Phthalates Induce Androgenic Effects at Exposure Levels That Can Be

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Ecotoxicology and Human Environmental Health

Phthalates induce androgenic effects at exposure levels that can be environmentally relevant in humans Meiping Tian, Liangpo Liu, Heng Wang, Xiaofei Wang, Francis Luke Martin, Jie Zhang, Qingyu Huang, and Heqing Shen Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00138 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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Environmental Science & Technology Letters

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Phthalates Induce Androgenic Effects at Exposure Levels that

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can be Environmentally Relevant in Humans

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Meiping Tiana,b, Liangpo Liua, Heng Wanga, Xiaofei Wanga, Francis L.

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Martinc, Jie Zhanga, Qingyu Huanga, Heqing Shena*

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a

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Academy of Sciences, Xiamen 361021, China;

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b

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Beijing 100049, China;

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c

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Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese

College of Resources and Environment, University of Chinese Academy of Sciences,

School of Pharmacy and Biomedical Sciences, University of Central Lancashire

(UCLan), Preston PR1 2HE, UK

11 12 13 14 15 16

*

Corresponding Author: Heqing Shen, Institute of Urban Environment, Chinese

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Academy of Sciences, 1799 Jimei Road, Xiamen 361021, China; Telephone/Fax:

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(86)-592-6190771; E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Background: Although anti-androgenic activity of various lipophilic chain phthalate

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acid esters (PAEs) has been reported in high-dose animal studies, their male

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reproductive risk remains a matter of debate because of conflicting epidemiological

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observations. Recently, we showed that PAEs acted as a preventative factor in male

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infertility, which implies these chemicals are androgenic in human steroidogenesis.

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Methods: To verify the androgenic observation, a reproductive age healthy male

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cohort (n=84) was recruited by following a cross-sectional study design, in which the

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infertility or clinical selection-introduced bias was avoided. Urine was used for both

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PAEs exposure monitoring and androgen measurements and sampling uncertainty was

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greatly reduced. Eight selected metabolites (i.e., MMP, MEP, MBP, MEHP, MBzP,

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MEHHP, MECPP and MEOHP) and two androgens, i.e., androstenedione (ASD) and

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testosterone, were measured by using HPLC-MS/MS.

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Results: Except for MBzP, the selected phthalates can be detected in all samples at

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concentrations (median [5th-95th percentile]) of 36.4 [2.0-261.0], 36.7 [5.6-318.5],

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75.3 [13.1-301.0], 3.2 [1.1-10.2], 3.8 [0.6-11.9], 13.6 [1.6-51.1] and 7.4 [0.9-31.8]

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ng/mL for MMP, MEP, MBP, MEHP, MEOHP, MECPP and MEHHP, respectively.

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Urinary PAEs metabolites generally correlated with ASD and testosterone in positive

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ways; the trends are most significant for MMP, MEP, MBP and ∑DEHP versus ASD

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and for ∑DEHP versus testosterone.

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Conclusion: This study observes that the phenotypic effect of our participants’

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exposure to PAEs at the typical environmental relevant exposure level is androgenic,

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which counters the notion of the well-accepted anti-androgenic effect.

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Keywords: Androgen; Biphasic effect; Endocrine effect; Leydig cell; Phthalate

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1. Introduction

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Phthalate acid esters (PAEs) are a group of chemicals possessing the same core

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phthalic acid structure but differing lipophilic side chains. Their physicochemical

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properties are ideally suited to applications in industrial and consumer products,

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including plasticizers, food packaging materials, toys, personal care products and

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medical devices1, 2. The non-covalent binding of phthalates in many product matrices

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means they can easily leach out of these matrices thus becoming bioavailable to

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humans and the environment via a variety of exposure routes3-5. Many reports have

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confirmed that PAEs and their corresponding metabolites are measurable in human

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bodily fluids and tissues including blood, urine, saliva, amniotic fluid, breast milk,

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cord blood and placenta6-10.

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Consequent human health issues arise from the observation that many PAEs

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appear to be anti-androgenic endocrine disruptors, with some inducing marked effects

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on reproduction and development1, 5. PAEs’ alkanol moieties are associated with their

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endocrine disruption, which apparently showed structurally specific inhibition to

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testosterone production in testis11, 12. Although some epidemiological studies show

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that phthalate exposure is negatively associated with sex hormone secretion,

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anogenital distance (AGD) and semen quality parameters, there are some important

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inconsistencies in other human studies8, 13-16. Toxicological tests

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contradicting mechanism that underpins human observations. More recently some

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reports note that rather than inhibiting androgen production, some phthalates may be

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pro-androgenic and apparently induce earlier pubarche in boys, which does not reflect

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17-19

also suggest a


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an anti-androgenic mode of action20, 21. We also observed that urinary PAEs were

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positively associated with preventative metabolomics markers of male infertility

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Therefore, how environmentally relevant PAEs levels translate to risks of human male

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reproduction remains obscure.

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.

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The urinary matrix includes both sex hormones and PAEs residuals and has been

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employed for screening/diagnosis of disorders of steroidogenesis and human PAEs

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exposure monitoring22-25. Herein, some selected PAEs and androgens were measured

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using the same urine spot from the healthy participants, who may have been exposed

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to phthalates from a range of equivocal sources but not occupational. To verify human

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observations, phthalate-induced steroidogenesis change was mapped using a Leydig

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cell model, which were treated by both diester and monoester phthalate mixtures at

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environmentally relevant levels [see Supporting Information (SI)]. Associations

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between urinary PAEs and excreted steroid metabolites were investigated for mining

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PAEs’ risk translation to androgen reproduction.

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2. MATERIALS AND METHODS

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Population Demography and Sample Collection. All the participants come from the

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same institute who were taking part their annual physical examinations in a hospital in

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Xiamen, China; the reproductive age healthy male subjects were enrolled in the

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present study. After giving the informed consent, all the subjects (n=84) who had

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lifestyles, diet and an environment that remained unchanged for several months prior

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to the sample collection were involved. Their information, including demographics

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(age, height, weight, etc.), lifestyle habits (smoking, drinking and plastic tableware

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and/or disposable plastic cup use in daily life with value YES/NO) and education

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status were recorded during sampling and listed in Table 1. Participants were

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recommended fasting for at least 8 hours before physical examinations. All

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participants contributed one sanguinis urina sample each towards the urine spot test,

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in the middle part of a morning before eating or drinking. After collection, samples

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were stored in glass bottles at -80°C until further analysis of phthalate metabolites and

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selected androgens.

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PAE metabolites and steroid hormones measurements. Detailed information on

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chemicals and stock solutions for measurements are provided in Supporting

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Information SI-1. Urinary and MLTC-1 cell culture medium phthalate metabolites

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were

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chromatography-electrospray-ionization coupled with tandem mass spectrometry

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(LC-ESI-MS/MS; Applied Biosystems/MDS SCIEX, Singapore) method25. Eight

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phthalate metabolites, i.e., MMP, MEP, MBP, MBzP, MEHP and its oxidative

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metabolites MEHHP, MECPP and MEOHP, were measured. The contents of the total

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species (the conjugated monoester phthalates plus their free forms) were analysed by

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a modified method25. Briefly, urine samples were initially spiked with the

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mass-labelled internal standards and deconjugated using E. coli β-glucuronidase, then

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purified by a SPE cartridge (Oasis HLB column, Waters, USA) and finally analysed

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by the isotope dilution method using LC-ESI-MS/MS. Two method blanks, two

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quality control samples (human urine spiked with known amounts of phthalate

detected

using

the

published

isotope

5


dilution

liquid


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metabolites) and two sets of calibration standards were analysed concurrently with the

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unknown samples in each analytical batch or run. All details are provided in the

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Supporting Information (Table S1-4).

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Urinary and MLTC-1 cell medium androgens of androstenedione (ASD) and

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testosterone were detected by LC-ESI-MS/MS. In brief, each sample of 1 mL urine

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was diluted with 3 mL of ammonium acetate buffer (1 mol/L), and 20 µL of 100

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ng/mL D3-testosterone was added as the internal standard (ASD was semi-quantified).

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Then the diluted samples were extracted by adding 3 mL ethyl acetate and vortexing

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vigorously for 15 seconds in a glass tube. The liquid-liquid extraction was repeated 3

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times. Phase separation was achieved by centrifugation at 1500 rpm for 10 min. The

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ether phase was transferred to another glass tube with a Pasteur pipette. The

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three-time extract was combined and evaporated under a gentle stream of nitrogen gas

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at 40oC. The residue was reconstituted with 200 µL of methanol/water (50:50, v:v) by

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vortexing vigorously for 15 seconds, and transferred into a HPLC vial. The sample

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was stored at -20oC until LC-MS-MS analysis. Two quality control samples and two

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sets of standards were analysed together with the unknown samples in each analytical

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batch.

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Statistical analysis. Statistical analysis was performed using SPSS 19.0 statistical

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package (SPSS Inc., Chicago, IL, USA). The external individual exposure to diester

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PAEs is calculated on the related urinary monoester counterparts. For example, DEHP

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is reconstructed by using the summed molar concentrations of MEHP, MEOHP,

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MECPP and MEHHP and then expressed in mass concentrations and indicated as

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ΣDEHP. ΣPAEs was the mass sum of individual phthalate metabolite of MMP, MEP,

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MBP, MEHP, MEOHP, MECPP and MEHHP. Given the low detection frequency

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(64.3%) of MBzP, it was not used in the further statistics. Due to urinary phthalates

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and testosterone steroid hormone data were not normally distributed, the logarithmic

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transformation was applied before the further analysis. Then the transformed data

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were assessed by Kolmogorov-Smirnov test and normal Q-Q plot to verify their

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distribution normality (Figure S1). Multiple linear regression was used to assess the

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association between PAEs and steroid hormones, while the relevant confounders of

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age, BMI, smoking, alcohol drinking and plastic usage were adjusted. To show

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dose-dependent steroid hormone changes, phthalates data were grouped into tertiles

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and Welch’s t-test was applied to check the statistical hypothesis. For all tests,

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p-values < 0.05 were considered as significant.

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3. RESULTS AND DISCUSSION

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Human urinary phthalate metabolite excretion. Eight urinary metabolites of

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diester phthalates, i.e., MMP, MEP, MBP, MBzP, MEHP, MEOHP, MECPP and

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MEHHP, were measured as indicators of the participants’ internal exposure to

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phthalates (Table 2). Except the MBzP, the selected phthalate metabolites were

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detected in all 84 samples with concentrations in range from 1.02 to 1432 ng/mL.

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Consistent with the previous reported exposure levels5, 8, 21, 26, 27, our data show that

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while non-occupational PAEs exposure in the general population is low, it is to the

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multiple phthalates simultaneously. Meanwhile, the short straight alkyl chain MMP,

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MEP or MBP concentration were higher than the branched alkyl chain MEHP and

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aromatic alkyl chain MBzP, mapped their different environmental exposure risk.

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Environmental relevant phthalate exposure induced male androgenic effects.

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Multiple linear regression was used to predict the androgens, in which PAE

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metabolites were treated as tertile category variables and continuous variables,

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respectively. These PAE predictors were adjusted by age, BMI, alcohol intake,

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smoking and plastic usage in both category models and continuous models. For the

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category models (Figure S2), ASD in the 3rd tertiles of MMP, MEP, MBP, MEHP and

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∑PAEs are significant higher than in the 1st tertiles, respectively, which appears to be

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dose-dependently increased across the tertiles. With regards to testosterone, only

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positive associations with ∑DEHP and ∑PAEs are observed (p-values for trend are

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0.005 and 0.002, respectively). For the continuous models (Figure 1), MMP, MEP,

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MBP, ∑DEHP and ∑PAEs were all positively associated with ASD, while only

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∑DEHP was positively associated with testosterone (Table S5).

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Although previous human studies had demonstrated the associations between the

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increased phthalate exposure (i.e., MBP and MEHP) and reduced serum androgens in

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reproductive age males8,

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exposure (i.e., MBP, MEHP and MBzP) and serum reproductive hormones27, while

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others even suggest that phthalate exposure (i.e., MBP and MBzP) in boys positively

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correlates with testosterone and the earlier age at pubarche21. We also suspected that

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the male infertility risk arising from exposure to PAEs remains inconclusive when

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these chemicals are assessed as anti-androgens22, 25.

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26

, other studies show no association between phthalate

Previous studies mainly focused on the production of testosterone measured in

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serum,

while

the

equally

important

male

developmental

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dihydrotestosterone (DHT) and ASD were neglected28. Because urine contains the

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precursors, intermediates and end-products of sex and corticosteroid hormone23, our

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study examined the production of testosterone with considering its key upstream

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intermediate of ASD. To the best of our knowledge, the present study is the first

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demonstration of positive associations between urinary PAEs and ASD in urine.

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Because only associations between ∑DEHP versus testosterone were positively

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observed in the same urine samples, we suggest that ASD is more sensitive than

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testosterone as a response to phthalate exposure. These data are in line with urinary

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cadmium (Cd) excretion associated with increased synthesis of urinary sex steroids in

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an environmentally-relevant low-dose Cd exposure population study23. The present

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observations imply that PAEs’ endocrine effects do not appear to be anti-androgenic,

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but instead androgenic with increasing steroid hormone production in urine. More

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interestingly, in addition to testosterone, our data indicates that ASD, the upstream

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intermediate of testosterone, is a biomarker to indicate PAEs’ effects on the human

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endocrine system, the results suggested its potential use in risk assessment.

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Phthalate-related androgenic effects in human can be illustrated by an initial

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phase action in a biphasic effect on steroidogenesis in Leydig cells. Because

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Leydig cells are the primary cells of androgen production in male23; to verify the

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observed androgenic effect in humans, we tested their response to phthalates by using

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a MLTC-1 model, which is mouse Leydig tumour cells; these have been widely used

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in steroidogenesis assessments29. In camparsion to the relevant rat cell line, the

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androgens

of


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MLTC-1 model appears to be more similar to human Leydig cells in their responses

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to PAEs stimulation29. A conceptual workflow of matching human observations and

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laboratory tests on metabolic phenotype is suggested (Figure S3). Our in vitro results

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were obtained when the cells were treated with PAEs at doses exhibiting no

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cytotoxicity (Figure S4). High-dose phthalates (100 µM) generated anti-androgenic

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effects with low-dose exposure (0.1-10 µM) stimulating androgen production; the

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biphasic action of DBP on steroidogenesis has been clearly profiled.

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The present in vitro biphasic action agrees with previous reports. For example,

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DEHP and MEHP stimulated testosterone production and advanced onset of puberty

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at low-doses, whereas they were anti-androgenic at high-dose exposures30. At

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hypothetical serum levels of phthalates corresponding to a toxic dose on target Leydig

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cells, the maximum levels that serum phthalate metabolites can be converted occurs

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according

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serum-to-urine ratios (S/U ratios) of phthalate concentrations are: MEP = 0.036, MBP

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= 0.239, and MEHP = 0.786. Therefore, all the present participants were exposed to

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phthalates

Phthalates Induce Androgenic Effects at Exposure Levels That Can Be

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