(NON SULUS
ELSEVIER
Chemosphere
2
CHEMOSPHERE ENVIRONMENTAL TOXICOLOGY AND RISK ASSESSMENT
Effects of polar oil related hydrocarbons on steroidogenesis in vitro in H295R cells
CrossMark
Anne Christine Knaga,*, Steven Verhaegen b, Erik Ropstad b, Ian Mayer a,b, Sonnich Meier℃
a Department of Biology, University of Bergen, P.O. Box 7803, N-5020 Bergen, Norway
b Norwegian School of Veterinary Science, P.O. Box 8146, Dep, N-0033 Oslo, Norway
” The Institute of Marine Research, P.O. Box 1870, Nordnes, N-5817 Bergen, Norway
HIGHLIGHTS
· H295r cells were exposed to polar hydrocarbons.
· Alkylphenols, naphthenic acids and produced water induced E2 and P4 production.
· Exposure to naphthenic acids caused a decrease in testosterone.
· All compounds cause an up-regulation in CYP1A.
ARTICLE INFO
Article history: Received 18 August 2012 Received in revised form 24 January 2013 Accepted 17 February 2013 Available online 2 April 2013
Keywords: Naphthenic acids Alkylphenols PAHs Produced water Endocrine disruption
ABSTRACT
Oil pollution from various sources, including exploration, production and transportation, is a growing global concern. Of particular concern is the environmental impact of produced water (PW), the main waste discharge from oil and gas platforms. In this study, we have investigated the potential of polar hydrocarbon pollutants to disrupt or modulate steroidogenesis in vitro, using a human adrenocortical car- cinoma cell line, the H295R assay. Effects of two of the major groups of compounds found in the polar fraction of crude oil and PW; alkylphenols (C2- and C3-AP) and naphthenic acids (NAS), as well as the polar fraction of PW as a whole has been assessed. Endpoints include hormone (cortisol, estradiol, pro- gesterone, testosterone) production at the functional level and key genes for steroidogenesis (17ß- HSD1, 17-HSD4, 3B-HSD2, ACTHR, CYP11A1, CYP11B1, CYP11B2, CYP17, CYP19, CYP21, DAX1, EPHX, HMGR, SF1, STAR) and metabolism (CYP1A) at the molecular level. All compounds induced the produc- tion of both estradiol and progesterone in exposed H295R cells, while the C3-AP and NAs decreased the production of testosterone. Exposure to C2-AP caused an up-regulation of DAX1 and EPHX, while exposure to NAs caused an up-regulation of ACTHR. All compounds caused an up-regulation of CYP1A1. The results indicated that these hydrocarbon pollutants, including PW, have the potential to disrupt the vitally important process of steroidogenesis.
@ 2013 Elsevier Ltd. All rights reserved.
1. Introduction
There is increasing public concern over the environmental im- pact of hydrocarbon pollutants resulting from oil exploration, pro- duction and transport (Ivanov, 2011). Of particular concern is the potential impact of both produced water (PW), the main waste dis- charge from offshore oil production facilities, and crude oil result- ing from oil-leakages and spills, on aquatic wildlife. Crude oil and PW both contain a complex mixture of hydrocarbons, several of which are known to cause adverse biological effects. Among these compounds are PAHs (polyaromatic hydrocarbons), as well as sev- eral polar compounds, for example alkylphenols (APs) and naph- thenic acids (NAS) (Røe Utvik, 1999).
In the literature APs are widely reported as estrogenic com- pounds, with their potency depending on the molecular structure (Routledge and Sumpter, 1997). The majority of APs present in PW are the more water soluble short-chained APs (C1-C3), reported to have ER agonistic properties in vitro (Thomas et al., 2004; Tollef- sen and Nilsen, 2008).
NAs are a highly complex mixture of polar organic carboxylic acids, composed of a carbon backbone of between 9 and 20 carbon atoms, and with a molecular weight between 120 and 700+. NAs are a natural component of all fossil fuels (Whitby, 2010), and may account for as much as 4% of the weight of crude petroleum (Clemente and Fedorak, 2005; Whitby, 2010). As such, NAs are also a major component of oil spills and oil production discharges (Rowland et al., 2011a). Further, NAs are also present in bitumen and most of the acute toxicity associated with oil sand process water (OSPW) is believed to be explained by the presence of high
* Corresponding author. Tel .: +47 970 46 302; fax: +47 5558 4450. E-mail address: anne.knag@bio.uib.no (A.C. Knag).
levels of NAs (Dokholyan and Magomedov, 1983; Mackinnon and Boerger, 1986; Madill et al., 2001; Frank et al., 2009; He et al., 2010). Although the mode of action of its reported toxicity is still uncertain, (He et al., 2010), with a suggested primary mode of action being cell narcosis (Frank et al., 2009), adverse biological effects of NAs have been reported in several wildlife species (reviewed in Cle- mente and Fedorak (2005) and Whitby (2010)). Several studies have reported altered sex steroid levels in aquatic wildlife following exposure to OSPW. For example, yellow perch (Perca flavescens) ex- posed to OSPW displayed decreased plasma levels of sex steroids (van den Heuvel et al., 1999a,b, 2012). Similarly, plasma levels of both testosterone (T) and estradiol (E2), as well as cortisol, were sig- nificantly reduced in goldfish (Carassius auratus) exposed to OSPW (Lister et al., 2008). In the same study, OSPW also inhibited gonadal T production in vitro. Recently, studies have reported reduced plas- ma levels of T and 11-ketotestosterone (11kT), and reduced levels of E2 in male and female fathead minnow (Pimephales promelas) respectively when exposed to either OSPW or NAs extracts (Kava- nagh et al., 2011, 2012). Using the H295R steroidogenic assay, He et al. (2010) recently demonstrated that OSPW was capable of dis- rupting normal steroidogenesis in vitro, with a significant decrease in T and increase in E2 production. This supports earlier findings that PW extracts, containing NAs, displayed both estrogenic and anti- androgenic properties in vitro (Thomas et al., 2009).
A wide variety of organic pollutants, including PAHs and APs have now been shown to interfere with the cytochrome P450 enzymes, notably aromatase (CYP19), the key enzyme controlling the conver- sion of T to E2 (Kazeto et al., 2004; Hinfray et al., 2006; Meucci and Arukwe, 2006; Bonefeld-Jørgensen et al., 2007). Aromatase might be impaired directly through competitive ligand binding or indi- rectly through feedback loops increasing aromatase to compensate for change in T or E2 (Ung and Nagar, 2009). Similarly, APs are known to inhibit the enzymes CYP11A, CYP17 and CYP21B, causing a de- crease in dibutyryl-cAMP induced cortisol secretion by exposed H295R cells (Nakajin et al., 2001). Several in vivo studies have also found that exposure to long- (nonylphenol) and middle-chained (4-tert-pentylphenol) APs down-regulates the gene expression of CYP11A in testis of medaka (Oryzias latipes) (Yokota et al., 2005) and the brain of Atlantic salmon, Salmo salar (Arukwe, 2005).
There are still major knowledge gaps in our understanding of the mode of action of hydrocarbon pollutants, notably the NAs and short-chain APs found in high concentration in the polar frac- tion of crude oil and PW. As such, the primary aim of this study was to evaluate further the effects of these compounds, as well as the polar fraction of PW, on steroidogenesis, both at the functional (hormone production) and the molecular level (key steroidogene- sis and metabolism gene expression). To that end, H295R cells were exposed to test compounds/mixtures via the cell medium, and the effect of exposure on steroid hormone secretion evaluated. Further, in addition to CYP1A1, the expression of 15 key genes reg- ulating the steroidogenic pathway were analysed by quantitative RT-PCR. Together, these results would enable a more comprehen- sive biological risk assessment of exposure to hydrocarbon pollu- tants to aquatic wildlife, specifically indicating their potential to impact reproductive function via deleterious effects on the steroi- dogenic pathway.
2. Materials and methods
2.1. Experimental design
The impact of four groups of compounds (NAS, C2-AP, C3-AP and the polar phase of PW) on steroidogenesis was evaluated in vitro using the H295R cell bioassay; a human adrenocortical cell-line capable of full steroidogenesis (Gazdar et al., 1990). The major
end-points of this steroidogenesis assay were the measurement of the mRNA of key genes involved in the steroidogenic pathway (analysed with quantitative RT-PCR) and the quantitative mea- surement of the key steroid hormones cortisol, E2 and T (analysed by radioimmunoassay). The hypothesis that was tested was: polar organic hydrocarbons have the potential to alter hormone levels and mRNA levels of genes involved in the steroidogenic pathway.
2.2. Chemicals and oil compounds
Ethanol, dimethylsulfoxide (DMSO), forskolin (CASNR 66575- 29-9) 98%, dichloromethane (DCM) and acetonitrile were all pur- chased from Sigma-Aldrich (Oslo, Norway). PW was obtained from Statoil’s platform Oseberg C, located in the Norwegian sector of the North Sea (60°36’28” N, 2º46’28” E). The PW was transported (within 24 h after collection) from the platform to the Department of Biology, University of Bergen, where it was immediately ali- quoted into 10 L containers and frozen at -20 ℃. The organic frac- tion of the PW was extracted with DCM and the polar phase was separated from the nonpolar hydrocarbons by partitioning be- tween hexane and acetonitrile as described in Boitsov et al. (2007). In brief, 2 L of PW were extracted three times with DCM (150 mL, 50 mL, 50 mL) and the combined extracts were dried with MgSO4 and filtered through a glass filter funnel. The extracts were reduced to dryness at 20 ℃ by rotary evaporation. The residues was dissolved in 4 mL of hexane and shaken twice with 6 mL of acetonitrile saturated with hexane. The combined acetonitrile layer was evaporated to dryness by rotary vaporation. The residues were re-dissolved in DCM and transferred to a weighed glass tube and evaporated to dryness under nitrogen gas flow, and the resi- dues were dissolved in ethanol to concentration of 3.7 mg m-1L-1. Commercial petroleum derived NAs were purchased from Sigma- Aldrich (Oslo, Norway) and short chained APs were purchased from Chiron (Trondheim, Norway). Environmentally relevant artificial mixtures were formulated in the laboratory to closely mimic the composition C2- and C3-APs found in waste discharges from North Sea offshore oil platforms (Harman et al., 2009) (see Table 1 for chemical composition of stock solutions).
2.3. Chemical characterization
The stock solutions were characterized at the Laboratory for Environmental Chemistry at the Institute of Marine Research, Ber- gen, Norway. The AP and the PAH concentration in the PW was determined by chromatography-mass spectrometry GC-MS according to previously described methods (Boitsov et al., 2007, 2011). The composition of the different APs and the PAH in the PW fraction is shown in Fig. 1, and the isomers composition of the C2-AP and C3-AP solutions are given in Table 1.
The distribution of the different molecular size and ring struc- tures of the commercial petroleum derived NAs were tentatively analysed after derivatization with N-methyl-N-(t-butyldimethylsi- lyl)-trifluoroacetamide (MTBSTFA) using previously described GC- MS method (St. John et al., 1998; Young et al., 2010). The samples were analysed by full scan from 50 to 500 m/z and the integrated areas were obtained for clusters of all the theoretical possible masses for the [M + 57]+ ions for CnH2n + ZO2 (where n is the num- ber of carbon atoms and Z the hydrogen deficiency resulting from ring formation. There were scanned for n = 5-29 and Z = 0-12, in total 128 masses. Extracted ion mass and distribution of NAs shown in Tables 2A and 2B.
2.4. Cell culture and exposure
Human adrenocortical carcinoma H295R-cells obtained from ATCC (CRL-2128; ATCC, Manassas, VA, USA) were cultured and
| A | PW extract (µg m-1L-1) | C2-AP stock (mg m-1L-1) | C3-AP stock (mg m-1L-1) |
|---|---|---|---|
| Phenol | 424.9 ± 7.7 | – | – |
| o-Cresol | 423.9 ± 8.4 | – | – |
| m-Cresol | 288.7 ± 3.4 | – | – |
| p-Cresol | 220.8 ± 0.8 | – | – |
| Σ Cresol | 933.4 ± 11.8 | – | – |
| 2,6-Dimethylphenol | 18.2 ±3.0 | 4.0 | – |
| 2,5-Dimethylphenol | 159.2 ± 1.4 | 21.1 | – |
| 2,4-Dimethylphenol | 273.1 ± 3.7 | 34.7 – | |
| 2,3-Dimethylphenol | 65.9 ±0.8 | 1.7 – | |
| 3,4-Dimethylphenol | 78.0 ± 0.8 | 1.9 – | |
| 3,5-Dimethylphenol | 121.3 ± 0.7 | 2.9 – | |
| 2-Ethylphenol | 39.1 ± 0.8 | 1.2 – | |
| 4-Etyhylphenol | 46.0 ±0.7 | 9.0 | – |
| Σ C2 phenol | 800.8 ± 4.8 | 76.5 | – |
| 2,4,6-Trimethylphenol | 3.7 ±0.8 | – | 4.1 |
| 2,3,6-Trimethylphenol | 5.8 ±0.3 | 2.5 – | |
| 2,3,5-Trimethylphenol | 5.2 ± 0.1 | 12.6 – | |
| 3-Etyl4-methylphenol | 40.6 ± 0.5 | – | 8.2 |
| 2-iso-Propylphenol | 14.5 ±0.2 | – | 9.5 |
| 3-iso-Propylphenol | 18.1 ±0.5 | – | 11.0 |
| 4-iso-Propylphenol | 62.3 ± 0.6 | 9.5 – | |
| 2-n-Propylphenol | 3.5 ± 0.0 | 8.0 – | |
| 3-n-Propylphenol | 18.8 ± 2.1 | 6.6 – | |
| 4-n-Propylphenol | 6.2 ± 0.3 | 4.9 – | |
| Σ C3-phenol | 178.7 ± 1.6 | – | 76.7 |
| Σ C4-phenol | 32.4 ± 0.6 | – | – |
| EC5-C9-Phenol | 2.2 ±0.3 | – – | |
| B | PW extract (µg m-1L-1) |
|---|---|
| Naphthalene | 3.60 ± 0.40 |
| ΣMethylnaphthalene | 3.96 ± 0.08 |
| EDimethylnaphtalene | 1.08 ± 0.06 |
| >Trimethylnaphthalene | 0.43 ± 0.08 |
| ETetramethylnaphthalene | 0.06 ± 0.01 |
| >2-Rings PAHs | 14.60 ± 0.55 |
| Phenanthrene | 0.34 ± 0.02 |
| ΣMethylphenanthrene | 0.79 ± 0.01 |
| >Dimethylphenanthrene | 0.22 ± 0.02 |
| ETrimethylphenantrene | 0.11 ± 0.05 |
| 1,2,6,9-Tetramethylphenantrene | 0.00 ± 0.00 |
| Dibenzothiophene | 0.08 ± 0.01 |
| 4-methyldibenzotiophene | 0.10 ± 0.00 |
| 4-ethyldibenzotiophene | 0.02 ± 0.00 |
| 4-propyldibenzotiophene | 0.03 ± 0.01 |
| Acenaphthylene | 0.01 ± 0.00 |
| Acenaphthene | 0.02 ± 0.00 |
| Fluorene | 0.17 ± 0.02 |
| Anthracene | 0.00 ± 0.00 |
| >3-Rings PAHs | 3.01 ± 0.18 |
| _4-Rings PAHs | 0.17 ± 0.03 |
| >5-Rings PAHs | 0.02 ± 0.00 |
maintained as described previously (Gazdar et al., 1990; Hilscherova et al., 2004). Cultures were kept in 75 cm2 flasks at 37 ℃ and 5% CO2 in a humidified atmosphere, in Dulbecco’s modified Eagle’s medium with Ham’s F12 nutrient mixture (DMEM/F12) containing HEPES buffer, L-glutamine, pyridoxine HCl (Gibco Invitrogen, Paisley, UK), supplemented with 1% ITS + Premix (BD Bioscience, Bedford, MA) and 2.5% NuSerum (BD Bioscience, San Jose, CA, USA). The medium was changed three times a week and cells detached for sub-cultur- ing once a week using trypsin/EDTA (Gibco Invitrogen, Paisley, UK).
H295R cell suspensions were seeded at a density of 3 x 105 cell m-1L-1 in 24-well transparent flat bottom plates (Falcon, Franklin Lakes, NJ). 24 h post seeding, the medium was refreshed and cells were exposed to treatments (in triplicates) for 48 h. Eth- anol was used as the carrier solvent and did not exceed 0.1% v v-1. In the H295R assays, three concentrations (high, medium, and low) of the four test compounds were used for steroid hormone
analysis, while gene expression was only evaluated from the high exposure groups.
The concentrations of stock solutions and medium are given in 3. Test plates included triplicates of 0.1% ethanol as solvent control as well as 5 uM forskolin as positive control. At the end of each experiment the culture medium was transferred to a plastic tube and stored at -20 ℃ until hormone analysis (three exposure experiments), or the medium was discarded and the plates frozen on dry ice and stored at -80 ℃ prior to gene expression analysis (five exposure experiments).
2.5. Cell viability/cytotoxicity
After extraction of the medium for hormone analysis, cell viabil- ity was measured on the remaining cells using the alamarBlue™ (Serotec Ltd., Oxford, UK) cytotoxicity assay. After 48 h of exposure
1000
A
12
B
I
10
800
=
AP (µg/ml)
PAH (µg/ml)
8
600
6
400
I
4
200
2
I
0
0
Phenol
Σ Cresol
Σ C2 phenol
Σ C3 phenol
Σ C4-phenol
EC5-C9-Phenol
2-Rings PAHs
3-Rings PAHs
4-Rings PAHs
5-Rings PAHs
| Carbon number | Z family | ||||||
|---|---|---|---|---|---|---|---|
| 0 | -2 | -4 | -6 | -8 | -10 | -12 | |
| 5 | 159 | ||||||
| 6 | 173 | ||||||
| 7 | 187 | 185 | |||||
| 8 | 201 | 199 | |||||
| 9 | 215 | 213 | |||||
| 10 | 229 | 227 | 225 | ||||
| 11 | 243 | 241 | 239 | ||||
| 12 | 257 | 255 | 253 | 251 | |||
| 13 | 271 | 269 | 267 | 265 | |||
| 14 | 285 | 283 | 281 | 279 | 277 | ||
| 15 | 299 | 297 | 295 | 293 | 291 | ||
| 16 | 313 | 311 | 309 | 307 | 305 | 303 | |
| 17 | 327 | 325 | 323 | 321 | 319 | 317 | |
| 18 | 341 | 339 | 337 | 335 | 333 | 331 | 329 |
| 19 | 355 | 353 | 351 | 349 | 347 | 345 | 343 |
| 20 | 369 | 367 | 365 | 363 | 361 | 359 | 357 |
| 21 | 383 | 381 | 379 | 377 | 375 | 373 | 371 |
| 22 | 397 | 395 | 393 | 391 | 389 | 387 | 385 |
| 23 | 411 | 409 | 407 | 405 | 403 | 401 | 399 |
| 24 | 425 | 423 | 421 | 419 | 417 | 415 | 413 |
| 25 | 439 | 437 | 435 | 433 | 431 | 429 | 427 |
| 26 | 453 | 451 | 449 | 447 | 445 | 443 | 441 |
| 27 | 467 | 465 | 463 | 461 | 459 | 457 | 455 |
| 28 | 481 | 479 | 477 | 475 | 473 | 471 | 469 |
the cells were refreshed with 1 mL of complete medium, after which 100 µL of Alamar Blue was added to each well and the cells incubated at 37 ℃ and 5% CO2 in a humidified atmosphere for 3 h. A 100 µL aliquot from each well was collected into a 96-well trans- parent well plate (Falcon, Franklin Lakes, NJ), and absorbance was measured at 570 and 600 nm in a VICTOR3™ spectrophotometer (Perkin Elmer, Shelton, CT).
2.6. Hormone measurement
Incubation medium was thawed and divided in two technical replicates per well per hormone analysis. Concentrations of E2, T and cortisol were determined by solid-phase radioimmunoassay kits “Coat-a-Count R” from Diagnostic Products Corporation, Los Angeles, CA, USA (“Estradiol”, Cat #, TKE25; “Total Testosterone”, Cat #, TKTT5; “Cortisol”, Cat #, TKCO5). Progesterone (P4) concen- trations were determined by solid-phase radioimmunoassay kit “Spectria” from Orion Diagnostica, Espoo, Finland. The kit assays
were modified by replacing the standard curves in serum with standards diluted in cell culture medium. There were no difference between solvent exposed cells and blanks, so the blanks were used for comparisons.
2.7. RNA Isolation
Total RNA was isolated from the cells exposed to the highest dose of pollutant using the RNeasy Mini prep, RNA isolation kit (Qiagen, Crawley, UK). The standard protocol for cells was followed. Follow- ing thawing of plates, cells were lysed by addition of lysis buffer di- rectly to each well of the culture plate. The triplicate wells from each plate were pooled and transferred to a QIAshredder spin column, centrifuged and the flow through transferred to RNeasy spin col- umns. Samples were DNase (Qiagen, Crawley, UK) treated for 15 min at room temperature on the spin column. Total RNA was eluted from the RNeasy Mini columns with RNase-free water, and stored at -80 ℃ until use. RNA quantity and quality were measured using Nano-drop (Thermo-Scientific, Waltham, MA, USA) and an Agilent RNA 6000 Nano LabChip Kit run on a 2100 Bioanalyser (Agi- lent Technologies, CA, USA), respectively.
2.8. cDNA prep and messenger RNA quantification by RT-PCR
cDNA was synthesized using the SuperScript VILO cDNA Syn- thesis Kit with SYBR GreenER qPCR Supermix for ABI PRISM (Invit- rogen, Carlsbad, CA, USA). Controls with no reverse transcriptase and non-template controls were also added to the RT-reaction. The cDNA was synthesized in a Peltier Thermal Cycler-225 (MJRe- search, Waltham, MA, USA). For cDNA synthesis, ABgene PCR plates (Thermo Scientific) with Absolute QPCR Seal (Thermo Scientific, Surrey, UK cat AB 1170) were used.
The resulting cDNA was amplified by real-time, quantitative PCR using gene-specific primers for 16 different genes: 17ß-HSD1 and 170-HSD4 (17ß-hydroxysteroid dehydrogenase, types 1 and 4), 3ß-HSD2 (3ß-hydroxysteroid dehydrogenase), ACTHR (adreno- corticotropic hormone receptor), CYP11A (cholesterol side-chain cleavage), CYP11B1 (steroid 11ß-hydroxylase, CYP11B2 (aldoste- rone synthetase), CYP17 (steroid 17a-hydroxylase and/or 17,20 lyase), CYP19 (aromatase), CYP1A1 (aryl hydrocarbon hydroxy- lase), CYP21 (steroid 21-hydroxylase), DAX1 (nuclear receptor pro- tein), EPHX (epoxide hydrolase), HMGR (hydroxymethylgutaryl CoA reductase), SF1 (steroidogenic factor 1) and StAR (steroid acute regulatory protein).
Primers used are described in Zimmer et al. (2011). The assay was optimized with regard to cDNA concentration. In total
| Carbon number | Percentage of naphthenic acids by z number | Percentage by carbon number | ||||||
|---|---|---|---|---|---|---|---|---|
| 0 | 2 | 4 | 6 | 8 | 10 | 12 | ||
| 5 | 7.66 | 7.66 | ||||||
| 6 | 1.78 | 1.78 | ||||||
| 7 | 1.47 | 2.28 | 3.75 | |||||
| 8 | 2.73 | 2.45 | 5.19 | |||||
| 9 | 16.12 | 3.05 | 19.17 | |||||
| 10 | 9.25 | 2.91 | 0.42 | 12.58 | ||||
| 11 | 10.12 | 2.35 | 0.64 | 13.11 | ||||
| 12 | 3.16 | 1.36 | 0.92 | 0.10 | 5.54 | |||
| 13 | 2.52 | 1.19 | 1.09 | 0.26 | 5.06 | |||
| 14 | 2.04 | 0.97 | 1.07 | 0.41 | 0.11 | 4.60 | ||
| 15 | 1.93 | 0.76 | 0.89 | 0.46 | 0.12 | 4.16 | ||
| 16 | 0.85 | 0.46 | 0.52 | 0.34 | 0.11 | 0.03 | 2.31 | |
| 17 | 0.41 | 0.25 | 0.31 | 0.17 | 0.06 | 0.02 | 1.22 | |
| 18 | 0.59 | 0.19 | 0.16 | 0.07 | 0.02 | 0.02 | 0.04 | 1.10 |
| 19 | 6.45 | 0.48 | 0.08 | 0.03 | 0.01 | 0.01 | 0.05 | 7.10 |
| 20 | 0.99 | 0.08 | 0.03 | 0.00 | 0.00 | 0.01 | 0.49 | 1.61 |
| 21 | 0.16 | 0.01 | 0.00 | 0.01 | 0.00 | 0.02 | 0.09 | 0.29 |
| 22 | 0.28 | 0.01 | 0.00 | 0.00 | 0.11 | 0.96 | 0.03 | 1.39 |
| 23 | 0.07 | 0.00 | 0.00 | 0.00 | 0.00 | 1.24 | 0.02 | 1.33 |
| 24 | 0.00 | 0.00 | 0.00 | 0.00 | 0.01 | 0.06 | 0.00 | 0.07 |
| 25 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.93 | 0.00 | 0.93 |
| 26 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.06 | 0.00 | 0.07 |
| 27 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.01 | 0.00 | 0.02 |
| 28 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| Total | 68.57 | 18.82 | 6.12 | 1.86 | 0.54 | 3.37 | 0.72 | 100.00 |
| NA (µg dw m-1L-1) | C2-AP (µg dw m-1L-1) | C3 AP (µg dw m-1L-1) | PW (µg dw m-1L-1) | |
|---|---|---|---|---|
| Stock solution conc. | 102 150 | 76544 | 76 690 | 3710 |
| High exposure conc. | 10 | 10 | 10 | 4 |
| High medium exposure conc. | 2.2 | |||
| Medium exposure conc. | 1 | 1 | 1 | 1 |
| Low exposure conc. | 0.1 | 0.1 | 0.1 | 0.1 |
160 ng of cDNA when assuming full reverse transcriptase (RT) effi- ciency, was added to the two-step qRT-PCR reaction. Samples were run in triplicate on a Fast Optical 96-well Reaction Plate with Micro- Amp Optical Adhesive Film (Applied Biosystems, Foster City, CA, USA). qPCR reactions were carried out using “Applied Biosystems 7900HT fast real-time PCR system”, and it’s software SDS 2.3. qPCR MicroAmp. The PCR thermal cycle profile was (1) 50 ℃ for 2 min (UDG incubation), (2) 95 ℃ 10 min (enzyme activation), (3) 40 cy- cles of 95 ℃ in 15 s (denaturation) and (4) 60 ℃ in 60 s (annealing and elongation). Dissociation curves were applied to check for pri- mer dimers and unspecific amplification. PCR efficiencies were as- sessed by standard curves with serial dilutions of cDNA.
The GeNorm-method was used to predict the most stable refer- ence genes (Vandesompele et al., 2002). GeNorm human detection kit and software were obtained from PrimerDesign Ltd. (South- ampton, UK). Of the five potential reference genes tested (B2M, BACT, YWHAZ, ATP5B and GAPDH), the two most stable genes, YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) and B2M (ß2 microglobulin) were selected as reference genes in this study (average expression stability, M = 0, 1 724591).
2.9. Statistics
2.9.1. Cell viability
Viability as per cent of solvent control was plotted against con- centration (low, medium, high) of each compound according to manufacturer’s protocol. As the number of viable cells is directly
proportional to the mitochondrial reduction of alamarBlue (ob- served as colour/fluorescent change) a linear curve of reduction implies no cytotoxicity of the doses tested.
2.9.2. Hormones
Exposures were conducted in triplicate wells at triplicate time points, and each individual exposure was measured in duplicate. Log transformation of E2 and cortisol gave a better fit to the normal distribution according to Shapiro and Wilks Goodness of Fit Test, and were used in the statistical analyses using JMP, version 10.0.0 (SAS Institute Inc., 2012). Untransformed data for progester- one and T gave a satisfactory fit to the normal distribution. General linear models (GLMs) were used. Measured hormone concentra- tions or log-transformed hormone concentrations were dependent variables. Independent variables were experiment (n = 3) and dilu- tions exposure compounds in culture medium entered as discrete variables. Dose-response relationships were evaluated by entering dilutions of exposure compounds as a continuous variable. Differ- ences between mean hormone concentrations were evaluated with Tukey HSD test, with an alpha level of 0.05.
2.9.3. Genes
Exposures were conducted in triplicate wells at five time points, the triplicate wells combined and measured in triplicate. Data was normalized using the geometric mean of selected reference genes, and target specific amplification efficiencies. Targets were scaled to solvent control (EtOH) set as 1.00. The quantitative gene expres- sion assays were analysed in qbasePLUS software version 2.3,
(Biogazelle) as described previously (Hellemans et al., 2007). The qbasePLUS programme uses the Tukey-Kramer method for post hoc multiple testing correction and this controls for all pairwise comparisons.
3. Results
3.1. Chemical characterization of AP and PAH in PW extract
The selected doses of exposure were not associated with any acute cytotoxicity in the H295R assay.
The concentration of exposure compounds in stock solutions and exposure wells are summarized in Table 3. The concentration and composition of the APs in the polar extract of PW from Oseberg C are shown in Table 1A and Fig. 1A. The APs contribute 65% of the dry weight of the polar extract, and total AP composition is domi- nated by the low chain length APs (18% phenol, 39% cresol, 34% C2- AP, 7% C3-AP, 2% C4-AP and 0.1% C5-9-AP). Table 1B and Fig. 1B shows the concentration and composition of the PAHs in the polar extract of PW from Oseberg C. The PAH are dominated by 2 and 3 rings PAH and their alkyl homologues. The concentration of PAH in polar extract is 11 µg L-1 and this correspond 0.3% of the dry weight. The GC-MS analysis of the NAs shows an extremely com- plex mixture, and the m/z of the [M + 57]+ indicates that the com- mercial petroleum derived NAs from Sigma-Aldrich is dominated
by acyclic carboxylic acids (68%), followed by 1 ring NAs (19%) and bicyclic NAs (6%). However the complexity and the lack of standards and possibility of generating response-factors make this qualitative distribution highly tentative.
3.2. Hormones
3.2.1. 17-estradiol
There was a significant dose-response relationship in all expo- sures, with E2 production increasing with increasing concentration of test compounds (see Fig. 2A). A significant increase in E2 produc- tion was found in cells exposed to the highest dose of all com- pounds, both C2- and C3-APs exposed cells showed a significant increase also in the medium dose. See Fig. 2A: comparisons with medium collected from cells exposed to solvent control (EtOH).
3.2.2. Progesterone
A significant dose-response relationship was observed in all exposures, with progesterone production increasing with increas- ing concentration of test compounds (see Fig. 2B). Exposure to C3-AP caused a significant increase in P4 secretion in all doses, C2-AP exposed cells produces significantly more P4 in medium and high, while PW and NA caused a significant increase in produc- tion at the highest doses. See Fig. 2B: comparisons with medium collected from cells exposed to solvent control (EtOH).
200
A
3
*
B
*
*
*
150
*
2
*
*
*
100
*
*
1
50
*
*
*
0
0
2.0
C
200
D
1.5
150
*
*
1.0
100
.5
50
.0
0
L MMHH LMH L MH L MH
L M MHH L MH L
LMHLMHLMH
MB SC
PW
NA
C2-AP C3-AP
MB SC
PW
NA
C2-AP C3-AP
3.2.3. Testosterone
There was a significant dose-response relationship in NAs and C3-AP, with T production decreasing with increasing concentration of these test compounds. Exposure to the highest test concentra- tions of both NA and C3-AP gave significantly lower T production. See Fig. 2C: comparisons with medium collected from cells ex- posed to solvent control (EtOH).
3.2.4. Cortisol
There were no significant differences with solvent control in mean cortisol production in cells exposed to either of the hydrocar- bon pollutants. There was a significant dose effect in the cortisol secretion of C2-AP exposed cells. See Fig. 2D: comparisons with medium collected from cells exposed to solvent control (EtOH).
3.3. Gene expression
Several genes were significantly up-regulated with a signifi- cance level of >0.05 and a cut-off value for fold change >1.5 at the transcriptional level (Fig. 3). CYP1A1 (aryl hydrocarbon hydroxylase) was up-regulated by all exposure compounds (fold
change PW: 9.6; NAs: 2.3; C2-AP: 1.9; C3-AP: 2.2). DAX1 (a nuclear receptor protein) was up-regulated by C2-AP (fold change 1.7), ACTHR (adrenocorticotropic hormone receptor) was up-regulated by NAs (fold change 3.5), and EPHX (epoxide hydrolase) was up- regulated by C2-AP (fold change 1.5).
4. Discussion
Previous studies have shown that hydrocarbon pollutants such as PW and NAs can act as putative endocrine disrupters, and as such have the potential to disrupt normal reproductive function. However, it is increasingly being recognized that anthropogenic pollutants have the potential to disrupt reproductive and develop- mental processes via different mode of actions, one of which is their capacity to disrupt or modulate steroidogenesis, the essential biochemical pathway controlling reproductive function in all ver- tebrates (Kime, 1987). In this in vitro study we report that polar pollutants found in PW have the capacity to modulate this bio- chemical pathway, which potentially could result in deleterious reproductive effects.
17@HSD1
17BHSD4
3ßHSD2
ACTHR
3
1.5
6
8
Fold Change
*
Fold Change
Fold Change
Fold Change
2
1.0
4
6
4
1
0.5
2
2
0
EtOH
PW
0.0
NA
C2-AP
C3-AP
EtOH
PW
NA
C2-AP
C3-AP
0
EtOH
PW
NA
C2-AP
C3-AP
0
EtOH
PW
NA
C2-AP
C3-AP
CYP11A1
CYP11B1
CYP11B2
CYP17
2.0
20
20
4
Fold Change
1.5
Fold Change
15
Fold Change
15
Fold Change
3
1.0
10
10
2
0.5
5
5
1
0.0
0
EtOH
PW
C2-AP
C3-AP
PW
C2-AP
C3-AP
0
PW
C2-AP
C3-AP
0
NA
EtOH
NA
EtOH
NA
EtOH
PW
NA
C2-AP
C3-AP
CYP19
CYP1A1
CYP21
DAX1
4
20
*
2.5
2.5
*
Fold Change
3
Fold Change
15
Fold Change
2.0
Fold Change
2.0
2
10
1.5
1.5
1.0
1.0
1
5
*
*
*
0.5
0.5
0
0
EtOH
PW
NA
C2-AP
C3-AP
EtOH
PW
NA
C2-AP
C3-AP
0.0
EtOH
PW
NA
C2-AP
C3-AP
0.0
EtOH
PW
NA
C2-AP
C3-AP
ΕΡΗΧ
HMGR
SF1
STAR
2.5
1.5
2.0
2.0
Fold Change
2.0
*
Fold Change
Fold Change
1.5
Fold Change
1.5
1.5
1.0
1.0
1.0
1.0
0.5
0.5
0.5
0.5
0.0
0.0
0.0
0.0
EtOH
PW
A
C2-AP
C3-AP
EtOH
PW
NA
C2-AP
C3-AP
EtOH
PW
NA
C2-AP
C3-AP
EtOH
PW
NA
C2-AP
C3-AP
All tested pollutants increased the production of both E2 and progesterone in exposed H295R cells. This indicates that the MOA of these compounds, in increasing E2 levels, might be through increased aromatase (CYP19) activity. Aromatase gene-expression was not significantly up-regulated in treated cells, but it has previ- ously been shown that the activity of aromatase is not reflected on the gene expression level of CYP19 in the H295R cell line (Hilscher- ova et al., 2004; Zhang et al., 2005).
The increased production of E2 and P4 coincided with a decrease in T production in cells exposed to NAs and C3-AP. For E2 and P4 production there was a significant response positively correlated to dose in all exposures, an indication of an estrogenic effect caused by these polar hydrocarbon pollutants. NAs and C3-AP had a significant negative dose-response relationship with T pro- duction, implying either (a) reduced T production due to an in- creased conversion to E2 or (b) an anti-androgenic effect resulting in a decrease in T production, as well as a corresponding increase in E2 production. There was a significant dose/cortisol-re- sponse in C2-AP exposed cells. These hormone results (up-regu- lated E2 and down-regulated T) corresponds well with a study where H295R cells were exposed to oil spill contaminated sedi- ments (Ji et al., 2011).
All hydrocarbon pollutants tested caused an up-regulation of CYP1A1, which indicates effects on the Phase I biotransformation system and confirms previous studies demonstrating up-regula- tion of this gene as a valid indicator of exposure to hydrocarbon pollution (Livingstone, 1993). The largest effect on CYP1A was found in PW-treated cells, and this effect is probably explained by the alkylated PAH-content in PW acting as ligands for AhR. Also Atlantic cod (Gadus morhua) exposed to C4-C7 AP showed an up- regulation in CYP1A mRNA (Lie et al., 2009) and protein level (Has- selberg et al., 2004). However, exposure to oil pollutant had no ef- fect on CYP1A expression in zebrafish, Danio rerio (Holth et al., 2008), and further a down-regulation at the protein level has been reported in Atlantic cod (Hasselberg et al., 2004; Sturve et al., 2006). As such, the use of only CYP1A expression as the single end- point of exposure effects has been precautioned against (Kammann et al., 2008).
The adrenocorticotropic hormone receptor is specific for ACTH, and binding stimulates glucocorticoid (and also mineralocorticoid and androgen) production (via the PKA pathway) (Sanderson, 2006). The up-regulation of ACTHR caused by NAs may explain the stimulation of steroidogenesis as found by increased hormone production. DAX1 (dosage sensitive sex reversal) is an orphan nu- clear receptor protein described as a general repressor of steroido- genesis (Zwermann et al., 2005). The up-regulation of DAX1 caused by C2-AP exposure suggests a possible repression of steroid pro- duction, although the opposite is found in measured hormone lev- els. Epoxide hydrolase (EH) does not have a described role in the adrenal gland steroidogenesis (Papadopoulos et al., 1994), but the up-regulation of EPHX caused by C2-AP indicates a detoxifica- tion process. Previous studies indicates that human microsomal EH is involved in ovarian estrogen production, implying that the EPHX up-regulation found in H295r cells exposed to C2-AP might also ex- plain the increased production of E2 (Hattori et al., 2000). Part of the estrogenic effects invoked by NP in fish has been explained by modulation of the steroidogenic acute regulatory (StAR) protein expression (Arukwe, 2005; Kortner and Arukwe, 2007). However, this effect was not significant in the present study although there was a tendency towards an up-regulation of StAR mRNA from C2-AP to C3-AP.
While APs are widely described in the literature as being estro- genic most studies have only assessed the effect of long-chained APs on receptor-mediated mechanisms. Importantly, here we re- port that also the quantitatively more ubiquitous shorter chained APs have an effect on the steroidogenesis.
The exposure concentrations of PW that were used were not environmentally relevant in terms of concentrations found at sea. The extracted Oseberg C sample had a dry weight concentration of 7.4 mg dw L-1. The high exposure group received a dose of 4 µg PW mL and this had a AP concentration approximately corre- sponding to exposure to 100% PW (2.6 µg AP m-1L-1 > 2.6 mg L-1) (Boitsov et al., 2007). The relative profile of the AP in the up-con- centrated extract had higher levels of the middle chain APs (C2- C4) and lower levels of the short chain APs (phenols and cresol) compared with what has previously been reported from Oseberg C PW (Boitsov et al., 2007). The lowest exposure groups received a dose (0.1 µg PW mL) corresponding to a 40 times dilution of PW. This is considered as relevant to concentrations expected in the waters close to the platform discharge point (Neff, 2002). The acid extractable compounds of both PW and OSPW are often re- ferred to as NAs per se, but recent studies suggest that these as well as commercial mixtures of “NAs” are not pure sources of NAs but also contains non-NAs compounds like AP and PAH (Grewer et al., 2010; West et al., 2011; Rowland et al., 2011b; Tollefsen et al., 2012). The commercial NAs mixture used here is not re- garded as representative for oil sands NAs also known as the “acid extractable fraction”. Our GC-MS analysis indicated the commer- cial NA from Sigma-Aldrich that was used in this experiment, is dominated by acyclic carboxylic acids, and this may explain why Tollefsen et al. (2012) found this mixture to have lower toxicity compared with other commercial NA mixtures.
The H295R assay cannot assess the effect of xenobiotic expo- sure at the hypothalamic or pituitary level, and as such the extrap- olation of in vitro data to in vivo and ultimately a population effect is extremely complex. However, the current results clearly indicate that polar hydrocarbons have the ability to modulate steroidogen- esis, and the impact of the quantitatively dominant short-chained APs should not be disregarded in environmental impact assessments.
Acknowledgements
The Norwegian Research Council and the L. Meltzer Foundation supported the research financially. Oseberg C (Statoil) is thanked for providing for and the shipping of the produced water samples. Ellen Dahl, Karin Zimmer, Camilla Karlsson and Doreen Ndossi at the Norwegian School of Veterinary Science are thanked for their great assistance with the RIAs, qPCRs and general cell maintenance, as well as Ingrid Olsaker who designed the primers.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.02.046.
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