The presence of microplastics in the aquatic ecosystem represents a major issue for the environment and human health. The capacity of organic pollutants to adsorb onto microplastic particles raises additional concerns, as it creates a new route for toxic compounds to enter the food web. Current knowledge on the impact of pristine and/or contaminated microplastics on aquatic organisms remains insufficient, and we provide here new insights by evaluating their biological effects in zebrafish (Danio rerio). Zebrafish larvae were raised in ZEB316 stand-alone housing systems and chronically exposed throughout their development to polyethylene particles of 20-27 μm, pristine (MP) or spiked with benzo[α]pyrene (MP-BaP), supplemented at 1% w/w in the fish diet. While they had no effect at 30 days post-fertilization (dpf), MP and MP-BaP affected growth parameters at 90 and 360 dpf. Relative fecundity, egg morphology, and yolk area were also impaired in zebrafish fed MP-BaP. Zebrafish exposed to experimental diets exhibited an increased incidence of skeletal deformities at 30 dpf as well as an impaired development of caudal fin/scales, and a decreased bone quality at 90 dpf. An intergenerational bone formation impairment was also observed in the offspring of parents exposed to MP or MP-BaP through a reduction of the opercular bone in 6 dpf larvae. Beside a clear effect on bone development, histological analysis of the gut revealed a reduced number of goblet cells in zebrafish fed MP-BaP diet, a sign of intestinal inflammation. Finally, exposure of larvae to MP-BaP up-regulated the expression of genes associated with the BaP response pathway, while negatively impacting the expression of genes involved in oxidative stress. Altogether, these data suggest that long-term exposure to pristine/contaminated microplastics not only jeopardizes fish growth, reproduction performance, and skeletal health, but also causes intergenerational effects.

Bone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3days initiated at 3days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose-dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.

Bone disorders affect millions of people worldwide and treatments currently available often produce undesirable secondary effects or have limited efficacy. It is therefore of the utmost interest for patients to develop more efficient drugs with reduced off-target activities. In the long process of drug development, screening and preclinical validation have recently gained momentum with the increased use of zebrafish as a model organism to study pathological processes related to human bone disorders, and the development of zebrafish high-throughput screening assays to identify bone anabolic compounds. In this review, we provided a comprehensive overview of the literature on zebrafish bone-related assays and evaluated their performance towards an integration into screening pipelines for the discovery of mineralogenic/osteogenic compounds. Tools available to standardize fish housing and feeding procedures, synchronize embryo production, and automatize specimen sorting and image acquisition/analysis toward faster and more accurate screening outputs were also presented.

Among the many anthropogenic chemicals that end up in the aquatic ecosystem, heavy metals, in particular cadmium, are hazardous compounds that have been shown to affect developmental, reproductive, hepatic, hematological, and immunological functions in teleost fish. There is also evidence that cadmium disturbs bone formation and skeletal development, but data is scarce. In this work, zebrafish was used to further characterize the anti-osteogenic/osteotoxic effects of cadmium and gain insights into underlying mechanisms. Upon exposure to cadmium, a reduction of the opercular bone growth was observed in 6-days post-fertilization (dpf) larvae and an increase in the incidence of skeletal deformities was evidenced in 20-dpf post-larvae. The extent and stiffness of newly formed bone was also affected in adult zebrafish exposed to cadmium while regenerating their caudal fin. A pathway reporter assay revealed a possible role of the MTF-1 and cAMP/PKA signaling pathways in mechanisms of cadmium osteotoxicity, while the expression of genes involved in osteoblast differentiation and matrix production was strongly reduced in cadmium-exposed post-larvae. This work not only confirmed cadmium anti-osteogenic activity and identified targeted pathways and genes, but it also suggested that cadmium may affect biomechanical properties of bone.