Adrenal Corticosteroid Perturbation by the Endocrine Disruptor BDE-47 in a Human Adrenocortical Cell Line and Male Rats

Abstract

Polybrominated diphenyl ethers (PBDEs) have been previously shown to alter various endocrine biosynthetic pathways. Growing epidemiological evidence suggests that PBDEs alter cardiovascular function. The goal of this study was to examine the effects of BDE-47 on adrenal corticosteroid pathways that play vital roles in cardiovascular homeostasis and pathophysiology. The effect of BDE-47 on aldosterone and cortisol secretion was characterized in a human adrenocortical cell line. HAC15 cells were exposed to various concentrations of BDE-47 (1 nM to 100 μM). Cell viability, corticosteroid secretion, gene expression of enzymes involved in corticosteroid synthesis, and metabolic activity was examined. Additionally, Sprague Dawley male rats were orally exposed to BDE-47 (10 or 100 µg/kg), 5 days per week for 16 weeks. Organ weights and plasma corticosteroid levels were measured. In HAC15 cells, basal and stimulated aldosterone and cortisol secretion was significantly increased by BDE-47. Gene expression of several enzymes involved in corticosteroid synthesis and mitochondrial metabolism also increased. In Sprague Dawley rats, adrenal but not heart, kidney, or liver weights, were significantly increased in BDE-47 treatment groups. Plasma corticosterone levels were significantly increased in the 100 µg BDE-47/kg treatment group. No change in plasma aldosterone levels were observed with BDE-47 exposure. These data indicate that BDE-47 disrupts the regulation of corticosteroid secretion and provides further evidence that PBDEs are potential endocrine disruptors. Future studies will determine the underlying molecular mechanism of altered corticosteroid production and examine whether these alterations result in underlying cardiovascular disease in our rodent model of 16-week BDE-47 exposure.

Endocrine disruptors are natural or manmade chemicals that interfere with carefully regulated endocrine pathways (1, 2). Due to the varied and essential roles of endocrine pathways, disruption can result in a diverse array of adverse effects that includes developmental, reproductive, neurological, and immune effects in wildlife and humans. One group of endocrine disruptors that is of concern are the polybrominated diphenyl ethers (PBDEs), which have been used as a flame retardant in consumer products such as electronics, plastics, and textiles since the 1970s (3, 4). Because these PBDEs are not covalently bound to these consumer materials, they leach into the environment. Due to their combined chemical properties of lipophilicity and resistance to degradation, PBDEs are now ubiquitous environmental contaminates and bioaccumulate in wildlife, livestock, and humans (5-14).

Due to these concerns about their persistence in the environment and their tendency to bioaccumulate along with the growing evidence of potential adverse health effects, the production of octaBDE and pentaBDE in the United States was voluntarily ceased in 2004 (15). Production and importation of decaBDE was voluntarily ceased in 2013. Despite these actions, PBDEs continue to persist in the environment and humans. A 2017 study examining mother-infant pairs in the Southeastern United States identified 5 PBDE congeners in maternal and infant sera, with higher levels of PBDEs in infant cord sera compared with maternal sera (16).

There is growing epidemiological evidence that suggests that PBDEs alter cardiovascular function. A study of children in upstate New York demonstrated a positive correlation between PBDE body burdens and cardiovascular risk factors and increased sympathetic nervous system activity (17). Firefighters who have a greater incidence of cardiovascular disease are exposed to levels of PBDEs that are greater than the general public (18). In addition, there are also numerous studies suggesting that PBDEs disrupt thyroid hormone homeostasis (19-25), which can impact various cardiovascular parameters including cardiac contractility, heart rate, and vascular resistance.

Experimental models suggest that the deleterious cardiovascular effects of PBDEs are multifaceted. Zebrafish larvae exposed to BDE-47 developed tachycardia and atrioventricular block arrhythmias (26). Perinatal exposure of Long-Evans rats to a technical mixture of PBDEs reduced vasopressin secretion and disrupted osmoregulation (27). Similarly, ex vivo tissue punches of rat supraoptic nuclei of the hypothalamus demonstrated attenuated somatodendritic vasopressin release (28). Moreover, cultured human endothelial cells exposed to various mixtures of PBDEs demonstrated impaired regulation of antioxidant pathways (29).

The goal of this study was to examine the effects of PBDEs on 2 endocrine pathways that play vital roles in cardiovascular homeostasis and pathophysiology: the hypothalamic-pituitary-adrenal (HPA) axis and the closely related renin-angiotensin-aldosterone system (RAAS). The HPA axis and cortisol serve a vital role in the adaptation to physical and psychosocial stressors, maintaining appropriate biological homeostasis in many organ systems (30). Excessive HPA activity and prolonged elevated levels of cortisol results in widespread deleterious effects including weight gain, diabetes mellitus, hypertension, immune suppression, reduced fertility, and atrophy of the central nervous system (31-35). Aldosterone is one of the primary endocrine signaling factors of the RAAS, which serves to regulate electrolyte balance, water homeostasis, and arterial blood pressure (36). Excess aldosterone production has a significant role in the pathophysiology of cardiovascular and kidney diseases ranging from hypertension and congestive heart failure to nephropathy (37).

Cortisol and aldosterone are both corticosteroids produced in the adrenal cortex and are central endocrine signaling molecules for the HPA axis and RAAS, respectively. There is some evidence that the adrenal gland may be a susceptible target organ to PBDE toxicity. In a study of polar bears in eastern Greenland, PBDE body burdens were positively correlated with cortisol levels (38). Tissue disposition studies in both mice and rats have identified the adrenal cortex as one of the primary sinks for PBDE accumulation (19, 39). Moreover, studies in cultured human adrenocortical cells have demonstrated altered expression of microsomal enzymes (40-44), which play an integral role in steroid hormone production in the adrenal cortex. To further characterize this potential association between PBDE exposure and disruption of adrenal steroidogenesis, we have examined the effect of BDE-47 on aldosterone and cortisol secretion in an ACTH-responsive human adrenocortical cell line. Additional experiments in an in vivo rodent model of chronic BDE-47 exposure were performed to further characterize these steroidogenic effects.

Methods

Chemicals and Reagents

All buffer reagents were purchased from Sigma Aldrich (St. Louis, MO). Cell culture media, collagen, and antibiotics were purchased from ThermoFisher Scientific (Waltham, MA). Cosmic calf serum was purchased from GE Healthcare Life Sciences (Logan, UT). Insulin/transferrin/selenium + (ITS+) Premix was purchased from Corning (Corning, NY). BDE-47 was purchased from Chem Service (West Chester, PA).

Cell Culture

HAC15 cells were kindly provided by Dr. William Rainey, University of Michigan, Ann Arbor (RRID: CVCL_S898) (45). Cells were cultured on plates or flasks that were coated with collagen at 37°C and 5% CO₂. Cells were cultured in a growth media consisting of DMEM/F12 medium supplemented with 10% cosmic calf serum, 1% ITS + Premix, and antibiotics. For BDE-47 exposures, HAC15 cells were cultured in steroidogenic media consisting of DMEM/F12 medium supplemented with 0.1% cosmic calf serum, 1% ITS + Premix, and antibiotics.

Cell Viability

HAC15 cells were plated on 24-well plates at a density of 2 × 10⁵ cells/well. After 48 hours, cells were exposed to vehicle (0.1% dimethyl sulfoxide [DMSO]) or BDE-47 (1 nM to 100 μM) for 72 hours. Cells were then lifted off the plate using a Puck’s EDTA cell dissociation solution and counted using a TC20 Automated Cell Counter (Bio-Rad Laboratories, Hercules, CA) in the presence of 0.2% Trypan Blue.

Cell Steroidogenesis

HAC15 cells were plated on 24-well plates at a density of 2 × 10⁵ cells/well. After 48 hours, cells were exposed to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 μM) for 24 to 96 hours. Following the BDE-47 exposure, cells were switched to fresh steroidogenic media, containing vehicle (0.1% ddH₂O) for basal steroidogenesis, or angiotensin II (Ang II) (100 nM), adrenocorticotropic hormone (ACTH) (100 nM), or KCl (15 mM) for stimulated steroidogenesis. After 24 hours, the supernatant was collected and stored at −20 °C for later analysis.

Aldosterone and Cortisol Enzyme-Linked Immunosorbent Assay

Both aldosterone and cortisol were measured in collected cell supernatants by enzyme-linked immunosorbent assay (ELISA). The aldosterone ELISA (RRID: AB_2892670) was performed as previously described (46, 47). The cortisol ELISA was performed using a commercially available kit (Arbor Assays, Ann Arbor, MI) (RRID: AB_2893032) (48).

Quantitative Polymerase Chain Reaction

HAC15 cells were plated on 6-well plates at a density of 1 × 10⁶ cells/well. After 48 hours, cells were exposed to vehicle (0.1% DMSO) or 10 μM BDE-47 for 72 hours. RNA was isolated from each well by salt-fractionation using a MasterPure RNA Purification Kit (Epicentre, Madison, WI). RNA was stored at −80 °C for later analysis. cDNA was synthesized using a MonsterScript 1st-Strand cDNA Synthesis Kit (Epicentre) utilizing both oligo(dT) and random nonamer primers. cDNA was stored at −20 °C for later analysis. Quantitative polymerase chain reaction (qPCR) was performed using an Illumina Eco (Illumina, San Diego, CA) with a reaction mixture comprised of PerfeCTa qPCR ToughMix (Quantabio, Beverly, MA), 500 nM for each primer, 250 nM for each probe, and 37.5 ng cDNA/well. PrimeTime qPCR probe–based assays (Integrated DNA Technologies, Coralville, IA) were used to analyze expression of each gene of interest in duplex with the reference gene GAPDH using probe fluorophores of FAM and HEX fluorophores respectively.

Western Blot

HAC15 cells were plated on T75 flasks at a density of 7.5 × 10⁶ cells/flask. After 48 hours, cells were exposed to vehicle (0.1% DMSO) or 10 μM BDE-47 for 72 hours. Cells were then rinsed with phosphate-buffered saline (PBS) and lysed with radioimmunoprecipitation assay (RIPA) buffer. Protein concentration was determined using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Next, 80 μg of each sample was diluted in ddH₂O with Bolt LDS Sample Buffer and Bolt Reducing Agent (ThermoFisher Scientific) to a final volume of 40 μL. Diluted samples were then heat-denatured at 70 °C for 10 minutes and placed on ice. Each sample was loaded into a respective well of a Bolt 10% Bis-Tris Plus Mini Gel, and the gel was run at a constant voltage of 200 V for 30 minutes in Bolt MES SDS Running Buffer (ThermoFisher Scientific). After electrophoresis, the gel was removed from the cassette and protein was transferred to an iBlot 2 PVDF Mini Membrane using the following voltage sequence on an iBlot 2 Dry Blotting System (ThermoFisher Scientific): 20 V for 1 minute, 23 V for 4 minutes, 25 V for 2 minutes. The PVDF membrane was then simultaneously probed for CYP11B2 and GAPDH overnight at 4 °C using an iBind Western System (ThermoFisher Scientific) according to the manufacturer’s protocol. The primary antibodies used were an anti-human CYPB11B2 (41-13 mouse monoclonal antisera, RRID: AB_2650562, a kind gift from Dr. Celso Gomez-Sanchez, University of Mississippi Medical Center) (1:250 dilution) (49, 50) and an anti-human GAPDH (mouse monoclonal, RRID: AB_10977387, ThermoFisher Scientific) (1:2500 dilution) (51). The secondary antibody was an anti-mouse IgG horseradish peroxidase conjugate (goat polyclonal, Jackson ImmunoResearch Laboratories) (1:2500 dilution). The following day the membrane was developed utilizing a chemiluminescent substrate (SuperSignal West Pico, ThermoFisher Scientific) and photographed and analyzed on a BioRad ChemiDoc XRS + Imaging System.

MTT Assay

HAC15 cells were plated on 24-well plates at a density of 2 × 10⁵ cells/well. After 48 hours, cells were exposed to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 μM) for 72 hours. Following the BDE-47 exposure, cells were switched to fresh steroidogenic media containing 1 mg/mL MTT and incubated for 3.5 hours at 37 °C and 5% CO₂. The media was then removed and replaced with 4 mM HCL, 0.1% Nondet P-40 isopropanol solution. The plate was then covered with aluminum foil and agitated on an orbital plate shaker for 15 minutes. Absorbance at 590 nm was read on a Beckman Coulter DTX 880 Multimode Detector.

ATP Production

HAC15 cells were plated on 96-well plates at a density of 1 × 10⁴ cells/well. After 48 hours, cells were exposed to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 μM) for 72 hours. Following the BDE-47 exposure, ATP production was measure by a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) according to the manufacturer’s protocol.

Mitochondrial Biogenesis

HAC15 cells were plated on 96-well plates at a density of 1 × 10⁴ cells/well. After 48 hours, cells were exposed to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 μM) for 72 hours. Following the BDE-47 exposure, the protein levels of 2 mitochondrial proteins, succinate dehydrogenase complex, subunit A (SDH-A, subunit of mitochondrial Complex II) and cytochrome c oxidase (COX-I, subunit of mitochondrial Complex IV) were measured by a quantitative immunocytochemistry assay (MitoBiogenesis In-Cell ELISA Kit, Abcam, Cambridge, MA) according to the manufacturer’s protocol.

Rodent Study

Twenty-three male Sprague Dawley rats were purchased from Envigo (Somerset, NJ). At 8 weeks of age, rats were treated with vehicle (DMSO), 10 μg BDE-47/kg, or 100 μg BDE-47/kg, orally 5 days per week for 16 weeks. Oral dosing was achieved utilizing small treats of transgenic dough diet (Bio-Serv, Flemington, NJ) that were inoculated with the respective treatment. After 16 weeks of treatment, rats were euthanized with overexposure to CO₂. All animals were euthanized in the morning hours of 9 to 10 am in order to avoid confounding normal circadian rhythm fluctuations in circulating steroid hormones. Plasma was collected from EDTA-treated blood, divided into aliquots, and frozen at −20 °C for later analysis. Aldosterone was measured by ELISA as described above. A corticosterone ELISA was performed using a commercially available kit (Arbor Assays, Ann Arbor, MI) (RRID :AB_2877626) (52). Adrenal glands, liver, and kidneys were removed, weighed, and frozen at −80 °C. All animal procedures were approved by and performed according to the Institutional Animal Care and Use Committee of Midwestern University in accordance with the Guide for the Care and Use of Laboratory Animals (53).

Statistics

Data from cell viability, concentration response, MTT, ATP, and mitochondrial biogenesis assays were analyzed by 1-way ANOVA with Holm-Sidak post hoc comparisons. Data from time course, qPCR, Western assays, organ weights, and plasma ELISAs were analyzed by multiple t tests using the Holm-Sidak method. A P < 0.05 was considered significant.

Results

BDE-47 and Cell Viability

HAC15 cells were exposed to vehicle (0.1% DMSO) or BDE-47 (1 nM to 100 μM) for 72 hours. BDE-47 decreased cell viability only at the highest concentration of 100 μM, which resulted in a 90% reduction in cell numbers (Fig. 1).

Figure 1.

HAC15 cell viability following 72-hour exposure to vehicle (0.1% DMSO) or BDE-47 (1 nM to 100 µM). Each value represents the mean ± SEM, N = 4-8. *Significantly different from respective control, P < 0.05.

BDE-47 Time Course on Aldosterone Secretion

HAC15 cells were exposed to vehicle (0.1% DMSO) or BDE-47 (10 μM) for 24, 48, 72, or 96 hours. Following BDE-47 exposure, both basal and Ang II-stimulated aldosterone secretion were examined. Basal aldosterone secretion was unchanged after 24 or 48 hours of BDE-47 exposure. However, after 72 and 96 hours of exposure, basal aldosterone secretion was significantly increased by 3-fold (72 hours, vehicle: 9.6 ± 1.7 pg/200k cells, BDE-47: 29.5 ± 2.7 pg/200k cells, P < 0.001; 96 hours, vehicle: 10.7 ± 1.5 pg/200k cells, BDE-47: 30.7 ± 1.8 pg/200k cells, P < 0.001) (Fig. 2A). Ang II-stimulated aldosterone secretion followed a similar time course response. Ang II-stimulated secretion was unchanged after 24 or 48 hours of BDE-47 exposure, while 72 and 96 hours of exposure resulted in a significant increase in Ang II-stimulated aldosterone secretion by 1.5-fold (72 hours, vehicle: 495.6 ± 9.1 pg/200k cells, BDE-47: 773.1 ± 15.9 pg/200k cells, P < 0.001; 96 hours, vehicle: 492.4 ± 22.3 pg/200k cells, BDE-47: 742.4 ± 22.4 pg/200k cells, P < 0.001) (Fig. 2B).

Figure 2.

HAC15 cell aldosterone production following vehicle (0.1% DMSO) or BDE-47 exposure (10 µM) for 24-96 hours. Aldosterone production was examined under both A, basal conditions and B, Ang II stimulation (100 nM). Each value represents the mean ± SEM, N = 4. *Significantly different from respective control, P < 0.05.

BDE-47 Concentration Response on Aldosterone Secretion

Based on the BDE-47 time course, a BDE-47 concentration response (1 nM to 10 μM) was performed with a 72-hour exposure. The 100 μM concentration of BDE-47 was not included in the concentration response due to overt cytotoxicity. Following BDE-47 exposure, basal, Ang II-stimulated, ACTH-stimulated, and K⁺-stimulated aldosterone secretion were examined. Basal aldosterone secretion was significantly increased by 100 nM to 10 μM BDE-47, with maximal secretion at 10 μM (vehicle: 10.2 ± 0.5 pg/200k cells, 10 μM BDE-47: 28.1 ± 1.5 pg/200k cells, P < 0.001) (Fig. 3A). Similarly, Ang II-stimulated aldosterone secretion was significantly increased by 10 nM to 10 μM BDE-47, with maximal secretion at 10 μM (vehicle: 249.3 ± 15.9 pg/200k cells, 10 μM BDE-47: 614.4 ± 35.7 pg/200k cells, P < 0.001) (Fig. 3B). ACTH-stimulated aldosterone secretion was significantly increased by 1 to 10 μM BDE-47, with maximal secretion at 10 μM (vehicle: 18.6 ± 1.5 pg/200k cells, 10 μM BDE-47: 67.5 ± 3.2 pg/200k cells, P < 0.001) (Fig. 3C). K⁺-stimulated aldosterone secretion was significantly increased by 10 nM to 1 μM BDE-47, with maximal secretion at 100 nM (vehicle: 189.2 ± 12.7 pg/200k cells, 1 μM BDE-47: 278.1 ± 18.8 pg/200k cells, P < 0.001) (Fig. 3D). Unlike the other conditions, 10 μM BDE-47 did not significantly increase K⁺-stimulated aldosterone secretion.

Figure 3.

HAC15 cell aldosterone production following 72-hour exposure to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 µM). Aldosterone production was examined under A, basal conditions; B, Ang II stimulation (100 nM); C, ACTH stimulation (100 nM); and D) K⁺ stimulation (15 mM). Each value represents the mean ± SEM, N = 8-28. *Significantly different from respective control, P < 0.05.

BDE-47 Concentration Response on Cortisol Secretion

Due to the overlap in the synthetic pathways of aldosterone and cortisol, a BDE-47 concentration response (1 nM to 10 μM) was also performed on cortisol secretion. Following BDE-47 exposure, basal and ACTH-stimulated cortisol secretion were examined. Basal cortisol secretion was significantly increased by 100 nM to 10 μM BDE-47, with maximal secretion at 10 μM (vehicle: 69.4 ± 4.6 ng/200k cells, 10 μM BDE-47: 138.2 ± 5.6 ng/200k cells, P < 0.001) (Fig. 4A). ACTH-stimulated cortisol secretion was significantly increased by 1-10 μM BDE-47, with maximal secretion at 10 μM (vehicle: 87.3 ± 3.1 ng/200k cells, 10 μM BDE-47: 137.8 ± 7.9 ng/200k cells, P < 0.001) (Fig. 4B). ACTH-stimulation of cortisol secretion was minimal in this cell line (vehicle: 69.4 ± 4.6 ng/200k cells, 100 nM ACTH: 87.3 ± 3.1 ng/200k cells) in comparison to the stimulatory effect of BDE-47.

Figure 4.

HAC15 cell cortisol production following 72-hour exposure to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 µM). Cortisol production was examined under A, basal conditions and B, ACTH stimulation (100 nM). Each value represents the mean ± SEM, N = 8. *Significantly different from respective control, P < 0.05.

mRNA Expression of the Corticosteroid Synthesis Pathway

mRNA expression of the steroidogenic enzymes of the corticosteroid pathway (CYP11A1, HSD3B2, CYP21A2, CYP11B1, CYP11B2), coenzymes (FDXR and FDX1), the cholesterol transport protein StAR, and the receptors AGTR1 and MC2R was examined in HAC15 cells following 72-hour exposure to vehicle or 10 μM BDE-47. The steroidogenic enzymes HSD3B2, CYP11B1, and CYP11B2 were significantly upregulated, with CYP11B2 having the highest level of induction (fold induction: 3.73 ± 0.36, P < 0.001) (Fig. 5). Expression of StAR and the cofactors FDXR and FDX1 were unchanged. Additionally, expression of the Ang II receptor AGTR1 was unchanged, while the ACTH receptor MC2R was upregulated 2-fold (fold induction: 2.05 ± 0.28, P < 0.001).

Figure 5.

mRNA expression of steroidogenic enzymes, coenzymes, and receptors in HAC15 cells following 72-hour exposure to vehicle (0.1% DMSO) or BDE-47 (10 µM). Each value represents the mean ± SEM, N = 6. *Significantly different from respective control, P < 0.05.

CYP11B2 Protein Expression

CYP11B2 protein levels were assessed following exposure to vehicle or 10 μM BDE-47. A doublet at 52 kDa was detected, which is consistent with previous reports of a precursor (upper band) and a mature mitochondrial form (lower band) of CYP11B2 (54). While total CYP11B2 (precursor and mature forms combined) was unchanged by BDE-47 exposure, BDE-47 decreased the amount of precursor CYP11B2 and increased the amount of mitochondrial CYP11B2 (Fig. 6).

Figure 6.

CYP11B2 protein levels following 72-hour exposure to vehicle (0.1% DMSO) (lanes 1-3) or BDE-47 (10 µM) (lanes 4-6). Values are expressed as CYP11B2 band intensity normalized to respective GAPDH band intensity. A doublet at 52 kDa was detected, representing a precursor (upper band) and a mature mitochondrial form (lower band) of CYP11B2. Each value represents the mean ± SEM, N = 3. *Significantly different from respective control, P < 0.05.

BDE-47 Increases Metabolic Activity

The metabolic activity of HAC15 cells was assessed following exposure to vehicle or BDE-47 (1 nM to 10 μM) for 72 hours. Metabolic activity of HAC15 cells was assessed by both a traditional MTT assay and a commercial assay that quantifies ATP production. An MTT assay is traditionally used as an indirect method to determine cell viability. However, the direct measure of an MTT assay is mitochondrial reductase activity, and therefore mitochondrial activity. MTT reduction, and therefore mitochondrial activity, was significantly increased by 100 nM to 10 μM BDE-47, with maximal induction at 10 μM BDE-47 (in Abs units/200k cells, vehicle: 0.0097 ± 0.0014, 10 μM BDE-47: 0.0392 ± 0.0004, P < 0.001) (Fig. 7). Similarly, ATP production was significantly increased by 100 nM to 10 μM BDE-47, with maximal induction at 10 μM BDE-47 (in Luminescence units/200k cells, vehicle: 10790 ± 309, 10 μM BDE-47: 15726 ± 672, P < 0.001) (Fig. 8).

Figure 7.

Mitochondrial reductase activity as measured by MTT reduction in HAC15 cells following 72-hour exposure to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 µM). Each value represents the mean ± SEM, N = 12. *Significantly different from respective control, P < 0.05.

Figure 8.

ATP production in HAC15 cells following 72-hour exposure to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 µM). Each value represents the mean ± SEM, N = 4. *Significantly different from respective control, P < 0.05.

BDE-47 Increases Mitochondrial Biogenesis

The protein levels of 2 mitochondrial proteins, SDH-A (subunit of mitochondrial Complex II) and COX-I (subunit of mitochondrial Complex IV), were assessed in HAC15 cells following exposure to vehicle or BDE-47 (1 nM to 10 μM) for 72 hours. Protein levels of SDH-A were unchanged (Fig. 9A). Protein levels of COX-I were significantly increased in both the 1 and 10 μM BDE-47 treatment groups (vehicle: 100.00 ± 3.04%, 1 μM BDE-47: 115.92 ± 2.58%, 10 μM BDE-47: 118.15 ± 4.47%, P < 0.05) (Fig. 9B).

Figure 9.

Protein levels of A, SDH-A (subunit of mitochondrial Complex II) and B, COX-I (subunit of mitochondrial Complex IV) in HAC15 cells following 72-hour exposure to vehicle (0.1% DMSO) or BDE-47 (1 nM to 10 µM). Each value represents the mean ± SEM, N = 8. *Significantly different from respective control, P < 0.05.

Chronic BDE-47 Exposure Increases Adrenal Weight and Plasma Corticosterone Levels

Sprague Dawley rats were exposed to BDE-47 for 16 weeks to determine the effects of chronic exposure on steroidogenesis in an in vivo model. Normalized adrenal, but not heart, kidney, or liver weights, were significantly increased in both the 10 and 100 μg BDE-47/kg treatment groups (adrenal weight in mg/kg of body weight, vehicle: 0.101 ± 0.003, 10 μg BDE-47/kg: 0.112 ± 0.005, 100 μg BDE-47/kg: 0.112 ± 0.003) (Table 1). In addition, plasma corticosterone levels were significantly increased in the 100 μg BDE-47/kg treatment group but not the 10 μg BDE-47/kg treatment group (in ng/mL, vehicle: 32.7 ± 7.9, 10 μg BDE-47/kg: 43.5 ± 16.8, 100 μg BDE-47/kg: 115.3 ± 47.7) (Fig. 10A). In contrast, no change in plasma aldosterone levels were observed with BDE-47 exposure (in pg/mL, vehicle: 3360 ± 398, 10 μg BDE-47/kg: 3167 ± 403, 100 μg BDE-47/kg: 3658 ± 303) (Fig. 10B).

Table 1.

Body and tissue weights of male Sprague Dawley rats following oral exposure to vehicle (0.1% DMSO) or BDE-47 (10 or 100 µg) for 16 weeks, 5 days per week

Weight (g) (mg/kg body weight)	Vehicle (n = 8)	BDE-47 (10 µg) (n = 7)	BDE-47 (100 µg) (n = 8)
Body	454.86 ± 13.14	438.86 ± 12.91	456.50 ± 11.94
Adrenals	0.0467 ± 0.0015 (0.1006 ± 0.0031)	0.0491 ± 0.0026 (0.1119 ± 0.0049)*	0.0509 ± 0.0020 (0.1119 ± 0.0027)*
Heart	1.2969 ± 0.0405 (2.8510 ± 0.0296)	1.2701 ± 0.0557 (2.8896 ± 0.0563)	1.3205 ± 0.0507 (2.9042 ± 0.0715)
Kidneys	2.8041 ± 0.1092 (6.1593 ± 0.1272)	2.8079 ± 0.1476 (6.3817 ± 0.1945)	2.8913 ± 0.0907 (6.3699 ± 0.1417)
Liver	15.9038 ± 0.7544 (34.8530 ± 0.8196)	15.4577 ± 0.6953 (35.1636 ± 0.8661)	15.7783 ± 0.4728 (34.7651 ± 0.7380)

Organ weights are expressed as weight in grams and normalized weight in mg/kg body weight in parentheses.

*Significantly different from respective control, P < 0.05

Figure 10.

A, Corticosterone and B, aldosterone plasma levels in male Sprague Dawley rats following oral exposure to vehicle (0.1% DMSO) or BDE-47 (10 or 100 µg) for 16 weeks, 5 days per week. Each value represents the mean ± SEM, N = 7-8. *Significantly different from respective control, P < 0.05.

Discussion

PBDEs are ubiquitous environmental contaminants that disrupt both endocrine and cardiovascular homeostasis (3, 4). Previous studies have demonstrated that PBDEs induce the expression of microsomal enzymes in various endocrine tissues (40-44, 55-58). Moreover, PBDEs accumulate in the adrenal cortex (19, 39), the site for the production of aldosterone and cortisol, 2 central endocrine hormones of the RAAS and HPA axis, respectively. Our current study demonstrates that BDE-47, one of the most common and environmentally persistent PBDE congeners, increases both aldosterone and cortisol secretion in a human adrenocortical cell line.

BDE-47 increases aldosterone and cortisol secretion in a concentration-dependent manner. Induction of corticosteroid secretion was observed at concentrations as low as 100 nm, with maximal effect observed at 10 μM. There are currently no data on the concentrations of BDE-47 or other PBDEs in human tissue other than sera, so it is unfortunately not possible to put the concentrations used in our studies in context. Still, tissue disposition studies in both mice and rats have identified the adrenal cortex as one of the primary sinks for PBDE accumulation (19, 39), which would suggest that levels of PBDEs in the adrenal cortex would be considerably greater in comparison with serum levels.

BDE-47 was able to induce corticosteroid secretion in the absence (basal) or presence of steroidogenic stimuli (Ang II, ACTH, and K⁺). While BDE-47 increased basal and stimulated aldosterone and cortisol secretion in a concentration-dependent manner in most conditions tested, K⁺ stimulation of aldosterone secretion was a notable exception. Ang II and ACTH stimulation of aldosterone secretion is dependent on activation and downstream signaling of a G-protein coupled receptor, while K⁺ stimulation of aldosterone secretion functions independent of a receptor by depolarizing cellular membrane potential through altered potassium efflux (59). BDE-47 has been shown to alter calcium homeostasis in other cell models (60-64). Increased intracellular calcium could alter the ability of K⁺ to depolarize the adrenocortical cell membrane due to increased levels of the positively charged calcium ion in the intracellular compartment. Additional studies would be necessary to explore this mechanism in adrenocortical cells.

The downstream mechanism by which BDE-47 increases corticosteroid secretion appears to be via increased expression of the steroidogenic enzymes in the corticosteroid synthesis pathway. Of particular note is the nearly 4-fold increase in CYP11B2, also known as aldosterone synthase, a key rate-limiting enzyme in the production of aldosterone. This increase in CYP450 expression is consistent with several other studies that demonstrated that PBDEs induced steroidogenic enzyme expression in other endocrine cell models (40-44, 55-58, 65).

Interestingly, the ability of BDE-47 to increase both aldosterone and cortisol secretion in HAC15 cells was time-dependent and required an exposure of at least 72 hours. The initial explanation for this delay in response relates to the likely mechanism by which BDE-47 appears to be increasing aldosterone and cortisol secretion, via increased expression of steroidogenic enzymes. The upregulation of mRNA and resulting increase in protein levels of these enzymes would typically take at least 24 hours. In addition to this transcriptional and translational delay, it is also possible that it is a metabolite of BDE-47 that exerts these changes in steroidogenic enzyme expression. This observation would be consistent with a previous study that demonstrated hydroxylation and methoxylation metabolites of BDE-47 differentially induced the expression of steroidogenic enzymes in H295R cells, another adrenocortical cell line (42).

Increased mitochondrial activity may be an additional effect by which BDE-47 increases corticosteroid production. BDE-47 increased MTT reduction and ATP production in HAC15 cells. While both of these assays are commonly used as an indicator of cell proliferation, they are technically a direct measure of cellular metabolic and mitochondrial activity. Additionally, BDE-47 significantly increased protein levels of COX-I, a subunit of mitochondrial Complex IV. Since corticosteroid production is mediated by a series of mitochondrial and microsomal enzymes, such changes in mitochondrial function could contribute to the potentiation of corticosteroid production. Several published studies demonstrate that PBDEs have effects on mitochondrial energetics (66-75). However, while we observed evidence of an increase in mitochondrial activity, the majority of these studies demonstrate impaired mitochondrial bioenergetics, increased reaction oxygen species production, and apoptosis. The reason for the differential effect in our cellular model is unclear. It is worth noting, however, these other published studies were performed in hepatic (70), spermatocyte (69), and neuroblastoma cell lines (71, 75) as well as isolated mitochondria (71-73). Despite potential differences in response due to cell type, we also observed a substantial reduction in cell viability at the 100 μM BDE-47 concentration, which roughly correlates with the reduced viability and apoptosis that was observed in several of these published studies (67, 68, 70, 75).

Finally, in order to confirm our observations in the HAC15 cell line, we performed a study of chronic BDE-47 exposure in Sprague Dawley rats. Oral dosing was chosen as the route of exposure for our study. Both inhalation and ingestion of house dust is a major route of PBDE exposure in humans, with dietary sources being a secondary route of oral exposure (76). These dosing regimens would result in an estimated steady state body burden of 189 and 1,890 μg/kg for the 10 and 100 μg BDE-47/kg doses, respectively, by Week 11. The resulting body burden of the 10 μg BDE-47/kg dosing regimen is within the range of samples from highly exposed humans (76). Both adrenal weight and plasma corticosterone levels were significantly elevated in the BDE-47 treated rats at the end of the chronic study. These data support the hypothesis that BDE-47 is an endocrine disruptor and that the adrenal cortex is a target of this activity. Interestingly, plasma aldosterone levels were not altered in the BDE-47 treated rats. These data suggest that the zona fasciculata, not the zona glomerulosa, is the primary cell type of the adrenal cortex that is affected by BDE-47 in vivo. While our current study did not address whether the zona reticularis and adrenal androgen production was affected by BDE-47 exposure, previous work by others has demonstrated that androgen steroidogenesis is increased in isolated primary rat Leydig cells following PBDE exposure (58).

In summary, PBDEs such as BDE-47 are endocrine disruptors that may also affect the HPA axis. In cultured human adrenocortical cells, BDE-47 increases aldosterone and cortisol secretion in a concentration- and time-dependent manner. Increased expression of steroidogenic enzymes and cellular metabolism contribute to these effects on corticosteroid production. These corticosteroid effects are additionally seen in a rodent model of chronic BDE-47 exposure, with corticosterone being the primary corticosteroid altered by BDE-47. Based on these data, the adrenal cortex is an additional toxicological target for endocrine disruption by BDE-47. Our future studies will: (1) determine the underlying molecular mechanism by which BDE-47 alters corticosteroid production and (2) examine whether these alterations in the HPA axis result in underlying pathophysiology or a predisposition to cardiovascular disease in our rodent model of chronic BDE-47 exposure.

Glossary

Abbreviations

ACTH

adrenocorticotropic hormone

Ang II

angiotensin II

COX

cytochrome c oxidase

DMSO

dimethyl sulfoxide

ELISA

enzyme-linked immunosorbent assay

HPA

hypothalamic-pituitary-adrenal

PBDE

polybrominated diphenyl ether

qPCR

quantitative polymerase chain reaction

RAAS

renin-angiotensin-aldosterone system

SDH

succinate dehydrogenase

SEM

standard error of the mean

Additional Information

Disclosures: None

Data Availability

Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.