Assessment of environmental contaminants' neurotoxicity : in vitro and in vivo experimental studies

Accumulating evidence over several decades shows that environmental contaminants may lead to neurodevelopmental alterations. An essential prerequisite to prevention is recognition of a chemical‟s ability to harm the developing nervous system. Knowledge that environmental contaminants are neurotoxic can prompt efforts to restrict its use and to control exposure. Many studies on environmental toxicants have been performed so far, but the knowledge available on the effects of exposures at low environmentally relevant doses and on cell-specific modes of action is still very limited. The work included in this thesis, is based on an experimental strategy including in vitro studies on cellular models and behavioral analyses in mice aimed at investigating the potential neurotoxic effects of selected food contaminants, such as methylmercury (MeHg), polychlorinated biphenyls (PCBs) and perfluorinated compounds (PFCs). Using in vitro models, we found that MeHg and PCBs cause cell death in the HT22 hippocampal cell line via a parallel activation of calpains and lysosomal proteases, with no involvement of caspases. Oxidative stress does not play a major role in PCB toxicity opposite to MeHg. Co-exposure to MeHg and PCBs shows mostly antagonistic interactions. We have also investigated the effects of PCBs and MeHg on primary cultures of rat neural stem cells (NSCs), focusing on the non-dioxin like (NDL)-PCBs 153 and 180. Both PCBs affect spontaneous neuronal differentiation with a significant increase in the number of neurite-bearing Tuj1 positive cells and a concomitant decrease in proliferating cells. In agreement, measurements of spontaneous Ca2+ oscillations showed a decreased number of cells with Ca2+ activity after PCB exposure, further confirming the increase in neuronal cells. Conversely, exposure to methylmercury (MeHg), which we evaluated in parallel, led to an increased number of cells with Ca2+ activity, in agreement with the previously observed inhibition of neuronal differentiation. Analyses by q-PCR of the Notch pathway revealed that PCBs have a repressive action on Notch signaling, whereas MeHg activates it. All together, the data indicate that nM concentrations of the selected NDL-PCBs and MeHg interfere in opposite directions with neuronal spontaneous differentiation of NSCs through Notch signalling. Combined exposures to PCBs and MeHg results in an induction of apoptosis and an antagonistic interaction on spontaneous neuronal differentiation. In the in vivo study performed in the frame of this project, mice offspring in utero exposed to perfluorooctanesulfonate (PFOS) or perfluorooctanoic acid (PFOA) were assessed for changes in motor function, circadian activity and emotion-related behaviour. Exposure to PFOS results in decreased locomotion in a novel environment and reduced muscle strength only in male offspring. Prenatal exposure to PFOA is associated with changes in exploratory behavior in male and female offspring, and increased home cage global activity only in males. In conclusion, our studies show that the developing nervous system is very susceptible to the neurotoxic effects of the selected food contaminants and that a combined approach with both in vitro and in vivo experimental models is most valuable for developmental neurotoxicity testing

The relevance of developmental neurotoxicity research in Malaysia

The developing brain is found greatly vulnerable towards the exposure of different environmental chemicals/drugs, even at concentrations, that are normally considered harmless in the mature brain. The developing central nervous system (CNS) is a ‘work under progress’ system, constantly undergoing remodeling, where active proliferation, differentiation, migration, synaptogenesis, and circuitry establishment take place within a tightly controlled time frame. An accumulating body of evidence pointing to links between toxic chemicals, including food contaminants with different neuropsychiatric and neurological disorders such as autism, dyslexia, attention deficit hyperactive disorder, cerebral palsy, schizophrenia, Parkinson’s disease, Alzheimer’s disease and IQ deficits. The impact of children’s IQ deficits is sometimes dismissed as unimportant because the magnitude of the impairments is considered to be clinically insignificant. Noteworthy, total economic loss in Asia in 2011 due to childhood Lead exposure was estimated around RM 26 trillion. This represents a very substantial value to the society where the largest burden of lead exposure is now borne by low- and middle-income countries. Thus, testing of compounds for neurotoxicity has become increasingly important in recent years. In Malaysia, developmental neurotoxicity research is a new field. To date, a large body of evidence showing that our environment and food was contaminated with pollutants. Also, the number of neurodevelopmental disorders and IQ deficits are keep on increasing. Taken together, we already have adequate reasons to start research works related to the developmental neurotoxicity research in Malaysia. However, the effort should be orchestrated together with researchers from different fields with a proper planning and research agendas to guarantee an achievable final goal, protecting our future generation from the ‘silent damage’ of those toxic chemicals. Developmental neurotoxicity research pivotal for formulating effective guidelines and strategies to limit the exposure to hazardous chemicals, especially towards the developing nervous system

Chronic embryonic exposure of arsenic trioxide induced alteration on motor functions and anxiety-like esponses in zebrafish (Danio rerio) larvae

Arsenic trioxide (As2O3) is a heavy metal that is widely used for therapeutic purpose and is found ubiquitously in the environment. Currently, information about the adverse effects associated with exposure to low levels of inorganic arsenic on the developing organisms is limited. We examined the effects of embryonic exposure of As2O3 on motor functions and anxiety-like behaviors in the developing zebrafish. The embryos were exposed to 4 different concentrations (20, 30, 40, 50 µM) starting from 5 hpf until hatching (72 hpf) in a semi-static condition. The control groups were maintained in the embryo media. The mortality rate increased in a dose dependent manner. We found that chronic embryonic exposure to 30 and 40 µM decreased the number of tail coiling, heartbeat, and swimming activity. Meanwhile, exposure 20 µM did not produce any significant alteration in these parameters. To further understand the effects ofAs2O3 interferences on the development of anxiety-related behavior, we chose 30 µM As2O3 exposed larvae since the 40 µM As2O3 achieved 100% mortality before the behavioral recording was conducted on 6 dpf. However, we found no alterations on thigmotaxis, avoidance response, speed and percentage of resting of 6 dpf larvae. Since we found no alterations in all parameters measured for anxiety related behavior, we evaluated the apoptosis in the eye of the larvae using acridine orange. We found a significant increase in the apoptotic signal particularly in the eye region. These results showed that exposure to microconcentration of As2O3 do not alter the anxiety related responses, yet, inducing apoptosis in the retina and lens, which may contribute to visual deficit. The potential underlying mechanisms and implication of As2O3 on visual acuity, motor performance and anxiety-like responses need further investigation

Chronic embryonic exposure to ethanol induced apoptosis and impairments of behavioral activities in the zebrafish larvae

Decades of research have pinpointed the pathological consequences of ethanol and we focused to understand the developmental neurotoxicity effects of ethanol on the zebrafish nervous system. The zebrafish embryos were exposed to different concentrations of ethanol (0.25%, 0.5%, 0.75%, 1.5% and 2.00%), in a semi static condition from 5 hours post fertilization (hpf), with daily renewal of the medium until hatching. We have measured the behavior of individual larva; spontaneous locomotor behavior, tail coiling at 24 hpf and locomotor behavior, swimming activity at 6 days post fertilization. We also measured anxiety related behavior in the group of 5 larvae at 6 dpf. Besides that, we also evaluate apoptosis in the zebrafish embryo at 24 hpf. We found that, chronic embryonic exposure to ethanol significantly decreased the number of spontaneous tail coiling as the ethanol concentration increased. The swimming activity in the individual zebrafish zebrafish larva at 6 dpf was significantly increase increased at 0.75%. Analysis of the anxiety related behavior has shown that exposure to ethanol and stimulation with aversive stimulus have significantly decreased in the percentage of outward preference in the larvae. The exposure to ethanol has induced significant decrease in the percentage of edge and down preference of the larvae in the presence of aversive stimulus as compared to control. The exposure to ethanol significantly decreased the percentage of right preference while the swimming speed of the larvae was not affected. The number of apoptotic cells was significantly increased in 1.5% ethanol as compared to the control at 24 hpf. This finding showed that chronic embryonic exposure to ethanol induced behavioral alterations in the zebrafish larvae and also increased apoptosis in the embryo. Further study is needed to understand the mechanism that underlie the behavioral alterations and the induction of apoptosis

Developmental neurotoxic effects of chronic endosulfan exposure in the zebrafish larvae

Endosulfan was listed as a member of Persistent Organic Pollutants (POP) and the usage was banned, yet this pesticide is still widely used in many developing countries. The usage of this pesticide can reach aquatic system through runoff from agricultural areas. Due to their persistent characteristic in the environment, human population are posed to continuous exposure to endosulfan. Consequently, endosulfan was found in the cord blood and breast milk of pregnant women, thus may induced adverse effects to the developing nervous system. In this study, we focused to determine the developmental neurotoxicity effects of chronic embryonic exposure to endosulfan in the zebrafish larvae. The zebrafish embryos were exposed to several concentrations of endosulfan in a semi static condition, where half of the medium was renewed for every 24 h from 5 hours post fertilization (hpf) until hatching. We measured spontaneous tail coiling at 24 hpf; heart rate at 48 hpf and mortality rate at 72 hpf. We found that, endosulfan decreased the spontaneous tail coiling, heart rate and increased the total percentage of mortality as the concentration increased. At 6 days post fertilization (dpf), we measured the swimming activity of individual larva and found that endosulfan significantly decreased the swimming activity behaviour at 40nM-200nM. The zebrafish larvae exposed to endosulfan higher than 200nM were unable to survive until 6 dpf. We further evaluated anxiety behaviour in the group of 5 larvae at 6 dpf exposed to 200nM endosulfan, with and without visual stimuli. Caffeine (100mg/L) was used as an anxiogenic drug. Caffeine increased the anxiety behaviour while endosulfan decreased the anxiety behaviour in the zebrafish larvae. Our result showed that zebrafish embryos can be an alternative research model to study the developmental neurotoxicity testing at high-throughput screenings

Perfluorooctane sulfonate induces neuronal and oligodendrocytic differentiation in neural stem cells and alters the expression of PPARγ in vitro and in vivo

Perfluorinated compounds are ubiquitous chemicals of major concern for their potential adverse effects on the human population. We have used primary rat embryonic neural stem cells (NSCs) to study the effects of perfluorooctane sulfonate (PFOS) on the process of NSC spontaneous differentiation. Upon removal of basic fibroblast growth factor, NSCs were exposed to nanomolar concentrations of PFOS for 48 h, and then allowed to differentiate for additional 5 days. Exposure to 25 or 50 nM concentration resulted in a lower number of proliferating cells and a higher number of neurite-bearing TuJ1-positive cells, indicating an increase in neuronal differentiation. Exposure to 50 nM also significantly increased the number of CNPase-positive cells, pointing to facilitation of oligodendrocytic differentiation. PPAR genes have been shown to be involved in PFOS toxicity. By q-PCR we detected an upregulation of PPARγ with no changes in PPARα or PPARδ genes. One of the downstream targets of PPARs, the mitochondrial uncoupling protein 2 (UCP2) was also upregulated. The number of TuJ1- and CNPase-positive cells increased after exposure to PPARγ agonist rosiglitazone (RGZ, 3 μM) and decreased after pre-incubation with the PPARγ antagonist GW9662 (5 μM). RGZ also upregulated the expression of PPARγ and UCP2 genes. Meanwhile GW9662 abolished the UCP2 upregulation and decreased Ca2 + activity induced by PFOS. Interestingly, a significantly higher expression of PPARγ and UCP3 genes was also detected in mouse neonatal brain after prenatal exposure to PFOS. These data suggest that PPARγ plays a role in the alteration of spontaneous differentiation of NSCs induced by nanomolar concentrations of PFOS

Developmental neurotoxicity assessment of arsenic: evaluation of apoptosis and behavioural response in the zebrafish larvae

Developmental Neurotoxicity research (DNT) aims to understand the adverse effects of xenobiotic exposure to the developing nervous system. The presence of a large number of yet untested chemicals in commerce, together with the increasing numbers of neurodevelopmental disorders worldwide, the pressure to the scientific community to develop testing strategies that can speed up the process of DNT testing become more critical. Behaviour of laboratory animal such as zebrafish has become a golden key for high-throughput screening in DNT testing owing to special characteristics of the zebrafish such as high fecundity, transparent eggs and fully characterized genome and behaviour phenotypes. In this study, we have assessed the zebrafish larvae behaviour in terms of sensorimotor response, motor response and social behaviour after exposure to arsenic (0μM, 2500μM, 3500μM) from 5 hours post fertilization until hatching in a semi-static condition. Arsenic was used as the model chemical because it is already classified as neurotoxic to human development. We also evaluated the apoptosis in the eye and brain area at 24hpf after exposure to arsenic (0μM, 2000μM, 4000μM). We found that exposure to arsenic significantly affect the right preference of the larvae at 6 days post fertilization as compared to the control by using our semi-high-throughput behavioural testing system. No significant difference in other parameters (edge and down preference, outward and clockwise orientation, distance between the larvae and swimming speed). In addition, exposure to 4000μM arsenic increase (35%) the percentage of apoptotic cells in the brain area as compared to the control. This study has shown that zebrafish is a powerful tool that can used to assess developmental neurotoxicity effects of toxicant at the behavioural and cellular level. Further study to understand the mechanisms that underlie the behavioural alterations and induction of apoptosis is necessary

Optimal method to introduce faeces sample for olfactory-cues studies in Malayan tapir (Tapirus indicus)

Studying the hidden meaning behind the behaviours of wildlife can provide plenty of information for the betterment of their conservation. In this research, a preliminary study had been conducted to find out whether Malayan tapir (Tapirus indicus) (1) sniffs on the faeces or not and (2) to identify the optimal method of presenting the faeces samples. Five individuals of Malayan tapirs were contributed to this research as the samples donors and/or the subject. Basically, for this experiment several replicates of faeces samples were collected from the donors and frozen at -20˚C, and the thawed samples were introduced to the subjects at different time slots (morning, afternoon and night). The results showed that tapirs exhibited both investigation and identification behaviours when sniff on faeces samples of other individuals. However, the sniffing was observed only during early in the morning and night when they were active due to their nocturnal characteristic. We found that, the location and the sample nature (i.e., thawed at sufficient period of time) had influenced the attractiveness of presented faeces sample to tapirs. Presenting the samples at feeding and sleeping areas increased the chance for the tapirs to sniff on samples and well thawed samples which emits strong odour drawn tapir’s attention. Thus, it is recommended to take into account the above factors for further investigation related to the individual recognition through olfactory cues in Malayan tapir. The findings of this study could be incorporated in ex-situ conservation mainly during the selection of mating partners prior to physical introduction of the animals (strategy to avoid any injuries to tapir due to fighting and inbreeding in captivity)

PFOS induces behavioral alterations, including spontaneous hyperactivity that is corrected by dexamfetamine in zebrafish larvae

Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae

Acute toxicity test of copper pyrithione on Javanese medaka and the behavioural stress symptoms

This study was conducted to investigate the median lethal concentration (LC50) of copper pyrithione (CuPT) at 96-hr exposure on adult Javanese medaka (Oryzias javanicus) in revealing toxicological effects of CuPT contamination in the tropical area. Wild stock fishes were acclimatized for 14-days prior analysis. Triplicate of test tanks for seven test concentrations were placed with ten fishes each, this includes two control tanks. The behaviour of the tested fishes was manually observed through a camera. The LC50 of CuPT at 96-h was found to be 16.58 mg/L. Tested fishes swam slowly in vertical movement and swam fast towards food during feeding time as the sign of stress behaviour. Meanwhile, fishes in the two control groups swam actively in a horizontal manner and no excitement during feeding time. No mortality in control groups. Results indicate CuPT to be toxic to Javanese medaka at low concentration and caused behavioural stress