Afshan Dean

Prolonged exposure to acetaminophen reduces testosterone production by the human fetal testis in a xenograft model

Prolonged exposure to therapeutic doses of acetaminophen reduces testosterone production by human fetal testis xenografts in mice. A risk of prolonged acetaminophen use The analgesic acetaminophen is one of the most commonly used medications worldwide. Although it has a good safety profile, previous human studies have found an association between the use of acetaminophen in pregnancy and cryptorchidism in male offspring. Now, van den Driesche et al. confirmed and refined this observation in a xenograft model using human fetal testicular tissue. The authors found that a single therapeutic dose of acetaminophen had no effect on testicular development, but continued dosing of acetaminophen for a week at clinically relevant doses reduced fetal testosterone and markers of androgen exposure. Further work will be needed to determine a safe dose and duration of acetaminophen exposure, but the current findings suggest a need for caution. Most common male reproductive disorders are linked to lower testosterone exposure in fetal life, although the factors responsible for suppressing fetal testosterone remain largely unknown. Protracted use of acetaminophen during pregnancy is associated with increased risk of cryptorchidism in sons, but effects on fetal testosterone production have not been demonstrated. We used a validated xenograft model to expose human fetal testes to clinically relevant doses and regimens of acetaminophen. Exposure to a therapeutic dose of acetaminophen for 7 days significantly reduced plasma testosterone (45% reduction; P = 0.025) and seminal vesicle weight (a biomarker of androgen exposure; 18% reduction; P = 0.005) in castrate host mice bearing human fetal testis xenografts, whereas acetaminophen exposure for just 1 day did not alter either parameter. Plasma acetaminophen concentrations (at 1 hour after the final dose) in exposed host mice were substantially below those reported in humans after a therapeutic oral dose. Subsequent in utero exposure studies in rats indicated that the acetaminophen-induced reduction in testosterone likely results from reduced expression of key steroidogenic enzymes (Cyp11a1, Cyp17a1). Our results suggest that protracted use of acetaminophen (1 week) may suppress fetal testosterone production, which could have adverse consequences. Further studies are required to establish the dose-response and treatment-duration relationships to delineate the maximum dose and treatment period without this adverse effect.

Metformin Reverses Development of Pulmonary Hypertension via Aromatase Inhibition.

Females are more susceptible to pulmonary arterial hypertension than males, although the reasons remain unclear. The hypoglycemic drug, metformin, is reported to have multiple actions, including the inhibition of aromatase and stimulation of AMP-activated protein kinase. Inhibition of aromatase using anastrazole is protective in experimental pulmonary hypertension but whether metformin attenuates pulmonary hypertension through this mechanism remains unknown. We investigated whether metformin affected aromatase activity and if it could reduce the development of pulmonary hypertension in the sugen 5416/hypoxic rat model. We also investigated its influence on proliferation in human pulmonary arterial smooth muscle cells. Metformin reversed right ventricular systolic pressure, right ventricular hypertrophy, and decreased pulmonary vascular remodeling in the rat. Furthermore, metformin increased rat lung AMP-activated protein kinase signaling, decreased lung and circulating estrogen levels, levels of aromatase, the estrogen metabolizing enzyme; cytochrome P450 1B1 and its transcription factor; the aryl hydrocarbon receptor. In human pulmonary arterial smooth muscle cells, metformin decreased proliferation and decreased estrogen synthesis by decreasing aromatase activity through the PII promoter site of Cyp19a1. Thus, we report for the first time that metformin can reverse pulmonary hypertension through inhibition of aromatase and estrogen synthesis in a manner likely to be mediated by AMP-activated protein kinase.

Analgesic exposure in pregnant rats affects fetal germ cell development with inter-generational reproductive consequences

Analgesics which affect prostaglandin (PG) pathways are used by most pregnant women. As germ cells (GC) undergo developmental and epigenetic changes in fetal life and are PG targets, we investigated if exposure of pregnant rats to analgesics (indomethacin or acetaminophen) affected GC development and reproductive function in resulting offspring (F1) or in the F2 generation. Exposure to either analgesic reduced F1 fetal GC number in both sexes and altered the tempo of fetal GC development sex-dependently, with delayed meiotic entry in oogonia but accelerated GC differentiation in males. These effects persisted in adult F1 females as reduced ovarian and litter size, whereas F1 males recovered normal GC numbers and fertility by adulthood. F2 offspring deriving from an analgesic-exposed F1 parent also exhibited sex-specific changes. F2 males exhibited normal reproductive development whereas F2 females had smaller ovaries and reduced follicle numbers during puberty/adulthood; as similar changes were found for F2 offspring of analgesic-exposed F1 fathers or mothers, we interpret this as potentially indicating an analgesic-induced change to GC in F1. Assuming our results are translatable to humans, they raise concerns that analgesic use in pregnancy could potentially affect fertility of resulting daughters and grand-daughters.

Prostaglandins, Masculinization and Its Disorders: Effects of Fetal Exposure of the Rat to the Cyclooxygenase Inhibitor- Indomethacin

Recent studies have established that masculinization of the male reproductive tract is programmed by androgens in a critical fetal ‘masculinization programming window’ (MPW). What is peculiar to androgen action during this period is, however, unknown. Studies from 20 years ago in mice implicated prostaglandin (PG)-mediation of androgen-induced masculinization, but this has never been followed up. We therefore investigated if PGs might mediate androgen effects in the MPW by exposing pregnant rats to indomethacin (which blocks PG production by inhibiting cyclooxygenase activity) during this period and then examining if androgen production or action (masculinization) was affected. Pregnant rats were treated with indomethacin (0.8 mg/kg/day; e15.5–e18.5) to encompass the MPW. Indomethacin exposure decreased fetal bodyweight (e21.5), testis weight (e21.5) and testicular PGE2 (e17.5, e21.5), but had no effect on intratesticular testosterone (ITT; e17.5) or anogenital index (AGI; e21.5). Postnatally, AGI, testis weight and blood testosterone were unaffected by indomethacin exposure and no cryptorchidism or hypospadias occurred. Penis length was normal in indomethacin-exposed animals at Pnd25 but was reduced by 26% (p<0.001) in adulthood, an effect that is unexplained. Our results demonstrate that indomethacin can effectively decrease intra-testicular PGE2 level. However, the resulting male phenotype does not support a role for PGs in mediating androgen-induced masculinization during the MPW in rats. The contrast with previous mouse studies is unexplained but may reflect a species difference.

Role of the Aryl Hydrocarbon Receptor in Sugen 5416–induced Experimental Pulmonary Hypertension

Abstract Rats dosed with the vascular endothelial growth factor inhibitor Sugen 5416 (Su), subjected to hypoxia, and then restored to normoxia have become a widely used model of pulmonary arterial hypertension (PAH). However, the mechanism by which Su exacerbates pulmonary hypertension is unclear. We investigated Su activation of the aryl hydrocarbon receptor (AhR) in human pulmonary artery smooth muscle cells (hPASMCs) and blood outgrowth endothelial cells (BOECs) from female patients with PAH. We also examined the effect of AhR on aromatase and estrogen levels in the lung. Protein and mRNA analyses demonstrated that CYP1A1 was very highly induced in the lungs of Su/hypoxic (Su/Hx) rats. The AhR antagonist CH223191 (8 mg/kg/day) reversed the development of PAH in this model in vivo and normalized lung CYP1A1 expression. Increased lung aromatase and estrogen levels in Su/Hx rats were also normalized by CH223191, as was AhR nuclear translocator (ARNT [HIF‐1&bgr;]), which is shared by HIF‐1&agr; and AhR. Su reduced HIF‐1&agr; expression in hPASMCs. Su induced proliferation in BOECs and increased apoptosis in human pulmonary microvascular ECs and also induced translocation of AhR to the nucleus in hPASMCs. Under normoxic conditions, hPASMCs did not proliferate to Su. However, when grown in hypoxia (1%), Su induced hPASMC proliferation. In combination with hypoxia, Su is proliferative in hPASMCs and BOECs from patients with PAH, and Su/Hx‐induced PAH in rats may be facilitated by AhR‐induced CYP1A1, ARNT, and aromatase. Inhibition of AhR may be a novel approach to the treatment of pulmonary hypertension.

4-Sodium phenyl butyric acid has both efficacy and counter-indicative effects in the treatment of Col4a1 disease

Abstract Mutations in the collagen genes COL4A1 and COL4A2 cause Mendelian eye, kidney and cerebrovascular disease including intracerebral haemorrhage (ICH), and common collagen IV variants are a risk factor for sporadic ICH. COL4A1 and COL4A2 mutations cause endoplasmic reticulum (ER) stress and basement membrane (BM) defects, and recent data suggest an association of ER stress with ICH due to a COL4A2 mutation. However, the potential of ER stress as a therapeutic target for the multi-systemic COL4A1 pathologies remains unclear. We performed a preventative oral treatment of Col4a1 mutant mice with the chemical chaperone phenyl butyric acid (PBA), which reduced adult ICH. Importantly, treatment of adult mice with the established disease also reduced ICH. However, PBA treatment did not alter eye and kidney defects, establishing tissue-specific outcomes of targeting Col4a1-derived ER stress, and therefore this treatment may not be applicable for patients with eye and renal disease. While PBA treatment reduced ER stress and increased collagen IV incorporation into BMs, the persistence of defects in BM structure and reduced ability of the BM to withstand mechanical stress indicate that PBA may be counter-indicative for pathologies caused by matrix defects. These data establish that treatment for COL4A1 disease requires a multipronged treatment approach that restores both ER homeostasis and matrix defects. Alleviating ER stress is a valid therapeutic target for preventing and treating established adult ICH, but collagen IV patients will require stratification based on their clinical presentation and mechanism of their mutations.

06-P031 Development of the male reproductive system: Potential mediators of androgen action during the male programming window

By E16.5, the secondary lens fibre cells were abnormally arranged with poor lens suture formation. Apoptotic cells were found in the centre of the lens as shown by TUNEL assay, the cytoskeleton and cell adhesion in the lens centre were disturbed as shown in immunohistochemistry analysis. By western blotting we found that mutant c-crystallins were reduced in amount and enriched in the insoluble fraction, suggesting that mutant ca-crystallins were misfolded and protein aggregates were formed. We found that the expression of genes involved in the unfolded protein response (UPR) pathways including BiP, CHOP and spliced variant of XBP-1 were all up-regulated significantly in E14.5 and 16.5 mutant lenses. Therefore, the mutant cA-crystallin appeared to trigger UPR and cell death in the fibre cells. The mutant cells lost their normal cell adhesion, failed to maintain the proper lens architecture, leading to cataract formation.