Test birds, treatment and sampling. White Leghorn hens from Cornell strain K, 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 /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 heavy duty aluminum foil. The eggs were collected either from the uterus or after they were laid. The eggs were cooked in Cryovac bags by placing the bags in boiling water for five minutes. The egg shell membranes, egg white and yolk were separated, lyophilized and stored at 2 C. until analyzed. Three hens were sacrificed at one, four or seven days following the treatment by administering 5 to 7 ml. of chloroform orally. Blood samples (15 ml.) were taken by cardiac puncture using a heparinized syringe while the bird was under anaesthesia. Immediately after each sacrifice, various tissues and the digestive tract along with its contents and other organs were excised under conditions which prevent cross contamination by blood, body fluid or dissecting instruments. Highly vascular organs were removed last. All the samples were freeze dried and stored at 2 C. until analyzed. Preparation of samples. Duplicate subsamples of 100 mg. of freeze dried material were transferred to 20 ml. glass counting vials with foil lined caps provided with a 0.3 inch teflon lining (The Chemical 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

3 1304 D. S. SAWHNEY, D. V. VADEHRA AND R. C. BAKER 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 at room temperature in the dark for one day before counting. Determination of 14 C activity. Radioactivity was determined by the use of a Nuclear Chicago well counter model The scintillation medium was a toluene solution containing gm. of 2,5 diphenyloxadole (PPO) and 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 by using an internal 14 C standard. Counting efficiency of 50% 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 % for various samples. Quantitation of aflatoxins. The quantitation of aflatoxins or their metabolites was done on the basis that 14 C in the various samples was of equivalent nature to that of the parent compound or their toxic metabolites. The estimation limit of this procedure for various samples was 1.74 p.p.b. of 14 C 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. 14 C Combined urinary-fecal excretions of activity. Figure 1 and Table 1 show the rate of combined urinary-fecal excretion of 14 C 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 14 C ac-

4 AFLATOXINS METABOLISM 1305 ACCUMULATED DAILY > o z u DAYS, FOLLOWING SINGLE DOSE OF,4 C AFIATOXIN FIG. 1. Daily rate and accumulated total urinary-fecal excretion of 14 C 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 14 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 14 C activity when compared to those at ten hours after ovulation. The activity de- TABLE 1. Urinary-fecal excretion of u C 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 Total DPM/gm. 9,040 5,924 5,651 2,161 22,776 1 Dry weight basis. Mg. of aflatoxins or their metabolites per gm Total dose of aflatoxins eliminated (%) 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 U C activity. The data obtained by radiochemical analysis of the tissues from the hens fed acetate- 14 C- labelled 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 intervals after a single oral dose of li C labeled aflatoxins Component Egg white Yolk Shell membrane Time after ovulatio l 10 hr DPM/gm.i 14 hr.* Ovi position time 24 hr. 48 hr Freeze dried samples. 2 Average counts for two eggs. Others are the average of three

5 1306 D. S. SAWHNEY, D. V. VADEHRA AND R. C. BAKER TABLE 3. The amount 1 of aflatoxins or their metabolites in the egg white, yolk and membranes of the eggs at different intervals after a single dose of U C labeled aflatoxins (11.26 mg., 0.29 nd.) Component Time after ovulation - 10 hr. Mg./gm. Egg white 4.98 Yolk 5.10 Shell membranes i 3.23 Hhr Oviposition time 24, hr - 48 hr. ng./gm 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 U C labeled aflatoxins Gizzard Reproductive organs Large ova (> 10 mm.) Small ova (< 10 mm.) Spleen Kidney Liver Bile Wing muscle Breast muscle Leg muscle Adipose tissue Skin Pancreas Heart Lungs and trachea Adrenal and thyroid Total DPM % of total dose/gm. Gastrointestinal tract Contents Crop and gizzard Digestive tract Total DPM % of total dose DPM/gm. of tissue 1 day , , ,175 5,050 8, days 7 days N.A , , ,565 6, ,931 6,400 12, ,801 2,428 4, 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 14 C 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 amount 1 of aflatoxins, or their metabolites in the tissues of hens 1, 4 and 7 days after a single oral dose of 14 C labeled aflatoxins Sample Aflatoxins or their metabolites /jg./gm. 1 day 4 days Gizzard Reproductive organs Large ova (> 10 mm.) Small ova (< 10 mm.) Spleen and kidney Liver. Bile Wing muscle Breast muscle Leg muscle Adipose tissue Skin Pancreas Heart Lungs and trachea Adrenal and thyroid Gastrointestinal tract Contents Crop and gizzard Digestive tract days N.A Calculated equivalents of aflatoxins on their metabolites. 2 N. A. = not available.

6 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, indicating 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

7 1308 D. S. SAWHNEY, D. V. VADEHRA AND R. C. BAKER 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 14 C 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 C- aflatoxins 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 70-80% 24 hours after the intraperitoneal administered dose (Wogan et al., 1967). The differences in results could be due to the mode of administration or species and/or both. The high concentration of 14 C 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 14 C 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. In Figure 2, the half-life of total aflatoxins equaled 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 that 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 and/or metabolites in the muscles and livers of only 15 out of 45 broilers when mg. to 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 that all the components of the egg and edible parts of the carcass had different concentrations of the aflatoxins and/or 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 that 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-

8 AFLATOXINS METABOLISM 1309 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 14 C activity and from the dispersal of feces of chickens that have ingested anatoxins should not be disregarded. REFERENCES Adye, J., and R. I. Mateles, Incorporation of labeled compounds into aflatoxins. Biochem. Biophys. Acta, 86: Asao, T., G. Buchi, M. M. Abdel-Kadar, S. B. Chang, E. L. Wick and G. N. Wogan, Aflatoxins B and G. J. Am. Chem. Soc. 85: Allcroft, R., and R. B. A. Carnaghan, Groundnut toxicity: An examination for toxin in human food products from animals fed toxic groundnut meal. Vet. Rec. 75: Carnaghan, R. B. A., R. D. Hartley and J. O'Kelley, Toxicity and fluorescence properties of the aflatoxins. Nature, 200: delongh, H., R. O. Vies and J. G. van Pelt, Milk of mammals fed an aflatoxin-containing diet. Nature, 202: Falk, H. L., S. J. Thompson and P. Kotin, Metabolism of aflatoxin Bi in the rat. Proc. Am. Assoc. Cancer Res. 6: 18. Hsieh, D. P. H., and R. I. Mateles, Preparation of labeled aflatoxins with high specific activities. Appl. Micro. 13: Millburn, P., P. L. Smiyh and R. T. Williams, Biliary excretion of foreign compounds, biphenyl, stilbestrol and phenolpthalein in rat: Molecular weight, polarity and metabolism as factors in biliary excretion. Biochem. J. 104: Plantonow, N., Investigation of the possibility of the presence of aflatoxin in meat and liver of chickens fed toxic groundnut meal. Vet. Rec. 77: Purchase, I. F. M., Fungal metabolites as potential carcinogens, with particular reference to their role in aetiology of hepatoma. S. African Med. J. 41: Robinson, P., Infantile cirrhosis of the liver in India. Clin. Pediatrics, 6: Sargeant, K., A. Sheridan, J. O'Kelley and R. B. A. Carnaghan, Toxicity associated with certain samples of groundnuts. Nature, 192: Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, Production of aflatoxin on rice. Appl. Microbiol. 14: Sims, W. M., Jr:, D. C. Kelley and P. E. Sanford, A study of aflatoxicosis in laying hens. Poultry Sci. 49: Sporn, M. B., C. W. Dingman and H. L. Phelps, Aflatoxin Bi: Binding to DNA in vitro and alteration of RNA metabolism in vivo. Science, 49: Sturkie, P. D., Avian Physiology. 2nd ed., p. 474, Comstock Publ., Ithaca, New York Van Zytveld, W. A., D. C. Kelley and S. M. Dennis, Aflatoxins or their metabolites in livers and skeletal muscles of chicken. Poultry Sci. 49: Williams, R. T., P. Millburn and R. L. Smith, The influence of enterohepatic circulation on toxicity of drugs. Ann. New York Acad. Sci. 123: Wiseman, H. G., Personal communications. Div. Vet. Research, Bureau Vet. Med. Dept. Health, Education and Welfare, Agricultural Research Center, Beltsville, Maryland Wogan, G. N., G. S. Edwards and R. C. Shank, Excretion and tissue distribution of radioactivity from aflatoxin Bi- 14 C in rats. Cancer Res. 27: OCTOBER 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.