CONCLUSIONS AND FUTURE RESEARCH
Within the United States, approximately one out of six children has a developmental disability, most of which affect the nervous system (Grandjean and Landrigan, 2006). Treatment of neurodevelopmental disorders is difficult, and the resulting disabilities may often be permanent and result in high damage and costs to family and society. Moreover, in the last few decades, the observed increase in neurodevelopmental disorders in children is thought to be partly due to increased environmental exposure to potential neurotoxicants. In fact, roughly 3% of neurodevelopmental disabilities may be due to exposure to environmental toxicants, while 25% may arise due to gene-environment interactions (Giordano and Costa, 2012). Although social factors are also believed to contribute to this increasing trend, there is mounting evidence that demonstrates associations between environmental chemicals and neurodevelopmental disorders. Consequently, DNT has now become an issue of growing concern around the world, and the efforts of scientists and regulators are focused on understanding the potential hazards of these chemical agents to help fill existing DNT data gaps (National Research Council, 2000). As a result, there is general agreement regarding the need to develop alternative test methods that are rapid and can be validated and used across multiple laboratories. While these methods must not only help generate reliable data that are predictive and effective, they should also accommodate screening
DNT that emphasizes the use of alternative animal models and can be used within a regulatory context is an important priority.
The first study (Chapter 2) demonstrated that HCS assays that rely on neurobehavioral endpoints in zebrafish embryos can be used to 1) screen and prioritize potential developmental neurotoxicants for further testing in other animal models and 2) provide a foundation for developing hypothesis-driven mechanistic studies in zebrafish embryos. For example, using the HCS assay described in Chapter 2, we identified two chemicals - abamectin and emamectin benzoate – that significantly decreased spontaneous activity in the absence of effects on survival and total body area. Although spontaneous activity in zebrafish occurs a very early developmental stage (which cannot be easily assessed in utero in mammals) and may not have direct phenotypic relevance to rodent models or humans, these results can help predict potential neurotoxic effects of chemicals that may have similar molecular targets in zebrafish and humans. In fact, similar behavioral assays in zebrafish have been developed as ways to promote drug discovery and even screen chemicals for neurotoxicity (Kokel et al., 2010).
Even though the HCS assay described in Chapter 2 was successful in identifying potential developmental neurotoxicants during early zebrafish embryogenesis, the assay had a few limitations. First, because spontaneous behavior is naturally variable and occurs within a narrow time window during normal embryonic development, we were only able to image 192 embryos (one well at a time) over an 80-minute time period using a 2x objective. However, future research can begin to optimize the assay using a 1x objective, which allows imaging of four wells together. Therefore, 384 embryos could be imaged in just half the time (40 minutes) required to image 192 embryos using a 2x
objective. This would also increase the sample size from 16 to 32 embryos per treatment (one to two columns), allowing for multiple plates to be imaged per day. Ideally, this would help control for the natural variation in spontaneous activity while also increasing throughput.
Furthermore, the two positive hits identified in our screen had similar modes-of- action, suggesting that this HCS assay may be biased towards identifying chemicals with only specific neurotoxic modes-of-action, particularly at this early embryonic stage of development. In addition, we only identified chemicals that resulted in hypo- rather than hyper-activity. However, this initial screen comprised only a small subset of chemicals, and the observed hit rate (1 out of 15, or 6.7%) is consistent with the hit rate of another zebrafish behavioral screening assay (Kokel et al., 2010). In the future, increasing the throughput of this assay would allow a larger battery of chemicals to be screened and help determine whether this assay could potentially be used to group chemicals that share common mechanisms of action and chemical structures (Kokel et al., 2010). Nonetheless, these results support the use of this HCS assay to help identify potential developmental neurotoxicants within a regulatory context.
The identification of abamectin as a potential developmental neurotoxicant led us to develop hypothesis-driven studies to begin investigating the mechanism of abamectin- induced hypoactivity. The second study (Chapter 3) demonstrated that 1) abamectin did not affect outgrowth of spinal motoneurons, 2) abamectin-induced hypoactivity was rapid and reversible, 3) abamectin-induced hypoactivity was blocked by pretreatment with two GABA receptor antagonists, and 4) GABA receptor subunits within zebrafish and mammals are homologous, suggesting that this has potential relevance to mammalian
systems. Based on the observed behavioral effect and known effects of GABA, we then hypothesized that abamectin-induced hypoactivity occurs due to an influx of chloride ions into interneurons, resulting in decreased membrane potential. However, initial attempts within zebrafish embryos to assess voltage changes using the FLIPR assay were unsuccessful. Future studies should rely on whole-cell patch clamp recording techniques to assess voltage changes of zebrafish spinal cord neurons in vehicle and abamectin- exposed embryos with or without addition of fipronil or endosulfan (GABA antagonists). The addition of exogenous GABA should be used as a positive control in these studies. In fact, the patch clamp technique has been used to successfully evaluate voltage changes of interneurons, motoneurons, and sensory neurons in vivo within the spinal cord of 18-26 hpf zebrafish embryos (Saint-Amant and Drapeau, 2003).
In addition, it is still unclear whether abamectin-induced hypoactivity occurs by direct chemical binding to the GABA receptor (as an agonist or antagonist), or by stimulating production of GABA, which is then released into the presynaptic inhibitory terminals (Novelli et al., 2012). Due to the rapid recovery of this behavior, we hypothesize that abamectin potentiates the effect of GABA by binding to a site on GABA receptors different from the active site. This would produce an effect similar to the sedative, anxiolytic behavior observed with diazepam (Valium), a benzodiazepine drug (Christian et al., 2013). Therefore, future studies should rely on 1) high-performance liquid chromatography (HPLC) to quantify the amount of GABA neurotransmitter after exposure to abamectin, 2) qPCR to determine whether genes involved in synthesizing or transporting GABA are altered, and 3) ligand-binding assays. Together, these
experiments would provide more detailed information about the mechanism of abamectin-induced hypoactivity.
Additional follow-up studies could also be conducted to determine whether abamectin exposure during early embryonic development has effects on other locomotive behaviors, such as touch-response or swimming, potential long-term effects on cognition, such as learning and memory, or multigenerational effects. Though our results suggest that abamectin-induced hypoactivity is likely reversible, some industrial chemicals have been found to exhibit “silent neurotoxicity” – a term that indicates the presence of subclinical effects which are not monitored within health statistics. One example of this is lead, a known developmental neurotoxicant, which is thought to affect IQ scores in the absence of visible health effects, resulting in high economic costs to society (Grandjean and Landrigan, 2006). Therefore, future work should aim to understand the effect of early abamectin exposure on behavior and cognition at later stages (juvenile or adult) in order to assess whether in utero exposure during early development has potential long-term effects on the nervous system.
Finally, although zebrafish is a valuable in vivo model for understanding how chemicals act within an intact organism, future in vitro studies using human cell lines would complement these data, enhancing human relevance. In fact, there are numerous human cell lines already available that are specific to the nervous system. For example, human umbilical cord blood stem cells have been used to generate neural stem cell lines, and human neural progenitor cells have been grown as neurospheres, allowing both the embryonic and fetal periods of neurodevelopment to be explored in vitro (Bal-Price et al., 2012; Smirnova et al., 2014). Neurospheres from different species can also be used to
compare various toxicity endpoints across species (Smirnova et al., 2014). Recently, human embryonic stem cells were even differentiated in vitro for DNT testing using methylmercury (Bal-Price et al., 2012). Within a tiered testing strategy, the use of in vivo data from zebrafish HCS/HTS assays or rodent modelsin combination with in vitro data from human cell lines will help improve species extrapolation to humans. Moreover, many imaging systems, including our ImageXpress Micro High-Content Imaging System, already comprise established protocols to assess endpoints such as cell viability and neurite outgrowth.
Overall, the work presented in this dissertation uses zebrafish as an alternative animal model to help identify and prioritize chemicals for high-cost, low-throughput DNT assays in rodent models, while also exploring the potential mechanism of action of abamectin – a chemical identified as a positive hit in the HCS assay - within early zebrafish embryos. Ultimately, a combination of assays with different endpoints will be needed to effectively predict in vivo DNT, as assays in cell lines and small animal models are not sufficient predictors of risk to humans on their own. The development of HCS/HTS assays to screen and prioritize chemicals and the use of zebrafish for follow-up mechanistic studies will reduce animal use as well as provide a means of defining hazard for the thousands of chemicals in commerce with unknown toxicity data, helping fill the current DNT data gaps. These data can then be used to help guide regulatory decisions and support the registration of chemicals for use in commerce.
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