Application of Caenorhabditis elegans (nematode) and Danio rerio embryo (zebrafish) as model systems to screen for developmental and reproductive toxicity of Piperazine compounds

Abstract

A B S T R A C TTo enable selection of novel chemicals for new processes, there is a recognized need for alternative toxicity screening assays to assess potential risks to man and the environment. For human health hazard assessment these screening assays need to be translational to humans, have high throughput capability, and from an animal welfare perspective be harmonized with the principles of the 3Rs (Reduction, Refinement, Replacement).In the area of toxicology a number of cell culture systems are available but while these have some predictive value, they are not ideally suited for the prediction of developmental and reproductive toxicology (DART). This is because they often lack biotransformation capacity, multicellular or multi-organ complexity, for example, the hypothalamus pituitary gonad (HPG) axis and the complete life cycle of whole organisms.To try to overcome some of these limitations in this study, we have used Caenorhabditis elegans (nematode) and Danio rerio embryos (zebrafish) as alternative assays for DART hazard assessment of some candidate chemicals being considered for a new commercial application. Nematodes exposed to Piperazine and one of the analogs tested showed a slight delay in development compared to untreated animals but only at high concentrations and with Piperazine as the most sensitive compound. Total brood size of the nematodes was also reduced primarily by Piperazine and one of the analogs. In zebrafish Piperazine and analogs showed developmental delays. Malformations and mortality in individual fish were also scored. Significant malformations were most sensitively identified with Piperazine, significant mortality was only observed in Piperazine and only at the higest dose. Thus, Piperazine seemed the most toxic compound for both nematodes and zebrafish.The results of the nematode and zebrafish studies were in alignment with data obtained from conventional mammalian toxicity studies indicating that these have potential as developmental toxicity screening systems. The results of these studies also provided reassurance that none of the Piperazines tested are likely to have any significant developmental and/or reproductive toxicity issues to humans when used in their commercial applications.

Piperazine designer drugs elicit toxicity in the alternative in vivo model Caenorhabditis elegans

“…In the present study, the reduction in the body area of the worms after exposure to piperazine designer drugs is an indicative of neurotoxicity. In a previous study, nematodes exposed to piperazine and piperazine analogs showed a slight delay in development, compared with untreated worms (Racz et al, ). The C. elegans body size is influenced by environmental conditions, and the nervous system plays a crucial role in the development of nematodes.…”

An International Perspective on the Tools and Concepts for Effluent Toxicity Assessments in the Context of Animal Alternatives: Reduction in Vertebrate Use

“…There are other recent toxicity model developments that offer potential as alternatives. For example, the preliminary developmental and reproductive toxicity assay was developed (National Centre for the Replacement Refinement & Reduction of Animals in Research ) with 2 models based on nematodes and zebrafish as alternatives to mammals for product screening (Racz et al ). More recently, the zebrafish model was used to screen contaminated groundwater samples for reproductive toxicity based on phenotypic changes (developmental delays and malformations) during the first 96 h in the developing zebrafish larvae (G. Whale, personal communication).…”

“…There are other recent toxicity model developments that offer potential as alternatives. For example, the preliminary developmental and reproductive toxicity assay was developed (National Centre for the Replacement Refinement & Reduction of Animals in Research 2017) with 2 models based on nematodes and zebrafish as alternatives to mammals for product screening (Racz et al 2017). More recently, the zebrafish model was used to screen contaminated groundwater samples for reproductive toxicity based on phenotypic changes (developmental delays Detection/testing of estrogen-, androgen-, and thyroid-active chemicals in various systems (e.g., cell lines, primary cells, fish/frog embryos, yeast, and cell-free systems) Alternative assays that measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis In vitro assays for testing acute toxicity Cytotoxicity tests with established fish cell lines Tests to explore toxicity pathways at the molecular and cellular levels PBTK models In vitro assays for testing genotoxicity Cellular/DNA damage exhibited as mutagenicity In vitro biomarker assays Cell lines (lower biological organizational level).…”

How omics technologies can enhance chemical safety regulation: perspectives from academia, government, and industry

“…For example, the potential of some of these test systems has recently been investigated as part of a National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs; UK) challenge (National Centre for the Replacement, Refinement & Reduction of Animals in Research). Using research from this challenge from nematode ( Caenorhabditis elegans ) and zebrafish models have been shown to have potential to screen for mammalian relevant pre–development and reproductive toxicity phenotypic endpoints (Racz et al ). The pre–development and reproductive toxicity challenge omics results also indicated development and reproductive toxicity–specific responses on RNA and protein level and that 4‐RNAi in C. elegans indicated critical genes for toxicological response, although these results are yet to be published.…”