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Chemosphere
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Chemosphere
Transcriptional changes in steroidogenesis by perfluoroalkyl acids (PFOA and PFOS) regulate the synthesis of sex hormones in H295R cells
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Jae Soon Kang ª, Jin-Soo Choi ª, June-Woo Park a, b, *
a Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology, Jin-Ju, Gyeongnam, Republic of Korea
b Human and Environmental Toxicology Program, Korea University of Science and Technology (UST), Daejeon, Republic of Korea
HIGHLIGHTS
. PFOA and PFOS induce a weak antagonistic ER transactivation.
· PFOA and PFOS exhibit strong estro- genic potential by increasing E2 production.
· Aromatase induction by PFOA and PFOS altered the sex hormone levels.
· PFOS may be involved in aldosterone synthesis.
ARTICLE INFO
Article history: Received 25 November 2015 Received in revised form 18 April 2016
Accepted 19 April 2016 Available online 30 April 2016
Handling Editor: I. Cousins
Keywords: Perfluorooctanoic acid Perfluorooctane sulfonate ER/AR transactivation Steroidogenesis Aromatase Endocrine disruptor chemical
GRAPHICAL ABSTRACT
PFOA PFOS
Steroidogenesis
OH
H
A
A
Endocrine disruption
HO
17B-estradiol
OH
H
Antagonistic ER transactivation
A
A
O
Testosterone
ABSTRACT
Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are two of the most widely used perfluoroalkyl acids (PFAAs). Because of their strong persistence, they have become widely distributed throughout the environment and human bodies. PFOA and PFOS are suspected to disrupt the endocrine system based upon many in vivo studies, but the underlying mechanisms are currently unclear. In this study, we investigated the endocrine-related effects of PFOA and PFOS using in vitro estrogen receptor (ER) and androgen receptor (AR) transactivation assays and steroidogenesis assay. The results showed that PFOA and PFOS exhibited weak antagonistic ER transactivation but did not exhibit agonistic ER or AR transactivation. In the steroidogenesis assay, PFOA and PFOS induced 170-estradiol (E2) level and reduced testosterone level, which would be caused by the induction of aromatase activity. The qPCR analysis of genes involved in steroidogenesis indicates that PFOA and PFOS associate with sex hormone synthesis by the transcriptional induction of two genes, cyp19 and 36-hsd2. Moreover, the transcriptional induction of cyp11b2 by PFOS suggests that this chemical may underlie the disruption of several physiological functions related to aldosterone. The results of the current study suggest that PFOA and PFOS are po- tential endocrine disrupting chemicals (EDCs) and provide information for further studies on the mo- lecular events that initiate the adverse endocrine effects.
@ 2016 Elsevier Ltd. All rights reserved.
* Corresponding author. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology, 660-844, Jinju, Republic of Korea. E-mail address: jwpark@kitox.re.kr (J .- W. Park).
1. Introduction
Perfluoroalkyl acids (PFAAs) are characterized by a fully fluori- nated hydrophobic linear carbon chain attached to various
hydrophilic heads (Buck et al., 2011). Because PFAAs have unique properties, such as anti-wetting and surfactant properties, they have been extensively used in industry and consumer products including oil and water repellents, coatings for cookware, carpets and textiles. PFAAs have strong carbon-fluorine bonds that do not break down naturally, which allows them to persist in the envi- ronment (Du et al., 2013b). Thus, many PFAAs have been detected in nearly all environmental areas, indicating that they are widespread throughout the ecosystem and human blood and tissues.
Among the PFAAs, perfluorooctanoic acid (PFOA) and per- fluooctane sulfonate (PFOS) have been found to be predominant in the some environmental samples (Giesy and Kannan, 2001). In particular, the two PFAAs were found in humans, from ng/L to µg/L levels (Olsen et al., 2007b; Kunacheva et al., 2012) and reported to have an approximately 4- and 5-year half-life in human serum, respectively (Olsen et al., 2007a), which has led to a rising concern about their possible adverse effects in humans such as hepatotox- icity, developmental toxicity, reproductive toxicity and carcino- genic potency (Lau et al., 2003; Thibodeaux et al., 2003; Guruge et al., 2006). PFOA and PFOS are suspected as endocrine disrupt- ing chemicals (EDCs), which are defined as chemicals that can interfere with the endocrine system (Jensen and Leffers, 2008). A 10% increase in 170-estradiol (E2) was detected in high PFOA- exposed workers (Olsen et al., 1998) and a lower than normal sperm count was observed in young Danish men with high PFOA and PFOS levels (Joensen et al., 2009). Moreover, maternal PFOA concentration was inversely related with birth weight, length and abdominal circumference (Fei et al., 2007, 2008). In mouse studies, PFOA accelerated puberty in males and delayed puberty in females (Lau et al., 2003) and reduced uterine weight (Yang et al., 2009). In fish studies, PFOS increased the hepatic vitellogenin (VTG) level in fish species including Oreochromis niloticus and Oncorhynchus mykiss (Liu et al., 2007; Benninghoff et al., 2011). Moreover, PFOS altered the sex ratio (i.e., increased the female proportion) and decreased larval survival in the F1 offspring of zebrafish (Wang et al., 2011).
To understand the endocrinal toxicity of PFOA and PFOS, it is important to identify the initiation events at the molecular and cellular levels triggered by them. Evidence suggests that PFOA and PFOS have endocrine disrupting potency, but the underlying mode of action remains unclear. To explain the underlying mechanisms of PFOA and PFOS, a few studies have been conducted in in vitro screening systems such as estrogen receptor (ER)- or androgen receptor (AR)-mediated transactivation and steroidogenesis assays in several human carcinoma cells (Wang et al., 2012; Du et al., 2013a, 2013b; Kjeldsen and Bonefeld-Jorgensen, 2013; Gorrocha- tegui et al., 2014; Wang et al., 2015); however, the results of endocrine disrupting potency of PFOA and PFOS in these studies was inconsistent depending on the applied methods and condi- tions. Moreover, because there is little information to explain the mechanistic reasoning of endocrine disruption by two chemicals suggested from the in vivo assays, more experimental evidence is required at the molecular and cellular level.
In the current study, the underlying mechanisms for endocrine disruption of PFOA and PFOS were evaluated using human carci- noma cell lines and the following assays were performed: an ER transactivation assay in MVLN, an AR transactivation assay in 22Rv1/MMTV and steroidogenesis assay in H295R. The transcrip- tional levels of genes involved in steroidogenesis after exposure to PFOA and PFOS were estimated using quantitative real-time PCR (qPCR); based on the results, the underlying mechanisms of endocrine disruption were suggested.
2. Materials and methods
2.1. Chemicals
PFOA (CAS No. 335-67-1, purity: 96%), PFOS (CAS No. 1763-23-1, purity: ≥98%), E2 (CAS No. 50-28-2, purity: >98%), dihy- drotestosterone (DHT) (CAS No. 521-18-6, purity: >97.5%), pro- chloraz (CAS No. 67747-09-5, purity: 98.6%) and forskolin (CAS No. 66575-29-9, purity: ≥98%) were purchased from Sigma-Aldrich (St Louis, MO, USA). All of the chemicals were dissolved in DMSO (Sigma-Aldrich) before the experiment.
2.2. Cell lines
For the EDC assays with PFOA and PFOS, three human carcinoma cell lines were used: MVLN cell line for ER transactivation assays, 22Rv1/MMTV cell line for AR transactivation assays and H295R cell line for steroidogenesis assay and qPCR. MVLN cells and 22Rv1/ MMTV cells were donated from Prof. Kyungho Choi at Seoul Na- tional University (Seoul, Republic of Korea) and Dr. Hee Seok Lee at National Institute of Food and Drug Safety Evaluation (Osong, Re- public of Korea), respectively. H295R cell line was purchased from ATCC (Manassas, VA, USA).
MVLN cell line is MCF-7 cell line, a human breast carcinoma cell line, stably transfected with a luciferase reporter gene plasmid consisting of a Xenopus laevis vitellogenin promoter region, which contains four estrogen responsive elements with the herpes sim- plex thymidine kinase promoter upstream of the firefly luciferase reporter gene (Demirpence et al., 1993). MVLN cells were cultured in Dulbecco’s Modified Eagle’s Media (DMEM): Ham’s F12 nutrient mixture without phenol red (Sigma-Aldrich, St Louis, MO, USA) containing sodium bicarbonate (Amresco, Solon, Ohio, USA), 10% FBS (Hyclone, Logan, UT, USA), 1% Sodium pyruvate (Sigma- Aldrich), 0.5 ml of insulin (1 mg/ml, Sigma-Aldrich) and 1% (v/v) antibiotic solution (penicillin and streptomycin, Invitrogen, Carls- bad, CA, USA). 22Rv1/MMTV cell line is 22Rv1 cell line, a human prostate carcinoma cell line, stably transfected with a pGL4/hyg luciferase vector, which has the LTR region of murine mammary tumour virus containing the androgen response element inserted into its promoter region (Sun et al., 2016). 22Rv1/MMTV cells were cultured in RPMI1640 media containing 10% FBS. H295R cell line, a human adrenal corticocarcinoma cell line, was cultured in DMEM containing 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenium, 1.25 mg/ml bovine serum albumin, 5.35 µg/ml linoleic acid and 2.5% Nu-Serum.
All three cell lines were incubated at 37 ℃ in a 5% humidified CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA).
2.3. ER and AR transactivation assay
An agonistic ER transactivation assay using MVLN cell line was performed as previously described (Kang et al., 2014). Briefly, 1 x 104 cells/well were plated on a 96-well white plate and incu- bated for 24 h. The cells were exposed to several concentrations of PFOA and PFOS (10 pM-10 µM, final DMSO 0.1% v/v), which were serially diluted in medium containing 5% charcoal-stripped FBS (DCC-FBS) for 48 h. DMSO (0.1%, v/v) and E2 were used as vehicle and positive controls, respectively. To evaluate ER antagonistic transactivation of PFOA and PFOS, the cells were exposed to two chemicals serially diluted in the medium containing 10 pM of E2 (E2-EC50) for 48 h. DMSO (0.1%, v/v), E2 and tamoxifen (Sigma- Aldrich) were used as vehicle, agonistic positive and antagonistic positive controls, respectively.
The agonistic and antagonistic AR transactivation assay using 22Rv1/MMTV cell line was performed as previously described (Sun
et al., 2016). For an agonistic ER transactivation assay, 3 × 104 cells/ well were plated on a 96-well white plate and incubated for 48 h. The cells were exposed to PFOA and PFOS (10 pM-10 uM, final DMSO 0.1% v/v) serially diluted in phenol-red free RPMI1640 me- dium containing 5% DCC-FBS and exposed for 24 h. DMSO (0.1%) and DHT were applied as vehicle and positive controls, respectively. For an antagonistic AR transactivation assay, 3 x 105 cells/ml were exposed to PFOA and PFOS serially diluted in the medium (same concentrations of ER transactivation assays) containing 1 nM dihydrotestosterone (DHT) (DHT-EC30) for 24 h. DMSO (0.1%), DHT and bicalutamide (Sigma-Aldrich) were used as vehicle, agonistic positive and antagonistic positive controls, respectively.
A One-Glo+Tox Luciferase Reporter and Cell Viability Assay (Promega, Madison, WI, USA) was used for luciferase activity induced in all transactivation assays and cell viability using Synergy H1 microplate reader (Biotek, Winooski, VT, USA). At the tested concentrations, cell death by PFOA and PFOS exposure was not observed in either of the cell lines (data not shown).
2.4. Steroidogenesis assay
To estimate the effect of PFOA and PFOS on steroidogenesis, the amount of E2 and testosterone produced after an exposure to the two PFAAs were measured in H295R cells. The exposure concen- trations (10 and 100 µM) of two PFAAs were referred to the pre- vious studies (Kraugerud et al., 2011; Rosenmai et al., 2013). In preliminary test, cell viability of H295R cells (1 x 104 cells/well on 96-well clear plate) exposed to two concentrations of PFOA and PFOS for 24 h was measured using CellTiter 96-AQueous One So- lution Cell Proliferation Assay (Promega) and no cell death was observed in the tested concentrations (data not shown). The ste- roidogenesis assay was performed according to the OECD Test Guideline 456, H295R Steroidogenesis Assay (OECD, 2011). Briefly, 5 x 106 cells/well were acclimated on a 6-well plate for 24 h and exposed to 10 and 100 µM of both PFOA and PFOS for 48 h. Forskolin and prochloraz known to induce and inhibit steroidogenesis, respectively, were applied as a positive and negative control and 0.1% DMSO was used as a vehicle control. The hormones released in the medium were extracted by diethyl ether. The amount of E2 and testosterone was measured using an Estradiol EIA Kit (Cayman Chemical, Ann Arbor, MI, USA) and Testosterone EIA Kit (Cayman Chemical) according to the protocol supplied from the manufac- turer, respectively. In addition, to investigate the effect of PFOA and PFOS on different type of estrogens, estrone which is regulated by aromatase, estrone level was estimated in the H295R cells exposed in the same way described above. The estrone level was measured using an Estrone ELISA Kit (Abnova, Taipei, Taiwan) according to the protocol supplied by the manufacturer.
2.5. Quantitative real-time PCR (qPCR)
H295R cells (5 x 106 cells/well on a 6-well plate) exposed to 10 µM PFOA and PFOS for 24 h were harvested. Total RNA was extracted from the harvested cells using an RNeasy mini kit (Qia- gen, Hilden, Germany) and the quality was confirmed (2.0 ± 0.05, 260/280 nm) using a Synergy H1 microplate reader (Biotek). The cDNA was synthesized from total RNA using the Superscript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA), and the concentration was measured with a Synergy H1 microplate reader (Biotek). For qPCR, each PCR mixture was composed of 10 ul of 2 x GoTaq qPCR Master Mix (Promega), 10 pmol of primers and 20 ng of cDNA. The primer information is summarized in Table 1. PCR amplification was performed as fol- lows: 45 cycles of 95 ℃ for 20 s, 55 ℃ for 20 s, 72 ℃ for 20 s and fluorescence reading. The qPCR was conducted using the Agilent
| Gene name | Primer sequence | |
|---|---|---|
| Forward | Reverse | |
| gapdh | 5'-gtcaaggctgagaacgggaa-3' | 5'-aaatgagccccagccttctc-3' |
| hmgr | 5'-ttcaggttccaatggcaaca-3' | 5'-gccacgagtcatcccatctg-3' |
| star | 5'-atgagtaaagtggtcccaga-3' | 5'-accttgatctccttgacatt-3' |
| cyp11a1 | 5'-gagatggcacgcaacctgaa-3' | 5'-cttagtgtctccttgatgct-3' |
| cyp17 | 5'-agccgcacaccaactatcag-3' | 5'-tcaccgatgctggagtcaac-3' |
| 36-hsd2 | 5'-tgccagtcttcatctacacc-3' | 5'-ttcccagaggctcttcttcg-3' |
| 170-hsd1 | 5'-ctccctctgaccagcaacc-3' | 5'-tgtgtctcccacgcaatctc-3' |
| 170-hsd4 | 5'-tgcgggatcacggatgatct-3' | 5'-gccaccattctcctcacaac-3' |
| cyp19 | 5'-aggtgctattggtrcatctt-3' | 5'-tggtggaatcgggtctttat-3' |
| cyp21 | 5'-acctcagtttctcctttatt-3' | 5'-agagccagggtccttcac-3' |
| cyp11b1 | 5'-ggtttgccaggctaagc-3' | 5'-caaactgcccagaggacag-3' |
| cyp11b2 | 5'-tccaggtgtgttcagtagtt-3' | 5'-gaagccatctctgaggtctg-3' |
Mx3005P qPCR system (Agilent Technologies). The qPCR confi- dence was confirmed by a melting curve analysis (data not shown). The relative transcriptional levels of each gene were estimated according to the 2-ACt method (Pfaffl, 2001).
2.6. Statistical analysis
The differences among each chemical were analysed by one- way analysis of variance (ANOVA) followed by the post hoc com- parison Least Significant Difference (LSD) test using SPSS software (SPSS v20, IBM, Armonk, NY, USA). The differences were considered statistically significant at p < 0.05. The data represented the mean values of three independent experiments. Each independent experiment was triplicated.
3. Results
3.1. ER and AR transactivation
In the presence of 10 pM of E2, both PFOA and PFOS showed a weak antagonistic ER transactivation by showing the reduced luciferase activity compared to 10 pM E2 (Fig. 1). About 5% (p> 0.05) and 20% decreases (p < 0.05) to 10 pM E2 in the luciferase activity
1.1
Relative luciferase activity to E2
I
₮
F
1.0
±
=
1
2
0.9
*
0.8
0.7
PFOA
PFOS
0.6
0.5
-11
-10
-9
-8
-7
-6
-5
Log[concentration] (M)
were observed at 1 µM and 10 uM for both of the chemicals, respectively. Little difference was detected between PFOA and PFOS. Agonistic ER and AR, and antagonistic AR transactivations were not observed at the tested concentrations (data not shown).
3.2. Steroidogenesis assay
To estimate the involvement in steroidogenesis of PFOA and PFOS, levels of two sex hormones including E2 and testosterone were measured using EIA methods. Levels of sex hormones of H295R cells exposed to each chemical were compared to a vehicle control. In E2 level, both PFOA and PFOS showed the estrogenic result by significantly increasing E2 level (p < 0.05). E2 level was approximately 2-fold increased at 10 and 100 µM of PFOA but only 100 µM of PFOS (Fig. 2). On the other hand, testosterone level was decreased at 100 µM of PFOA and PFOS (p < 0.05), but not 10 µM of two PFAAs (Fig. 3).
To investigate the involvement of aromatase in steroidogenesis changed by the two PFAAs, level of estrone which is another es- trogen regulated by aromatase was additionally measured using EIA method. As a result, estrone level was significantly (p < 0.05) increased compared to a vehicle control at both PFAAs treatments (Fig. 4). In steroidogenesis, prochloraz and forskolin used as a negative and positive control decreased and increased the levels of three sex hormones, respectively (p < 0.05). PFOA slightly increased more estrone level than PFOS, but not statistically different.
3.3. Transcriptional expression of genes related with steroidogenesis
To analyse the steroidogenesis-related genes expression changes after exposure to PFOA and PFOS, eleven genes involved in steroidogenesis were selected and their transcriptional levels were estimated using qPCR (Fig. 5). The gene expression of 30-hsd2 and cyp19 involved in sex hormone biosynthesis were increased over 2- fold by PFOA and PFOS (p < 0.05), and cyp17 was 1.5-2-fold tran- scriptionally activated (p < 0.05). The gene expression of cyp11b2 related to corticoid biosynthesis was over 2-fold increased (p < 0.05) by only PFOS. The other genes were not significantly transcriptionally changed by PFOA or PFOS.
Relative 17ß-estradiol level to DMSO
30
*
20
10
5
4
3
*
*
*
2
1
*
T
0
Prochloraz
Forskolin
PFOA 10 uM
PFOA 100 uM
PFOS 10 uM
PFOS 100 uM
10.0
Relative testosterone level to DMSO
*
5.0
2.0
1.5
T
1.0
*
T
*
T
*
T
0.5
0.0
Prochloraz
Forskolin
PFOA 10 uM
PFOA 100 uM
PFOS 10 uM
PFOS 100 uM
70
Relative estrone level to DMSO
60
*
50
20
*
15
*
* T
10
* T
5
*
0
Prochloraz
Forskolin
PFOA 10 uM
PFOA 100 uM
PFOS 10 uM
PFOS 100 uM
4. Discussion
PFOA and PFOS are of great concern among toxicologists because of their wide distribution and strong persistence in the environment and animals, including humans (Florentin et al., 2011; Kunacheva et al., 2012). The two PFAAs were reported to generate adverse effects and, in particular, are suspected EDCs (Olsen et al., 1998; Lau et al., 2003; Thibodeaux et al., 2003; Joensen et al., 2009). Their endocrine disruptions have been demonstrated through several in vivo tests using mouse, rat and fish models (Lau et al., 2003; Liu et al., 2007; Yang et al., 2009; Benninghoff et al., 2011; Wang et al., 2011); however, the underlying mechanisms at the cellular and molecular level are unclear. In the current study, we evaluated the endocrine disruption potentials of PFOA and PFOS using the following in vitro screening systems: ER and AR
Relative trancription level to DMSO
12
*
Prochloraz
10
Forskolin
*
PFOA
8
PFOS
*
*
*
6
*
4
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3
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*
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*
*
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2
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:
L
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T
T
T
T
1
I
T
T
0
hmgr
star
cyp11a1
cyp17
3b-hsd2
17b-hsd1
17b-hsd4
cyp19
cyp21
cyp11b1
cyp11b2
Initiation
Sex hormones
Corticoids biosynthesis
biosynthesis
Cholesterol
Steroid hormones biosynthesis
biosynthesis
transactivation, steroidogenesis assays, and investigated underly- ing endocrine disrupting mode of action at transcriptional levels.
In the ER and AR transactivation assays, PFOA and PFOS showed no ER and AR agonistic transactivation at the tested concentrations, indicating that these chemicals are likely to have low binding af- finity to the two hormone receptors. In antagonistic ER trans- activation assay, however, PFOA and PFOS exhibited the reducing luciferase activity at 10 uM (Fig. 1). In previous studies, it was shown that PFOA and PFOS had no or weak binding affinity to ER or AR. The competition assay using [3H]-estradiol in trout liver cyto- solic protein showed a weak binding affinity of PFOA and PFOS to ER (Benninghoff et al., 2011). Other study using MVLN cells showed that PFOA did not induce agonistic ER transactivation activity at 0.1 mM (Wang et al., 2012). In Kjeldsen and Bonefeld-Jorgensen study, however, they conducted ER- and AR-transactivation assays using the MVLN cell line and CHO-K1 cell line and reported that PFOA and PFOS have weak ER-agonistic transactivation activity at 10 µM and more and AR-antagonistic transactivation activity at 1 µM and more (Kjeldsen and Bonefeld-Jorgensen, 2013). Although Kjeldsen and Bonefeld-Jorgensen used MVLN cells in ER trans- activation assay like current study, the different results were observed, which is probably due to different exposing concentra- tions of two PFAAs and E2. Likely observed in our study, the agonistic ER transactivation activity was not observed in 10 uM of two PFAAs in Kjeldsen and Bonefeld-Jorgensen study. In antago- nistic ER transactivation assay, they used the lower concentration of E2 (EC20, 25 pM) as a co-exposed chemical than current study (EC50, 1 nM). Du et al. (2013a,b) showed that PFOA and PFOS have little direct binding affinity for ER in the absence of E2, but enhanced the ER-transactivation in the presence of E2 in CV-1 cells transfected with pERE-TATA-Luc+, rERa/pCI and phRL-tk. According to the previous studies, ER or AR transactivation activity of PFOA and PFOS were variable depending on the tested cell lines and experimental methods, which would be caused by the low affinity of the two PFAAs to both ER and AR. The low binding affinity of PFOA and PFOS to ER or AR was supported by the binding prediction studies using molecular docking analysis (Cheng et al., 2010; Benninghoff et al., 2011; Gao et al., 2013). These studies showed that PFOA and PFOS
had a higher binding free energy than bisphenol A (BPA), which is known to be a weak binding chemical to ER and AR. The high binding free energy results in PFOA and PFOS being unable to generate the appropriate interaction with the binding pocket of hormone receptors.
In contrast to their low potential as EDCs to bind to ER or AR, PFOA and PFOS revealed significant endocrine disrupting potency in steroidogenesis assays using enzyme immunoassay method. In the steroidogenesis assay, PFOA and PFOS increased E2 level and decreased testosterone level (Figs. 2 and 3), corresponding with the results reported by Du et al., which measured E2 and testosterone levels in H295R cells exposed to PFOA and PFOS (Du et al., 2013a, 2013b) using radioimmunoassay. The increasing E2 level in H295R cells exposed to PFOA and PFOS was also observed in other studies using radio- or fluoro immunoassays (Kraugerud et al., 2011; Rosenmai et al., 2013). The previous and current studies showed the consist results regarding steroidogenesis, suggesting that PFOA and PFOS induce the endocrine disruption by being involved in steroidogenesis. Although different measurement methods, the E2 induction and testosterone reduction were observed in our and previous studies. The increased E2 and decreased testosterone levels would be mainly caused by aroma- tase, which is an enzyme responsible for the biosynthesis of es- trogens (i.e., estrone and E2) from androgens (i.e., androstenedione and testosterone). Significantly increasing estrone level and gene expression level of cytochrome p450 superfamily 19 (cyp19) encoding CYP19 (aromatase) supported the involvement of aro- matase induction in steroidogenesis change by PFOA and PFOS (Figs. 4 and 5). Previous studies investigated the change of aro- matase activity by PFOA and PFOS (Kraugerud et al., 2011; Gorrochategui et al., 2014). Kraugerud et al. reported that PFOA and PFOS did not affect the aromatase enzyme activity in H295R cell whereas Gorrochategui et al. showed that PFOA inhibited the aromatase activity in JEG-3 cell. These studies showed that aro- matase activity was not enhanced by PFOA and PFOS. In particular, Kraugerud et al. reported that the increased E2 level was due to the increased activity of upstream enzymes rather than aromatase it- self in steroidogenesis and no significant changes in transcriptional
level of genes in steroidogenesis were not observed. In current study, however, 3ß-hydroxysteroid dehydrogenase 2 (36-hsd2) and cyp17 encoding 36-HSD2 and CYP17 were approximately 2-fold increased by PFOA and PFOS. The 36-HSD2 and CYP17 enzymes catalyse the biosynthesis of intermediate steroid hormones (4 progestogens and 3 androgens, not including testosterone and dihydrotestosterone), thus the overexpression of their genes may increase the production of androgens (i.e., androstenedione and testosterone). Finally, up-regulated cyp19 may lead to decreased testosterone and increased E2 (Fig. 6). Aromatase is known to irreversibly synthesize estrogens from androgen and regulate the ratio of estrogen and androgen, thus the breakdown of sex hor- mone balanced by aromatase is likely to disrupt the endocrine system and generate adverse outcomes.
Considering all of our results, PFOA and PFOS may have both estrogenic and anti-estrogenic potencies, which was shown in other studies (Liu et al., 2007; Henry and Fair, 2013). In particular, Liu et al. reported that PFOA and PFOS had an estrogenic activity shown by induction of the hepatic VTG level VTG induction, whereas their binary mixture with E2 had an anti-estrogenic ac- tivity as the hepatic VTG reduction in tilapia males. The VTG is an egg yolk precursor protein found in females of most oviparous animals and widely used as a biomarker of estrogenic EDCs (Sumpter and Jobling, 1995). This hepatic VTG reduction may be caused by an interference of E2 binding to ER via binding compe- tition. The previous studies (Liu et al., 2007; Henry and Fair, 2013), in addition to current study, suggested that PFOA and PFOS have both estrogenic and anti-estrogenic activities, which could induce endocrine disruption such as E2 induction and antagonistic ER binding. However the adverse effects on the reproductive endo- crine system by PFOA and PFOS would be actually more governed by estrogenic activity via E2 induction than by an anti-estrogenic
activity in the environment. This hypothesis was supported by in vivo studies using fish species (Liu et al., 2007; Benninghoff et al., 2011; Wang et al., 2011). The VTG induction in male fish is an early adverse effect and a cellular or organ response caused by an E2 increase (Liu et al., 2007). Previous studies reported that VTG production or gene expression was significantly increased by PFOA and PFOS (Liu et al., 2007; Du et al., 2009; Benninghoff et al., 2011). Although these studies did not provide the molecular initiations (i.e., ER binding or aromatase induction), VTG induction might be caused by aromatase induction stimulated by PFOA and PFOS.
In the current study, with an increase in E2 level, PFOA and PFOS decreased testosterone level in response to aromatase induction. Previous studies reported that the exposure of adult male rats to PFOA caused testosterone reduction concomitantly with estradiol induction (Cook et al., 1992; Biegel et al., 1995). These studies suggested that this sex hormone alteration, in particular testos- terone reduction, induced Leydig cell hyperplasia and adenomas in the testes. Joensen et al. reported that the PFOA and PFOS levels in human serum were negatively associated with testosterone levels, which might affect sperm morphology and motility (Joensen et al., 2009, 2013). In a fish study, PFOS exposure impaired the male gonad development and reduced sperm density and motility in zebrafish (Wang et al., 2011). Moreover, sex alteration (i.e., increase in the female-to-male ratio) in a dose-dependent manner was observed in zebrafish exposed to PFOS until sexual maturity from embryos (Du et al., 2009; Wang et al., 2011). Testosterone and E2 are known to be involved in the development of the reproductive tissues and secondary sexual characteristics in males and females, respectively (Ryan, 1982; Mooradian et al., 1987).
As well as the alteration of sex hormones, PFOS may be related to aldosterone synthesis. PFOS significantly increased the gene expression of cyp11b2 encoding CYP11B2 (aldosterone synthase),
Cholesterol StAR CYP11A
Aldosterone
CYP11B2
(1.0/2.5)
Pregnenolone
Progesterone
CYP21
Deoxy- corticosterone
CYP11B1
Corticosterone
CYP17
CYP17
17a-hydroxyl- pregnenolone
11-deoxycortisol
Cortisol
3B-HSD
17a-hydroxyl progesterone
CYP17
CYP17
(2.0/2.5)
Dehydropi- androsterone
Androstenedione
Estrone
(12.3/9.9)
CYP19
(2.0/2.2)
CYP17
17B-HSD
17B-HSD
Androstenediol
Testosterone
17B-estradiol
(2.0/2.2)
(0.7/0.7)
which is an enzyme involved in the biosynthesis of aldosterone. Aldosterone is a mineralocorticoid that plays a role in the regula- tion of blood pressure by increasing the reabsorption of ions and water in the kidney. Thus, the dysregulation of aldosterone can cause cardiovascular and renal disease (Hu et al., 2012). Moreover, the transcriptional levels of other genes related to corticoid biosynthesis (i.e., cyp21 and cyp11b1) were also slightly increased by PFOS (Fig. 5), indicating that corticoids including aldosterone were increased by PFOS. Little information exists regarding the relationship between PFOS and aldosterone, but some studies have reported that PFOS increases the transcriptional level of cyp11b2 in H295R cells (Du et al., 2013a; van den Dungen et al., 2015). Recently, Rogers et al. reported that PFOS raised systolic blood pressure in the offspring of gestation 2-6 weeks rats (Rogers et al., 2014); however, no association between PFOS and hypertension was observed in other studies (Lin et al., 2011; Geiger et al., 2014). In this study, the experiment to evaluate the relationship between PFAAs exposure and changes in aldosterone level was not conducted, which could be important topic for PFAAs toxicity study.
In summary, this study showed that PFOA and PFOS exert both anti-estrogenic activity by presumably interfering with E2 binding to ER, and estrogenic activity by increasing estrogens (E2 and estrone) and reducing testosterone levels via regulation of key genes expression in steroidogenesis. Interestingly, PFOS increased the gene expression of aldosterone, suggesting this chemical affects the physiological functions regulated by aldosterone. Because there is little knowledge of the relationship between PFOS and aldosterone-related functions, future studies will provide novel information on PFOS-induced environmental toxicity. This study has provided information for the underlying molecular mecha- nisms of endocrine disruption induced by PFOA and PFOS and is also expected to contribute to the development of a toxicological knowledge framework of endocrine disruption.
Acknowledgements
This study was supported by Grant KK-1607 from the Korea Institute of Toxicology. We appreciate Prof. Kyungho Choi at Seoul National University and Dr. Hee Seok Lee at National Institute of Food and Drug Safety Evaluation for donating MVLN and 22Rv1/ MMTV cell lines.
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