Adverse effects of endocrine disruptors on the foetal testis development: focus on the phthalates.

There are great concerns about the increasing incidence of abnormalities in male reproductive function. Human sperm counts have markedly dropped and the rate of testicular cancer has clearly augmented over the past four decades. Moreover, the prevalence rates of cryptorchidism and hypospadias are also probably increasing. It has been hypothesized that all these adverse trends in male reproduction result from abnormalities in the development of the testis during foetal and neonatal life. Furthermore, many recent epidemiological, clinical and experimental data suggest that these male reproductive disorders could be due to the effects of xenobiotics termed endocrine disruptors, which are becoming more and more concentrated and prevalent in our environment. Among these endocrine disruptors, we chose to focus this review on the phthalates for different reasons: 1) they are widespread in the environment; 2) their concentrations in many human biological fluids have been measured; 3) the experimental data using rodent models suggesting a reprotoxicity are numerous and are the most convincing; 4) their deleterious effects on the in vivo and in vitro development and function of the rat foetal testis have been largely studied; 5) some epidemiological data in humans suggest a reprotoxic effect at environmental concentrations at least during neonatal life. However, the direct effects of phthalates on human foetal testis have never been explored. Thus, as we did for the rat in the 1990s, we recently developed and validated an organ culture system which allows maintenance of the development of the different cell types of human foetal testis. In this system, addition of 10-4 M MEHP (mono-2-ethylhexyl phthalate), the most produced phthalate, had no effect on basal or LH-stimulated production of testosterone, but it reduced the number of germ cells by increasing their apoptosis, without modification of their proliferation. This is the first experimental demonstration that phthalates alter the development of the foetal testis in humans. Using our organotypic culture system, we and others are currently investigating the effect of MEHP in the mouse and the rat, and it will be interesting to compare the results between these species to analyse the relevance of toxicological tests based on rodent models

Ontogenesis of testicular function in humans.

The two major functions of the testis, steroidogenesis and gametogenesis, take place during fetal life. These two functions have been extensively studied in rodents and adult humans. However, their onset during fetal life is poorly documented in humans. In the first part of this work we presented both our experimental data and some data of literature concerning the development of the human fetal testis. In the second part of this article, using the organ culture system we previously developed, we have investigated the regulations or perturbations of fetal testis development both in rodent and human models. Our findings provide important insight into the potential role of exposure to environmental pollutants (physical factors, in particular ionizing radiation, cadmium and endocrine disruptors such as phthalates) during fetal testicular development and their potential deleterious effects on male fertility in adulthood. Our results highlight the specificity of the human model compared with rodent models

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC

BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients

Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors.

International audienceFetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk

Polluants environnementaux et développement du testicule foetal humain : effets et mécanismes des phtalates

Since the last decades, an increase in several abnormalities of the male reproductive function has been progressively evidenced in industrialized countries. According to these observations, it was hypothesized that exposure to some environmental pollutants may impair the fetal testis development, and therefore be at the origins of those abnormalities. Among incriminated compounds, phthalates are molecules highly produced worldwide. These compound are classified as reprotoxic molecules, as they disrupt the development of the germ cell lineage in different species and lead to a decrease in testosterone production in rat. Nevertheless, very few data are available concerning their effects in human. In this study we analyzed the effects of one phthalate, the MEHP, on the human fetal testis development during the first trimester of pregnancy. It was performed using an organotypic culture system that allows the preservation of the different testis structures. We first demonstrated that MEHP (10-4M) does not affect testosterone production of the human fetal testis, in opposition to the results described in rat. We also have demonstrated that MEHP exposure triggers apoptosis in the fetal germ cells, leading to a quick decrease in the total number of these cells. Following those results, we tested the effects of lower doses of MEHP that are close to the highest doses measured in human biological fluids. We therefore demonstrated that fetal germ cells are altered by exposure to this dose of MEHP (10-5M). Finally, in the third part of this work, we focused on the mechanisms of action of phthalate toxicity. Different studies, mostly in the liver, report the involvement of the nuclear receptor superfamilly in the effect of those compounds. Thus, it seemed important to investigate their implication in the effect of phthalates on the human fetal testis. We demonstrated that LXRα is certainly implicated in these effects as its transcriptional level is increased. Moreover, this nuclear receptor regulates two metabolic pathways: Cholesterol and fatty acid synthesis pathways, that seemed to by both modulated by phthalate exposure in the human fetal testis. We also showed that the modulation of these two metabolic pathways is a common process to both the male and female gonads. Nevertheless, the germ cell decrease we evidenced in the human fetal testis was never observable in the fetal ovary. In conclusion, this work contributed to improve our knowledge about the effects of phthalate exposure on the establishment and the development of the human fetal reproductive system. We also have evidenced a new mechanism of these compounds that involves members of the nuclear receptors superfamilly, as well as cholesterol and fatty acid synthesis.Au cours des dernières décennies, nous avons progressivement vu augmenter un certain nombre d’anomalies de la fonction de reproduction masculine dans les pays industrialisés. Ces constatations ont fait émerger l’hypothèse selon laquelle certains polluants de notre environnement pourraient altérer le développement du testicule fœtal et ainsi être responsables de ces anomalies. Parmi les composants incriminés se trouvent les phtalates, largement répandus dans l’environnement. Ces composés ont été décrits comme reprotoxiques, ils altèrent le développement de la lignée germinale dans différentes espèces et entraînent une diminution de la production de testostérone chez le rat. Toutefois, très peu de données sont disponibles quant à leurs effets chez l’Homme. Dans cette étude, nous avons analysé les effets d’un phtalate, le MEHP, sur le développement du testicule fœtal humain au premier trimestre de la grossesse, dans un modèle de culture organotypique qui permet le maintien des différentes structures de l’organe. Nous avons tout d’abord démontré que le MEHP (10-4M) n’altère pas la production de testostérone du testicule fœtal humain, contrairement aux résultats décrits chez le rat. En revanche, nous avons montré que l’exposition au MEHP entraîne une rapide diminution du nombre de cellules germinales par apoptose. A la suite de ces résultats, nous avons testé l’effet de doses plus faibles de MEHP afin de se placer à des concentrations de phtalates ayant été mesurées dans les liquides biologiques. Nous avons ainsi démontré que les cellules germinales du testicule fœtal humain sont altérées suite à l’exposition à des doses de MEHP de 10-5M. Enfin, dans la 3ème partie de ce travail, nous nous sommes intéressés aux mécanismes d’action des phtalates. Différentes études, notamment dans le foie, démontrent l’implication des récepteurs nucléaires dans les effets de ces composés. Il nous a donc semblé important de rechercher leur implication dans les effets des phtalates sur le testicule fœtal. Nous avons démontré que LXRα est très certainement impliqué ces effets puisque l’expression des ARNm de ce récepteur est augmentée. Par ailleurs, ce récepteur nucléaire contrôle deux voies métaboliques, la synthèse de cholestérol et la synthèse des acides gras qui semblent toutes deux modulées par les phtalates dans le testicule fœtal humain. Enfin, nous avons montré que l’implication de ces voies métaboliques est commune entre la gonade mâle et la gonade femelle, sans pour autant que l’effet sur les cellules germinales mâles ai pu être mis en évidence dans l’ovaire fœtal. En conclusion, cette étude a contribué à caractériser les effets des phtalates sur la mise en place des fonctions de reproduction chez le fœtus humain. Nous avons également pu mettre en évidence un nouveau mécanisme de ces composés, impliquant la superfamille des récepteurs nucléaires ainsi que la synthèse du cholestérol et des acides gras

Environmental pollutants and human fetal testis development : phthalates effects and mechanisms of action

Au cours des dernières décennies, nous avons progressivement vu augmenter un certain nombre d’anomalies de la fonction de reproduction masculine dans les pays industrialisés. Ces constatations ont fait émerger l’hypothèse selon laquelle certains polluants de notre environnement pourraient altérer le développement du testicule fœtal et ainsi être responsables de ces anomalies. Parmi les composants incriminés se trouvent les phtalates, largement répandus dans l’environnement. Ces composés ont été décrits comme reprotoxiques, ils altèrent le développement de la lignée germinale dans différentes espèces et entraînent une diminution de la production de testostérone chez le rat. Toutefois, très peu de données sont disponibles quant à leurs effets chez l’Homme. Dans cette étude, nous avons analysé les effets d’un phtalate, le MEHP, sur le développement du testicule fœtal humain au premier trimestre de la grossesse, dans un modèle de culture organotypique qui permet le maintien des différentes structures de l’organe. Nous avons tout d’abord démontré que le MEHP (10-4M) n’altère pas la production de testostérone du testicule fœtal humain, contrairement aux résultats décrits chez le rat. En revanche, nous avons montré que l’exposition au MEHP entraîne une rapide diminution du nombre de cellules germinales par apoptose. A la suite de ces résultats, nous avons testé l’effet de doses plus faibles de MEHP afin de se placer à des concentrations de phtalates ayant été mesurées dans les liquides biologiques. Nous avons ainsi démontré que les cellules germinales du testicule fœtal humain sont altérées suite à l’exposition à des doses de MEHP de 10-5M. Enfin, dans la 3ème partie de ce travail, nous nous sommes intéressés aux mécanismes d’action des phtalates. Différentes études, notamment dans le foie, démontrent l’implication des récepteurs nucléaires dans les effets de ces composés. Il nous a donc semblé important de rechercher leur implication dans les effets des phtalates sur le testicule fœtal. Nous avons démontré que LXRα est très certainement impliqué ces effets puisque l’expression des ARNm de ce récepteur est augmentée. Par ailleurs, ce récepteur nucléaire contrôle deux voies métaboliques, la synthèse de cholestérol et la synthèse des acides gras qui semblent toutes deux modulées par les phtalates dans le testicule fœtal humain. Enfin, nous avons montré que l’implication de ces voies métaboliques est commune entre la gonade mâle et la gonade femelle, sans pour autant que l’effet sur les cellules germinales mâles ai pu être mis en évidence dans l’ovaire fœtal. En conclusion, cette étude a contribué à caractériser les effets des phtalates sur la mise en place des fonctions de reproduction chez le fœtus humain. Nous avons également pu mettre en évidence un nouveau mécanisme de ces composés, impliquant la superfamille des récepteurs nucléaires ainsi que la synthèse du cholestérol et des acides gras.Since the last decades, an increase in several abnormalities of the male reproductive function has been progressively evidenced in industrialized countries. According to these observations, it was hypothesized that exposure to some environmental pollutants may impair the fetal testis development, and therefore be at the origins of those abnormalities. Among incriminated compounds, phthalates are molecules highly produced worldwide. These compound are classified as reprotoxic molecules, as they disrupt the development of the germ cell lineage in different species and lead to a decrease in testosterone production in rat. Nevertheless, very few data are available concerning their effects in human. In this study we analyzed the effects of one phthalate, the MEHP, on the human fetal testis development during the first trimester of pregnancy. It was performed using an organotypic culture system that allows the preservation of the different testis structures. We first demonstrated that MEHP (10-4M) does not affect testosterone production of the human fetal testis, in opposition to the results described in rat. We also have demonstrated that MEHP exposure triggers apoptosis in the fetal germ cells, leading to a quick decrease in the total number of these cells. Following those results, we tested the effects of lower doses of MEHP that are close to the highest doses measured in human biological fluids. We therefore demonstrated that fetal germ cells are altered by exposure to this dose of MEHP (10-5M). Finally, in the third part of this work, we focused on the mechanisms of action of phthalate toxicity. Different studies, mostly in the liver, report the involvement of the nuclear receptor superfamilly in the effect of those compounds. Thus, it seemed important to investigate their implication in the effect of phthalates on the human fetal testis. We demonstrated that LXRα is certainly implicated in these effects as its transcriptional level is increased. Moreover, this nuclear receptor regulates two metabolic pathways: Cholesterol and fatty acid synthesis pathways, that seemed to by both modulated by phthalate exposure in the human fetal testis. We also showed that the modulation of these two metabolic pathways is a common process to both the male and female gonads. Nevertheless, the germ cell decrease we evidenced in the human fetal testis was never observable in the fetal ovary. In conclusion, this work contributed to improve our knowledge about the effects of phthalate exposure on the establishment and the development of the human fetal reproductive system. We also have evidenced a new mechanism of these compounds that involves members of the nuclear receptors superfamilly, as well as cholesterol and fatty acid synthesis

Polluants environnementaux et développement du testicule foetal humain (effets et mécanismes des phtalates.)

Au cours des dernières décennies, nous avons progressivement vu augmenter un certain nombre d anomalies de la fonction de reproduction masculine dans les pays industrialisés. Ces constatations ont fait émerger l hypothèse selon laquelle certains polluants de notre environnement pourraient altérer le développement du testicule fœtal et ainsi être responsables de ces anomalies. Parmi les composants incriminés se trouvent les phtalates, largement répandus dans l environnement. Ces composés ont été décrits comme reprotoxiques, ils altèrent le développement de la lignée germinale dans différentes espèces et entraînent une diminution de la production de testostérone chez le rat. Toutefois, très peu de données sont disponibles quant à leurs effets chez l Homme. Dans cette étude, nous avons analysé les effets d un phtalate, le MEHP, sur le développement du testicule fœtal humain au premier trimestre de la grossesse, dans un modèle de culture organotypique qui permet le maintien des différentes structures de l organe. Nous avons tout d abord démontré que le MEHP (10-4M) n altère pas la production de testostérone du testicule fœtal humain, contrairement aux résultats décrits chez le rat. En revanche, nous avons montré que l exposition au MEHP entraîne une rapide diminution du nombre de cellules germinales par apoptose. A la suite de ces résultats, nous avons testé l effet de doses plus faibles de MEHP afin de se placer à des concentrations de phtalates ayant été mesurées dans les liquides biologiques. Nous avons ainsi démontré que les cellules germinales du testicule fœtal humain sont altérées suite à l exposition à des doses de MEHP de 10-5M. Enfin, dans la 3ème partie de ce travail, nous nous sommes intéressés aux mécanismes d action des phtalates. Différentes études, notamment dans le foie, démontrent l implication des récepteurs nucléaires dans les effets de ces composés. Il nous a donc semblé important de rechercher leur implication dans les effets des phtalates sur le testicule fœtal. Nous avons démontré que LXRa est très certainement impliqué ces effets puisque l expression des ARNm de ce récepteur est augmentée. Par ailleurs, ce récepteur nucléaire contrôle deux voies métaboliques, la synthèse de cholestérol et la synthèse des acides gras qui semblent toutes deux modulées par les phtalates dans le testicule fœtal humain. Enfin, nous avons montré que l implication de ces voies métaboliques est commune entre la gonade mâle et la gonade femelle, sans pour autant que l effet sur les cellules germinales mâles ai pu être mis en évidence dans l ovaire fœtal. En conclusion, cette étude a contribué à caractériser les effets des phtalates sur la mise en place des fonctions de reproduction chez le fœtus humain. Nous avons également pu mettre en évidence un nouveau mécanisme de ces composés, impliquant la superfamille des récepteurs nucléaires ainsi que la synthèse du cholestérol et des acides gras.Since the last decades, an increase in several abnormalities of the male reproductive function has been progressively evidenced in industrialized countries. According to these observations, it was hypothesized that exposure to some environmental pollutants may impair the fetal testis development, and therefore be at the origins of those abnormalities. Among incriminated compounds, phthalates are molecules highly produced worldwide. These compound are classified as reprotoxic molecules, as they disrupt the development of the germ cell lineage in different species and lead to a decrease in testosterone production in rat. Nevertheless, very few data are available concerning their effects in human. In this study we analyzed the effects of one phthalate, the MEHP, on the human fetal testis development during the first trimester of pregnancy. It was performed using an organotypic culture system that allows the preservation of the different testis structures. We first demonstrated that MEHP (10-4M) does not affect testosterone production of the human fetal testis, in opposition to the results described in rat. We also have demonstrated that MEHP exposure triggers apoptosis in the fetal germ cells, leading to a quick decrease in the total number of these cells. Following those results, we tested the effects of lower doses of MEHP that are close to the highest doses measured in human biological fluids. We therefore demonstrated that fetal germ cells are altered by exposure to this dose of MEHP (10-5M). Finally, in the third part of this work, we focused on the mechanisms of action of phthalate toxicity. Different studies, mostly in the liver, report the involvement of the nuclear receptor superfamilly in the effect of those compounds. Thus, it seemed important to investigate their implication in the effect of phthalates on the human fetal testis. We demonstrated that LXRa is certainly implicated in these effects as its transcriptional level is increased. Moreover, this nuclear receptor regulates two metabolic pathways: Cholesterol and fatty acid synthesis pathways, that seemed to by both modulated by phthalate exposure in the human fetal testis. We also showed that the modulation of these two metabolic pathways is a common process to both the male and female gonads. Nevertheless, the germ cell decrease we evidenced in the human fetal testis was never observable in the fetal ovary. In conclusion, this work contributed to improve our knowledge about the effects of phthalate exposure on the establishment and the development of the human fetal reproductive system. We also have evidenced a new mechanism of these compounds that involves members of the nuclear receptors superfamilly, as well as cholesterol and fatty acid synthesis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF