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. 2023 Oct 5;40(1):153–161. doi: 10.1007/s43188-023-00212-1

Comparative study on estrogen receptor alpha dimerization and transcriptional activity of parabens

Jong-Yeon Kim 1, Yooheon Park 2, Seok-Hee Lee 2, Eun‐Jung Park 3,4,✉, Hae‐Jeung Lee 1,3,4,5,6,✉
PMCID: PMC10786792  PMID: 38223674

Abstract

Parabens are used as preservatives in various household products, including oral products, cosmetics, and hair/body washes. In recent years, the widespread use of parabens has raised concerns due to the potential health risks associated with their estrogenic effects. In the present study, we evaluated and compared the estrogenic activity of parabens using two cell-based in vitro tests: (1) bioluminescence resonance energy transfer (BRET)-based estrogen receptor alpha (ERα) dimerization using HEK293 cells that were stably transfected with ERα‐fused NanoLuc luciferase (Nluc) and HaloTag (HT) expression vector, and (2) stably transfected transcriptional activation (STTA) assays using ERα-HeLa9903 cells. The following parabens were tested using the BRET‐based ERα dimerization assay and showed estrogenic activity (PC20 values): methyl paraben (MP, 5.98 × 10−5 M), ethyl paraben (EP, 3.29 × 10−5 M), propylparaben (PP, 3.09 × 10−5 M), butyl paraben (BP, 2.58 × 10−5 M), isopropyl paraben (IsoPP, 1.37 × 10−5 M), and isobutyl paraben (IsoBP, 1.43 × 10−5 M). Except MP, all other parabens tested using the STTA assay also showed estrogenic activity: EP, 7.57 × 10−6 M; PP, 1.18 × 10−6 M; BP, 3.02 × 10−7 M; IsoPP, 3.58 × 10−7 M; and IsoBP, 1.80 × 10−7 M. Overall, EP, PP, BP, IsoPP, and IsoBP tested positive for estrogenic activity using both assays. These findings demonstrate that most parabens, albeit not all, induce ERα dimerization and possess estrogenic activity.

Keywords: Paraben, Estrogenic activity, Bioluminescence resonance energy transfer (BRET)-based estrogen receptor alpha dimerization assay, Stably transfected transcriptional activation assay

Introduction

Parabens are alkyl esters homologous to 4-hydroxybenzoic acid and form a class of preservatives with antibacterial and antifungal properties [1]. Since the 1920s, they have been widely used in cosmetics, oral products, and pharmaceuticals. The most common parabens are methyl, ethyl, propyl, and butylparaben (MP, EP, PP, and BP, respectively) [2]. Although parabens possess favorable properties, such as high efficiency, low cost, and stability over a wide pH range, there have been controversies and uncertainties regarding their safety over the past 20 years due to the potential endocrine-disrupting effects [1, 3]. Endocrine-disrupting chemicals (EDCs) interfere with the endocrine system by mimicking or suppressing endogenous hormones [4]. Parabens could mimic estrogenic physiological activity and interfere with production of steroid hormones in vitro and [5, 6]. In addition, several in vivo studies have demonstrated that parabens change the weight of adrenal glands, thyroid, and reproductive organs [6–9]. These are the reasons parabens are considered EDCs.

Over the past two decades, the Organization for Economic Cooperation and Development (OECD) has been working to formulate new guidelines for screening and testing chemicals with endocrine-disrupting activity [10]. The OECD conceptual framework for the testing and assessment of EDCs comprises five levels, with an in vitro method providing mechanistic data in level 2 [11]. To evaluate the estrogenic effects of EDCs, estrogen receptor (ER) binding and ER transactivation assays are used. The OECD Test Guideline 455 (OECD TG 455) is related to the detection of ER agonists and antagonists using a stably transfected transcriptional activation (STTA) assay. In this assay, estrogenic activity is determined by measuring the extent to which a chemical substance that functions as a ligand can bind ERα and induce transcription to exhibit luciferase activity [12].

Subsequently, a bioluminescence resonance energy transfer (BRET)-based ERα dimerization assay was developed to effectively explore the estrogen signaling pathway [13]. Although OECD TG 493 is based on the ability of the radiolabeled ligand [3 H]17β-estradiol to bind ERα [14], this method can easily lead to radioactive contamination, which is difficult to mitigate. The BRET-based ERα dimerization assay, which can overcome certain drawbacks of the OECD TG 493 method, confirms the dimerization affinity of chemical substances for ERα; this method has been validated based on reliability and relevance data [13].

The European Union authorities have introduced regulations restricting the use of parabens as preservatives. Parabens in cosmetic products should not exceed a 0.4% concentration for a single ester and 0.8% concentration for a mixture of ester. In case of BP and PP, the sum of the individual concentration of them should not exceed 0.14% [3, 15, 16]. Likewise, the Ministry of Food and Drug Safety (MFDS) of South Korea has strictly managed the use of parabens, such as restricting MP and PP in toothpaste to 0.2% or less [17]. Nonetheless, the application of parabens remains controversial. Therefore, the present study explored the estrogenic activity and potential endocrine-disrupting effects of parabens by comparing their ERα dimerization and transcriptional activity.

Materials and methods

Test and reference chemicals

Parabens were used as the test chemicals. MP, EP, PP, BP, IsoPP, and IsoBP were purchased from Sigma-Aldrich (St. Louis, MO, USA). 17β-Estradiol (E2) and 4‐hydroxytamoxifen (OHT) were purchased from Sigma-Aldrich and used as positive controls (PCs). 17α‐Estradiol (αE2), corticosterone, and digitonin were purchased from Wako (Chuo‐Ku, Osaka, Japan) and used as reference chemicals. Stock solutions of all test and reference chemicals were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich) (Table 1).

Table 1.

Parabens and reference chemicals in BRET-based ERα dimerization and STTA assays

No. Chemical name CAS no. Stock concentration (M) Treatment concentration (M)
BRET-based ERα dimerization assay STTA assay
1 Methyl paraben (MP) 99-76‐3 10−1 10−10–10−4 10−10–10−4
2 Ethyl paraben (EP) 120-47‐8 10−1 10−10–10−4 10−10–10−4
3 Propyl paraben (PP) 94-13‐3 10−1 10−10–10−4 10−10–10−4
4 Butyl paraben (BP) 94-26‐8 10−1 10−10–10−4 10−10–10−4
5 Isopropyl paraben (IsoPP) 4191-73‐5 10−1 10−10–10−4 10−10–10−4
6 Isobutyl paraben (IsoBP) 4247-02‐3 10−1 10−10–10−4 10−10–10−4
7 17β-Estradiol (E2) 50-28‐2 10−2 10−13–10−7 10−14–10−8
8 4-Hydroxytamoxifen (OHT) 68392-35‐8 10−1 10−10–10−6 –
9 17α-Estradiol (αE2) 57-91‐0 10−2 – 10−12–10−6
10 Corticosterone 50-22‐6 10−1 10−10–10−4 10−10–10−4
11 Digitonin 11024-24‐1 10−1 10−4 –
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Cell culture

HEK293 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). In our previous study, we prepared a human ERα (hERα)-HEK293 cell line stably transfected with an ERα-fused NanoLuc luciferase (Nluc) and Tag (HT) expression vector [13]. hERα‐HEK293 cells were cultured in minimum essential medium (MEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) and 1% antibiotic-antimycotic (Gibco BRL) at 37 °C under 5% CO2. The cells were used within 20 passages to maintain the BRET response. The hERα‐HeLa9903 cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The cells were cultured in Eagle’s minimum essential medium (EMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% dextran‐coated charcoal treated FBS (DCC‐FBS; Access Biologicals LLC, Vista, CA, USA), sodium bicarbonate (7.5 w/v%; Gibco BRL), and l‐glutamine (200 mM; Gibco BRL) at 37 °C under 5% CO2. The cells were used within 40 passages.

BRET-based ERα dimerization assay

First, MEM without phenol red and containing 4% DCC-FBS and 1% antibiotic-antimycotic was prepared as the assay medium. hERα-HEK293 cells were seeded in 96-well white cell culture plates at the density of 2.5 × 105 cells·mL−1 in 80 µL of assay media and 10 µL of ligand (HaloTag® NanoBRET™ 618, Promega Madison, WI, USA). The cells were incubated for 1 h at 37 °C under 5% CO2, and serially diluted test and reference chemical solutions (10 µL; Table 1) were added to the cells. Opti‐MEM (Gibco BRL, Grand Island, NT, USA) containing 1% NanoBRET™ Nano‐Glo® Substrate (Promega, Wisconsin, USA) was prepared as the detection medium. After 1 h of incubation, 25 µL of the detection medium was added to the cells. Luminescence and fluorescence were measured using the GloMax multimode plate reader (Promega, Madison, WI, USA). Cytotoxicity of the chemicals was assessed using the CellTiter‐Glo® 2.0 Reagent (Promega, Madison, WI, USA) with the GloMax multimode plate reader (Promega, Madison, WI, USA).

STTA assay

hERα-HeLa9903 cells were seeded in 96-well white cell culture plate at the density of 1 × 104 cells per well. After 3 h of pre-incubation, serially diluted test and reference chemical solutions were added to the cells. The concentration ranges are shown in Table 1. After 20–24 h of incubation, the medium was removed, and 50 µL of luciferase assay solution (Steady‐Glo® Luciferase Assay System, Promega, Madison, WI, USA) was dispensed into each well. After incubating for 10 min in the dark, luciferase activity was measured using the GloMax multimode plate reader (Promega, Madison, WI, USA).

Statistical analysis

All data are presented as mean ± standard deviation of at least three independent experiments. In the BRET-based ERα dimerization assay, the paraben that accounted for 20% or more of the response induced by PC was classified as positive when it appeared more than two times in three independent experiments. The relative BRET unit (RBU) of the parabens was calculated as follows to assess dimerization activity: RBU (% to PC) = (mean background − corrected BRET unit of paraben/mean background − corrected BRET unit of PC) × 100. In the STTA assay, the paraben that accounted for 10% or more of the response induced by PC was classified as positive when it appeared more than two times in three independent experiments.

Results

ERα dimerization activity of parabens

To evaluate the ER dimerization activity of parabens, we conducted a BRET-based ERα dimerization assay. For the proficiency test, E2, OHT, and corticosterone were used as reference chemicals, and their fold induction passed the acceptable criteria (Table 2; Fig. 1).

Table 2.

Acceptable criteria of reference chemicals for BRET-based ERα dimerization assay

Chemical LogPC20 (M) Log PC20 (M) LogPC50 (M) Log PC50 (M)
Acceptable criteria Result Acceptable criteria Result
17β-Estradiol (E2) − 10.5 to − 9.1 − 9.82 – –
4-Hydroxytamoxifen (OHT) − 10.1 to − 9.2 − 9.74 − 9.6 to − 8.4 − 9.15
Corticosterone – – – –
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PC20, paraben concentration accounting for 20% of the response induced by the positive control OHT; PC50, Parabens concentration accounting for 50% of the response induced by the positive control OHT

Fig. 1.

Fig. 1

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Proficiency test results of BRET‐based ERα dimerization assay. The relative BRET unit of reference chemicals was expressed as the percentage of activity of the positive control (10−8 M OHT). All data are presented as mean ± SD. BRET, bioluminescence resonance energy transfer; OHT, 4- hydroxytamoxifen

All parabens (MP, EP, PP, BP, IsoPP, and IsoBP) showed ER dimerization activity, albeit weaker than OHT (Fig. 2), with the respective PC20 values of approximately 5.98 × 10−5, 3.29 × 10−5, 3.09 × 10−5, 2.58 × 10−5, 1.37 × 10−5, and 1.43 × 10−5 M (Table 3). Moreover, PC50 values for BP, IsoPP, and IsoBP were respectively 9.03 × 10−5, 8.65 × 10−5, and 8.52 × 10−5 M (Table 3).

Fig. 2.

Fig. 2

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Estrogen receptor dimerization activity of parabens in BRET‐based ERα dimerization assay. The relative BRET unit of A 4‐hydroxytamoxifen (OHT), B methyl paraben (MP), C ethyl paraben (EP), D propyl paraben (PP), E butyl paraben (BP), F isopropyl paraben (IsoPP), and G isobutyl paraben (IsoBP) was expressed as the percentage of activity of the positive control (10−8 M OHT). All data are presented as mean ± SD. BRET, bioluminescence resonance energy transfer

Table 3.

PC20 and PC50 values of parabens in BRET-based ERα dimerization assay

Chemical PC20 (M) PC50 (M)
4-Hydroxytamoxifen (OHT) 1.22 × 10−10 8.13 × 10−10
Methyl paraben (MP) 5.98 × 10−5 –
Ethyl paraben (EP) 3.29 × 10−5 –
Propyl paraben (PP) 3.09 × 10−5 –
Butyl paraben (BP) 2.58 × 10−5 9.03 × 10−5
Isopropyl paraben (IsoPP) 1.37 × 10−5 8.65 × 10−5
Isobutyl paraben (IsoBP) 1.43 × 10−5 8.52 × 10−5
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PC20, paraben concentration accounting for 20% of the response induced by the positive control OHT; PC50, paraben concentration accounting for 50% of the response induced by the positive control OHT

Estrogen receptor transcriptional activity of parabens

To evaluate the ER transcriptional activity of parabens, we conducted an STTA assay according to the OECD guidelines [12]. First, proficiency tests were performed with reference chemicals, namely E2, αE2, and corticosterone, and all passed the criteria (Table 4; Fig. 3). The performance standard curves of E2 and αE2, namely strong and weak estrogen, assumed a sigmoid shape, indicating the presence of estrogenic activities. Corticosterone did not exhibit any estrogenic activity.

Table 4.

Acceptable criteria of reference chemicals for STTA assay

Chemical LogPC10 (M) Log PC10 (M)  LogPC50 (M) Log PC50 (M)
Acceptable criteria Result Acceptable criteria Result
17β-Estradiol (E2) < − 11 − 11.6 − 11.4 to − 10.1 − 10.4
17α-Estradiol (αE2) − 10.7 to -9.3 − 9.6 − 9.6 to − 8.1 − 8.6
Corticosterone – – – –
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PC10, parabens concentration accounting for 10% of the response induced by the positive control E2; PC50, paraben concentration accounting for 50% of the response induced by the positive control E2

Fig. 3.

Fig. 3

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Proficiency test results of STTA assay. The luciferase activity of reference chemicals was expressed as the percentage of activity of the positive control (10−9 M E2). All data presented as mean ± SD. STTA assay, stably transfected transcriptional activation assay; E2, 17β‐estradiol

As PC, 1 nM E2 was used, and the ER transcriptional activity of the parabens was assessed based on PC10 or PC50 values. EP, PP, BP, IsoPP, and IsoBP, but not MP, showed estrogen receptor transcriptional activity, albeit weaker than E2 (Fig. 4); their respective PC10 values were approximately 7.57 × 10−6, 1.18 × 10−6, 3.02 × 10−7, 3.58 × 10−7, and 1.80 × 10−7 M, and their respective PC50 values were approximately 2.25 × 10−5, 2.69 × 10−6, 1.48 × 10−6, 1.59 × 10−6, and 1.22 × 10−6 M (Table 5). As a result, EP, PP, BP, IsoPP, and IsoBP were shown weaker estrogenic activity, which were approximately 5,300,000-fold, 830,000-fold, 210,000-fold, 250,000-fold, 130,000-fold, in comparison to E2.

Fig. 4.

Fig. 4

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Estrogen receptor transcriptional activity of parabens in STTA assay. The luciferase activity of A 17β‐estradiol (E2), B methyl paraben (MP), C ethyl paraben (EP), D propyl paraben (PP), E butyl paraben (BP), F isopropyl paraben (IsoPP), and G isobutyl paraben (IsoBP) was expressed as the percentage of activity of the positive control (10−9 M E2). All data are presented as mean ± SD. STTA assay, stably transfected transcriptional activation assay

Table 5.

PC10 and PC50 values of parabens in STTA assay

Chemical PC10 (M) PC50 (M)
17β-Estradiol (E2) 1.42 × 10−12 1.99 × 10−11
Methyl paraben (MP) – –
Ethyl paraben (EP) 7.57 × 10−6 2.25 × 10−5
Propyl paraben (PP) 1.18 × 10−6 2.69 × 10−6
Butyl paraben (BP) 3.02 × 10−7 1.48 × 10−6
Isopropyl paraben (IsoPP) 3.58 × 10−7 1.59 × 10−6
Isobutyl paraben (IsoBP) 1.80 × 10−7 1.22 × 10−6
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PC10, paraben concentration accounting for 10% of the response induced by the positive control E2; PC50, parabens concentration accounting for 50% of the response induced by the positive control E2

Comparison of ERα dimerization and estrogen receptor transcriptional activity to determine the estrogenic effects of parabens

We evaluated parabens that exhibited 20% or 10% activity relative to OHT and E2, respectively, as estrogenic activity-positive parabens and as final estrogenic activity-positive parabens if their PC20 and PC10 values appeared more than two times in three independent experiments.

While all parabens showed ER dimerization activity in the BRET-based ERα dimerization assay, five parabens, except MP, showed ER transcriptional activity in the STTA assay (Fig. 5).

Fig. 5.

Fig. 5

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Comparison of the estrogenic activity of parabens using BRET‐based ERα dimerization and STTA assays. Parabens that exhibited 20% or 10% activity relative to OHT or E2, respectively, were classified as positive for estrogenic activity. BRET, bioluminescence resonance energy transfer; ER, estrogen receptor; STTA assay, stably transfected transcriptional activation assay; OHT, 4-hydroxytamoxifen; E2, 17β‐estradiol

Discussion

To date, only a few studies have investigated the signaling pathway information gap between the binding of parabens to ERα and transcriptional activation, although many studies have investigated the estrogenic activity of parabens based on ERα binding and transcription assays. Therefore, in the present study, the ERα dimerization and transcriptional activity of six parabens were compared.

Parabens possess antifungal and antibacterial properties and are used as preservatives in food, cosmetics, and pharmaceuticals. According to the analysis of numerous available food ingredients and additives, MP, EP, and PP were detected predominantly compared to BP [18]. In addition, MP, EP, and PP are the predominant compounds detected in pharmaceuticals [19]. Parabens flowed into the body through skin absorption, food intake, and inhalation are readily transported into the bloodstream [20]. Although parabens are known to have relatively short half-life and are easily excreted through urine, there are some concerns due to endocrine disturbance effects, such as mimicking endogenous estrogens [20, 21]. Previous studies demonstrate that parabens show estrogenic activity as EDCs. Parabens acted as sulfotransferase (SULT) substrates, inhibited estrogen SULT, and increased estrogen levels, showing estrogenic effects in vitro study [22]. Animal studies have reported that mRNA expression level of Calbindin-D9K, which are associated with direct effects of E2, are increased in the uterus of rats exposed to MP, EP, PP, BP, IsoPP and IsoBP [23]. Moreover, a previous study showed the increased levels of BP in urine of pregnant women are correlated with decreased levels of E2 [24]. Simultaneously, sex hormone binding globulin levels increased as MP levels increased in urine [24]. In addition to endocrine disturbance effects, excessive exposure of parabens through the skin could be related to a risk of skin diseases such as malignant melanoma, itching, and atopic eczema [25–27].

E2, the most powerful estrogen, is involved in the overall physiological activity of the body and is mediated by ERα and ERβ [28]. ER binds estrogen in the cytosol and undergoes a conformational change to form a dimer [29]. The ligand–receptor complex is delivered to the nucleus, where it directly interacts with the estrogen response element (ERE), ultimately activating transcription [30]. OHT is a metabolite of tamoxifen, an antiestrogen, and is regarded as a selective ER modulator that functions as an antiestrogenic activator by competing with E2 for binding ERs [31, 32].

The BRET-based ERα dimerization assay was designed to measure the BRET signal generated when ERα binds a ligand, and its accuracy has been demonstrated in a previous study [13]. In contrast, the STTA assay is designed to detect the degree of luciferase activity when a chemical substance bind ERα in the HeLa 9903 cell line, which is transfected with hERα and firefly luciferase reporter genes containing five tandem EREs [11, 12].

Using BRET-based ERα dimerization assay, we confirmed that all tested parabens, including MP, showed weaker estrogenic activity than OHT. These findings are consistent with previous reports that parabens present weak estrogenic activity and compete for binding to human ERα [4, 33]. The STTA assay revealed that all parabens except MP produced weak estrogenic effects, and their PC10 values were in the order of IsoBP > BP > IsoPP > PP > EP. Similar patterns have been reported in other studies. As such, in transfected CHO‐K1 cells, ERα agonistic activity was in the order of IsoBP > BP > IsoPP > PP > EP [34]. Other studies have found that the estrogenic potency of parabens increases with increasing length of the alkyl side chain [34–36].

Furthermore, we comprehensively compared ERα dimerization and transcription assay results for parabens and found that the results of the two assays were inconsistent. MP induced ERα dimerization but did not transactivate ERα. Previous studies have reported different results regarding the relative binding affinity of MP. Specifically, the in vitro binding affinity of MP was lower than that of EP, and the IC50 (the concentration of parabens required to inhibit the binding of E2 to ERα by 50%) for MP was very low to calculate [4]. In contrast, Blair et al. reported an IC50 value of 2.45 × 10–4 M for MP in a competitive binding assay to E2 [36]. Furthermore, MP has been reported to exhibit stronger estrogenic activity in silico [37]. However, our results indicate that distinguishing estrogen agonists based solely on physical binding is difficult, and this requires further clarification.

Humans are primarily exposed to parabens through personal care products (PCPs). Parabens (detection frequency for MP, EP, PP, and BP were > 90, 50–70, 100, and 17–67%, respectively) were detected on PCP from China, including face cream, body/hand lotion, and face cleanser [38]. Parabens exposure in the body are usually detected in urine. The geometric concentrations of parabens detected in urine of Korean adults are 165 ng/mL (MP), 46.0 ng/mL (EP), 17.7 ng/mL (PP), and 1.96 ng/mL (BP) [39]. Concentrations at which estrogenic activity was exhibited in this study were higher than geometric concentrations detected in urine in previous study. However, concentrations used in vitro study may not accurately convey the effects of parabens to clinically detected concentrations because metabolism may vary depending on the exposure route [40]. Moreover, test methods proposed by the OECD were evaluated for the purpose of selecting and prioritizing EDC, the concentration of paraben used in vitro estrogenic assay cannot be directly compare with the permissible limit of the product, which is a limitation of this study.

In summary, we compared the estrogenic activity of parabens using BRET-based ERα dimerization and STTA assays. All parabens induced ERα dimerization, although not all parabens that induced ERα dimerization also showed transcriptional activity. These findings may contribute to a better understanding of the estrogenic activity and potential endocrine-disrupting effects of parabens via the ER signaling pathway.

Funding

This work was supported by “Cooperative Research Program of Center for Companion Animal Research (project no. PJ01398403), Rural Development Administration, Republic of Korea.

Data availability

The data could be obtained upon reasonable request to the corresponding author.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Eun‐Jung Park, Email: ejpark@gachon.ac.kr.

Hae‐Jeung Lee, Email: skysea1010@gmail.com, Email: skysea@gachon.ac.kr.

References

  • 1.Engeli RT, Rohrer SR, Vuorinen A, Herdlinger S, Kaserer T, Leugger S, Schuster D, Odermatt A. Interference of paraben compounds with estrogen metabolism by inhibition of 17beta-hydroxysteroid dehydrogenases. Int J Mol Sci. 2017;18:2007. doi: 10.3390/ijms18092007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Costa JR, Campos MS, Lima RF, Gomes LS, Marques MR, Taboga SR, Biancardi MF, Brito PVA, Santos FCA. Endocrine-disrupting effects of methylparaben on the adult gerbil prostate. Environ Toxicol. 2017;32:1801–1812. doi: 10.1002/tox.22403. [DOI] [PubMed] [Google Scholar]
  • 3.Nowak K, Ratajczak-Wrona W, Gorska M, Jablonska E. Parabens and their effects on the endocrine system. Mol Cell Endocrinol. 2018;474:238–251. doi: 10.1016/j.mce.2018.03.014. [DOI] [PubMed] [Google Scholar]
  • 4.Okubo T, Yokoyama Y, Kano K, Kano I. ER-dependent estrogenic activity of parabens assessed by proliferation of human breast cancer MCF-7 cells and expression of ERalpha and PR. Food Chem Toxicol. 2001;39:1225–1232. doi: 10.1016/s0278-6915(01)00073-4. [DOI] [PubMed] [Google Scholar]
  • 5.Mitra P, Chatterjee S, Paul N, Ghosh S, Das M (2021) An overview of endocrine disrupting chemical paraben and search for an alternative–a review. In: Proceedings of the zoological society, vol 4. Springer, pp 479–493. 10.1007/s12595-021-00418-x
  • 6.Vo TT, Yoo YM, Choi KC, Jeung EB. Potential estrogenic effect(s) of parabens at the prepubertal stage of a postnatal female rat model. Reprod Toxicol. 2010;29:306–316. doi: 10.1016/j.reprotox.2010.01.013. [DOI] [PubMed] [Google Scholar]
  • 7.Chuffa LG, Lupi-Junior LA, Costa AB, Amorim JP, Seiva FR. The role of sex hormones and steroid receptors on female reproductive cancers. Steroids. 2017;118:93–108. doi: 10.1016/j.steroids.2016.12.011. [DOI] [PubMed] [Google Scholar]
  • 8.Oishi S. Effects of butylparaben on the male reproductive system in rats. Toxicol Ind Health. 2001;17:31–39. doi: 10.1191/0748233701th093oa. [DOI] [PubMed] [Google Scholar]
  • 9.Oishi S. Lack of spermatotoxic effects of methyl and ethyl esters of p-hydroxybenzoic acid in rats. Food Chem Toxicol. 2004;42:1845–1849. doi: 10.1016/j.fct.2004.06.015. [DOI] [PubMed] [Google Scholar]
  • 10.Beekhuijzen M, Rijk JCW, Meijer M, de Raaf MA, Pelgrom S. A critical evaluation of thyroid hormone measurements in OECD test guideline studies: is there any added value? Reprod Toxicol. 2019;88:56–66. doi: 10.1016/j.reprotox.2019.07.014. [DOI] [PubMed] [Google Scholar]
  • 11.Lee HS, Park EJ, Han S, Oh GY, Kim MH, Kang HS, Suh JH, Oh JH, Lee KS, Hwang MS, Moon G, Hong JH, Hwang IG. In vitro OECD test methods applied to screen the estrogenic effect of chemicals, used in Korea. Food Chem Toxicol. 2016;95:121–127. doi: 10.1016/j.fct.2016.06.014. [DOI] [PubMed] [Google Scholar]
  • 12.OECD (2021) Test No. 455: Performance-based test guideline for stably transfected transactivation in vitro assays to detect estrogen receptor agonists and antagonists. 10.1787/9789264265295-en
  • 13.Lee SH, Seo H, Seo H, Lazari M, D’Agostino M, Byrd N, Yoon KS, Lee HS, Park Y. An in vitro dimerization assay for the adverse outcome pathway approach in risk assessment of human estrogen receptor alpha-mediated endocrine-disrupting chemicals. Chemosphere. 2022;290:133267. doi: 10.1016/j.chemosphere.2021.133267. [DOI] [PubMed] [Google Scholar]
  • 14.OECD (2015) Test No. 493: Performance-based test guideline for human recombinant estrogen receptor (hrER) in vitro assays to detect chemicals with ER binding affinity. 10.1787/9789264242623-en
  • 15.Union P. Regulation (EC) no 1223/2009 of the european parliament and of the council. Off J Eur Union L. 2009;342:59. [Google Scholar]
  • 16.Authority E Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to para hydroxybenzoates (E 214–219) EFSA J. 2004;2:83. doi: 10.2903/j.efsa.2004.83. [DOI] [Google Scholar]
  • 17.Lim S. The associations between personal care products use and urinary concentrations of phthalates, parabens, and triclosan in various age groups: the Korean National Environmental Health Survey cycle 3 2015–2017. Sci Total Environ. 2020;742:140640. doi: 10.1016/j.scitotenv.2020.140640. [DOI] [PubMed] [Google Scholar]
  • 18.Liao C, Chen L, Kannan K. Occurrence of parabens in foodstuffs from China and its implications for human dietary exposure. Environ Int. 2013;57–58:68–74. doi: 10.1016/j.envint.2013.04.001. [DOI] [PubMed] [Google Scholar]
  • 19.Ma WL, Zhao X, Lin ZY, Mohammed MO, Zhang ZF, Liu LY, Song WW, Li YF. A survey of parabens in commercial pharmaceuticals from China and its implications for human exposure. Environ Int. 2016;95:30–35. doi: 10.1016/j.envint.2016.07.013. [DOI] [PubMed] [Google Scholar]
  • 20.Fransway AF, Fransway PJ, Belsito DV, Yiannias JA. Paraben toxicology. Dermatitis. 2019;30:32–45. doi: 10.1097/DER.0000000000000428. [DOI] [PubMed] [Google Scholar]
  • 21.Nowak K, Jablonska E, Garley M, Iwaniuk A, Radziwon P, Wolczynski S, Ratajczak-Wrona W. Investigation of estrogen-like effects of parabens on human neutrophils. Environ Res. 2022;214:113893. doi: 10.1016/j.envres.2022.113893. [DOI] [PubMed] [Google Scholar]
  • 22.Prusakiewicz JJ, Harville HM, Zhang Y, Ackermann C, Voorman RL. Parabens inhibit human skin estrogen sulfotransferase activity: possible link to paraben estrogenic effects. Toxicology. 2007;232:248–256. doi: 10.1016/j.tox.2007.01.010. [DOI] [PubMed] [Google Scholar]
  • 23.Vo TT, Jeung EB. An evaluation of estrogenic activity of parabens using uterine calbindin-d9k gene in an immature rat model. Toxicol Sci. 2009;112:68–77. doi: 10.1093/toxsci/kfp176. [DOI] [PubMed] [Google Scholar]
  • 24.Aker AM, Watkins DJ, Johns LE, Ferguson KK, Soldin OP, Del Anzalota LV, Alshawabkeh AN, Cordero JF, Meeker JD. Phenols and parabens in relation to reproductive and thyroid hormones in pregnant women. Environ Res. 2016;151:30–37. doi: 10.1016/j.envres.2016.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kim S, Carson KA, Chien AL. Methyl paraben may increase risk of pruritus in African Americans whereas Triclosan is inversely associated with pruritus and eczema. Dermatitis. 2021;32:124–130. doi: 10.1097/DER.0000000000000495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Matwiejczuk N, Galicka A, Zareba I, Brzoska MM. The protective effect of rosmarinic acid against unfavorable influence of methylparaben and propylparaben on collagen in human skin fibroblasts. Nutrients. 2020 doi: 10.3390/nu12051282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Handa O, Kokura S, Adachi S, Takagi T, Naito Y, Tanigawa T, Yoshida N, Yoshikawa T. Methylparaben potentiates UV-induced damage of skin keratinocytes. Toxicology. 2006;227:62–72. doi: 10.1016/j.tox.2006.07.018. [DOI] [PubMed] [Google Scholar]
  • 28.Yasar P, Ayaz G, User SD, Gupur G, Muyan M. Molecular mechanism of estrogen-estrogen receptor signaling. Reprod Med Biol. 2017;16:4–20. doi: 10.1002/rmb2.12006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Negi A, Kesari KK, Voisin-Chiret AS. Estrogen receptor-alpha targeting: PROTACs, SNIPERs, Peptide-PROTACs, antibody conjugated PROTACs and SNIPERs. Pharmaceutics. 2022 doi: 10.3390/pharmaceutics14112523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Seo H, Seo H, Lee HY, Lee SH, Park Y. Comprehensive analysis of cellular estrogen signaling in representative estrogen receptor ligands. Chem Biol Interact. 2023;369:110303. doi: 10.1016/j.cbi.2022.110303. [DOI] [PubMed] [Google Scholar]
  • 31.Borgna JL, Rochefort H. High-affinity binding to the estrogen receptor of [3H]4-hydroxytamoxifen, an active antiestrogen metabolite. Mol Cell Endocrinol. 1980;20:71–85. doi: 10.1016/0303-7207(80)90095-7. [DOI] [PubMed] [Google Scholar]
  • 32.Katzenellenbogen BS, Katzenellenbogen JA. Estrogen receptor transcription and transactivation: estrogen receptor alpha and estrogen receptor beta: regulation by selective estrogen receptor modulators and importance in breast cancer. Breast Cancer Res. 2000;2:335–344. doi: 10.1186/bcr78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Byford JR, Shaw LE, Drew MG, Pope GS, Sauer MJ, Darbre PD. Oestrogenic activity of parabens in MCF7 human breast cancer cells. J Steroid Biochem Mol Biol. 2002;80:49–60. doi: 10.1016/s0960-0760(01)00174-1. [DOI] [PubMed] [Google Scholar]
  • 34.Watanabe Y, Kojima H, Takeuchi S, Uramaru N, Ohta S, Kitamura S. Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor alpha and beta and androgen receptor. Food Chem Toxicol. 2013;57:227–234. doi: 10.1016/j.fct.2013.03.036. [DOI] [PubMed] [Google Scholar]
  • 35.Routledge EJ, Parker J, Odum J, Ashby J, Sumpter JP. Some alkyl hydroxy benzoate preservatives (parabens) are estrogenic. Toxicol Appl Pharmacol. 1998;153:12–19. doi: 10.1006/taap.1998.8544. [DOI] [PubMed] [Google Scholar]
  • 36.Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, Tong W, Shi L, Perkins R, Sheehan DM. The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol Sci. 2000;54:138–153. doi: 10.1093/toxsci/54.1.138. [DOI] [PubMed] [Google Scholar]
  • 37.Guadarrama P, Fomine S, Salcedo R, Martinez A. Construction of simplified models to simulate estrogenic disruptions by esters of 4-hydroxy benzoic acid (parabens) Biophys Chem. 2008;137:1–6. doi: 10.1016/j.bpc.2008.06.001. [DOI] [PubMed] [Google Scholar]
  • 38.Guo Y, Wang L, Kannan K. Phthalates and parabens in personal care products from China: concentrations and human exposure. Arch Environ Contam Toxicol. 2014;66:113–119. doi: 10.1007/s00244-013-9937-x. [DOI] [PubMed] [Google Scholar]
  • 39.Kang HS, Kyung MS, Ko A, Park JH, Hwang MS, Kwon JE, Suh JH, Lee HS, Moon GI, Hong JH, Hwang IG. Urinary concentrations of parabens and their association with demographic factors: a population-based cross-sectional study. Environ Res. 2016;146:245–251. doi: 10.1016/j.envres.2015.12.032. [DOI] [PubMed] [Google Scholar]
  • 40.Calafat AM, Ye X, Wong LY, Bishop AM, Needham LL. Urinary concentrations of four parabens in the U.S. population: NHANES 2005–2006. Environ Health Perspect. 2010;118:679–685. doi: 10.1289/ehp.0901560. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Data Availability Statement

The data could be obtained upon reasonable request to the corresponding author.

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