Persisting neurobehavioral consequences of daily or intermittent paternal cannabis administration in F1 and F2 Rats.

Repeated paternal preconception exposure to Δ9-tetrahydrocannabinol (Δ9-THC) alone or together with the other constituents in a cannabis extract has been shown in our earlier studies in rats to cause significant neurobehavioral impairment in their offspring. In the current study, we compared the effects of daily cannabis extract (CE) exposure to cannabis on two consecutive days per week, modeling weekend cannabis use in human. The CE contained Δ9-THC as well as cannabidiol and cannabinol. We also extended the investigation of the study to cross-generational effects of grand-paternal cannabis exposure on the F2 generation and included testing the effects of paternal cannabis exposure on responding for opiate self-administration in F1 and F2 generation offspring. We replicated the findings of neurobehavioral impairment in F1 offspring of male rats exposed to cannabis extract containing 4 mg/kg/day of Δ9-THC daily for four weeks prior to mating with drug naïve females. The 4-week cannabis extract exposure caused a significant decrease in weight gain in the male rats exposed daily. In contrast, their offspring showed significantly greater body weights and anogenital distances (AGD) in the third to fourth weeks after birth. The behavioral effects seen in the F1 generation were increased habituation of locomotor activity in the figure-8 maze in female offspring and increased lever pressing for the opiate drug remifentanil in male offspring. The F2 generation showed significantly impaired negative geotaxis and an elimination of the typical sex-difference in locomotor activity, with effects not seen in the F1 generation. This study shows that daily paternal cannabis exposure for four weeks prior to mating causes significant neurobehavioral impairment in the F1 and F2 offspring. Intermittent exposure on two consecutive days per week for four weeks caused comparable neurobehavioral impairment. In sum, there should be concern about paternal as well as maternal exposure to cannabis concerning neurobehavioral development of their offspring.

Developmental exposure to pesticides that disrupt retinoic acid signaling causes persistent retinoid and behavioral dysfunction in zebrafish.

Early developmental exposure to environmental toxicants may play a role in the risk for developing autism. A variety of pesticides have direct effects on retinoic acid (RA) signaling and as RA signaling has important roles in neurodevelopment, such compounds may cause developmental neurotoxicity through an overlapping adverse outcome pathway. It is hypothesized that a pesticide's embryonic effects on retinoid function may correspond with neurobehavioral disruption later in development. In the current studies, we determined the effects of RA-acting pesticides on neurobehavioral development in zebrafish. Buprofezin and imazalil caused generalized hypoactivity in the larval motility test, whereas chlorothalonil and endosulfan I led to selective hypoactivity and hyperactivity, respectively. With buprofezin, chlorothalonil, and imazalil, hypoactivity and/or novel anxiety-like behaviors persisted in adulthood and buprofezin additionally decreased social attraction responses in adulthood. Endosulfan I did not produce significant adult behavioral effects. Using qPCR analyses of adult brain tissue, we observed treatment-induced alterations in RA synthesis or catabolic genes, indicating persistent changes in RA homeostasis. These changes were compound-specific, with respect to expression directionality, and potential patterns of homeostatic disruption. Results suggest the likely persistence of disruptions in RA signaling well into adulthood and may represent compensatory mechanisms following early life stage exposures. This study demonstrates that early developmental exposure to environmental toxicants that interfere with RA signaling causes short as well as long-term behavioral disruption in a well-established zebrafish behavioral model and expand upon the meaning of the RA adverse outcome pathway, indicating that observed effects likely correspond with the nature of underlying homeostatic effects.

Oxytetracycline changes the behavior of zebrafish larvae by inhibiting NMDA receptors.

Oxytetracycline (OTC), a tetracycline antimicrobial, is one of the antimicrobial drugs frequently used in the aquaculture and livestock industries. Due to its extensive usage and emissions, OTC has been identified as a significant new emerging pollutant (EP) in a number of environments. OTC frequently causes toxic effects on the central nervous system, but it can be challenging to monitor, and it is still unclear how these toxicities are caused. We used bioinformatic analysis techniques to screen for OTC targets and discovered that NMDA receptors are potential targets of OTC neurotoxicity. To confirm this finding, we exposed zebrafish embryos to 5 mg/L OTC-containing rearing water from 2-hour post fertilization (hpf) to 8-day post fertilization (dpf), performed spontaneous movement and light-dark stimulation assays at 6 and 8 dfp, and discovered that OTC inhibited locomotor activity and attenuated anxiety-like responses in zebrafish larvae. Meanwhile, the qPCR and immunofluorescence staining results suggested that OTC inhibited the expression of multiple subtypes of NMDA receptors (grin1a, grin1b, grin2bb, grin2ca) and induced apoptosis in the brains of zebrafish embryos. Simultaneous administration of NMDA, an NMDA receptor agonist, completely antagonized the inhibitory neurobehavioral changes in zebrafish larvae, as well as the downregulation of N-methyl-D-aspartate (NMDA) receptor expression and apoptosis in the embryonic brains caused by OTC exposure. In conclusion, OTC exhibited significant inhibitory neurobehavioral toxicity in zebrafish larvae during early development, which may be dependent on its suppression of NMDA receptor activity and expression. Furthermore, OTC-induced neurodevelopmental toxicity may be associated with NMDA receptor-regulated neuronal apoptosis.

Embryonic exposure to PFAS causes long-term, compound-specific behavioral alterations in zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are commonly used as surfactants and coatings for industrial processes and consumer products. These compounds have been increasingly detected in drinking water and human tissue, and concern over their potential effects on health and development is growing. However, relatively little data are available for their potential impacts on neurodevelopment and the degree to which different compounds within this class may differ from one another in their neurotoxicity. The present study examined the neurobehavioral toxicology of two representative compounds in a zebrafish model. Zebrafish embryos were exposed to 0.1-100uM perfluorooctanoic acid (PFOA) or 0.01-1.0uM perfluorooctanesulfonic acid (PFOS) from 5 to 122 h post-fertilization. These concentrations were below threshold for producing increased lethality or overt dysmorphologies, and PFOA was tolerated at a concentration 100× higher than PFOS. Fish were maintained to adulthood, with behavioral assessments at 6 days, 3 months (adolescence) and 8 months of age (adulthood). Both PFOA and PFOS caused behavioral changes in zebrafish, but PFOS and PFOS produced strikingly different phenotypes. PFOA was associated with increased larval motility in the dark (100uM), and enhanced diving responses in adolescence (100uM) but not adulthood. PFOS was associated with a reversed light-dark response in the larval motility test (0.1-1uM), whereby the fish were more active in the light than the dark. PFOS also caused time-dependent changes in locomotor activity in the novel tank test during adolescence (0.1-1.0uM) and an overall pattern of hypoactivity in adulthood at the lowest concentration (0.01uM). Additionally, the lowest concentration of PFOS (0.01uM) reduced acoustic startle magnitude in adolescence, but not adulthood. These data suggest that PFOS and PFOA both produce neurobehavioral toxicity, but these effects are quite distinct from one another.

Embryonic exposure to benzo[a]pyrene causes age-dependent behavioral alterations and long-term metabolic dysfunction in zebrafish.

Polycyclic aromatic hydrocarbons (PAH) are products of incomplete combustion which are ubiquitous pollutants and constituents of harmful mixtures such as tobacco smoke, petroleum and creosote. Animal studies have shown that these compounds exert developmental toxicity in multiple organ systems, including the nervous system. The relative persistence of or recovery from these effects across the lifespan remain poorly characterized. These studies tested for persistence of neurobehavioral effects in AB* zebrafish exposed 5-120 h post-fertilization to a typical PAH, benzo[a]pyrene (BAP). Study 1 evaluated the neurobehavioral effects of a wide concentration range of BAP (0.02-10 µM) exposures from 5 to 120 hpf during larval (6 days) and adult (6 months) stages of development, while study 2 evaluated neurobehavioral effects of BAP (0.3-3 µM) from 5 to 120 hpf across four stages of development: larval (6 days), adolescence (2.5 months), adulthood (8 months) and late adulthood (14 months). Embryonic BAP exposure caused minimal effects on larval motility, but did cause neurobehavioral changes at later points in life. Embryonic BAP exposure led to nonmonotonic effects on adolescent activity (0.3 µM hyperactive, Study 2), which attenuated with age, as well as startle responses (0.2 µM enhanced, Study 1) at 6 months of age. Similar startle changes were also detected in Study 2 (1.0 µM), though it was observed that the phenotype shifted from reduced pretap activity to enhanced posttap activity from 8 to 14 months of age. Changes in the avoidance (0.02-10 µM, Study 1) and approach (reduced, 0.3 µM, Study 2) of aversive/social cues were also detected, with the latter attenuating from 8 to 14 months of age. Fish from study 2 were maintained into aging (18 months) and evaluated for overall and tissue-specific oxygen consumption to determine whether metabolic processes in the brain and other target organs show altered function in late life based on embryonic PAH toxicity. BAP reduced whole animal oxygen consumption, and overall reductions in total basal, mitochondrial basal, and mitochondrial maximum respiration in target organs, including the brain, liver and heart. The present data show that embryonic BAP exposure can lead to neurobehavioral impairment across the life-span, but that these long-term risks differentially emerge or attenuate as development progresses.

Neurobehavioral anomalies in zebrafish after sequential exposures to DDT and chlorpyrifos in adulthood: Do multiple exposures interact?

A sequence of different classes of synthetic insecticides have been used over the past 70 years. Over this period, the widely-used organochlorines were eventually replaced by organophosphates, with dichlorodiphenyltrichloroethane (DDT) and chlorpyrifos (CPF) as the principal prototypes. Considerable research has characterized the risks of DDT and CPF individually, but little is known about the toxicology of transitioning from one class of insecticides to another, as has been commonplace for agricultural and pest control workers. This study used adult zebrafish to investigate neurobehavioral toxicity following 5-week chronic exposure to either DDT or CPF, to or their sequential exposure (DDT for 5 weeks followed by CPF for 5 weeks). At the end of the exposure period, a subset of fish were analyzed for brain cholinesterase activity. Behavioral effects were initially assessed one week following the end of the CPF exposure and again at 14 months of age using a behavioral test battery covering sensorimotor responses, anxiety-like functions, predator avoidance and social attraction. Adult insecticide exposures, individually or sequentially, were found to modulate multiple behavioral features, including startle responsivity, social approach, predator avoidance, locomotor activity and novel location recognition and avoidance. Locomotor activity and startle responsivity were each impacted to a greater degree by the sequential exposures than by individual compounds, with the latter being pronounced at the early (1-week post exposure) time point, but not 3-4 months later in aging. Social approach responses were similarly impaired by the sequential exposure as by CPF-alone at the aging time point. Fleeing responses in the predator test showed flee-enhancing effects of both compounds individually versus controls, and no additive impact of the two following sequential exposure. Each compound was also associated with changes in recognition or avoidance patterns in a novel place recognition task in late adulthood, but sequential exposures did not enhance these phenotypes. The potential for chemical x chemical interactions did not appear related to changes in CPF metabolism to the active oxon, as prior DDT exposure did not affect the cholinesterase inhibition resulting from CPF. This study shows that the effects of chronic adult insecticide exposures may be relevant to behavioral health initially and much later in life, and that the effects of sequential exposures may be unpredictable based on their constituent exposures.

Aluminum-Induced Cognitive Impairment and PI3K/Akt/mTOR Signaling Pathway Involvement in Occupational Aluminum Workers.

Epidemiological studies indicate that long-term occupational exposure to aluminum (Al) causes neurotoxicity and cognitive impairment. While the molecular underpinnings associated with workers' cognitive impairment is unclear, one mechanism may involve Al-induced PI3K/Akt/mTOR activation and neuronal cell death, which impairs learning and memory in rats. Here, we sought to determine whether PI3K/Akt/mTOR is also associated with cognitive impairment in Al-exposed occupational workers. Cognitive function was screened by Mini-Mental State Examination (MMSE) and Clock-Drawing Test (CDT), and serum Al and PI3K/Akt/mTOR-associated gene expression was quantified. A negative correlation between serum Al and scores of MMSE and CDT was found, which might relate with downregulation of PI3K/Akt/mTOR. To determine the role of the PI3K/Akt/mTOR pathway cognitive function, we treated zebrafish with Al and observed a profound impairment in learning and memory. Increased brain Al levels was associated with decreased expression of PI3K/Akt/mTOR in Al-exposed zebrafish. Finally, rapamycin, an mTOR inhibitor, was added to isolate the role of mTOR specifically in the Al exposed zebrafish. The results suggested that Al induces learning and memory deficits by downregulating PI3K, Akt, and mTOR1 expression and inducing neuronal cell death like rapamycin group. This study indicates that aluminum exposure can cause cognitive impairment through PI3K/Akt/mTOR pathway, with mTOR activity being a critical player involved in this mechanism. Future studies are necessary to further characterize the role of PI3K/Akt/mTOR1 signaling in Al-induced neurocognitive decline among Al occupational workers. These findings draw attention to Al risk exposure among occupational workers and the need to implement novel safety and protective measures to mitigate neurocognitive health risks in the Al industrial workspace.

Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish.

Farmers are often chronically exposed to insecticides, which may present health risks including increased risk of neurobehavioral impairment during adulthood and across aging. Experimental animal studies complement epidemiological studies to help determine the cause-and-effect relationship between chronic adult insecticide exposure and behavioral dysfunction. With the zebrafish model, we examined short and long-term neurobehavioral effects of exposure to either an organochlorine insecticide, dichlorodiphenyltrichloroethane (DDT) or an organophosphate insecticide chlorpyrifos (CPF). Adult fish were exposed continuously for either two or 5 weeks (10-30 nM DDT, 0.3-3 µM CPF), with short- and long-term effects assessed at 1-week post-exposure and at 14 months of age respectively. The behavioral test battery included tests of locomotor activity, tap startle, social behavior, anxiety, predator avoidance and learning. Long-term effects on neurochemical indices of cholinergic function were also assessed. Two weeks of DDT exposure had only slight effects on locomotor activity, while a longer five-week exposure led to hypoactivity and increased anxiety-like diving responses and predator avoidance at 1-week post-exposure. When tested at 14 months of age, these fish showed hypoactivity and increased startle responses. Cholinergic function was not found to be significantly altered by DDT. The two-week CPF exposure led to reductions in anxiety-like diving and increases in shoaling responses at the 1-week time point, but these effects did not persist through 14 months of age. Nevertheless, there were persistent decrements in cholinergic presynaptic activity. A five-week CPF exposure led to long-term effects including locomotor hyperactivity and impaired predator avoidance at 14 months of age, although no effects were apparent at the 1-week time point. These studies documented neurobehavioral effects of adult exposure to chronic doses of either organochlorine or organophosphate pesticides that can be characterized in zebrafish. Zebrafish provide a low-cost model that has a variety of advantages for mechanistic studies and may be used to expand our understanding of neurobehavioral toxicity in adulthood, including the potential for such toxicity to influence behavior and development during aging.

Developmental exposure to low concentrations of two brominated flame retardants, BDE-47 and BDE-99, causes life-long behavioral alterations in zebrafish.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) were widely used as flame retardants until the early 2000s, mainly in home furnishings and electronics. The persistence of PBDEs in the environment leads to continued ubiquitous exposure to low levels, with infants and children experiencing higher exposures than adults. Accumulating evidence suggest that low-level exposures during early life stages can affect brain development and lead to long-term behavioral impairments. We investigated the effects of zebrafish exposure to low doses of the two prominent PBDEs; 2,2',4,4',5,-Pentabromodiphenyl ether (BDE-99) and 2,2',4,4',-Tetrabromodiphenyl ether (BDE-47), during embryo-development on short- and long-term behavioral endpoints. We included the organophosphate pesticide chlorpyrifos (CPF) due to its well documented neurotoxicity across species from zebrafish to humans. METHODS: Zebrafish embryos were exposed to the following individual treatments; 0.1% DMSO (vehicle control); 0.3µM CPF; 0.01, 0.03, 0.1, 0.3µM BDE-47; 0.003, 0.03, 0.3, 1, 3, 10, 20µM BDE-99 from 5 until 120h post fertilization (hpf). Low exposure levels were determined as those not causing immediate overt toxicity, and behavior assays were conducted in the low-level range. At 144 hpf the larvae were tested for locomotor activity. At approximately 6 months of age adult zebrafish were tested in a behavioral battery including assays for anxiety-related behavior, sensorimotor response and habituation, social interaction, and predator avoidance. RESULTS: In the short-term, larval locomotor activity was reduced in larvae treated with 0.3µM CPF and 0.1µM BDE-47. BDE-99 treatment caused non-monotonic dose effects, with 0.3µM causing hyperactivity and 1µM or higher causing hypoactivity. In the long-term, adult anxiety-related behavior was reduced in all treatments as measured in both the novel tank dive test and tap test. DISCUSSION: We show that exposure of zebrafish embryos to low concentrations of the brominated flame retardants BDE-47 and BDE-99, and the organophosphate pesticide CPF, caused both short- and long-term behavioral impairments. Interestingly, we also found that at very low exposure concentrations, where there were no visible effects on larval activity, adult behavior was still strongly affected.

Meta-analysis on occupational exposure to pesticides--neurobehavioral impact and dose-response relationships.

While the health impact of high exposures to pesticides is acknowledged, the impact of chronic exposures in the absence of acute poisonings is controversial. A systematic analysis of dose-response relationships is still missing. Its absence may provoke alternative explanations for altered performances. Consequently, opportunities for health prevention in the occupational and environmental field may be missed. Objectives were (1) quantification of the neurotoxic impact of pesticides by an analysis of functional alterations in workers measured by neuropsychological performance tests, (2) estimates of dose-response relationships on the basis of exposure duration, and (3) exploration of susceptible subgroups. The meta-analysis employed a random effects model to obtain overall effects for individual performance tests. Twenty-two studies with a total of 1758 exposed and 1260 reference individuals met the inclusion criteria. At least three independent outcomes were available for twenty-six performance variables. Significant performance effects were shown in adults and referred to both cognitive and motor performances. Effect sizes ranging from dRE=-0.14 to dRE=-0.67 showed consistent outcomes for memory and attention. Relationships between effect sizes and exposure duration were indicated for individual performance variables and the total of measured performances. Studies on adolescents had to be analyzed separately due to numerous outliers. The large variation among outcomes hampered the analysis of the susceptibility in this group, while data on female workers was too scant for the analysis. Relationships exist between the impact of pesticides on performances and exposure duration. A change in test paradigms would help to decipher the impact more specifically. The use of biomarkers appropriate for lower exposures would allow a better prevention of neurotoxic effects due to occupational and environmental exposure. Intervention studies in adolescents seem warranted to specify their risk.

Neurotoxicology and development: human, environmental and social impacts.

The 12th International symposium of the Scientific Committee on Neurotoxicology and Psychophysiology, International Commission on Occupational Health was held in Cape Town, South Africa on March 24-27, 2013. Reflecting the meeting aiming to build greater focus on challenges facing working populations and communities in developing countries, the Symposium theme was Neurotoxicology and Development: Human, Environmental and Social Impacts. A total of 23 countries were represented with strong participation from 5 African countries. In addition to the more traditional topics of these Symposia, like metal, solvents and pesticides neurotoxicity, the conference embraced several new themes including affective disorders arising from chemical exposure, neurodevelopmental impacts in early life and novel approaches to genetic and epigenetic biomarkers for the assessment of neurotoxic impact. The theme of the conference prompted extensive discussions, which have laid the basis for a number of new directions for research, advocacy and capacity building to prevent and manage chemical neurotoxicity in workplace and community settings across the globe.

Toward better research practice--shortcomings decreasing the significance of epidemiological studies in the toxicological field.

Neurobehavioral studies do not always gain the impact they should have, neither in the scientific nor in the regulatory field of neurotoxicology. Among others, shortcomings and inconsistencies across epidemiological studies may contribute to this situation. Examples were compiled to increase awareness of obstacles for conclusions. Meta-analyses were exploited since they sometimes allow the detection of deficits that are not obvious from individual studies. Exposure assessment, performance measures, and confounding were scrutinized among 98 primary studies included in meta-analyses on mercury, solvents, manganese and pesticides. Inconsistent and hardly comparable markers of exposure were found; figures, units or sampling periods were not always provided. The contribution of test materials to differences in test outcomes across studies could sometimes not be evaluated due to the insufficient description of the employed tests. Hypotheses for the selection of performance variables often remained undisclosed. Matching procedures prevailed with respect to the confounder age; the comparability of groups with respect to intelligence and gender remained more elusive. 8% and 16% of the studies did not even mention confounding from intelligence and gender, respectively. Only one third of the studies provided adjusted means for group comparisons; the proportion was slightly larger for studies published 2000-2010. While 50% of the studies considered confounders for their dose-response assessment, only 29% reported results for the total of test variables. The outlined deficits impede, among others, the assessment of exposure-effect relationships and confounding across studies; thereby they limit the use of the studies for toxicological risk assessment and future prevention. Some shortcomings also impede a deeper insight into the mechanisms of toxicity: tests like the Digit Symbol show that something is affected, but not what is affected. Thorough description of measures employed is among the first consequences from the data. The consideration of mechanistic insights from research on animals and neurobiology may further help to increase the significance of epidemiological studies.