Microplastics from miscellaneous plastic wastes: Physico-chemical characterization and impact on fish and amphibian development.

Microplastic pollution represents a global problem with negative impacts on aquatic environment and organisms' health. To date, most of the laboratory toxicological studies on microplastics (MPs) have made use of single commercial micro and nano-polymers, which do not reflect the heterogeneity of environmental MPs. To improve the relevance of the hazard assessment, micrometer-sized plastic particles of miscellaneous non-reusable waste plastics, with size <100 µm and <50 µm (waste microplastics, wMPs), were characterized by microscopic and spectroscopic techniques and tested on developing zebrafish and Xenopus laevis by FET and FETAX assays respectively. Moreover, the modalities of wMP interaction with the embryonic structures, as well as the histological lesions, were explored by light and electron microscopy. We have shown that wMPs had very heterogeneous shapes and sizes, were mainly composed of polyethylene and polypropylene and contained metal and organic impurities, as well as submicrometric particle fractions, features that resemble those of environmental occurring MPs. wMPs (0.1-100 mg/L) caused low rate of mortality and altered phenotypes in embryos, but established species-specific biointeractions. In zebrafish, wMPs by adhering to chorion were able to delay hatching in a size and concentration dependent manner. In Xenopus embryos, which open stomodeum earlier than zebrafish, wMPs were accumulated in intestinal tract, where produced mechanical stress and stimulated mucus overproduction, attesting an irritation response. Although wMP biointeractions did not interfere with morphogenesis processes, further studies are needed to understand the underlying mechanisms and long-term impact of these, or even smaller, wMPs.

Iron nanoparticle bio-interactions evaluated in Xenopus laevis embryos, a model for studying the safety of ingested nanoparticles.

Iron nanoparticles (NPs) have been proposed as a tool in very different fields such as environmental remediation and biomedical applications, including food fortification against iron deficiency, even if there is still concern about their safety. Here, we propose Xenopus laevis embryos as a suitable model to investigate the toxicity and the bio-interactions at the intestinal barrier of Fe3O4 and zerovalent iron (ZVI) NPs compared to Fe(II) and (III) salts in the 5 to 100 mg Fe/L concentration range using the Frog Embryo Teratogenesis Assay in Xenopus (FETAX). Our results demonstrated that, at concentrations at which iron salts induce adverse effects, both iron NPs do not cause acute toxicity or teratogenicity even if they accumulate massively in the embryo gut. Prussian blue staining, confocal and electron microscopy allowed mapping of iron NPs in enterocytes, along the paracellular spaces and at the level of the basement membrane of a well-preserved intestinal epithelium. Furthermore, the high bioaccumulation factor and the increase in embryo length after exposure to iron NPs suggest greater iron intake, an essential element for organisms. Together, these results improve the knowledge on the safety of orally ingested iron NPs and their interaction with the intestinal barrier, useful for defining the potential risks associated with their use in food/feed fortification.

Effects of TCDD on spermatogenesis related factor-2 (SRF-2): gene expression in Xenopus.

2,3,7,8-Tetra-chlorodibenzo-p-dioxin (TCDD) is one of the most toxic dioxins belonging to the wide family of Endocrine Disruptors (EDs), environmental chemicals that adversely interfere with endocrine processes and upset normal function of some target systems. It has been hypothesized that EDs enter cellular cytosol, bind to the Aryl Hydrocarbon Receptor (AhR) and form a heterodimer with the AhR nuclear translocator; this complex binds xenobiotic responsive elements that drive activation of the so-called "Ah gene battery". Spermatogenesis Related Factor-2 (SRF-2) is one of the most recently cloned genes involved in germ cell division and differentiation, whose expression seems to be affected by treatment with TCDD. With the aim to try to clarify the underlying mechanism of TCDD and to investigate if SRF-2 gene represents a good biomarker for ED exposure, we used Xenopus laevis as an animal model, considered to be almost insensitive toward TCDD effects. In this study we reported the partial cloning of SRF-2 cDNA in X. laevis; we then evaluated the SRF-2 expression in embryos exposed to TCDD 0.62 microM by real-time PCR. We also analyzed SRF-2 expression in several adult control tissues and in testis after perilymphatic injection of a single dose of 10 microg/kg body weight. Although SRF-2 expression does not seem to be affected by the treatment, exposed embryos died within 15 days. In the light of these results, we can conclude that SRF-2 is not a good candidate in signalling EDs exposure and that the molecular mechanism for TCDD toxicity in Xenopus likely involves the AhR signalling cascade, as in other vertebrate species.

Differential modulation of cytochrome P-450 1A and P-glycoprotein expression by aryl hydrocarbon receptor agonists and thyroid hormone in Xenopus laevis liver and intestine.

Several defence mechanisms, such as cytochrome P450 1A (CYP1A) enzymes and P-glycoprotein (Pgp), may influence the intracellular concentration and consequently the toxicity of xenobiotics. The parallel expression of CYP1A and Pgp has been investigated in mammals and, to a lesser extent in fish, in search for evidence of co-ordinated responses to xenobiotic exposure. The aryl hydrocarbon receptor (AHR) agonists are well known CYP1A inducers but some of them resulted not to have a uniquely defined action on Pgp levels in mammalian and fish species. To the best of our knowledge, no detailed studies have been carried out so far on amphibians Xenopus laevis. For this reason, in this work, the time dependent responses of the hepatic CYP1A and Pgp, to the prototypical CYP1A inducers, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in X. laevis have been assessed at the protein level and compared. The responsiveness of Xenopus intestinal Pgp to these compounds has also been analysed, as the epithelial cells lining the lumen of intestine represent another preferential site of Pgp expression. In addition, since the thyroid hormone has been demonstrated to down regulate the mdr gene during Xenopus development and in primary culture of Xenopus intestinal epithelial cells, the effects of 3,3',5-triiodo-L-thyronine (T(3)) on CYP1A and Pgp protein levels have been investigated in adult organisms. Western blot evidenced that a single injection of B(a)P (100 mg/kg), 3MC (20 mg/kg), and TCDD (3 microg/kg) elicited a statistically significant induction of hepatic CYP1A at all time points considered (72, 120 and 168 h) which decreased in time. The same trend of liver CYP1A induction was observed in T(3) treated Xenopus (15 microg/kg). Unlike CYP1A induction, the modulation of hepatic and intestinal Pgp expression exhibits an heterogeneous pattern. The basal levels of hepatic and intestinal Pgp were not statistically significant affected by treatments. In particular, the hepatic Pgp levels seem not to be induced by TCDD and T(3) at all times considered in comparison to control. For the first time the modulation of CYP1A and Pgp levels by B(a)P, 3MC and in particular by TCDD and T(3) in Xenopus has been demonstrated and the results herewith indicate that the two target defence mechanisms respond to AHR agonists in a dissimilar way in terms of proteins induction in Xenopus. Moreover, these data suggest additional experiments in order to clarify the complex mechanism, which adjusts the parallel expression of CYP1A and Pgp in Xenopus.