In search of a comprehensible set of endpoints for the routine monitoring of neurotoxicity in vertebrates: sensory perception and nerve transmission in zebrafish (Danio rerio) embryos

Abstract

In order to develop a test battery based on a variety of neurological systems in fish, three sensory systems (vision, olfaction, and lateral line) as well as nerve transmission (acetylcholine esterase) were analyzed in zebrafish (Danio rerio) embryos with respect to their suitability as a model for the screening of neurotoxic trace substances in aquatic ecosystems. As a selection of known or putative neurotoxic compounds, amidotrizoic acid, caffeine, cypermethrin, dichlorvos, 2,4-dinitrotoluene, 2,4-dichlorophenol, 4-nonylphenol, perfluorooctanoic acid, and perfluorooctane sulfonic acid were tested in the fish embryo test (OECD test guideline 236) to determine EC values, which were then used as maximum test concentration in subsequent neurotoxicity tests. Whereas inhibition of acetylcholinesterase was investigated biochemically both in vivo and in vitro (ex vivo), the sensory organs were studied in vivo by means of fluorescence microscopy and histopathology in 72- or 96-h-old zebrafish embryos, which are not regarded as protected developmental stages in Europe and thus - at least de jure - represent alternative test methods. Various steps of optimization allowed this neurotoxicity battery to identify neurotoxic potentials for five out of the nine compounds: Cypermethrin and dichlorvos could be shown to specifically modulate acetylcholinesterase activity; dichlorvos, 2,4-dichlorophenol, 4-nonylphenol, and perfluorooctane sulfonic acid led to a degeneration of neuromasts, whereas both vision and olfaction proved quite resistant to concentrations ≤ EC of all of the model neurotoxicants tested. Comparison of neurotoxic effects on acetylcholinesterase activity following in vivo and in vitro (ex vivo) exposure to cypermethrin provided hints to a specific enzyme-modulating activity of pyrethroid compounds. Enhancement of the neuromast assay by applying a simultaneous double-staining procedure and implementing a 4-scale scoring system (Stengel et al. 2017) led to reduced variability of results and better statistical resolution and allowed to differentiate location-dependent effects in single neuromasts. Since acetylcholinesterase inhibition and neuromast degeneration can be analyzed in 72- and 96-h-old zebrafish embryos exposed to neurotoxicants according to the standard protocol of the fish embryo toxicity test (OECD TG 236), the fish embryo toxicity test can be enhanced to serve as a sensitive neurotoxicity screening test in non-protected stages of vertebrates.

Investigating the Predictive Power of Three Potential Sublethal Endpoints for the Fathead Minnow Fish Embryo Toxicity Test: Snout-Vent Length, Eye Size, and Pericardial Edema

“…For example, Cd has been shown to impact behaviors such as place preference and hyperactivity . Likewise, PFOS has been shown to negatively impact the average number of neuromast cells present along the lateral line of zebrafish embryos, which likely impacts C-start behavior, and is known to alter C-start responses of juvenile qingbo (Spinibarbus sinensis) . Embryonic exposure to DCA (500 μg DCA/L) resulted in decreased locomotor activity in zebrafish larvae .…”

“…It has also been adopted as a standardized OECD test method for evaluating chemical toxicity (OECD 2013); however, the data derived from the zebrafish FET test alone cannot be used to satisfy the regulatory requirements of REACH and thus must be combined with other data in a weight-of-evidence approach . Further adoption and enhanced use of the FET test as a standard method for assessing toxicity has been limited due, in part, to two primary factors: (1) unlike other fish-based test types, the FET test lacks endpoints capable of estimating sublethal adverse effects , and (2) the FET test has been found to be less sensitive than other test types for some chemicals, particularly those classified as neurotoxicants. , The development of sublethal endpoints for the FET test has been proposed to overcome these limitations and to broaden its appeal. − A variety of potential sublethal endpoints have been proposed, including the expression of genes related to development, , embryonic behaviors, and the presence of developmental abnormalities. ,,,, Of these proposed sublethal endpoints, developmental abnormalities may offer the most promise for inclusion as standard FET test endpoints. In general, developmental abnormalities can be easily visualized during routine mortality assessments conducted as part of the FET test, making them feasible with regard to time, cost, and equipment required.…”

An ecotoxicological view on neurotoxicity assessment

“…In order to assess the full neurotoxic potential of environmental pollutants, a combination of tests and the assessment of multiple sensory systems are necessary to precisely localize effects within the nervous system [2, 200]. Future studies should strive to increase our mechanistic understanding of chemical neurotoxicity, which would help predicting eco-neurotoxicological effects.…”

“…Furthermore, as discussed above, a variety of neurotoxicity assays are needed to capture relevant endpoints [283]. Relevant neurotoxicity assays of varying sophistication and suitability for high-throughput studies include microelectrode arrays [128, 284], yeast assays [285, 286], assays using zebrafish [200, 287] and neural organoids derived from three-dimensional stem cell cultures [130].…”

First study on response of astrocytes in alevines of red-bellied pacu (Piaractus brachypomus) to subchronic exposure to chlorpyrifos and trichlorfon

“…Zebrafish (Danio rerio) has been used as a model for monitoring the central nervous system in vivo [63,64], and other species of teleosts have been studied for changes in behavior and learning processes after exposure to CPF [65]. However, our study is the first that is designed with the goal of understanding the mechanisms of astrocyte response against exposure to oxon-type OPs in fish and the first evidence-based proposal for an alternative to environmental screening, that is, the use of P. brachypomus as an animal model in the study of neurodegenerative diseases that affects humans.…”