ELSEVIER
Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Toxicology Letters
TL
Perfluorinated compounds differentially affect steroidogenesis and viability in the human adrenocortical carcinoma (H295R) in vitro cell assay
Marianne Kraugerud a,*, Karin E. Zimmerb, Erik Ropstadª, Steven Verhaegen ª
a Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, Postboks 8146 Dep, 0033 Oslo, Norway
b Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Postboks 8146 Dep, 0033 Oslo, Norway
ARTICLE INFO
Article history: Received 28 March 2011 Received in revised form 2 May 2011 Accepted 3 May 2011 Available online 27 May 2011
Keywords:
Perfluorooctane sulfonate Perfluorooctanoic acid Perfluorononanoic acid Apoptosis Endocrine disruption H295R
ABSTRACT
Perfluorinated compounds (PFCs) comprise a large class of man-made chemicals of which some are persistent and present throughout the ecosystem. This raises concerns about potential harmful effects of such PFCs on humans and the environment. In order to investigate the effects of potentially harmful PFCs on steroid hormone production, human adrenocortical H295R cells were exposed to three persistent PFCs including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) at six different concentrations (6 nM to 600 p.M) for 48 h. Exposure to 600 p.M PFOS resulted in a dose-responsive increase in oestradiol as well as a smaller dose-responsive increase in progesterone and testosterone secretion measured using radioimmunoassay. The aromatase activity was not significantly altered by PFOS. Only small changes in hormone secretion were detected following exposure to PFOA and PFNA. Gene expression of CYP11A, quantified using qRT-PCR was decreased by all exposure doses of PFOA, whereas HMGR expression was decreased by 60 nM PFNA. The viability markedly decreased by exposure to 600 p.M of PFOA or PFNA, but not PFOS. Flow cytometric analysis demonstrated a significant increase in apoptosis following exposure to PFNA at the highest concentration. We conclude that PFOS is capable of altering steroidogenesis in the H295R in vitro model by a mechanism other than changes in gene expression or activity of aromatase. Additionally, PFCs appear to differentially affect cell viability with induction of cell death via apoptosis at high doses of PFNA.
@ 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Perfluorinated compounds (PFCs) are a diverse group of man-made chemicals used as ingredients in a wide variety of appli- cations including stain repellents and lubricants as well as in the production of fluoropolymers used in non-stick cookware. Per- fluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) are chemicals within this groups that are, due to their persistence and widespread use, detectable throughout all compartments of the ecosystem including air, water, sediments, animals and humans (Fromme et al., 2009; Nakata et al., 2006; Yamashita et al., 2005) even in remote places such as the Arctic (AMAP, 2009). Recently, much attention has been directed towards the potential harmful effects on humans and the envi- ronment associated with the long-term exposure to certain PFCs. As a result, the United Nations Environment Programme (UNEP) made the decision to list the PFC (PFOS) and its salts as persistent organic pollutants (POPs) at the Stockholm Convention in Geneva, May 2009.
PFCs consist of a fully fluorinated carbon backbone of typically 4-14 carbons attached to a charged functional moiety, giving rise to a large variety of molecules. Due to the stability of the C-F bond, some PFCs are slowly degradable, and human half-lives as long as 5.4 years and 3.8 years for PFOS and PFOA, two of the most abundant PFCs in environmental samples, have been reported (Olsen et al., 2007).
Although the evidence of adverse effects of PFOS, PFOA and PFNA in human epidemiological studies has been a subject of debate, some studies have found that increasing human plasma PFOS and PFOA are correlated with a lower birth weight, head and abdom- inal circumference (Andersen et al., 2010; Apelberg et al., 2007; Fei et al., 2008). Recent reported mean serum levels in the US general population were 13.2 ng/ml (PFOS), 4.13 ng/ml (PFOA) and 1.49 ng/ml (PFNA) (Kato et al., 2011). Studies on laboratory animals using exposure doses considerably higher than detected concentra- tions in the human general population, suggest that PFOS increases the risk of stillbirth, decreases foetal weight and causes develop- mental defects only at high doses (Case et al., 2001; Lau et al., 2003; Thibodeaux et al., 2003). Nevertheless, this is of great concern, con- sidering that clearance rates of some PFCs are much higher in some of the laboratory species than in humans. For example, in female rats half-lives of 0.08 and 2.44 days for PFOA and PFNA, respectively, have been reported (Ohmori et al., 2003).
* Corresponding author. Tel .: +47 22597049; fax: +47 22597081. E-mail address: Marianne.Kraugerud@nvh.no (M. Kraugerud).
It is also becoming increasingly recognized that certain PFCs including PFOS, PFOA and PFNA can have endocrine disrupting effects in rodent models and in vitro systems. Several in vivo stud- ies typically report decreased plasma levels of testosterone and of the thyroid hormone, thyroxin. In addition, increases in oestra- diol and cortisol levels following exposure to PFCs including PFOS and PFOA have been reported (Biegel et al., 1995, 2001; Lau et al., 2003; Oakes et al., 2004; Thibodeaux et al., 2003). Studies have also identified that PFOA increased incidence of hepatic and Ley- dig cell adenomas in rats (Biegel et al., 1995, 2001). Whereas the hepatic adenoma induction is mediated though peroxisome prolif- erator receptor (PPAR) binding, it is postulated that the adenomas of Leydig cells are caused, at least partially, by elevated oestradiol levels (Biegel et al., 2001). This is of great concern due to the possi- ble link between oestrogenic effects of environmental pollution and the increased prevalence of human testicular cancer (Skakkebaek et al., 2001).
To date, little research has focussed on the steroidogenesis path- way as a potential target for PFOS, PFOA or PFNA. Furthermore, previous studies have invariably been carried out on tissues and cell cultures of animal origin. Considering the great differences in response to these compounds and their elimination between some species, it is evident that inter-species extrapolation of these results would be inaccurate.
The human adrenocortical cell line, H295R, shows characteris- tics of an undifferentiated foetal adrenal cortex and is capable of full steroidogenesis (Gazdar et al., 1990; Skakkebaek et al., 2001). Furthermore, it has been employed as an in vitro model for studying endocrine disruptors and it is currently being evaluated by the US- EPA and OECD as a Tier 1 screening assay for effects on production of oestradiol and testosterone (Hecker et al., 2007).
The aim of this study was to investigate how three structurally different PFCs commonly found in environmental samples affect steroidogenesis in doses ranging from low, environmentally rel- evant doses, to doses proven to induce effects on the endocrine system in rodent models and in vitro cell cultures, using the human H295R in vitro cell model. The compounds include the eight car- bon chain molecules PFOS and PFOA in addition to the nine carbon chain molecule PFNA.
2. Materials and methods
2.1. Chemicals
Tetrabutylammonium heptadecafluorooctanesulfonate, perfluorooctanoic acid and perflouorononanoic acid were purchased in powder form from Sigma-Aldrich (St Louis, MO, USA) with a purity of >95%, >90% and >97% respectively. Chemicals were dissolved in dimethyl sulfoxide (DMSO) to 600 mM stock solutions and stored in aliquots at -20 ℃.
2.2. H295R cell culture
The H295R cell line was obtained from the American Type Culture Collection (ATCC CRL-2128, ATCC, Manassas, VA, USA) and cultured in 75 cm2 flasks in Dul- becco’s modified Eagle medium/HamF12 (DMEM/F12) containing HEPES buffer, L-glutamine and pyridoxine HCI (Gibco, Invitrogen, Paisley, UK). The medium was further supplemented with 1% ITS + premix and 2.5% NuSerum (BD Biosciences, Bed- ford, MA). Cells were incubated at 37 ℃ with 5% CO2 in a humidified atmosphere. Medium was changed every 2-3 days and cells passaged at approximately 80% confluence by a brief exposure to 0.25% trypsin/0.53 mM EDTA (Gibco, Invitrogen) followed by centrifugation and reseeding. The cells were used between passages 5-13.
2.3. Exposure studies for cell viability assay and radioimmunoassay
For experiments, cells were seeded at 3 x 105 cells/well in 24-well cell culture plates (Falcon, Franklin Lakes, NJ, USA). Cells were allowed to attach 24h prior to exposure to the compounds.
H295R cells were exposed for 48 h, according to OECD recommendations, to six concentrations (6 nM, 60 nM, 600 nM, 6 µM, 60 M or 600 p.M) of PFOS, PFOA or PFNA of in triplicates. Each plate also contained triplicate wells of 600 pM tetra- butylammoniumchloride (TbACl) dissolved in DMSO as solvent control for PFOS or
0.1% DMSO as solvent control for PFOA and PFNA in addition to 10 p.M forskolin as positive control. Forskolin stimulates cyclic AMP (cAMP) production in H295R cells and has similar effects to adrenocortiotrophic hormone (ACTH), the physiolog- ical stimulant of adrenocortical steroidogenesis (Gracia et al., 2006). Exposure doses were selected to cover environmental exposure doses as well as higher doses pre- viously found to cause endocrine disrupting effects in vitro (Biegel et al., 1995; Liu et al., 2007). The experiment was conducted independently three times for hormone analysis and cell viability assay.
2.4. Cell viability assay
Cell viability was estimated using Alamar blue assay. At the end of exposure, medium was collected and stored at -20 ℃ for subsequent hormone analysis. Each well received 1 ml fresh medium containing 10% AlamarBlue (Invitrogen, Carlsbad, CA). Plates were incubated for a further 3 h, then a 100 ul sample from each well was transferred in duplicates to a fresh 96-well ELISA plate (Falcon) and read in a Victor3TM spectrophotometer (Perkin Elmer, Shelton, USA) at 570 nm and 600 nm. Viability was expressed as percentage of solvent control.
2.5. Radioimmunoassay
Oestradiol-17ß, testosterone and cortisol secreted in the cell culture medium were measured using Coat-a-count radioimmunoassay (RIA) kits (Siemens, LA, CA, USA). Progesterone was measured using Spectria Progesterone RIA kit (Orion Diag- nostica, Espoo, Finland). All kits were used according to manufacturer’s instructions. The only modification was the use of fresh standard solutions prepared in medium from same batch as the cell cultures, rather than the supplied standards. The limits of detection were 20 pg/ml, 0.8 ng/ml, 0.10 ng/ml and 3.0 ng/ml and inter-assay coef- ficients of variation were 6.3%, 9.5%, 8.85% and 8.55% for oestradiol, progesterone, testosterone and cortisol respectively.
2.6. Aromatase assay
Aromatase activity was determined according to a method described by Lephart and Simpson (1991). H295R cells between passages 7 and 13 were seeded (3 x 105 cells suspended in 1 ml medium/well) in 24-wells plates and incubated for 24h at 37 ℃ in a humidified atmosphere with 5% CO2. The cells were then exposed in trip- licates to 0.1% DMSO or TbACI (solvent control), 5 p.M forskolin (treatment control), 1 µM formestane (Sigma-Aldrich) (negative control) or 60 p.M, 600 nM or 6 nM of PFOS, PFOA or PFNA. After 48 h exposure, medium was removed and cells washed with 1 ml 1 x phosphate buffered saline, and added 500 pl 1-3H-androstenedione (0.1 µCi/well, dissolved in blank medium) (Perkin Elmer, Boston, MA, USA). The cells were then incubated for another 1.5 h before the reaction was terminated by plac- ing the plate on ice. The medium was transferred to glass tubes and 500 M of distilled H2O was added to each sample. The samples were extracted three times with 2 ml, 2 ml and 1 ml chloroform (Merck, Darmstadt, Germany), respectively. From the water phase 500 pl was transferred to scintillation vials and 8 ml of scin- tillation cocktail (Ultima Gold, Perkin Elmer) was added. Each sample was counted in a Packard Tri-Carb® 2100TR Liquid Scintillation Analyzer (Perkin Elmer) for three minutes. The whole experiment was repeated three times.
2.7. Flow cytometric detection of cell cycle distribution and cell death by propidium iodide staining
H295R cells between passages 7 and 13 were seeded (3 x 105 cells suspended in 1 ml medium/well) in 24-wells plates and allowed to attach for 24h at 37 ℃ in a humidified atmosphere with 5% CO2. The cells were then exposed in triplicate wells to 0.1% DMSO or TbACI (solvent control) or 600 p.M, 60 p.M or 600 nM of PFOS, PFOA or PFNA. After 48 h exposure, the supernatant was collected and cells exposed briefly to 0.25% trypsin/0.53 mM EDTA (Gibco, Invitrogen). Trypsinized cells were mixed with the supernatant. Cells were washed twice and cells from each well resuspended in 3 ml PBS with 0.1% BSA. Pelleted cells were fixed by dropwise adding 2 ml 70% ice-cold ethanol while vortexing. Cells were kept for 1 h at ice before storage at 4 ℃ until processed for propidium iodide staining on the day of flow cytometric analysis. The experiment was repeated three times.
On the day of analysis, fixed cells were kept on ice, washed twice in PBS and incubated with 1 ml propidium iodide (PI) (50 µg/ml) (Sigma-Aldrich) in PBS, with RNase A (50 ng/ml) (Qiagen, Crawley, UK) for 3h at 4℃. Samples were analysed using a Coulter EPICS XL flow cytometer (Beckman Coulter Ltd., Brea, CA, USA). Forward and light scatter data were collected in a linear mode. Fluorescence data was collected in the FL3 channel on a linear scale. Doublets were excluded using gating. Side- and forward-light scatter parameters were used to identify the cell events and 10 000 cells per sample were collected.
2.8. Cytospin preparations
H295R cells between passages 7 and 13 were seeded (3 x 105 cells suspended in 1 ml medium/well) in 24-wells plates and incubated for 24h at 37℃ in a humidified atmosphere with 5% CO2. The cells were then exposed in triplicates to 0.1% DMSO or TbACI (solvent control) or 600 µM or 60 p.M of PFOS, PFOA or
PFNA. After exposure, the supernatant was collected and cells exposed briefly to 0.25% trypsin/0.53 mM EDTA (Gibco, Invitrogen). Trypsinized cells were mixed with the supernatant and a 200 ul aliquot was centrifuged at 300 rpm for 5 min onto SuperFrost Plus® microscope slides (Menzel-Gläzer, Braunschweig, Germany) and allowed to air-dry. Cells were stained with a modified Wright’s Stain (Hema-Tek Stain Pak) using a Hema Tek® automated hematologic slide stainer (Siemens, Medi- cal Solutions Diagnostics, IN, USA) according to manufacturers instructions. Cytospin preparations were investigated by light microscopy at ×400 magnification using a Leica DM2000 microscope equipped with a Leica EC3 digital camera and Leica Application Suite 2.4.0 software for image capture (Leicha Microsystems, Wetzlar, Germany).
2.9. RNA preparation
H295R cells between passages 7 and 13 were seeded (3 x 105 cells suspended in 1 ml medium/well) in 24-wells plates and allowed to attach for 24h at 37℃ in a humidified atmosphere with 5% CO2. The cells were then exposed for 48 h in triplicate wells to 0.1% DMSO or TbACI (solvent control) or 60 p.M, 600 nM or 6 MM of PFOS, PFOA or PFNA. Total RNA was isolated from cell culture plates using Qiagen RNeasy mini-kit (Qiagen) according to manufacturer’s recommendations. Lysis buffer was added to wells and cells detached by scraping the bottom of the well with the pipette tip. Prior to RNA extraction, cell lysates were then centrifuged through Qiagen shredder spin columns (Qiagen). Samples were treated with DNase (RNase-Free DNase Set, Qiagen) on-column for 15 min at room temperature. After isolation, samples were eluted in RNase-free H2O and stored at -70℃. RNA concen- tration and quality was determined using NanoDrop (Thermo-Scientific, Waltham, MA, USA) and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) respectively. The experiment was repeated five times.
2.10. Quantitative RT-PCR
GeNorm human detection kit and software (PrimerDesign Ltd., Southampton, UK) was used to predict the most stable reference genes. Out of the five genes tested (B2M, B-actin, YWHAZ, ATP5B, GAPDH), YWHAZ and ATP5B were the most stable and were thus selected as reference genes in this study.
Primer sequences for CYP11A, CYP11B2, CYP17, CYP19, CYP21,3BHSD2, 17BHSD1, 17 BHSD4, StAR and HMGR were obtained from (Hilscherova et al., 2004). Primers for CYP11B1 were designed by PrimerDesign Ltd. (Southampton, UK). Primer sequences for SF-1, DAX-1 and ACTH-R were designed in house using PrimerExpress version 1.5 (Applied Biosystems, Foster City, CA, USA). Primer sequences have been previously reported (Zimmer et al., 2011). Specificities for all primer pairs were checked using nucleotideBLAST and primerBLAST (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi). All primers were used in a working concentration of 200 nM. The primers were tested with regard to annealing temperature. PCR products were analysed on an ethidium bromide agarose gel to ensure the expected product was formed and to check for potential primer artefacts.
Synthesis of cDNA and quantitative PCR was performed using Superscript Platinum III Two-Step qRT-PCR with SYBR Green (Invitrogen) according to man- ufacturer’s instructions. The cDNA synthesis was performed using a Peltier Thermal Cycler-225 (MJResearch, Waltham, MA, USA) and qRT-PCR was carried out using a DNA Engine Thermal Cycler with Chromo 4 Real-Time Detector (MJResearch) and its software, Opticon Monitor 3, (Bio-Rad Laboratories, Hercules, CA, USA) according to previously described methods (Zimmer et al., 2011).
2.11. qPCR data analysis
Data was transferred from Opticon Monitor 3 software into an Excel spreadsheet for further processing. The ACt was calculated from the difference in expression between the gene of interest and the mean expression of the two reference genes. The 44Ct was calculated from the difference in ACt between cells exposed to solvent control and the chemical of interest. Fold change was calculated using 2-44Ct(Livak and Schmittgen, 2001).
2.12. Statistical analysis
Statistical analysis was performed using JMP software (SAS Institute Inc., Cary, NC, USA). The distribution of data was tested using the Shapiro-Wilk test. Data that did not fit the normal distribution were log- or square root transformed prior to analysis. Differences in hormone secretion between solvent control and exposed cells were evaluated using Student’s t-test. Dose-response relationships were assessed in General Linear Models (GLM) where measured hormone concen- trations were included as dependent variables and experiment (n=3) and dose of the relevant test compounds were entered as continuous variables. The hor- mone data from the highest exposure doses of PFOA and PFNA (600 p.M) were excluded from statistical analyses due to cytotoxic effects on the cell viability assay. Differences in gene expression were evaluated by Student’s t-test by com- paring the log 2 transformed 2-AACt value of each exposure to solvent control. Differences in % gated cells in the apoptotic, G0/G1, G2M and M phases were inves- tigated using Tukey HSD test. Aromatase data expressed as % of solvent control
140.0
% of solvent control
120.0
T
T
T
-
100.0
T
Y
T
T
-
=
T
80.0
60.0
PFOS
40.0
PFOA
PFNA ☐
20.0
0.0
solvent control
6 nM
60 nM
600 nM
6 μΜ
60 μΜ
600 μΜ
exposure
were compared using Signed-Rank test. P values <0.05 were considered statistically significant.
3. Results
3.1. Cell viability assay
Cell viability assessed by Alamar blue conversion indicated a via- bility of >90% in all tested doses of PFOS. Exposure to PFOA or PFNA at 600 p.M resulted in a dramatic decrease in viability, whereas cells exposed to the other doses of these two compounds had a viability of >90% (Fig. 1).
3.2. Hormone analyses
Exposure to forskolin (positive control) significantly increased mean oestradiol concentration by 1214%, cortisol by 264%, proges- terone by 132% and testosterone by 175%. All three test compounds affected hormone production in H295R cells (Fig. 2). Oestradiol was significantly elevated with the highest test dose (600 µM) of PFOS compared to solvent control. In addition, oestradiol lev- els followed a significantly positive dose-response relationship with increasing doses of PFOS. PFNA exposure between 0 and 60 p.M also resulted in a significantly positive dose-response relationship on oestradiol. At 60 p.M exposure, PFNA gave a numer- ically higher, though not statistically significant, concentration of oestradiol. Cells treated with PFOA did not show any significant differences in oestradiol secretion to cells treated with solvent control.
A positive dose-response relationship between progesterone and PFOS exposure was identified. Furthermore, the highest dose of PFOS (600 µM) resulted in a significant increase in progesterone compared to solvent control. Although PFOA and PFNA exposure also resulted in a positive dose-response relationship on proges- terone, no significant differences were seen when each dose was compared to the solvent control.
Testosterone levels were influenced by all three compounds. Cells exposed to PFOS showed a significant positive relation- ship between testosterone concentration and increasing exposure doses. When comparing each dose to control, exposure to PFOS only caused a small but significant increase in testosterone in the highest exposure dose. PFOA mildly elevated testosterone in the 6 pM and 600 nM exposure doses. In contrast, PFNA expo- sure led to a significant negative dose-response relationship on testosterone. Additionally, the highest non cytotoxic dose of PFNA (60 MM) resulted in a small, but significant reduction of testos- terone (Fig. 3).
Cortisol was not significantly altered by exposure to PFOS and PFOA, although a negative dose-response relationship was identi- fied with exposure to PFNA.
400
Oestradiol
*
Progesterone
350
7
*
pg/ml oestradiol
300
ng/ml progesterone
6
250
5
PFNA
200
4
- PFOA
₸
150
3
PFOS
100
2
50
1
ΟμΜ
0,006μΜ
0,06μΜ
0,6μΜ
6μΜ
60μΜ
600μΜ
ΟμΜ
0,006μΜ
0,06μΜ
0,6μΜ
6μΜ
60μΜ
600μΜ
100
3.5
Testosterone
Cortisol
*
90
ng/ml testosterone
3
*
I
*
ng/ml cortisol
80
I
₮
*
2.5
70
2
60
İ
İ
±
50
1.5
40
1
30
ΟμΜ
0,006μΜ
0,06μΜ
0,6μΜ
6μΜ
60μΜ
600μΜ
ΟμΜ
0,006μΜ
0,06μΜ
0,6μΜ
6μΜ
60μΜ
600μΜ
3.3. Aromatase assay
Forskolin significantly increased activity of aromatase by 1075%. PFOA at 600 nM significantly increase aromatase activity by 267%. No changes in aromatase activity were observed with exposure to the other test doses of PFOA, PFNA or PFOS (data not shown).
PFOS
CYP11A
PFOA
PFNA
1.4
1.2
T
T
1
T
T
-
T
Fold change
0.8
*
T
*
*
0.6
7
T
T
0.4
0.2
0
0μ.Μ
0,006μ.Μ
0,6μ.Μ
60μ.Μ
1.4
HMGR
1.2
T
T
T
*
T
T
T
Fold change
1
T
0.8
0.6
0.4
0.2
0
Ομ.Μ
0,006μ.Μ
0,6μ.Μ
60μ.Μ
3.4. Flow cytometric analysis of cell cycle distribution and cell death by propidium iodide staining
Table 1 summarizes the percentage of cells in each cell cycle phase G0/G1, S, G2/M as well as the apoptotic fraction (A0) for cells exposed to the three compounds and the respective controls. Expo- sure to PFOS did not influence the apoptotic fraction with any of the concentrations tested. The 600 µM dose of PFOA had a numer- ically greater, but not significantly different number of cells in the apoptotic fraction (Fig. 4A). Exposure to 600 µM PFNA, however, resulted in 96.7% of the cells being in the apoptotic fraction, with the number of cells in G0/G1, S or G2/M phase being lower than 2% (Fig. 4C). The S and G2M phases were not significantly altered by any of the exposures.
| Apoptotic | G0/G1 | S | G2/M | |
|---|---|---|---|---|
| PFOS | ||||
| Control | 10.71 (4.9) | 56.27 (9.50) | 22.04 (0.83) | 5.62 (4.25) |
| 600 nM | 13.07 (5.21) | 51.28 (3.09) | 25.52 (4.59) | 7.07 (2.32) |
| 60 μ.Μ | 13.75 (5.68) | 51.37 (4.08) | 25.20 (4.14) | 6.49 (2.22) |
| 600 μ.Μ | 18.39 (7.72) | 47.78 (2.47) | 24.52 (6.13) | 6.39 (1.00) |
| PFOA | ||||
| Control | 12.79 (2.13) | 48.64 (2.05) | 27.67 (2.72) | 7.77 (2.49) |
| 600 nM | 14.09 (6.56) | 54.55 (8.31) | 22.60 (2.19) | 6.01 (2.74) |
| 60 μ.Μ | 11.95 (5.56) | 54.98 (5.04) | 24.89 (3.55) | 6.16 (2.31) |
| 600 μ.Μ | 40.83 (16.82) | 40.80 (13.00) | 14.39 (5.07) | 2.79 (1.23) |
| PFNA | ||||
| Control | 12.79 (2.13) | 48.64 (2.05) | 27.67 (2.72) | 7.77 (2.49) |
| 600 nM | 10.67 (4.00) | 51.08 (3.10) | 25.83 (2.89) | 8.42 (2.50) |
| 60 μ.Μ | 17.50 (0.78) | 31.00 (15.20) | 31.44 (9.80) | 12.74 (2.25) |
| 600 μ.Μ | 96.68 (2.61)* | 1.02 (0.58) | 1.67 (1.47) | 0.27 (0.23) |
* Significantly different from control.
.
GOG1
=
A0
GO/G1
:
Ao
A0
S
S
G2/M
A
G2/M
B
C
W
0
1023
0
1023
0
1023
FL3 LIN
FL3 LIN
FL3 LIN
:
GO/G1
:
GO/G1
A0
A0
S
S
G2/M
D
G2/M
E
0
r
1023
0
T
1023
FL3 LIN
FL3 LIN
3.5. Cystospin preparations
Cytospin preparations from cells exposed to PFNA and PFOA (60 µM) showed a cell population present with the typical mor- phology of apoptosis (shrunken cells with pyknotic nuclei) (Fig. 5). This population was absent in the respective controls (TbACl and DMSO).
3.6. Gene expression
Only PFOA and PFNA induced changes in gene expression in the current study (Fig. 3). All three test doses of PFOA decreased CYP11A levels. The other two test compounds did not affect mRNA levels of CYP11A. PFNA, however, significantly reduced HMGR expression, although only at 60 p.M. The expression of the other genes tested was not significantly altered by exposure to the test compounds.
4. Discussion
In the current study we demonstrated that PFOS has the capa- bility to modulate steroidogenesis in the human adrenocortical H295R cell assay. Small changes in steroidogenesis were detected with exposure to PFOA and PFNA. However, these rarely followed a concentration-response relationship and their biological signifi- cance is therefore uncertain.
Whereas an increase in oestradiol was observed with the high- est test dose (600 [M) of PFOS, the PFOA and PFNA exposure at the same concentration gave a sharp decrease of all hormones mea- sured due to cytotoxicity. At the second highest dose level tested (60 µM) the oestradiol level appeared increased with PFNA expo- sure, however this was not statistically significant. Oestradiol was not altered by PFOA. In contrast, some previous in vitro studies on
rat Leydig cells have reported a characteristic increase in oestra- diol secretion induced by 10-500 p.M PFOA (Biegel et al., 1995; Liu et al., 1996). In addition, elevated plasma oestradiol levels following PFOA exposure in rats has been detected (Cook et al., 1992). Pre- vious investigations of the ability of other fluorochemicals, such as PFOS, to elevate oestradiol is limited to one in vivo report where PFOS increases serum oestradiol secretion in fathead minnows (Pimephales promelas), however the response appeared not to be dose-dependent and the results are therefore difficult to inter- pret (Oakes et al., 2005). Our findings, however, suggest oestradiol secretion can also be increased by PFOS. A possible explanation for the lack of response in oestradiol production with PFOA exposure in the current study could be the narrow response range limited by cytotoxicity in this particular in vitro model.
Interestingly, we observed no significant changes in mRNA lev- els of CYP 19, the gene encoding the P450 aromatase enzyme responsible for conversion of testosterone to oestradiol, with expo- sure to any of the test chemicals. Hence, the PFOS-induced increase in oestradiol levels could be a result of a different mechanism than control of gene expression. Additionally, the activity the aromatase enzyme was not significantly altered with PFOS exposure. This sug- gests that oestradiol concentration could be increased as a result of an alternative mechanism, such as increased activity of steroido- genic enzymes upstream of aromatase or an alteration in oestradiol metabolism.
In the current study, the exposure compounds had a small effect on medium testosterone concentration which showed different patterns. Whereas PFNA decreased testosterone, PFOS and PFOA exposure led to an increase in testosterone. Previous studies on rats have demonstrated a non significant negative dose response rela- tionship between PFOA and testosterone, which became significant when rats were challenged by hCG (Biegel et al., 2001). Perfluorodo-
TbACI
DMSO
DMSO
-
PFOS 60 µM
PFOA 60 µM
PFNA 60 μM
-
decanoic acid (PFDoA) exposed rats exhibited lower testosterone levels in the absence of hCG (Shi et al., 2007). In vitro, differences in testosterone production was seen by exposure to 500 or 50 p.M PFOA in Leydig cells where testosterone levels were normal in the absence of hCG and dramatically reduced when hCG was present (Biegel et al., 1995; Liu et al., 1996). Although immature rainbow trout (Oncorhynchus mykiss) had an elevated plasma testosterone following PFOS exposure, this was not seen in other fish species (Oakes et al., 2005). The increase in testosterone in vitro with the highest dose of PFOS observed in this study has, to the best of our knowledge, not been previously reported.
Certain perfluorinated compounds, including PFOA have previ- ously been suggested to interfere with fatty acid metabolism and lipid transport in the livers of rare minnows (Gobiocypris rarus) (Wei et al., 2008) and appear to inhibit the transport of choles- terol into the mitochondria in rat Leydig cell cultures (Boujrad et al., 2000). However, such effects would lead to a reduced steroidoge- nesis due to a lack of substrate and could therefore not explain the elevated testosterone observed in the current study. Significantly elevated progesterone, in addition to oestradiol and testosterone, suggests that PFOS could affect the steroid synthesis upstream of progesterone in our study.
The expression of CYP11A, which encodes the cholesterol side- chain cleavage enzyme regulating the first and rate limiting step in steroidogenesis, was significantly decreased by all exposure doses of PFOA. Although the expression showed a slight decrease between the lowest and highest dose, no significant dose-response relationship was detected. A possible explanation could be that the response had reached a plateau even at the lowest test dose. A decrease in concentration of all steroid hormones investigated would be expected with a decreased expression of CYP11A. Such a response was not detected with PFOA, possibly due to an insuf- ficient suppression of CYP11A expression or due to compensatory mechanisms.
In the current study, no significant effect on cortisol production was observed by any of the three test compounds. Little research has been attributed to the effect PFOS, PFOA or PFNA on cortisol pro- duction. Two studies in which mice were exposed to PFNA, reported increased plasma cortisol levels (Fang et al., 2008, 2009). In one of these studies, an elevated level of ACTH was also found, suggesting that these compounds may affect cortisol production upstream of the adrenal gland in the hypothalamic-pituitary-adrenal axis. This
could explain the lack of effects on cortisol synthesis observed in our study.
This study demonstrated that PFOS induce limited cytotox- icity at the highest exposure dose (600 M). In contrast, PFOA showed higher effects on cytotoxicity, and PFNA severely affected cell viability at this dose. The flow cytometry and cytology demonstrated that the cytotoxic response was largely due to apoptosis. An increase in apoptosis with exposure to various PFCs have been reported (Feng et al., 2009; Fernandez et al., 2008; Hu and Hu, 2009). In these studies, apoptosis is typi- cally induced by the intrinsic pathway triggered by oxidative stress inducing both caspase-dependent and independent path- ways.
Levels of PFOS as high as 13 µg/ml have been reported in human blood (Olsen et al., 1998), although most reports are considerably lower, with a means of 18 ng/ml and 13.2 ng/ml reported in the general population by two studies (Kato et al., 2011; Monroy et al., 2008). Reported levels for PFOA and PFNA are somewhat lower with one study reporting a mean of 2.54 ng/ml and 0.86 ng/ml respec- tively (Monroy et al., 2008). Although direct comparisons of plasma levels with cell culture medium concentrations are problematic due to the difference in lipid content, it is evident that only the low- est concentrations used in this study are representative of human exposure levels.
In summary, PFOS is capable of modulating steroid secretion in human adrenocortical H295R cells. Oestradiol, progesterone and testosterone was elevated by the highest dose (600 µM) of PFOS. Exposure to PFOA and PFNA, but not PFOS, induced cytotoxicity mediated by apoptosis at the highest doses. We propose that the changes in hormone secretion by PFOS are mediated by mecha- nisms other than changes in aromatase activity or modulation of gene expression of the steroidogenic enzymes investigated.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by the Research Council of Norway (158849/I10 and 175098/V40). The authors would like to thank
Ellen Dahl, Kristine von Krogh, Else Britt Gondrosen and Camilla Almås for excellent technical assistance. Further, we would like to acknowledge Karin Waterhouse for advice regarding flow cytome- try. S.V. would like to thank Dr. Christine Nelleman and Dr. Anders Elleby Engel-Kofoed at the National Food Institute, Denmark, for the chance of visiting their lab and for assisting us in establishing and standardizing the H295R steroidogenesis assay.
References
AMAP, 2009. Arctic Pollution 2009. Arctic Monitoring and Assessment Programme, Oslo. xi+83 pp.
Andersen, C.S., Fei, C., Gamborg, M., Nohr, E.A., Sorensen, T.I., Olsen, J., 2010. Prenatal exposures to perfluorinated chemicals and anthropometric measures in infancy. Am. J. Epidemiol. 172, 1230-1237.
Apelberg, B.J., Witter, F.R., Herbstman, J.B., Calafat, A.M., Halden, R.U., Needham, L.L., Goldman, L.R., 2007. Cord serum concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in relation to weight and size at birth. Environ. Health Perspect. 115, 1670-1676.
Biegel, L.B., Hurtt, M.E., Frame, S.R., O’Connor, J.C., Cook, J.C., 2001. Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats. Toxicol. Sci. 60, 44-55.
Biegel, L.B., Liu, R.C., Hurtt, M.E., Cook, J.C., 1995. Effects of ammonium perfluorooc- tanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies. Toxicol. Appl. Pharmacol. 134, 18-25.
Boujrad, N., Vidic, B., Gazouli, M., Culty, M., Papadopoulos, V., 2000. The peroxisome proliferator perfluorodecanoic acid inhibits the peripheral-type benzodiazepine receptor (PBR) expression and hormone-stimulated mitochondrial choles- terol transport and steroid formation in Leydig cells. Endocrinology 141, 3137-3148.
Case, M.T., York, R.G., Christian, M.S., 2001. Rat and rabbit oral developmental toxi- cology studies with two perfluorinated compounds. Int. J. Toxicol. 20, 101-109.
Cook, J.C., Murray, S.M., Frame, S.R., Hurtt, M.E., 1992. Induction of Leydig cell adenomas by ammonium perfluorooctanoate: a possible endocrine-related mechanism. Toxicol. Appl. Pharmacol. 113, 209-217.
Fang, X., Feng, Y., Shi, Z., Dai, J., 2009. Alterations of cytokines and MAPK signal- ing pathways are related to the immunotoxic effect of perfluorononanoic acid. Toxicol. Sci. 108, 367-376.
Fang, X., Zhang, L., Feng, Y., Zhao, Y., Dai, J., 2008. Immunotoxic effects of perfluo- rononanoic acid on BALB/c mice. Toxicol. Sci. 105, 312-321.
Fei, C., Mclaughlin, J.K., Tarone, R.E., Olsen, J., 2008. Fetal growth indicators and perfluorinated chemicals: a study in the Danish National Birth Cohort. Am. J. Epidemiol. 168, 66-72.
Feng, Y., Shi, Z., Fang, X., Xu, M., Dai, J., 2009. Perfluorononanoic acid induces apo- ptosis involving the Fas death receptor signaling pathway in rat testis. Toxicol. Lett ..
Fernandez, F.P., Perez Martin, J.M., Herrero, O., Peropadre, A., de la, P.E., Hazen, M.J., 2008. In vitro assessment of the cytotoxic and mutagenic potential of perfluo- rooctanoic acid. Toxicol. In Vitro 22, 1228-1233.
Fromme, H., Tittlemier, S.A., Volkel, W., Wilhelm, M., Twardella, D., 2009. Perfluori- nated compounds-exposure assessment for the general population in Western countries. Int. J. Hyg. Environ. Health 212, 239-270.
Gazdar, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., Triche, T.J., Myers, C.E., Chrousos, G.P., Brennan, M.F., Stein, C.A., La Rocca, R.V., 1990. Establishment and charac- terization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 50, 5488-5496.
Gracia, T., Hilscherova, K., Jones, P.D., Newsted, J.L., Zhang, X., Hecker, M., Higley, E.B., Sanderson, J.T., Yu, R.M., Wu, R.S., Giesy, J.P., 2006. The H295R system for evaluation of endocrine-disrupting effects. Ecotoxicol. Environ. Saf. 65, 293-305.
Hecker, M., Akahori, Y., Murphy, M., Nelleman, C., Highley, E., Newsted, J.L., Wu, R., Lam, P., Lasley, B., Buckalew, A., Grund, S., Nakai, M., Timm, G., Giesy, J.P., 2007. The OECD validation program of the H295R steroidogenesis assay for the identification of in vitro inhibitors and inducers of testosterone and estradiol production. Phase 2: inter-laboratory pre-validation studies. Environ. Sci. Pollut. Res. 14, 23-30.
Hilscherova, K., Jones, P.D., Gracia, T., Newsted, J.L., Zhang, X., Sanderson, J.T., Yu, R.M., Wu, R.S., Giesy, J.P., 2004. Assessment of the effects of chemicals on the expression of ten steroidogenic genes in the H295R cell line using real-time PCR. Toxicol. Sci. 81, 78-89.
Hu, X.Z., Hu, D.C., 2009. Effects of perfluorooctanoate and perfluorooctane sulfonate exposure on hepatoma Hep G2 cells. Arch. Toxicol. 83, 851-861.
Kato, K., Wong, L.Y., Jia, L.T., Kuklenyik, Z., Calafat, A.M., 2011. Trends in Exposure to Polyfluoroalkyl Chemicals in the U.S. Population: 1999-2008. Environ. Sci. Technol. [E publication ahead of print].
Lau, C., Thibodeaux, J.R., Hanson, R.G., Rogers, J.M., Grey, B.E., Stanton, M.E., Buten- hoff, J.L., Stevenson, L.A., 2003. Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II: postnatal evaluation. Toxicol. Sci. 74, 382-392. Lephart, E.D., Simpson, E.R., 1991. Assay of aromatase activity. Methods Enzymol. 206, 477-483.
Liu, C., Yu, K., Shi, X., Wang, J., Lam, P.K., Wu, R.S., Zhou, B., 2007. Induction of oxida- tive stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Aquat. Toxicol. 82, 135-143.
Liu, R.C., Hahn, C., Hurtt, M.E., 1996. The direct effect of hepatic peroxisome prolif- erators on rat Leydig cell function in vitro. Fundam. Appl. Toxicol. 30, 102-108. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402-408.
Monroy, R., Morrison, K., Teo, K., Atkinson, S., Kubwabo, C., Stewart, B., Foster, W.G., 2008. Serum levels of perfluoroalkyl compounds in human maternal and umbil- ical cord blood samples. Environ. Res. 108, 56-62.
Nakata, H., Kannan, K., Nasu, T., Cho, H.S., Sinclair, E., Takemurai, A., 2006. Per- fluorinated contaminants in sediments and aquatic organisms collected from shallow water and tidal flat areas of the Ariake Sea, Japan: environmental fate of perfluorooctane sulfonate in aquatic ecosystems. Environ. Sci. Technol. 40, 4916-4921.
Oakes, K.D., Sibley, P.K., Martin, J.W., MacLean, D.D., Solomon, K.R., Mabury, S.A., Van Der Kraak, G.J., 2005. Short-term exposures of fish to perfluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids. Environ. Toxicol. Chem. 24, 1172-1181.
Oakes, K.D., Sibley, P.K., Solomon, K.R., Mabury, S.A., Van Der Kraak, G.J., 2004. Impact of perfluorooctanoic acid on fathead minnow (Pimephales promelas) fatty acyl-CoA oxidase activity, circulating steroids, and reproduction in outdoor microcosms. Environ. Toxicol. Chem. 23, 1912-1919.
Ohmori, K., Kudo, N., Katayama, K., Kawashima, Y., 2003. Comparison of the toxicoki- netics between perfluorocarboxylic acids with different carbon chain length. Toxicology 184, 135-140.
Olsen, G.W., Burris, J.M., Ehresman, D.J., Froehlich, J.W., Seacat, A.M., Butenhoff, J.L., Zobel, L.R., 2007. Half-life of serum elimination of perfluorooctanesulfonate, perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ. Health Perspect. 115, 1298-1305.
Olsen, G.W., Gilliland, F.D., Burlew, M.M., Burris, J.M., Mandel, J.S., Mandel, J.H., 1998. An epidemiologic investigation of reproductive hormones in men with occupational exposure to perfluorooctanoic acid. J. Occup. Environ. Med. 40, 614-622.
Shi, Z., Zhang, H., Liu, Y., Xu, M., Dai, J., 2007. Alterations in gene expression and testosterone synthesis in the testes of male rats exposed to perfluorododecanoic acid. Toxicol. Sci. 98, 206-215.
Skakkebaek, N.E., Rajpert-De, M.E., Main, K.M., 2001. Testicular dysgenesis syn- drome: an increasingly common developmental disorder with environmental aspects. Hum. Reprod. 16, 972-978.
Thibodeaux, J.R., Hanson, R.G., Rogers, J.M., Grey, B.E., Barbee, B.D., Richards, J.H., Butenhoff, J.L., Stevenson, L.A., Lau, C., 2003. Exposure to perfluorooctane sul- fonate during pregnancy in rat and mouse. I: maternal and prenatal evaluations. Toxicol. Sci. 74, 369-381.
Wei, Y., Liu, Y., Wang, J., Tao, Y., Dai, J., 2008. Toxicogenomic analysis of the hepatic effects of perfluorooctanoic acid on rare minnows (Gobiocypris rarus). Toxicol. Appl. Pharmacol. 226, 285-297.
Yamashita, N., Kannan, K., Taniyasu, S., Horii, Y., Petrick, G., Gamo, T., 2005. A global survey of perfluorinated acids in oceans. Mar. Pollut. Bull. 51, 658-668.
Zimmer, K.E., Montano, M., Olsaker, I., Dahl, E., Berg, V., Karlsson, C., Murk, A.J., Skaare, J.U., Ropstad, E., Verhaegen, S., 2011. In vitro steroidogenic effects of mixtures of persistent organic pollutants (POPs) extracted from burbot (Lota lota) caught in two Norwegian lakes. Sci. Total Environ. 409, 2040-2048.