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Bull Vet Inst Pulawy 49, 93-96, 2005

EFFECTS OF DIETARY AFLATOXIN AND SODIUM

BENTONITE ON SOME HORMONES IN BROILER CHICKENS

GOKHAN ERASLAN1, DINC ESSIZ2, MEHMET AKDOGAN3, FATMA SAHINDOKUYUCU4,LEVENT ALTINTAS5 AND SAHVER EGE HISMIOGULLARI6

1Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,Erciyes University, Kayseri 38080, Turkey

2Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,Kafkas University, Kars 36100, Turkey

3Faculty of Medicine, Department of Biochemistry,Suleyman Demirel University, Isparta 32260, Turkey

4Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,

Akdeniz University, Burdur 15100, Turkey5Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,Ankara University, Ankara 06110, Turkey

6Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,Mustafa Kemal University, Hatay 31040, Turkey

e-mail: geraslan38@hotmail.com

Received for publication June 06, 2004.

Abstract

In this study, 72 one-day-old, male, Avian race, broiler chicks were used. The birds were divided into 6 equalgroups. While the first group was kept as a control, groups 2,3, 4, 5 and 6 received for 45 d the feed containing 0.25% of sodium bentonite, 0.5% of sodium bentonite, 1 ppm of aflatoxin (AF) (approximately, 85% B1, 10% B2, 3% G1 and2% G2), 0.25% of sodium bentonite with 1 ppm of AF, and0.5% of sodium bentonite with 1 ppm of AF, respectively. Atthe end of the experiment, the animals were sacrificed, bloodwas collected and plasma was separated. Plasmatriiodothyronine (T3), thyroxine (T4), thyroid-stimulatinghormone (TSH), testosterone, and growth hormone (GH)levels were measured. A significant decrease was detected in plasma T3 and T4 levels in groups 2, 3, 4, 5 and 6, and asignificant increase in plasma testosterone level in groups 2, 4,5 and 6, compared to the control group. FA and sodium bentonite, which were given alone, were found to be effectiveon T3, T4, and testosterone contents. In the case of their combination, there was no certain evidence that the adsorbentameliorated the effects of AF. Therefore these parameterscould not be used inin vivo studies of binding the FA withsodium bentonite in broiler chickens.

Key words : broiler chickens, aflatoxin,sodium bentonite, hormones.

Aflatoxins (AFs) are poisonous compounds

synthesised by genus of Aspergillus fungi (2, 11). Thespecies of the fungi, frequently isolated from the feedand feedstuffs, are Aspergillus flavus and Aspergillus

parasiticus (7). These species are also called the

toxigenic fungi (19). In the presence of optimalconditions regarding temperature, humidity, O2 and CO2

in the feed, the fungi rapidly produce toxins andcontaminate the feed (3, 15). The ingestion of such feed by poultry causes many adverse effects such as decreasein body weight gain, decrease in feed consumption andfeed conversion rate, and damage of organs (8-10, 16,18, 23, 26, 29). In order to prevent such negative effects,there has recently been a common practice of addingadsorbents into animal feed. Moreover, thesecompounds may be added into animal feed at certainrates and given to animals for protective purposes (12,16, 18). The basic quality of these compounds is thehigh binding capacity due to their physico-chemicalstructures and irreversible binding properties (5, 31). On

the other hand, certain amounts of AFs may be absorbedfrom the digestive tract because of incomplete binding(5). Sodium bentonite is one of the aluminosilicatecompounds, used as adsorbent (29). Higher bindingefficacy of this compound makes it the most preferredone, compared to others (5).

The objective of this study was to determine theeffects of AF and bentonite, administered separately or in combination, on thyroid hormones, testosterone, andgrowth hormone. There have been few studies about thetopic. However, we could not come across any study,concerning the effect of sodium bentonite given alone or in combination with FA. The obtained results will be a

guide for in vivo binding efficacy trials with thiscompound in broiler chickens. It will be seen if these parameters could be used or not as references in thechickens.

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Material and Methods

In this study, 72 one-day-old, male, Avian racechicks were used. The animals were weighed anddivided into 6 groups with 12 animals in each byconsidering a homogenous weight distribution. A 24 h

lighting system was applied and the animals had anaccess to feed and water ad libitum. While the first group was fed the broiler ration,

the groups 2, 3, 4, 5 and 6 were fed the ration containing0.25% of sodium bentonite, 0.5% of sodium bentonite, 1 ppm of AF, 0.25% of sodium bentonite with 1 ppm of AF, 0.5% of sodium bentonite with 1 ppm of AF,respectively, for 45 d. At the end of the experiment, theanimals were sacrificed and their plasma levels of triiodothyronine (T3), thyroxine (T4), thyroid-stimulatinghormone (TSH), testosterone and growth hormone (GH)were determined. The analyses of hormones were done by electrochemiluminescense techniques, which were

performed on Elecsys 2010 immunoassay analyser withBoehringer Manheim hormone kit. The method of Demet et al. was used in AF production (4), which is based on report of Shotwellet al. (32). The strain of Aspergillus parasiticus (NRLL 2999) was used to produce AF (4). Total AF levels in rice were detected byELISA with Ridascreen kit and its method. AF contentin rice was determined by Nabney and Nesbits method

(20). Subsequently, 85% B1, 10% B2, 3% G1 and 2% G2 were detected in rice flour, in average.

The data were evaluated as arithmetic means andstandard deviation. A one-way-variance analysis wasused and the significant differences between groupswere determined by Duncan test. The statistical analysis

was done on the SPSS 10.0 for Windows package program.

Results

The groups 2-6 which received sodium bentonite and AF in combination or alone displayed thedecreases in the levels of T3, and T4, and increases inGH and testosterone contents. The changes in TSH leveloccurred as either an increase or a decrease. Thesignificant decreases were found in plasma T3 and T4 levels in groups 2, 3, 4, 5 and 6, compared to the control

chickens (P

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On the other hand, the other mechanismconnected with the decreases of both hormone levelsmay be related to the changes which probably developduring the transport of these hormones in the bloodstream. An example for that is a significantreduction of plasma protein content by AF. (13). This

decrease may trigger another decrease concerning therates of protein-bounded T3 and T4, therefore, may leadto increased free hormone levels in the blood. Freehormones (not bounded by plasma proteins) diffusequickly into tissues and are rapidly eliminated by theliver as soon as they come (28). In both cases, the plasma levels of the mentioned hormones may decline.This may be primary reason for the decrease which wasmuch more higher in the group that received only AF.Yet, the decrease did not occur rapidly in the groupswhich received sodium bentonite only (groups 2 and 3)and in the groups which received sodium bentonite as acombination with AF. The absence of statistically

significant difference among three groups proved thatthis effect is not the primary mechanism for the decreasein the hormone levels.

Another mechanism is the change in themetabolic rate of the hormones in tissues. T4 isconverted into T3 via 5-deiodinase in tissues (1). By thisconversion, T4 level progressively decreases, incontrary, T3 level increases in bloodstream. In fact, mostof T3 in blood is formed as a consequence of thisconversion (1, 21, 22). The decreases in T3 and T4 levelsin all trial groups, renders the suspected hypothesisconcerning the changes in tissue-enzyme level,compared to the control group. If both compounds

which are added into feed alone or combined acceleratethe conversion rate of these hormones (this may result inthe increase in 5-deiodinase activities, involved in thisconversion), there should be a decrease in blood T4 level(it was detected in the present study). In contrary, the T3 level should display an increase. However, this increasewas not seen in the present study. On the other hand,regarding the decrease in the mentioned enzymeactivity, there should be an increase in T4 and a decreasein T3 levels. The results showed that none of themhappened. Consequently, it could be suggested thatneither of the components had a direct effect on theconversion of the hormones which takes place in thetissues (conversion of T4 into T3).Moreover, parallel to changes concerning T3-T4 hormones, no significant difference was observed in plasma levels of TSH. In fact, the decrease in plasmalevels of T3 and T4 also causes an increase in thesynthesis and release of TSH (22, 30). However, theresults of the present study did not confirm that (nostatistically significant difference was found between thegroups). The emphasised reason may be related to T3-sensitive nuclear receptors in the group which receivedAF, because any situation may inhibit the stimulation of T3-sensitive nuclear receptors in tissues and the thyroidgland, subsequently, disrupts TSH secretion by theadenohypophysis. AFs are known to cause lipid peroxidation (10, 27). Essentially, intermediate and end products harmful for the cell membrane and other intracellular organelles may be formed following this

reaction. If this reaction is severe enough to be notcompensated by intracellular defense mechanism, it mayresult in damage to the cell and, unfortunately, lead to itsdeath. This is the effect, which may reduce thesensitivity of the above-mentioned receptors of T3.Structural alterations in the receptor, which originates

from this peroxidation, may be responsible for this pathway. It is not possible to suggest the same situationfor the groups which received only sodium bentonite inthe feed. Although adsorbent significantly reduced theabsorption of vitamin E in the digestive tract (25), thereis no evidence to suggest that it causes the peroxidationand cellular damage directly.

LH-RH and LH, which are secreted by thehypothalamus and pituitary gland, respectively, controlthe synthesis of testosterone and its levels in blood.Testosterone is synthesized in the Leydigs cells fromcholesteryl esters and cholesterol. Cholesterol isconverted into pregnanolon by protein kinases, finally,

other microsomal enzymes and various intermediatereactions contribute to it in order to transformtestosterone (22). Therefore, the increases in enzymeactivity and in the level of the primary products(cholesteryl esters, cholesterol) may also indirectlychange the level of this hormone in blood. However, thechange is also necessary concerning the other mechanisms (LH-RH, LH) which control its synthesisand release, otherwise, this change could not take place.An increase in testosterone level was detected in thestudy. This increase was observed in all trial groups. Itwas reported that AF reduced testosterone level bydamaging testicular cells (24). Intriguingly, a decrease,

not an increase, was observed in our study. Thisincrease, although not definite, may be originated fromthe possible increase of protein kinase activity caused byAF. These changes were observed in the animals whichreceived sodium bentonite only. The reason for thatcould not be clarified. The increase was even muchmore distinctive in the group 6 which receivedcombination of both compounds.

The growth hormone is effective in increasingthe protein synthesis (22). In the present study, nosignificant difference was found, regarding the growthhormone in all groups, compared to control. This clearly proved that AF given separately or in combination withsodium bentonite did not make either a positive or negative effect on the mentioned hormone.

In conclusion, these hormones should not betaken into consideration as parameters which are used todetect the efficacy of such AF binding agents as sodium bentonite in in vivo trials on broiler chicks. Another conclusion is that the binding agents should not beadded into animal feed in arbitrary intention. Again, thelack of similar studies which are related to this topic,does not allow to evaluate comparatively of the obtaineddata. However, the present study may serve as areference for future investigations. Nevertheless, theobtained results revealed that many detailedinvestigations have to be done to clarify the effect of both studied compounds on these hormones as well astheir mechanisms in broiler chickens.

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