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Brominated flame retardants (BFRs) and heavy metals (HMs) are two main types of pollutants in electronic waste recycling sites, which are also ubiquitously detectable in environmental media and human tissues. However, the adverse health effects of exposure to the mixture of these types of pollutants are unknown. In this study, we investigated the reproductive toxicity of a mixture of decabromodiphenyl ether (BDE-209), tetrabromobisphenol A, cadmium chloride, and lead acetate (PbAc) at the environmental relevant levels. Zebrafish were waterborne and exposed to chemical mixtures for one generation. The reproductive effects were evaluated for F0 adults and F1 offspring. Chemical residues were also analyzed in the exposed adults and their eggs at the end of exposure. Our findings demonstrated that exposure to the chemical mixture for 150 days had no effect on the survival rate of zebrafish, but it decreased body length and weight in females and increased body weight and condition factor in males. The mixture exposure resulted in a female-biased sex ratio in adults and decreased sperm density and motility in males and egg production in females. For the F1 offspring, decreased fertilization, delayed hatching, and increased malformation were found in all exposure groups. In conclusion, chronic co-exposure to BFRs and HMs at the environmental relevant levels not only affected growth, sex ratio, and sperm quantity/quality and egg production in adults but also reduced the reproductive success in the offspring, implying that multi-pollutants in the environmental media may pose a public health risk to other exposed organisms or human beings.

Perfluorooctane sulfonic acid (PFOS), as a potential endocrine disrupting chemical, is widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on thyroid in aquatic organisms and the underlying mechanisms are largely unknown. The present study assessed the effect of chronic PFOS exposure on thyroid structure and function using zebrafish model. Zebrafish at 8 h post fertilization (hpf) were exposed to PFOS (250 µg/l) until 120 d post fertilization (dpf). Thyroid hormone (T3 and T4) level, thyroid morphology and thyroid function related gene expression were evaluated in zebrafish at 120 dpf. Our findings demonstrated that chronic PFOS exposure altered thyroid hormone level, thyroid follicular cell structure and thyroid hormone related gene expression, suggesting the validity of zebrafish as an alternative model for PFOS chronic toxicity screening.

Benzene exposure affects the hematopoietic system and leads to the occurrence of various types of leukemia and hematotoxicity. It has been confirmed that active metabolites of benzene, including 1,4-benzoquinone (1,4-BQ), can induce reactive oxygen species (ROS) and apoptosis in the bone marrow, and recent studies have also suggested that benzene exposure can affect mitochondrial function in both experimental animals and cell lines. However, the potential relationship among ROS production, mitochondrial damages, and subsequent apoptosis following benzene exposure has not been well studied in detail. In the present study, we utilized HL-60 cells, a well-characterized human myeloid cell line, as an in vitro model and examined the effects of 1,4-BQ on intracellular ROS formation, mitochondria damage, and the occurrence of apoptotic events with or without using the ROS scavenger N-acetyl-l-cysteine (NAC). The results demonstrated that 1,4-BQ could dose-dependently induce production of ROS and mitochondrial damage as characterized by mitochondrial membrane potential disruption, mitochondrial ultrastructure alteration, and induced apoptosis and activated caspase-3 and caspase-9. Preincubation of HL-60 cells with NAC prior to 1,4-BQ treatment could block 1,4-BQ-induced production of ROS and the occurrence of apoptosis. These results demonstrated that 1,4-BQ induced apoptosis in HL-60 cells through a ROS-dependent mitochondrial-mediated pathway.

Perfluorooctanesulfonic acid (PFOS), a ubiquitous contaminant, has been used in various industrial applications. Currently few studies have documented the effects of chronic PFOS exposure on lipid metabolism, especially in aquatic organisms. The present study defined the effects of chronic exposure to low level of PFOS on lipid metabolism in F0 adult zebrafish and F1 offspring. Our findings revealed a severe fatty degeneration in the liver of F0 males treated with 0.5 μM PFOS and significant ultrastructure changes associated with substance transport or metabolism in liver and intestines (abnormal mitochondria and endoplasmic reticulum, disordered arrangement of inner microvilli within intracellular canaliculus). To address the potential trans-generational effects of PFOS exposure, the early gene expression related to lipid metabolism was measured by real-time quantitative polymerase chain reaction in F1 derived from chronically exposed parental fish. The results indicated that lepa (leptin α), kiss1 (kisspeptins), xdh (xanthine dehydrogenases), and insr (insulin receptor) were significantly upregulated in F1 while dgat1b (diacylglycerol O-acyltransferase), hb9 (motor neuron/pancreas homeobox), and Apoa1 (apolipoprotein A-I) were downregulated. These findings provided evidence that PFOS chronic exposure adversely impacts lipid metabolism in both F0 and F1 and demonstrated the validity of using zebrafish as an alternative model for PFOS chronic toxicity screening.

Perfluorooctane sulfonate (PFOS), one persistent organic pollutant, has been widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on lipid metabolism, especially in aquatic organisms. The underlying mechanisms of hepatotoxicity induced by chronic PFOS exposure are still largely unknown. The present study defined the effects of chronic exposure to low level of PFOS on lipid metabolism using zebrafish as a model system. Our findings revealed a severe hepatic steatosis in the liver of males treated with 0.5μM PFOS as evidenced by hepatosomatic index, histological assessment and liver lipid profiles. Quantitative PCR assay further indicated that PFOS significantly increase the transcriptional expression of nuclear receptors (nr1h3, rara, rxrgb, nr1l2) and the genes associated with fatty acid oxidation (acox1, acadm, cpt1a). In addition, chronic PFOS exposure significantly decreased liver ATP content and serum level of VLDL/LDL lipoprotein in males. Taken together, these findings suggest that chronic PFOS exposure induces hepatic steatosis in zebrafish via disturbing lipid biosynthesis, fatty acid β-oxidation and excretion of VLDL/LDL lipoprotein, and also demonstrate the validity of using zebrafish as an alternative model for PFOS chronic toxicity screening.

© The Author 2016. Polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants associated with adverse neurodevelopmental outcomes in children and preclinical models; however, the mechanisms by which PBDEs cause developmental neurotoxicity remain speculative. The structural similarity between PBDEs and nondioxin-like (NDL) polychlorinated biphenyls (PCBs) suggests shared toxicological properties. Consistent with this, both NDL PCBs and PBDEs have been shown to stabilize ryanodine receptors (RyRs) in the open configuration. NDL PCB effects on RyR activity are causally linked to increased dendritic arborization, but whether PBDEs similarly enhance dendritic growth is not known. In this study, we quantified the effects of individual PBDE congeners on not only dendritic but also axonal growth since both are regulated by RyR-dependent mechanisms, and both are critical determinants of neuronal connectivity. Neuronal-glial co-cultures dissociated from the neonatal rat hippocampus were exposed to BDE-47 or BDE-49 in the culture medium. At concentrations ranging from 20pM to 2 μM, neither PBDE congener altered dendritic arborization. In contrast, at concentrations ≥ 200 pM, both congeners delayed neuronal polarization resulting in significant inhibition of axonal outgrowth during the first few days in vitro. The axon inhibitory effects of these PBDE congeners occurred independent of cytotoxicity, and were blocked by pharmacological antagonism of RyR or siRNA knockdown of RyR2. These results demonstrate that the molecular and cellular mechanisms by which PBDEs interfere with neurodevelopment overlap with but are distinct from those of NDL PCBs, and suggest that altered patterns of neuronal connectivity may contribute to the developmental neurotoxicity of PBDEs.

As a persistent organic contaminant, perfluorooctanesulphonic acid (PFOS) has been widely detected in the environment, wildlife, and humans. The present study revealed that zebrafish embryos exposed to 16 µM PFOS during a sensitive window of 48–96 hour post-fertilization (hpf) disrupted larval morphology at 120 hpf. Malformed zebrafish larvae were characterized by uninflated swim bladder, less developed gut, and curved spine. Histological and ultrastructural examination of PFOS-exposed larvae showed structural alterations in swim bladder and gut. Whole genome microarray was used to identify the early transcripts dysregulated following exposure to 16 µM PFOS at 96 hpf. In total, 1,278 transcripts were significantly misexpressed (p < 0.05) and 211 genes were changed at least two-fold upon PFOS exposure in comparison to the vehicle exposed control group. A PFOS-induced network of perturbed transcripts relating to swim bladder and gut development revealed that misexpression of genes were involved in organogenesis. Taken together, early life stage exposure to PFOS perturbs various molecular pathways potentially resulting in observed defects in swim bladder and gut development.

Bisphenol A (BPA) is a ubiquitous contaminant in environment and human body. The reproductive and developmental effects of BPA exposure in aquatic and laboratory animals have been extensively studied. However, BPA exposure on the nervous system and motor behavior development are not well understood. In this study, we utilized zebrafish embryo as a model system to investigate the effect of developmental BPA exposure on larval teratology, motor behaviors, axonal growth of spinal motoneurons and muscle structure at various developmental stages. Our findings revealed that BPA exposure altered spontaneous movement, significantly decreased touch response and swimming speed in response to light stimulation in developing zebrafish. These effects were observed at the concentrations that did not yield any significant teratogenic effects. Correlated with those changes in swimming activity, BPA-induced axial muscle damage occurred at the same concentration range (1-15 μM), but disruption of axonal growth of primary and secondary motoneuron occurred only at higher concentration (15 μM). BPA-induced apoptotic cell death subsequent to initial ROS formation and oxidative DNA damage may be the underlying mechanism for axial muscle damage, suggesting the functional relevance of muscle structural changes and the observed deficits in swimming activity.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental pollutants detected in a wide variety of environmental matrixes and pose a significant public health concern. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is one of the most predominant PBDE congeners in environmental media, biota and human tissues. However, few studies have explored the BDE-47 developmental neurotoxicity and underlying mechanisms. In this study, zebrafish (Danio rerio) embryos were waterborne exposed to BDE-47 at 1.25, 5, 20 μM starting from 6h post-fertilization (hpf). Motor behavior development and swimming behavior in response to light-to-dark photoperiod stimulation were studied at various developmental stages. Our data indicate that BDE-47 exposure significantly affected spontaneous movement, decreased touch response and free swimming speed, altered larvae swimming behavior in response to light stimulation in developing zebrafish. Consistent with these motor deficits, BDE-47 significantly inhibited axonal growth of primary and secondary motor neurons during the early developmental stages, suggesting the functional relevance of structural changes. Our findings demonstrate that the altered patterns of neuronal connectivity may contribute to motor behavior deficits, indicating the relevance of zebrafish as a model for studying toxicant developmental neurotoxicity.

Background: Aroclor 1254 (A1254) interferes with normal dendritic growth and plasticity in the developing rodent brain, but the mechanism(s) mediating this effect have yet to be established. Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) enhance the activity of ryanodine receptor (RyR) calcium ion (Ca2+) channels, which play a central role in regulating the spatiotemporal dynamics of intracellular Ca2+ signaling. Ca2+ signaling is a predominant factor in shaping dendritic arbors, but whether PCB potentiation of RyR activity influences dendritic growth is not known. Objective: We determined whether RyR activity is required for PCB effects on dendritic growth. Methods and Results: Golgi analysis of hippocampi from weanling rats confirmed that developmental exposure via the maternal diet to NDL PCB-95 (2,2´,3,5´6-pentachlorobiphenyl), a potent RyR potentiator, phenocopies the dendrite-promoting effects of A1254. Dendritic growth in dissociated cultures of primary hippocampal neurons and in hippocampal slice cultures is similarly enhanced by PCB-95 but not by PCB-66 (2,3,4´,4-tetrachlorobiphenyl), a congener with negligible effects on RyR activity. The dendrite-promoting effects of PCB-95 are evident at concentrations as low as 2 pM and are inhibited by either pharmacologic blockade or siRNA knockdown of RyRs. Conclusions: Our findings demonstrate that environmentally relevant levels of NDL PCBs modulate neuronal connectivity via RyR-dependent effects on dendritic arborization. In addition, these findings identify RyR channel dysregulation as a novel mechanism contributing to dysmorphic dendritogenesis associated with heritable and environmentally triggered neurodevelopmental disorders.

Polybrominated diphenyl ethers (PBDEs) have been used extensively in electrical and electronic products, but little is known about the exposure level in the electrical appliance factories workers and nearby local residents. In this study, we assessed body burdens of PBDEs in 194 Chinese injection workers from electrical appliance factories and also measured 205 blood samples from catering workers, leather factory workers, umbilical cord and infertile men for comparison. Twelve PBDE congeners in serum samples were measured by GC–MS. The highest concentration for total PBDEs was found in injection workers, which is positively correlated to employment duration. BDE-209 was the most dominant congener followed by BDE-47, 28 and 99. We also found the presence of all twelve PBDEs in cord blood, suggesting an evidence of fetal exposure. Concentration of BDE-47 was particularly higher in serum samples from infertile men in comparison with that of catering workers and leather factory workers.

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants around the world. Because of large production volumes, widespread usage and persistence, PBDEs are now ubiquitous environmental pollutants detected in a wide variety of environment media and human samples and therefore pose a significant public health concern. Deca-PBDE (BDE-209) is the only commercial PBDE mixture still allowed for use at present, and has been recently detected at high levels in human samples. However, few studies explore its effect on development, reproduction or neurobehavior with animal models. In particular, studies with long-term chronic exposure at relatively low doses are lacking. In this study, we utilize the zebrafish model to explore the developmental, reproductive, and behavioral toxicities associated with long-term chronic exposure to deca-PBDE (BDE-209). Our findings revealed that long-term chronic exposure to low dose of deca-BDE (ranging from 0.001 to 1 μM) affected overall fitness (measured by condition factor), gonad development, male gamete quantity and quality in F0 parental fish. For F1 offspring without continuous exposure to BDE-209, parental BDE treatment led to delayed hatch and motor neuron development, loose muscle fiber, slow locomotion behavior in normal conditions, and hyperactivity when subjected to light-dark photoperiod stimulation. In conclusion, parental chronic low dose BDE-209 exposure not only affects F0 growth and reproduction, but also elicits neurobehavior alternations in F1 offspring.

BACKGROUND:Neurodevelopmental disorders are associated with altered patterns of neuronal connectivity. A critical determinant of neuronal connectivity is the dendritic morphology of individual neurons, which is shaped by experience. The identification of environmental exposures that interfere with dendritic growth and plasticity may, therefore, provide insight into environmental risk factors for neurodevelopmental disorders. OBJECTIVE:We tested the hypothesis that polychlorinated biphenyls (PCBs) alter dendritic growth and/or plasticity by promoting the activity of ryanodine receptors (RyRs). METHODS AND RESULTS:The Morris water maze was used to induce experience-dependent neural plasticity in weanling rats exposed to either vehicle or Aroclor 1254 (A1254) in the maternal diet throughout gestation and lactation. Developmental A1254 exposure promoted dendritic growth in cerebellar Purkinje cells and neocortical pyramidal neurons among untrained animals but attenuated or reversed experience-dependent dendritic growth among maze-trained littermates. These structural changes coincided with subtle deficits in spatial learning and memory, increased [3H]-ryanodine binding sites and RyR expression in the cerebellum of untrained animals, and inhibition of training-induced RyR upregulation. A congener with potent RyR activity, PCB95, but not a congener with negligible RyR activity, PCB66, promoted dendritic growth in primary cortical neuron cultures and this effect was blocked by pharmacologic antagonism of RyR activity. CONCLUSIONS:Developmental exposure to PCBs interferes with normal patterns of dendritic growth and plasticity, and these effects may be linked to changes in RyR expression and function. These findings identify PCBs as candidate environmental risk factors for neurodevelopmental disorders, especially in children with heritable deficits in calcium signaling.

Neonatal factors can cause latent functional changes that increase susceptibility to disease and/or dysfunction later in life. This hypothesis of the “developmental origins of adult disease” was originally derived from studies that identified stroke as one of several cardiovascular diseases associated with low birth weight (McMillen and Robinson 2005; Morley 2006). Subsequently, brief separations of infant mice from their mothers were demonstrated to increase their sensitivity to cerebral ischemic injury as adults, an effect mediated by functional changes in the hypothalamic–pituitary axis and inflammatory response (Craft et al. 2006). Recent evidence linking developmental exposure to environmental contaminants of concern to neurodegenerative diseases (Barlow et al. 2007; Bolin et al. 2006; Zawia and Basha 2005) suggests a third category of neonatal factors that influences susceptibility of the adult brain to injury, but whether these factors influence adult sensitivity to stroke has not been investigated. Polychlorinated biphenyls (PCBs) are organic compounds that were used mostly as coolants and lubricants from the 1930s until 1977, when they were banned. Despite the ban, PCBs persist in environmental samples, and high residue levels are still detected in foods, particularly fish (Kostyniak et al. 2005; Stewart et al. 1999), and in human tissue samples (Chu et al. 2003; Covaci et al. 2002; DeCaprio et al. 2005; Jursa et al. 2006). Epidemiologic and laboratory evidence suggests that PCBs increase the incidence of risk factors for stroke such as diabetes (Everett et al. 2007; Longnecker and Daniels 2001; Vasiliu et al. 2006) and vascular disease (Carpenter 2006; Hennig et al. 2005); also, living near hazardous waste sites contaminated with PCBs was recently linked to increased stroke incidence (Shcherbatykh et al. 2005). However, these studies did not address whether PCBs influence stroke outcome (the extent of damage occurring after a stroke) nor did they consider the contribution of developmental exposure to PCBs in determining adult susceptibility to cerebral ischemia. The developing nervous system is particularly sensitive to PCBs (Carpenter 2006; Schantz et al. 2003), and in experimental models, exposure of the developing brain to PCBs alters gene expression (Basha et al. 2006) and seizure susceptibility (Pessah IN, personal communication) in the adult brain. Factors known to influence stroke outcome include estrogen (Hurn et al. 2005) and preconditioning, a well-described phenomenon in which exposure to a mild ischemic or noxious stress up-regulates endogenous anti-oxidant and anti-inflammatory mechanisms to protect the brain against subsequent more severe ischemic injury (Koerner and Alkayed 2007). Although mechanisms of preconditioning are not completely understood, de novo gene expression in the brain is clearly required. Bcl2 (B-cell leukemia/lymphoma 2) and Cyp2C11 (cytochrome P450 2C11) are two genes whose up-regulation has been functionally implicated in preconditioning (Koerner and Alkayed 2007). Bcl2 protects neurons against ischemic injury via its anti-apoptotic activity (Kitagawa 2007; Mattson 2007), and CYP2C11 metabolizes arachidonic acid to expoxyeicosatrienoic acids, which are thought to protect against cerebral ischemia via vasodilatory, anti-inflammatory and antioxidant activities (Koerner and Alkayed 2007). Developmental PCB exposure modulates systemic (Meerts et al. 2004) and brain (Colciago et al. 2006) levels of estrogen, increases Bcl2 expression in adult testes (Hsu et al. 2007), and increases expression and activity of hepatic Cyp2C11 (Chen et al. 1992; Kramer et al. 1999). These observations suggest that developmental exposure to PCBs may alter stroke outcome. We tested this hypothesis using a well-established animal model of focal cerebral ischemia to quantify brain infarction in rats exposed throughout gestation and lactation to the commercial PCB mixture Aroclor 1254 (A1254). Although our findings are paradoxical in that developmental PCB exposure was observed to protect against stroke damage, they are of public health significance in that they identify developmental exposures to environmental contaminants as potentially significant determinants of adult susceptibility to cerebral ischemia.

Perfluorooctanesulfonate (PFOS) has been widely detected in the environment, wildlife and humans, but few studies have ever examined its mutagenic effect in vivo. In the present study, we use a transgenic fish model, the λ transgenic medaka, to evaluate the potential mutagenicity of PFOS in vivo following a subchronic exposure of 30 days. The mutant frequency of cII target gene was 3.46 × 10−5 in liver tissue from control fish, which increased by 1.4-fold to 4.86 × 10−5 in fish exposed to 6.7 μg/L PFOS, 1.55-fold to 5.36 × 10−5 in fish exposed to 27.6 μg/L PFOS, and 2.02-fold to 6.99 × 10−5 in fish exposed to 87.6 μg/L PFOS. This dose-dependent increase of mutant frequency was also accompanied with mutational spectrum changes associated with PFOS exposure. In particular, PFOS-induced mutation was characterized by +1 frameshift mutations, which increased from 0% in control fish to 13.2% in fish exposed to 27.6 μg/L PFOS and 14.6% in fish exposed to 87.6 μg/L PFOS. Our findings provide the first evidence of PFOS’s mutagenicity in an aquatic model system. Given the fact that most conventional mutagenic assays were negative for PFOS, we propose that PFOS-induced mutation in liver tissue of λ transgenic medaka may be mediated through compromised liver function.

Objective Perinatal exposure to polychlorinated biphenyls (PCBs) is associated with decreased IQ scores, impaired learning and memory, psychomotor difficulties, and attentional deficits in children. It is postulated that these neuropsychological deficits reflect altered patterns of neuronal connectivity. To test this hypothesis, we examined the effects of developmental PCB exposure on dendritic growth. Methods Rat dams were gavaged from gestational day 6 through postnatal day (PND) 21 with vehicle (corn oil) or the commercial PCB mixture Aroclor 1254 (6 mg/kg/day). Dendritic growth and molecular markers were examined in pups during development. Results Golgi analyses of CA1 hippocampal pyramidal neurons and cerebellar Purkinge cells indicated that developmental exposure to PCBs caused a pronounced age-related increase in dendritic growth. Thus, even though dendritic lengths were significantly attenuated in PCB-treated animals at PND22, the rate of growth was accelerated at later ages such that by PND60, dendritic growth was comparable to or even exceeded that observed in vehicle controls. Quantitative reverse transcriptase polymerase chain reaction analyses demonstrated that from PND4 through PND21, PCBs generally increased expression of both spinophilin and RC3/neurogranin mRNA in the hippocampus, cerebellum, and cortex with the most significant increases observed in the cortex. Conclusions This study demonstrates that developmental PCB exposure alters the ontogenetic profile of dendritogenesis in critical brain regions, supporting the hypothesis that disruption of neuronal connectivity contributes to neuropsychological deficits seen in exposed children.

Background Exposure to dichlorvos (DDVP), an organophosphorus pesticide, is known to result in neurotoxicity as well as other metabolic perturbations. However, the molecular causes of DDVP toxicity are poorly understood, especially in cells other than neurons and muscle cells. To obtain a better understanding of the process of non-neuronal DDVP toxicity, we exposed zebrafish to different concentrations of DDVP, and investigated the resulting changes in liver histology and gene transcription. Results Functional enrichment analysis of genes affected by DDVP exposure identified a number of processes involved in energy utilization and stress response in the liver. The abundance of transcripts for proteins involved in glucose metabolism was profoundly affected, suggesting that carbon flux might be diverted toward the pentose phosphate pathway to compensate for an elevated demand for energy and reducing equivalents for detoxification. Strikingly, many transcripts for molecules involved in β-oxidation and fatty acid synthesis were down-regulated. We found increases in message levels for molecules involved in reactive oxygen species responses as well as ubiquitination, proteasomal degradation, and autophagy. To ensure that the effects of DDVP on energy metabolism were not simply a consequence of poor feeding because of neuromuscular impairment, we fasted fish for 29 or 50 h and analyzed liver gene expression in them. The patterns of gene expression for energy metabolism in fasted and DDVP-exposed fish were markedly different. Conclusion We observed coordinated changes in the expression of a large number of genes involved in energy metabolism and responses to oxidative stress. These results argue that an appreciable part of the effect of DDVP is on energy metabolism and is regulated at the message level. Although we observed some evidence of neuromuscular impairment in exposed fish that may have resulted in reduced feeding, the alterations in gene expression in exposed fish cannot readily be explained by nutrient deprivation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1941-2) contains supplementary material, which is available to authorized users.

© 2015 Harrill et al.; licensee BioMed Central. Background: Synaptogenesis is a critical neurodevelopmental process whereby pre- and postsynaptic neurons form apposed sites of contact specialized for chemical neurotransmission. Many neurodevelopmental disorders are thought to reflect altered patterns of synaptic connectivity, including imbalances between excitatory and inhibitory synapses. Developing rapid throughput approaches for assessing synaptogenesis will facilitate toxicologic and drug screening studies of neurodevelopmental disorders. The current study describes the use of high-content imaging to quantify the ontogeny of excitatory and inhibitory synapses using in vitro models of neurodevelopment. These data are compared to biochemical and functional measures of synaptogenesis. Results: The ontogenetic patterns of synapse formation were compared between primary rodent hippocampal and cortical neurons over 28 days in vitro (DIV). As determined by ELISA, the increase in synaptophysin expression levels as cultures matured was similar between hippocampal and cortical cultures. High-content imaging of immunoreactivity of excitatory and inhibitory synaptic biomarkers demonstrated an overall greater number of synapses in hippocampal relative to cortical neurons with marked differences in the pattern of inhibitory synapse development between these two neuronal cell types. Functional assays revealed that both the mean firing rates and mean bursting rates were significantly increased in cortical cultures relative to hippocampal cultures. This difference may reflect decreased inhibitory synaptic tone in cortical versus hippocampal cultures. Conclusions: These data demonstrate differences and similarities in the ontogeny of synaptogenesis between hippocampal and cortical neurons, depending on the biological level examined. Assessment of synaptophysin protein levels by ELISA showed a general increase in synapse formation in both cell types with increasing time in culture, while high-content imaging was able to delineate cell type-dependent differences in formation of excitatory versus inhibitory synapses. The functional significance of differences in the balance of excitatory to inhibitory synapses was confirmed by the assessment of network activity using microelectrode arrays. These results suggest that high-content imaging and microelectrode arrays provide complementary approaches for quantitative assessment of synaptogenesis, which should provide a robust readout of toxicologic and pharmacologic effects on this critical neurodevelopmental event.

Schwann cells form basal laminae (BLs) containing laminin-2 (Ln-2; heterotrimer alpha2beta1gamma1) and Ln-8 (alpha4beta1gamma1). Loss of Ln-2 in humans and mice carrying alpha2-chain mutations prevents developing Schwann cells from fully defasciculating axons, resulting in partial amyelination. The principal pathogenic mechanism is thought to derive from structural defects in Schwann cell BLs, which Ln-2 scaffolds. However, we found loss of Ln-8 caused partial amyelination in mice without affecting BL structure or Ln-2 levels. Combined Ln-2/Ln-8 deficiency caused nearly complete amyelination, revealing Ln-2 and -8 together have a dominant role in defasciculation, and that Ln-8 promotes myelination without BLs. Transgenic Ln-10 (alpha5beta1gamma1) expression also promoted myelination without BL formation. Rather than BL structure, we found Ln-2 and -8 were specifically required for the increased perinatal Schwann cell proliferation that attends myelination. Purified Ln-2 and -8 directly enhanced in vitro Schwann cell proliferation in collaboration with autocrine factors, suggesting Lns control the onset of myelination by modulating responses to mitogens in vivo.

Gene, sequence and probe information for Quantigene probes. (PDF 1561Â kb)

Functional analysis of differentially expressed genes. (PDF 2394Â kb)

The mortality curve for zebrafish exposed to DDVP for 96Â h was determined in range finding studies. (PDF 813Â kb)

Replication of microarray data using Quantigene assays. (PDF 1546Â kb)

We recently demonstrated that polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substitutions sensitize ryanodine receptors (RyRs), and this activity promotes Ca²⁺-dependent dendritic growth in cultured neurons. Many ortho-substituted congeners display axial chirality, and we previously reported that the chiral congener PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) atropselectively sensitizes RyRs. Here, we test the hypothesis that PCB 136 atropisomers differentially alter dendritic growth and other parameters of neuronal connectivity influenced by RyR activity. (-)-PCB 136, which potently sensitizes RyRs, enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. The dendrite-promoting activity of (-)-PCB 136 is observed at concentrations ranging from 0.1 to 100 nM and is blocked by pharmacologic RyR antagonism. Neither atropisomer alters axonal growth or cell viability. Quantification of PCB 136 atropisomers in hippocampal cultures indicates that atropselective effects on dendritic growth are not due to differential partitioning of atropisomers into cultured cells. Imaging of hippocampal neurons loaded with Ca²⁺-sensitive dye demonstrates that (-)-PCB 136 but not (+)-PCB 136 increases the frequency of spontaneous Ca²⁺ oscillations. Similarly, (-)-PCB 136 but not (+)-PCB 136 increases the activity of hippocampal neurons plated on microelectrode arrays. These data support the hypothesis that atropselective effects on RyR activity translate into atropselective effects of PCB 136 atropisomers on neuronal connectivity, and suggest that the variable atropisomeric enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure.

To investigate the molecular mechanism of intermediate myasthenia syndrome (IMS), we analyzed the toxic effects of the representative organophosphate dimethoate on the function and expression of the nicotinic acetylcholine receptor (nAChR) in primary skeletal muscle cell culture. The results showed that the expression of nAChR on the muscle cell membrane was significantly increased after cells were exposed to dimethoate (130 microM). AChR function measured by carbachol-induced (22)Na+ influx demonstrated that dimethoate may inhibit the nAChR function either by binding to a noncompetitive site and changing the conformational state of nAChR or by blocking the nAChR channel directly. This study also demonstrated that dimethoate could rapidly induce the expression of c-fos, with a maximal effect at about 40 min, and c-fos might act as a transcriptional factor in regulating the expression of nAChR in the primary skeletal muscle cell culture following organophosphate exposure.

While both epidemiological and experimental animal studies have demonstrated that perinatal exposure to polychlorinated biphenyls (PCBs) negatively impacts cognitive and psychomotor function, there remains considerable uncertainty regarding mechanisms by which PCBs cause these functional deficits. In vitro studies have shown that PCBs can trigger apoptosis in cultured neurons and suggest this effect is mediated in part by increased levels of reactive oxygen species (ROS). However, whether PCBs cause similar effects in vivo in the developing brain has yet to be reported. In this study, rat pups were exposed to the commercial PCB mixture Aroclor 1254 (A1254) at 0.1 or 1.0 mg/kg/d in the maternal diet throughout gestation and lactation. Apoptosis and oxidative damage were quantified in three brain regions within several days after birth and at weaning. Caspase-3 activity was significantly increased in the cortex, hippocampus and cerebellum of newborn but not weanling rats exposed to A1254 at 1.0 mg/kg/d in the maternal diet. The most prominent effect was observed in the cerebellum, and PCB-induced apoptosis in this brain region was confirmed by TUNEL. Western blotting revealed that developmental A1254 exposure also increased levels of 3-nitrotyrosine and 4-hydroxynonenal levels in the cerebellum of new-born rats, indicating increased oxidative damage of proteins and lipids, respectively. These findings provide the first in vivo data in support of the hypothesis that PCB-induced oxidative stress alters spatiotemporal profiles of apoptosis, and suggest that this is an important mechanism contributing to the developmental neurotoxicity of PCBs.

The toxicity of exposure to polycyclic aromatic hydrocarbons (PAHs) or phytoestrogen is relatively well characterized. However, the toxicity of combined exposure to PAHs and phytoestrogen is not well investigated. In the present study, benzo(a)pyrene (B(a)P) and benzo(k)fluorathene (B(k)F), genistein, along with 17β-estradiol (E2), were investigated for their single and combined developmental toxicity using zebrafish embryos as model system. We demonstrated that two representative PAHs, both B(a)P (≥1 μM) and B(k)F (≥10 μM), can cause significant malformation and mortality in developing zebrafish embryos. The toxicity effect of B(a)P was in general higher than that of B(k)F. Developmental exposure to high level of genistein (>20 μM) or E2 (>10 μM), also caused significant malformation and mortality in zebrafish larvae at 120 hours post fertilization (hpf). However, different toxic effects were observed for the combined exposure to PAHs and phytoestrogen in zebrafish. Lower doses of genistein (1 and 10 μM) and E2 (0.1 and 1 μM), when used in combination with high concentration of B(a)P (1 μM) or B(k)F (20 μM), can significantly suppress the toxicity effect of B(a)P and B(k)F in developing zebrafish embryos. The beneficial effect of genistein may be due to the inhibition of cytochrome P450 enzymes via directly interacting with aryl-hydrocarbon receptor (AhR) pathway, or disturbing the AhR pathway through interacting with estrogen receptor pathway.

Background: Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) promote dendritic growth in hippocampal neurons via ryanodine receptor (RyR)-dependent mechanisms; however, downstream signaling events that link enhanced RyR activity to dendritic growth are unknown. Activity-dependent dendritic growth, which is a critical determinant of neuronal connectivity in the developing brain, is mediated by calcium ion (Ca2+)-dependent activation of Ca2+/calmodulin kinase-I (CaMKI), which triggers cAMP response element binding protein (CREB)-dependent Wnt2 transcription. RyRs regulate the spatiotemporal dynamics of intracellular Ca2+ signals, but whether RyRs promote dendritic growth via modulation of this signaling pathway is not known. Objective: We tested the hypothesis that the CaMKI–CREB–Wnt2 signaling pathway couples NDL PCB-enhanced RyR activity to dendritic arborization. Methods and Results: Ca2+ imaging of dissociated cultures of primary rat hippocampal neurons indicated that PCB-95 (2,2´,3,5´6-pentachlorobiphenyl; a potent RyR potentiator), enhanced synchronized Ca2+ oscillations in somata and dendrites that were blocked by ryanodine. As determined by Western blotting and quantitative polymerase chain reaction, PCB-95 also activated CREB and up-regulated Wnt2. Blocking CaMKK, CaMKIα/γ, MEK/ERK, CREB, or Wnt2 prevented PCB-95–induced dendritic growth. Antagonism of γ-aminobutyric acid (GABA) receptors with bicuculline (BIC) phenocopied the dendrite-promoting effects of PCB-95, and pharmacological antagonism or siRNA knockdown of RyR blocked BIC-induced dendritic growth in dissociated and slice cultures of hippocampal neurons. Conclusions: RyR activity contributes to dynamic remodeling of dendritic architecture in response to NDL PCBs via CaMKI–CREB–Wnt2 signaling in rats. Our findings identify PCBs as candidate environmental risk factors for neurodevelopmental disorders, especially in children with heritable deficits in calcium signaling associated with autism.

Axonal morphology is a critical determinant of neuronal connectivity, and perturbation of the rate or extent of axonal growth during development has been linked to neurobehavioral deficits in animal models and humans. We previously demonstrated that the organophosphorus pesticide (OP) chlorpyrifos (CPF) inhibits axonal growth in cultured neurons. In this study, we used a zebrafish model to determine whether CPF, its oxon metabolite (CPFO), or the excreted metabolite trichloro-2-pyridinol (TCPy) alter spatiotemporal patterns of axonal growth in vivo. Static waterborne exposure to CPFO, but not CPF or TCPy, at concentrations ≥ 0.03 μM from 24- to 72-h post fertilization significantly inhibited acetylcholinesterase, and high-performance liquid chromatography detected significantly more TCPy in zebrafish exposed to 0.1 μM CPFO versus 1.0 μM CPF. These data suggest that zebrafish lack the metabolic enzymes to activate CPF during these early developmental stages. Consistent with this, CPFO, but not CPF, significantly inhibited axonal growth of sensory neurons, primary motoneurons, and secondary motoneurons at concentrations ≥ 0.1 μM. Secondary motoneurons were the most sensitive to axonal growth inhibition by CPFO, which was observed at concentrations that did not cause mortality, gross developmental defects, or aberrant somatic muscle differentiation. CPFO effects on axonal growth correlated with adverse effects on touch-induced swimming behavior, suggesting the functional relevance of these structural changes. These data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of CPF and demonstrate the relevance of zebrafish as a model for studying OP developmental neurotoxicity.

It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 μM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration.

It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 μM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration.

The mechanism underlying intermediate myasthenia syndrome (IMS) following acute organophosphate poisoning remains largely unknown. Previous studies indicated that the mechanism of myasthenia in rats and IMS patients is most likely due to a postsynaptic neurotransmission blocking at neuromuscular junctions (NMJ). Nicotinic acetylcholine receptor (nAChR) is a key postsynaptic component at NMJ. Whether functional changes of nAChR are related to the development of myasthenia has not been demonstrated and addressed in vivo so far. In this study, we attempted to investigate temporal and spatial changes of nAChR in the blood lymphocyte, muscle and brain of rats during the course of myasthenia after acute dimethoate poisoning by using radioligand-binding assay. We found that specific nAChR binding activity in the gastrocnemius muscle and blood lymphocytes of myasthenia rats was significantly increased at 48 h after dimethoate poisoning. However, no changes of nAChR binding activity were found in the lymphocytes and muscle of non-myasthenia rats which were sacrificed at 1 h after intoxication. Interestingly, no changes of nAChR and muscarinic acetylcholine receptor (mAChR) binding activity were found in the cerebrum and cerebellum of all rats after dimethoate intoxication either at 1 or 48 h. The change of nAChR specific binding activity in the lymphocytes is parallel with that in the skeletal muscle during the development of myasthenia. This implied that the changes of nAChR receptor binding activity in the skeletal muscle and lymphocytes are highly associated with the development of myasthenia. The functional changes of nAChR at NMJ might play an important role in the paralysis of skeletal muscle following acute organophosphates (OPs) poisoning.

Evidence that children are widely exposed to organophosphorus pesticides (OPs) and that OPs cause developmental neurotoxicity in animal models raises significant concerns about the risks these compounds pose to the developing human nervous system. Critical to assessing this risk is identifying specific neurodevelopmental events targeted by OPs. Observations that OPs alter brain morphometry in developing rodents and inhibit neurite outgrowth in neural cell lines suggest that OPs perturb neuronal morphogenesis. However, an important question yet to be answered is whether the dysmorphogenic effect of OPs reflects perturbation of axonal or dendritic growth. We addressed this question by quantifying axonal and dendritic growth in primary cultures of embryonic rat sympathetic neurons derived from superior cervical ganglia (SCG) following in vitro exposure to chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and trichloropyridinol (TCP). Axon outgrowth was significantly inhibited by CPF or CPFO, but not TCP, at concentrations > or =0.001 microM or 0.001 nM, respectively. In contrast, all three compounds enhanced BMP-induced dendritic growth. Acetylcholinesterase was inhibited only by the highest concentrations of CPF (> or =1 microM) and CPFO (> or =1 nM); TCP had no effect on this parameter. In summary, these compounds perturb neuronal morphogenesis via opposing effects on axonal and dendritic growth, and both effects are independent of acetylcholinesterase inhibition. These findings have important implications for current risk assessment practices of using acetylcholinesterase inhibition as a biomarker of OP neurotoxicity and suggest that OPs may disrupt normal patterns of neuronal connectivity in the developing nervous system.

In order to investigate the cellular mechanism of muscular weakness in the Intermediate Myasthenia Syndrome (IMS) following acute organophosphate poisoning, we studied the cytotoxicity of dimethoate and its effects on the activity of acetylcholine esterase (AChE), Na + –K + -ATPase, succinate dehydrogenase (SDH), and Ca 2+ -ATPase in primary cultured skeletal muscle cells. The results showed that the activity of AChE was significantly inhibited in a dose and time-dependent manner when cells were exposed to dimethoate for 2 h, but the expression of heat-shock protein (HSP70) in muscle cells was significantly increased in a time-dependent manner following dimethoate exposure. Dimethoate can significantly increase the activity of Na + –K + -ATPase in the mitochondrial and cytoplasm fraction of muscle cells, and inhibit the activity of Ca 2+ -ATPase. This study suggests that the disruption of intracellular homeostasis and energy metabolism of the muscle cells may play a role in the etiology of IMS.

The function of the neuromuscular transmission in rats dosed with phoxim (P), methomyl (M), fenvalerate (F), and mixtures of P+M and P+F was studied by using both the stimulation single fiber electromyography (SSFEMG) and repetitive nerve stimulations (RNS) to determine the single muscle fiber action potential and compound muscle action potential (CMAP) respectively. The results showed that the mean consecutive difference (MCD) in SSFEMG was significantly prolonged in P, P+M and P+F intoxicated rats during the presence of myasthenia, but not in rats dosed with F and M when stimuli were given at 10 Hz or 20 Hz, thus indicating a transmission blocking at the neuromuscular junction (NMJ) induced by P. The frequency of neuromuscular transmission abnormalities detected by SSFEMG was significantly higher than those detected by RNS. This study demonstrated that the neuromuscular junction (NMJ) blocking is more frequently seen in P, P+M and P+F poisoning than in M and F poisoning, and that SSFEMG is a more sensitive electrophysiological method than RNS for detecting neuromuscular transmission blockage in myasthenia rats with acute insecticides poisoning.

Effects of DDVP exposure on non-neural tissues. (PDF 7021Â kb)

Effects of DDVP exposure on non-neural tissues. (PDF 7021Â kb)

RefSeq IDs for Fig. 3a. (PDF 26 kb)

RefSeq IDs for Fig. 3a. (PDF 26 kb)