Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis

Toxicology Letters ELSEVIER

Toxicology

Letters

89 (1996) 115122

Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M, in turkey poults. Lack of effect by activated charcoal on aflatoxicosis’ T.S. Edrington”, “U.S.Department

A.B. Sarrb, L.F. Kubenaa,

R.B. Harvey”,*,

T.D. Phillipsb

of .4griculture, Agricultural Research Service, Food Animal Protection Research Laboratory, 2881 F & B Road, College Station, TX 77845, USA bCollege of Veterinary Medicine, Texas A & M University, College Station, TX 77843, USA Received

28 February

1996; accepted

1 July 1996

Abstract In one experiment, the effect of inorganic sorbents on the metabolic fate of aflatoxin B, (AFB,) was studied in turkey poults. At 5 weeks of age, female poults were surgically colostomized and 9 days later orally dosed with 0.75 mg AFB,/kg BW. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal (AC) were tested, by concomitant administration with AFB,. Urine was collected up to 48 h post-dosing and analyzed for aflatoxin M, (AFM:,) which was the major metabolite found in all treatment groups. Hydrated sodium calcium aluminosilicate, previously proven beneficial in alleviating aflatoxicosis in farm animals, reduced urinary AFM, output when orally dosed simultaneous with AFB,. Also, acidic HSCAS and AC significantly decreased AFM, excretion when administered concomitantly with AFB,. A second experiment was conducted to evaluate the ability of two types of AC to modify aflatoxicosis when added to aflatoxin (AF)-contaminated (from culture material) diets of turkey poults. Although AC was able to decrease AFM, excretion in the first experiment, no protective effects from AF toxicity were observed in the feeding study. Keywords:

Aflatoxin; Inorganic sorbents; Activated charcoal; Metabolism;

* Corresponding author. Tel.: + 409 2609259; fax: + 409 2609377: email: [email protected]. .’ Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its ap-

proval to the exclusion of other products that may be suitable. 037%4274/96/$15.00

G 1996 El sevier Science Ireland

PII SO378-4274(96)03795-2

Ltd. All rights

Turkeys

1. Introduction Aflatoxins (AF), a group of closely related, biologically active mycotoxins, are produced by strains of AspergillusJEavus and A. parasiticus and reserved

116

T.S. Edringtan

et al. 1 Toxicology

occur naturally in a number of important animal feeds. Aflatoxins damage the liver, kidney, and thymus resulting in a variety of effects including decreased growth rates, poor productivity, immunosuppression, and disruption of carbohyprotein, and lipid metabolism [I]. drate, Additionally, AF have been shown to be potent carcinogens, strong mutagens, and potential teratogens [2-41. Lactating animals fed aflatoxin B, (AFB,) secrete into the milk a carcinogenic and mutagenic metabolite, aflatoxin M, (AFM,) [S71. The metabolic fate of AF is not fully understood. Aflatoxins are absorbed easily from the gastro-intestinal tract, are extensively metabolized, and are eliminated rapidly from the body, however, substantial variation exists among species [l]. The metabolic fate of AF in livestock and poultry is important for two reasons: (1) metabolism or activation of AF accounts for much of the toxicity of AF, and (2) systemic distribution of AF or their metabolites within food-producing animals can result in hazardous residues in products intended for use by humans

VI. As a result of the widespread occurrence of AF in feedstuffs and their deleterious effects on food animals, recent detoxification efforts have included the use of nutrient modifications [8-lo] and the dietary inclusion of chemisorbent compounds to render AF unavailable for absorption from the digestive tract [ 1 l- 131. In an evaluation of various inorganic sorbent materials for AF inactivation, Phillips et al. [14] reported a hydrated sodium calcium aluminosilicate (HSCAS) that adsorbed more than 80% of AFB, in solution. Incorporation of HSCAS into the diet proved beneficial in alleviating the negative effects of aflatoxin in growing lambs [15], broilers [1618], turkey poults [19], and barrows [20]. The secretion of AFM, into milk was reduced when HSCAS was added to the diet of dairy cows consuming AFB, [21]. In this study, we examined the effects of HSCAS, acidic HSCAS, or activated characoal (AC) on the metabolic profile of AFB, in the urine of surgically colostomized turkey poults. Additionally, we examined the potential of two types of AC for alleviating AF-depressed

Letters

89 (1996) 115-122

performance poults.

when fed to intact, growing turkey

2. Methods Female turkey poults (Nicholas large whites) were housed in commercial grower batteries under continuous fluorescent lighting with commercial turkey feed (CTF) and water available ad libitum. Five-week-old poults were surgically colostomized. Briefly, birds were anesthetized with a combination of RompunO (xylazine hydrochloride, Miles Inc., Shawnee Mission, KS) at 2 mg/ kg BW, and KetasetO (ketamine hydrochloride, Aveco Co. Inc., Ft. Dodge, IA) at 2 mg/kg BW injected i.m. into the left breast. Feathers were removed from the abdomen and the area was cleansed and disinfected. An oval 2-cm incision was made 5-8 cm cranial to the vent and 1 cm to the right of midline. The lower colon was brought to the incision, ligated near the rectum, severed, and sutured to the skin surface with simple interrupted 00 silk sutures. The area was flushed with saline and 0.5 ml of penicillin G and dihydrostreptomycin administered i.m. Approximately 1 week following the colostomy, birds were anesthetized as above and the vent area cleaned and disinfected. A disposable pipette bulb with the end cut off was sutured directly to the vent. Following surgery, birds were individually caged with feed and water available ad libitum. Care, use, and handling of experimental animals was pre-approved by the Animal Care and Use Committee of the Food Animal Protection Research Laboratory, USDA. 2.1. Experiment I Two days following bulb placements, poults were randomly assigned to the following groups and orally dosed via gelatin capsule: (1) Negative AFB, control (no AFB,, no adsorbant; 2 birds); (2) 0.75 mg AFBJkg BW (positive AFB, control; 3 birds); (3) 0.75 mg AFB, plus 0.5% charcoal D (AC-D, 3 birds); (4) 0.75 mg AFB,/kg BW plus 0.5% HSCAS (4 birds); or (5) 0.75 mg AFB,/kg BW plus 0.5% acidic HSCAS (2 birds). Aflatoxin

T.S. Edrington et al. / Toxicology Letters 89 (1996) 115-122

B, was dissolved in methanol, applied to an amount of sorbent (HSCAS, acidic HSCAS, or charcoal D) equal to 0.5% of the daily feed intake and placed in a gelatin capsule. Immediately following dosing, a rubber balloon was attached to the bulb and urine output collected at 2 h, and thereafter 3 h output collections were made at 5, 8, 11, 24, 36 and 48 h. Feed and water were available ad libitum for the duration of the study. Urine volume was measured and the urine stored frozen ( - 20°C) for later analysis. Aflatoxin B, was purchased from Sigma (St. Louis, MO). The HSCAS (NovaSilTM) and acidic HSCAS were obtained from Engelhard Corp. (Cleveland, OH). Charcoal D (Darco decolorizing carbon G-60) was purchased from J.T. Baker Inc. (Phillipsburg, NJ). 2.2. Experiment

2

Because of the iability of AC to bind AFB, in the first experiment, a second experiment was conducted to evaluate two ACs, one of which (AC-D) was used in Exp. 1, to alleviate toxicosis in growing turkey poults when included in a diet containing AF. Because of the possibility that AC from different manufacturers might have different binding capacities. the ACs used in this experiment were purchased from two different sources. One-hundred eighty day-old female turkey poults (Nicholas large whites) were purchased from a commercial hatchery, individually weighed, wingbanded, and houlsed in heated batteries under continuous fluorescent lighting. Poults were fed a CTF starter ration and randomly assigned to the following treatment groups: (1) basal diet only (control); (2) 0.5% (w/w) charcoal S (AC-S); (3) 0.5% (w/w) charcoal D (AC-D); (4) 0.75 mg AF/ kg of diet; (5) 0.5% (w/w) AC-S + 0.75 mg AFjkg of diet; (6) 0.5% (w/w) AC-D + 0.75 mg AFjkg of diet. Each treatment consisted of six pens of five birds/pen. Feed and water were provided for ad libitum consumption. Feed consumption and BW were recorded weekly and pen feed conversion calculated. At 21 days of age, the study was terminated and 18 birds (six replicates of three each) from each treatment group were bled by cardiac puncture for serum biochemical analysis

117

and hematological determinations (six replicates of two each, 12 birds/treatment). After bleeding, birds were killed by cervical dislocation and the liver, left kidney, spleen, pancreas, proventriculus, gizzard, heart, and bursa of Fabricus were removed, weighed, and calculated as relative organ weights (g/l00 g BW). Aflatoxin was produced via fermentation of rice by Aspergillus parasiticus NRRL 2999 [22,23]. Fermented rice was autoclaved and ground and the AF content measured by spectrophotometric analysis [24]. Of the total AF content in the rice powder, 79% was AFB,, 16% was AFG,, 4% was AFB,, and 1% was AFG,. The rice powder was incorporated into the basal diet and confirmed by HPLC to provide the desired level of 0.75 mg AFjkg of diet. Charcoal S (activated charcoal, untreated powder, 100-400 mesh) was purchased from Sigma (St. Louis, MO). 2.3. Sample preparation and analysis A modified affinity chromatography purification procedure was used [25]. Briefly, phosphate buffered saline (PBS) was added at the top of an aflatoxin monoclonal antibody immunoaffinity column (VICAM, Watertown, MA) and was allowed to flow until the meniscus reached the top of the resin. Urine (1 ml) was added and after elution by gravity, the affinity column was washed with 5 ml of PBS. Aflatoxins were eluted from the column with 1 ml of 50% (v/v) dimethylsulfoxide in PBS solution and the eluates analyzed by HPLC as described by Groopman et al. [26]. Spectral grade solvents (Burdick and Jackson, Muskegon, MI) were used. Because urine volumes and AF concentrations were highly variable, data are presented as total AF (ng) excreted during each sampling time as opposed to AF concentration (ng/ml). In Exp. 1, data were subjected to ANOVA and if the F-test was significant (P < 0.05) means were separated by Bonferroni tstatistics. All procedures were computed using the GLM procedure of SAS [27]. In Exp. 2, data (pen means) were subjected to ANOVA [28] using the GLM procedure in the PC-SAS version 6.02 statistical software [27]. Variable means for treatments showing significant differences in the

T.S. Edrington et al. / Toxicology Letters 89 (1996) 115-122

118 600 T

500 f

;~

400 1

‘;

4\

‘*, ‘b

2

+ ‘.\

..

-f-

ACIDIC HSCAS

.._q.‘.

CHARCOAL

./+_

\ 200 1

HSCAS

-&--

‘\ G ; 300 +j:

- - - CONTROL

-.._. \\

-..

-.

+-.

--.___

__

..x_ .-.

10

15

20

25

30

35

40

45

50

Time (h)

Fig. I. Urinary excretion of AFM, (ng) in female turkey poults dosed with 0.75 mg AFB,/kg BW alone or in combination with HSCAS, charcoal D, and acidic HSCAS (Exp. 1).

ANOVA were compared using the Fisher’s protected least significant difference procedure [28]. All statements of significance are based on the 0.05 level of probability.

3. Results Aflatoxin M, was found to be the major metabolite in all treatment groups of this study and in rat urine of a previous study [29], therefore, AFM, was the biomarker used in the present study. The CTF was negative for AF and AFM, was not detected in the urine of the negative AFB, control turkeys (no AFB, or adsorbent).

undetectable in AFB,-positive control birds and were very low (21, 17, and 13 ng) in HSCAS, acidic HSCAS, and AC-D treated birds, respectively. The percentage reduction was calculated as the ratio of the difference of AFM, output in AFB,-control and treated groups over the AFM, output in AFB,-control birds; however, because total urine output was not collected, the calculation represents only the reduction seen at each sampling time, independent of previous AFM, output. All three adsorbent compounds lowered AFM, output and the greatest reduction was seen in the AC-D (72%) and acidic HSCAS (71%) treatments. Percentage reduction in the HSCAS treatment ranged from 29-80%, and averaged 52% over the entire experimental period.

3.1. Experiment 1 3.2. Experiment 2 Aflatoxin M, output over a 48 h sampling period is presented in Fig. 1. All three sorbents (HSCAS, acidic HSCAS and AC-D) significantly lowered the output of AFM, in urine at each sampling period; AC-D and the acidic HSCAS produced the greatest decrease in bioavailability. Aflatoxin M, output decreased over the 48-h sampling period in treated birds when compared with AFB,-positive control. The highest AFM, output was observed for all treatments during the first collection period (O-2 h). Quantities of AFM, dropped considerably by 8 h and by 48 h were

The effects of AF on performance and relative organ weights of poults fed the two ACs and AF are presented in Table 1. Body weights on day 1 were not different among treatment groups, however BW was markedly decreased for 3 week-old birds fed AF. No benefits in weight gain were observed when ACs were added to control diets, nor were the ACs effective in alleviating the BW loss induced by AF when added to AF-contaminated diets. Feed intake over the 3-week experimental period was not affected by charcoal

T.S. Edrington et al. / Toxicology Letters 89 (1996) 115-122

119

Table 1 Effects of 0.5% activated charcoal and 0.75 mg AFjkg of diet on body weights, feed conversion, and relative organ weights of growing turkey poults ~(Exp.2) Treatment* Item

Conrrol

Charcoal-S

Charcoal-D

AF

Charcoal-S + AF

Charcoal-D

LSD

+AF Body weight (g) Day 1 Day 21 Total gain Change from control (%)

58 :h 0.4” 541 :h 11.0” 483 :h 11.3” 0

58 + 0.2” 520 k 10.4” 462 k 10.3” -4

58 f 0.2” 544* 11.1” 486k 11.0” 0

58 + 0.3” 349 + 10.7b 291 + 10.6b -35

51+ 0.5” 344* 11.7b 287 + 12.0b -36

57 * 0.5” 356 i 9.8b 299 + 9.7b -34

Feed: gain (g/g)

1.45 + 0.07”

1.30 + 0.02”

1.38 f 0.09”

1.38 + 0.04”

1.38 f 0.03”

1.38 f 0.07”

2.48 + 0.45 f 0.11 + 0.39 f. 3.16 k 0.22 *

2.48 + 0.44 f 0.10 + 0.36 + 3.14 f 0.22 f

1.93 & 0.06b 0.62 k 0.02” 0.12 + 0.009a,b 0.51 + 0.02” 3.42 k 0.13” 0.26 f 0.013”

1.80 + 0.59 f 0.13 + 0.51 + 3.36 k 0.24 +

Organ weight (g/100 g BW) Liver 2.42 f 0.05” Kidney 0.45 f 0.02b Spleen 0.10~0.004’ Pancreas 0.35 + 0.02b Gizzard 3.08 k 0.07d Bursa of 0.22 * 0.004’ Fabricus

0.06” O.Olb 0.005b~C 0.02b 0.07b,c,d 0.010’

0.06” O.Olb 0.004b*C 0.01 b O.lOc,d 0.006”

O&lb 1.88 + 0.01” 0.57 f 0.005a 0.11 f 0.02” 0.55 f 0.04”,b,c 3.41 + O.O1O”*b*c0.25 +

0.06b 0.02” 0.005a*b 0.02” O.lOa,b 0.017”,b

1.1 31.2 31.2

0.17 0.165 0.047 0.016 0.05 0.26 0.032

*Values shown are means + S.E.M. LSD, least significant difference. a,b,c,dMeans within rows with the same superscript are not significantly different (P
treatment, but was decreased by the addition of AF to the diet (data not shown). Efficiency of feed utilization was not different among treatments. Treatment with AF (with and without AC) decreased relative l.iver and increased relative kidney weights compared with control and charcoaltreated birds. Relative pancreas, gizzard, spleen and bursa of Fabricus weights were increased by dietary AF. With the exception of marginal protection of bursal weight by AC-S, the ACs did not protect against organ weight changes induced by AF. No differences were noted in proventriculus or heart weight (d,ata not shown). The effects of dietary treatments on selected serum constituents are presented in Table 2. Serum glucose was decreased by AF fed alone or by AF + AC-D, b-ut was not different from control when AC-S was fed with AF. All diets containing AF decreased serum triglyceride, cholesterol and total protein concentrations when compared with controls. Blood urea nitrogen was elevated in turkeys fed the three AF diets, while AF alone and AF + AC-D significantly decreased

serum calcium when compared with controls. Serum calcium of birds fed AF + AC-S was intermediate between the values of AF alone and control. Increased serum enzyme activities were observed for lactic dehydrogenase (LDH), and creatine kinase (CK) in all birds fed dietary AF, with or without AC. Aspartate aminotransferase activities were elevated in birds fed AF + AC-S compared with controls. No differences were observed for serum activities of alanine aminotransferase or alkaline phosphatase, or for concentrations of albumin, uric acid, creatinine, inorganic phosphorus, or for hematologic measurements (data not shown).

4. Discussion Results from recent research demonstrated the ability of HSCAS to selectively chemisorb AF in vitro, diminish the normal uptake of AF in the blood and the subsequent distribution to target organs, prevent aflatoxicosis in chickens, turkeys,

120

T.S. Edrington

Table 2 Effects of 0.5% activated poults (Exp. 2)

charcoal

et al. 1 Toxicology

and 0.75 mg AFjkg

Letters

89 (1996) 115-122

of diet on selected serum constituents

and enzyme activities

in growing

turkey

Treatment* Item

Control

Charcoal-S

Charcoal-D

AF

Charcoal-S +AF

Serum constituent Glucose (mg/

300 & 4.4”

305 * 4.8”

294 + 9.7”

249 k 8.6’

dl) Triglycerides

Charcoal-D

LSD

+AF

281 -+ 7.0”.”

263 -+ 16. I ‘J

26.9 18.3

75 * 5.8”

88 k 5.4”

72 k 7.0”

26 * 3.9b

37 & 4.7b

41 * 9.5b

(mg/dl) Total protein

2.50 k 0.12”

2.65 i 0.06”

2.73 + 0.63”

0.86 & 0.07b

I .24 k 0.08b

1.24 + 0.21b

0.81

(g/dl) Urea nitrogen

I .18 f 0.03’

I.21 f 0.08’

1.37 _+ O.lOb~’

1.81 + 0.08”

1.76+0.15”

1.59 _ + 0.09”.b

0.28

(mgidl) Cholesterol

I21 k 4.4”

(mgidl) Calcium

15.0 * 1.12”

(mgi

I08 f 8.3”

43 * 3.1’

59 _ + 3.4b.’

60 k 8.4b

17.0

13.3 -+ 0.67”,’

10.0 + 1.14’

2.3

dl) Serum enzymes AST LDH CK

(tU/L)d I86 + 3.O”. 562 : 45b 578 k 70b

175 f 2.6’ 529 + 20b 477 k 87b

I78 + 5.3’ 592 + 70b 507 * 7Sb

197 _+ 12.7a,b.c 930 + 75” 1067 + 109”

219+ 11.6” 922 + 71” 1033 + I II”

210 + 16.8”.b 901; 40” lll7k 132”

29.4 163 290

*Values are shown as mean i: S.E.M. LSD, least significant difference. “.b.‘Means within rows with the same superscript are not significantly different (PcO.05) dAST, aspartate aminotransferase; LDH, lactic dehydrogenase; CK, creatine kinase.

swine, and sheep, and decrease the level of AFM, in the milk of lactating dairy cattle [30]. Further research suggested that the mechanism for this action may be related to chelation via the B-dicarbony1 group of AF and trace metal sites on HSCAS 131,321. In the present study (Exp. l), HSCAS and acidic HSCAS were effective in binding AFB, and reducing its bioavailability in turkey poults as evidenced by a reduction in urinary AFM, output. These findings are in agreement with a preceding rodent study in which HSCAS significantly decreased AFM, urinary output in rats dosed with AFB, and HSCAS [29]. In that study, AFM, was the primary AFB, metabolite in urine, the AFB,-complex was not significantly dissociated in vivo, and importantly, no additional AFB, metabolites were found. Reduction of urinary AFM, associated with AC may be attributed partly to the in vitro constraints of our dosing model, in which AF and AC were combined prior to administration to poults. Also,

the higher surface densities and surface areas associated with AC versus HSCAS may have played a role in the increased binding by AC that was observed, and AFB, dissolved in methanol would be more evenly disributed and have more frequent access to AC binding sites. In an earlier study in vivo, Kubena et al. [17] demonstrated that AC-S did not protect growing chicks against the effects of aflatoxins. In the present study (Exp. 2), we confirmed this finding in turkey poults and also demonstrated that neither form of AC (S or D) provided any protection. The discrepency between binding of AF by AC (Exp. 1 vs. Exp. 2) may be due to the nonspecific nature of adsorption by AC. In other words, adsorption sites on AC may be saturated (and inactivated) by components in animal feed, preventing significant binding of AF in the GI tract. Reduction of urinary AFM, associated with acidic HSCAS (Exp. 1) may have resulted from AF interaction with acid functional groups and

T.S. Edrington et al. 1 Toxicology

binding at surface sites on acidic HSCAS particles. These conclusions are supported by previous studies in vitro showing the importance of clay surface density and pH on the adsorption of mycotoxins possessing ketoenol functional groups [33]. The toxicity of AF in poultry has been welldocumented [1,14.,16-19,34-371. In Exp. 2, inclusion of 0.75 mg AFjkg of diet significantly decreased feed intake and BW gain, disrupted protein metabolism, and caused liver and kidney damage, as evidenced by increased or decreased organ weights and altered serum activities of LDH and CK and altered concentrations of calcium, cholesterol, glucose, triglycerides, total protein and urea nitrogen. The efficacy of AC as an oral antidote in the treatment of poisoning is well recognized. Charcoals act by binding chlemicals in the gastrointestinal tract, thereby decreasing their absorption. However, the addition of AC in Exp. 2 showed no beneficial effects in alleviating aflatoxicosis as reflected by BW gain and organ weights. With few exceptions, AC had no effect on selected serum constituents. Serum glucose concentrations were improved to control levels in birds fed AF + AC-S; however, this apparent benefit due to AC is probably inconsequential when the overall performance and health of the birds are examined. There are reports in the literature that AC in the diet of growing chickens can improve feed intake, BW gain, and other toxic responses associated with a&toxicosis [35-371. Our findings, or those of Kubena et al. [17] do not support these conclusions. Addition of two types of AC did not appear to have any beneficial effects against aflatoxicosis in growing turkey poults. However, earlier studies [35,37] and in Exp. 1 of our study, pure AFB, was used. In Exp. 2 of our study, we utilized a crude mixture of four naturally-produced aflatoxins (i.e. B,, G,, B, and GJ. It is possible that the crude m:ixture of aflatoxins may have competed for adsorption sites on charcoal, thus decreasing its abiility to bind aflatoxin B, and prevent toxicity. Further studies are warranted to delineate this effect.

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89 (1996) 115-122

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5. Conclusions Methods to detoxify mycotoxin-containing feedstuffs on a large scale and in a cost-effective manner are not currently available. Results from this study support earlier findings that certain inorganic, non-nutritive adsorbents can effectively bind AFB,. Two HSCAS compounds and AC, administered concomitantly with AFB,, were shown to adsorb AFB,, thereby limiting AFB, bioavailability and reducing urinary ‘excretion of AFM, in turkey poults. Activated charcoal reduced AFMl output in Exp. 1, but failed to alleviate toxicosis when included in an AF-contaminated diet fed to growing turkey poults (Exp. 2). These findings suggest that adsorption by AC may occur via nonspecific binding of AFB, and may be altered in the presence of competing ligands.

References

PI Cheeke, P.R. and Shull, L.R. (1985) Natural Toxicants in Feeds and Poisonous Plants. AVI Publishing, Westport, CT, pp. 393-477. PI Enomoto, M. and Saito, M. (1972) Carcinogens produced by fungi. Annu. Rev. Microbial. 26, 279-312. 131Ciegler, A. (1975) Mycotoxins: occurrence, chemistry, biological activity. Lloydia 38, 21-35. [41Patterson, D.S.P. (1976) Structure, metabolism and toxicity of the aflatoxins [Review]. Can. Nutr. Diet 2, 71-76. [51Patterson, D.S.P., Glancy, E.M. and Roberts, B.A. (1980) The ‘carry-over’ of aflatoxin M, into the milk of cows fed rations containing a low concentration of aflatoxin B,. Food Cosmet. Toxicol. 18, 35-37. Fl Loury, D.N. and Hsieh, D.P.H. (1984) Effects of chronic exposure to aflatoxin B, and aflatoxin M, on the in vivo covalent binding of aflatoxin B, to hepatic macromolecules. J. Toxicol. Environ. Health 13, 575-587. [71Wong, J.J. and Hsieh, D.P.H. (1976) Mutagenicity of aflatoxins related to their metabolism and carcinogenic potential. Proc. Natl. Acad. Sci. USA 73, 2241-2244. PI Edrington, T.S., Harvey, R.B. and Kubena, L.F. (1994) Effect of aflatoxin in growing lambs fed ruminally degradable or escape protein sources. J. Anim. Sci. 72, 1274-1281. [91Coffey, M.T., Hagler, Jr., W.M. and Cullen, J.M. (1989) Influence of dietary protein, fat and amino acids on the response of weanling swine to aflatoxin B,. J. Anim. Sci. 67, 465-472. 1101Smith, J.W., Hill, C.H. and Hamilton, P.B. (1971) The effect of dietary modifications on aflatoxicosis in the broiler chicken. Poult. Sci. 50, 768-774.

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[I I] Schell, T.C., Lindemann, M.D., Kornegay, E.T. and Blodgett, D.J. (1993). Effects of feeding aflatoxin-contaminated diets with and without clay to weanling and growing pigs on performance, liver function, and mineral metabolism. J. Anim. Sci. 71, 120991218. [12] Schell, T.C., Lindemann, M.D., Kornegay, E.T., Blodgett, D.J. and Doerr, J.A. (1993) Effectiveness of different types of clay for reducing the detrimental effects of aflatoxincontaminated diets on performance and serum profiles of weanling pigs. J. Anim. Sci. 71, 122661231. [13] Phillips, T.D. (1987) Novel approaches to detection and detoxification of mycotoxins. In: Recent Developments in the Study of Mycotoxins, Kaiser Chemicals, Cleveland, OH, pp. C3-Cll. [14] Phillips, T.D., Kubena, L.F., Harvey, R.B., Taylor, D.R. and Heidelbaugh, N.D. (1988) Hydrated sodium calcium aluminosilicate: a high affinity sorbent for aflatoxin. Poult. Sci. 61, 243-241. [IS] Harvey, R.B., Kubena, L.F., Phillips, T.D., Corrier, D.E., Ehssalde, M.H. and Huff, W.E. (1991) Diminution of aflatoxin toxicity to growing lambs by dietary supplementation with hydrated sodium calcium aluminosilicate. Am. J. Vet. Res. 52, 1522156. [16] Kubena, L.F., Harvey, R.B., Huff, W.E., Elissalde, M.H., Yersin, A.G., Phillips, T.D. and Rottinghaus, G.E. (1993) Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and diacetoxyscirpenol. Poult. Sci. 72, 51-59. [17] Kubena, L.F., Harvey, R.B., Phillips, T.D., Corrier, D.E. and Huff, W.E. (1990) Diminution of aflatoxicosis in growing chickens by the dietary addition of a hydrated, sodium calcium aluminosilicate. Poult. Sci. 69, 727135. [18] Kubena, L.F., Harvey, R.B., Huff, W.E., Corrier, D.E., Phillips, T.D. and Rottinghause, G.E. (1990) Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and T-2 toxin. Poult. Sci. 69, lO781086. [19] Kubena, L.F., Huff, W.E., Harvey, R.B., Yersin, A.G., Elissalde, M.H., Witzel, D.A., Giroir, L.E., Phillips, T.D. and Peterson, H.D. (1991) Effects of a hydrated sodium calcium aluminosilicate on growing turkey poults during aflatoxicosis. Poult. Sci. 70, 1823-1830. [20] Harvey, R.B., Kubena, L.F., Phillips, T.D., Huff, W.E. and Corrier, D.E. (1989) Prevention of aflatoxicosis by addition of hydrated sodium calcium aluminosilicate to the diets of growing barrows. Am. J. Vet. Res. 50, 416420. [21] Harvey, R.B., Phillips, T.D., Ellis, J.A., Kubena, L.F., Huff, W.E. and Peterson, H.D. (1991) Effects on aflatoxin M, residues in milk by addition of hydrated sodium calcium aluminosilicate to aflatoxin-contaminated diets of dairy cows. Am. J. Vet. Res. 52, 1556-1559. [22] Shotwell, O.L., Hesseltine, C.W., Stubblefield, R.D. and Sorenson, W.G. (1966) Production of aflatoxin on rice. Appl. Microbial. 14, 425-428. [23] West, S., Wyatt, R.D. and Hamilton, P.B. (1973) In-

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[24]

[25]

[26]

[27] [28]

[29]

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