Biomarker toxicodynamics linked to BPA toxicokinetics in three-spined stickleback: a PBPK-TD model

International audienceBisphenol A (BPA) is considered a substance of very serious concern due toits endocrine disruptor function, high production volume, and persistence in the aquaticenvironment. In this context, physiologically-based toxicokinetic models coupled withtoxicodynamics (PBTK-TD) have proven to be valuable tools. They allow proposingmore actual exposure scenarios as the exposure may vary over time and they alsoenable linking internal concentrations to various effects that are often decorrelated toexternal dose. Recently, a PBTK has been developed and adapted to BPA ADME(Absorption, Distribution, Metabolization, and Excretion) processes in the three-spinedstickleback. Thus, this work aimed to develop a PBTK-TD model that would permit tobetter understand the impact of BPA on the different functions of the fish, includingdefense capacities and reproduction. In particular, a precise knowledge of the internaldoses in the target organs would allow to i) clearly identify whether the transientbiomarker responses are linked to toxicokinetics, ii) identify the compounds (parentand/or metabolites) responsible for the biomarker responses, and iii) to identifynon-monotonic dose dependencies of biological responses to external exposures thatwould be non-monotonic. In detail, the PBTK was extended to include thetoxicodynamics of various biomarkers measured during two exposures to BPA, one ofseven-day exposure and seven-day depuration, and one of 21-day exposure.Biomarkers including non-specific cellular immunity, metabolic detoxification, oxidativestress, and reproductive parameters were analyzed to select a set of parametersvarying when exposed to BPA. Since it was supposed that the biomarker responseswere produced by indirect mechanisms, indirect toxicodynamic models were chosen todescribe the response. Consequently, mechanism-based equations describing theresponses of biomarkers over time were implemented in the existing PBTK specific toBPA. Then, TD data were divided into two sets, one to allow calibration of parametersrelated to biomarker toxicodynamics and the other to enable evaluation on an unusedcollection of TD data. This study demonstrates the relevance of the three-spinedstickleback as EDC sentinel species and the approach using a PBTK-TD to explore thedynamics of the EDC effects

Modélisation de la dynamique de biomarqueurs : application d'un modèle PBTK-TD chez l'épinoche exposée au bisphénol A.

National audienc

PBTK-TD model of the phagocytosis activity in three-spined stickleback exposed to BPA

International audienceDue to the high production volume and persistence in the environment of bisphenol A (BPA) and its substitutes, realistic exposure scenarii were proposed in some species to better understand the relationship between external and internal concentrations. For example, a recent PBTK model has been developed and adapted to BPA ADME (Absorption, Distribution, Metabolization, and Excretion) processes in three-spined stickleback. These substances have an impact on organism physiology including reproductive and immune functions. In this context, physiologically-based toxicokinetic models coupled with toxicodynamics (PBTK-TD) have proven to be valuable tools to fill the knowledge gap between external exposure and effect dynamics. The aim of the current work was to explain the impact of BPA on the immune response by determining its temporality. In addition, the relationship between BPA dose and these responses was investigated using a PBTK-TD model. Two experiments were performed on stickleback to characterize their biomarker responses, (i) a short exposure (14 days) at 0, 10 and 100 µg/L, including a depuration phase (7 days), and (ii) a long exposure (21 days) at 100 µg/L to measure the immunomarker dynamic over a long period. The fish spleens were sampled to analyze immune responses of stickleback at various times of exposure and depuration: leucocyte distribution, phagocytic capacity and efficiency, lysosomal presence and leucocyte respiratory burst index. At the same date, blood, muscle, and liver were sampled to quantify BPA and their metabolites (BPA monoglucuronide and BPA monosulfate). All these data enabled the development of the indirect pharmacodynamic models (PBTK-TD) by implementing the responses of biomarkers in the existing BPA PBTK of stickleback. The results shown a high induction of phagocytosis activity by BPA in the two exposure conditions. Furthermore, the immunomarkers exhibit very different temporal dynamics. This study demonstrates the need of a thorough characterization of biomarker response for a further use in Environmental Biomonitoring

Application in a biomonitoring context of three-spined stickleback immunomarker reference ranges

International audienceThe relevance of a biomarker for biomonitoring programs was influenced both by the knowledge on biomarker natural inter-individual and site variabilities and by the sensitivity of the biomarker towards environmental perturbations. To minimize data misinterpretation, robustness reference values for biomarkers were important in biomonitoring programs. Specific three-spined stickleback, Gasterosteus aculeatus, immune reference ranges for field studies had been determined based on laboratory data and one reference station (Contentieuse river at Houdancourt). In this study, data obtained in one uncontaminated and three contaminated sites were compared to these reference ranges as a validation step before considering them for larger scale biomonitoring programs. When the field reference range were compared to data from the uncontaminated station (Béronelle), only few deviations were shown. In this way, data coming from uncontaminated station (Béronelle) was integrated in the field reference ranges to improve the evaluation of site variability. The new field reference ranges provided better discrimination of sites and spanned a larger range of fish lengths than the initial reference ranges. Furthermore, the results suggest lysosomal presence during several months and phagocytosis capacity in autumn may be the most relevant immunomarkers towards identifying contaminated sites. In the future, combining this reference value approach with active biomonitoring could facilitate the obtention of data in multiple stream conditions

Exposure and hazard of bisphenol A, S and F: a multi-biomarker approach in three-spined stickleback

International audienceDue to the oestrogenic behaviour of bisphenol (BP) A, industries have developed many substitutes, such as BPS and BPF. However, due to their structural similarities, adverse effects on reproduction are currently observed in various organisms, including fish. Even if new results have shown impacts of these bisphenols on many other physiological functions, their mode of action remains unclear. In this context, we proposed to better understand the impact of BPA, BPS and BPF on immune responses (leucocyte sub-populations, cell death, respiratory burst, lysosomal presence and phagocytic activity) and on biomarkers of metabolic detoxification (ethoxyresorufin-O-deethylase, EROD, and glutathione S transferase, GST) and oxidative stress (glutathione peroxidase, GPx, and lipid peroxidation with thiobarbituric acid reactive substance method, TBARS) in an adult sentinel fish species, the three-spined stickleback. In order to enhance our understanding of how biomarkers change over time, it is essential to determine the internal concentration responsible for the observed responses. Therefore, it is necessary to explore the toxicokinetics of bisphenols. Thus, sticklebacks were exposed either to 100 µg/L of BPA, BPF or BPS for 21 days, or for seven days to 10 and 100 µg/L of BPA or BPS followed by seven days of depuration. Although BPS has very different TK, due to its lower bioaccumulation compared to BPA and BPF, BPS affect oxidative stress and phagocytic activity in the same way. For those reasons, the replacement of BPA by any substitute should be made carefully in terms of risk assessment on aquatic ecosystems

Effets combinés de l’éthinylestradiol et du réchauffement climatique sur la physiologie et la dynamique de population de l’épinoche à trois-épines

National audienc

Combined effects of climate change and éthinylestradiol on the life cycle and physiology of three-spined stickleback

International audienc

Application of the Fpg-modified comet assay on three-spined stickleback in freshwater biomonitoring: toward a multi-biomarker approach of genotoxicity

International audienceAquatic species are exposed to a wide spectrum of substances, which can compromise their genomic integrity by inducing DNA damage or oxidative stress. Genotoxicity biomarkers as DNA strand breaks and chromosomal damages developed on sentinel species have already proved to be relevant in aquatic biomonitoring. However, these biomarkers do not reflect DNA oxidative lesions, i.e., the 8-oxodG, recognized as pre-mutagenic lesion if not or mis-repaired in human biomonitoring. The relevance to include the measure of these lesions by using the Fpg-modified comet assay on erythrocytes of the three-spined stickleback was investigated. An optimization step of the Fpg-modified comet assay considering enzyme buffer impact, Fpg concentration, and incubation time has been performed. Then, this measure was integrated in a battery of genotoxicity and cytotoxicity biomarkers (considering DNA strand breaks, DNA content variation, and cell apoptosis/necrosis and density) and applied in a freshwater monitoring program on six stations of the Artois Picardie watershed (3-week caging of control fish). These biomarkers allowed to discriminate the stations regarding the genotoxic potential of water bodies and specifically by the measure of oxidative DNA lesions, which seem to be a promising tool in environmental genotoxicity risk assessment

Development of a multi-biomarker approach of genotoxicity on the three spined stickleback for aquatic biomonitoring application

International audienceThe aquatic environment is considered as the receptacle of anthropogenicpressures, including chemical contamination as genotoxic compounds, which canimpact the health of aquatic species. Genotoxic substances may directly or indirectlyaffect the integrity of cell’s genetic material, which can lead to long-term mutagenic,carcinogenic or teratogenic effects. In this context, the measure of damages at differentgenomic scales may be relevant and provide an integrated view of the genotoxic risk ofexposed organism. The finality of our work is to propose their use as early-warningsignals of potential long-term alterations to population and ecosystem health.Genotoxicity assessment was performed on erythrocytes of the three- spinedstickleback by combining the measure of DNA integrity by the alkaline and Fpg(formamidopyrimidines DNA glycosylase)-modified comet assays and the measure ofvariations in nuclear DNA content by flow cytometry (FCM). In this sense, blood cells offish offer many advantages as cellular models as their ease of collection, gives a highdensity of erythrocytes already dissociated allowing to obtain multiple assays on thesame sample. The alkaline comet assay has been largely used with erythrocytes of fishto detect alkali-labile sites, DNA-double- and single- strand breaks. The Fpg-modifiedcomet assay has been optimized in the present study, which expand the comet assaysensitivity to oxidized pyrimidines. Furthermore, FCM may be applied to assess thevariations in the nuclear DNA content of a large population of cells and has alreadydemonstrated its relevance for detecting chromosomal damages in blood cells of fish.The presentation will detail results regarding the definition of baseline levels ofgenotoxicity biomarkers (DNA strand breaks, Fpg-sensitive sites, DNA content,erythrocyte mortality), which are useful in an ecotoxicological context. These biomarkershave been measured in various field context (active biomonitoring) and integrated in theexisting multi-biomarker approach of the three-spined stickleback (Gasterosteusaculeatus L., 1758)

The comet assay and the Fpg-modified comet assay: optimization and field-application on erythrocytes of the three-spined stickleback

International audienc