Many putative endocrine disruptors inhibit prostaglandin synthesis

Abstract

Background: Prostaglandins (PGs) play key roles in development and maintenance of homeostasis of the adult body. Despite these important roles, it remains unclear whether the PG pathway is a target for endocrine disruption. However, several known endocrine-disrupting compounds (EDCs) share a high degree of structural similarity with mild analgesics.

Objectives and methods: Using cell-based transfection and transduction experiments, mass spectrometry, and organotypic assays together with molecular modeling, we investigated whether inhibition of the PG pathway by known EDCs could be a novel point of endocrine disruption.

Results: We found that many known EDCs inhibit the PG pathway in a mouse Sertoli cell line and in human primary mast cells. The EDCs also reduced PG synthesis in ex vivo rat testis, and this reduction was correlated with a reduced testosterone production. The inhibition of PG synthesis occurred without involvement of canonical PG receptors or the peroxisome proliferator-activated receptors (PPARs), which have previously been described as targets of EDCs. Instead, our results suggest that the compounds may bind directly into the active site of the cyclooxygenase (COX) enzymes, thereby obstructing the conversion of arachidonic acid to PG precursors without interfering with the expression of the COX enzymes. A common feature of the PG inhibitory EDCs is the presence of aromatic groups that may stabilize binding in the hydrophobic active site of the COX enzymes.

Conclusion: Our findings suggest a hitherto unknown mode of action by EDCs through inhibition of the PG pathway and suggest new avenues to investigate effects of EDCs on reproductive and immunological disorders that have become increasingly common in recent decades.

Figure 1

COX-2 enzyme expression and PGD2 secretion in the SC5 juvenile mouse Sertoli cell line. (A) Membrane-bound COX-2 enzyme located in the endoplasmic reticulum and nuclear envelope (weak 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining); bar = 50 μm. Inhibition of PGD2 secretion from mouse SC5 cells by ASA (B), Ace (C), Ibu (D), and Indo (E), normalized (norm) to control values. Data are mean ± SE for three experiments performed in triplicate.

Figure 2

Endocrine disruptors inhibit PG synthesis in the SC5 juvenile mouse Sertoli cell line. DBP (A), BPa (B), BP3 (C), and BPA (D) dose-dependently inhibit secretion of PGD2 after incubation for 24 hr [normalized (norm) to control values]. (E) Incubation for 24 hr with MBP and MEHP, monoesters of DBP and DEHP, showed that they had no inhibitory effect on PGD2 secretion from SC5 cells. (F) LC-MS/MS analysis for MBP and MEHP after exposure to parental compounds DBP and DEHP and the monoesters revealed that MBP and MEHP were not taken up by SC5 cells. (G) Electroporation with MBP showed that the compound has inhibitory effect on PGD2 secretion from SC5 cells. (H) PGE2 is dose-dependently inhibited by DBP, BPa, BP3, and BPA in SC5 cells after 24 hr incubation. Data are mean ± SE for three experiments performed in triplicate. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with controls by two-tailed Student’s t-test.

Figure 3

Inhibition of PG synthesis in fetal testes from GD14.5 rats (A–E) and SC5 juvenile mouse Sertoli cells (F). (A) MBP (10 μM) inhibits secretion of PGD2 after 24 hr culture. (B) MEHP (10 μM) weakly inhibits PGD2 secretion after 24 hr. (C) The inhibitory action of MEHP is evident after stimulation of PGD2 synthesis with 100 μM AA for all time points, also implying that the inhibition is downstream from AA. The CYP17 inhibitor ketoconazole reduced testosterone production (D) but did not affect PGD2 synthesis (E). (F) Stimulation of PGD2 secretion from SC5 cells with 1 and 100 μM AA is inhibited by DBP, BPa, BP3, and BPA, normalized (norm) to control values. Similar action is seen with pharmaceutical inhibitors ASA, Ace, and Ibu, indicating that DBP, BPa, BP3, and BPA are inhibiting the COX enzymes. Data are mean ± SE for three experiments performed in triplicate. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with controls by two-tailed Student’s t-test.

Figure 4

Endocrine disruptors share structural features with mild analgesics and inhibit PGD2 secretion from primary human mast cells. (A) Secretion of PGD2 from primary human mast cells after stimulation with IgE and anti-IgE is dose-dependently inhibited by exposure to DBP, BPa, BP3, BPA, ASA, and Ace (n = 8). (B and C) Secretion of PGD2 after exposure to different compounds (at 10 μM), indicating that compounds with an isobutyl side chain (DiBP and iBPa) cause the most potent inhibition (n = 3). Data are normalized (norm) to control values. (D) Phthalates share structural similarities with salicylates, here exemplified with valeryl salicylate and MBP. Data are mean ± SE. Abbreviations: BzPa, benzylparaben; DBzP, dibenzyl phthalate; DEHP, diethylhexyl phthalate; DEP, diethyl phthalate; DiBP, diisobutyl phthalate; DiNP, di-isononyl phthalate; DMP, dimethyl phthalate; DNP, di-n-nonyl phthalate; DPeP, di-n-pentyl phthalate; DPP, di-n-propyl phthalate; EPa, ethylparaben; HBa, 4-hydroxy benzoic acid; iBPa, isobutylparaben, MPa, methylparaben, NPa, n-nonylparaben; PePa, n-pentylparaben; PPa, n-propylparaben. *p < 0.05, **p < 0.01, compared with controls by two-tailed Student’s t-test.