Time- and Dose-Related Effects of Di-(2-ethylhexyl) Phthalate and Its Main Metabolites on the Function of the Rat Fetal Testis in Vitro

International audienceBACKGROUND: Endocrine-disrupting effects of phthalates are understood primarily from in utero exposures within the fetal rat testis. Nevertheless, their path of action, dose-response character, and cellular target(s) within the fetal testis are not known. OBJECTIVES: In this study we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP), and several of their metabolites on the development of organo-cultured testes from rat fetus. METHODS: We removed testes from 14.5-day-old rat fetuses and cultured them for 1-3 days with or without DEHP, MEHP, and the metabolites. RESULTS: DEHP (10(-5) M) produced a proandrogenic effect after 3 days of culture, whereas MEHP disrupted testis morphology and function. Leydig cells were the first affected by MEHP, with a number of them being inappropriately located within some seminiferous tubules. Additionally, we found a time- and dose-dependent reduction of testosterone. By 48 hr, gonocyte proliferation had decreased, whereas apoptosis increased. Sertoli cell number was unaffected, although some cells appeared vacuolated, and production of anti-Müllerian hormone decreased in a time- and dose-dependent manner. The derived metabolite mono-(2-ethyl-5-hydroxyhexyl) phthalate was the only one to cause deleterious effects to the rat fetal testis in vitro. CONCLUSION: We hope that this in vitro method will facilitate the study of different phthalate esters and other endocrine disruptors for direct testicular effects

Many Putative Endocrine Disruptors Inhibit Prostaglandin Synthesis

International audienceBACKGROUND: 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

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

correction de l'article correspondant à la notice https://prodinra.inra.fr/record/425677International audienceConcern has been raised over increased male reproductive disorders in the Western world, and the disruption of male endocrinology has been suggested to play a central role. Several studies have shown that mild analgesics exposure during fetal life is associated with antiandrogenic effects and congenital malformations, but the effects on the adult man remain largely unknown. Through a clinical trial with young men exposed to ibuprofen, we show that the analgesic resulted in the clinical condition named "compensated hypogonadism," a condition prevalent among elderly men and associated with reproductive and physical disorders. In the men, luteinizing hormone (LH) and ibuprofen plasma levels were positively correlated, and the testosterone/LH ratio decreased. Using adult testis explants exposed or not exposed to ibuprofen, we demonstrate that the endocrine capabilities from testicular Leydig and Sertoli cells, including testosterone production, were suppressed through transcriptional repression. This effect was also observed in a human steroidogenic cell line. Our data demonstrate that ibuprofen alters the endocrine system via selective transcriptional repression in the human testes, thereby inducing compensated hypogonadism

Intrauterine exposure to mild analgesics is a risk factor for development of male reproductive disorders in human and rat

International audienceBACKGROUND: More than half of pregnant women in the Western world report intake of mild analgesics, and some of these drugs have been associated with anti-androgenic effects in animal experiments. Intrauterine exposure to anti-androgens is suspected to contribute to the recent increase in male reproductive problems, and many of the anti-androgenic compounds are like the mild analgesics potent inhibitors of prostaglandin synthesis. Therefore, it appears imperative to further investigate the potential endocrine disrupting properties of mild analgesics. METHODS: In a prospective birth cohort study, 2297 Danish and Finnish pregnant women completed a questionnaire and 491 of the Danish mothers participated in a telephone interview, reporting on their use of mild analgesics during pregnancy. The testicular position of newborns was assessed by trained paediatricians. In rats, the impact of mild analgesics on anogenital distance (AGD) after intrauterine exposure was examined together with the effect on ex vivo gestational day 14.5 testes. RESULTS: In the Danish birth cohort, the use of mild analgesics was dose-dependently associated with congenital cryptorchidism. In particular, use during the second trimester increased the risk. This risk was further increased after the simultaneous use of different analgesics. The association was not found in the Finnish birth cohort. Intrauterine exposure of rats to paracetamol led to a reduction in the AGD and mild analgesics accordingly reduced testosterone production in ex vivo fetal rat testes. CONCLUSION: There was an association between the timing and the duration of mild analgesic use during pregnancy and the risk of cryptorchidism. These findings were supported by anti-androgenic effects in rat models leading to impaired masculinization. Our results suggest that intrauterine exposure to mild analgesics is a risk factor for development of male reproductive disorders

Mono-(2-ethylhexyl) phthalate directly alters the expression of Leydig cell genes and CYP17 lyase activity in cultured rat fetal testis.

International audienceExposure to phthalates in utero alters fetal rat testis gene expression and testosterone production, but much remains to be done to understand the mechanisms underlying the direct action of phthalate within the fetal testis. We aimed to investigate the direct mechanisms of action of mono-(2-ethylhexyl) phthalate (MEHP) on the rat fetal testis, focusing on Leydig cell steroidogenesis in particular. We used an in vitro system based on the culture for three days, with or without MEHP, of rat fetal testes obtained at 14.5 days post-coitum.Exposure to MEHP led to a dose-dependent decrease in testosterone production. Moreover, the production of 5 alpha-dihydrotestosterone (5α-DHT) (-68%) and androstenedione (-54%) was also inhibited by 10 µM MEHP, whereas 17 alpha-hydroxyprogesterone (17α-OHP) production was found to increase (+41%). Testosterone synthesis was rescued by the addition of androstenedione but not by any of the other precursors used. Thus, the hormone data suggested that steroidogenesis was blocked at the level of the 17,20 lyase activity of the P450c17 enzyme (CYP17), converting 17α-OHP to androstenedione. The subsequent gene expression and protein levels supported this hypothesis. In addition to Cyp17a1, microarray analysis showed that several other genes important for testes development were affected by MEHP. These genes included those encoding insulin-like factor 3 (INSL3), which is involved in controlling testicular descent, and Inha, which encodes the alpha subunit of inhibin B.These findings indicate that under in vitro conditions known to support normal differentiation of the fetal rat testis, the exposure to MEHP directly inhibits several important Leydig cell factors involved in testis function and that the Cyp17a1 gene is a specific target to MEHP explaining the MEHP-induced suppression of steroidogenesis observed

Parallel assessment of BPA and BPA-substitutes on the human Leydig cell function

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Paracetamol, aspirin, and indomethacin display endocrine disrupting properties in the adult human testis in vitro.

International audienceSTUDY QUESTION: Do mild analgesics affect the endocrine system of the human adult testis? SUMMARY ANSWER: Mild analgesics induce multiple endocrine disturbances in the human adult testis in vitro. WHAT IS KNOWN ALREADY: Mild analgesics have recently been incriminated as potential endocrine disruptors. Studies of the effects of these widely used molecules on the androgenic status of men are limited and somewhat contradictory. This prompted us to investigate whether these compounds could alter the adult human testicular function. We therefore assessed in parallel the effects of paracetamol, aspirin and indomethacin on organo-cultured adult human testis and on the NCI-H295R steroid-producing human cell line. STUDY DESIGN, SIZE, DURATION: Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with 10(-4) or 10(-5) M paracetamol, aspirin or indomethacin for 24-48 h. The effect of 10(-5) M ketoconazole, used as an anti-androgenic reference molecule, was also assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testes were obtained from prostate cancer patients, who had not received any hormone therapy. The protocol was approved by the local ethics committee of Rennes, France and informed consent was given by the donors. Only testes displaying spermatogenesis, as assessed by transillumination, were used in this study. Hormone levels in the culture media were determined by radioimmunoassay (testosterone, insulin-like factor 3), Enzyme-Linked Immunosorbent Assay (inhibin B) or Enzyme Immunosorbent Assay [prostaglandin (PG) D2, and PGE2]. Tissues were observed and cells counted using classical immunohistochemical methods. MAIN RESULTS AND THE ROLE OF CHANCE: The three mild analgesics caused multiple endocrine disturbances in the adult human testis. This was particularly apparent in the interstitial compartment. Effective doses were in the same range as those measured in blood plasma following standard analgesic treatment. The production of testosterone and insulin-like factor 3 by Leydig cells was altered by exposure to all these drugs. Inhibin B production by Sertoli cells was marginally affected by aspirin only. Our experiments also revealed that mild analgesics display direct anti-PG activity, which varied depending on the drug used, the dose and the duration of exposure. Nevertheless, associations between the alteration of the PG and testosterone profiles were not systematically observed, suggesting that a combination of mechanisms of endocrine disruption is at play. LIMITATIONS, REASONS FOR CAUTION: Our studies were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS: We provide the first evidence that direct exposure to mild analgesics can result in multiple endocrine disturbances in the human adult testis. Caution, concerning the consumption of mild analgesics by men, should be strengthened, particularly in high-risk population subgroups such as elite athletes

In utero and lactational exposure to low-doses of the pyrethroid insecticide cypermethrin leads to neurodevelopmental defects in male mice-An ethological and transcriptomic study.

Accumulating evidence suggests that developmental exposure to environmental chemicals may modify the course of brain development, ultimately leading to neuropsychiatric / neurodegenerative disorders later in life. In the present study, we assessed the impact of one of the most frequently used pesticides in both residential and agricultural applications - the synthetic pyrethroid cypermethrin (CYP) - on developmental neurotoxicity (DNT). Female mice were perinatally exposed to low doses of CYP (5 and 20 mg/kg body weight) from gestation to postnatal day 15. Behavioral analyses were performed during the offspring's early life and during adulthood. Postnatal analyses revealed that perinatal exposure to CYP disturbed motor development without modifying sensory and communicative skills. We found that later in life, CYP-exposed offspring expressed maladaptive behaviors in response to highly challenging tasks and abnormal sociability. Transcriptomic analyses performed in the offspring's brain at the end of the exposure, highlighted mitochondrial dysfunction as a relevant pathomechanism underlying CYP-induced DNT. Interestingly, several genes involved in proteostasis maintenance were also shown to be dysregulated suggesting that alterations in biogenesis, folding, trafficking and degradation of proteins may significantly contribute to CYP-related DNT. From a regulatory perspective, this study highlights that behavioral and transcriptomic analyses are complementary tools providing useful direction for better DNT characterization, and as such, should be used together more systematically