Innovations Thérapeutiques et Résistances

Mutations in SMARCAL1 (HARP) cause Schimke immunoosseous dysplasia (SIOD). The mechanistic basis for this disease is unknown. Using functional genomic screens, we identified SMARCAL1 as a genome maintenance protein. Silencing and overexpression of SMARCAL1 leads to activation of the DNA damage response during S phase in the absence of any genotoxic agent. SMARCAL1 contains a Replication protein A (RPA)-binding motif similar to that found in the replication stress response protein TIPIN (Timeless-Interacting Protein), which is both necessary and sufficient to target SMARCAL1 to stalled replication forks. RPA binding is critical for the cellular function of SMARCAL1; however, it is not necessary for the annealing helicase activity of SMARCAL1 in vitro. An SIOD-associated SMARCAL1 mutant fails to prevent replication-associated DNA damage from accumulating in cells in which endogenous SMARCAL1 is silenced. Ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) phosphorylate SMARCAL1 in response to replication stress. Loss of SMARCAL1 activity causes increased RPA loading onto chromatin and persistent RPA phosphorylation after a transient exposure to replication stress. Furthermore, SMARCAL1-deficient cells are hypersensitive to replication stress agents. Thus, SMARCAL1 is a replication stress response protein, and the pleiotropic phenotypes of SIOD are at least partly due to defects in genome maintenance during DNA replication.

Antimicrobial resistance (AMR) is a major global public health concern, shared by a large number of human and animal health actors. Within the framework of a One Health approach, actions should be implemented in the environmental realm, as well as the human and animal realms. The Government of France commissioned a report to provide policy and decision makers with an evidential basis for recommending or taking future actions to mitigate AMR in the environment. We first examined the mechanisms that underlie the emergence and persistence of antimicrobial resistance in the environment. This report drew up an inventory of the contamination of aquatic and terrestrial environments by AMR and antibiotics, anticipating that the findings will be representative of some other high-income countries. Effluents of wastewater treatment plants were identified as the major source of contamination on French territory, with spreading of organic waste products as a more diffuse and incidental contamination of aquatic environments. A limitation of this review is the heterogeneity of available data in space and time, as well as the lack of data for certain sources. Comparing the French Measured Environmental Concentrations (MECs) with predicted no effect concentrations (PNECs), fluoroquinolones and trimethoprim were identified as representing high and medium risk of favoring the selection of resistant bacteria in treated wastewater and in the most contaminated rivers. All other antibiotic molecules analyzed (erythromycin, clarithromycin, azithromycin, tetracycline) were at low risk of resistance selection in those environments. However, the heterogeneity of the data available impairs their full exploitation. Consequently, we listed indicators to survey AMR and antibiotics in the environment and recommended the harmonization of sampling strategies and endpoints for analyses. Finally, the objectives and methods used for the present work could comprise a useful example for how national authorities of countries sharing common socio-geographic characteristics with France could seek to better understand and define the environmental dimension of AMR in their particular settings.

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

Abstract Background Titanium dioxide (TiO 2 ) is broadly used in common consumer goods, including as a food additive (E171 in Europe) for colouring and opacifying properties. The E171 additive contains TiO 2 nanoparticles (NPs), part of them being absorbed in the intestine and accumulated in several systemic organs. Exposure to TiO 2 -NPs in rodents during pregnancy resulted in alteration of placental functions and a materno-foetal transfer of NPs, both with toxic effects on the foetus. However, no human data are available for pregnant women exposed to food-grade TiO 2 -NPs and their potential transfer to the foetus. In this study, human placentae collected at term from normal pregnancies and meconium (the first stool of newborns) from unpaired mothers/children were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopy (STEM) coupled to energy-dispersive X-ray (EDX) spectroscopy for their titanium (Ti) contents and for analysis of TiO 2 particle deposition, respectively. Using an ex vivo placenta perfusion model, we also assessed the transplacental passage of food-grade TiO 2 particles. Results By ICP-MS analysis, we evidenced the presence of Ti in all placentae (basal level ranging from 0.01 to 0.48 mg/kg of tissue) and in 50% of the meconium samples (0.02–1.50 mg/kg), suggesting a materno-foetal passage of Ti. STEM-EDX observation of the placental tissues confirmed the presence of TiO 2 -NPs in addition to iron (Fe), tin (Sn), aluminium (Al) and silicon (Si) as mixed or isolated particle deposits. TiO 2 particles, as well as Si, Al, Fe and zinc (Zn) particles were also recovered in the meconium. In placenta perfusion experiments, confocal imaging and SEM-EDX analysis of foetal exudate confirmed a low transfer of food-grade TiO 2 particles to the foetal side, which was barely quantifiable by ICP-MS. Diameter measurements showed that 70 to 100% of the TiO 2 particles recovered in the foetal exudate were nanosized. Conclusions Altogether, these results show a materno-foetal transfer of TiO 2 particles during pregnancy, with food-grade TiO 2 as a potential source for foetal exposure to NPs. These data emphasize the need for risk assessment of chronic exposure to TiO 2 -NPs during pregnancy.

The measurement of bisphenol-S (BPS) and its glucurono-conjugate (BPSG) in urine may be used for the biomonitoring of exposure in populations. However, this requires a thorough knowledge of their toxicokinetics. The time courses of BPS and BPSG were assessed in accessible biological matrices of orally and dermally exposed volunteers. Under the approval of the Research Ethics Committee of the University of Montreal, six volunteers were orally exposed to a BPS-d8 deuterated dose of 0.1 mg/kg body weight (bw). One month later, 1 mg/kg bw of BPS-d8 were applied on 40 cm2 of the forearm and then washed 6 h after application. Blood samples were taken prior to dosing and at fixed time periods over 48 h after treatment; complete urine voids were collected pre-exposure and at pre-established intervals over 72 h postdosing. Following oral exposure, the plasma concentration–time courses of BPS-d8 and BPSG-d8 over 48 h evolved in parallel, and showed a rapid appearance and elimination. Average peak values (±SD) were reached at 0.7 ± 0.1 and 1.1 ± 0.4 h postdosing and mean (±SD) apparent elimination half-lives (t½) of 7.9 ± 1.1 and 9.3 ± 7.0 h were calculated from the terminal phase of BPS-d8 and BPSG-d8 in plasma, respectively. The fraction of BPS-d8 reaching the systemic circulation unchanged (i.e. bioavailability) was further estimated at 62 ± 5% on average (±SD) and the systemic plasma clearance at 0.57 ± 0.07 L/kg bw/h. Plasma concentration–time courses and urinary excretion rate profiles roughly evolved in parallel for both substances, as expected. The average percent (±SD) of the administered dose recovered in urine as BPS-d8 and BPSG-d8 over the 0–72 h period postdosing was 1.72 ± 1.3 and 54 ± 10%. Following dermal application, plasma levels were under the lower limit of quantification (LLOQ) at most time points. However, peak values were reached between 5 and 8 h depending on individuals, suggesting a slower absorption rate compared to oral exposure. Similarly, limited amounts of BPS-d8 and its conjugate were recovered in urine and peak excretion rates were reached between 5 and 11 h postdosing. The average percent (±SD) of the administered dose recovered in urine as BPS-d8 and BPSG-d8 was about 0.004 ± 0.003 and 0.09 ± 0.07%, respectively. This study provided greater precision on the kinetics of this contaminant in humans and, in particular, evidenced major differences between BPA and BPS kinetics with much higher systemic levels of active BPS than BPA, an observation explained by a higher oral bioavailability of BPS than BPA. These data should also be useful in developing a toxicokinetic model for a better interpretation of biomonitoring data.

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity.

Resistance to the anthelmintic macrocyclic lactone ivermectin (IVM) has a great impact on the control of parasitic nematodes. The mechanisms by which nematodes adapt to IVM remain to be deciphered. We have identified NHR-8, a nuclear hormone receptor involved in the xenobiotic response in Caenorhabditis elegans, as a new regulator of tolerance to IVM. Loss-of-function nhr-8(ok186) C. elegans mutants subjected to larval development assays and electropharyngeogram measurements, displayed hypersensitivity to IVM, and silencing of nhr-8 in IVM-resistant worms increased IVM efficacy. In addition, compared to wild-type worms, nhr-8 mutants under IVM selection pressure failed to acquire tolerance to the drug. In addition, IVM-hypersensitive nhr-8(ok186) worms displayed low transcript levels of several genes from the xenobiotic detoxification network and a concomitant low Pgp-mediated drug efflux activity. Interestingly, some pgp and cyp genes known to impact IVM tolerance in many nematode species, were down regulated in nhr-8 mutants and inversely upregulated in IVM-resistant worms. Moreover, pgp-6 overexpression in nhr-8(ok186) C. elegans increased tolerance to IVM. Importantly, NHR-8 function was rescued in nhr-8(ok186) C. elegans with the homolog of the parasitic nematode Haemonchus contortus, and silencing of Hco-nhr-8 by RNAi on L2 H. contortus larvae increased IVM susceptibility in both susceptible and resistant H. contortus isolates. Thus, our data show that NHR-8 controls the tolerance and development of resistance to IVM in C. elegans and the molecular basis for this relates to the NHR-8-mediated upregulation of IVM detoxification genes. Since our results show that Hco-nhr-8 functions similarly to Cel-nhr-8, this study helps to better understand mechanisms underlying failure in drug efficacy and open perspectives in finding new compounds with NHR-8 antagonist activity to potentiate IVM efficacy.

Bisphenol S (BPS) is widely used as a substitute for Bisphenol A in consumer products. Despite its potential endocrine-disrupting effects and widespread exposure, toxicokinetic data, particularly during the critical period of pregnancy, are not available for BPS. The objectives of our study were to evaluate the mechanisms determining fetal exposure to BPS and to BPS glucuronide (BPSG) and to compare them with those prevailing for BPA. The disposition of BPS and BPSG was evaluated in the materno-fetal unit of the catheterized pregnant ewe model, following intravenous administrations of BPS and BPSG to mothers and their fetuses. In a second experiment, the rate of BPS accumulation in the fetal compartment was determined under steady-state conditions after repeated intravenous BPS administrations to the mother. In the maternal compartment, BPS was mainly metabolized into BPSG and totally eliminated in urine. Only 0.40% of the maternal dose was transferred to the fetus. However, once in the fetal compartment, 26% of the fetal dose was rapidly eliminated through placental transfer, while 46% of BPS was metabolized into BPSG which remained trapped in the fetal compartment. Thus, the elimination of BPSG from the fetal compartment required its back-conversion into bioactive BPS, leading to an 87% enhancement of the fetal BPS exposure. Our findings demonstrate that, despite the low materno-fetal placental transfer of BPS, this substitute for BPA is able to accumulate in the fetal compartment after repeated maternal exposure, leading to chronic fetal exposure to BPS in a range of concentrations similar to those of BPA.

Quantitative data on fecal shedding of antimicrobial-resistant bacteria are crucial to assess the risk of transmission from dogs to humans. Our first objective was to investigate the prevalence of quinolone/fluoroquinolone-resistant and beta-lactam-resistant Enterobacteriaceae in dogs in France and Spain. Due to the particular concern about possible transmission of extended-spectrum cephalosporin-resistant isolates from dogs to their owners, we characterized the ESBL/AmpC producers collected from dogs. Rectal swabs from 188 dogs, without signs of diarrhea and that had not received antimicrobials for 4 weeks before the study, were quantified for total and resistant Enterobacteriaceae on selective media alone or containing relevant antibiotic concentrations. Information that might explain antimicrobial resistance was collected for each dog. Extended-spectrum cephalosporin-resistant isolates were subjected to bacterial species identification (API20E), genetic lineage characterization (MLST), ESBL/pAmpC genes identification (sequencing) and plasmid characterization (pMLST). Regarding beta-lactam resistance, amoxicillin- (AMX) and cefotaxime- (CTX) resistant Enterobacteriaceae were detected in 70% and 18% of the dogs respectively, whereas for quinolone/fluoroquinolone-resistance, Nalidixic acid- (NAL) and ciprofloxacin- (CIP) resistant Enterobacteriacea were detected in 36% and 18% of the dogs, respectively. Medical rather than preventive consultation was a risk marker for the presence of NAL and CIP resistance. CTX resistance was mainly due to a combination of specific ESBL/pAmpC genes and particular conjugative plasmids already identified in human patients: blaCTX-M-1/IncI1/ST3 (n = 4), blaCMY-2/IncI1/ST12 (n=2) and blaCTX-M-15/IncI1/ST31 (n=1). blaSHV-12 (n=3) was detected in various plasmid lineages (InI1/ST3, IncI1/ST26, and IncFII). ESBL/AmpC plasmids were located in different genetic lineages of E. coli, with the exception of 2 strains in France (ST6998) and 2 in Spain (ST602). Our study highlights dogs as a potential source of Q/FQ-resistant and ESBL/pAmpC-producing bacteria that might further disseminate to humans, and notably a serious risk of future acquisition of CTX-M-1 and CMY-2 plasmids by the owners of dogs.

was licensed, with the objective of avoiding stability deficiencies of the old formulation. Before launching this new formulation, an average bioequivalence trial, based on European Union recommended guidelines, was performed. The implicit rationale was the assumption that the two products, being bioequivalent, would also be switchable, allowing substitution of the new for the old formulation, thus avoiding the need for individual calibration of the dosage regimen of thyroxine, using the thyroid-stimulating hormone level as the endpoint, as required for a new patient on initiating treatment. Despite the fact that both formulations were shown to be bioequivalent, adverse drug reactions were reported in several thousands of patients after taking the new formulation. In this opinion paper, we report that more than 50% of healthy volunteers enrolled in a successful regulatory average bioequivalence trial were actually outside the a priori bioequivalence range. Therefore, we question the ability of an average bioequivalence trial to guarantee the switchability within patients of the new and old levothyroxine formulations. We further propose an analysis of this problem using the conceptual framework of individual bioequivalence. This involves investigating the bioavailability of the two formulations within a subject, by comparing not only the population means (as established by average bioequivalence) but also by assessing two variance terms, namely the within-subject variance and the variance estimating subject-by-formulation interaction. A higher within individual variability for the new formulation would lead to reconsideration of the appropriateness of the new formulation. Alternatively, a possible subject-by-formulation interaction would allow a judgement on the ability, or not, of doctors to manage patients effectively during transition from the old to the new formulation.

Abstract In S. aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) and are highly tolerant to antimicrobial drugs. We thus sought to identify non-antibiotic substances with broad-spectrum activity able to destroy the EPS matrix and enhance the effect of antibiotics on embedded biofilm bacteria. Among eight substances tested, subtilisin A (0.01 U/mL) and calcium gluconate (CaG, Ca 2+ 1.25 mmol/L) significantly reduced the biomass of biofilms formed by at least 21/24 S. aureus isolates. Confocal laser scanning microscopy confirmed that they both eliminated nearly all the proteins and PNAG from the matrix. By contrast, antibiotics alone had nearly no effect on biofilm biomass and the selected one (oxytetracycline-OTC) could only slightly reduce biofilm bacteria. The combination of OTC with CaG or subtilisin A led to an additive reduction (average of 2 log 10 CFU/mL) of embedded biofilm bacteria on the isolates susceptible to OTC (MBC < 10 μg/mL, 11/24). Moreover, these two combinations led to a reduction of the embedded biofilm bacteria higher than 3 log 10 CFU/mL for 20–25% of the isolates. Further studies are now required to better understand the factors that cause the biofilm produced by specific isolates (20–25%) to be susceptible to the combinations.

Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding fourteen adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment.

SMARCAL1 is an ATPase in the SNF2 family that functions at damaged replication forks to promote their stability and restart. It acts by translocating on DNA to catalyze DNA strand annealing, branch migration, and fork regression. Many SNF2 enzymes work as motor subunits of large protein complexes. To determine if SMARCAL1 is also a member of a protein complex and to further understand how it functions in the replication stress response, we used a proteomics approach to identify interacting proteins. In addition to the previously characterized interaction with replication protein A (RPA), we found that SMARCAL1 forms complexes with several additional proteins including DNA-PKcs and the WRN helicase. SMARCAL1 and WRN co-localize at stalled replication forks independently of one another. The SMARCAL1 interaction with WRN is indirect and is mediated by RPA acting as a scaffold. SMARCAL1 and WRN act independently to prevent MUS81 cleavage of the stalled fork. Biochemical experiments indicate that both catalyze fork regression with SMARCAL1 acting more efficiently and independently of WRN. These data suggest that RPA brings a complex of SMARCAL1 and WRN to stalled forks, but that they may act in different pathways to promote fork repair and restart.

The PK/PD cut-off (PK/PDCO ) value of florfenicol for calf pathogens was determined for long acting formulations (MSD Nuflor® and a bioequivalent generic product). PK/PDCO is one of the three MICs considered by VetCAST, a sub-committee of the European Committee on Susceptibility Testing, to establish a Clinical Breakpoint for interpreting Antimicrobial Susceptibility Testing. A population model was built by pooling three pharmacokinetic data sets, obtained from 50 richly sampled calves, receiving one of two formulations (the pioneer product and a generic formulation). A virtual population of 5000 florfenicol disposition curves was generated by Monte Carlo Simulations over the 96 h of the assumed duration of action of the formulations. From this population, the maximum predicted MIC, for which 90% of calves can achieve some a priori selected critical value for two PK/PD indices, AUC/MIC and T>MIC, was established. Numerical values were established for two bacterial species of the bovine respiratory disease complex, Pasteurella multocida and Mannheimia haemolytica. It was concluded that the PK/PDCO of florfenicol for both AUC/MIC and T>MIC was 1 mg/L.

Due to restriction of the use of BPA, several structural analogues such as BPS and BPF have been proposed for its replacement in many consumer products. This has increased the prevalence of BPS and BPF in urine from tested cohorts. However, these substitutes have similar endocrine disrupting properties to BPA, particularly on reproductive and metabolic functions, which suggests that fetal exposure to these analogues could be of concern for human health. Bisphenols (BPs) are mainly metabolized to glucuronides (BP-Gs), which are considered as inactive but provide a relevant marker of fetal exposure during pregnancy. In most instances, these metabolites are indirectly quantified after hydrolysis and measurement of the corresponding native BPs, which may lead to bias due to spurious BPs contamination during blood collection and/or analyses. We have developed a new method for direct quantification of BP-Gs, which has the advantage of not being affected by errors related to the presence of BPs. First, BP-Gs were extracted from plasma by anion exchange solid phase extraction. They were then labelled with dansyl chloride, using experimentally-optimized incubation conditions, after which the dansyl derivatives were injected into an on-line SPE-UHPLC/MS/MS system. The performance of the method, in terms of sensitivity, precision and accuracy, was evaluated in plasma over a concentration range of 0.05-5 ng/mL. The intra- and inter-day CV% precision were lower than 20% with accuracies ranging from 93% to 115%. The limit of quantification was set at 0.05 ng/mL. The method was then applied to measure BP-Gs in forty-four cord plasma samples. Although no BPF-G was found, BPA-G and BPS-G was determined in almost half of the cord plasma samples with concentration ranges nd-0.089 ng/mL and nd-0.586 ng/mL, respectively.

Combining currently available antibiotics to optimize their use is a promising strategy to reduce treatment failures against biofilm-associated infections. Nevertheless, most assays of such combinations have been performed in vitro on planktonic bacteria exposed to constant concentrations of antibiotics over only 24h and the synergistic effects obtained under these conditions do not necessarily predict the behavior of chronic clinical infections associated with biofilms. To improve the predictivity of in vitro combination assays for bacterial biofilms, we first adapted a previously-described Hollow-fiber (HF) infection model by allowing a Staphylococcus aureus biofilm to form before drug exposure. We then mimicked different concentration profiles of amikacin and vancomycin, similar to the free plasma concentration profiles that would be observed in patients treated daily over 5 days. We assessed the ability of the two drugs, alone or in combination, to reduce planktonic and biofilm-embedded bacterial populations, and to prevent the selection of resistance within these populations. Although neither amikacin nor vancomycin exhibited any bactericidal activity on S. aureus in monotherapy, the combination had a synergistic effect and significantly reduced the planktonic bacterial population by -3.0 to -6.0 log10 CFU/mL. In parallel, no obvious advantage of the combination, as compared to amikacin alone, was demonstrated on biofilm-embedded bacteria for which the addition of vancomycin to amikacin only conferred a further maximum reduction of 0.3 log10 CFU/mL. No resistance to vancomycin was ever found whereas a few bacteria less-susceptible to amikacin were systematically detected before treatment. These resistant bacteria, which were rapidly amplified by exposure to amikacin alone, could be maintained at a low level in the biofilm population and even suppressed in the planktonic population by adding vancomycin. In conclusion, by adapting the HF model, we were able to demonstrate the different bactericidal activities of the vancomycin and amikacin combination on planktonic and biofilm-embedded bacterial populations, suggesting that, for biofilm-associated infections, the efficacy of this combination would not be much greater than with amikacin monotherapy. However, adding vancomycin could reduce possible resistance to amikacin and provide a relevant strategy to prevent the selection of antibiotic-resistant bacteria during treatments.

Bisphenol (BP) structural analogues of BPA are widely used. Previous studies showed similar effects of BPA and BPS on reproduction in several species including human. We hypothesised that the similar effects of several bisphenols (BPs) could accumulate in granulosa cells (GCs) and affects steroidogenesis. This study investigated the effects of seven BP analogues and their equimolar cocktail on human granulosa cells (hGC) and assessed BPA, BPS, BPF and BPAF level exposures in the follicular fluid of 277 women undergoing Assisted Reproductive Technology. The hGCs were recovered after women oocyte punctures and treated with the seven BP analogues (BPS, BPA, BPAF, BPF, BPAP, BPE and BPB) or their equimolar cocktail of 7 × 1.43 or 7 × 7.14 μM for each of the seven BPs, the sum of BPs reaching 10 ("∑BPs 10 μM"), or 50 μM ("∑BPs 50 μM"), respectively. Oestradiol and progesterone secretion, cell proliferation, viability and expression of steroidogenic enzymes were investigated. Progesterone secretion was decreased by 6 BPs 10 μM and the cocktail "∑BPs 10 μM", (-17.8 to -41.3%) and by all seven BPs 50 μM and "∑BPs 50 μM" (-21.8 to -84.2%). Oestradiol secretion was decreased only by 50 μM BPAF and BPAP (-37.8% and -44%, respectively), with corresponding decreases in CYP17A1 and CYP19A1 gene expression. Cellular proliferation was decreased after treatment with 50 μM BPAF (-32.2%), BPAP (-29%), BPB (-24%) and the equimolar cocktail "∑BPs 50 μM" (-33.1%). BPB (50 μM) and the cocktail "∑BPs 50 μM" increased HSD3B2 mRNA expression. At least one BP was detected in 64 of 277 (23.1%) women follicular fluids. Similar effects of the seven BPs or their cocktail were observed on progesterone secretion and/or on cell proliferation, suggesting cumulative effects of BPs. Our results highlight the urge to consider all BPs simultaneously and to further investigate the potential additive or synergistic effects of several BPs.

Bisphenol A (BPA), an endocrine disruptor, has been replaced by structural analogues including bisphenol S (BPS). BPA and BPS exhibited similar effects regarding reproductive functions. Moreover, metabolic status and lipid metabolism are related to female fertility and could worsen BPS effects. The objective was to determine BPS in vivo effects on folliculogenesis and embryo production after chronic exposure through diet, and the influence of metabolic status in adult ewes. Sixty primiparous 2.5 year-old ewes, undergoing a restricted or well fed diet, were exposed to BPS (0, 4 or 50 µg/kg/day) for at least three months. After hormonal oestrus synchronisation and ovarian stimulation, ewes were subjected to ovum pick-up (OPU) procedures to collect immature oocytes, that underwent in vitro maturation, fertilisation and embryo production. Body weight, body condition score and plasma glucose were higher in well-fed compared to restricted ewes, while plasma NEFA was lower during the 4-5 months after the beginning of the diets. Plasma progesterone levels increased on day 5 before OPU session in well-fed compared to restricted ewes. No effect of BPS dose was observed on follicle population, plasma AMH levels and embryo production numbers and rates. However, a significant diet x BPS dose interaction was reported for cleaved embryos, > 4-cell embryos, blastocyst and early blastocyst numbers, and plasma triiodothyronine levels. Our study showed that a contrasted diet did not affect follicle population nor embryo production in adult ewes but could affect the quality and progesterone secretion of the corpus luteum. Chronic low BPS exposure had no effect on follicular population and oocyte competence. Nevertheless, the significant diet x dose interactions observed on embryo production suggest that BPS effect is modulated by metabolic status. Further studies are required to assess the risk of BPS exposure for public reproductive health.

activity against cyathostomin free-living stages observed in the past. We analysed the effect of a sainfoin-rich diet on cyathostomin population and the efficacy of oral ivermectin treatment. Two groups of 10 naturally infected horses were enrolled in a 78-day experimental trial. Following a 1-week adaptation period, they were either fed with dehydrated sainfoin pellets (70% of their diet dry matter) or with alfalfa pellets (control group) for 21-days. No difference was found between the average fecal egg counts (FECs) of the two groups, but a significantly lower increase in larval development rate was observed for the sainfoin group, at the end of the trial. Quantification of cyathostomin species abundances with an ITS-2-based metabarcoding approach revealed that the sainfoin diet did not affect the nemabiome structure compared to the control diet. Following oral ivermectin treatment of all horses on day 21, the drug concentration was lower in horses fed with sainfoin, and cyathostomin eggs reappeared earlier in that group. Our results demonstrated that short-term consumption of a sainfoin-rich diet does not decrease cyathostomin FEC but seems to slightly reduce larval development. Consumption of dehydrated sainfoin pellets also negatively affected ivermectin pharmacokinetics, underscoring the need to monitor horse feeding regimes when assessing ivermectin efficacy in the field.

Due to the restrictions of its use, Bisphenol A (BPA) has been replaced by many structurally related bisphenols (BPs) in consumer products. The endocrine disrupting potential similar to that of BPA has been described for several bisphenols, there is therefore an urgent need of toxicokinetic (TK) data for these emerging BPs in order to evaluate if their internal exposure could increase the risk of endocrine disruption. We investigated TK behaviors of eleven BPA substitutes (BPS, BPAF, BPB, BPF, BPM, BPZ, 3-3BPA, BP4-4, BPAP, BPP, and BPFL) by intravenous and oral administrations of mixtures of them to piglets and serial collection of blood over 72 h and urine over 24 h, to evaluate their disposition. Data were analyzed using nonlinear mixed-effects modeling and a comparison was made with TK predicted by the generic model HTTK package. The low urinary excretion of some BPs, in particular BPM, BPP and BPFL, is an important aspect to consider in predicting human exposure based on urine biomonitoring. Despite their structural similarities, for the same oral dose, all BPA analogues investigated showed a higher systemic exposure (area under the plasma concentration-time curve (AUC) of the unconjugated Bisphenol) than BPA (2 to 4 fold for 3-3BPA, BPAF, BPB and BPZ, 7-20 fold for BP4-4, BPAP, BPP, BPFL, BPF and BPM and 150 fold for BPS) due mainly to a considerable variation of oral bioavailability (proportion of BP administered by oral route that attains the systemic circulation unchanged). Given similarities in the digestive tract between pigs and humans, our TK data suggest that replacing BPA with some of its alternatives, particularly BPS, will likely lead to higher internal exposure to potential endocrine disruptive compounds. These findings are crucial for evaluating the risk of human exposure to these emerging BPs.