Neural alterations from lead exposure in zebrafish

NTT-06486; No of Pages 9 Neurotoxicology and Teratology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera

Neural alterations from lead exposure in zebrafish

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Nicole M. Roy ⁎, Sarah DeWolf, Alexius Schutt, Ashia Wright

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Department of Biology, Sacred Heart University, Fairfield, CT, United States

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Article history: Received 20 May 2014 Received in revised form 26 August 2014 Accepted 27 August 2014 Available online xxxx

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Keywords: Zebrafish Development Lead Neural Vasculature

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Lead was used extensively as a gas additive and pesticide, in paints, batteries, lead shot, pipes, canning and toy manufacturing. Although uses of lead have been restricted, lead persists in our environment especially in older homes, and generally in soil and water. Although extensive studies have determined that fetal and childhood exposures to lead have been associated with childhood and adolescent memory impairments and learning disabilities, there are limited studies investigating early neural and morphological effects that may lead to these behavioral and learning abnormalities. Here we utilize the zebrafish vertebrate model system to study early effects of lead exposure on the brain. We treat embryos with 0.2 mM lead for 24, 48 and 72 h and analyze neural structures through live imagery and transgenic approaches. We find structural abnormalities in the hindbrain region as well as changes in branchiomotor neuron development and altered neural vasculature. Additionally, we find areas of increased apoptosis. We conclude that lead is developmentally neurotoxic to a specific region of the brain, the hindbrain and is toxic to branchiomotor neurons residing in rhombomeres 2 through 7 of the hindbrain and hindbrain central artery vasculature. © 2014 Elsevier Inc. All rights reserved.

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Although lead has been banned in the United States since the 1970s, lead is persistent in our environment (Tong et al., 2000). Soil remains contaminated with lead from old paint or past emissions of leaded gasoline. In addition, pollution from operating or abandoned industrial sites and smelters contributes to soil contamination. Leaded water pipes, found in many homes built before 1930 and soil erosion into water bodies contribute to water exposure (Philp, 2001; Tong et al., 2000). Although copper has replaced lead in newer construction, old leaded pipes are still found in homes and commercial buildings and soft water can cause increased leaching (Philp, 2001). In fact, a study performed in Washington DC schools in 2008 demonstrated that tap water in the school drinking water fountains contained up to 41% lead (Triantafyllidou et al., 2009). A number of activities and hobbies can expose humans to lead including pottery, stained glass and home repairs. Food crops may be contaminated by lead in agricultural water or soil. Because of the long term and widespread contamination of lead in the environment, lead exposure is a global concern (Tong et al., 2000). Lead exposure is particularly toxic to the brain. Blood lead levels (BLLs) and the effects on learning and behavior have been studied (Needleman, 2004) and CNS (central nervous system) effects can occur at very low BLL. For children, an overall decrease in cognitive ability including reduced IQ (Intelligence Quotient), increased

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1. Introduction

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⁎ Corresponding author at: Sacred Heart University, Biology Department, 5151 Park Ave, Fairfield, CT 06825, Unites States. Tel.: +1 203 365 4772; fax: +1 203 365 4785. E-mail address: [email protected] (N.M. Roy).

hyperactivity, ADHD (Attention Deficit Hyperactivity Disorder), and memory and behavioral disorders are among the main effects of lead neurotoxicity (Lidsky and Schneider, 2003, Needleman, 2004). Lead can easily pass through the placenta and affect the developing fetal brain and produce slow body growth. Based on a wealth of studies that demonstrated that even very low levels of lead can cause lifelong health effects, the Centers for Disease Control (CDC) changed its “blood lead level of concern” to 10 μg/dl in January 2012 (CDC, 2012). Zebrafish are often used as a model of vertebrate development and toxicity studies (Dai et al., 2014, Hill et al., 2005, Parng et al., 2007, Teraoka et al., 2003). Sequencing of the zebrafish genome has shown that 70% of human genes have an analogous gene in the zebrafish (Howe et al., 2013). This similarity to humans and other vertebrates allows for the modeling of toxin and disease mechanisms. Details of the morphological and physiological characteristics are known for all stages of zebrafish development. Embryos develop ex utero and are transparent through the early stages of development allowing morphometric analysis (Padilla et al., 2012). The availability of fluorescent transgenic zebrafish, coupled with embryonic transparency, has allowed real time, in vivo analysis to be completed on toxin exposed embryos (Dai et al., 2014; Higashijima, 2008). This offers an advantage to traditional techniques like in situ hybridization or immunolocalizations where embryos must be fixed in time allowing only certain time windows of gene expression to be analyzed. Previous studies in zebrafish found that a 72 hour lead exposure resulted in gene alterations associated with neurodevelopment (Peterson et al., 2011). Specifically lead exposed zebrafish embryos were analyzed for global transcriptional alterations at 72 and 120 hour post fertilization (hpf) time points using microarray approaches.

http://dx.doi.org/10.1016/j.ntt.2014.08.008 0892-0362/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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(Struzynska et al., 2001). Impaired neuronal morphogenesis has been noted in tadpoles (Cline et al., 1996). Many questions remain on how lead affects the brain, specifically in the early neural developmental stages. In the lab setting, zebrafish embryos are commonly exposed to chemicals at high concentrations during early life stages to screen for potential toxicity (Hill et al., 2005). In fact, the EPA (Environmental Protection Agency) has developed the ToxCast™ program which treats embryos at early developmental stages (6–8 h) with toxic chemicals at relatively high doses to assess overt and organismal toxicity (Padilla et al., 2012) to support predictive human modeling of developmental toxicity. Because zebrafish follow the vertebrate developmental plan and share the same developmental signaling pathways as higher order mammals, including humans, they can provide information relevant to overt and organismal toxicity (Padilla et al., 2012). Furthermore, in vivo toxicity can be assessed because zebrafish develop metabolic

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Comprehensive studies on specific alterations in neural morphology have been limited. However, lead mediated alterations in specific axon tracts and the role of axonogenesis related genes demonstrated decreased axonal density in axon tracts of the midbrain and forebrain concomitant with down-regulation of axonogenesis related genes before 24 hpf (Zhang et al., 2011). Dou and Zhang (2011) reported that a reduction in nerve cells due to increased apoptosis of neuron and glial cells leads to impaired neurogenesis (Dou and Zhang, 2011). Behaviorally, lead exposure has been linked to behavioral alterations, learning and memory deficits in adult zebrafish (Chen et al., 2012) as well as sensorimotor deficits (Rice et al., 2011). However, other than the few studies mentioned, lead developmental toxicity has not been fully characterized in zebrafish. Early acute lead exposure has been studied in rat models and resulted in impaired cognitive function, learning impairments and behavioral abnormalities

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Fig. 1. Developmental time course of lead exposure in live embryos. (A, C, E) Control and (B, D, F) lead treated shown in lateral views. (A, B) 24 h, (C, D) 48 h and (E, F) 72 h. fb: forebrain, mb: midbrain, hb: hindbrain, ov: otic vesicle. Asterisk denotes lead treatment value of less than 0.05.

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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2. Methods

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2.1. Adult and embryo handling

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Wild-type AB strain and transgenic adult zebrafish were housed in a ZMOD (Zebrafish Module) System (Aquatic Habitats Inc.) on a 14:10 hour light:dark cycle. Adults were fed brine shrimp daily. Water quality was tested daily and daily water changes preformed. Transgenic fish islet-1 gfp (green fluorescent protein) were attained from the Linney Lab (Duke University Medical Center) and fli-1 gfp transgenic fish as a generous gift from the Lawson Lab (University of Massachusetts Medical Center). Embryos were generated by natural pair-wise mating in zebrafish mating boxes (Westerfield, 1993). Embryos were placed in Petri dishes in 30% Danieau Buffer diluted from a 50× stock solution.

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2.3. Microscopy and measurements

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Live images were taken under a Leica dissection microscope attached to a digital camera using QCapture Software. Embryos were placed in 3% methylcellulose for positioning purposes in a depression slide. Tricaine methanesulfonate (MS-222) (Westerfield, 1993) was

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Embryos were collected and transferred to control (30% Danieau Buffer) or 0.2 mM lead acetate (Sigma Aldrich, St. Louis, MO) diluted in 30% Danieau Buffer at 12 hpf and treated continuously until 24, 48 or 72 hr time points when they were released from lead exposure to 30% Danieau Buffer. Doses were chosen based on previous studies (Dou and Zhang, 2011) and further explained in the Results section. Embryos were raised at 28.5 °C in standard Petri dishes. Developmental toxicity was monitored daily, however at the dose tested, 0.2 mM, embryos demonstrated 100% survival at 24 hpf, 48 hpf and 72 hpf.

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2.2. Lead acetate solutions and exposure protocols

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The 50× Danieau's solution contains 169.475 g NaCl, 2.61 g KCl, 4.93 g MgSO4 7H2O, 7.085 g Ca(NO3)2 4H2O, and 0.5 M Hepes at a pH of 7.6. Water is added to a final volume of 1 l. The solution is then autoclaved. A 30% Danieau's solution was prepared by mixing 6 ml of the 50× concentrated solution into 1 l of dH2O at 28 °C. Zebrafish were staged in accordance with standard staging series (Kimmel et al., 1995). All treatments were approved and meet ethical standards by the Sacred Heart University IACUC (Institutional Animal Care and Use Committees) committee.

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organs like the liver and share similar metabolic enzymes (Padilla et al., 2012). These procedures are also commonly performed for heavy metals, pesticides and other environmental contaminants (Chow et al., 2008). Using this rationale, we sought to treat zebrafish embryos during early neural developmental time windows (12–72 hpf) to assess the developmental toxicity of lead. We hypothesize that lead exposure will negatively impact the general brain morphology and vascularization as well as neuron development and migration. Here, we seek to investigate the effects of lead looking at gross brain morphology and using a transgenic approach to analyze branchiomotor neuron development and neural vasculature. Here, we find alterations in neural ventricle formation, hindbrain branchiomotor neurons, neural vasculature and increased apoptosis.

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Fig. 2. Brain ventricle measurements at 24, 48 and 72 hpf. Measurements are in millimeters. Each value represents means ± standard deviation of a total of 30 embryos (three independent experiments with an n of 10 each for control and lead treated were performed). Asterisk denotes lead treatment value of less than 0.05. A plus symbol denotes a significant effect of time (p b 0.05).

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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Acridine Orange (Sigma Aldrich, A6014) was prepared in a stock concentration of 1 mg/ml. Embryos were treated in 1 μg/ml diluted in 30% Danieau Buffer and incubated for 1–2 h after which they were washed extensively in 30% Danieau Buffer prior to imaging. Embryos were analyzed using a fluorescent microscope under the FITC (Fluorescein Isothiocyanate) filter as described above in Section 2.3. A total of ten embryos for control and lead treated were analyzed for apoptosis.

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2.4. Apoptosis staining

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used to anesthetize highly mobile 72 hpf embryos. Fluorescent images were taken using the above embryo protocols using a Nikon Eclipse E400 fluorescent microscope. Transgenics were treated with 1× phenylthiourea to prevent melanin formation which would obscure fluorescent signal (Westerfield, 1993). A total of ten embryos for control and lead treated were analyzed for all live and transgenic embryos for three independent experiments. Embryos were randomly selected from the control or lead treated dishes. Images were analyzed in Photoshop and measurements were taken utilizing the ruler function.

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Fig. 3. Branchiomotor neuron development as visualized by islet-1 gfp transgenics. (A, C) Control and (B, D, E) lead treated all in dorsal views. 48 h (A, B) and 72 h (C–E). Control embryos at 48 and 72 hpf demonstrate normal development and migration of branchiomotor neurons in the hindbrain and normal axonal projections. (r) rhombomere, nV, trigeminal neurons, nVII facial neurons, nX, vagal neurons. (B) Lead exposed embryos at 48 hpf show no alterations. (D, E) By 72 hpf, lead exposed embryos show decreased and disorganized neural clusters.

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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Embryos were randomly selected from the control or lead treated dishes.

determined that this dose leads to altered swimming movements and 216 Q7 slowed escape responses in larval fish. 217

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3.2. General brain and body morphology

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3. Results

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3.1. Determination of concentration

Although previous studies have investigated changes to neural gene expression in lead exposure by in situ hybridization and immunostaining these techniques involve embryo fixation or homogenization (PCR, polymerase chain reaction). No live images of lead treatments at a magnified diagnostic level were found in the literature and fixation can alter visual structures. To investigate general in vivo neural structural changes, embryos were examined at 24, 48 and 72 hpf. No differences in mortality or hatching numbers were seen at any time point. No visual changes were seen in the developing embryos at 24 and 48 hpf (Fig. 1A–D) in the fore, mid, or hindbrain regions and the size of the brain ventricles was not affected by lead exposure (Fig. 2). A 2-way ANOVA detected no effect of lead in the forebrain (p = 0.349) or midbrain (p = 0.818), but a change in the size of the forebrain was detected over time (p ≤ 0.001), consistent with brain growth. By 72 h of lead exposure, alterations in brain architecture became apparent (Fig. 1E, F). The 2-way ANOVA showed a significant effect of lead treatment in the hindbrain (p ≤ 0.001), in addition to the growth of the hindbrain over time (p ≤ 0.001). There was a significant interaction between time and lead treatment (p ≤ 0.001). It appears that lead had the greatest effect after 72 hpf (Fig. 2).

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For each trial, a total of 10 embryos in the control and lead treatment were imaged and measured at 24, 48 or 72 hpf. A total of three independent trials were performed. Thus, the total n for control and lead treatment at the three time points tested was 30. Embryos were chosen at random from the treatment dishes. The PASW (Predictive Analytics Software) statistics program was utilized. A Shapiro–Wilk normality test and a Levene's homogeneity of variance test were performed prior to conducting a 2-way ANOVA with factors of time (24, 48, 72 hpf) and treatment (control or lead). No post-hoc comparisons were run to determine which time points differed from each other, due to significant interactions in the 2-way ANOVAs (Quinn and Keough, 2002). A post-hoc comparison was not required to determine the effects of lead because there were only 2 levels of lead treatment (control and lead). A lead treatment p-value less than 0.05 is indicated with asterisk and a plus symbol is used to indicate a significant effect of time (p b 0.05).

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An initial LD50 experiment was performed at 72 hpf utilizing a high concentration of 2 mM and decreasing concentrations including 1.6, 1.2, 0.8, 0.4, 0.2 and 0 mM lead control. Treatments began at 12 hpf and continued until 72 hpf. The 72 hpf LD50 was determined to be 0.37 mM, which produced 50% death. However, the 50% of surviving embryos were grossly malformed demonstrating massive cardiac edema, yolk sac edema and extensive spinal bending. At the next lowest dose tested, 0.2 mM, embryos demonstrated 100% survival at 24 hpf, 48 hpf and 72 hpf, but demonstrated neural and vascular alterations. Thus, the 0.2 mM dose was chosen for the remainder of the experiments. At the 72 hpf time point, embryos were released from lead exposure into the embryo medium and raised in 30% Danieau Buffer. The 0.2 mM dose was also utilized by Dou and Zhang (2011) who

3.3. Hindbrain islet-1 green fluorescent protein transgenic neurons

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Analysis of branchiomotor neurons using islet-1 gfp embryos demonstrated no changes at 48 h (Fig. 3A–B), but noticeable changes in branchiomotor neuron cell bodies and axonal projections were seen at 72 hpf (Fig. 3C–E). At 48 and 72 hpf control embryos showed normal clustering of nV trigeminal neuronal cell bodies in rhombomeres (r) r2 and r3 with axons exiting r2 through the mandibular arch innervating the jaw. The nVII facial neurons have migrated caudally and were clustered in r6 and r7 with axons exiting r4 through the hyoid arch innervating the face (Fig. 3A, C). Lead treated embryos were normal at 48 hpf (Fig. 3B), but showed noticeable loss of cell bodies as well as loss of organization of the cell body clusters at 72 h (Fig. 3D, E). It was

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Fig. 4. Branchiomotor neuron measurements from rhombomeres 2–7. Measurements are in millimeters. Each value represents means ± standard deviation of a total of 30 embryos (three independent experiments with an n of 10 each for control and lead treated were performed). Asterisk denotes lead treatment value of less than 0.05. A plus symbol denotes a significant effect of time (p b 0.05).

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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3.4. Brain vascularization

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The fli-1 gfp transgenic was utilized to assess vascular changes in the brain. No changes were seen by 48 hpf in control or lead exposed embryos (Fig. 5A, B) looking at general vascular morphology. The midcerebral vein (MCeV) was strong and apparent in both, as was the central artery (CtA) network which was beginning to sprout. By 72 h, noticeable loss of vasculature was seen in lead treatments. Notably, severe changes in the central artery (CtA) were seen, including loss of

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Acridine orange live staining of control embryos demonstrated no apoptotic cells in the brain tissue at 48 or 72 hpf (Fig. 7A, C, E). Lead treated embryos were comparative to control at 48 h (Fig. 7B), but demonstrated clusters of apoptotic cells in the brain by 72 h (Fig. 7D, F). A

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vasculature, disorganized vasculature and general loss of patterning compared to control, but the mid-cerebral vein was apparent (Fig. 5C– F). Length measurements were made along the rostral–caudal axis at 48 and 72 h. A 2-way ANOVA detected no change in the length of the CtA (p = 0.615) over time. The 2-way ANOVA showed a significant effect of lead treatment in the CtA (p ≤ 0.001). There was a significant interaction between time and lead treatment (p ≤ 0.001). It appears that lead had the greatest effect after 72 hpf (Fig. 6). Total lengths for the CtA measurements were less than the length of the entire hindbrain (Fig. 2) as only the CtA network within the hindbrain was measured and not the entire hindbrain vasculature network which was too complex to accurately measure without a confocal study.

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difficult to assess nV from nVII neurons due to the disorganization and loss of the canonical pattern. A 2-way ANOVA detected a change in the length of rhombomeres 2–7 within the hindbrain over time (p ≤ 0.001). The 2-way ANOVA showed a significant effect of lead treatment in rhombomeres 2–7 (p ≤ 0.001). There was a significant interaction between time and lead treatment (p ≤ 0.001). It appears that lead had the greatest effect after 72 hpf (Fig. 4). Total lengths for branchiomotor measurements were less than the length of the entire hindbrain (Fig. 2) as the green fluorescent islet signal only fluoresces in a portion of the hindbrain.

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Fig. 5. Neural vasculature as visualized with fli-1 gfp transgenics. (A, C, E) Control, (B, D, F) lead in lateral (A–D) or dorsal (E, F) views. (A, B) Control and lead brains at 48 h demonstrate a strong mid-cerebral vein (MCeV) and developing central arteries (CtA) are apparent. (C, E) Control at 72 h demonstrates a more developed and complex web of the CtA vascular network. (D, F) Lead exposed embryos demonstrate an overall decrease in vasculature, the MCeV is apparent, but the CtAs are stunted and few.

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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qualitative assessment was made given the three-dimensionality of the brain and the limitations of a non-confocal microscope.

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Zebrafish are a particularly useful model for studying developmental toxicity (Hill et al., 2005; Teraoka et al., 2003). Development follows the same pathways as higher order vertebrates and in particular, neural development is highly conserved with the human brain (Zhao et al., 2006). Additionally, the ability to generate transgenic embryos has allowed real time, in vivo examination of changes that can occur in response to toxin challenge (Dai et al., 2014). In this study, we demonstrated that lead exposure caused structural defects in the brain, changed vasculature in the brain, altered branchiomotor neuron patterning in the hindbrain and increased areas of apoptosis. Relatively few studies have been conducted on lead and zebrafish. Of these, behavior in relation to startled response, memory deficits, swimming patterns and escape actions has been studied (Chen et al., 2012; Dou and Zhang, 2011; Rice et al., 2011). Global gene and neurological gene alterations were assessed through microarray technologies (Peterson et al., 2013, 2011). Decreases in axonal density and changes in axonal guidance genes have been examined (Zhang et al., 2011) and impaired neurogenesis and increased neural apoptosis have been seen (Dou and Zhang, 2011). However, much is left to be investigated. Interestingly, none of these studies have closely examined live images at magnified levels to assess structural changes. Most assess alterations using in situ hybridization, immunostaining or PCR technologies. These traditional approaches to gene expression or protein localization require tissue fixation or embryo homogenization. Our developmental neurotoxicity assessments showed no alterations in development at 24 or 48 hpf. However, by 72 hpf, defects in the brain were seen. Control embryos clearly showed well-defined forebrain, midbrain and hindbrain ventricles by 24, 48 and 72 hpf. Lead exposed embryos appeared visually normal at 24 and 48 h and measurements of the fore, mid and hindbrain ventricles demonstrated no change compared to control. However, by 72 hpf, lead exposed embryos demonstrated decreased hindbrain ventricle size with a p-value b 0.05 (Figs. 1 and 2). As a decrease in the hindbrain ventricle was observed under gross morphology, we utilized the islet-1 gfp transgenic fish which expresses specifically within the hindbrain region (Higashijima et al., 2000). During normal development, the fifth cranial motor neuron (trigeminal

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nerve) cell bodies arise in rhombomeres (r) 2 and 3 and their axons exit out of r2 through the mandibular arch and innervate the muscles for mastication. The seventh cranial motor neuron (facial nerve) cell bodies are born in r4 and migrate caudally into r6 and r7 with axonal projections exiting r4 through the hyoid arch innervating the face. The tenth cranial motor neuron (vagal nerve) cell bodies are generated exclusively in caudal hindbrain and innervate gill muscles (Chandrasekhar, 2004, 1997; Higashijima et al., 2000). In control embryos, cell bodies, caudal migration, clustering and axonal projections were clearly seen by 48 h (Fig. 3A). By 72 h, the same pattern was seen visually, cell bodies and clusters were imaged, but axonal projections were less clear in images due to increased brain thickness (Fig. 3C). In lead treated embryos, the same 48 hr neuronal pattern was seen with no noticeable changes from controls (Fig. 3B). By 72 hpf, a loss of cell bodies and a general disorganization of cell clusters was seen (Fig. 3D, E). The length of r2–r7 was decreased significantly (Fig. 4). No other studies have examined live neurons in this way, however, Zhang et al. (2011) have investigated time point specific alterations of axonal density using immunofluorescence staining. Using a 100 ppb dose, they have found that lead developmental neurotoxicity was partially mediated through alterations in neuronal growth and changes in axon tracts through 36 hpf. Interestingly however, they found that later stages demonstrate axonal densities similar to control (Zhang et al., 2011). The differences between our studies are most likely due to the concentration of lead, but bring up interesting questions regarding the mechanisms of lower and more toxic doses of lead. To investigate if a loss of blood flow could be contributing to the alterations in the brain, fli-1 gfp transgenic fish were analyzed. fli-1 is an endothelial marker and the fli-1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis (Lawson and Weinstein, 2002). At 48 h, no changes in the developing vasculature were detected between control and lead treated embryos (Fig. 5A, B). By 72 hpf, in control embryos, the vasculature was strong and pronounced in the fore, mid and hindbrain regions. Specifically, the hindbrain's CtA demonstrated well developed formation in the rhombomeres (Fig. 5C, E) (Ulrich et al., 2011). However, lead treated embryos demonstrated a loss of intrarhombomeric vasculature. There was a significant decrease of the CtA along the rostral–caudal axis (Fig. 6). Remaining arteries were disorganized and unidentifiable, but the mid-cerebral vein was present (Fig. 5D, F). To fully analyze loss and changes of the vasculature, a

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Fig. 6. Neural vascular measurements at 48 and 72 hpf. Measurements of the CtA along the rostral–caudal axis are in millimeters. Each value represents means ± standard deviation of a total of 30 embryos (three independent experiments with an n of 10 each for control and lead treated were performed). Asterisk denotes lead treatment value of less than 0.05.

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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Fig. 7. Apoptosis in the neural tissue. (A, B) 48 h and (C–F) 72 h. (A, C, E) Control and (B, D, F) lead treated. (A, C, E) Control brains show no apoptotic spots at 48 or 72 hpf in lateral (A, C) or dorsal (E) views. Lead treated embryos in lateral view demonstrate no apoptotic spots in the brain ventricles at 48 h (B), but show numerous apoptotic spots by 72 h in lateral (D) or dorsal (E) views.

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confocal study should be performed, however, this was beyond the scope of this study. Dou and Zhang (2011) have utilized TUNEL staining to assess neural apoptosis and they found increased neural apoptosis in the mid and hindbrain regions at 24 hpf (Dou and Zhang, 2011). Alterations at later stages of development were not studied. Interestingly, we did not detect any changes in neural morphology, neural vasculature or branchiomotor neuron development and migration until 72 hpf using the same 0.2 mM dose of lead. Although Dou and Zhang (2011) detect apoptosis by 24 hpf, we did not detect any apoptotic cells until 72 hpf using acridine orange staining (Fig. 7). Peterson et al. (2013) have also

investigated apoptosis, but they did not see a change in apoptotic cells in the brain up to 96 hpf, although they used a lower dose of lead (Peterson et al., 2013). The apoptosis we detected by 72 h is not restricted to only the hindbrain, and may reflect general developmental neurotoxicity of lead by this later time point. There have been limited in situ hybridization studies to assess changes in gene expression in response to lead treatment during early time windows. In a 2011 study, gfap expression domains were reduced in the embryonic midbrain and hindbrain at 24 hpf and huC expression was reduced, but only in the midbrain (Dou and Zhang, 2011). Genes involved in neurogenesis ngn1 and crestin have also been studied and

Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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Nothing declared.

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Transparency document

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The Transparency document associated with this article can be found, in the online version.

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We would like to thank the Linney Lab and the Lawson lab for donated transgenic fish and Denise Zannino and Charles Sagerstrom for their assistance. We would also like to thank Dr. Latina Steele for assistance with the statistical analysis.

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showed no changes in gene expression patterns compared to control (Dou and Zhang, 2011). Reelin glycoprotein plays a critical role during neuronal development. However, lead exposure did not affect the expression of reln in cellular populations from 24–96 hpf (Peterson et al., 2013). One of the problems with an in situ approach to assessing changes in response to lead exposure is that the hindbrain is transient and undergoes numerous changes in gene expression during its development due to signaling molecules and upregulation of various transcription factors. In situ hybridizations for early hindbrain development markers hoxb1a, hoxa2b, krox-20 were performed prior to 24 h when they are expressed, but control and lead showed no differences (data not shown). This seems consistent with the fact that there were no alterations at 24 h in hindbrain structure, vasculature, or apoptosis. However, these markers do not express at later developmental stages. We performed in situ hybridizations with markers of the hindbrain in later stages nkx6.1, pax6 and pax7. No changes were seen when they are strongly expressed at 48 h, again consistent with no changes detected in gross morphology, vasculature or branchiomotor expression at this time point. Although these markers are expressed later, their expression is much fainter in the hindbrain. For example, pax6 by 72 h becomes shifted to the midbrain and the retina and is only faintly expressed in the hindbrain so it was very difficult to assess if there was a change or a loss in lead given the expression is faint in the hindbrain in control embryos. Because the bulk of hindbrain research is focused on early patterning phases when rhombomeres are forming and the neurons developing within those rhombomeres, there are fewer tools to assess the hindbrain at later stages. A complete study of hindbrain markers would be very complex and requires hundreds of in situ probes and many more time windows as genes change extensively during hindbrain development due to different signaling centers, positive and negative feedback loops and up or down-regulation of genes. These experiments are beyond the scope of this study, but would provide interesting data on gene expression changes during hindbrain development. The zebrafish model is a widely accepted model for vertebrate developmental toxicity. It is particularly useful to detect neural tube defects (NTDs) from early-life exposures (Lee and Freeman, 2014) and relevant given the organization of the major brain structures is highly conserved with the human brain (Zhao et al., 2006). Environmental lead exposure is a global concern given the years of environmental pollution and thus, understanding the mechanism of lead developmental neurotoxicity is important to elucidate. The EPA's current lead action level in drinking water is 15 ppb, considerably lower than the 0.2 mM dose utilized in this study. However, our approach was not to mimic an infant's low dose exposure over a long period of time from daily exposure, but rather to follow the EPA's ToxCast™ approach to model developmental toxicity as described in the Introduction. Here we provide evidence that lead exposure leads to altered brain ventricle formation and neurotoxicity associated with neural vasculature and branchiomotor neuron development. Furthermore, increased neural apoptosis was noted. Although this study provides preliminary information on the toxicity of lead, much remains to be studied to elucidate the mechanisms of lead toxicity in zebrafish.

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Please cite this article as: Roy NM, et al, Neural alterations from lead exposure in zebrafish, Neurotoxicol Teratol (2014), http://dx.doi.org/ 10.1016/j.ntt.2014.08.008

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