Acute effects of hexabromocyclododecane on Leydig cell cyclic nucleotide signaling and steroidogenesis in vitro

Abstract

Hexabromocyclododecane (HBCDD), an additive brominated flame retardant routinely added to various consumer products, was reported to have toxic effects upon biota, including endocrine disruption. In this study, the potential toxicity of HBCDD was tested in peripubertal rat Leydig cells in vitro during 6h exposure. HBCDD inhibited human chorionic gonadotropin- and forskolin-supported cAMP accumulation and steroidogenesis. It also inhibited basal cAMP production, but elevated basal steroidogenesis. The expression of several cAMP-dependent genes, including steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase, was also inhibited by HBCDD treatment. Nevertheless, this was not accompanied by a decrease in steroidogenic acute regulatory protein expression, as documented by western blot analysis, and activity of steroidogenic enzymes, as documented by unaffected steroidogenesis in the presence of permeable 22(R)-hydroxycholesterol. However, HBCDD caused significant decrease in mitochondrial membrane potential in untreated and human chorionic gonadotropin-treated cells. This indicates that HBCDD acute toxicity in Leydig cells reflects changes in mitochondrial membrane potential-dependent cAMP production and basal and cAMP-regulated cholesterol transport. This in turn facilitates basal but inhibits cAMP-dependent steroidogenesis. Acute effects of HBCDD treatment on transcription are also indicative of its sustained effects on Leydig cell function.

Fig. 1

Concentration-dependent effects of HBCDD on basal and hCG-supported androgen, progesterone, cAMP and cGMP production in Leydig cells. (A and B) Effects on basal (A) and hCG-stimulated (B) androgen (T+DHT) and progesterone (PROG) accumulation in the culture medium. (C and D) Effects on basal (C) and hCG-supported (D) cAMP accumulation in culture medium. Notice the lack of effects on cGMP levels. In this and following figures, cells (250000 in 500 μl/well) were seeded in 24-well plates, and treated during 6 h with increasing HBCDD concentrations in the absence or presence of 0.125 ng/ml hCG. Data shown represents mean ± SEM from 3–4 independent experiments. * p < 0.05 vs. corresponding control (0).

Fig. 2

Effects of HBCDD on androgen and cAMP accumulation in culture medium of Leydig cells with inhibited PDEs by 1 mM IBMX. (A) Effects on basal androgen and cAMP accumulation in the incubation medium. (B) Effects on hCG (0.125 ng/ml)-supported androgen and cAMP accumulation in the incubation medium. (C) HBCDD effects on cAMP levels when normalized to corresponding control. Results shown are means ± SEM from three independent experiments. * p < 0.05 vs. corresponding control (0).

Fig 3

Effects of HBCDD on androgen and cAMP accumulation in culture medium of Leydig cells treated with forskolin, an activator of ACs. (A and C) Effects of forskolin (0.1, 1 and 10 μM) on basal (A) and hCG (0.125 ng/ml)-supported (C) cAMP accumulation in the incubation medium. (B and D), Effects on basal (B) and hCG-supported (D) androgen production. Results shown are means ± SEM from three independent experiments. * p < 0.05 vs. corresponding control (0).

Fig. 4

Effects of HBCDD on expression of genes controlling steroidogenesis. (A and B). Gene expression in basal (A) and hCG (0.125 ng/ml)-supported (B) conditions. The mRNA transcripts were analyzed with qRT-PCR. Relative gene expression was calculated by equation RQ=2^−ΔΔCt, whereas Enpp1 was used as an endogenous control. Results shown are means ± SEM from three independent experiments. * p < 0.05 vs. corresponding control (white bars).

Fig. 5

Effect of 10 μM HBCDD on Leydig cell steroidogenesis in the absence and presence of 20 μM cholesterol and 20 μM 22R-hydroxycholesterol. (A and B) Effects on basal androgen (A) and progesterone (B) production. (C and D) Effects on hCG (0.125 ng/ml) supported androgen (C) and progesterone (D) production. Results shown are means ± SEM from three independent experiments. * p < 0.05 vs. corresponding control (0).

Fig. 6

Independence of HBCDD action on basal Leydig cell steroidogenesis of COX-2 expression and intracellular calcium. (A) Effects of celecoxib (5 μM and 15 μM), a COX-2 inhibitor, on in vitro steroidogenesis. Cells were treated during 6 h with 10 μM HBCDD in the absence and presence of celecoxib (B) HBCDD effects on Leydig cell basal and hCG (0.125 ng/ml)-supported androgen production when challenged with BAPTA-AM (20 μM), an intracellular Ca²⁺ chelator. Notice that BAPTA-AM did not diminish HBCDD toxic effects. Data points represent means ± SEM in one out of three similar experiments. * p < 0.05 vs. corresponding control (0).