Elsevier

Toxicology

Volume 232, Issue 3, 11 April 2007, Pages 248-256
Toxicology

Parabens inhibit human skin estrogen sulfotransferase activity: Possible link to paraben estrogenic effects

https://doi.org/10.1016/j.tox.2007.01.010Get rights and content

Abstract

Parabens (p-hydroxybenzoate esters) are a group of widely used preservatives in topically applied cosmetic and pharmaceutical products. Parabens display weak associations with the estrogen receptors in vitro or in cell based models, but do exhibit estrogenic effects in animal models. It is our hypothesis that parabens exert their estrogenic effects, in part, by elevating levels of estrogens through inhibition of estrogen sulfotransferases (SULTs) in skin. We report here the results of a structure-activity-relationship of parabens as inhibitors of estrogen sulfation in human skin cytosolic fractions and normal human epidermal keratinocytes. Similar to reports of paraben estrogenicity and estrogen receptor affinity, the potency of SULT inhibition increased as the paraben ester chain length increased. Butylparaben was found to be the most potent of the parabens in skin cytosol, yielding an IC50 value of 37 ± 5 μM. Butylparaben blocked the skin cytosol sulfation of estradiol and estrone, but not the androgen dehydroepiandrosterone. The parabens were also tested as inhibitors of SULT activity in a cellular system, with normal human epidermal keratinocytes. The potency of butylparaben increased three-fold in these cells relative to the IC50 value from skin cytosol. Overall, these results suggest chronic topical application of parabens may lead to prolonged estrogenic effects in skin as a result of inhibition of estrogen sulfotransferase activity. Accordingly, the skin anti-aging benefits of many topical cosmetics and pharmaceuticals could be derived, in part, from the estrogenicity of parabens.

Introduction

Parabens (p-hydroxybenzoate esters) are a group of preservatives widely used in cosmetic, food and pharmaceutical products (Fig. 1). These compounds have a broad spectrum of antimicrobial activity. Toxicology studies in rodents and humans indicate that parabens are practically non-toxic, non-irritating and non-sensitizing (Elder, 1984). Parabens are approved as additives in topical products by the United States Food and Drug Administration (FDA, 1979). Until recently, paraben potency, stability and worldwide regulatory acceptance made them an attractive choice as preservatives in topical cosmetics and pharmaceuticals. However, many manufacturers are reducing the use of parabens because of a growing body of evidence that parabens are estrogenic (Reisch, 2005). The topical formulation industry became even more averse to using parabens after researchers reported that parabens were present in breast cancer tissue (Darbre et al., 2004).

Various in vitro and cell based assays have shown parabens can compete with estrogens for binding to the estrogen receptors. Other assays have shown parabens to be estrogenic, albeit much less potent than estradiol. Each study reported an increase in estrogenicity with increasing chain length of paraben ester. Parabens were also shown to stimulate the proliferation of MCF-7 breast cancer cells in culture (Okubo et al., 2001, Byford et al., 2002). The most compelling support for paraben estrogenicity comes from an in vivo experiment where rats were dosed both orally and subcutaneously with butylparaben (Routledge et al., 1998). The orally dosed parabens were inactive, most likely due to rapid ester hydrolysis during first-pass metabolism. Orally ingested parabens are readily absorbed through the gut, hydrolyzed, conjugated and then excreted in the urine without significant accumulation in the body (Soni et al., 2005). Only the hydrolysis product p-hydroxybenzoic acid, which has no estrogenic activity, and its conjugates were identified in the blood and urine (Elder, 1984). However, subcutaneous injection of butylparaben did cause a significant estrogenic response.

The above stated differences in paraben in vivo activity are suggested to result from evasion of first-pass metabolism by subcutaneous injection (Routledge et al., 1998). Topical application of parabens would also evade first-pass metabolism encountered during oral administration. The physicochemical properties allow parabens to readily penetrate the stratum corneum of the skin, and then be retained in the epidermal layers of the skin, because of their low aqueous solubility. Epidermal tissue does contain esterase activity, but at much lower levels than the liver (Prusakiewicz et al., 2006). Human skin epidermal extracts are able to hydrolyze parabens, although at rates much lower than other tissues (Lobemeier et al., 1996). Therefore, the increased stability of parabens in skin would stimulate the endogenous estrogen signaling cascades. Paraben containing products may be chronically applied over a period of years. Thus, the frequency and duration of paraben exposure and estrogenicity may be continuous.

Estrogenic signal transduction plays a multitude of roles in skin physiology. The benefits of continuous estrogenic stimuli in the skin can be evaluated in women taking hormone replacement therapies. Studies of post-menopausal women suggest that epidermal estrogens function to retain collagen, water content, elasticity, thickness and promote wound healing (Shah and Maibach, 2001). Skin expresses the enzymatic machinery needed to convert circulating androgens and estrogen sulfates into active estradiol. As a result, this organ generates most of the extraglandular estrogen (Nelson and Bulun, 2001). Estrogen metabolizing enzymes, such as sulfotransferases (SULTs), are also expressed in the skin (Dooley et al., 2000).

It is well known that SULT enzymes can be inhibited by various xenobiotics and environmental chemicals (Wang and James, 2006). Many of the “natural” compounds that exhibit inhibitory potency towards SULTs are dietary chemicals such as flavonoids and catechins. Fruit juices and extracts of red wine, which contain high levels of phenols and quercetin, have been shown to contain SULT inhibitory activity (Gibb et al., 1987, De Santi et al., 2002). SULT enzymes play a major role in facilitating the biotransformation of many pharmaceuticals. However, some drugs such as mefenamic acid, diclofenac and salicylic acid inhibit SULT activity (Vietri et al., 2000). Contact with other SULTs inhibitors can come from environmental exposure to polychlorinated biphenyls and other polyhalogenated aromatic hydrocarbons (i.e., dioxins). These compounds are normally poor substrates, but potent inhibitors. Hydroxylated metabolites of polyhalogenated aromatic hydrocarbons contain a phenolic moiety similar to parabens, and are well-known endocrine disrupters with estrogenic effects. Hydroxylated polychlorinated byphenyls have low affinity for the estrogen receptors, but are potent inhibitors of estrogen SULTs (Kester et al., 2000, Kester et al., 2002).

Although parabens display weak associations with the estrogen receptors, they still exhibit estrogenic effects. It is our hypothesis that parabens inhibit estrogen SULTs in skin and exert their estrogenic effects, in part, by elevating levels of estrogens. We report here the results of a structure-activity-relationship of parabens as inhibitors of estrogen sulfation in human skin and liver. The potency of SULT inhibition increased as the ester chain length increased. The parabens were inhibitors of SULT activity in a cellular system with normal human epidermal keratinocytes (NHEKs). Thus, chronic topical application of parabens may lead to prolonged estrogenicity in skin through inhibition of estrogen SULT activity.

Section snippets

Reagents

Unless otherwise stated chemical reagents, chromatographic materials and recombinant SULT1A1*2 were purchased from Sigma–Aldrich (St. Louis, MO). The internal standard estradiol-sulfate-d4 was obtained from CDN Isotopes (Pointe-Claire, Quebec, Canada). Pooled human liver cytosolic fraction was obtained from BD Genetest (San Jose, CA).

Preparation of skin fractions

Light female human skin from reduction mammoplasty procedures was obtained from the University of Michigan Section of Plastic and Reconstructive Surgery (Ann

Inhibition of estradiol sulfation by parabens in vitro

Preliminary experiments were performed to titrate the protein and substrate concentrations, while optimizing reaction times to ensure pseudo-first order kinetics (<10% substrate conversion). We observed substrate-dependent inhibition at micromolar concentrations of estradiol, which is in agreement with the previous reports (Hobkirk et al., 1989, Gamage et al., 2005). Therefore, we used a substrate concentration of 250 nM, which gave maximal enzyme velocity and is consequently near published Km

Discussion

Estrogens are very important hormonal regulators in the skin. They function to preserve collagen content, which supports and maintains elasticity. Stimuli from estrogens also promote thickening of the skin, and initiate wound healing (Shah and Maibach, 2001). In fact, skin tissue contains all of the enzymes needed to process androgens and estrogen sulfates into estradiol, the most active endogenous estrogen (Fig. 7). Estrogen SULTs are also present in skin, and act to down-regulate the levels

Conflict of interest statement

There are no conflicts of interest for any authors.

Acknowledgements

We would like to thank Pfizer PDM Dermatology members, especially Arun Agrawal, Ann Payne and Gwen Fate for helpful discussions and critical examination of the manuscript.

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