Ibuprofen alters human testicular physiology to produce a state of compensated hypogonadism

Ibuprofen inhibits Leydig cell hormone production in human testicular explants. (A) Dose effect of ibuprofen for 24 and 48 h on testosterone production by the adult human testis. Values are means ± SEM of five independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. The slopes and P values of these results were calculated with Spearman correlation. (B) Effect of 10⁻⁵ and 10⁻⁴ M ibuprofen on the Δ4 and Δ5 steroidogenic pathways. Steroid precursors were measured by GC-MS/MS. Values are means ± SEM of three independent experiments from different donors. Dose effects were analyzed for significance with the Mann–Whitney U test. DHEA, dehydroepiandrosterone. (C) Effect of 10⁻⁵ and 10⁻⁴ M ibuprofen on the gene expression of steroidogenic enzymes after 48-h exposure. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. (D) Dose effect of ibuprofen for 24 and 48 h on INSL3 production by the adult human testis. Values are means ± SEM of three independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. (E) Specific Leydig cell hormone gene expression. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. BZW1, basic leucine zipper and W2 domains 1; CYP11A1, cytochrome P450 family 11 subfamily A member 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; GUSB, β-glucuronidase; INSL3, insulin-like factor 3; HSD17B3, hydroxysteroid 17-β dehydrogenase 3; HSD3B2, hydroxy-δ-5-steroid dehydrogenase 3 β- and steroid δ-isomerase 2; LHCGR, luteinizing hormone/choriogonadotropin receptor; StAR, steroidogenic acute regulatory protein; TSPO: translocation protein. *P ≤ 0.05, **P ≤ 0.01.

The steroid screen identifies ibuprofen as an inhibitor of steroidogenesis in the human NCI-H295R cell line. (A) Effects of ibuprofen (red trace; n = 9–18) and abiraterone (blue trace; n = 6–15) exposure on the relative steroidogenic hormone production in the dose ranges of 10⁻⁶–10⁻³ M and 3.15 × 10⁻¹¹–3.15 × 10⁻⁶ M (x axis), respectively, in the human NCI-H295R cell line, according to OECD standards. The position of each graph corresponds to the position of that particular steroid hormone in steroidogenesis. Steroid concentrations (y axis; % of control) are depicted as mean ± SEM with key enzymes shown in blue boxes. For statistics see Table S1. (B–D) Quantitative RT-PCR screen of steroidogenic gene expression in NCI-H295R cells after 48 h of culture with 10⁻⁷–10⁻³ M ibuprofen. Values are means ± SEM of three independent experiments analyzed with one-way ANOVA followed by a post hoc Dunnett’s test. CYP11A1, cytochrome P450 family 11 subfamily A member 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; HSD17B3, hydroxysteroid 17-β dehydrogenase 3; HSD3B2, hydroxy-δ-5-steroid dehydrogenase 3 β- and steroid δ-isomerase 2; StAR, steroidogenic acute regulatory protein; TSPO, translocation protein. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Ibuprofen affects Sertoli cell activity in human testicular explants. (A and B) Dose effect of ibuprofen on the production of inhibin B after 24 and 48 h (A) and anti-Müllerian hormone (AMH) after 48 h (B) by the adult human testis. Values are means ± SEM of three independent experiments from different donors. Slopes and P values of Spearman correlation are indicated. (C) Quantitative RT-PCR performed after 48 h of culture treated with 10⁻⁵ and 10⁻⁴ M ibuprofen for specific Sertoli cell gene expression. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. AMH, anti-Müllerian hormone; BZW1, basic leucine zipper and W2 domains 1; FSHR, follicle-stimulating hormone receptor; GUSB, β-glucuronidase; INHBB, inhibin B subunit B; LAMA5, laminin subunit α5. *P ≤ 0.05, **P ≤ 0.01.

Ibuprofen decreases gene expression in peritubular cells but does not affect germ cells or morphology in human testicular explants. (A and B) Quantitative RT-PCR performed after 48 h of culture treated with 10⁻⁵ and 10⁻⁴ M ibuprofen for gene expression in peritubular cells (A) and germ cells (B). Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Values are means ± SEM of five independent experiments from different donors. A Mann–Whitney U test was performed. (C) Number of apoptotic germ cells. Values are means ± SEM of caspase⁺ cells in three independent experiments from different donors. (D) Immunostaining of apoptotic germ cells in testis explants cultured for 48 h in the presence of DMSO (control) or 10⁻⁵ or 10⁻⁴ M ibuprofen. Each micrograph shows representative areas of ibuprofen-induced morphology compared with corresponding area. (Scale bars: 50 µm.) ACTA2, actin α2 smooth muscle aorta; ALPP, alkaline phosphatase, placental; BZW1, basic leucine zipper and W2 domains 1; GUSB, β-glucuronidase; KCNIP4, potassium voltage-gated channel interacting protein 4; MYH11, myosin heavy polypeptide 11, smooth muscle; PGK2, phosphoglycerate kinase 2; PRM2, protamine 2; THY1, Thy-1 cell-surface antigen. **P ≤ 0.01.

Ibuprofen decreases PGE2 and PGD2 production and PTGS gene expression in human testicular explants. (A and B) Dose effect of ibuprofen exposure after 24 and 48 h on PGD2 (A) and PGE2 (B) production by adult human testicular explants. Values are means ± SEM of five independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. Slopes and P values of Spearman correlation are indicated. (C) Quantitative RT-PCR was performed after 48 h of culture treated with 10⁻⁵ and 10⁻⁴ M ibuprofen. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Values are means ± SEM of five independent experiments from different donors. Differences in gene expression were analyzed with a Mann–Whitney U test. BZW1, basic leucine zipper and W2 domains 1; GUSB, β-glucuronidase; PTGS, prostaglandin-endoperoxide synthase. *P ≤ 0.05, **P ≤ 0.01.