ToxSci Advance Access originally published online on July 22, 2008
Toxicological Sciences 2008 106(1):206-213; doi:10.1093/toxsci/kfn148
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Do Parabens Have the Ability to Interfere with Steroidogenesis?
* National Food Institute, Technical University of Denmark, Department of Toxicology and Risk Assessment, DK-2860 Søborg, Denmark
Department of Growth and Reproduction, Copenhagen University Hospital, Rigshospitalet, DK-2100 Copenhagen, Denmark
1 To whom correspondence should be addressed at National Food Institute, Technical University of Denmark, Dept. of Toxicology and Risk Assessment, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark. E-mail: camta{at}food.dtu.dk.
Received May 30, 2008; accepted July 17, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The effects of ethyl and butyl paraben on steroidogenesis were evaluated in rats exposed in utero. Pregnant Wistar rats were dosed from gestational day (GD) 7 to GD 21, followed by examination of the dams, and the fetuses. Additionally, both parabens were tested in vitro in the H295R steroidogenesis assay and in the T-screen assay, the later to test for their ability to act as thyroid hormone receptor agonist or antagonist. In the in utero exposure toxicity study, neither ethyl nor butyl paraben showed any treatment-related effects on testosterone production, anogenital distance, or testicular histopathology. However, butyl paraben caused a significant decrease in the mRNA expression level of estradiol receptor-beta in fetal ovaries, and also significantly decreased the mRNA expression of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the adrenal glands. In vitro butyl paraben increased the proliferation of the GH3 cells in the T-Screen assay, thereby acting as a weak thyroid hormone receptor agonist. In the adrenal H295R steroidogenesis assay both ethyl and butyl paraben caused a significant increase in the progesterone formation. Overall, the results indicate that butyl paraben might have the ability to act as endocrine disruptor by interfering with the transport of cholesterol to the mitochondrion, thereby interfering with steroidogenesis, but also that the two tested parabens do not show clear endocrine disrupting capabilities in our short-term in vivo experiment.
Key Words: butyl paraben; ethyl paraben; endocrine disruption; h295r assay; t-screen; steroidogenesis; developmental rat study.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Parabens are a group of alkyl esters of p-hydroxybenzoic acid and typically include methyl paraben, ethyl paraben, propyl paraben, butyl paraben, isobutyl paraben, isopropyl paraben, and benzyl paraben. The parabens or their salts are widely used as preservatives in cosmetics, toiletries, food, and pharmaceuticals. Humans are exposed to parabens via personal care products and cosmetics among others, and thorough knowledge on their possible adverse effects is still lacking. Parabens have been studied in a number of in vitro and in vivo systems, and many of the parabens were shown to have weak estrogenic activity, and some, including butyl paraben, also caused reduction in testosterone levels and in sperm production in rats (Byford et al., 2002
; Oishi, 2001, 2002; Okubo et al., 2001; Pedersen et al., 2000; Routledge et al., 1998).
In vitro screening data can determine the nature and extent to which substances interact with, for example, a hormone receptor, as well as solve mechanistic issues. However, only in vivo data can provide evidence of how hormonally active substances are influenced by absorption, distribution, metabolism, feed back mechanisms, and excretion in an intact animal. Therefore, a combined in vitro/in vivo approach is a useful way to gain a complete understanding of the activities of the compound in question. In the current study, the question was: Do parabens have a mechanism of action that resembles that of phthalates? Phthalates are known to impair steroidogenesis in fetal male rats and thereby impair the production of testosterone (Lehmann et al., 2004; Thompson et al., 2004). To assess whether parabens affect fetal steroidogenesis, pregnant rats were dosed with butyl paraben or ethyl paraben from gestational day (GD) 7 to GD 21, followed by examination of the dams and the fetuses.
Because most paraben-containing products are topically applied to the skin, dermal absorption is particularly important with respect to estimating doses and potential effects. Thus, in the current study, the parabens were administered by subcutaneous injections. In combination with the in vivo study, we also tested butyl paraben and ethyl paraben in two selected in vitro assays. The parabens were run in the H295R assay to test for their ability to interfere with steroid hormone biosynthesis, and in the T-screen assay. The later assay is a proliferation assay used to detect binding and activation of the thyroid receptor (TR), thereby determining the ability of a compound to be a thyroid hormone receptor agonist or antagonist. The measured endpoints were selected based on suspected effects of the parabens. Limited material such as blood was available from the fetuses, restricting number of endpoints. The selected in vitro assays were chosen, because the endpoints in these assays were relevant to the in vivo study.
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MATERIALS AND METHODS |
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Test Compounds
The following test compounds were used: ethyl paraben 99% (CAS no. 120-47-8) and butyl paraben 99% pure (CAS no. 94-26-8), both from Sigma-Aldrich.
Test compounds were dissolved in sterile peanut oil supplied by the Royal Veterinary Agriculture Pharmacy, Copenhagen, Denmark.
Animals and Exposure
Sixty time-mated, young adult Wistar rats (HanTac: WH, Taconic M&B, Ejby, Denmark) were supplied day 3 after mating. The animals were distributed, housed and handled as previously described in Vinggaard et al. (2005). The day after arrival, that is, GD 4, animals were weighed and assigned to four groups with similar weight distributions, a control group of 18 animals, one ethyl paraben group, and two butyl paraben groups each with 14 animals. An acclimatization period of 4 days was allowed before starting exposure. The rats were dosed by subcutaneous injections, with vehicle (peanut oil) or 400 mg/kg/day ethyl paraben, 200 mg/kg/day butyl paraben, or 400 mg/kg/day butyl paraben, respectively, from GD 7 to GD 21. The animals were dosed the last time on GD 21, 90 min before they were sacrificed.
Health Status of Dams
In the control group, 18 out of 18 time-mated animals were pregnant, in the ethyl- and the two butyl paraben groups 10, 13, and 13 out of 14 animals were pregnant, respectively. The females were observed daily for signs of toxicity. Body weights were recorded on GD 4 and daily during the entire dosing period. The maternal weight gain from GD 7 to GD 21 was calculated.
Caesarian Sections GD 21
At GD 21 dams were weighed, anesthetized in CO2/O2 and decapitated, and caesarean sections were performed as previously described in Vinggaard et al. (2005). Trunk blood was collected into heparin-coated vials from all fetuses for hormone analysis; one pool per litter was made for all male and female fetuses, respectively. Body weight and anogenital distance (AGD) was measured for all fetuses. Fetal adrenals, ovaries and testes were excised and sampled for hormone analyses, histopathology or gene expression studies.
Hormone Levels
Testosterone and progesterone levels were analyzed in serum from dams and fetuses at GD 21, as were ex vivo testosterone and progesterone productions as described in Laier et al. (2006). After extraction with heptane, steroid hormones (testosterone and progesterone) were analyzed in testis, estradiol was analyzed in ovaries and cortisol levels were measured in adrenal glands. Testes, ovaries or adrenal glands were placed in vials containing 100 µl of water and 0.5 ml of heptane. The tissues were homogenized and the vials placed in a tub consisting of dry ice and acetone until the water-fraction was frozen. The heptane-fraction was transferred to a clean vial, the procedure was repeated, and the two extracts were pooled and evaporated. Before analyzing, the samples were resuspended in 100 µl of Diluent 1 (PerkinElmer, Turku, Finland) and incubated over night at 4°C. At the day of analysis the samples were vortexed and incubated for 10 min at 45°C, before the hormones were measured by use of a Delfia time-resolved fluorescence kit (PerkinElmer) and measured by use of a Wallac Victor 1420 multilabel counter (PerkinElmer Life Sciences). The cortisol levels were analyzed using the HitHunter Cortisol Assay Kit (Discoverx, Amersham Biosciences, Uppsala, Sweden). Serum levels of 17
-hydroxyprogesterone in the dams were analyzed by use of a 17-hydroxyprogesterone-enzyme immunoassay kit from Assay Designs according to manufacturer's instructions (Electra-Box Diagnostica Aps, Denmark).
The plasma level of thyroid hormones triiodothyronine (T3) and thyroxine (T4) were analyzed using a modified Delfia T3 and T4 (cat. no. 1244-029 and 1244-030, respectively) time-resolved fluoroimmunoassay from PerkinElmer (Wallac Oy, Turku, Finland). Instead of the T3 and T4 standards and the T3 and T4 antibody supplied in the Delfia kits, rat T3 and T4 standards in T3 and T4-free rat serum (cat. no. 30042 and 30041, respectively), as well as biotinylated rat T3 (30040) and rat T4 (30039) antibody from Biovian, Ltd, Finland were used. The assay was run as outlined in the protocol supplied by Biovian, Ltd, using streptavidin microtitration strips 8 x 12 wells (4009-0010) and T3 or T4 assay buffer (1244-029 or 1244-111) from PerkinElmer. The measurements were performed by use of a Wallac Victor 1420 multilabel counter (PerkinElmer Life Sciences).
Cortisol levels were measured in adrenal glands as well as supernatants from incubated adrenal glands. The adrenal glands were incubated in a shaking water bath at 37°C for 5 h, in 0.5 ml of Dulbecco's modified Eagle's medium (DMEM/F12) (+ 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid + L-glutamine) medium. After incubation vials were centrifuged at 4000 x g for 10 min and the supernatants were stored at –80°C until cortisol levels were analyzed using the HitHunter Cortisol Assay Kit (Discoverx, Amersham Biosciences).
Gene Expression Levels Determined by Real-Time reverse transcription–PCR
The organs were homogenized and total RNA was isolated using RNeasy-mini kit and RNase-Free DNase set (Qiagen, Ballerup, Denmark). cDNA was synthesized from 0.5 µg total RNA using the Omniscript Reverse Transcription kit (Qiagen) with T16 oligos and an 18S ribosomal RNA (18S rRNA) primer. cDNA samples were quantified on the 7900HT fast real-time PCR System (Applied Biosystems) by standard TaqMan technology and using the primer and probe sets listed in Table 1. The primer and probe sets were generated using the Primer Express Software v2.0, from Applied Biosystems. Expression levels of the following genes were quantified, in ovaries: complement component 3 (Compl. C3), insulin-like growth factor 1 (IGF-1), estradiol receptor (ER)-alpha, ER-beta, the aromatase gene, and 18S rRNA. In testis: scavenger receptor class B, member 1 (ScarB1), steroidogenic acute regulatory protein (StAR), cytochrome P450-side-chain cleavage (P450scc), Cyp17a1 (P450c17), ER-alpha and beta, the aromatase gene and 18S rRNA. In adrenal glands, peripheral benzodiazepine receptor (PBR/Bzrp), StAR, P450scc, P450c17, and 18S rRNA were evaluated. All genes were quantified from standard curves of cloned fragments, and expression levels of each target gene were normalized to the expression level of the housekeeping gene 18S rRNA.
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Histopathology and Immunohistochemistry
Testes were fixed in Bouin's fixative and adrenals were fixed in neutral buffered formalin and embedded in paraffin. Hematoxylin and eosin stained sections were evaluated by an observer blinded to treatment group. Immunohistochemical staining for StAR, P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), and PBR/Bzrp was performed on testis sections as described in Borch, Metzdorff et al. (2006)
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Cell Culture
The T-Screen assay.
The rat pituitary cell line GH3 obtained from American Type Culture Collection (ATCC) were cultured in a humid atmosphere at 37°C and 95% air/5% CO2 in phenol-red free DMEM/F12 (Gibco-Invitrogen, Paisley, UK) supplemented with 1% Antibiotic/Antimycotic (PSF) and 10% (vol/vol) fetal calf serum (FCS) (Gibco). For the thyroid hormone-responsive cell proliferation assay (T-screen), cells were grown in test medium containing 10% (vol/vol) T3-depleted dextran-charcoal treated FCS (DC-FCS). Removal of thyroid hormone from DC-FCS was performed by treatment with Bio-Rad (Copenhagen, Denmark) AG-1X8 resin as described in Samuels et al., (1979) modified by changing the resin three times after 6, 18, and 6 h of centrifugation at 37°C by centrifugation and adding fresh resin. Finally the last resin was removed by centrifugation at 27,000 x g for 10 min and the serum was sterilized by filtration through a 0.2-µm polyethersulfone (PES) filter.
Forty-eight hours prior to plating the cells into 96-well microplates (Costar, Fisher Scientific Biotech Line) standard culture media was changed to test media containing 10% (vol/vol) T3- and T4-depleted DC-FCS. After 48 h in T3/T4-depleted DC-FCS, which was changed once after 24 h, the GH3 cells were harvested using a cell scraper and seeded in 96-well black, clear bottom microplates, 50 µl of cell suspension and 50 µl of test compound per well, at a density of 2500 cells per well. All compounds were tested in triplicate (0, 0.01, 0.375, 1, 3, 10, and 30µM) and were tested both in the absence or presence of 0.22nM T3 (T3-EC50) to test for agonistic and antagonistic potency. Control wells contained cells and test medium with the same amount of dimethyl sulfoxide (DMSO [0.1%]) as the exposed cells. The plates were incubated for 96 h, and then cell growth was measured using the dye resazurine (O'Brien et al., 2000). Enzymes in the mitochondria of the GH3 cells reduce resazurine from an almost non-fluorescent oxidized form into its highly fluorescent reduced from resorufin. The fluorescence is a measure for the amount of viable cells present (Schriks et al., 2006). Following the 96-h exposure 100 µl, of a 0.005 mg/ml resazurine solution in phosphate buffered saline, was added to each well, and the plates were incubated 3 h at 37°C, protected from light. Subsequently the plates were analyzed by measuring fluorescence (excitation wavelength 560 nm/emission. 590 nm) on a Wallac Victor 1420 multilabel counter (PerkinElmer Life Sciences).
The H295R steroid syntheses assay.
The H295R human adrenocortical carcinoma cell line obtained from the ATCC (CRL-2128; ATCC Manassas, VA) was grown in 24-well culture plates (Costar, Corning, NY) at 37°C humidified atmosphere of 5% CO2/air. Each well, contained 1 ml DMEM/F12 medium (GibcoBRL Life Technologies) supplemented with 2.0% Nu-serum (BD Sciences, Denmark), Insulin-transferrin-sodium selenite + premix (containing 6.25 µg/ml insulin, 6.25 µg/ml transferin, 6.25 µg/ml selenium, 1.25 µg/ml bovine serum albumin, and 5.35 µg/ml linoic acid) and 100 U/ml penicillin, 100 mg/ml streptomycin and 250 ng/ml amphotericin B (Fungizone). The cells were plated at a density of 2 x 105 cells per well and allowed to settle for 24 h. Culture media was removed and new media containing paraben dissolved in DMSO was added to the cells in triplicates (0.0001, 0.001, 0.01, 0.1, 0.3, 1, 3, 10, and 30µM, ethyl paraben or butyl paraben). Control wells contained the same amount of DMSO (0.1%) as exposed cells. After incubation for 48 h, the media was removed and stored at –20°C until measured for testosterone, progesterone and estradiol levels. Samples of 800 µl were concentrated on IST Isolute SPE columns (100 mg, C18, 1 ml; IST, Hengoed, UK), and the steroid hormones were measured using Delfia kits (PerkinElmer Life Sciences).
After exposure, the cells were incubated with resazurin solution for 3 h to test for cytotoxicity, as for the T-screen assay. Media from these wells (200 µl) was transferred to black microplates (Costar, Corning) before fluorescence was measured.
Statistical Analyses
Nonprocessed and logarithmically transformed hormone and gene expression data were examined for normal distribution and homogeneity of variance. Data transformations showing normal distribution and homogeneity of variance were analyzed by one-way ANOVAs and, if significant, followed by the post hoc test, Dunnett's test. Significance was judged at p < 0.05. For statistical evaluation of pregnancy and litter data, the litter was considered as the statistical unit and the alpha level was 0.05. The results were analyzed by ANOVA, and in order to adjust for litter effects, litter was included in the analysis of variance as a nested random factor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The present study aims at clarifying the question of whether or not parabens have a mechanism of actions that resembles that of phthalates, that is, whether parabens have the ability to interfere with steroidogenesis. To assess whether parabens affect steroidogenesis, pregnant rats were dosed with from GD 7 to GD 21, as this previously have been shown to be a good time point for studying effects on steroidogenesis (Borch et al., 2006
; Laier et al., 2006).
In Vivo Effects
Pregnancy and litter data.
Neither ethyl paraben nor butyl paraben had any statistically significant effects on AGD, body weight or any other of the endpoints listed in Table 2.
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No significant toxic effects on fetuses or mothers were observed, except for one dam out of 10 in the ethyl paraben group that showed black amniotic fluid, and gave rise to 5% late resorptions (Table 2).
Hormone levels.
Plasma levels of progesterone, 17-hydroxyprogesterone, T3, and T4 were analyzed in the dams at GD 21, but neither ethyl paraben nor butyl paraben showed any statistically significant effects on any of the measured hormones (Table 3). Fetal plasma levels of T4 (data not shown), progesterone and testosterone were measured in both male and females, but there were no statistically significant effects for any of the tested parabens (Table 4). Testicular testosterone and progesterone levels, as well as ex vivo testicular testosterone and progesterone production were analyzed in male fetuses, but no effects were observed for either ethyl paraben or butyl paraben (Table 5). The level of estradiol was analyzed in fetal ovaries at GD 21, but no effects were found (data not shown).
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Cortisol levels were measured in adrenal glands from male fetuses (data not shown) as well as in supernatant fluid from incubated adrenal glands from both male and female fetuses. There were no significant effects observed for any of the measured cortisol levels, however, butyl paraben showed a tendency towards causing a dose-dependent reduction in the cortisol levels in the supernatant fluid from female adrenal glands (Table 4).
Gene expression.
Butyl paraben administration led to a statistically significant decrease in the expression level of ER-beta in ovaries from female fetuses (Fig. 1A) as well as a decrease in the expression of StAR and Bzrp in adrenal glands also from female fetuses (Fig. 1B). However, for Bzrp the decrease was only significant in the lowest dose group. In fetal ovaries there was a tendency of butyl paraben to cause a dose-dependent increase in the expression level of Compl.C3 (Fig. 1A), but this effect was not statistically significant. No effects were observed on the expression of any of the genes investigated in the testis (data not shown), nor were any effects observed on the gene expression in the adrenal glands from the male fetuses (Fig. 1B).
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Histopathology and immunohistochemistry.
Testes, adrenals and ovaries from paraben exposed fetuses and thyroids from exposed dams were histologically similar to controls. In testis sections, no differences were observed in the intensity of immunostaining for StAR, P450scc, 3β-HSD, and PBR/Bzrp (data not shown).
In Vitro Effects
The T-Screen assay.
The T-Screen assay is based on the T3 dependent proliferation of GH3 rat pituitary tumor cells. The assay is used to test a compounds ability to bind and activate the TR, thereby leading to proliferation of the GH3 cells.
The effect on proliferation of GH3 cells in vitro by ethyl and butyl paraben is illustrated in Figure 2. Ethyl paraben had no significant effect on GH3 cell growth. Butyl paraben was able to increase the effect of T3 as well as acting agonistic on its own. Butyl paraben increased the cell proliferation significantly from 10nM to approximately 300% of solvent control at 3000nM. Above 10µM both ethyl and butyl paraben caused a significant decrease in the cell proliferation due to cytotoxicity.
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H295R steroid synthesis assay.
To look for effects on steroidogenesis in vitro, we tested butyl paraben and ethyl paraben in the H295R steroid synthesis assay. Both butyl and ethyl paraben significantly increased the progesterone production at the highest concentration (30µM) tested (Fig. 3). Neither butyl paraben nor ethyl paraben had any effect on the production of testosterone or estradiol in H295R cells, although butyl paraben showed a tendency to decrease testosterone and estradiol production (Fig. 3). Cytotoxicity was not observed at any of the concentrations tested.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The objective of the current study was to address whether parabens have the ability to interfere with steroidogenesis. We focused on ethyl- and butyl paraben, as they represent a short and a long chain alkyl paraben. Several in vitro and in vivo studies have shown that many of the parabens are weakly estrogenic compounds, and some of the parabens, including butyl and propyl paraben, have also been shown to cause reduction in testosterone levels and sperm production in rats (Byford et al., 2002
; Okubo et al., 2001; Oishi, 2001, 2002; Pedersen et al., 2000; Routledge et al., 1998). It has been hypothesized that in utero exposure to weakly endocrine-active chemicals might be associated with a range of adverse effects on the developing fetuses, including abnormalities on the reproductive organs in males and increased risk of breast, testicular, or prostate cancer later in life. Kang et al. (2002) demonstrated that maternal exposure to butyl paraben during gestation and lactation affected the development of the reproductive organs and sperm count of the offspring. In the presented study the effects of butyl and ethyl paraben were investigated in an in utero exposure study, in which pregnant rats were dosed by subcutaneous injections from GD 7 to GD 21. There were no treatment-related effects of either ethyl paraben or butyl paraben on body weights, AGD or hormone levels. However, significant effects on gene expression in the female fetuses were observed. In the ovaries, butyl paraben exposure led to a significant decrease in the expression level of ER-beta as well as a tendency to an increase in Compl. C3 and IGF-1, that could all be a response to an estrogenic effect (Sundstrom et al., 1989; Suzuki et al., 2007). Butyl paraben also significantly decreased the expression of StAR and Bzrp in adrenal glands. This finding is supported by Bzrp being significantly down regulated by 30µM butyl paraben, in the H295R human adrenocortical carcinoma cells used in the H295R assay, after 48 h exposure (unpublished data). Both StAR and Bzrp are genes that code for proteins involved in regulation of the movement of cholesterol across the mitochondrial membranes, the rate-determining step in steroidogenesis (Papadopoulos et al., 1997; Stocco and Clark, 1997). A current model suggest that StAR binds cholesterol in the cytosol and transports it to the mitochondrial membrane where Bzrp is involved in the transport of cholesterol from the outer to the inner mitochondrial membrane (Niswender, 2002). Our results suggest that butyl paraben might have the ability to act as endocrine disruptor by interfering with the transport of cholesterol to the mitochondrion thereby interfering with steroidogenesis, but the results on gene expression needs to be confirmed on the protein level. Supporting the ability of butyl paraben to exert adverse effects is the study by Oishi et al. (2001) where butyl paraben administered to 3-week-old male rats caused reduced testosterone and sperm production.
Measurements of the levels of ethyl and butyl paraben in maternal plasma and amniotic fluids demonstrated the presence of both parabens, and in amniotic fluid both ethyl and butyl paraben were excreted in a dose-dependent manner (Frederiksen et al., 2008). This later result, in particular, contradicts the conclusion made by Hoberman et al. (2008), who conclude that parabens are not reproductive toxicants when administered by a relevant route, because they are completely metabolized by enzymes in the skin. In vitro in the adrenal H295R steroidogenesis assay, both ethyl and butyl paraben caused a significant increase in the progesterone formation. However, the increase in progesterone formation was only seen at the highest concentration tested (30µM), and that could explain why we do not see any increase in progesterone in the in utero study. In the T-screen assay, starting at a very low concentration, butyl paraben significantly increased the proliferation of the GH3 cells, thereby possibly acting as a weak thyroid hormone receptor agonist. However, no effects on thyroid hormone levels were observed in vivo. To explain the effects found in vitro and in vivo, more studies on the mechanism of action of the parabens are needed. Currently, it looks like the parabens could have multiple mechanisms of action, and that different mechanisms might be involved in different tissues. Our findings suggest that the female fetuses might be more sensitive to the two parabens than the male fetuses, but other time points of investigation, such as puberty, and dosing during lactation, should be performed. Further studies investigating the effects on the female reproductive system and on breast development in the female offspring would be interesting. Even though the current study show little sign of endocrine disrupting effects of ethyl- and butyl paraben, humans are exposed to several different chemicals simultaneously, and the combined effects induced by these different compounds might be additive (Hass et al., 2007; Rajapakse et al., 2002; Silva et al., 2002). Finally we can conclude that, ethyl and butyl paraben, do not resemble phthalates when it comes to potency in the selected in vitro and in vivo set-up.
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FUNDING |
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The Danish Environmental Protection Agency (grants no. 7041-0335, and 1231-0065).
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ACKNOWLEDGMENTS |
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We are indebted to Birgitte Møller Plesning, Heidi Letting, Morten Andreasen, Dorte Hansen, Ulla El-Baroudy and Lillian Sztuk for excellent technical assistance.
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