Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish

Pyrethroids are generally considered to be a safer alternative to other insecticides because they exhibit low mammalian toxicity and low environmental persistence (Demoute, 1989). Consequently, their use and popularity is on the rise (Grube et al., 2011). Human exposure is well documented, with 70% of urine samples collected from the 1999–2002 National Health and Nutrition Examination Survey cohort containing detectable levels of the pyrethroid metabolite 3-phenoxybenzoic acid (3PBA) (Barr et al., 2010). Because it is widely recognized that children are potentially more sensitive to the adverse effects of pesticides (National Research Council (U.S.). Committee on Pesticides in the Diets of Infants and Children, 1993), a troubling statistic highlighted by this survey was the fact that levels of 3PBA were significantly higher in children than adolescents or adults (Barr et al., 2010). Other surveys reported finding pyrethroid metabolites in the urine of pregnant women (Berkowitz et al., 2003; Qi et al., 2012; Whyatt et al., 2002) and children (Babina et al., 2012; Bradman et al., 2007; Couture et al., 2009; Fortin et al., 2008; Heudorf et al., 2004; Morgan et al., 2007; Richardson et al., 2015) Thus, it is important to understand the consequences of pyrethroid exposure during development. An increasing number of studies point to toxicant exposure during critical developmental periods as having long-term effects, including contributing to neurological disease in adulthood (Fox et al., 2012). Currently, there is limited evidence in rodents indicating that developmental exposure to pyrethroids results in persistent changes in neurochemical, behavioral, and cognitive endpoints, reviewed in Shafer et al. (2005). Increased locomotor activity was observed in adult mice that had been exposed to nontoxic doses of deltamethrin (0.7 mg/kg) between PND10-16 (Eriksson and Fredriksson, 1991). In addition to increased locomotor activity, behavioral effects such as increased impulsivity and working memory deficits have also been observed in 6-week-old male mice that had been developmentally exposed to deltamethrin at levels below the no observed adverse effect level (NOAEL) (3 mg/kg) (Richardson et al., 2015). In rats, gestational exposure to deltamethrin resulted in decreased locomotor activity at 3 and 6 weeks (Husain et al., 1992). Similar findings have been reported using even lower doses (0.08–0.5 mg/kg) of commercial deltamethrin formulations and persisting to longer timepoints (6–9 weeks) (Johri et al., 2006), including adulthood (Lazarini et al., 2001). Together, this data suggest that deltamethrin may be a developmental neurotoxicant that results in neurobehavioral deficits. Because the dopamine (DA) system plays an essential role in mediating many of the observed behavioral and locomotor effects, it is possible that disruption of DA neurotransmission during a phase of developmental plasticity is a mode of action for pyrethroid-induced locomotor changes. A recent study in mice demonstrates that hyperactivity resulting from developmental exposure to deltamethrin at doses below the NOAEL (3 mg/kg) is associated with increased protein levels of the DA transporter and D1 dopamine receptor, and decreased levels of extracellular DA. Importantly, deltamethrin-induced hyperactivity was attenuated by treatment with D1 receptor antagonists and D2 receptor agonists, implicating the role of the DA system in mediating these effects (Richardson et al., 2015). Acutely, pyrethroid exposure influences the kinetics of DA transmission by altering DA uptake in synaptosomes prepared from pyrethroid-treated mice (Bloomquist et al., 2002; Elwan et al., 2006; Karen et al., 2001; Kirby et al., 1999). Similarly, in vivo microdialysis analysis of conscious rats demonstrates that both DA reuptake and release are affected by acute pyrethroid exposure (Hossain et al., 2006). Pyrethroid-induced changes in DA uptake has been linked to immediate (Bloomquist et al., 2002; Elwan et al., 2006; Kirby et al., 1999) and persistent (Gillette and Bloomquist, 2003; Richardson et al., 2015) changes in DA transporter expression and DA uptake activity in mice. In addition, pyrethroid exposure was associated with changes in the concentrations of striatal DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat models (Doherty et al., 1988; Hudson et al., 1986). Thus, if these disturbances occur during a critical period, such as neurodevelopment, this could cause disruption of normal development, leading to persistent changes in function. The zebrafish has gained popularity as a model organism due to its small size, high fecundity, optical transparency, and genetic similarity to humans. In addition, the availability of an annotated genome and molecular genetic tools allows for the study of basic vertebrate biology at the molecular level (Driever et al., 1994). The zebrafish model is also being used for pharmacological and toxicological studies due to the high level of conservation of functional substrate binding domains between the zebrafish and humans (Renier et al., 2007). Together, these attributes make the zebrafish a powerful model of human disease (Dooley and Zon, 2000). We previously characterized the developmental toxicity associated with an acute exposure to 6 different pyrethroid pesticides and found that type II pyrethroids were more toxic than type I pyrethroids, based on LC50 values. In addition, at higher concentrations, zebrafish embryos presented with tremors and spasms. Finally, it was determined that the tremors and spasms were the result of sodium channel activation since sodium channel blockade ameliorated these effects (DeMicco et al., 2010). The effects of pyrethroid exposure observed in the zebrafish embryo model are consistent with that reported in the mammalian literature, thus validating this model system. Here, the vertebrate zebrafish model was utilized to recapitulate the persistent motor deficits caused by embryonic exposure to the pyrethroid pesticide deltamethrin, and to test the hypothesis that dopaminergic dysfunction is responsible for mediating these effects. Zebrafish embryos were exposed to deltamethrin at concentrations below the lowest observed adverse effect level (LOAEL) (0.33–0.50 μg/l). At these concentrations, mortality, dysmorphogenesis, or acute signs of toxicity (tremors or spasms) do not occur. The data reveal that developmental exposure [3–72 h postfertilization (hpf)] to deltamethrin, led to increases in locomotor activity following a transition into darkness at the larval stage. Decreased DA receptor transcript levels and increased HVA levels accompanied these locomotor effects. Using methylphenidate, a DA transporter inhibitor, and a morpholino (MO) to knockdown DA transporter gene (slc6a3) expression, we demonstrated that DA dysfunction is a possible mechanism for the long-term locomotor deficits caused by developmental deltamethrin exposure.