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Effects of long-term feeding of ammoniated, aflatoxin-contaminated corn to Fischer 344 rats
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
Effects of Long-Term
WILLIAM
70,96-104
(1983)
Feeding of Ammoniated, Aflatoxin-Contaminated Corn to Fischer 344 Rats
P. NORRED’
AND RICHARD
E. MORRISSEY
Richard B. Russell Agricultural Research Center, U.S. Department of Agriculture, Agricultural Research Service, Athens, Georgia 30613
Received December 20. 1982; accepted March 25. 1983 Effects of Long-Term Feeding of Ammoniated, Aflatoxin-Contaminated Corn to Fischer 344 Rats. Norred, W. P., and Morrissey, R. E. ( 1983). Toxicol. Appl. Pharmacol. 70,96-104. The effectivenessof ammonia treatment in reducing the chronic toxicity of aflatoxin-contaminated corn was determined. Fischer 344 rats were fed semi-purified rations containing 20% w/w corn that was either free of aflatoxin or naturahy contaminated with 880 &kg total aflatoxin and was either treated with ammonia gas or was not treated. Therefore the rats that were fed the aflatoxin-contaminated diet received 176 ppb total aflatoxins. Body weight and food consumption were recorded throughout the study; hematological measurements were made after 87 weeks of feeding; and after 9 1 weeks the rats were killed and histopathological abnormalities were noted. Signs of chronic toxicosis in rats fed aflatoxincontaminated corn included increased mortality, decreased hematocrit and hemoglobin levels, elevated serum alkaline phosphatase activities, and a 100% incidence of liver neoplasia. These signs did not occur in rats in the other dietary treatment groups, including those fed ammoniated, aflatoxinumtaminated corn. The results provide further evidence that the atmospheric ammoniation process effectively reduces the toxicity of aflatoxincontaminated corn.
The use of ammonia, either in gaseous or liquid form, at elevated heat and pressure or under ambient conditions, has been described by a number of investigators as a detoxification procedure for aflatoxin-contaminated commodities (Masri et al., 1969; Gardner, 197 1; McKinney et al., 1973; Thiesen, 1977). Treatment of aflatoxin-contaminated corn with ammonia gas at atmospheric pressure appears to be the most effective and economically feasible method of salvaging the corn for use as animal feed (Bagley, 1979; Key1 and Norred, 1979; Norred, 1982). The process (Brekke et al., 1977a, 1978, 1979) reduces the content of aflatoxin in naturally contaminated corn to less than 20 ppb; it results in corn which is accepted and efficiently utilized by swine (Jensen et al., 1977); it prevents the production of hepatoma in rainbow trout (Brekke et al.,
Aflatoxins are toxic metabolites produced by molds (Aspergillusflavus, A. parasiticus, and Penicillium puberulum) which can invade food and feed ingredients (Wogan, 1966; Schoental, 1967). Contamination of corn in particular has caused severe economic problems for livestock producers and may create potential health risks by the transmission of aflatoxins and/or their metabolites from livestock to humans through consumption of meat, milk, or eggs. Conditions favorable to mold growth have occurred periodically, particularly in the southeastern United States; the severe weather conditions and insect damage in the summer of 1977 resulted in widespread aflatoxin contamination with resultant losses (Wilson et al., 1979; Bagley, 1979). ’ To whom correspondence should be addressed. 96 OO41OO8X/83 $3.00
AMMONIATED,
AFLATOXIN-CONTAMINATED
1977b); it eliminates signs of acute toxicity which result from administration of corn fortified with high levels of aflatoxin B, (Norred, 1979); and it decreases the uptake from the gastrointestinal tract, enhances the excretion, and reduces the binding to liver of carbon-14 after po administration to rats of 14C-aflatoxin B,-containing corn (Norred, 198 1). Relay feeding studies, although not yet completed, have indicated no deleterious effects to rats fed egg, pork, poultry, or beef from farm animals fed ammoniated, aflatoxin-contaminated corn (Norred, 1982). In the present study the effectiveness of ammonia treatment of corn naturally contaminated with aflatoxin was further evaluated by feeding the corn directly to rats in a chronic study. Treated corn was prepared under field conditions at the Northern Regional Research Center (NRRC), ARS, USDA, Peoria, Ill., and was fed, along with appropriate control diets, to Fischer rats for a 23-month period. This paper reports the histological and hematological findings of this investigation. METHODS Animals. Three-week-old Fischer 344 rats (reared in our facility from breeding stock obtained from Charles River Breeding Labs, Wilmington, Mass.)* were randomly assigned by sex to one of five treatment groups, with 12 males and I2 females in each group. The rats were identified by ear notch and housed two per cage in suspended wire cages in racks equipped with automatic watering systemsand automatic flushing. Temperature was maintained at 22 rf: 3°C and relative humidity at 50 to 70%. Fluorescent lighting was turned on at 6:00 AM and off at 6:00 PM to provide a 12-hr light-dark cycle. Powdered diets were provided ad libitum. All rats were weighed weekly for the first 15 weeks and monthly thereafter. Food consumption was determined during a 1 week period each month. Diets. Diets fed to the rats were either lab chow (LC) (Teklad Mouse/Rat Chow, Winfield, Iowa, ground to pass a 3-mm sieve) or semi-purified ration (Table I) containing 20% w/w of one of four types of corn. The dietary treatment groups were identified based on the type of corn fed, where
’ The mention of this product, equipment, or company name does not imply recommendation over others that may be suitable for use.
97
CORN TABLE I
Ingredient
% w/w
Casein Dextrin Corn oil Cellulose Mineral mix, Jones-Foster Vitamin mix, Teklad Corn, ground*
12 58 1 4 4 1 20
’ All ingredients except corn were blended by Teklad Test Diets, Madison, Wisconsin. ’ One of the four types of corn (GC, GCA, AC, ACA) as defined under Methods was ground to pass a 3-mm sieve, and blended for 20 min with the basal ration in a Reynolds mixer. GC (good corn) was corn with no detectable levels of aflatoxin, ochratoxin, or zearalenone; AC (aflatoxin-contaminated) was corn naturally contaminated with 880 rg/ kg of total aflatoxins (750 of B, ,90 of B2, and 40 of G,); GCA (good corn ammoniated) was GC corn that had been ammoniated, and ACA (aflatoxin corn, ammoniated) was AC corn that had been ammoniated. Therefore rats fed AC diet received 176 ppb total aflatoxins, whereas those fed ACA diet received an equivalent amount of ammoniated aflatoxin by-products. The corn was obtained from the Northern Regional Research Center, ARS, USDA, and ammoniated samples had been treated as described by Brekke ef al. (1979). The corn was ground (3-mm sieve), and blended with the semi-purified ration. The blended rations were stored at 4°C and feed hoppers were replaced weekly with fresh ration. Fresh diets were prepared monthly. Histopathology. A necropsy was performed on each rat that died during the study or was killed when the study was terminated at 92 weeks. Tissues (stomach, duodenum, jejunum, ileum. cecum, colon, liver, pancreas, salivary gland, lungs, trachea, esophagus, thyroid, parathyroid, adrenals, kidneys, urinary bladder, testes, epididymides, seminal vesicles, prostate, spleen, heart, brain, pituitary, eyes,sternum, ovaries, uterus, and mammary gland) were preserved in 10% buffered Formalin, and routinely prepared paraffin sections were stained with hematoxylin and eosin and examined by light microscopy. Liver tumors were classified according to criteria established by the National Academy of Science ( 1980). Hemafo/ogy. Blood (1 to 2 ml) was collected by cardiac puncture from lightly anesthetized (ether) male and female rats (five of each) selected randomly from each dietary treatment group. Collections were made 87 weeks after treatments began. Serum was separated from clotted blood by centrifuging at 5OOg. The activities of alkaline phos-
98
NORRED
AND MORRISSEY TABLE 2
EFFECT OF AMMONIA
TREATMENT OF NEOPLASTK
OF AFLATOXIN-CONTAMINATED LESIONS IN MALE FISCHER
CORN RATS”
ON INCIDENCE
Dietary treatmentb Gc
GCA
AC
ACA
LC
Stomach Malignant lymphoma
O/l2
O/11
l/l 1
O/l2
o/12
Duodenum Hepatocellular carcinoma, metastatic
o/12
o/10
l/10
O/l2
o/12
Liver Hepatocellular carcinoma Neoplastic nodule Fibrosarcoma Malignant lymphoma
o/12 o/12 O/l2 O/l2
o/11 o/11 o/11 O/11
12112’ 6112’ l/12 l/12
o/12 o/12 o/12 O/l2
o/12 O/l2 O/l2 O/l2
Pancreas Islet cell adenoma Malignant lymphoma
l/l2 O/l2
l/10 o/10
o/11 l/11
l/12 o/12
2/11 o/11
Salivary gland Malignant lymphoma
o/12
O/IO
l/12
o/10
o/10
Lungs Hepatocellular carcinoma, metastatic Malignant lymphoma
O/l1 O/l1
O/l1 O/II
2112 l/12
o/12 O/l2
o/12 o/12
Adrenals Cortical adenoma, unilateral Pheochromocytoma, unilateral
O/II o/11
O/l0 O/l0
l/10 o/10
l/12 o/12
O/l2 l/l2
Kidneys Malignant lymphoma, bilateral
O/l2
O/l1
l/l2
O/l2
o/12
Testes Interstitial cell adenoma, bilateral Interstitial cell adenoma, unilateral
l/l 1 8/ll
O/IO 8110
3112 8/12
6112 S/12’
4112 l/l2
Spleen Malignant lymphoma
o/12
l/12
o/12
O/l2
Pituitary Chromophobe adenoma
O/IO
O/4
016
l/9
Skin Keratoacanthoma
111
o/o
Thyroid C cell carcinoma, unilateral
O/l1
016
o/o
W3
O/9
a Values are number of rats affected/number of rats examined. b Treatment designations are as defined under Methods. c Significantly different from CC treatment group (p < 0.05). phatase (AP) and asparatate aminotransferase (AST) were determined in the serum with kits (Hycel and Dade, respectively) purchased from Scientific Products, Stone
Mountain, Georgia. Blood (0.3 to 0.5 ml) was also collected from the same rats into syringesrinsed with EDTA solution (Sequester-Sol, Fort Lauderdale, Fla.) to prevent clotting.
AMMONIATED,
AFLATOXIN-CONTAMINATED
99
CORN
TABLE 3 EFFECT OFAMMONIATREATMENTOFAFLATOXIN-CONTAMMATEDCORNONINCIDENCE OFNEOPLASTICLESIONSINFEMALEFISCHERRAW Dietary tmatmentb tic
GCA
AC
ACA
LC
Liver Hepatocelhnar carcinoma Neoplastic nodules Fibrosarcoma
o/12 o/12 o/12
o/11 o/11 o/11
1 l/12’ 10/12c l/12
o/12 o/12 o/12
o/12 o/12 o/12
Lungs Hepatocellular carcinoma, metastatic Malignant lymphoma
o/12 o/12
o/12 l/12
2112 o/12
Of12 o/12
o/12 o/12
Adrenals Pheochromocytoma, unilateral
O/11
O/11
1/l 1
o/12
o/12
Pituitary Chromophobe adenoma
214
O/9
w
l/4
319
Skin Malignant lymphoma
o/12
l/12
o/12
o/12
o/12
Uterus Leiomyoma
o/12
o/11
l/12
l/12
o/12
Thyroid C cell carcinoma, unilateral
Of7
O/9
O/7
2111
O/IO
Mammary gland Adenocarcinoma Adenoma
o/12 o/12
o/11 O/11
o/12 o/12
o/12 l/12
l/12 o/12
’ Values are number of rats affected/number ofrats examined. ’ Treatment designations are as defined under Methods. ’ Significantly different from CC treatment group (p < 0.05). White cell and red cell counts were determined in these samples with a Coulter ZBI counter; hemoglobin content was measured with a Coulter hemoglobinometer; and hematocrit and differential white cell counts were determined by conventional means. Statistical analysis. One-way analysis of variance was used to determine significant differences (p i 0.05) between treatment groups (Dixon and Massey, 1969). Duncan’s multiple range test was used for determining mean seg aration in cases where there was significant variation among sample means. Chi Square with Yates’ correction was used to determine significance of incidences of histological lesions, with values observed in rats fed CC used as controls.
RESULTS All rats gained weight during the study; however, male rats fed lab chow were heavier
(8 to 15%) than rats fed the corn-containing semi-purified rations. Both sexes consumed greater quantities of lab chow (3 to 5 g/rat/ day more) than did rats fed the corn-containing semi-purified rations. There were no significant differences in body weight or food consumption for male or female rats fed GC, GCA, AC, or ACA. However, after 60 to 65 weeks of feeding, rats fed AC corn weighed 3 to 5% less than rats in the other corn treatment groups.
After 80 weeks of feeding, 6 of 12 male rats and 2 of 12 female rats fed aflatoxin-contaminated corn had died or were moribund. At necropsy these rats all had grossly visible liver nodules. In the ACA group three rats were accidentally killed by heart puncture during
100
NORRED
AND MORRISSEY
FIG. 1. Photomicrograph of a liver section from a Fischer 344 rat showing a normal appearing central vein (CV) and surrounding parenchyma. This animal consumed the ACA diet (ammoniated corn) for 627 days. H and E; bar is 100 pm.
collection of blood samples. Other rats which died or were moribund prior to termination included 2 of 12 male and 2 of 12 female rats in the GCA group, and 2 of 12 male rats fed lab chow. No liver lesions were observed in these animals at necropsy. The remainder of the rats were killed after 91 to 92 weeks of continuous feeding. Grossly visible nodules were found in livers of all rats fed diets that contained AC corn, but there were no grossly visible nodules in livers of rats fed the other diets. Livers of the rats fed ACA appeared normal and were indistinguishable from those of rats in the GC, GCA, or LC groups. Tables 2 and 3 summarize the neoplastic lesions observed. Liver neoplasia was observed in all rats fed AC, but in none of the rats fed the other diets (Fig. 1). Distinct foci of hypertrophied hepatocytes containing a clear cytoplasm were noted in the AC group but not
in other groups. Metastases of hepatocellular carcinoma were found in lung and duodenum of some rats fed diet that contained AC corn. Fibrosarcoma was observed in the liver of one male and one female rat in the AC dietary treatment group. Malignant lymphoma occurred in one male rat fed AC and in one female rat fed GCA. Thyroid carcinoma was noted in 1 of 8 males and 2 of 11 females fed the ammoniated, aflatoxin-contaminated (ACA) diet. Testicular interstitial cell adenomas occurred in most of the male rats, regardless of dietary treatment. No neoplastic lesions were found in the ileum, cecum, salivary gland, trachea, esophagus, parathyroid, brain, or epididymus in any animal. All female rats had histologically normal stomach, duodenum, jejunum, urinary bladder, pituitary, and bone marrow. Bile duct proliferation was observed in some rats in all
AMMONIATED,
AFLATOXIN-CONTAMINATED
CORN
101
FIG. 2. Photomicrograph of a liver section from a Fischer 344 rat with hepatocellular carcinomma. This animal was fed the AC (880 &kg total atlatoxin) diet for 624 days. The normal pattern of hepatic triads is absent from most of this Iiver and is replaced by a neoplasm in which abnormal trabeculae predominate. Trabeculae (T) vary from one to several cells in thickness, lack a pattern, and are separated by sinusoids that vary in width. H and E; bar is 100 pm.
dietary treatment groups, but severe bile duct proliferation occurred only in some rats in the AC group. Pulmonary foreign body granulomas occurred in 5 of 12 male rats fed AC diet and 7 of 12 fed ACA diet, whereas these lesions did not occur in males in other treatment groups or in female rats. Hematocrit and hemoglobin were significantly lower in female rats fed the AC diet and tended to be lower in male rats (Table 4). Values for alkaline phosphatase activity were increased in both male and female rats fed the aflatoxin-contaminated diet. Values for the activity of AST tended to be higher in male and female rats fed aflatoxin-contaminated diet than in any of the other treatment groups. Hematological abnormalities produced by feeding aflatoxin-contaminated corn were not observed in rats fed aflatoxin-con-
taminated corn that had been ammoniated. Differential white blood cell counts (Table 5) were essentially the same in rats from each of the dietary groups. DISCUSSION The results reported here demonstrate that ammonia treatment of aflatoxin-contaminated corn as developed by Brekke et al. ( 1977a, 1978, 1979) eliminates .aIIatoxin-induced hepatic carcinogenesis in Fischer 344 rats. The liver tumors produced in rats fed rations prepared from aIIatoxin-contaminated corn were not present in rats fed similarly contaminated corn which had been ammoniated. In addition, clear cell foci were observed only in rats fed AC diets. Foci of the type observed have been reported to be de-
102
NORRED
AND
MORRISSEY
TABLE
TREATMENTS ON HEMATOLOGICAL PARAMETERS=
EFFECT OF DIETARY
Dietary treatment’
RBC/mm’ (x10-q
4
WBC/mm’ (x10-3)
Hematocrit (%)
Hemoglobin (mg/lOO ml)
AP W/t)
AST (U/l)
Males Gc GCA AC ACA
LC
6.6 5.7 6.6 6.3 6.1
f 0.8 f 0.1 + 0.3 + 0.4 z!z 0.5
8.3 + 1.8 7.5 + 1.0
10.1 f 4.5 7.3 + 0.7 5.8 f 1.0
45.8 44.6 43.6 45.6 46.4
+ + ++ f
4.2 0.9 2.8 0.6 3.4
15.8 15.0 14.4 15.3 15.6
+ + + k f
1.2 0.1 1.2 0.2 1.1
22.6 22.2 38.5 23.3 17.5
k 2.9 It 3.1 + 32.9 + 0.9 + 2.7
93.4 89.2 98.6 84.2 83.4
f 6.7 + 22.9 f 19.9 _+ 8.4 f 5.0
15.7 15.1 14.0 15.7 15.8
f + f + f
0.7 0.6 0.9’ 0.2 0.7
12.6 12.5 18.1 15.8 14.0
1?I l.7g k 0.9B + 2.3 + 2.3* k 3.2Lh
72.2 70.2 87.0 77.6 81.2
f 3.4 + 8.0 + 12.7 f 6.9 f 15.2
Females Gc GCA AC ACA
LC
5.9 5.6 5.7 6.4 6.4
f f + + f
0.6c,d 0.3’ 0.4’ 0.4d 0.4
4.5 4.8 5.4 4.6 7.1
+ f f + +
0.8 0.8 0.6
1.1 2.8
46.8 45.6 42.0 47.2 47.2
+ 1.6 f 1.3 + 2.1’
+ 1.1 + 1.5
’ Values are X +- SD of determinations made from five rats/treatment group. ’ Treatment designations are as defined under Methods. ch Values with different superscripts within each sex-parameter group differ significantly (p < 0.05).
ficient in certain enzymes, to have abnormal glycogen storage, and to have relevance to the production of hepatocellular carcinomas (Williams et al., 1976; Bannasch, 1968). The sensitivity of the Fischer 344 rat to aflatoxin-induced hepatocellular carcinomas has previously been reported (Wogan et al., 1974). As little as 1 ppb of aflatoxin B, produced a 9% tumor incidence after 109 weeks of feeding, while higher incidences were observed at higher doses (up to 100 ppb) and shorter feeding durations (54 to 88 weeks). In the present study a 100% tumor incidence occurred after 87 weeks of feeding of diets containing 176 ppb total ahatoxins. Chemical analyses of the corn following the ammoniation procedure indicated destruction of greater than 99% of the a&toxin (Brekke et al., 1979). Our results provide evidence of the effectiveness of this destruction, in that a well-known biological property of aflatoxin, i.e., hepatic tumor formation in rats, was eliminated. Elimination of other biological effects of aflatoxin-containing corn by ammoniation has previously been demonstrated, including pre-
vention of hepatic tumors in rainbow trout (Brekke et al., 1977b) and elimination of signs of acute toxicity in rats (Norred, 1979). Feeding ammoniated, aflatoxin-contaminated corn (ACA) was not associated with any signs of chronic toxicity. Body weight change, food consumption, and hematologic parameters were similar in rats fed either the GC, GCA, ACA, or LC diets. Non-neoplastic lesions were, for the most part, common to Fischer rats of the age used in our study (Coleman et al., 1977; Goodman et al., 1979). These included bile duct proliferation, diffuse congestion (liver, lungs, spleen, and kidneys), nephritis, and extramedullary hematopoiesis. Testicular interstitial cell adenomas observed in many of the male rats in our study are also commonly found in Fischer rats (Coleman et al., 1977; Goodman et al., 1979). The important finding was that the lesions associated with feeding the AC diet, including hepatic neoplasia, depressed hematocrit and hemoglobin values in female rats, and the trend toward elevated alkaline phosphatase levels did not occur in rats fed the ACA diet.
AMMONIATED,
AFLATOXIN-CONTAMINATED
103
CORN
TABLE 5 EFFECT OF DIETARY TREATMENTS ON DIFFERENTIAL WHITE CELL COUNTS’ ?h SD Dietary treatments*
Lymphocytes
Segmented neutrophils
Monocytes
Eosinophils
Basophils
Males CC GCA AC ACA LC
62.8 72.6 61.0 65.6 62.6
+ +
9.2 1.4
rf: 10.1 f f
4.5 3.8
35.0 25.6 36.6 30.4 34.8
+ 1.1 1.0 * 1.7 1.2 + 1.1
+- 8.5 f 7.8 f 10.6 k 6.0 k 5.9
1.4
2.6 + 2.1 1.8 + 2.2
0.8 0.8 1.6 0.8 0.5
0 0 0 0.2 + 0.5 0
0.6 + 0.9 1.2 f 0.8 1.6 + 1.1
0.2 f 0.5 0 0.2 f 0.5 0 0.2 * 0.5
0.8 0.8 1.2 1.2 0.8
f f f f k
Females CC GCA AC ACA LC
71.4 f 10.7 69.4 63.4 65.6 66.2
+ 12.6 + 7.0 + 6.4 f 8.4
26.8 29.0 32.8 31.6 30.4
iz 10.3 k 12.1 k 7.7 + 6.3 zk 7.8
1.0 + l.2c,d 0.4 2.0 1.4 2.2
k f * f
0.6' 1.2d 0.9c,d 0.5d
1.4 + 0.6 1.0 + 1.2
’ Values are X + SD of percentages of each type cell observed in five rats/treatment group. Blood was collected at the 87th week of feeding. * Treatment designations are as defined under Methods. c,dValues with different superscripts differ significantly (p < 0.05).
In conclusion, these results provide additional evidence of the effectiveness of the ammoniation process. However, prior to governmental approval of the process or recommendations for its use, the results of relay feeding studies must be elevated, and other toxicologic investigations may be needed. ACKNOWLEDGMENTS The excellent technical assistance of Jency L. Showker, Philip C. Stancel, Joyce L. Lanier, and Norma B. Hix is acknowledged. Ammoniation was conducted at the Northern Regional Research Center under the direction of 0. L. Brekke and E. B. Bagley. The allatoxin assays were conducted by Dr. 0. Shotwell and E. Vandegraft, Northern Regional Research Center, USDA-AR& Peoria, Ill.
REFERENCES BAGLEY, E. B. (1979). Decontamination of corn containing a5atoxin by treatment with ammonia. J. Amer. Oil Chem. Sot. 56, 808-811.
BANNASCH, P. (1968). The cytoplasm of hepatocytes during carcinogenesis. Recent Results Cancer Rex 19, l100. BREKKE, 0. L., PEPLINSKI, A. J., AND LANCASTER, E. B. (1977a). Aflatoxin inactivation in corn by aqua ammonia. Trans. ASAE 20, 1160-l 168. BREKKE, 0. L., SINNHUBER, R. O., PEPLINSKI, A. J., WALES, J. H., PUTNAM, G. B., LEE, D. J., AND CIEGLER, A. (1977b). Aflatoxin in corn: Ammonia inactivation and bioassay with rainbow trout. Appl. Environ. Microbiol. 34, 34-37. BREKKE, 0. L., STRINGFELLOW, A. C., AND PEPLINSKI, A. J. (1978). Aflatoxin inactivation in corn by ammonia gas: Laboratory trials. J. Agr. Food Chem. 26, 13831389. BREKKE, 0. L., PEPLINSKI, A. J., NOFSINGER, G. W., CONWAY,H. F., STRINGFELLOW,A. C., MONTGOMERY, R. R., SILMAN, R. W., SOHNS, V. E., AND BAGLEY, E. B. (1979). Aflatoxin inactivation in corn by ammonia gas: A field trial. Trans. ASAE 22,425-432. COLEMAN, G. L., BARTHOLD, S. W.. OSBALDISTON, G. W., FOSTER,S. J., AND JONAS,A. M. (1977). Pathological changes during aging in barrier-reared Fischer 344 male rats. J. Gerontology 32, 258-278. DIXON, W., AND MASSEY, F. (1969). Introduction to Statistical Analysis. McGraw-Hill, New York. GARDNER, H. K. ( 197 1). Inactivation of a5atoxins in pea-
104
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nut and cottonseed meals by ammoniation. J. Amer. Oil Chem. Sk. 48, 70-73. GOODMAN, D. G., WARD, J. M., SQUIRE, R. A., CHU, K. C., AND LINHART, M. S. (1979). Neoplastic and non-neoplastic lesions in aging F344 rats. Toxicol. Appl. Pharmacol. 48,237-248. JENSEN, A. H., BREJCKE,0. L., FRANK, G. R., AND PEPLINSIU, A. J. (1977). Acceptance and utilization by swine of aflatoxin-contaminated corn treated with aqueous or gaseous ammonia. J. Anim. Sci. 45, 8-12. KEYL, A. C., AND NORRED, W. P. (1979). Utilization of atlatoxin-contaminated feed. In Interactions of Mycotoxins in Animal Production. pp. 185, 195. National Academy of Sciences, Washington, DC. MASRI, M. S., VIX, H. L. E., AND GOLDBLA~, L. A. (1969). Process for detoxifying substances contaminated with aflatoxin. U.S. Patent No. 3,429,709 of February 25th, 1969. MCKINNEY, J. D., CAVANAUGH, G. C., BELL, J. T., HOVERSLAND, A. S., NELSON, D. M., PEARSON, J., AND SELKIRK, R. J. (1973). Effects of ammoniation of aflatoxins in rations fed lactating cows. J. Amer. Oil Chem. Sot. 50, 79-84. NATIONAL ACADEMY OF SCIENCES (1980). Histologic typing of liver tumors of the rat. J. Nat. Cancer Inst. 64. 179-206.
MORRISSEY
NORRED, W. P. (1979). Effect of ammoniation on the toxicity of corn artificially contaminated with atlatoxin B,. Toxicol. Appl. Pharmacol. 51, 41 I-416. NORRED, W. P. ( 198 I ). Excretion and distribution of ammoniated “C-labelled aflatoxin Bt-containing corn. Fed. Proc. 40, 694. NORRED, W. P. (1982). Use of ammonia treatment to destroy aflatoxins in corn. J. Food Protect. 45, 972976.
SCHOENTAL,R. ( 1967). Aflatoxins. Annu. Rev.Pharmacol. 7, 343-356.
THIESEN, J. (1977). Detoxification of aflatoxins in groundnut meal. Anim. Feed Sci. Technol. 2,67-75. WILLIAMS, G. M., KLAIBER, M., PARKER, S. E., AND FARBER, E. (1976). Nature of early appearing, carcinogen-induced liver lesions resistant to iron accumulation. J. Nat. Cancer Inst. 57, 157-165. WILSON, D. M., MCMILLIAN, W. W., AND WIDSTROM, N. W. (1979). Field aflatoxin contamination of corn in South Georgia. J. Amer. Oil Chem. Sot. 56,798-799. WOGAN, G. N. (1966). Chemical nature and biological effects of the aflatoxins. Bacterial. Rev. 30, 460-470. WOGAN, G. N., PAGLIALUNGA, S., AND NEWBERNE, P. M. (1974). Carcinogenic effects of low dietary levels of aflatoxin B, in rats. Fd. Cosmet. Toxicol. 12, 681685.
AND
APPLIED
PHARMACOLOGY
Effects of Long-Term
WILLIAM
70,96-104
(1983)
Feeding of Ammoniated, Aflatoxin-Contaminated Corn to Fischer 344 Rats
P. NORRED’
AND RICHARD
E. MORRISSEY
Richard B. Russell Agricultural Research Center, U.S. Department of Agriculture, Agricultural Research Service, Athens, Georgia 30613
Received December 20. 1982; accepted March 25. 1983 Effects of Long-Term Feeding of Ammoniated, Aflatoxin-Contaminated Corn to Fischer 344 Rats. Norred, W. P., and Morrissey, R. E. ( 1983). Toxicol. Appl. Pharmacol. 70,96-104. The effectivenessof ammonia treatment in reducing the chronic toxicity of aflatoxin-contaminated corn was determined. Fischer 344 rats were fed semi-purified rations containing 20% w/w corn that was either free of aflatoxin or naturahy contaminated with 880 &kg total aflatoxin and was either treated with ammonia gas or was not treated. Therefore the rats that were fed the aflatoxin-contaminated diet received 176 ppb total aflatoxins. Body weight and food consumption were recorded throughout the study; hematological measurements were made after 87 weeks of feeding; and after 9 1 weeks the rats were killed and histopathological abnormalities were noted. Signs of chronic toxicosis in rats fed aflatoxincontaminated corn included increased mortality, decreased hematocrit and hemoglobin levels, elevated serum alkaline phosphatase activities, and a 100% incidence of liver neoplasia. These signs did not occur in rats in the other dietary treatment groups, including those fed ammoniated, aflatoxinumtaminated corn. The results provide further evidence that the atmospheric ammoniation process effectively reduces the toxicity of aflatoxincontaminated corn.
The use of ammonia, either in gaseous or liquid form, at elevated heat and pressure or under ambient conditions, has been described by a number of investigators as a detoxification procedure for aflatoxin-contaminated commodities (Masri et al., 1969; Gardner, 197 1; McKinney et al., 1973; Thiesen, 1977). Treatment of aflatoxin-contaminated corn with ammonia gas at atmospheric pressure appears to be the most effective and economically feasible method of salvaging the corn for use as animal feed (Bagley, 1979; Key1 and Norred, 1979; Norred, 1982). The process (Brekke et al., 1977a, 1978, 1979) reduces the content of aflatoxin in naturally contaminated corn to less than 20 ppb; it results in corn which is accepted and efficiently utilized by swine (Jensen et al., 1977); it prevents the production of hepatoma in rainbow trout (Brekke et al.,
Aflatoxins are toxic metabolites produced by molds (Aspergillusflavus, A. parasiticus, and Penicillium puberulum) which can invade food and feed ingredients (Wogan, 1966; Schoental, 1967). Contamination of corn in particular has caused severe economic problems for livestock producers and may create potential health risks by the transmission of aflatoxins and/or their metabolites from livestock to humans through consumption of meat, milk, or eggs. Conditions favorable to mold growth have occurred periodically, particularly in the southeastern United States; the severe weather conditions and insect damage in the summer of 1977 resulted in widespread aflatoxin contamination with resultant losses (Wilson et al., 1979; Bagley, 1979). ’ To whom correspondence should be addressed. 96 OO41OO8X/83 $3.00
AMMONIATED,
AFLATOXIN-CONTAMINATED
1977b); it eliminates signs of acute toxicity which result from administration of corn fortified with high levels of aflatoxin B, (Norred, 1979); and it decreases the uptake from the gastrointestinal tract, enhances the excretion, and reduces the binding to liver of carbon-14 after po administration to rats of 14C-aflatoxin B,-containing corn (Norred, 198 1). Relay feeding studies, although not yet completed, have indicated no deleterious effects to rats fed egg, pork, poultry, or beef from farm animals fed ammoniated, aflatoxin-contaminated corn (Norred, 1982). In the present study the effectiveness of ammonia treatment of corn naturally contaminated with aflatoxin was further evaluated by feeding the corn directly to rats in a chronic study. Treated corn was prepared under field conditions at the Northern Regional Research Center (NRRC), ARS, USDA, Peoria, Ill., and was fed, along with appropriate control diets, to Fischer rats for a 23-month period. This paper reports the histological and hematological findings of this investigation. METHODS Animals. Three-week-old Fischer 344 rats (reared in our facility from breeding stock obtained from Charles River Breeding Labs, Wilmington, Mass.)* were randomly assigned by sex to one of five treatment groups, with 12 males and I2 females in each group. The rats were identified by ear notch and housed two per cage in suspended wire cages in racks equipped with automatic watering systemsand automatic flushing. Temperature was maintained at 22 rf: 3°C and relative humidity at 50 to 70%. Fluorescent lighting was turned on at 6:00 AM and off at 6:00 PM to provide a 12-hr light-dark cycle. Powdered diets were provided ad libitum. All rats were weighed weekly for the first 15 weeks and monthly thereafter. Food consumption was determined during a 1 week period each month. Diets. Diets fed to the rats were either lab chow (LC) (Teklad Mouse/Rat Chow, Winfield, Iowa, ground to pass a 3-mm sieve) or semi-purified ration (Table I) containing 20% w/w of one of four types of corn. The dietary treatment groups were identified based on the type of corn fed, where
’ The mention of this product, equipment, or company name does not imply recommendation over others that may be suitable for use.
97
CORN TABLE I
Ingredient
% w/w
Casein Dextrin Corn oil Cellulose Mineral mix, Jones-Foster Vitamin mix, Teklad Corn, ground*
12 58 1 4 4 1 20
’ All ingredients except corn were blended by Teklad Test Diets, Madison, Wisconsin. ’ One of the four types of corn (GC, GCA, AC, ACA) as defined under Methods was ground to pass a 3-mm sieve, and blended for 20 min with the basal ration in a Reynolds mixer. GC (good corn) was corn with no detectable levels of aflatoxin, ochratoxin, or zearalenone; AC (aflatoxin-contaminated) was corn naturally contaminated with 880 rg/ kg of total aflatoxins (750 of B, ,90 of B2, and 40 of G,); GCA (good corn ammoniated) was GC corn that had been ammoniated, and ACA (aflatoxin corn, ammoniated) was AC corn that had been ammoniated. Therefore rats fed AC diet received 176 ppb total aflatoxins, whereas those fed ACA diet received an equivalent amount of ammoniated aflatoxin by-products. The corn was obtained from the Northern Regional Research Center, ARS, USDA, and ammoniated samples had been treated as described by Brekke ef al. (1979). The corn was ground (3-mm sieve), and blended with the semi-purified ration. The blended rations were stored at 4°C and feed hoppers were replaced weekly with fresh ration. Fresh diets were prepared monthly. Histopathology. A necropsy was performed on each rat that died during the study or was killed when the study was terminated at 92 weeks. Tissues (stomach, duodenum, jejunum, ileum. cecum, colon, liver, pancreas, salivary gland, lungs, trachea, esophagus, thyroid, parathyroid, adrenals, kidneys, urinary bladder, testes, epididymides, seminal vesicles, prostate, spleen, heart, brain, pituitary, eyes,sternum, ovaries, uterus, and mammary gland) were preserved in 10% buffered Formalin, and routinely prepared paraffin sections were stained with hematoxylin and eosin and examined by light microscopy. Liver tumors were classified according to criteria established by the National Academy of Science ( 1980). Hemafo/ogy. Blood (1 to 2 ml) was collected by cardiac puncture from lightly anesthetized (ether) male and female rats (five of each) selected randomly from each dietary treatment group. Collections were made 87 weeks after treatments began. Serum was separated from clotted blood by centrifuging at 5OOg. The activities of alkaline phos-
98
NORRED
AND MORRISSEY TABLE 2
EFFECT OF AMMONIA
TREATMENT OF NEOPLASTK
OF AFLATOXIN-CONTAMINATED LESIONS IN MALE FISCHER
CORN RATS”
ON INCIDENCE
Dietary treatmentb Gc
GCA
AC
ACA
LC
Stomach Malignant lymphoma
O/l2
O/11
l/l 1
O/l2
o/12
Duodenum Hepatocellular carcinoma, metastatic
o/12
o/10
l/10
O/l2
o/12
Liver Hepatocellular carcinoma Neoplastic nodule Fibrosarcoma Malignant lymphoma
o/12 o/12 O/l2 O/l2
o/11 o/11 o/11 O/11
12112’ 6112’ l/12 l/12
o/12 o/12 o/12 O/l2
o/12 O/l2 O/l2 O/l2
Pancreas Islet cell adenoma Malignant lymphoma
l/l2 O/l2
l/10 o/10
o/11 l/11
l/12 o/12
2/11 o/11
Salivary gland Malignant lymphoma
o/12
O/IO
l/12
o/10
o/10
Lungs Hepatocellular carcinoma, metastatic Malignant lymphoma
O/l1 O/l1
O/l1 O/II
2112 l/12
o/12 O/l2
o/12 o/12
Adrenals Cortical adenoma, unilateral Pheochromocytoma, unilateral
O/II o/11
O/l0 O/l0
l/10 o/10
l/12 o/12
O/l2 l/l2
Kidneys Malignant lymphoma, bilateral
O/l2
O/l1
l/l2
O/l2
o/12
Testes Interstitial cell adenoma, bilateral Interstitial cell adenoma, unilateral
l/l 1 8/ll
O/IO 8110
3112 8/12
6112 S/12’
4112 l/l2
Spleen Malignant lymphoma
o/12
l/12
o/12
O/l2
Pituitary Chromophobe adenoma
O/IO
O/4
016
l/9
Skin Keratoacanthoma
111
o/o
Thyroid C cell carcinoma, unilateral
O/l1
016
o/o
W3
O/9
a Values are number of rats affected/number of rats examined. b Treatment designations are as defined under Methods. c Significantly different from CC treatment group (p < 0.05). phatase (AP) and asparatate aminotransferase (AST) were determined in the serum with kits (Hycel and Dade, respectively) purchased from Scientific Products, Stone
Mountain, Georgia. Blood (0.3 to 0.5 ml) was also collected from the same rats into syringesrinsed with EDTA solution (Sequester-Sol, Fort Lauderdale, Fla.) to prevent clotting.
AMMONIATED,
AFLATOXIN-CONTAMINATED
99
CORN
TABLE 3 EFFECT OFAMMONIATREATMENTOFAFLATOXIN-CONTAMMATEDCORNONINCIDENCE OFNEOPLASTICLESIONSINFEMALEFISCHERRAW Dietary tmatmentb tic
GCA
AC
ACA
LC
Liver Hepatocelhnar carcinoma Neoplastic nodules Fibrosarcoma
o/12 o/12 o/12
o/11 o/11 o/11
1 l/12’ 10/12c l/12
o/12 o/12 o/12
o/12 o/12 o/12
Lungs Hepatocellular carcinoma, metastatic Malignant lymphoma
o/12 o/12
o/12 l/12
2112 o/12
Of12 o/12
o/12 o/12
Adrenals Pheochromocytoma, unilateral
O/11
O/11
1/l 1
o/12
o/12
Pituitary Chromophobe adenoma
214
O/9
w
l/4
319
Skin Malignant lymphoma
o/12
l/12
o/12
o/12
o/12
Uterus Leiomyoma
o/12
o/11
l/12
l/12
o/12
Thyroid C cell carcinoma, unilateral
Of7
O/9
O/7
2111
O/IO
Mammary gland Adenocarcinoma Adenoma
o/12 o/12
o/11 O/11
o/12 o/12
o/12 l/12
l/12 o/12
’ Values are number of rats affected/number ofrats examined. ’ Treatment designations are as defined under Methods. ’ Significantly different from CC treatment group (p < 0.05). White cell and red cell counts were determined in these samples with a Coulter ZBI counter; hemoglobin content was measured with a Coulter hemoglobinometer; and hematocrit and differential white cell counts were determined by conventional means. Statistical analysis. One-way analysis of variance was used to determine significant differences (p i 0.05) between treatment groups (Dixon and Massey, 1969). Duncan’s multiple range test was used for determining mean seg aration in cases where there was significant variation among sample means. Chi Square with Yates’ correction was used to determine significance of incidences of histological lesions, with values observed in rats fed CC used as controls.
RESULTS All rats gained weight during the study; however, male rats fed lab chow were heavier
(8 to 15%) than rats fed the corn-containing semi-purified rations. Both sexes consumed greater quantities of lab chow (3 to 5 g/rat/ day more) than did rats fed the corn-containing semi-purified rations. There were no significant differences in body weight or food consumption for male or female rats fed GC, GCA, AC, or ACA. However, after 60 to 65 weeks of feeding, rats fed AC corn weighed 3 to 5% less than rats in the other corn treatment groups.
After 80 weeks of feeding, 6 of 12 male rats and 2 of 12 female rats fed aflatoxin-contaminated corn had died or were moribund. At necropsy these rats all had grossly visible liver nodules. In the ACA group three rats were accidentally killed by heart puncture during
100
NORRED
AND MORRISSEY
FIG. 1. Photomicrograph of a liver section from a Fischer 344 rat showing a normal appearing central vein (CV) and surrounding parenchyma. This animal consumed the ACA diet (ammoniated corn) for 627 days. H and E; bar is 100 pm.
collection of blood samples. Other rats which died or were moribund prior to termination included 2 of 12 male and 2 of 12 female rats in the GCA group, and 2 of 12 male rats fed lab chow. No liver lesions were observed in these animals at necropsy. The remainder of the rats were killed after 91 to 92 weeks of continuous feeding. Grossly visible nodules were found in livers of all rats fed diets that contained AC corn, but there were no grossly visible nodules in livers of rats fed the other diets. Livers of the rats fed ACA appeared normal and were indistinguishable from those of rats in the GC, GCA, or LC groups. Tables 2 and 3 summarize the neoplastic lesions observed. Liver neoplasia was observed in all rats fed AC, but in none of the rats fed the other diets (Fig. 1). Distinct foci of hypertrophied hepatocytes containing a clear cytoplasm were noted in the AC group but not
in other groups. Metastases of hepatocellular carcinoma were found in lung and duodenum of some rats fed diet that contained AC corn. Fibrosarcoma was observed in the liver of one male and one female rat in the AC dietary treatment group. Malignant lymphoma occurred in one male rat fed AC and in one female rat fed GCA. Thyroid carcinoma was noted in 1 of 8 males and 2 of 11 females fed the ammoniated, aflatoxin-contaminated (ACA) diet. Testicular interstitial cell adenomas occurred in most of the male rats, regardless of dietary treatment. No neoplastic lesions were found in the ileum, cecum, salivary gland, trachea, esophagus, parathyroid, brain, or epididymus in any animal. All female rats had histologically normal stomach, duodenum, jejunum, urinary bladder, pituitary, and bone marrow. Bile duct proliferation was observed in some rats in all
AMMONIATED,
AFLATOXIN-CONTAMINATED
CORN
101
FIG. 2. Photomicrograph of a liver section from a Fischer 344 rat with hepatocellular carcinomma. This animal was fed the AC (880 &kg total atlatoxin) diet for 624 days. The normal pattern of hepatic triads is absent from most of this Iiver and is replaced by a neoplasm in which abnormal trabeculae predominate. Trabeculae (T) vary from one to several cells in thickness, lack a pattern, and are separated by sinusoids that vary in width. H and E; bar is 100 pm.
dietary treatment groups, but severe bile duct proliferation occurred only in some rats in the AC group. Pulmonary foreign body granulomas occurred in 5 of 12 male rats fed AC diet and 7 of 12 fed ACA diet, whereas these lesions did not occur in males in other treatment groups or in female rats. Hematocrit and hemoglobin were significantly lower in female rats fed the AC diet and tended to be lower in male rats (Table 4). Values for alkaline phosphatase activity were increased in both male and female rats fed the aflatoxin-contaminated diet. Values for the activity of AST tended to be higher in male and female rats fed aflatoxin-contaminated diet than in any of the other treatment groups. Hematological abnormalities produced by feeding aflatoxin-contaminated corn were not observed in rats fed aflatoxin-con-
taminated corn that had been ammoniated. Differential white blood cell counts (Table 5) were essentially the same in rats from each of the dietary groups. DISCUSSION The results reported here demonstrate that ammonia treatment of aflatoxin-contaminated corn as developed by Brekke et al. ( 1977a, 1978, 1979) eliminates .aIIatoxin-induced hepatic carcinogenesis in Fischer 344 rats. The liver tumors produced in rats fed rations prepared from aIIatoxin-contaminated corn were not present in rats fed similarly contaminated corn which had been ammoniated. In addition, clear cell foci were observed only in rats fed AC diets. Foci of the type observed have been reported to be de-
102
NORRED
AND
MORRISSEY
TABLE
TREATMENTS ON HEMATOLOGICAL PARAMETERS=
EFFECT OF DIETARY
Dietary treatment’
RBC/mm’ (x10-q
4
WBC/mm’ (x10-3)
Hematocrit (%)
Hemoglobin (mg/lOO ml)
AP W/t)
AST (U/l)
Males Gc GCA AC ACA
LC
6.6 5.7 6.6 6.3 6.1
f 0.8 f 0.1 + 0.3 + 0.4 z!z 0.5
8.3 + 1.8 7.5 + 1.0
10.1 f 4.5 7.3 + 0.7 5.8 f 1.0
45.8 44.6 43.6 45.6 46.4
+ + ++ f
4.2 0.9 2.8 0.6 3.4
15.8 15.0 14.4 15.3 15.6
+ + + k f
1.2 0.1 1.2 0.2 1.1
22.6 22.2 38.5 23.3 17.5
k 2.9 It 3.1 + 32.9 + 0.9 + 2.7
93.4 89.2 98.6 84.2 83.4
f 6.7 + 22.9 f 19.9 _+ 8.4 f 5.0
15.7 15.1 14.0 15.7 15.8
f + f + f
0.7 0.6 0.9’ 0.2 0.7
12.6 12.5 18.1 15.8 14.0
1?I l.7g k 0.9B + 2.3 + 2.3* k 3.2Lh
72.2 70.2 87.0 77.6 81.2
f 3.4 + 8.0 + 12.7 f 6.9 f 15.2
Females Gc GCA AC ACA
LC
5.9 5.6 5.7 6.4 6.4
f f + + f
0.6c,d 0.3’ 0.4’ 0.4d 0.4
4.5 4.8 5.4 4.6 7.1
+ f f + +
0.8 0.8 0.6
1.1 2.8
46.8 45.6 42.0 47.2 47.2
+ 1.6 f 1.3 + 2.1’
+ 1.1 + 1.5
’ Values are X +- SD of determinations made from five rats/treatment group. ’ Treatment designations are as defined under Methods. ch Values with different superscripts within each sex-parameter group differ significantly (p < 0.05).
ficient in certain enzymes, to have abnormal glycogen storage, and to have relevance to the production of hepatocellular carcinomas (Williams et al., 1976; Bannasch, 1968). The sensitivity of the Fischer 344 rat to aflatoxin-induced hepatocellular carcinomas has previously been reported (Wogan et al., 1974). As little as 1 ppb of aflatoxin B, produced a 9% tumor incidence after 109 weeks of feeding, while higher incidences were observed at higher doses (up to 100 ppb) and shorter feeding durations (54 to 88 weeks). In the present study a 100% tumor incidence occurred after 87 weeks of feeding of diets containing 176 ppb total ahatoxins. Chemical analyses of the corn following the ammoniation procedure indicated destruction of greater than 99% of the a&toxin (Brekke et al., 1979). Our results provide evidence of the effectiveness of this destruction, in that a well-known biological property of aflatoxin, i.e., hepatic tumor formation in rats, was eliminated. Elimination of other biological effects of aflatoxin-containing corn by ammoniation has previously been demonstrated, including pre-
vention of hepatic tumors in rainbow trout (Brekke et al., 1977b) and elimination of signs of acute toxicity in rats (Norred, 1979). Feeding ammoniated, aflatoxin-contaminated corn (ACA) was not associated with any signs of chronic toxicity. Body weight change, food consumption, and hematologic parameters were similar in rats fed either the GC, GCA, ACA, or LC diets. Non-neoplastic lesions were, for the most part, common to Fischer rats of the age used in our study (Coleman et al., 1977; Goodman et al., 1979). These included bile duct proliferation, diffuse congestion (liver, lungs, spleen, and kidneys), nephritis, and extramedullary hematopoiesis. Testicular interstitial cell adenomas observed in many of the male rats in our study are also commonly found in Fischer rats (Coleman et al., 1977; Goodman et al., 1979). The important finding was that the lesions associated with feeding the AC diet, including hepatic neoplasia, depressed hematocrit and hemoglobin values in female rats, and the trend toward elevated alkaline phosphatase levels did not occur in rats fed the ACA diet.
AMMONIATED,
AFLATOXIN-CONTAMINATED
103
CORN
TABLE 5 EFFECT OF DIETARY TREATMENTS ON DIFFERENTIAL WHITE CELL COUNTS’ ?h SD Dietary treatments*
Lymphocytes
Segmented neutrophils
Monocytes
Eosinophils
Basophils
Males CC GCA AC ACA LC
62.8 72.6 61.0 65.6 62.6
+ +
9.2 1.4
rf: 10.1 f f
4.5 3.8
35.0 25.6 36.6 30.4 34.8
+ 1.1 1.0 * 1.7 1.2 + 1.1
+- 8.5 f 7.8 f 10.6 k 6.0 k 5.9
1.4
2.6 + 2.1 1.8 + 2.2
0.8 0.8 1.6 0.8 0.5
0 0 0 0.2 + 0.5 0
0.6 + 0.9 1.2 f 0.8 1.6 + 1.1
0.2 f 0.5 0 0.2 f 0.5 0 0.2 * 0.5
0.8 0.8 1.2 1.2 0.8
f f f f k
Females CC GCA AC ACA LC
71.4 f 10.7 69.4 63.4 65.6 66.2
+ 12.6 + 7.0 + 6.4 f 8.4
26.8 29.0 32.8 31.6 30.4
iz 10.3 k 12.1 k 7.7 + 6.3 zk 7.8
1.0 + l.2c,d 0.4 2.0 1.4 2.2
k f * f
0.6' 1.2d 0.9c,d 0.5d
1.4 + 0.6 1.0 + 1.2
’ Values are X + SD of percentages of each type cell observed in five rats/treatment group. Blood was collected at the 87th week of feeding. * Treatment designations are as defined under Methods. c,dValues with different superscripts differ significantly (p < 0.05).
In conclusion, these results provide additional evidence of the effectiveness of the ammoniation process. However, prior to governmental approval of the process or recommendations for its use, the results of relay feeding studies must be elevated, and other toxicologic investigations may be needed. ACKNOWLEDGMENTS The excellent technical assistance of Jency L. Showker, Philip C. Stancel, Joyce L. Lanier, and Norma B. Hix is acknowledged. Ammoniation was conducted at the Northern Regional Research Center under the direction of 0. L. Brekke and E. B. Bagley. The allatoxin assays were conducted by Dr. 0. Shotwell and E. Vandegraft, Northern Regional Research Center, USDA-AR& Peoria, Ill.
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BANNASCH, P. (1968). The cytoplasm of hepatocytes during carcinogenesis. Recent Results Cancer Rex 19, l100. BREKKE, 0. L., PEPLINSKI, A. J., AND LANCASTER, E. B. (1977a). Aflatoxin inactivation in corn by aqua ammonia. Trans. ASAE 20, 1160-l 168. BREKKE, 0. L., SINNHUBER, R. O., PEPLINSKI, A. J., WALES, J. H., PUTNAM, G. B., LEE, D. J., AND CIEGLER, A. (1977b). Aflatoxin in corn: Ammonia inactivation and bioassay with rainbow trout. Appl. Environ. Microbiol. 34, 34-37. BREKKE, 0. L., STRINGFELLOW, A. C., AND PEPLINSKI, A. J. (1978). Aflatoxin inactivation in corn by ammonia gas: Laboratory trials. J. Agr. Food Chem. 26, 13831389. BREKKE, 0. L., PEPLINSKI, A. J., NOFSINGER, G. W., CONWAY,H. F., STRINGFELLOW,A. C., MONTGOMERY, R. R., SILMAN, R. W., SOHNS, V. E., AND BAGLEY, E. B. (1979). Aflatoxin inactivation in corn by ammonia gas: A field trial. Trans. ASAE 22,425-432. COLEMAN, G. L., BARTHOLD, S. W.. OSBALDISTON, G. W., FOSTER,S. J., AND JONAS,A. M. (1977). Pathological changes during aging in barrier-reared Fischer 344 male rats. J. Gerontology 32, 258-278. DIXON, W., AND MASSEY, F. (1969). Introduction to Statistical Analysis. McGraw-Hill, New York. GARDNER, H. K. ( 197 1). Inactivation of a5atoxins in pea-
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nut and cottonseed meals by ammoniation. J. Amer. Oil Chem. Sk. 48, 70-73. GOODMAN, D. G., WARD, J. M., SQUIRE, R. A., CHU, K. C., AND LINHART, M. S. (1979). Neoplastic and non-neoplastic lesions in aging F344 rats. Toxicol. Appl. Pharmacol. 48,237-248. JENSEN, A. H., BREJCKE,0. L., FRANK, G. R., AND PEPLINSIU, A. J. (1977). Acceptance and utilization by swine of aflatoxin-contaminated corn treated with aqueous or gaseous ammonia. J. Anim. Sci. 45, 8-12. KEYL, A. C., AND NORRED, W. P. (1979). Utilization of atlatoxin-contaminated feed. In Interactions of Mycotoxins in Animal Production. pp. 185, 195. National Academy of Sciences, Washington, DC. MASRI, M. S., VIX, H. L. E., AND GOLDBLA~, L. A. (1969). Process for detoxifying substances contaminated with aflatoxin. U.S. Patent No. 3,429,709 of February 25th, 1969. MCKINNEY, J. D., CAVANAUGH, G. C., BELL, J. T., HOVERSLAND, A. S., NELSON, D. M., PEARSON, J., AND SELKIRK, R. J. (1973). Effects of ammoniation of aflatoxins in rations fed lactating cows. J. Amer. Oil Chem. Sot. 50, 79-84. NATIONAL ACADEMY OF SCIENCES (1980). Histologic typing of liver tumors of the rat. J. Nat. Cancer Inst. 64. 179-206.
MORRISSEY
NORRED, W. P. (1979). Effect of ammoniation on the toxicity of corn artificially contaminated with atlatoxin B,. Toxicol. Appl. Pharmacol. 51, 41 I-416. NORRED, W. P. ( 198 I ). Excretion and distribution of ammoniated “C-labelled aflatoxin Bt-containing corn. Fed. Proc. 40, 694. NORRED, W. P. (1982). Use of ammonia treatment to destroy aflatoxins in corn. J. Food Protect. 45, 972976.
SCHOENTAL,R. ( 1967). Aflatoxins. Annu. Rev.Pharmacol. 7, 343-356.
THIESEN, J. (1977). Detoxification of aflatoxins in groundnut meal. Anim. Feed Sci. Technol. 2,67-75. WILLIAMS, G. M., KLAIBER, M., PARKER, S. E., AND FARBER, E. (1976). Nature of early appearing, carcinogen-induced liver lesions resistant to iron accumulation. J. Nat. Cancer Inst. 57, 157-165. WILSON, D. M., MCMILLIAN, W. W., AND WIDSTROM, N. W. (1979). Field aflatoxin contamination of corn in South Georgia. J. Amer. Oil Chem. Sot. 56,798-799. WOGAN, G. N. (1966). Chemical nature and biological effects of the aflatoxins. Bacterial. Rev. 30, 460-470. WOGAN, G. N., PAGLIALUNGA, S., AND NEWBERNE, P. M. (1974). Carcinogenic effects of low dietary levels of aflatoxin B, in rats. Fd. Cosmet. Toxicol. 12, 681685.