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Exposure to the Three Structurally Different PCB Congeners (PCB 118, 153, and 126) Results in Decreased Protein Expression and Altered Steroidogenesis in the Human Adrenocortical Carcinoma Cell Line H295R
Nina Hårdnes Tremoenª, Paul A. Fowler”, Erik Ropstadª, Steven Verhaegenª & Anette Krogenæsª a
a Department of Production Animal Sciences, Norwegian School Veterinary Science, Oslo
b Institute of Medical Sciences, Division of Applied Medicine, University of Aberdeen, Aberdeen, Scotland, United Kingdom Published online: 22 Apr 2014.
To cite this article: Nina Hårdnes Tremoen, Paul A. Fowler, Erik Ropstad, Steven Verhaegen & Anette Krogenæs (2014) Exposure to the Three Structurally Different PCB Congeners (PCB 118, 153, and 126) Results in Decreased Protein Expression and Altered Steroidogenesis in the Human Adrenocortical Carcinoma Cell Line H295R, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:9-11, 516-534, DOI: 10.1080/15287394.2014.886985
To link to this article: http://dx.doi.org/10.1080/15287394.2014.886985
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EXPOSURE TO THE THREE STRUCTURALLY DIFFERENT PCB CONGENERS (PCB 118, 153, AND 126) RESULTS IN DECREASED PROTEIN EXPRESSION AND ALTERED STEROIDOGENESIS IN THE HUMAN ADRENOCORTICAL CARCINOMA CELL LINE H295R
Nina Hårdnes Tremoen1, Paul A. Fowler2, Erik Ropstad1, Steven Verhaegen1, Anette Krogenæs1
1 Department of Production Animal Sciences, Norwegian School Veterinary Science, Oslo
2 Institute of Medical Sciences, Division of Applied Medicine, University of Aberdeen, Aberdeen, Scotland, United Kingdom
Polychlorinated biphenyls (PCB), synthetic, persistent organic pollutants (POP), are detected ubiquitously, in water, soil, air, and sediments, as well as in animals and humans. PCB are associated with range of adverse health effects, such as interference with the immune system and nervous system, reproductive abnormalities, fetotoxicity, carcinogenicity, and endocrine disruption. Our objective was to determine the effects of three structurally dif- ferent PCB congeners, PCB118, PCB 126, and PCB 153, each at two concentrations, on the steroidogenic capacity and proteome of human adrenocortical carcinoma cell line cultures (H295R) . After 48 h of exposure, cell viability was monitored and estradiol, testosterone, cortisol and progesterone secretion measured to quantify steroidogenic capacity of the cells. Two-dimensional (2D) gel-based proteomics was used to screen for proteome alterations in H295R cells in response to the PCB. Exposure to PCB 118 increased estradiol and cortisol secretion, while exposure to PCB 153 elevated estradiol secretion. PCB 126 was the most potent congener, increasing estradiol, cortisol, and progesterone secretion in exposed H295R cells. Seventy-three of the 711 spots analyzed showed a significant difference in normalized spot volumes between controls (vehicle only) and at least one exposure group. Fourteen of these protein spots were identified by liquid chromatography with mass spectroscopy (LC-MS/MS). Exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. This study represents an initial analysis of the effects on proteins and hormones in the H295R cell model, and addi- tional studies are required in order to obtain a more complete understanding of the pathways disturbed by PCB congeners in H295R cells. Overall, alterations in protein regulation and steroid hormone synthesis suggest that exposure to PCB disturbs several cellular processes, including protein synthesis, stress response, and apoptosis.
Polychlorinated biphenyls (PCB) are syn- thetic industrial chemicals that are persistent in nature, bioaccumulate in food chains, and were widely used between the 1930s and 1970s. The potential harmful effects of the chemicals were recognized in the late 1970s and the production of PCB was banned. Due to their lipophilic and stable properties, PCB
bioaccumulate and biomagnify, which provides the basis for human exposure to PCB 40 noted years after their banning. PCB are associ- ated with a range of adverse health effects such as interference with the immune sys- tem (Nakanishi et al., 1985; Van Oostdam et al., 2005), the nervous system (Seegal, 1996; Fonnum and Mariussen, 2009), repro-
Address correspondence to Anette Krogenæs, Department of Production Animal Sciences, Norwegian School Veterinary Science, P.b. 8146 Dep., 0033 Oslo, Norway. E-mail: anette.krogenaes@nmbu.no
ductive abnormalities, fetal toxicity, carcino- genicity, and the endocrine system, includ- ing steroidogenesis (Safe, 1994; Fischer et al., 1998; Ropstad et al., 2006). In addition, there are reports of associations between PCB, obe- sity, and diabetes (Fierens et al., 2003; Tabb and Blumberg, 2006; Codru et al., 2007; Newbold et al., 2008; Lyche et al., 2010; Airaksinen et al., 2011).
A PCB consists of two phenyl rings con- nected by a carbon-carbon bond and a varying degree of chlorination (Safe, 1994). There are 10 available carbon atoms, and the difference in number and placement of chlorine sub- stitutions resulted in 209 different congeners (Voie et al., 2000). Based on the positions of chlorine substitution, PCB can be divided into three groups. Congeners with no chlorine substitutions in the ortho position are known as coplanar PCBs. Due to their resemblance to dioxins, they are can be aryl hydrocarbon receptor (AhR) agonists (Hestermann et al., 2000). The mono-ortho-substituted PCB con- sist of one chlorine substitution in the ortho position and may appear as coplanar, and congeners that contain two or more chlorine substitutions in the ortho position are non- coplanar (Fischer et al., 1998). Due to the non- and mono-ortho-substituted congeners’ ability to bind AhR, these agents are able to produce steroidogenic system changes (Lyche et al., 2010), such as induction of CYP1A1 enzymes, and are also associated with carcinogenicity
(Safe, 1994). The ortho-substituted congeners, on the other hand, with low affinity for the Ah receptor, are associated with effects on the nervous system (Seegal, 1996). The ortho- substituted congeners are the PCB detected mainly in blood and tissue of humans and animals (Fischer et al., 1998). In this study all three groups of congers are represented: PCB126 as a non-ortho-substituted congener, PCB118 as a mono-ortho-substituted congener, and PCB153 as an ortho-substituted congener (Figure 1).
The adrenal gland is critical both for (1) syn- thesis of steroid hormones as part of the sympathetic nervous system and (2) accu- mulating PCB efficiently due to the high lipid content and vascularity (Brandt, 1977). The human adrenocortical carcinoma cell line H295R maintains expression of genes and gene products (enzymes) necessary for producing steroid hormones (Gazdar et al., 1990). The H295R model reduces the need for lab animals and is a useful tool for in vitro assessment of endocrine-disrupting com- pounds (EDC) (Gracia et al., 2006; Zimmer et al., 2009; Kraugerud et al., 2010). This model has several possibilities, including study of hormone production (Hecker et al., 2006), gene expression (Hilscherova et al., 2004), and activity of steroidogenic enzymes (Staels et al., 1993). Further, there has been an increas- ingly strong interest in applying proteomics to provide a better understanding of disease
A. PCB 118
B. PCB 126
CI
CI
CI
CI
CI
CI
CI
CI
CI
CI
C. PCB 153
CI
CI
CI
CI
CI
processes and mechanisms of action related to endocrine disruption. Proteome changes were reported in the H295R steroidogenesis model induced by mitotane (Stigliano et al., 2008), the mycotoxin zearalenone, and two of its metabo- lites (Busk et al., 2011, 2012). In addition, the cell line has been Organization for Economic Cooperation and Development (OECD) vali- dated (Hecker et al., 2011).
The objective of the present study was to investigate effects of the three structurally dif- ferent PCB congeners,PCB118, PCB126, and PCB153 on proteome and steroidogenic capac- ity of human adrenocortical carcinoma cell line H295R. Investigation of the proteome was designed to increase the understanding of the three different congeners’ mode of action.
MATERIAL AND METHODS
Test Chemicals
3,3’,4,4’,5-Pentaclorobiphenyl (IUPAC no. 126) in powder form (98.9%) was acquired from LGC (Middlesex, UK). 2,2’,4,4’,5,5’- hexachlorobiphenyl (IUPAC no. 153) and 2,3’, 4,4’,5-pentachlorobiphenyl (IUPAC no. 118) in pure (99%) powder form were acquired from Accustandard (New Haven, CT). PCB 153 was purified over charcoal to remove any traces of dioxins and furans that might have been formed during synthesis. Briefly, the powder was dissolved in hexane and the solution cleaned in a 15-cm column with charcoal and Celite 520 (Sigma-Aldrich, St. Louis, MO) (ratio 20:80). PCB 118 and PCB 126 were dissolved in cyclohexane, but not purified, as the chem- ical synthesis is different and does not lead to the formation of unwanted by-products. To the solutions was then added dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO), and cyclohexane was evaporated under a nitrogen gas stream for at least 12 h. A small volume of DMSO was diluted in cyclohexane and verified for the presence of PCB in the solutions by gas chromatography with electron-capture detection (GC-ECD) according to Haave et al. (2003). Final stock concentrations of 7.1 mM (PCB 118), 6.4 mM (PCB 153), and 10 mM
(PCB 126) were stored at room temperature in sintered glass.
Cell Line, Cell Culture, and Experimental Design
H295R cells (American Type Culture Collection, ATCC number CRL-2128, ATCC, Manassas, VA) were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture (DMEM/F12) sup- plemented with 5 ml/L of ITS+ Premix, and 12.5 ml/L of BD Nu-Serum as previously described (Gracia et al., 2006; Hecker et al., 2006). Medium was refreshed thrice a week and subcultured once a week to maintain cell density and viability. Cells were used between passages 5 and 13. Prior to exposure, cells were seeded at a density of 150,000 cells/cm2. The medium was refreshed and cells were exposed 24 h post seeding for 48 h. Final concentrations used for the PCB were 4 uM as the low concen- tration for all the congeners and 10 uM, 12.76 uM, and 14.1 µM as the high concentrations for PCB 126, 153, and 118, respectively. The solvent controls contained the same amount of DMSO as the PCB dilutions. The concen- trations of PCB were based on previous stud- ies conducted in vitro in which the included concentrations yielded effects in hormone pro- duction and gene expression (Kraugerud et al., 2010).
Viability
Cell viability was monitored visually (×10 objective magnification), both 24 and 48 h postexposure. The alamarBlue Assay (Invitrogen, Carlsbad, CA) was also performed to monitor cell viability. Forty-eight hours postexposure, the medium was collected and frozen at -80℃. Cells were incubated in 3 h under the same conditions as previously described with 11.25 ml fresh medium con- taining 1.25 ml alamarBlue, and the medium was then transferred to 96-well plates (Falcon, Franklin Lakes, NJ), and read in a Victor3TM spectrophotometer (Perkin Elmer, Shelton, USA) at 570 nm and 600 nm. The percent
viability was calculated according to the man- ufacturer’s instructions. The viability assay was conducted in a separate experiment due to the risk of the reagent in the assay influencing the cells to be isolated.
Isolation of Proteins
After 48 h, the medium was collected and stored at -80℃ for future hormone analysis. The cells were detached by adding 2 ml 0.25% trypsin/0.53 mM ethylenediamine tetraacetic acid (EDTA) to each flask, then medium was added to inactivate the trypsin/EDTA, and the cells were pelleted and kept on ice until isola- tion. Isolation of proteins was performed using AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Crawley, UK) according to the manufacturer protocol with two modifications: (i) inclusion of Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO) in the lysis buffer (RLT) to maxi- mize protein integrity and yield, and (ii) protein pellets were resuspended in Modified Reswell Solution (MRS; 7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 0.3% [w/v] dithiothreitol [DTT]; all reagents electrophoresis grade), which is not included in the kit. MRS facilitates pro- tein solubilization and is compatible with pro- tein assays and subsequent two-dimensional electrophoresis analysis. The proteins were quantified using RC DC Protein Assay (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) and stored at -80°C.
Hormone Quantification
To measure hormone concentrations in cell-conditioned medium, solid-phase radioim- munoassay (RIA) kits were used. For measure- ment of estradiol, testosterone, and cortisol, Coat-A-Count kits (Siemens Medical Solutions Diagnostics, Los Angeles, CA) were used. Progesterone was measured using the Spectria Progesterone RIA kit (Orion Diagnostica, Espoo, Finland). Standards were made by diluting the hormone for each hormone assay in cell culture medium. All standards, controls, and samples were duplicated and an average was calcu- lated for each sample. The limits of detection
(LOD) were 10 pg/ml, 0.08 ng/ml, 2.5 ng/ml, and 0.22 ng/ml and the interassay coeffi- cients of variation were 10%, 7.1%, 10%, and 2.6% for estradiol, testosterone, cortisol, and progesterone, respectively.
Sample Preparation for Two-Dimensional and One-Dimensional Electrophoresis
Equal quantities of protein from each repli- cate culture were pooled for each treatment group, yielding 800 µg protein per pool. Salts and other contaminants were removed from the protein pools using ReadyPrep 2-D Cleanup Kit (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) according to the manufac- turer’s instructions.
One-Dimensional Electrophoresis and Western Blots
The protein pools were electrophoresed in triplicate in a random order (30 µg protein/lane) on 26-lane one-dimensional NuPage Novex Midi Bis-Tris Gel (Invitrogen, Carlsbad, CA) under reducing conditions and transferred to immobilon-FL membrane (Milipore (UK) Ltd, Watford, UK). Odyssey Two-Color Protein Molecular Weight Marker (LI-COR Biosciences, Lincoln, NE) was elec- trophoresed in the first lane and every 11th lane in every gel. After transfer, the membranes were treated with Odyssey Blocking Buffer (LI- COR Biosciences, Lincoln, NE) diluted 1:1 with phosphate-buffered saline (PBS) and incubated overnight at 4℃ with primary antibodies (in blocking buffer): BAX (1:200, Santa Cruz, cat- alogue number sc-493), CYP17 (1:5000, a kind gift of immune serum from Dr. Anita Payne [dec.], Stanford University), HSP27 (0.2 ug/ml, AbCam, catalogue number ab78436), StAR (1:500, a kind gift from Prof. Douglas M. Stocco, Texas Tech University), and vimentin (1:200, AbCam, catalogue number ab7752). All antibodies were combined with an anti-ß- actin loading control (rabbit, 1:10000 ab8227, mouse, 1:5000 ab6276, both from AbCam Ltd, Cambridge, UK). The protein bands were visu- alized using an Odyssey infrared fluorescence
imager (LI-COR). The electronic images were analyzed using Phoretic-1D Advanced software (Nonlinear Dynamics Ltd, Newcastle upon Tyne, UK) to determine band volumes and molecular weights. B-Actin band volumes were independent of treatments validating the use of this molecule as a load control.
Proteomics
Two-Dimensional Gel Electrophoresis
Proteins were analysed by two-dimensional gel electrophoresis (2-DE) as previously described (Fowler et al., 2008). Each protein pool (100 µg of total protein) was electrophoresed in quadruplicate gels. Proteins were detected by staining the gels with colloidal Coomassie blue G250. The gels were scanned wet using an ImageScanner III (GE Healthcare, Uppsala, Sweden). The images were transferred to Progenesis Samespots v. 3.3 (Nonlinear Dynamics Ltd, Newcastle, UK). The auto- mated and interactive routines available in this software were used to detect and quantify protein spots, as well as to match the profiles across a gel series. Individual spot volumes are expressed as normalized volumes relative to the total detected spot volume separately for each gel, minimizing potential analytical artifacts from variations in protein loading and migration. The software was used to combine the quadruplicate gels/treatment and calculated fold changes and significance (by analysis of variance [ANOVA] of log-normalized values). Molecular mass values and pIs of spots of interest were estimated from separate gels electrophoresed with pH and MW markers. The most robustly matched, clearest protein spots demonstrating a significant difference of ≥1.2-fold, p < . 05, between at least two groups were excised from the gels and iden- tified by liquid chromatography with mass spectroscopy (LC-MS/MS).
Mass Spectroscopic Protein Identification Proteins were identified using LC-MS/MS, as described previously (Fowler et al., 2008). Only proteins showing reliable agreement between the molecular mass and pI on the two- dimensional (2D) gels and calculated values,
along with statistically significant MOWSE scores (Pappin et al., 1993) and good sequence coverage, were considered to be positive iden- tifications (Fowler et al., 2008).
Ingenuity Pathway Analysis (IPA)
Ingenuity Pathway Analysis (IPA) software was used to make a functional network based on the gene names of the affected proteins. The software does not consider the origin of the tissue but provides an illustration of the inter- actions between the proteins identified in this study.
Statistics
JMP8 software (SAS Institute, Inc., Cary, NC) was used for all statistical analyses. Normality of data distribution was tested with the Shapiro-Wilk W-test. In the case of nonnor- mal distribution a logarithmic transformation was performed. Data were tested for homo- geneous variance by Levene’s test. Differences between PCB-treated cells and the solvent con- trol were evaluated using ANOVA followed by Tukey-Kramer HSD, Student’s t-test or Wilcoxon/Kruskal-Wallis test; p < . 05 was con- sidered significant. Fold change values were calculated to be able to compare each PCB congener to the DMSO control, setting the DMSO control as 1.
RESULTS
Effects of Three PCB Congeners on H295R Cell Viability
Cell viability, as determined using the ala- marBlue assay, remained >95% at all concen- trations of the three PCB tested, demonstrating that there was no direct adverse effect of PCB on the cells. Visual inspection of the cells also revealed no indication of toxicity.
Effects of Three PCB Congeners on Steroidogenesis in H295R Cells
All three congeners of PCB induced effects on hormone production in H295R
A.
450
Oestradiol
B.
120
Testosterone
400
% of DMSO control
350
*
% of DMSO control
100
300
80
250
+PCB118
++PCB118
*
200
-*- PCB126
60
-*- PCB126
150
*
— PCB153
40
-+-PCB153
100
50
20
0
0
0
Low
High
0
Low
High
Exposure dose
Exposure dose
C.
200
Progesterone
D.
180
Cortisol
180
160
% of DMSO control
160
*
% of DMSO control
:
140
140
120
++-PCB118
120
-+-PCB118
100
*
PCB126
100
— PCB126
80
-PCB153
80
60
-PCB153
60
40
40
20
20
0
0
0
Low
High
0
Low
High
Exposure dose
Exposure dose
cells (Figure 2, a-d). In cells exposed to PCB 118, there was a significant increase in estradiol production at the highest concen- tration. In addition, the highest concentra- tion of PCB 118 was associated with a sig- nificant decrease in testosterone production and a significant rise in cortisol production. In cells exposed to PCB 153, estradiol pro- duction and progesterone production were significantly elevated and testosterone produc- tion was significantly lowered at the highest concentration. In cells exposed to PCB 126, at both concentrations a significant increase in estradiol and progesterone production was noted. In addition, the highest concentra- tion produced a significant rise in cortisol production.
Effects of Three PCB Congeners on the H295R Cell Proteome
Of the 711 distinct, reproducible pro- tein spots included in the analysis (Figure 3), 73 showed significantly different normalized spot volumes between at least two treatment groups, independent of DMSO control, based on a minimum change of ≥1.2-fold. Of the
73 significant spots, 12 were upregulated and 61 were downregulated by exposure to PCB. There were no absent or unique spots. Fourteen of the most abundant spots show- ing the largest and most consistent differences were excised and identified by LC-MS/MS (Table 1). Of the identified proteins, four are structural proteins, four are involved in tran- scription and/or protein synthesis, and two are involved in proteolysis. In addition, one protein involved in insulin signaling, one trans- port protein, one stress-related protein, and one protein involved in protein repair were identified.
Individual fold change values between each specific PCB congener and DMSO control were analyzed. There were differences in the up- or downregulation of proteins depending on PCB congener used for exposure. PCB126 was most potent, indicated by higher fold changes compared to other congeners (Figure 4). It was evident that the general trend, especially for PCB126, was for the PCB to reduce protein expression where there was a statistically sig- nificant difference between at least one PCB and DMSO controls. Of the 14 identified pro- teins, 8 proteins were downregulated, 4 were
3
Non-linear pH gradient
10
191
550
527
97
732
548
705
64
935
1059
1096
51
1125
1112
1162
1257
39
1189
1452
28
19
14
upregulated, and 2 proteins showed no change in expression following exposure to PCB 126 (Table 1). Exposure to PCB 153 resulted in eight downregulated proteins, five5 upregulated pro- teins, and one protein with no alteration in expression. However, the up- and downreg- ulation were not similar to the case of PCB 126-exposed cells. Exposure to the last con- gener, PCB 118, resulted in upregulation in expression of six proteins and downregulation of expression in eight proteins. This illustrates the different modes of action between the three congeners.
Analysis of Individual Proteins
The antibodies used for Western blot were selected based on identification of proteins from the 2D gels (vimentin) and known functions in stress response (HSP27), steroidogenesis (CYP1A1 and CYP17), and apoptosis (Bax). Exposure to PCB118 signif- icantly altered expression of vimentin and HSP27 (Figure 5).
Pathway Analysis of PCB Effects on H295R Cells
All proteins identified were connected in the same network with the three proteins nuclear factor (NF)-KB (complex), MAPK3, and TRAF6 in central positions (Figure 6). This network is associated with cell death, lipid metabolism, protein synthesis, and cellular assembly and organization and is detailed in Table 2. Canonical pathways and biological/disease functions affected by PCB exposure are shown in Supplementary Figure 1. There was no enrichment of toxicological path- ways above two proteins and data are therefore not shown.
DISCUSSION
The present study demonstrated that three structurally different PCB congeners, PCB 118, PCB 126, and PCB 153, have the potential to disturb both steroidogenesis and diverse bio- logical processes by altering protein expression, using the H295R in vitro model. The results
| Spot | Protein | Mass kD [from gel] | Gene name | Function | Fold change vs. control | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| pI | MOWSE score | Subcellular location | PCB118 | PCB126 | PCB153 | |||||
| Intercellular signaling | ||||||||||
| 550 | Insulin-degrading enzyme | 119 [98] | 6.3 | 88 | IDE | Degradation of insulin, glucagon and other polypeptides. Plays a role in intercellular peptide signaling. | Cytoplasm | 1.01 | 0.73 | 0.92 |
| Transport | ||||||||||
| 527 | Albumin | 30 [97] | 6.97 | 47 | ALB | Main function is the regulation of the colloidal osmotic pressure of blood. Transport. | Secreted | 0.90 | 0.67 | 1.03 |
| Stress 935 | Heat-shock 60-kD protein (chaperonin) | 61 [58] | 5.7 | 247 | HSPD1 | Mitochondrial protein import and macromolecular assembly, may facilitate correct folding of imported proteins, may prevent misfolding and promote the refolding and proper assembly of unfoldedpolypeptides generated under stress conditions in the mitochondrial matrix. | Mitochondrial matrix | 0.98 | 0.85 | 0.96 |
| Structure 1162 | Septin 2 | 42 [42] | 6.15 | 291 | SEPT2 | Normal organization of the actin cytoskeleton, biogenesis of polarized columnar-shaped epithelium, efficient vesicle transport, impeding MAP4 binding to tubulin, required for the progression through mitosis, may be required for chromosome segregation and spindle elongation during anaphase. | Cytoplasm/ cytoskeleton | 0.90 | 0.64 | 1.05 |
| 1189 | Beta actin | 42 [41] | 5.29 | 139 | ACTB | Involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells. | Cytoplasm/ cytoskeleton | 1.04 | 1.46 | 0.99 |
| 732 | Valosin-containing protein | 90 [70] | 5.14 | 231 | VCP | Vesicle transport and fusion, assembly of peroxisomes, spindle pole body function, ubiquitin-dependent protein degradation. | Cytoplasm/ Nucleus | 1.03 | 1.27 | 0.99 |
| 1125 | Vimentin | 54 [43] | 5.03 | 294 | VIM | Class III intermediate filaments involved in cell motion and interspecies interaction between organisms. | Cytoplasm | 1.05 | 1.78 | 0.89 |
(Continued)
| Spot | Protein | Fold change vs. control | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Mass kD [from gel] | pI | MOWSE score | Gene name | Function | Subcellular location | PCB118 | PCB126 | PCB153 | |||
| Proteolysis | |||||||||||
| 1112 | Ses2 protein | 44 [44] | 5.82 | 81 | Ses2 | Proteolysis | Cytoplasm | 0.93 | 1.00 | 0.80 | |
| 1257 | Proteasome 26S subunit, non-ATPase 14 | 35 [38] | 6.06 | 118 | PSMD14 | Metalloprotease component of the 26S proteasome that specifically cleaves 'Lys-63'-linked polyubiquitin chains | Proteasomes | 0.81 | 0.77 | 0.94 | |
| Protein repair | |||||||||||
| 1452 | Protein-L-isoaspartate(D- | 25 [27] | 6.05 | 105 | PCMT1 | Catalyzes the methyl esterification of | Cytoplasm | 0.89 | 1.03 | 0.80 | |
| aspartate) O-methyltransferase | L-isoaspartyl and D-aspartyl residues, | ||||||||||
| catalyzes the methyl esterification of L-isoaspartyl and D-aspartyl residues. Plays a role in repair and/or degradation of damaged proteins | |||||||||||
| Transcription/protein synthesis | |||||||||||
| 705 | Threonyl-tRNA synthetase | 85 [78] | 6.23 | 401 | TARS | Protein biosynthesis | Cytoplasm | 1.08 | 0.68 | 1.00 | |
| 1112 | TAR DNA-binding protein | 45 [45] | 5.85 | 89 | TARDBP | DNA and RNA-binding protein which regulates transcription and splicing. Involved in the regulation of CFTR splicing. | Nucleus | 0.93 | 1.00 | 0.80 | |
| 1096 | Elongation factor Tu | 50 [49] | 7.7 | 602 | TUFM | Promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis. | Mitochondria | 1.03 | 0.87 | 1.45 | |
| 548 | Transcription intermediary factor 1-beta | 81 [96] | 5.67 | 65 | TRIM28 | Transcription, transcription regulation. | Nucleus | 0.87 | 0.76 | 0.93 | |
Note. Proteins were identified by LC/MS-MS. Data shown include proteins mass (kD) from NCBI, approximate mass from gel, isoelectric pH (pI), MOWSE score, gene names used for network analysis, functions, subcellular location, and fold change. Fold change of treatments was expressed relative to control (DMSO). Those in bold are statistically different from control, p < . 05.
Downloaded by [George Mason University] at 12:49 27 December 2014
A. PCB118
2.0
1.0
0.5
B. PCB153
PCB/DMSO
2.0
1.0
0.5
C. PCB126
2.0
1.0
0.5
Spot #
D. Cluster analysis of protein spots with significantly different volumes vs DMSO
*DMSO
·DMSO
*OMSO
*DMSO
*DMSO
·CB118
·CB118
*PCB118
*DMSO
·PCB152
·PCB152
*CB152
YCB126
*PCB126
*PC#126
LY
Spot #
indicated that the three PCB congeners affected at least three fundamental cellular processes: protein synthesis, structural cellular conforma- tion in terms of changes in the cytoskeleton, and stress response.
The direct comparison of PCB levels in plasma and cell culture medium is problem- atic; nevertheless, it is important to estab- lish cellular in vitro exposures to attain more
information regarding effects of potential EDC. There are many reasons why conclusions from in vitro studies need to be carefully extrap- olated to in vivo conditions. More in-depth mechanistic research is needed to further explore whether these effects are modified in vivo by effects in other parts of the axis or other organs influencing the HPA axis in vivo. Another important issue concerning in
A. Vimentin
B. HSP27
C. CYP1A1
D. CYP17
E. BAX
58 kDa
23 kDa
56 kDa
58 kDa
23 kDa
42 kDa
42 kDa
42 kDa
42 kDa
42 kDa
160
160
*
140
300
160
140
140
120
140
% of control/B-actin
% of control/B-actin
% of control/B-actin
% of control/B-actin
250
120
120
% of control/B-actin
100
200
120
100
100
80
100
80
80
150
80
60
60
60
100
60
40
40
40
20
50
40
20
20
20
0
0
0
0
0
0.1% DMSO
PCB 118 14,1 µM
PCB 126 10UM
PCB 153 12,76UM
0.1% DMSO
PCB 118 14,1µM
PCB 126 10UM
PCB 153 12,76uM
0.1% DMSO
PCB 118 14,1µM
PCB 126 10AM
PCB 153 12,76uM
0.1% DMSO
PCB 118 14,1µM
PCB 126 10Μ
PCB 153 12,76uM
0.1% DMSO
PCB 118 14,1 µM
PCB 126 10μΜ
PCB 153 12,76uM
ATPase
IDE
ALE
HSPD1
NEKB (complex)
Insulin
TARDBP
VCP
MĄPK3
VIM
Caspase
CTB
TRAP6
PCMT1
TUFM
SEPT2
PAKZ
TRIM28
TARS
PSMD14
vitro studies is the lack of consideration for metabolism of the chemical(s) under investiga- tion, although this can be addressed by using metabolites of the environmental pollutant(s)
in vitro rather than parent compounds. Taking this into account, it is still interesting to make careful comparisons of exposure levels for in vitro studies and levels reported in humans and
| Molecules in network | Score | Focus molecules | Top diseases and functions |
|---|---|---|---|
| 26s Proteasome, ACTB, Actin, Akt, ALB, ATPase, C16orf82, CARNS1, caspase, CLEC4A, DNA2, DQX1, EEF1A1, ERK1/2, Fgf18, Hsp70, HSPD1, IDE, Insulin, Jnk, mt-Atp8, MYO16, NFKB (complex), Ntp, P38 MAPK, Pkc(s), PSMD14, Psmd5, SES2, SWSAP1, TARDBP, TRIM28, Ubiquitin, VCP, VIM ANKFY1, APEH, C12orf44, COG4, COPA, COPE, CORO1B, CORO1C, DEGS1, DYRK2, EXOC4, FAM114A1, FAM129B, HN1L, PAPOLA, PCMT1, PIK3C2A, PRKAG1, PRKAG2, SEPT1, SEPT2, SEPT3, SEPT4, SEPT5, SEPT6, SEPT7, SEPT8, SEPT9, SEPT11, SNX24, TARS2, TARS, TARSL2, UBC, UGP2 | 31 6 | 11 3 | DNA replication, recombination, and repair, energy production, nucleic acid metabolism Cell cycle, cellular movement, cell morphology |
Note. Proteins identified in the present study are shown in bold text.
wildlife. The levels of PCB 153 were reported to be 3.74-4.32 ng/g lipid (3.74-4.32 ppb) in Norwegian women (Kvalem et al., 2009). A comprehensive global literature review of blood levels of dioxins and dioxin-like com- pounds by Consonni et al. (2012) noted mean levels of 21.1 pg/g lipid for PCB 126 and 5910 pg/g lipid for PCB 118. Higher con- centrations are found in wildlife, such as 1130-2800 ng/g lipid (1130-2800 ppb) in adi- pose tissue from polar bears (_PCB) (Braune et al., 2005). In comparison, the concentra- tions used in this study (1400-9600 ppb for PCB 153) were higher than representative of human exposure levels and higher than con- centrations detected in wildlife. However, the concentrations of PCB used in the present study exerted no marked effect on cell viability or expression of protein levels of the represen- tative pro-apoptotic marker BAX. In light of these results, exposure concentrations utilized in this study are appropriate to investigate some of the cellular responses of the adrenal gland to PCB, at the hormonal and proteome level. The reported serum levels are representative of humans exposed through a lifetime (Kvalem et al., 2009), compared to our study where cells were only exposed for 48 h.
Alterations in Hormone Production
All three congeners resulted in an altered hormone production. For PCB 153 and PCB 118, only the highest dose yielded signifi- cantly elevated levels of estradiol, while PCB
126 induced an increased estradiol production at both low and the high exposure concentra- tions. Elevated levels of estradiol were previ- ously reported following exposure to the same PCB congeners in the same cell line (Li, 2007; Kraugerud et al., 2010). Testosterone produc- tion was reduced in cells exposed to the highest concentrations of both PCB 118 and PCB 153. Several previous studies noted reduced produc- tion of testosterone both in vitro and in vivo fol- lowing exposure to several PCB congeners, and this decrease might be due to the PCB ability to inhibit testosterone biosynthesis by diminish- ing expression of genes encoding steroidogenic enzymes (Andric et al., 2000; Wojtowicz et al., 2005b; Gregoraszczuk et al., 2008; Murugesan et al., 2008; Goncharov et al., 2009; Kraugerud et al., 2010). In addition to this, the fall in testosterone combined with increased produc- tion of estradiol reported in this study suggests that testosterone might have been a substrate for elevated estradiol. Kraugerud et al. (2010) showed that increased levels of estradiol follow- ing exposure to PCB 126 were accompanied by a rise in CYP19 mRNA levels. This expo- sure pattern was previously observed in the same cell line and supports the suggestion of testosterone being a substrate for estradiol pro- duction in exposed cells (Li, 2007). However, the highest exposure concentration of PCB 126 did not alter testosterone levels, suggest- ing that there might be other mechanisms involved in addition to CYP19-induced transfor- mation to estradiol. Another explanation might be attributed to PCB ability to change the
cell-membrane permeability, producing leak- age of steroid into the medium and giving a falsely higher level of testosterone.
Only PCB 126 significantly affected progesterone (Figure 2c). Kraugerud et al. (2010) demonstrated an increase in expres- sion of several steroidogenic genes following exposure to PCB 126, including StAR, HMGR, 17 B-HSD, CYP 19, and 3 -HSD. Data suggest that PCB 126 might thus amplify steroidogenesis at several steps, and that this congener is the most potent of the ones tested.
In the present study, H295R cells exposed to the highest doses of PCB 118 and PCB 126 enhanced the synthesis of cortisol. An in vivo study conducted in goats showed lower basal cortisol concentrations in goat offspring following exposure to PCB 126 (Zimmer et al., 2009). Increased levels of cortisol in the H295R cell-line were reported following exposure to PCB, and the findings in the current study supports the suggestion of elevated cortisol lev- els being produced by lack of hypothalamic feedback in vitro (Kraugerud et al., 2010).
Alterations in Protein Expression
Of the 73 statistically significantly expressed proteins, 61 were downregulated and 12 were upregulated, a clear bias of an effect follow- ing PCB exposure, which was to reduce syn- thesis of a range of proteins rather than an approximately equal mixture of increased and reduced expression of proteins seen in other EDC studies that employed proteomic analyses (Fowler et al., 2008; Bellingham et al., 2013). Four of the identified proteins are known to be involved in protein synthesis and all were downregulated in the present study. This demonstrates that PCB exposure, to a varying degree, diminished protein synthesis. A study performed using cod supports PCB ability to affect protein synthesis (Berg et al., 2011). The functions, processes, and pathways that might be affected are described more in detail in the following.
The significantly downregulated proteins following PCB 126 exposure were a fragment of albumin (ALB), insulin-degrading enzyme
(IDE), and septin2 (SEPT2). ALB is a transport protein with a main function to regulate col- loidal pressure of blood. In addition, ALB is involved in the distribution of PCB in tissues, which may explain the lower levels of ALB in exposed cells (Borlakoglu et al., 1990). IDE is a multifunctional protein involved in sev- eral basic cellular processes including degrada- tion of insulin (Fernandez-Gamba et al., 2009). Previously, an involvement of PCB in devel- opment of metabolic syndrome was indicated (Ruzzin et al., 2010). The decreased levels of SEPT2 suggest reduced proliferation of cells since this protein is involved in cell cycle reg- ulation, cell division, and mitosis (Schmidt and Nichols, 2004).
The significantly upregulated proteins fol- lowing PCB 126 exposure were vimentin (VIM) and B-actin (ACTB). Both VIM and ACTB are structural proteins included in the cytoskele- ton. Previous studies on different cell cultures demonstrated alterations in structural proteins following exposure to PCB153 (Lasserre et al., 2009; 2012; Zhang et al., 2013). Evidence indi- cates that PCB changes the ability of cells to move and proliferate. In an in vivo study where sheep were exposed to PCB 153 and 118, VIM was upregulated in testis (A. Krogenæs, per- sonal communication). However, since ACTB quantified by Western blot was not markedly altered by PCB, this 2D gel finding is most likely a change in a single isoform of ACTB not associated with overall levels of protein.
Exposure to PCB 118 resulted in two significantly downregulated proteins: 26S proteasome non-ATPase regulatory subunit 14 (PSMD14) and protein-L-isoaspartate (D- aspartate) O-methyltransferase (PCMT1). While PSMD14 is involved in degradation of proteins (Spataro et al., 1997; Byrne et al., 2010), PCMT1 is involved in initiation of repair of proteins (DeVry and Clarke, 1999). Decreased levels of both of these proteins suggest a reduction in cell proliferation induced by PCB exposure. In addition, Western blot analysis showed a significant rise in expression of both VIM and heat-shock protein 27 (HSP27) fol- lowing exposure to PCB 118. As previously above, in an in vivo study performed in sheep,
VIM was upregulated in testis after exposure to PCB 153 and 118 (A. Krogenæs, personal communication). The heat-shock proteins (HSP) are a family of stress-inducible proteins and aid in the restoration of structure and function of denatured proteins (Sharp et al., 1999; Leal et al., 2007). Overexpression was found to protect against apoptotic cell death, suggesting that the cells are stressed in response to the exposure to PCB 118 (Parcellier et al., 2003).
Exposure to PCB153 resulted in one signifi- cantly upregulated protein, elongation factor Tu (TUFM), and one significantly downregulated protein, protein-L-isoaspartate (D-aspartate) O- methyltransferase (PCMT1). TUFM is involved in the translation elongation of the protein biosynthesis in mitochondria (Ling et al., 1997). Data suggest increased protein synthesis following exposure; however, this is unlikely, given that the other findings indicate the opposite. A more likely explanation might be that elevated levels of TUFM may be attributed to a delayed response in the transcription machinery.
The proteins identified following PCB exposure were not as directly connected to steroidogenesis as might be expected, but were involved in more general cellular pro- cesses. There might be proteins connected to steroidogenesis, which are significantly altered, but these have not been isolated and identi- fied. This indicates that further studies need to be done, specifically to be able to completely understand the effects on steroidogenesis fol- lowing exposure to the three PCB congeners in the H295R cell line. However, this study has contributed to an understanding of the possible mode of action of these PCB congeners in this cell line at a general level.
The coplanar congeners used in this study, PCB 118 and 126, were found to both bind AhR and induce CYP1A1 (Safe, 1994). However, in this study CYP1A1, as measured by Western blot, was not significantly altered compared to DMSO control, but a decreas- ing trend following exposure to PCB 118 and 126 was seen (Figure 5). The may be due to a delayed response where the transcription of the
gene and thereby the translation of the protein have yet to reach their peak. A study comparing AhR expression in porcine theca and granulosa cells following exposure to PCB 126 showed a difference in AhR responsiveness and that the connection probably is the difference in these cells’ estradiol secretion and different prolif- eration potential (Wojtowicz et al., 2005a). In addition, mechanisms other than AhR path- way may be involved in the PCB-induced toxi- city (Duffy and Zelikoff, 2006). Taking this into consideration, further studies need to be done to completely understand the mechanisms of exposure to coplanar PCB in H295R cells when it comes to induction of AhR.
Ingenuity Pathway Analysis (IPA)
Using functional network analysis may improve the understanding of mechanisms involved in exposures by examining protein- protein interactions. Based on altered pro- teomes, IPA provided a network that indicated that exposures were producing effects that had some level of connection. However, as the network analysis compiles connections of paired protein-protein interactions into larger networks, these are likely to span over a high number of different processes. In addition, while no toxicological pathways were enriched with more than two of the identified proteins, several canonical signaling pathways and cell death pathways were enriched (Supplementary Figure 1). The fact that there were no marked effects of the PCB on cell viability suggests that these findings demonstrate a reduced cellular health.
The IPA-identified network contained three proteins in central positions: NF-KB (complex), MAPK3, and TRAF6 (Figure 6). The nuclear fac- tor kappa-light-chain-enhancer of activated B cells (NF-KB) is a protein complex that controls transcription of DNA. NF-KB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, free radi- cals, and reactive oxygen species (ROS). When activated, NF-KB is transported to the nucleus, binds to target genes, and alters their expres- sion, thus potentially inducing cell proliferation
(Chandel et al., 2000, Karin and Ben-Neriah, 2000). PCB 153 induces the formation of ROS, and thus activation of NF-KB (Lu et al., 2003). Cell proliferation rates were not quantified in this study because of the acute exposure period used. However, Lu et al. (2003) previously indi- cated that exposure to PCB did not markedly affect cell proliferation.
Mitogen-activated protein (MAP) kinases are serine/threonine-specific protein kinases that respond to extracellular stimuli such as heat shock and contribute to the regulation of cellular activities, including gene expression, proliferation, and apoptosis (Pearson et al., 2001). Estradiol activates MAPK inducing cell proliferation (Lucas et al., 2010). The increased levels of estradiol production in cells exposed to PCB in the present study may be associated with enhanced cell proliferation. However, other findings suggest that proliferation was decreased. This indicates that activation of MAPK by estradiol is not sufficient for increased cell proliferation. Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) is a cyto- plasmic protein that mediates signal trans- duction from members of the TNF receptor superfamily. TRAF6 activates transcription fac- tors such as NF-KB (Naito et al., 2002). The central position of TRAF6 provided by IPA sup- ports the postulation of PCB involvement in the NF-KB pathway. Considering that all three pro- teins with central positions in the IPA-generated network are reported to be involved in cell proliferation, it is likely that PCB most likely ini- tiated the processes that, with more time, might have resulted in cell proliferation in the present study.
In addition to the three proteins just men- tioned, IPA also included insulin signalling in the network. Exposure to PCB is associated with an increased prevalence of diabetes type 2 and metabolic syndrome in humans (Tabb and Blumberg, 2006; Codru et al., 2007; Newbold et al., 2008; Ruzzin et al., 2010; Airaksinen et al., 2011; Ibrahim et al., 2011). However, the mechanism underlying the rela- tionship between PCB and diabetes is not known. Longnecker et al. (2001) suggested either that the PCB may produce diabetes, or
alternatively that patients with diabetes may have an altered pharmacokinetics of PCB lead- ing to higher internal concentrations of PCB. In addition, diabetes may affect CYP enzymes, and thereby the metabolism of PCB. Patients with diabetes may also have dysregulation of their fat metabolism, potentially affecting the distribution and elimination of lipophilic com- pounds such as PCB (Fierens et al., 2003). Considering the reported association between diabetes, obesity, and PCB, the relatively high levels of PCB still found in the environment are of great concern.
CONCLUSIONS
This study represents an analysis of the effects on proteins and steroid hormones in the H295R cell model, and more investiga- tions are required for a greater understanding regarding how these PCB congeners affect pro- cesses in H295R cells. However, exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. The alterations in protein regulation suggested that exposure to these compounds affected several cellular processes, including protein synthesis, stress response, and apoptosis.
FUNDING
We are grateful to Margaret Fraser, Evelyn Argo, and Elisabeth Stewart (University of Aberdeen) for their excellent technical assis- tance. This project was funded by the Research Council of Norway through the strategic pro- gram GenPOP “Mechanisms behind toxic effects of POPs,” grant 17098/V40. The authors declare they have no actual or potential competing financial interests.
SUPPLEMENTAL DATA
Supplemental data are available for this article at http://dx.doi.org/10.1080/15287394. 2014.886985
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