Side Chains of Parabens Modulate Antiandrogenic Activity: In Vitro

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Side Chains of Parabens Modulate Antiandrogenic Activity: In Vitro and Molecular Docking Studies Keke Ding, Xiaotian Kong, Jingpeng Wang, Liping Lu, Wenfang Zhou, Tingjie Zhan, Chunlong Zhang, and Shulin Zhuang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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Environmental Science & Technology

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Side Chains of Parabens Modulate Antiandrogenic Activity: In

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Vitro and Molecular Docking Studies

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Keke Ding†, Xiaotian Kong‡, Jingpeng Wang†, Liping Lu†, Wenfang Zhou§, Tingjie

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Zhan†, Chunlong Zhang⊥, Shulin Zhuang†, *

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†

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310058, China

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‡

College of Environmental and Resource Sciences, Zhejiang University, Hangzhou

Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University,

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Suzhou, Jiangsu 215123, P. R. China.

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§

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China

⊥

Department of Biological and Environmental Sciences, University of Houston-Clear

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Lake, 2700 Bay Area Blvd., Houston, Texas 77058, USA.

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* Corresponding author: College of Environmental and Resource Sciences, Zhejiang

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University, Hangzhou 310058, China.

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Email address: [email protected] (S Zhuang)

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ACS Paragon Plus Environment


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ABSTRACT: Parabens have been widely used in packaged foods, pharmaceuticals

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and personal-care products. Considering their potential hydrolysis, we herein

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investigated structural features leading to the disruption of human androgen receptor

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(AR) and whether hydrolysis could alleviate such effects using the recombinant yeast

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two-hybrid assay. Parabens with an aryloxy side chain such as benzyl paraben and

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phenyl paraben have the strongest antiandrogenic activity. The antiandrogenic activity

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of parabens with alkyloxyl side chains decreases as the side chain length increases

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from 1 to 4, and no antiandrogenic effect occurred for heptyl, octyl and dodecyl

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parabens with the number of alkoxyl carbon atoms longer than 7. The antiandrogenic

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activity of parabens correlates significantly with their binding energies (R2 = 0.84, p =

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0.01) and were completely diminished after the hydrolysis, particularly for parabens

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with aryloxy side chains. The Km for the hydrolysis of parabens with aromatic moiety

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side chain is one order of magnitude higher than that of the parabens with alkyl side

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chains. Both in vitro and in silico data, for the first time, suggest parabens with

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aromatic side chains are less prone to hydrolysis. Our results provide an insight into

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risk of various paraben and considerations for design of new paraben-related

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substitutes.

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INTRODUCTION

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Parabens (PBs) are 4-hydroxybenzoic acid (HbA) esters with aryloxy or alkoxyl

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moieties in different lengths as side chains (Table 1). They are widely used as

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anti-microbial preservatives in packaged foods, pharmaceuticals and personal-care

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products.1-3 The concentrations of total parabens in 215 cosmetic products were as

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high as 0.01% - 0.87% (w/w),4 with the concentration of up to 0.4% for a single

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paraben.5 Parabens are commonplace in our daily exposure and they can be absorbed

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through human skin (the dermal intake of total parabens is estimated to be 31.0

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µg/kg-bw/day for adult female in the U.S.)2 and gastrointestinal tract.6 Parabens can

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also be bioaccumulated and biomagnified through food chains due to the relatively

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high lipophilicity.7 The extensive use of parabens frequently leads to the detection of

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residues in multiple environmental matrices, including surface waters, soils,

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sediments, indoor air and dust. 8-10 Parabens were also frequently detected in breast

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milk, urine and serum with concentrations at µg/L levels.6, 11-14

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The ubiquitous presence of parabens in the environment has raised public concern

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about whether they potentially cause adverse reproductive effects and their safety has

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been widely discussed.15-17 Exposure to parabens was reported to cause estrogenic

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disrupting effects.18-23 Ethyl- and butyl parabens caused a significant increase in

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progesterone formation in the adrenal H295R steroidogenesis assay and interfered

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with the transport of cholesterol to the mitochondria.24 There has been a growing

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concern in recent years about the health risk of parabens to the androgen hormone

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system.25-27 Parabens induced an adverse effect on the male reproductive system

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through reduced testosterone concentrations and sperm production in rats,28

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potentially by influencing the biosynthesis, transport, and metabolism of steroid

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hormone.24 Evaluation of the androgen receptor (AR) disruption is thus of

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significance for better risk assessment of various paraben analogues.

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There are two major types of side chains of parabens including the alkyl and

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aromatic chains (Table 1). Whether the length of paraben side chains will affect their

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potential health risk to androgen system remains largely unexplored. Structural

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analogues of many environmental contaminants have been examined to determine

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how a minor change in structure governs their human health risk.24, 29-33 For example,

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minor changes in the bridged carbon of several bisphenol A substitutes correlated well

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with experimentally measured toxicities and hormonal activities.29, 34, 35 The binding

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affinity of twelve perfluorinated carboxylic acids to human liver fatty acid binding

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protein increased significantly with their carbon number from 4 to 11, and decreased

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slightly when the number was over 11.36 To date studies relevant to the risk of a

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homolog series of parabens are limited. 37, 38 The side chains of parabens can be

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removed via hydrolysis in human tissues and multiple matrices (air, water, soil, or

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sediment), possibly affecting their environmental risks. 8, 39, 40 Till now, much still

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remains unknown as to whether and how the hydrolysis of side chains of parabens

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disrupts human AR. The influence of the sizes of side chains on their hydrolysis and

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the associated kinetic parameters are essential to the risk assessment of parabens.

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Such information on the effect of different side chains before and after the hydrolysis

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is of significance for better health risk assessment of parabens.

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In the present study, we investigated the effect of parabens with different sizes of

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alkyl and aromatic side chains on their induction of AR disruption and the impact of

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hydrolysis on this effect through an in vitro removal of side chains via

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esterase-mediated hydrolysis. Molecular docking was conducted to elucidate the

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contribution of different side chains of parabens to their interactions with AR ligand

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binding domain (LBD). We revealed the significant role of aromatic moiety toward

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the AR disrupting effect of parabens. Our results provided a structure activity

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relationship (SAR) to decipher how hydrolytic metabolism and side chain change of

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parabens can exert considerable difference in activity and potential risk. Such

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information is crucial to assess the risk of various paraben-containing consumer

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products and design environmentally benign paraben substitutes by considering the

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multiple adverse effects induced by various local molecular moieties.

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MATERIALS AND METHODS

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Chemicals. Parabens (purity ≥ 98%) with different side chain moieties (Table 1)

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were purchased from J&K Chemicals Corporation (Shanghai, China). Dimethyl

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sulfoxide (DMSO, 99.5%) and dihydrotestosterone (DHT, 98%) were purchased from

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Sigma Chemical (St. Louis, MO, USA). Porcine liver esterase (PLE, Catalogue:

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E3019-20KU) was also purchased from Sigma Chemical (St. Louis, MO, USA).

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SD/-Leu/-Trp medium was purchased from Mobitec Company (Catalogue: 4823-6).

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The constituents in mobile phase including water, methanol and acetic acid were

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chromatographic grade. All other chemicals were analytical grade. All parabens were

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dissolved in DMSO and diluted using deionized water (18.2 MΩ, Millipore, Bedford, 5



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MA) for activity evaluation. All prepared solutions were stored at 4 oC in centrifuge

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tubes. Recombinant Yeast Two-Hybrid Assay. The recombinant two-hybrid yeast

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system employed yeast cells transformed with the human AR plasmid, coactive

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plasmid, and the reporter gene expressing β-galactosidase. pGBT9 AR plasmid was

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provided by Dr. Erik Jan Dubbink (Erasmus University, Holland) and the yeast

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transformed with the pGBT9AR plasmid was provided by Prof. Zijian Wang

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(Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,

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Beijing, China). The transfection efficiencies of the yeast and positive control samples

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were greater than 4.5 × 104 cfu / µg. The blank control was 0, and there was a

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significant difference between the positive and negative controls (p PnP > MeP > EtP > PrP > BuP >

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HbA. This agrees with the previous report that MeP, PrP and BuP inhibited

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testosterone-induced transcriptional activity by 40%, 33% and 19%, respectively, at a

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concentration of 10 µM using human embryonic kidney (HEK) 293 cells that lack

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critical steroid metabolizing enzymes.47 Although parabens have no predominant

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antiandrogenic effect at the low concentration, their bioaccumulation and

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biomagnification through food chains may lead to the higher concentration in humans,

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thereby inducing potentially higher adverse health risks.

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With the 10 analogues of parabens tested, some structural dependency of the

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antiandrogenic activity could be inferred. The antiandrogenic activity potency could

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be associated with the length of the ester alkyl side chain of parabens. The AR

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disrupting potency decreased with an increased alkyl side chain length from 1 to 4

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(MeP > EtP > PrP > BuP), whereas the lipophilicity and their penetration through

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human epidermis-dermis layers are in the reverse order (MeP < EtP < PrP < BuP).48

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No significant AR disrupting potency was found for HeP, OcP and DdP with the alkyl

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side chain length equals to 7, 8 and 12, respectively. This is an interesting observation

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for parabens with alkyl side chains, which implies that the adverse effects of MeP

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with its highest antiandrogenic activity can be reduced to the greatest extent by the

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nature of its low lipophilicity and dermal penetration. The parabens with aromatic

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side chain have distinct disrupting effect in comparison with parabens with alkyl side

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chain. Side chains with an aromatic moiety showed a stronger AR disrupting potency

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compared with the alkyl side chains. These parabens with the aromatic side chain are

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less abundant (e.g., 15% in baby care products) 2 than MeP, EtP, and PrP but their

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higher logKow values might potentially aggravate their adverse health hazards. This

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observed structural dependence agrees with an acute aquatic toxicity assessment of

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parabens toward Vibrio fischeri, in which the acute toxicity value (EC50) of benzyl

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paraben was 3.8 µg/L (0.017 µM), 2 to 3 orders of magnitude lower than that of

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parabens with alkyl side chains.49 Therefore, the different side chains of parabens

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have significant influence on its androgenic disrupting effect and the aromatic moiety

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of the side chain contributes predominantly to the observed toxicity.

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Side Chains Affect the Molecular Recognition toward AR LBD. To elucidate

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why the side chain of different sizes affects the AR disruption effect, the molecular

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docking was applied to examine their interactions with AR LBD at the atomic level.

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The ligand in the crystal structure (PDB entry: 1Z95) was extracted and re-docked

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into the binding pocket. The RMSD between the predicted binding pose and the

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respective experimental structure was calculated.50-54 The low RMSD (0.49 Å)

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suggests Glide docking can accurately reproduce the experimental binding poses. A

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series of parabens and DHT were then all automatically docked into the binding

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cavity of AR LBD. The docking free energy and three decomposed energy

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components including hydrophobic interactions (∆E ), electrostatic interactions

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(∆E

) and van der Waals interactions (∆E ) are listed in Table S1. DHT has the

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smallest docking score compared with the parabens, indicating its strongest binding

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affinities with AR LBD. The energy components of DHT to AR LBD including

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∆E , ∆E

and ∆E also have the most negative values. Besides DHT, the

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binding potency of parabens to AR LBD was in the order of BzP > PnP > MeP > EtP >

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PrP > BuP > HbA > HeP > OcP. The docking scores appear to be relevant to the

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antiandrogen of parabens corresponding to the structural variations of different side

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chains. Consequently, the correlation between Glide docking energy and the

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maximum antiandrogenic activity of parabens were performed. As shown in Figure 2,

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there exists a significant linear correlation between the docking energy and the

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antiandrogenic activity (R2 = 0.84, p = 0.01). The parabens with the smaller docking

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score have the stronger antiandrogenic activities, further indicating the agreement of

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our in vitro and in silico data based on yeast assay and molecular docking.

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We examined the surrounding amino acids and binding sites of parabens to AR

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LBD. Before we conducted the molecular docking of parabens to AR LBD, the

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natural androgen hormone DHT was first docked into the binding cavity of AR LBD.

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Our results suggested that two amino acids Thr877 and Asn705 were involved in

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forming hydrogen bonds between DHT and AR LBD (Figure S2). This agrees with a

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previous study on the interactions of DHT with AR LBD,55 hence validating our

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molecular docking approach. As shown in Figure 3, benzyl-, phenyl paraben and the

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alkoxyl parabens with the number of carbon atom equals to 1 to 4, 7 and 8 were

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docked into the AR LBD, while the larger side chain (e.g., dodecyl paraben) could not

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be docked into the AR LBD (data not shown). The reason why dodecyl paraben could

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not be docked into the AR LBD attributes to the relatively bulkier volume of dodecyl

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paraben which is too large to fit the binding cavity of AR LBD. The hydrogen bonds

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were also formed between the hydroxyl oxygen of parabens and two amino acids

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(Thr877 and Asn705), similar to the binding mode of DHT to AR LBD. The HbA

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formed the hydrogen bonds with the amino acids Gln711 and Arg752 of AR LBD and

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did not show any antiandrogenic effect. These results clearly suggested that the

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parabens capable of binding Thr877 and Asn705 of AR LBD can compete with DHT,

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thereby causing the antiandrogenic activity. The HbA and OcP formed hydrogen

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bonds with Arg752 and Gln711, but did not show any antiandrogenic activity. This

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further suggests that Thr877 and Asn705 are the amino acids vital to the induction of

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the antiandrogenic activity. It is worth noting that heptyl paraben did not show any

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antiandrogenic activity. Nevertheless, heptyl paraben was docked into the AR LBD

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and formed the hydrogen bond with Thr877 and Asn705. This could be explained by

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the fact that the yeast two-hybrid system is not sufficiently sensitive to respond to the

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very weak AR disrupting effect of heptyl paraben.

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The Hydrolysis Reduced Antiandrogenic Activities. It was reported that the

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metabolism of parabens by carboxylesterases in rat liver microsomes markedly

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reduced the estrogenic activity.56 As important Phase I metabolizing enzymes, the

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esterase enzymes play an important role in the hydrolysis of various exogenous

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contaminants. The porcine liver esterase was chosen in this study to determine

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whether the hydrolysis of parabens will influence the potential for androgen

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disruption in vitro. Six parent parabens with obvious AR disrupting effects were

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subjected to hydrolysis, including MeP, EtP, PrP, BuP, PnP and BzP. In the absence of

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porcine liver esterase, the antiandrogenic activity remained unchanged when

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incubating parent parabens with the incubation solutions, demonstrating the

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incubation solutions did not influence AR disruption. In the presence of porcine liver

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esterase, antiandrogenic activities were tested following the hydrolysis of six parabens

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at 5, 25 and 50 µM by comparing antiandrogenic activities before and after the

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hydrolysis. As shown in Figure 4, the antiandrogenic activities of parent parabens

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(benzyl, phenyl, methyl, ethyl, propyl and butyl paraben) at three concentrations of 5,

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25 and 50 µM were all significantly decreased after the incubation for 2 h. The

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metabolic products from the hydrolysis of six parent parabens did not show any

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noticeable AR inhibition, indicating a very weak antiandrogenic activity and a

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significant hydrolysis deactivation. A similar metabolic deactivation of paraben

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toward ERα and ERβ was confirmed by incubating butyl paraben with rat liver

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microsomes.56 Besides hydrolysis, parabens can also be metabolized via other

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pathways such as photochemical transformation, phase I and phase II metabolism.

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Parabens were reportedly detected in the conjugated form

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(glucuronidation/sulfonation) in vivo and their activities were markedly decreased.12

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The androgenic disrupting effect of other forms of paraben metabolites should be

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considered in future study.

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The Hydrolysis Products Identified by HPLC-UV and UPLC-MS/MS. To

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further identify the metabolites of parabens after hydrolysis, the products of parabens

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were firstly qualified using HPLC-UV and UPLC-MS/MS. Representative

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chromatograms of parent parabens, including MeP, EtP, PrP, BuP, BzP and PnP, are

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shown in Figure S3 (A) - (F), and the retention times of the corresponding parabens

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are 10.08, 11.35, 13.89, 18.64, 6.75 and 8.22 min, respectively. After a 2 h incubation

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with four individual parabens (MeP, EtP, PrP and BuP) and porcine liver esterase at 25

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o

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peak at the retention time of around 9.00 min, similar to the chromatogram of a

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standard containing HbA under the same chromatographic condition. The

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corresponding UV-Vis absorbance spectrum is characterized by a maximum

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absorbance wavelength at 254.6 nm, also similar to the UV-Vis spectrum of the

C, the peak of each parent paraben completely disappeared and all presented a new

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standard HbA. For PnP and BzP, however, after 2 h incubation with porcine liver

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esterase, the enzymatic hydrolysis products peaks can only be resolved by changing

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the mobile phase to 65% methanol and 35% acetic acid (0.01%, V/V). As shown in

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Figure S3 (e) and (f), the peak of hydrolysis products in the presence of porcine liver

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esterase was observed at the retention time of 8.98 and 9.00 min for the incubation of

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phenyl and benzyl paraben, respectively. The corresponding UV-Vis spectrum of

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hydrolysis product also has a maximum absorbance wavelength at 254.6 nm, similar

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to the UV-Vis spectrum of standard HbA. The chromatograms of six parabens

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obtained from UPLC-MS/MS were the same to the above HPLC-UV analysis with the

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metabolite peak at the retention time of around 9.00 min, validating the

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chromatographic separation of the products. The precursor ion of metabolite was [M -

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H]- with the m/z around 137 Da (Figure 5), one Da lower than HbA with a molecular

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weight of 138 Da. The m/z values of corresponding daughter ion were around 93 Da

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([M - H - 44]-), in agreement with the loss of a 44 Da (CO2). They matched the

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retention time and MS2 spectra with the standard HbA, indicating that only the ester

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bond was cleaved for these parabens. The peak at the retention time of about 9.00 min

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from the hydrolysis of six parent parabens can be identified as HbA.

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The hydrolysis products of six parabens did not display the antiandrogenic

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activity at the test concentrations, in line with the antiandrogenic activity of HbA

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standard (Figure 1). Our multiple lines of evidence indicate the formation of HbA

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product after the hydrolysis of BzP, PnP, MeP, EtP, PrP and BuP. These results were

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supported by the reported pathways of paraben hydrolysis using skin microsomes and

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cytosol from human and minipigs.57 The formation of HbA induced negligible

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antiandrogenic activities of paraben. The dissipated antiandrogenic activities after the

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hydrolysis mediated by porcine liver esterase are consistent with the observed

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cleavage of the side chain of parabens.

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The Hydrolysis Rate was Side Chain-Dependent. To determine the effects of

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parabens with different side chains on the rate of hydrolysis mediated by porcine liver

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esterase, the commonly used Lineweaver-Burk plot between 1/V0 and 1/S was applied

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to determine the maximum rate of reaction (Vmax) and the specificity of enzyme

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porcine liver esterase for parabens with different side chains. The Lineweaver-Burk

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plot displayed a good linear correlation based on the R2 values ranging from 0.9958 to

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0.9997 (Figure S4). The Km and Vmax calculated from the slopes and intercepts are

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shown in Table 2. The Vmax values were 0.09 - 0.19 mM/min for alkyl parabens and

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3.43 - 4.91 mM/min for parabens with aromatic side chains, suggesting the very quick

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hydrolysis rate mediated by porcine liver esterase for alkyl parabens. This high rate

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suggests that the rapid metabolism may prevent these parabens from accumulating in

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biological tissues. Hydrolysis rate in porcine liver esterase may differ from that in

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human plasma likely due to the different enzymatic concentration and enzyme

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specificity. The Km values for MeP, EtP, PrP, BuP, PnP and BzP incubated with

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porcine liver esterase were 0.40 ± 0.01, 0.33 ± 0.02, 0.27 ± 0.02, 0.17 ± 0.04, 6.00 ±

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0.28 and 8.23 ± 0.15 mM, respectively (Table 2). It was suggested that the Km values

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decreased with the increasing alkyl side chain length of parabens, indicating the

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parabens with the longer alkyl side chains have the lower hydrolysis rates. The Km

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values of parabens with aromatic moiety side chain have over one order of magnitude

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higher than that of the parabens with alkyl side chains, demonstrating that parabens

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with alkyl side chains are the preferred substrate compared with the parabens with

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aromatic side chains for porcine liver esterase. In contrast to parabens with aromatic

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side chain moieties, parabens with alkyl side chains are more susceptible to hydrolysis

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by porcine liver esterase. These results strongly indicated that the parabens with an

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aromatic moiety in their side chains tend to be bioaccumulated more readily and

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hence pose a greater threat to organisms.

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ENVIRONMENTAL IMPLICATIONS

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The present study for the first time provides both the in vitro and in silico evidence on

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the structure dependent human AR disrupting effects of parabens. It becomes

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increasingly clear that certain parabens can exert adverse biological activities and

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their environmental and human health risk should be of concern. Although direct

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extrapolation from such in vitro and in silico data should be cautious, the effect

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concentrations observed in this study should help further delineation of human health

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exposure. The parabens with aromatic side chain moieties have a stronger AR

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disrupting potency compared with the alkyl side chains, indicative of the aromatic

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moiety of parabens as the toxicophore toward AR disruption. Parabens with aromatic

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moieties in the side chains seem to have slower hydrolysis rates and could lead to a

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higher bioaccumulation and hence a greater threat to organisms. Insight into the

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structure dependence of antiandrogenic activities and other biological activities of

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parabens is valuable to formulate and design safe parabens and their substitutes for 18


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use in consumer products. Parabens-containing consumer products can be better

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formulated by modulating the compositions of the primary parabens (i.e., methyl-,

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ethyl- and propyl parabens) to ensure their safe uses. Our results will be beneficial to

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understand the environmental transformation and fate of paraben esters and provide

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the guideline for the production of paraben-containing products with low human

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toxicity and high efficiency in preservation as antibacterial agents.

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ASSOCIATED CONTENT

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Supporting Information

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Table S1: The decomposition of binding free energies. Table S2: The amino acids

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involved with hydrogen bonds and its binding energy between parabens and AR LBD.

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Figure S1: Androgenic activity of ten parabens evaluated by two-hybrid yeast assay.

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Figure S2: The binding mode of DHT to AR LBD. Figure S3: High performance

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liquid chromatogram of parent parabens and products from hydrolysis mediated by

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porcine liver esterase. Figure S4: The Lineweaver-Burk plots in describing the

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kinetics of hydrolysis of parabens incubated with porcine liver esterase. The

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Supporting Information is available free of charge on the ACS Publications website.

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AUTHOR INFORMATION

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Corresponding Author

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* E-mail: [email protected]. Phone: +86-571-8898-2344.

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ORCID

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Shulin Zhuang: 0000-0002-7774-7239

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416

Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

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The authors thank the National Natural Science Foundation of China (No. 21477113)

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and the Major Research Plan of the National Natural Science Foundation of China

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(No. 91643107). CZ acknowledged the Welch Foundation for partial support.

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606 607 608 609 610 611 612 613

28


Page 28 of 37

Page 29 of 37


614

615

Table 1. The CAS numbers, structures and Log Kow of the test parabens.

a

Structure

Log Kow a

Compound

CAS

4-hydroxybenzoic acid (HbA)

99-96-7

1.58

methyl paraben (MeP)

99-76-3

1.96

ethyl paraben (EtP)

120-47-8

2.47

propyl paraben (PrP)

94-13-3

3.04

butyl paraben (BuP)

94-26-8

3.57

heptyl paraben (HeP)

1085-12-7

4.83

octyl paraben (OcP)

1219-38-1

5.43

dodecyl paraben (DdP)

2664-60-0

7.40

phenyl paraben (PnP)

17696-62-7

3.21

benzyl paraben (BzP)

94-18-8

3.56

The values of Log Kow were calculated by the U.S. EPA EPI Suite version 4.1.

616 617 618 619 29



Page 30 of 37

620

Table 2. Parameters (Km, Vmax) of the Lineweaver-Burk plots for the hydrolysis

621

kinetics of parabens. Km (mM)

Vmax (mM/min)

R2

methyl paraben

0.40 ± 0.01

0.19 ± 0.02

0.9976

ethyl paraben

0.33 ± 0.02

0.17 ± 0.01

0.9981

propyl paraben

0.27 ± 0.04

0.13 ± 0.02

0.9958

butyl paraben

0.17 ± 0.04

0.09 ± 0.01

0.9675

phenyl paraben

6.00 ± 0.28

3.43 ± 0.10

0.9939

benzyl paraben

8.23 ± 0.15

4.91 ± 0.08

0.9997

Compounds

622 623 624 625 626 627 628 629 630 631 632 633 634 635 636

30


Page 31 of 37


637

Figure Legends

638

Figure 1. The antiandrogenic activity of paraben analogues measured by the yeast

639

two-hybrid assay. The antiandrogenic activity was expressed as the values of

640

β-galactosidase activity (U) for positive control substrate of the samples divided by

641

the values of U for the positive control. Values were measured from three independent

642

experiments and were presented as the mean ± standard error. The symbol * stands for

643

the significant difference in comparison with the control (p < 0.05).

644

Figure 2. The linear regression curves for the maximum antiandrogenic activity of

645

parabens vs. the calculated binding energy.

646

Figure 3. The binding mode of parabens to AR-LBD. The protein is shown in cartoon

647

form. Parabens and surrounding residues are represented in sticks form. The dashed

648

lines indicate the formation of hydrogen bonds.

649

Figure 4. The antiandrogenic activity of parabens before and after hydrolysis

650

mediated by porcine liver esterase. The symbol * stands for the significant difference

651

in comparison without the incubation with porcine liver esterase (p < 0.05).

652

Figure 5. Mass spectrometric fragmentation of metabolite formed after the hydrolysis

653

of parent parabens. (A) and (B) are the mass spectra in full-scan and MS2 modes,

654

respectively.

655 656 657

31



Page 32 of 37

658

Figure 1

659 660 661 662 663 664 665 666 667 668 669 670 671 672 673

32


Page 33 of 37


674

Figure 2

675 676 677 678 679 680 681 682 683 684 685 686 687 688

33



Page 34 of 37

689

690 691

Figure 3

692 693 694 695 696 697 698 699 700 701 702 703

34


Page 35 of 37


704

705

Figure 4

706 707 708 709 710

35



Page 36 of 37

711

Figure 5

712 713 714 715 716 717 718 719 720 721 722 723 36


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724

Table of Contents Graphic

725

726

37


Side Chains of Parabens Modulate Antiandrogenic Activity: In Vitro

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