Activité oestrogéniques de colorants alimentaires dans le Yeast Estrogen Screen

Nous sommes exposés tous les jours à de nombreuses substances de synthèse issues de l’industrie chimique par le biais de nombreuses sources dont notre alimentation. Certains additifs et colorants retrouvés dans les denrées alimentaires ont la capacité de perturber notre système hormonal. Comme nous l’avons vu dans l’article précédent, certains parabènes comme le propyl et le butyl parabène sont des conservateurs et des additifs utilisés dans les aliments qui ont la capacité de mimer l’oestrogène naturel. Par exemple, le propyl gallate (E310) utilisé comme anti-oxydant est un xéno-estrogène (Amadasi et al., 200). Parmi ces xéno-estrogènes se retrouvent également certains colorants comme la tartazine (E102), l’érytrosine B (E127) ou encore le Sudan (rouge de Sudan) qui n’est plus autorisé dans l’Union Européenne depuis 1995.

Ce volet de recherche a pour but de tester l’aptitude de cinq colorants alimentaires, à induire une réponse oestrogénique en utilisant le YES (Yeast Estrogen Screen) et de comparer cette activité à celle de cinq parabènes couramment utilisés (méthyl parabène (MeP), éthyl parabène (EtP), propyl parabène (PrP), butyl parabène (BuP), benzyl parabène (BzP)). Et dont l’activité oestrogénique est connue. Ces travaux ont été publiés dans Journal of Ecosystem & Ecography Volume 3 : 128. 2013, Pages 1-5.

Dans cette étude nous avons testé, pour la première fois, l’induction de l’activité oestrogénique de colorants alimentaires avec le YES. En effet, le YES a été développé pour l’identification et la caractérisation de composés capables de se lier au récepteur des oestrogènes (Roultdge et al., 1996). La spécificité de ce test au composé oestrogénique est expliquée par le nature du récepteur aux oestrogènes où le site de liaison est plus flexible (Bistan et al., 2012). De ce fait, le récepteur des oestrogènes devient une cible plus vulnérable à un large éventail de composés qui possèdent des analogies structurales avec le 17 β-oestradiol, comme les oestrogènes synthétiques, le bisphénol A, le nonylphénol et les phyto-oestrogènes.

Parmi les colorants testés, seuls deux composés ont la capacité d’induire une activité oestrogénique avec le YES, l’E171 (Dioxyde de titane) et l’E132 (l’indigotine). Le tableau 16 présente les activités oestrogéniques de différents colorants. L’E171 a une activité oestrogénique de 33% à une concentration de 1g/l. Cette activité oestrogénique est faible en comparaison de celle induite par les 17β-oestradiol et les parabènes. En effet, ce colorant est 106 fois moins oestrogénique que le 17 β-oestradiol,

Tableau 16 : Activité oestrogénique relative des cinq colorants alimentaires (1g/l).

Composés Activité oestrogénique agoniste (E2 %)

E2 100 (10-9M) E122 0 E104 0 E132 10 E 124 0.93 E171 33

L’inhibition par 4-OH-TAM de l’activité oestrogénique induite par le colorant dans le YES démontre que ce composé est un xéno-oestrogène comme les parabènes. Le 4-OH-TAM inhibe l’induction du ERE.

Research Article Open Access

a n e al , J Ecosys Ecograph 2013, 3:3 h p: o org 10 172 2157-7625 1000128

Research Article Open Access

Ecosystem & Ecography

Keywords: Food dyes; Paraben; Food additives; Estrogenic activity

Introduction

Food additives are artificial ingredients added to foods, medications and cosmetics in order to enhance flavour, colour, and texture. They facilitate the preparation and prevent spoiling. The most common food additives are: Aspartame Monosodium Glutamate (MSG), Sodium Nitrate, parabens and dyes like tartrazine (Yellow Dye #5). Although many of these individual additives are included in small amounts individual average consumption is estimated at about 5 pounds of synthetic additives in total each year. If we include sugar data - as it’s the most used additive by the food industry - then that number increases to 135 pounds a year. Between them we can find food dyes (like tartrazine the most widely used) and parabens (methylparaben, ethylparaben, propylparaben, butylparaben and isobutylparaben). The most common health issue that occurs from the intake of food additives is the worsening of asthmatic symptoms and development of allergies. But safety of some of them is well known. For example, the safety of parabens has been debated overlooked to male and female breast, testicular cancer and fertility, they are able to mimic estrogens [1-5]. Except quinolone, food colors are relatively weak mutagen and certified as safe additives despite reports that some people have allergic reactions towards them [6]. However, studies have revealed toxic effects of many colorants [7].

All these compounds are classified as xenoestrogens. Xenoestrogens are exogenous chemicals able to modulate endogenous estrogen activity through, for example, structural similarities leads to receptors interactions or estrogen metabolizing enzymes. A common mode of action is with xenoestrogens shows an affinity for Estrogen Receptors (ER) and subsequently causes endocrine disruption through this interaction [8]. Other chemicals may bind to other receptors related to ERs and modulate their functioning [9].

The Estrogen Receptor (ER) is the key factor that elicits estrogenic response in vertebrates. Several structural trends have been identified to be potential ligands for the ER. Various additives like parabens have been proven to be endocrine disruptor compounds using either in vivo or in vitro assay [10]. Recombinant Yeast Assays (RYA) (or Yeast Estrogen Screen) based on use of estrogen receptors of vertebrates are convenient functional assays to evaluate the potential endocrine disruption of a substance [11]. But few studies have focused on the

estrogenic activity of food dyes. Studies concern mainly food colors such as tartazine (E 102), sudan I and erythrosine B (E 127) which have effects on the chromosomal structure and can increase Estrogen Receptor (ER) site-specific DNA binding effect to Estrogen Response Element (ERE) in HTB 133 cells [12,13] and in the E-screen test [14]. Axon et al. [15] helped to identify several modulator of the human ER among food additives such as tartazine and sunset yellow.

In the current work, we sought to detect the presence of xenobiotic ER agonists between various food additives using the Yeast Estrogen Screen (YES).

Materials and Methods

Selected compounds

Selected compounds methylparaben (MP), ethylparaben (EP), n-propylparaben (PP), n-butylparaben (BP), and 17-β-estradiol (E2) were purchased from Sigma-Aldrich (Table 1). All stock solutions were prepared in methanol and serially diluted in distilled water to obtain the targeted concentrations. For each paraben the endocrine activity was assessed at various concentrations in the range of 10-9 to 10-3 M. The methanol concentration in the exposure solutions, including controls was 0.01% (v/v) in the tested solutions, which is a non-effective dose as estimated in preliminary tests.

A selection of currently used dyes was chosen for this study. These dyes were selected on the basis of their potential use food industries (http://www.omicsonline.org/2155-6199/2155-6199-1-110. php#Table1)

For each dye, a stock solution (0.1 M) was prepared by dissolving

*Corresponding author: I ngrid B azi n, Ecole des Mines d’ Alè s, LG EI center, 6 avenue de Claviè re 3 0 3 1 9 Alè s Cedex, F rance, E- mail: I ngrid. b az in@ mines- ales. f r

Received May 2 7 , 2 0 1 3 ; Accepted Ju ne 2 5 , 2 0 1 3 ; Published Ju ne 2 7 , 2 0 1 3 Citation: B azi n I , Hassine AI H, Mnif W , G onza lez C ( 2 0 1 3 ) YES as a T ool f or D etecting Estrogenic Activity of S ome F ood Additives Compounds: E 1 0 4 , E 1 2 2 , E 1 2 4 , E 1 3 2 and E 1 7 1 . J Ecosys Ecograph 3 : 1 2 8 . doi:1 0 . 4 1 7 2 / 2 1 5 7 - 7 6 2 5 . 1 0 0 0 1 2 8

Copyright: © 2 0 1 3 B azi n I , et al. T his is an open- access article distrib uted under the terms of the Creative Commons Attrib ution License, w hich permits unrestricted use, distrib ution, and reproduction in any medium, provided the original author and source are credited.

Abstract

T he toxi city ( mutagenic and carcinogenic ef f ects) of f ood dyes ( used like f ood additive) has b een w ell documented. However the endocrine disrupting effects of these dyes have been poorly investigated. We studied five commercial f ood dyes f or their agonistic estrogen activity and do a comparison w ith estrogenic activity of some parab en ( other f ood additives compounds) . T otal estrogenic activities w ere measured using the Yeast Estrogen S creen b ioassay ( YES ) . T he estrogenic potency of the f ood dyes w as measured b y response curves and compared to a dose-response curve of 17-β-estradiol (E2), (reference compound). Estrogen EC50 values have been calculated using standard linear regression methods. Among these compounds, three f ood dyes, indigotine ( E 1 3 2 ) , the monoazo dye Ponceau 4 R ( E 1 2 4 ) and the inorganic dye titanium dioxi de ( E 1 7 1 ) , revealed w eak estrogenic activity. N egative results w ere ob tained f or Q uinoline Yellow ( E 1 0 4 ) .

YES as a Tool for Detecting Estrogenic Activity of Some Food Additives

Compounds: E 104, E 122, E 124, E 132 and E 171

Ingrid Bazin1*, Aziza Ibn Hadj Hassine1, Wissem Mnif2 and Catherine Gonzalez1

1Ecole des Mines d’Alès, LGEI center, 6 avenue de Clavière 30319 Alès Cedex, France

Citation: B azi n I , Hassine AI H, Mnif W , G onza lez C ( 2 0 1 3 ) YES as a T ool f or D etecting Estrogenic Activity of S ome F ood Additives Compounds: E 1 0 4 , E 1 2 2 , E 1 2 4 , E 1 3 2 and E 1 7 1 . J Ecosys Ecograph 3 : 1 2 8 . doi:1 0 . 4 1 7 2 / 2 1 5 7 - 7 6 2 5 . 1 0 0 0 1 2 8

Page 2 of 5

in distilled water, followed by filtration through Whatmann No. 5 filter paper. All these dyes appeared perfectly soluble in water at this concentration as assessed by the absence of precipitate. For each dye, the endocrine activity was assessed at four concentrations in the range of 1×10−6 M to 1×10-2 M. The natural fluorescence of the dyes and their interference on the fluorescence emitted by yeast was determined beforehand in order to avoid spurious signals generated by the YES test in our experimental conditions. The fluorescence of each dye has been measured in the absence of yeast cells and subtracted from the data obtained for estrogenic tests. The cytotoxicity of each dye was evaluated by measuring yeast growth at an O.D. of 600 nm.

Yeast estrogen screen assay (YES)

Yeast strain BY4741 (MATa ura3D0 leu2D0 his3D1 met15D0) was obtained from Euroscarf, Frankfurt, Germany. Expression plasmid pH5HE0 contains the human estrogen cloned into the constitutive yeast expression vector pAAH5 [11]. Plasmid pVITB2x encompasses the reporter gene beta-galactosidase from Escherichia coli fused to the yeast CYC1 promoter and under the control of the pseudopalindromic Estrogen Responsive Element ERE2 from Xenopus laevis vitellogenin B1 gene (50-AGTCACTGTGACC-30 , two copies) [11]. The test measures β-galactosidase activity with 4-methylumbelliferone β-D-galactopyranoside (fluorescence at 460 nm and excitation at 355 nm) with a fluorimeter (Fluoroskan Twinkle LB 970, BERTHOLD Technologies) after 6 h of exposure to the compounds. Tests were performed in 96-well plates.

To determine the estrogen agonistic activity of studied compounds, E2 was used as a positive control and distillated water was used as a negative control. Stock solutions of MP, EP, PP BP (10-2 M), 17 β -estradiol (10-3 M), and dyes (0.1 M) are prepared in methanol and serially diluted in distilled water. The transformed yeast strain were grown overnight at 30°C in a non-selective medium (YPD: 5 g/L yeast extract, 10 g/L peptone, 20 g/L glucose, all from Sigma-Aldrich, France). The following day, 15 µl of saturated culture is added to 15 ml of selective media (SD, 6.7 g/L yeast nitrogen base without amino acids, DIFCO, Basel, Switzerland; 20 g/L glucose, supplemented with 0.1 g/L of prototrofic markers as required). The final culture was diluted in selective media and adjusted to an optical density (DO) between 0.1-0.2 at 600 nm. 45 µl aliquots were transferred into a 96-well polypropylene microtiter plate. 5 µl aliquots of each sample were then dispensed into wells in quadruplicate. This experimentation was carried out 3 times in order to obtain representative statistical data. After 6h incubation, 50 µl of YPER (Pierce, Rockford, IL, USA) were added to each well and incubated at 30°C for 30 min. 50 µl of buffer Z ( 60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7.0 plus 0.5% 2-mercaptoethanol (Fluka), 1% Triton X-100 (Sigma), 4-methylubelliferyl

β-D-Galactoside (Sigma)) were added to the cell lysat then incubated at room temperature for 30 min after a centrifugation at 1000 rpm for 1 min. The fluorescence was mesured at 460 nm (excitation at 355 nm and emission at 460 nm). Confirmation of human ER activation by xenobiotics was made with 4 Hydroxytamoxifen (4-OH-TAM). 0.5 µM of 4-OH-TAM was added to yeast cell prior to addition of potential ER activator. 4 OH-TAM competitively binds to estrogen receptors on tumor cells and other tissue targets, producing a nuclear complex that decreases DNA synthesis and inhibits estrogen effects. After 6h exposure to binary mixture, β-D-galactosidase activity was determined.

Calculation of EC₅₀ values: EC₅₀ values, defined as the concentration where transcriptional response reaches 50% of its value at saturating concentration of ligand, were calculated from dose-response assays, using 5 or 7 concentrations for parabens, 5 concentrations of dyes and 8 concentrations for 17-β-estradiol.

Results and Discussion

Natural and artificial azo dyes and parabens are widely used as coloring agents for food ingredients, drugs and cosmetics. These toxicological effects can vary from carcinogenicity and mutagenicity to genotoxicity. Many azo dyes are genotoxic in short-term tests and carcinogenic in laboratory animals [7]. The genotoxicity of these dyes are controversial, it were classified as genotoxic in one review [7] but not in another [16].

To our knowledge, food additives such as ponceau 4R, azorubine, titanium dioxyde or indigotine have not been examined and/or reported to be estrogenic chemicals. Accordingly, we investigated their potency of action in the yeast/(ERE) 2-pLacZ reporter gene assay. Figure 1 illustrates a typical dose–response for E2 on relative reporter gene expression in yeast cells transfected with (ERE) 2-pLacZ. In our experimental conditions the EC₅₀ is 3.77*10-10 M (22 ng/L). These data were used to calculate EC₅₀% values for a range of compounds. Table 2 and Figure 2 demonstrate that some formulation compounds of cosmetics such as paraben esters, are human ER agonists [17], in our assay. A correlation between the length of the paraben ester chain and the estrogenicity has been also pointed out [17,18]. In addition the cosmetic and food additives titanium dioxide and indigotine were also shown to be ER_ agonists (Figure 2). They induce a concentration-dependent response in yeast cell. As shown in Figure 2, the estrogenic potency of food dyes is very weak compared to that of E2 or some parabens. As previously reported [15], the food dye quinolone yellow

compound Code foodstuf CAS # Class Formula methylparab en E 2 1 8 9 9 - 7 6 - 3 - - - - C₈H₈O ₃

ethylparab en E 2 1 4 1 2 0 - 4 7 - 8 - - - - C₉H_{1 0}O ₃ propylparab en E 2 1 6 9 4 - 1 3 - 3 - - - - C_{1 0}H_{1 2}O ₃ b utylparab en - - - - 9 4 - 2 6 - 8 - - - - C_{1 1}H_{1 2}O ₃

Azo rub ine, E1 2 2 3 5 6 7 - 6 9 - 9 monoazo C_{2 0}H_{1 2}N ₂O ₇S ₂N a₂ Q uinoline Yellow ^{E1 0 4 8 0 0 4 - 9 2 - 0 q uinophtalone C}1 8H₉N O ₈S ₂N a₂ indigotine E1 3 2 8 6 0 - 2 2 - 0 indigoid C1 6 H8N 2O 8S 2N a2 Ponceau 4 R E 1 2 4 2 6 1 1 - 0 3 6 - 2 monoazo C2 0H1 1N 2O 1 0S 3N a3 T itanium D ioxyd e ^{E1 7 1 1 3 4 6 3 - 6 7 - 7 inorganic T iO} 2

Table 1: compounds analyze d in this study.

1 2 0 1 0 0 8 0 6 0 4 0 2 0 0 1 E- 1 3 1 E- 1 2 1 E- 1 1 1 E- 1 0 1 E- 0 9 1 E- 0 8 1 E- 0 7 1 E- 0 6 1 E- 0 5 1 E- 0 4 1 E- 0 3 E2 concentration ( M) Relative activity ( % )

Figure 1: D ose- response curve f or β estradiol ( E2 ) in distilled w ater . Average ± standard deviation ( n= 3 ) .

Citation: B azi n I , Hassine AI H, Mnif W , G onza lez C ( 2 0 1 3 ) YES as a T ool f or D etecting Estrogenic Activity of S ome F ood Additives Compounds: E 1 0 4 , E 1 2 2 , E 1 2 4 , E 1 3 2 and E 1 7 1 . J Ecosys Ecograph 3 : 1 2 8 . doi:1 0 . 4 1 7 2 / 2 1 5 7 - 7 6 2 5 . 1 0 0 0 1 2 8

Page 3 of 5 was not shown to be an ER agonist (Figure 2). Table 2 demonstrates that food colorings such as indigotine, ponceau Red 4 and titanium dioxide – the most potent food xenoestrogens examined in this study - were approximately 106 times less effective that E2 at activating the human ER in the Yeast cell. The most xenoestrogen between paraben used like food additive is butylparaben. In 2003, butylparaben was cleared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) for use as a flavoring agent “at very low levels” (not specified) [19]. Butylparaben is used as a food additive in Japan and as an additive to beer to retard microbial growth. Recently, a call for data regarding suggested limits for the use of butylparaben as a food additive [20].

Table 2 also demonstrates that food colorings are generally less potent activators of the human ER than parabens compounds.

We have analyzed if 4 OH-TAM (a classical ER antagonist) can inhibit the estrogenic activity induced by studied compounds. Inhibition of the induction of (ERE) 2-pLacZ reporter can confirm that studied compounds induce an estrogenic effect in yeast cells. Figure 3 demonstrates that (ERE) 2-pLacZ reporter gene induced activity using E2 as well as with benzylparaben, butylparaben, propylparaben ethylparaben, indigotine and titanium dioxide, were inhibited by 4 OH-TAM.

Our assays consist on an engineered yeast strain that harbors two foreign genetic elements: a vertebrate receptor, human Estrogen Receptor (hER), in our case and a reporter gene. Expression of reporter gene, LacZ, is dependent on the occurrence of estrogens. The final product concentration is easy to quantify, the beta-galactosidase activity. The mechanism of YES is shown in the Figure 4. This is a simplified version of the mechanisms used by natural estrogens to operate in vertebrates. The fundamental similarity of the transcriptional machinery in all eukaryotes ensures that it is similar also on yeast. In a strict sense, the YES assay detects only agonists of the ER, which are, substances that bind to ER and elicit transcriptional response in this simplified system. In conclusion some food dyes like titanium dioxide

Methyl Paraben (MP) Ethyl Paraben (EP) Propyl Paraben (PP) Butyl Paraben (BP) 17-β-Estradiol E2 Titanium Dioxyde Indigotine Ponceau 4R 4 8 + / - 1 8 mg. L- 1 ( 3 . 0 8 * 1 0 - 4 M) ^{7 . 8 + / - 2 mg. L} - 1 ( 4 . 7 3 * 1 0 - 5 M) ^{1 . 3 2 mg. L} - 1 + / - 8 0 µ g. L -1 ( 7 . 2 1 * 1 0- 6 M) ^{5 0 4 + / - 1 5 µ g. L} - 1 ( 2 . 5 9 * 1 0- 6 M) ^{1 0 3 + / - 2 2 ng. L} - 1) ( 3 . 7 7 * 1 0 - 1 0 M) ^{5 0 0 + / - 2 mg. L} - 1) ( 6 . 5 * 1 0 - 3 M) ^{4 7 1 + / - 1 5 mg. L} - 1) ( 1 . 8 * 1 0- 3 M) ^{4 0 3 + / - 8 mg. L} - 1) ( 2 . 4 * 1 0 - 3 M) Table 2: EC_{5 0} of 1 7 - β- Estradiol and f ood additives compounds in distilled w ater samples ob tained in YES test.

( EC_{5 0} values, defined as the concentration where transcriptional response reaches 50% of its value at saturating concentration of ligand, were calculated from dose-response assays, using 8 concentrations f or parab ens and f ood dyes and 1 0 concentrations f or 1 7 β- estradiol) .

120 100 80 60 40 20 0

1E-14 1E-12 1E-10 1E-08 0,000001 0,0001 0,01 1 concentration (M) BP PP MP EP E2 titanium dioxide indigotine % of E2 response

Figure 2: Response of the yeast estrogen screen to a range of parab ens of varying alkyl group size ( Methylparab en, Ethylparab en, Propylparab en and B utylparab en) , some f ood dyes ( titanium dioxi de and indigotine) and 1 7 β- estradiol ( E2 ) , dose response curves w ere normalize d to maxi mum E2 response. 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 Estrogenic activity ( % ) + 4 0 H- T AM E2 1 0 nM Methyl Parab em Ethyl Parab em Propyl Parab em B utyl Parab em Ponceau R4 titanium diox yde indigotine Q uinoline yellow Az orub ine 1 mM 10 M

Figure 3: Ef f ect of 4 - O H- T AM on estrogenic activity of selected f ood additives. T he selected concentrations are closed to EC_{5 0} values, 1 0 nM f or E2 , 1 0 µ M f or parab ens and 1 mM f or studied dyes. Average ± standard deviation ( n= 3 ) .

ERE Promotor LacZ Estrogen receptor Estrogen Activated receptor β galactosidase ERE Estrogen responsive element LacZ Reportrt gene hER Human Estrogen receptor Medium

Yeast cell Cytoplasma

nucleus

hER

Citation: B azi n I , Hassine AI H, Mnif W , G onza lez C ( 2 0 1 3 ) YES as a T ool f or D etecting Estrogenic Activity of S ome F ood Additives Compounds: E 1 0 4 , E 1 2 2 , E 1 2 4 , E 1 3 2 and E 1 7 1 . J Ecosys Ecograph 3 : 1 2 8 . doi:1 0 . 4 1 7 2 / 2 1 5 7 - 7 6 2 5 . 1 0 0 0 1 2 8

Page 4 of 5 and indigotine have weak estrogenic effect in yeast cell. Their activities

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Citation: B azi n I , Hassine AI H, Mnif W , G onza lez C ( 2 0 1 3 ) YES as a T ool f or D etecting Estrogenic Activity of S ome F ood Additives Compounds: E