Endocrine Abstracts

Introduction: Benzo[a]Pyrene (BaP) is an endocrine-disrupting chemical, which may impact reproduction. It is a polycyclic aromatic hydrocarbon generated by the incomplete combustion of organic compounds. BaP may be accumulated in the environment, achieving effective concentrations in the nanomolar range and exerting genotoxic effects in long-term exposed humans.

Aim: We evaluated the short-term impact of BaP on luteinizing-hormone (LH)/choriogonadotropin (hCG)-mediated functions in the steroidogenic mouse Leydig tumor cell line (mLTC1), in vitro.

Methods: mLTC-1 cells were treated with increasing BaP doses (range fM-µM), over 0-40 h, in the presence or in the absence of the maximal, non-saturating effective LH and hCG concentration (1500 and 300 pM, respectively). The maximal, non-cytotoxic BaP dose was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. 3-h intracellular cyclic adenosine monophosphate (cAMP) production was assessed by bioluminescence resonance energy transfer (BRET), in transfected cells overexpressing the specific biosensor. 10, 50 and 100 µM forskolin-treated cells served as controls. 15-min cAMP-response element binding protein (CREB), extracellularly-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38-MAPK) phosphorylation was evaluated by Western blotting. 8- and 24-h progesterone and testosterone levels were measured by immunoassay. Results were compared with those from gonadotropin- and BaP-untreated samples by Kruskal-Wallis test followed by Dunn’s post-hoc test (P<0.05; n = 6).

Results: 1 µM BaP was determined as the maximal, non-cytotoxic dose (P≥0.5) and was used for cell treatments, as well as the environmentally available dose of 1 nM. LH/hCG-induced intracellular cAMP accumulation was dampened by 1 µM BaP (2.0-fold compared to BaP-untreated; P<0.05), with no gonadotropin-specific differences, while 1 nM BaP did not produce any effect. 1 µM BaP failed in inhibiting 10-100 µM forskolin-induced cAMP (P≥0.5), excluding that the compound modulates adenylyl cyclase enzyme functioning. Consistently, BaP interfered with most of the downstream intracellular cAMP-dependent events. 1 µM BaP decreased LH/hCG-induced CREB phosphorylation (P<0.05) and increased basal ERK1/2 phosphorylation (P<0.05), while no perturbation of p38-MAPK phosphorylation was detected (P≥0.05). The modulation of signal transduction is linked to interfering effects on steroidogenesis. LH/hCG-mediated, 24-h progesterone synthesis (1.5-fold BaP-untreated; P<0.05), as well as 8-h testosterone production (2.0-fold BaP-untreated; P<0.05), decreased significantly in the presence of 1 µM BaP.

Conclusions: These results demonstrated that a micromolar BaP concentration inhibits short-term steroidogenic signals in a mammal Leydig cell line, through an unknown molecular mechanism. These data suggest that BaP may potentially interfere with endocrine signals, dysregulating male gonadal and reproductive functions.