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Evaluation of the possible endocrine disruptive effect of butylated hydroxyanisole, butylated hydroxytoluene and propyl gallate in immature female rats

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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.
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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
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(g)
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) 1721. 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.
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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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... ter doses of BHA may cause cancer in humans. BHT is known for its chemical name 3,5-di-tert-butyl-4-hydroxytoluene. In vitro investigations have shown that BHA has a modest estrogenic impact as well as anti-androgenic effects. BHT was less estrogenic than BHA and was included in the non-estrogenic chemicals list based on cell proliferation studies (Pop et. al., 2013) [3]. In vitro testing revealed that PG is one of the strongest ligands among the xenochemicals. According to studies estimating BHA and BHT daily consumption, the population can receive close to the ADI (acceptable daily intake) through an ordinary diet, and a small percentage population may be susceptible to higher dose compared to ADI ...
... BHT was less estrogenic than BHA and was included in the non-estrogenic chemicals list based on cell proliferation studies (Pop et. al., 2013) [3]. In vitro testing revealed that PG is one of the strongest ligands among the xenochemicals. ...
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... One original study (Pop et al., 2013) was qualified for a risk of bias, relevance of endpoint and weight of evidence evaluation. The results reported by Pop et al. (2013) did not indicate that the ADI established by EFSA needed to be revised. ...
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SCCS OPINION on Butylated Hydroxytoluene (BHT) - SCCS/1636/21 - Final version U. Bernauer, L. Bodin, Q. Chaudhry, P.J. Coenraads, M. Dusinska, J. Ezendam, E. Gaffet, C. L. Galli, B. Granum, E. Panteri, V. Rogiers, Ch. Rousselle, M. Stepnik, T. Vanhaecke, S. Wijnhoven, A. Koutsodimou, W. Uter, N. von Goetz The SCCS adopted this document by written procedure on 2 December 2021 Published:17 August 2022 Ed. Publications Office of the European Union, Luxembourg, Luxembourg (35 pages) ISSN : 1831-4767 ISBN : 978-92-76-54819-5 DOI : https://data.europa.eu/doi/10.2875/53206 Catalog Number : EW-AQ-22-016-EN-N https://op.europa.eu/fr/publication-detail/-/publication/ce2b5ca2-21c7-11ed-8fa0-01aa75ed71a1 https://op.europa.eu/fr/publication-detail/-/publication/ce2b5ca2-21c7-11ed-8fa0-01aa75ed71a1/language-en/format-PDF/source-264517273
... Recently, Park et al. (2019) have shown the potent cytotoxic activity of BHA on human astrocytes which may cause impairment of brain and nerve development. The BHA (300 mg/kg BW) and BHT (75 mg/kg b.w.) also result in the disruption of the endocrine system of Wistar rats (Pop et al., 2013). Moreover, several studies reported that BHA and BHT lead to urticaria (Thune and Granholt, 1975;Tosti et al., 1987;Fisherman and Cohen, 1977;Juhlin, 1981). ...
... The nongenotoxic and noncarcinogenic of propyl gallate has been reported under in vivo conditions (Aguilar et al., 2014). The adverse effects of PG (405 mg/kg BW) on the endocrine system of Wistar rats was reported by (Pop et al., 2013). The gallates induced skin and/or stomach irritation has also been reported in the literature (Winter, 1972;Dastychová et al., 2008). ...
Chapter
Food products containing fats and oils, such as meat, vegetable oil, dairy, and bakery products are susceptible to oxidation due to their chemistry. The unsaturated fatty acids are more vulnerable to rancidity due to less stability as compared to saturated fatty acids. Thus, during the storage, most of the time undesirable flavors and oxidized products are formed due to auto-oxidation. These oxidized products may result in various types of harmful effects, such as cytotoxicity, mutagenicity, peptic ulcers, atherosclerotic plaque, oxidative stress, etc. The auto-oxidation of food products depends upon various factors such as temperature, light, oxygen, humidity, ionizing radiations, catalysts, type of oil, microorganisms, and processing and/or storage conditions and can be prevented or blocked at various steps (processing, packaging, and storage). The antioxidants are added to food products to prevent auto-oxidation. However, the use of antioxidants has its advantages and disadvantages. Thus, the first part of this chapter focuses on the mechanism of the auto-oxidation process and the methods for the determination of auto-oxidation in food. The second part of the chapter deals with the factors involved in the auto-oxidation process and the toxic effects of food auto-oxidation. Finally, the chapter focuses on the measures which can be taken to prevent the auto-oxidation process, the antioxidants used in food industries, and current challenges faced during the prevention of food auto-oxidation.
... Ref. : Olsen 1986;Price 1994;EFSA 2012;ANSES 2016 The Norwegian Scientific Committee for Food and Environment performed systematic literature searches (from 2012 to 2018) to identify publications potentially indicating that the ADI established by EFSA needed to be revised. One original study (Pop et al., 2013) was qualified for a risk of bias, relevance of endpoint and weight of evidence evaluation. The results reported by Pop et al. (2013) did not indicate that the ADI established by EFSA needed to be revised. ...
... One original study (Pop et al., 2013) was qualified for a risk of bias, relevance of endpoint and weight of evidence evaluation. The results reported by Pop et al. (2013) did not indicate that the ADI established by EFSA needed to be revised. ...
Book
SCCS OPINION on Butylated Hydroxytoluene (BHT) - SCCS/1636/21 - Final version U. Bernauer, L. Bodin, Q. Chaudhry, P.J. Coenraads, M. Dusinska, J. Ezendam, E. Gaffet, C. L. Galli, B. Granum, E. Panteri, V. Rogiers, Ch. Rousselle, M. Stepnik, T. Vanhaecke, S. Wijnhoven, A. Koutsodimou, W. Uter, N. von Goetz The SCCS adopted this document by written proccedure on 2 December 2021 Mise en Ligne le 3 Décembre 2021 https://ec.europa.eu/health/sites/default/files/scientific_committees/consumer_safety/docs/sccs_o_257.pdf
... 11 The need to progressively replace synthetic antioxidants is emphasized by their side effects, such as endocrine disruption or even carcinogenesis. 12 A large variety of polyphenolic compounds is found in Vaccinium species, that is why plants from this family, like Vaccinium vitis-idaea L. or Vaccinium myrtillus L, commonly known as lingonberry or bilberry, respectively, are intensively studied thanks to their significant antioxidant and antimicrobial activities. 13 Wild bilberry (Vaccinium myrtillus L). is a spontaneous shrub found in mountain areas. ...
Article
Full-text available
Purpose In this study, wound dressings were designed using zinc-modified marine collagen porous scaffold as host for wild bilberry (WB) leaves extract immobilized in functionalized mesoporous silica nanoparticles (MSN). These new composites were developed as an alternative to conventional wound dressings. In addition to the antibacterial activity of classic antibiotics, a polyphenolic extract could act as an antioxidant and/or an anti-inflammatory agent as well. Methods Wild bilberry leaves extract was prepared by ultrasound-assisted extraction in ethanol and its properties were evaluated by UV-Vis spectroscopy (radical scavenging activity, total amount of polyphenols, flavonoids, anthocyanins, and condensed tannins). The extract components were identified by HPLC, and the antidiabetic properties of the extract were evaluated via α-glucosidase inhibitory activity. Spherical MSN were modified with propionic acid or proline moieties by post-synthesis method and used as carriers for the WB leaves extract. The textural and structural features of functionalized MSN were assessed by nitrogen adsorption/desorption isotherms, small-angle XRD, SEM, TEM, and FTIR spectroscopy. The composite porous scaffolds were prepared by freeze drying of the zinc-modified collagen suspension containing WB extract loaded silica nanoparticles. Results The properties of the new composites demonstrated enhanced properties in terms of thermal stability of the zinc-collagen scaffold, without altering the protein conformation, and stimulation of NCTC fibroblasts mobility. The results of the scratch assay showed contributions of both zinc ions from collagen and the polyphenolic extract incorporated in functionalized silica in the wound healing process. The extract encapsulated in functionalized MSN proved enhanced biological activities compared to the extract alone: better inhibition of P. aeruginosa and S. aureus strains, higher biocompatibility on HaCaT keratinocytes, and anti-inflammatory potential demonstrated by reduced IL-1β and TNF-α levels. Conclusion The experimental data shows that the novel composites can be used for the development of effective wound dressings.
... However, the utilization of synthetic antioxidants is questioned since they may cause harmful effects on human health. Previous studies have shown that synthetic antioxidants have potential carcinogenicity, cytotoxicity and endocrine disrupter effects (Pop et al. 2013;Saito et al. 2003). Indeed, the use of synthetic antioxidants has been limited in the USA, Canada, Japan and numerous European countries (Wang et al. 2018a). ...
Article
This study was conducted to evaluate the effectiveness of propolis in improving the oxidative stability of sunflower oil (SFO) in comparison to buthylated hydroxytoluene (BHT), a synthetic antioxidant, under simulated frying conditions by using Attenuated Total Reflection-Mid Infrared (ATR-MIR) spectroscopy. Control, two different concentrations of propolis (1500 and 2000 ppm) and BHT added SFOs were heated at 180 °C for 24 h (8 h per day) and changes in the spectra of these oils sampled every 2 h were evaluated. The results revealed that the areas of the infrared bands related to primary and secondary oxidation products (the bands at 3482 and 1745 cm−1) and to trans-unsaturated fatty acids (the bands at 987 and 965 cm−1) increased and the areas of the bands related to cis fatty acids (the bands at 3009 and 722 cm−1) decreased in the control SFO spectra after the heating process as a result of oxidation. 2000 ppm propolis delayed all these oxidation process, in a similar manner to BHT. Principal component analysis and chemical studies confirmed that propolis has a protective effect on the thermal oxidation of SFO. These results indicated that propolis could be recommended as an effective natural antioxidant and used instead of synthetic antioxidants in edible oil industry. This study also showed that ATR-MIR spectroscopy could be used as a fast and efficient technique to evaluate the oxidative stability of edible oils and the bands at 3482, 3009, 1745, 987, 965, 722 cm−1 can be used as biomarkers for oxidation.Graphical Abstract
... 139,140 The antiestrogenic activity of BHA was further confirmed in vivo as it caused delayed vaginal opening, reduced vaginal weight, and decreased uterine weight in immature rats. 52,141,142 BHA could exert its antiestrogenic effect by partially antagonizing 5-dihydrotestosterone (DHT), which is a potent androgen receptor (AR) agonist. 143 After coculture of BHA (0, 0.1, 1.0 and 10 μmol L −1 ) with PC-3LUC AR+ cells for 18 h, BHA antagonized the activation of ARs by DHT when the DHT concentration was 50 pmol L −1 . ...
Article
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Butylated hydroxyanisole (BHA) is mainly used as a food additive due to its antioxidant properties, which prevent or delay oxidation reactions and extend the storage life of products. The widespread use of BHA has led to its extensive presence in various environmental matrices and human tissues. Food intake is the main route of human exposure to BHA. Under different conditions, BHA can produce different metabolites, with tert‐butyl hydroquinone (TBHQ) being one of the major products. Several studies have shown that BHA could cause thyroid system damage, metabolic and growth disorders, neurotoxicity, and carcinogenesis. Mechanisms such as endocrine disruption, genotoxicity, disturbances of energy metabolism, reactive oxygen species (ROS) production, signaling pathways, and imbalances in calcium homeostasis appear to be associated with the toxic effects of BHA. Avoiding the toxic effects of BHA to the maximum extent possible is a top priority. Finding safe, non‐toxic and environmentally friendly alternatives to BHA should be the focus of subsequent research. In all, this review summarized the current situation related to BHA and might make recommendations for future research directions. © 2023 Society of Chemical Industry.
Article
Antioxidants are compounds that may neutralize free radicals and highly reactive molecules that might damage cells, thereby protecting the body from numerous ailments. A recent study found that high dosages of synthetic antioxidants increased the risk of cancer. This made researchers start to develop natural ingredients that can be used as natural antioxidants. One of the natural antioxidants that can be used is the avocado plant.Many phytochemicals, including phenolics, flavonoids, tannins, saponins, oxalates, phytic acids, and alkaloids, are present in seed wastes. Avocado seeds have been studied primarily as antioxidants in 70% and 100% ethanol solvents using the FRAP and DPPH methods. There is no detailed information on the above test using 96% ethanol with various antioxidant testing methods such as the ABTS method (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), which has been linked to active phenolic compounds, flavonoids, and proanthocyanidins as a high natural antioxidant chemical compound.According to research findings, the avocado seed extract in 95% ethanol contains secondary metabolites such as alkaloids, flavonoids, saponins, tannins, and steroids. It has 62.18%GAE±0.73 phenolics, 0.157%QE±0.01 flavonoids and 2.1841 mg/L±0.25 proanthocyanidins. With IC50 of 0.6419 mg/L±0.04 ascorbic acid and 3.2467 mg/L±0.27 and a sample. This antioxidant activity has a very strong category. Keyword : Antioxidants, Avocados, ABTS, Phenolics, Flavonoids, Proanthocyanidins
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Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of butylated hydroxytoluene (BHT) as a feed additive for all animal species. The additive BHT is considered safe for chickens for fattening and weaned piglets at the maximum proposed concentration of 150 mg/kg complete feed. This conclusion is extended to chickens reared for laying and extrapolated to pigs for fattening. In the absence of data, no conclusion on the safety for the other target species could be drawn. The exposure of the consumer to BHT from tissues and products of animals fed the additive ranged from 1% to 3% of the acceptable daily intake (ADI). The FEEDAP Panel concluded that the use of BHT as a feed additive at the proposed conditions of use is of no concern for the safety of the consumers. Exposure of the user to BHT via inhalation is likely; however, the Panel is not in the position to conclude on the potential inhalation toxicity of the additive. BHT is a skin and eye irritant, no conclusions can be drawn on the potential of the additive to be a skin sensitiser. In the absence of data, the FEEDAP Panel cannot conclude on the safety of BHT for the environment. The additive BHT is considered an efficacious antioxidant in feedingstuffs for all animal species.
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Betulinic acid (3β, hydroxy-lup-20(29)-en-28-oic acid, BA) is a pentacyclic triterpene previously tested on several cell lines of neuroectodermal origin and on some in vivo tumor models, such as the melanoma model. The aim of the present work was to test its activity by increasing its water soluble fraction with gamma type cyclodextrins (hydroxipropilgamma cyclodextrin). The study consisted in a preliminary in vitro evaluation on A431 and MCF-7 cell lines and in vivo tests on fertilized eggs. The dissolution capacity in presence of cyclodextrin was analysed by the phase diagram. The main conclusion was that betulinic acid possessed an important antiangiogenic activity and a significant cytotoxicity, especially on the A431 cell line. Rezumat Acidul betulinic (acidul 3β, hydroxy-lup-20(29)-en-28-oic, BA) este o triterpenă pentaciclică testată pe o serie de linii celulare de origine neuroectodermică şi pe unele modele tumorale in vivo, mai specific în studiile pe melanom. Scopul prezentului studiu a fost testarea activităţii compusului prin creşterea solubilităţii apoase prin încorporare în ciclodextrine de tip gamma (hidroxipropilgamma ciclodextrina). Studiul a constat în evaluarea preliminară in vitro pe liniile celulare A431 şi MCF-7 şi teste in vivo pe ou embrionat. Capacitatea de solubilizare cu ciclodextrină a fost analizată prin diagrama de fază. Concluzia principală a fost că acidul betulinic posedă o importantă capacitate antiangiogenică dar şi o citotoxicitate semnificativă în special pe celulele A431.
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Nuclear receptors function as ligand-regulated transcription factors and modulate the expression of sets of genes in response to varying concentrations of ligands. The ligand modulators can be endogenous metabolites that function as hormones, or they can be exogenous substances, such as pharmaceutical agents or environmental substances of natural or man-made origin, which in some cases can cause endocrine disruption. Ligands modulate nuclear receptor activity by binding to their ligand-binding domains and stabilizing conformations that lead either to transcriptional activation or repression. The ligand-binding pocket is somewhat flexible, and binding affinities can be measured over a 10-million-fold range (i.e., with equilibrium dissociation constant values ranging from ca. 0.01 nM to 100 muM). Thus, it is not surprising that by binding a large variety of structures, some nuclear receptors can appear to be promiscuous; however, when affinity is considered, the binding patterns are more restricted. The spectrum of ligands that bind to the estrogen receptor has been most thoroughly investigated. Those from natural sources include natural products in food, such as soy isoflavones and whole grain lignans, as well as microbial products and components from wood. Aside from pharmaceuticals, man-made estrogen ligands can be found in industrial products, such as alkyl phenols from nonionic detergents, bisphenols from plastics, indicator dye impurities, polymer chemicals, and chlorinated aromatics and pesticides. Exogenous ligands are also known for the androgen and progesterone receptors. While it is possible that endocrine disruption can result from exogenous chemicals acting directly as ligands for the nuclear receptors, endocrine disruption needs to be considered in the broader context; thus, compounds also need to be assessed for their effects at other levels, such as on endogenous hormone production, transport, metabolism, and clearance, and at points in signal transduction cascades that are beyond the ligand-receptor interaction.
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Evidence exists for adverse effects of endocrine disruptors on wildlife populations, but there is as yet only limited evidence that links environmental exposure to these substances and adverse effects on human health. It has been suggested that exposure to endocrine disrupting chemicals may be responsible for a possible decline in human sperm production and increases other male reproductive problems such as testicular cancer. This chapter reviews the historical development of work on endocrine disruption and provides a concise description of the state of present knowledge in regards to the ability of chemicals to disrupt a number of hormone systems. In addition, the potential for these compounds to affect animal and human health is discussed. The major potential targets for environmental endocrine disruption are the reproductive, neurological, thyroid, and immune systems. Other endocrine organs, such as stomach, pancreas, kidney, and adrenals, are also potential targets but less is known about their susceptibility. The classes of compound with endocrine-disrupting activity are as follows: estrogens, androgens and antiandrogens, progesterone, steroid receptors such as pregnane X receptors (PXR), polychlorinated biphenyl (PCB), and aryl hydrocarbon receptors.
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Long exposure to small concentrations of xenoestrogens (XEs) seems to be responsible for the increase of certain hormone-dependent diseases. The results of the research concerning the study of potentially synergic interactions between XEs are few and contradictory and need further studies. The oxidative stress is involved in the generation of several pathologies, such as cancer. We studied the oxidative stress induction in rats after exposure to bisphenol A (BPA) and/or methylparaben, two xenobiotics identified as XEs. Malondialdehyde (MDA), a lipid peroxidation end-product, and 2,3-dihydroxybenzoic acid (2,3-DHBA), a marker of in vivo hydroxyl radical production, were used to evaluate the oxidative stress generation. The levels of MDA in rat plasma were determined by HPLCUV (detection at 307 nm) after derivatization with dinitrophenylhydrazine. 2,3-DHBA was analyzed by LC-MS/MS in multiple reaction monitoring (MRM) mode using an ion trap mass spectrometer with electrospray negative ionization. The ion transition monitored was m/z 153 → m/z (109). The results indicated that both studied xenobiotics have the potential of oxidative stress induction in rats but simultaneous exposure does not increase the intensity of effects.
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Melatonin's pharmacologic and physiopathologic potential constitutes the object of modern research regarding organisms' integration mechanisms within the great informational variety of the environment. Nowadays melatonin's effects are considered both for the central neural structures and especially for the peripheric ones. The present study aims at investigating melatonin's effects upon the adrenergic innervation of the vas deferens isolated from mouse. The experiments were performed in vitro, using the organ bath, after incubating the isolated vas deferens with the physiological dose of melatonin. In this respect, we determined the neurogenic contractile response of the mouse vas deferens using the following conditions: electrical stimulation (trains of 5, 50 and 100 pulses of 0.5ms duration and a frequency of 10 Hz), neurotransmitter stimulation (noradrenaline 100μM) and α1-adrenergic stimulation (phenylephrine 10μM and 100 μM). The analysis criteria for all experimental data was based upon the maximum amplitude of the vas deferens' biphasic contractions:"twitch" (purinergic, fast) and "hump"(noradrenergic, slow). We also evaluated the contraction time variation for the vas deferens considered. The experimental results evoke the bound between melatonin and the neurogenic adrenergic component of contraction of the mouse vas deferens. In this respect, the pineal hormone reduces, statistically significant, both the vas deferens contraction force and also the time required for noradrenaline to be released from vesicles.
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We have combined several endpoints into a single 5-dayin vivoscreening procedure to identify estrogenic compounds and dopaminergic modulators, both of which play important roles in enhancing mammary tumorigenesis in rodents. The endpoints evaluated as markers of estrogenicity included increases in uterine fluid and vaginal cornification incidence, serum prolactin levels, uterine weight, uterine epithelial cell height, uterine stromal cell proliferation, and uterine progesterone receptor (PR) number and decreases in uterine estrogen receptor (ER) number. The endpoints evaluated for changes in dopamine regulation included increases in prolactin and decreases in growth hormone levels. The estrogen agonists estradiol (E2) and estriol (E3), the mixed estrogen agonist/antagonist tamoxifen (TAM), the full antiestrogen ICI-182,780 (ICI), and the dopamine modulators haloperidol (HAL) and reserpine (RES) were tested using a three-time/day (8-hr intervals) intraperitoneal dosing regimen in sexually mature ovariectomized female Crl:CD BR rats. All compounds were evaluated over a range of concentrations. Thisin vivobattery was used to evaluate the effects of different classes of endocrine modulators on the selected endpoints. For example, the estrogen receptor agonists E2 and E3 display a unique profile based on changes in the uterotrophic endpoints (estrus conversion, uterine fluid imbibition, increases in uterine weight, and uterine endometrial cell proliferation) where full and partial agonists can be distinguished by the magnitude of these responses. Both the estrogen receptor antagonist ICI and the dopamine modulators HAL and RES lack these uterotrophic responses. Dopamine modulators can be distinguished from estrogen receptor agonists by the profile of increased prolactin levels with no uterotrophic changes. Estrogen receptor antagonists can be distinguished from agonists by comparing their effects on ER, PR, and uterotrophic responses. For instance, the full estrogen receptor antagonist ICI decreased ER (to almost 0) and PR levels, but has no uterotrophic effects, while TAM decreases ER (to almost 0) and increases PR with uterotrophic effects. The most useful endpoints for distinguishing estrogen agonists and dopamine modulators were uterine fluid imbibition, uterine weight, uterine stromal cell proliferation, and serum prolactin levels. In order to distinguish an estrogen agonist from an antagonist, other endpoints, such as receptor levels, are necessary. The advantage of anin vivoscreen is that it utilizes a metabolically and physiologically defined system which is especially important with highly integrative systems such as the endocrine system. This battery can be used as a screening tool to identify potential endocrine modulators as well as to identify mode of action following adverse findings in toxicology studies. Last, additional endpoints may be added to identify other classes of endocrine modulators.
Article
A simple electrochemical method was developed for the single and simultaneous determination of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in food samples using square-wave voltammetry (SWV). A carbon composite electrode modified (MCCE) with copper (II) phosphate immobilized in a polyester resin was proposed. The modified electrode allowed the detection of BHA and BHT at potentials lower than those observed at unmodified electrodes. A separation of about 430mV between the peak oxidation potentials of BHA and BHT in binary mixtures was obtained. The calibration curves for the simultaneous determination of BHA and BHT demonstrated an excellent linear response in the range from 3.4x10(-7) to 4.1x10(-5)molL(-1) for both compounds. The detection limits for the simultaneous determination of BHA and BHT were 7.2x10(-8) and 9.3x10(-8)molL(-1), respectively. In addition, the stability and repeatability of the electrode were determined. The proposed method was successfully applied in the simultaneous determination of BHA and BHT in several food samples, and the results obtained were found to be similar to those obtained using the high performance liquid chromatography method with agreement at 95% confidence level.
Article
In the search for xenoestrogens within food additives, we have analyzed the Joint FAO-WHO expert committee database, containing 1500 compounds, using an integrated in silico and in vitro approach. This analysis identified 31 potential estrogen receptor alpha ligands that were reduced to 13 upon applying a stringent filter based on ligand volume and binding mode. Among the 13 potential xenoestrogens, four were already known to exhibit an estrogenic activity, and the other nine were assayed in vitro, determining the binding affinity to the receptor and biological effects. Propyl gallate was found to act as an antagonist, and 4-hexylresorcinol was found to act as a potent transactivator; both ligands were active at nanomolar concentrations, as predicted by the in silico analysis. Some caution should be issued for the use of propyl gallate and 4-hexylresorcinol as food additives.