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FARMACIA, 2013, Vol. 61, 1
51
EVALUATION OF THE POSSIBLE ENDOCRINE
DISRUPTIVE EFFECT OF BUTYLATED
HYDROXYANISOLE, BUTYLATED
HYDROXYTOLUENE AND PROPYL GALLATE
IN IMMATURE FEMALE RATS
ANCA POP1, CRISTIAN BERCE2, POMPEI BOLFA3*, ANDRAS
NAGY3, CORNEL CATOI3, ION-BOGDAN DUMITRESCU4,
LUMINITA SILAGHI-DUMITRESCU5, FELICIA LOGHIN1
1IuliuHaţieganu University of Medicine and Pharmacy, Faculty of
Pharmacy, Department of Toxicology, Cluj-Napoca, Romania
2University of Agricultural Sciences and Veterinary Medicine, Faculty of
Veterinary Medicine, Department of Animal Reproduction, Gynecology
and Obstetrics, Cluj-Napoca, Romania
3University of Agriculture Sciences and Veterinary Medicine, Faculty of
Veterinary Medicine, Department of Pathology, Cluj-Napoca, Romania
4Carol Davila University of Medicine and Pharmacy, Faculty of
Pharmacy, Department of Pharmaceutical Physics and Informatics,
Bucharest, Romania
5Babes-Bolyai University, Faculty of Chemistry and Chemical
Engineering, Department of Organic Chemistry, Cluj-Napoca, Romania
*corresponding author: pompei.bolfa@usamvcluj.ro
Abstract
We evaluated the possible endocrine disruptive effect of butylated
hydroxyanisole (BHA) (300mg/kg bw), butylated hydroxytoluene (BHT) (75mg/kg bw)
and propyl gallate (PG) (405mg/kg bw), compounds extensively used as antioxidants in
foods, food packaging, cosmetics and pharmaceuticals using the immature rat uterotrophic
assay. To investigate their estrogenic and/or antiestrogenic effect we used 17-21 days old
Wistar female rats that were given suspensions of the studied compounds for three
consecutive days. Absolute and relative uterus weights were significantly decreased by all
three compounds, while endometrial epithelium thickness was significantly affected only
by propyl gallate when compared to the negative control. The data obtained from the
present study suggests that BHA, BHT and PG indeed have endocrine disruptive effects on
Wistar prepubescent female rats, the most aggressive compound being PG.
Rezumat
S-a evaluat posibilul efect perturbator endocrin al butilhidroxianisolului
(300mg/kg corp), butilhidroxitoluenului (75mg/kg corp) şi propil galatului (405mg/kg
corp), compuşi folosiţi ca antioxidanţi în industria alimentară, a ambalajelor, cosmeticelor,
produselor farmaceutice, folosind testul uterotrofic la şobolani. Pentru a investiga efectele
estrogenice/antiestrogenice s-au folosit femele de şobolan Wistar de 17-21 de zile cărora li
s-au administrat suspensii ale compuşilor aflaţi în studiu timp de trei zile consecutive.
Greutăţile absolute şi relative ale uterului au scăzut semnificativ la toate loturile aflate în
studiu, iar grosimea epiteliului endometrului a scăzut semnificativ faţă de lotul control doar
FARMACIA, 2013, Vol. 61, 1
52
la animalele care au primit propil galat. Datele obţinute în acest studiu indică faptul că
BHA, BHT şi PG au efect perturbator endocrin la concentraţiile testate, efectul cel mai
pronunţat fiind în cazul propil galatului.
Keywords: BHA, BHT, PG, immature rat uterotrophic assay
Introduction
An endocrine disruptor is an exogenous substance or mixture that
alters the function(s) of the endocrine system and consequently causes
adverse health effects in an intact organism, or its progeny, or (sub)
populations. The family of endocrine disrupting chemicals (EDCs) includes
pharmaceutical estrogenic compounds, antioxidants, phytoestrogens,
pesticides, plastic manufacturing chemicals, detergents, heavy metals,
preservatives from cosmetics [2, 4-7, 14, 15, 25].
Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT)
and propyl gallate (PG) are extensively used as antioxidants in foods, food
packaging, cosmetics and pharmaceuticals. In the past years, all three
compounds raised concerns regarding their possible endocrine disrupting
effect. The existing in vitro studies indicate that BHA (E320) presents weak
estrogenic effect and also anti-androgenic properties. BHT (E321) was
proved to be less estrogenic than BHA, and based on the cell proliferation
assays it was included in the nonestrogenic chemicals list. After being tested
in vitro PG (E310) was found to be one of the strongest ligands among the
xenochemicals that are known as ERα binders, but without inducing any
transactivation activity at the concentrations tested. BHA was also found to
have antiestrogenic properties in one in vivo study. [1, 10, 12, 13,17, 18, 20,
24, 27, 29].
Studies that estimate the daily intake of BHA and BHT showed that
through an average diet the population can get close to the ADI (acceptable
daily intake) and that a fraction of the population might be exposed to doses
superior to ADI [11, 28].
The objective of this work was to assess the effects of BHA, BHT
and PG at concentrations higher than the average diet exposure on genital
female tract using the immature rat uterotrophic assay [8, 23].
Materials and Methods
Chemicals
BHA, BHT, PG, 17-β estradiol were purchased from Sigma-Aldrich
(USA), buffered formalin from Chempur (Poland).
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Animals and housing
17-21 days Wistar female rats were purchased from the Practical
Skills and Experimental Medicine Center of the ‘‘Iuliu Hatieganu’’
University of Medicine and Pharmacy, Romania. The rats were kept in
standard conditions of temperature, humidity, day/night cycle and they had
access to food and water ad libitum throughout the experiment. Body weight
and clinical signs were recorded daily.
Experimental protocol
The experimental protocol was in compliance with the institutional
and European guidelines for laboratory animal experiments, being approved
by the Ethics Committee of the University. The protocol included three
experimental groups and two control groups, each consisting of 10 animals.
Chemicals were administrated once per day for three consecutive days in the
morning between 9-10a.m. The doses studied represented three times
NOAEL (No Observable Adverse Effects Level) of each compound. The
tested compounds and the negative control (vehicle) were given as an oral
suspension at doses of 300 mg/kg bw for BHA, 75mg/kg bw for BHT and
405mg/kg bw for PG while 17-beta estradiol (positive control) was given by
subcutaneous injection into dorsal surface at a dose of 20µg/kg in sun flower
oil. The rats were weighed and sacrificed using diethyl ether at 24hrs after
the last treatment. Besides the genital tract, the liver, spleen and also
kidneys were removed and weighed.
Tissue collection
After removal, the genital tract (ovaries, oviduct, uterine horns,
body, cervix and vagina) was weighed and immediately immersed for
fixation in 10% buffered formalin for at least 24 hours. The sectioning and
trimming were performed in accordance with the goRENI standards [26].
Longitudinal sections through the uterus, the utero-cervical junction, cervix
and vagina (as a single unit) as well as transversal section through the
midhorn were prepared.
Tissues were embedded in paraffin according to standard
histological techniques [3].
Histopathological and morphometric assessment
Slides containing H&E (hematoxylin and eosin) stained tissue
samples were blindly evaluated by two pathologists for pathologic changes
using conventional light microscopy (Olympus BX 51 microscope equipped
with Olympus SP 350 digital camera). Pathological assessments were
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evaluated according to standardized National Toxicology Program (NTP)
pathology codes.
Morphometric analysis was performed on midhorn cross sections of
both uterine horns for all animals (n = 10 per treatment group) using
Olympus Stream Basic image analysis software. As previously described,
we quantified the length of basal lamina underlying the luminal epithelium
(LE) and corresponding areas of LE, stroma, and myometrium for multiple
representative sectors of each section [9, 16]. Total luminal and glandular
circumferences were also quantified.
Morphometric analysis was limited to LE cell height, stromal
thickness, myometrium thickness and full-thickness of uterine horns, as
these parameters capture the most sensitive histological endpoints in the
uterotrophic assay [9,16]. For each animal we averaged the cell
height/thickness of at least three locations on the H&E slides.
Statistical analysis on all morphometry data were performed using
Shapiro-Wilk normality test followed by one way ANOVA and by the two-
sample t-test, using R’ software. The results were expressed in
mean±standard error (S.E.M.).
Results and Discussion
Body weight gain, organ weights
Except for the controls, both negative and positive, no significant
increase or decrease in body weight gain was observed in the treated groups.
As seen in the Figure 1 a-c, the relative liver, kidneys and spleen
weights were not affected by the treatment with BHA, BHT and PG.
Relative uterine and ovaries weights (Figure 1d) were significantly
decreased by all the tested compounds compared to the negative control,
propyl gallate having the most visible effect.
(g)
NegativeControl BHA BHT PG
0
10
20
30
40
50
Relative liver weight
p=0,75
n.s
a
(g)
NegativeControl BHA BHT PG
0
2
4
6
8
10
12
Relative kidneys weight
b
Figure 1 a-b
The relative weight of: a) liver, b) kidneys after the 3 days treatment
FARMACIA, 2013, Vol. 61, 1
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(g)
NegativeControl BHA BHT PG
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Relative slpeen weight
p=0.75
n.s.
c
(g)
NegativeControl Estradiol BHA BHT PG
0
1
2
3
4
5
6
7
8
Relative uterine+ovaries weight
p=0.02 p=0.02
p=0.00112
d
Figure 1 c-d
The relative weight of: c) spleen and d) uterus+ovaries after the 3 days treatment
Histopathology
The histological sections from the negative control animals, showed
no estrogenic changes (Figure 2A and 2B). On the other hand, examination
of the uterus, cervix and vagina of estradiol treated rats, revealed several
characteristic, estrogenic changes (Figure 2C and 2D). In the uterus, there
was mild edema of the endometrium characterized by separation of the
endometrial stromal cells, accompanied by moderate thickening
(hyperplasia) of the uterine mucosal and endometrial glandular epithelia.
Apoptotic endometrial epithelial cells were also observed as well as mitoses
and some lymphocytic infiltrate in the same layer. In all estradiol treated
animals uterine luminal circumference was increased, with more
pronounced invaginations of endometrial epithelium. Infiltrating neutrophils
were present in the uterine and cervical stroma. The cervical and vaginal
epithelium responded to estradiol stimulation by squamous hyperplasia and
cornification of the epithelium.
Rats treated with BHA alone had minimal histopathologic changes in
the uterus, with no changes in the cervix and vagina. Thus, in one animal we
noticed moderate increase of luminal circumference, with more pronounced
invaginations of endometrial epithelium into the stroma as well as a minimal
endometrial stroma edema and minimal neutrophilic infiltrate (Figure 2E
and 2F). From the BHT treated female rats, just in one case we observed a
minimal endometrial stroma edema (Figure 2G and 2H), with no other
visible morphological changes in the evaluated reproductive tract segments.
There were no visible morphological changes in the uterine (Figure 2I and
2J), cervical and vaginal segments of the PG treated animals.
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Figure 2
Histopathologic alterations in the rat uterus in control, estadiol, BHA, BHT or PG treated animals for 3 days
from PND (post natal day) 17–21. Control Tissues (A and B): Note normal morphology of the uterus in a
control rat; Estradiol treatment: (C), note moderate increase of luminal and glandular circumference, with
increased invaginations of endometrial epithelium and increased number of uterine glands; (D), note moderate
edema of the endometrial stroma (ES), moderate hyperplasia of endometrial epithelium (EE) and endometrial
gland epithelium (EG), accompanied by myometrial (M) hypertrophy; a moderate transmural infiltrate with
neutrophils (eosinophilic aspect) is also observed; BHA treatment: (E), note moderate increase of luminal
circumference, with more pronounced invaginations of endometrial epithelium into the stroma; (F), note
minimal edema of endometrial stroma (ES), no hyperplasia of endometrial epithelium (EE) and endometrial
gland epithelium (EG), accompanied by minimal myometrial (M) hypertrophy; a minimal, transmural
neutrophilic infiltrate is also present; BHT treatment: (G), note no changes in the uterine morphology; (H),
note minimal edema of endometrial stroma (ES); PG treatment (I and J): absence of visible morphological
changes; Left column, H&E 4×, original magnification; Right column, H&E 20x, original magnification.
FARMACIA, 2013, Vol. 61, 1
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Morphometric analysis
Endometrial epithelial hyperplasia of the uterine mucosa, stroma,
myometrium and that of the uterine wall are considered histological markers
of endocrine action [19, 21, 22]. As expected, endometrial epithelium cell
height (EECH) was significantly increased in estradiol treated rat group, as
compared to the other four groups. Interestingly, EECH of animals from
BHT and PG treated groups was significantly decreased as compared to that
of BHA treated group. Endometrial stroma (ES) of estadiol treated animals
was significantly thicker than that of animals from BHA and PG treated
group. Moreover, the PG treated rats had a significantly thinner ES as
compared to that of BHT treated ones. Myometrial thickness was
significantly increased in the estradiol treated group and BHT treated group
as compared to the control group and to the PG group. The myometrium of
animals from the PG treated group was significantly reduced as compared to
that of the BHA treated group. A similar trend was observed regarding the
uterine wall thickness, where the animals from the estradiol, BHA and BHT
treated groups had significantly thicker walls as compared to the control
group and to the PG group.
ControlNegativ Estradiol BHA BHT PG
0
5
10
15
20
25
30
Endometrial epithelium thickness
a
p= 0.03662
b
b
µm
a
Controlnegativ Estradiol BHA BHT PG
0
20
40
60
80
100
120
140
160
µm
Endometrial stromal thickness
b
NegativeControl Estradiol BHA BHT PG
0
20
40
60
80
100
120
140
µm
Myometrial thickness
a
p=0.00149
b
p=0.00028
c
NegativeControl Estradiol BHA BHT PG
0
50
100
150
200
250
300
350
400
µm
Uterine wall thickness
a
p=0.00067
b
p=0.00212
b
p=0.01034
b
p=0.00020
d
Figure 3
Morphometric analysis of changes in uterine cellular and compartmental structures.
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Morphometric methods were used to quantify morphological indexes
in histological cross sections from the midhorn uterine wall of Wistar
female rats. Average endometrial epithelium cell height (EECH), stromal
thickness (ES), myometrial thickness (M) and uterine wall thickness (U)
were calculated for each animal. For EECH: (a) marked groups have
significantly lower values as compared to the negative control group; (b)
marked groups have significantly lower values as compared to the estradiol
treated rats. For ES: no significantly differences between the animals from
control groups, both positive and negative and the treated animals. For M:
(a) marked groups have significantly higher values as compared to the
untreated control group; (b) marked groups have significantly higher values
as compared to estradiol treated group. For U: (a) marked groups have
significantly higher values as compared to the untreated control group; (b)
marked groups have significantly lower values as compared to estradiol
treated control.
For all groups a confidence level of 95% (p < 0.05) was considered
significant.
Information about the in vivo effects of BHA, BHT and PG on
prepubescent female Wistar rats and uterine development is unknown or
sparse. Consistent with previously published data about the presumed
endocrine disrupting effects of the substances used in our study we conclude
from the present in vivo study that BHT, BHA and PG have the ability to
decrease the relative uterine weight of prepubescent female rats, PG having
the most severe effect (Figure 1d). The mentioned data may be correlated
with the morphometric analysis results. Thus, the endometrial epithelium
cell height was decreased in the groups treated with BHT and PG, PG
treated group being the one were the decrease was statistically significant
(p<0.05) (Figure 3a).
According to previously published studies [13], BHA was not
expected to have any influence on the endometrial epithelium cell height
when compared to control, but in our study this parameter was increased,
though not statistically significant, showing a possible estrogenic effect
(Figure 3a)[13].
Other results yielded by the morphometric analysis can be associated
with the decreased uterine weight such as the decreased endometrial stroma
in the PG treated group and the significantly reduced myometral thickness,
also in the PG treated group (Figure 3c and 3d).
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Conclusions
The data obtained from the present study suggest possible endocrine
disruptive effects at the dose of 3x NOAEL for BHA, BHT and PG in
Wistar prepubescent female rats, with statistically significant changes
showing the antiestrogenic properties of PG.
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Manuscript recieved: July 14th 2011