Download effects of dietary aflatoxin and sodium bentonite on some hormones

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
1
Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,
Erciyes University, Kayseri 38080, Turkey
2
Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,
Kafkas University, Kars 36100, Turkey
3
Faculty of Medicine, Department of Biochemistry,
Suleyman Demirel University, Isparta 32260, Turkey
4
Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,
Akdeniz University, Burdur 15100, Turkey
5
Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,
Ankara University, Ankara 06110, Turkey
6
Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology,
Mustafa Kemal University, Hatay 31040, Turkey
e-mail: [email protected]
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 equal
groups. 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 and
2% G2), 0.25% of sodium bentonite with 1 ppm of AF, and
0.5% of sodium bentonite with 1 ppm of AF, respectively. At
the end of the experiment, the animals were sacrificed, blood
was collected and plasma was separated. Plasma
triiodothyronine (T3), thyroxine (T4), thyroid-stimulating
hormone (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 a
significant 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 effective
on T3, T4, and testosterone contents. In the case of their
combination, there was no certain evidence that the adsorbent
ameliorated the effects of AF. Therefore these parameters
could not be used in in vivo studies of binding the FA with
sodium 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). The
species of the fungi, frequently isolated from the feed
and feedstuffs, are Aspergillus flavus and Aspergillus
parasiticus (7). These species are also called the
toxigenic fungi (19). In the presence of optimal
conditions regarding temperature, humidity, O2 and CO2
in the feed, the fungi rapidly produce toxins and
contaminate the feed (3, 15). The ingestion of such feed
by poultry causes many adverse effects such as decrease
in body weight gain, decrease in feed consumption and
feed 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 adding
adsorbents into animal feed. Moreover, these
compounds may be added into animal feed at certain
rates and given to animals for protective purposes (12,
16, 18). The basic quality of these compounds is the
high binding capacity due to their physico-chemical
structures and irreversible binding properties (5, 31). On
the other hand, certain amounts of AFs may be absorbed
from the digestive tract because of incomplete binding
(5). Sodium bentonite is one of the aluminosilicate
compounds, used as adsorbent (29). Higher binding
efficacy of this compound makes it the most preferred
one, compared to others (5).
The objective of this study was to determine the
effects of AF and bentonite, administered separately or
in combination, on thyroid hormones, testosterone, and
growth hormone. There have been few studies about the
topic. 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 this
compound in broiler chickens. It will be seen if these
parameters could be used or not as references in the
chickens.
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Material and Methods
In this study, 72 one-day-old, male, Avian race
chicks were used. The animals were weighed and
divided into 6 groups with 12 animals in each by
considering a homogenous weight distribution. A 24 h
lighting system was applied and the animals had an
access 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 containing
0.25% 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, the
animals were sacrificed and their plasma levels of
triiodothyronine (T3), thyroxine (T4), thyroid-stimulating
hormone (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 with
Boehringer Manheim hormone kit. The method of
Demet et al. was used in AF production (4), which is
based on report of Shotwell et al. (32). The strain of
Aspergillus parasiticus (NRLL 2999) was used to
produce AF (4). Total AF levels in rice were detected by
ELISA with Ridascreen® kit and its method. AF content
in rice was determined by Nabney and Nesbit’s 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 and
standard deviation. A one-way-variance analysis was
used and the significant differences between groups
were 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 the
decreases in the levels of T3, and T4, and increases in
GH and testosterone contents. The changes in TSH level
occurred as either an increase or a decrease. The
significant decreases were found in plasma T3 and T4
levels in groups 2, 3, 4, 5 and 6, compared to the control
chickens (P<0.05). The significant increases were
observed in plasma testosterone level in groups 2, 4, 5
and 6, compared to the control group (P<0.05). No
significant difference was found between the groups
concerning TSH and GH concentrations (P>0.05) (Table
1).
Table 1
Plasma T3, T4, TSH, testosterone and GH levels in control and trial groups
Parameters
Groups*
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
a, b, c
T3
(ng/mL)
1.32±0.13c
1.19±0.16b
1.07±0.13ab
0.91±0.05a
1.05±0.07a
0.94±0.17a
T4
(µg/dL)
2.34±0.23c
2.12±0.31b
1.91±0.27ab
1.65±0.12a
1.91±0.17a
1.62±0.30a
TSH
(µIU/ml)
0.36±0.03
0.34±0.02
0.41±0.08
0.41±9.29
0.36±0.01
0.41±0.06
Testosterone
(ng/dL)
11.83±2.18a
25.33±4.98b
16.45±2.09ab
19.90±1.78b
22.62±2.56b
31.43±8.01c
GH
(ng/mL)
2.40±0.36
2.66±0.33
2.71±0.30
2.89±0.66
3.02±0.24
2.50±0.20
differences are statistically significant in groups marked with different letters in the same column (P<0.05)
*group 1 - control; group 2 - sodium bentonite (0.25%); group 3 - sodium bentonite (0.5%); group 4 - AF; group 5 - sodium
bentonite (0.25%) with AF; group 6 - sodium bentonite (0.5%) with AF
Discussion
In the present study, the statistically significant
decreases were observed in the levels of blood T3 and T4
in all trial groups compared to the control group. No
changes were detected in TSH levels. The mechanism
concerning the decreases in plasma T3 and T4 levels may
be associated with the decrease in blood iodine level. It
is essentially significant in the synthesis of these
hormones (1, 6, 17). Interestingly, the decreases in both
hormone levels were not only observed in the group
with received aflatoxin only, it was also observed in the
groups which received feed with sodium bentonite and
in the groups which received both compounds. It is
obvious that AF causes damage in the digestive system
(14). The damage which developed in the epithelium of
the digestive tract, may reduce the absorption of dietary
iodine from the digestive tract. Thus, iodine levels in the
bloodstream may decline, compared with its normal
limits. This may indirectly hinder the synthesis of T3 and
T4 in the thyroid gland. The reason for these declines in
the hormone levels in the groups which received
adsorbent material alone could be the irreversible
binding of iodine by bentonite, subsequently, its
disrupted absorption from the digestive tract. In fact,
sodium bentonite is a compound that possesses high
binding capacity (5).
95
On the other hand, the other mechanism
connected with the decreases of both hormone levels
may be related to the changes which probably develop
during the transport of these hormones in the
bloodstream. An example for that is a significant
reduction of plasma protein content by AF. (13). This
decrease may trigger another decrease concerning the
rates of protein-bounded T3 and T4, therefore, may lead
to increased free hormone levels in the blood. Free
hormones (not bounded by plasma proteins) diffuse
quickly into tissues and are rapidly eliminated by the
liver 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 was
much more higher in the group that received only AF.
Yet, the decrease did not occur rapidly in the groups
which received sodium bentonite only (groups 2 and 3)
and in the groups which received sodium bentonite as a
combination with AF. The absence of statistically
significant difference among three groups proved that
this effect is not the primary mechanism for the decrease
in the hormone levels.
Another mechanism is the change in the
metabolic rate of the hormones in tissues. T4 is
converted into T3 via 5-deiodinase in tissues (1). By this
conversion, T4 level progressively decreases, in
contrary, T3 level increases in bloodstream. In fact, most
of T3 in blood is formed as a consequence of this
conversion (1, 21, 22). The decreases in T3 and T4 levels
in all trial groups, renders the suspected hypothesis
concerning the changes in tissue-enzyme level,
compared to the control group. If both compounds
which are added into feed alone or combined accelerate
the conversion rate of these hormones (this may result in
the increase in 5-deiodinase activities, involved in this
conversion), 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 increase
was not seen in the present study. On the other hand,
regarding the decrease in the mentioned enzyme
activity, there should be an increase in T4 and a decrease
in T3 levels. The results showed that none of them
happened. Consequently, it could be suggested that
neither of the components had a direct effect on the
conversion of the hormones which takes place in the
tissues (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 plasma
levels of T3 and T4 also causes an increase in the
synthesis and release of TSH (22, 30). However, the
results of the present study did not confirm that (no
statistically significant difference was found between the
groups). The emphasised reason may be related to T3sensitive nuclear receptors in the group which received
AF, because any situation may inhibit the stimulation of
T3-sensitive nuclear receptors in tissues and the thyroid
gland, subsequently, disrupts TSH secretion by the
adenohypophysis. 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 not
compensated by intracellular defense mechanism, it may
result in damage to the cell and, unfortunately, lead to its
death. This is the effect, which may reduce the
sensitivity 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 situation
for the groups which received only sodium bentonite in
the feed. Although adsorbent significantly reduced the
absorption of vitamin E in the digestive tract (25), there
is no evidence to suggest that it causes the peroxidation
and cellular damage directly.
LH-RH and LH, which are secreted by the
hypothalamus and pituitary gland, respectively, control
the synthesis of testosterone and its levels in blood.
Testosterone is synthesized in the Leydig’s cells from
cholesteryl esters and cholesterol. Cholesterol is
converted into pregnanolon by protein kinases, finally,
other microsomal enzymes and various intermediate
reactions contribute to it in order to transform
testosterone (22). Therefore, the increases in enzyme
activity and in the level of the primary products
(cholesteryl esters, cholesterol) may also indirectly
change the level of this hormone in blood. However, the
change is also necessary concerning the other
mechanisms (LH-RH, LH) which control its synthesis
and release, otherwise, this change could not take place.
An increase in testosterone level was detected in the
study. This increase was observed in all trial groups. It
was reported that AF reduced testosterone level by
damaging testicular cells (24). Intriguingly, a decrease,
not an increase, was observed in our study. This
increase, although not definite, may be originated from
the possible increase of protein kinase activity caused by
AF. These changes were observed in the animals which
received sodium bentonite only. The reason for that
could not be clarified. The increase was even much
more distinctive in the group 6 which received
combination of both compounds.
The growth hormone is effective in increasing
the protein synthesis (22). In the present study, no
significant difference was found, regarding the growth
hormone in all groups, compared to control. This clearly
proved that AF given separately or in combination with
sodium bentonite did not make either a positive or
negative effect on the mentioned hormone.
In conclusion, these hormones should not be
taken into consideration as parameters which are used to
detect 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 be
added into animal feed in arbitrary intention. Again, the
lack of similar studies which are related to this topic,
does not allow to evaluate comparatively of the obtained
data. However, the present study may serve as a
reference for future investigations. Nevertheless, the
obtained results revealed that many detailed
investigations have to be done to clarify the effect of
both studied compounds on these hormones as well as
their mechanisms in broiler chickens.
96
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