Metabolomics is becoming a more and more popular tool for toxicological and functional genomics studies, disease diagnosis and drug discovery. The conceptual framework of metabolomics has already been well established to study how organisms respond to various stimuli. In this work, a high-throughput liquid chromatography coupled to tandem electrospray mass spectrometry (LC-ESI-MS/MS) neurometabolomics platform based on marine medaka (Oryzais melastigma) is developed as a toxicological assessment tool for impacts of numerous environmental contaminants and bio-toxins on the central nervous system (CNS) of vertebrates. A special derivatization technique using dansyl chloride (Dan-Cl) for derivatization of classical neurotransmitters and their metabolites so as to improve their chargeability, and hence their detection sensitivity by mass spectrometry, is adopted in order to achieve pg (fmol) levels of detection sensitivity for these analytes. Moreover, this dansylation derivatization increases the metabolome coverage for our metabolomic profiling.
This neurometabolomics platform used a small-sized teleost fish, marine medaka (Oryzais melastigma) as the vertebrate model organism to study changes in the profiles of up to 43 neurotransmitters and their metabolites in the brain caused by selected external stimuli. With the high sensitivity of quantification, our method is capable of revealing the profile of neurotransmitters in individual brain of each tiny fish. This greatly enhances the correlation capability of the neurotransmitters metabolome of marine medaka with any external stimuli and environmental stresses. We demonstrated this by studying the neurometabolic changes induced by the exposure to BDE-47, an endocrine disrupting congener of polybrominated diphenyl ethers (PBDEs), and brevetoxins (PbTxs), sodium channel activating neurotoxins, on the CNS of marine medaka. The protein-normalized concentration of each neurotransmitter and their metabolites were analysed by multivariate statistical analysis. Multivariate analysis of variance (MANOVA) was applied to evaluate the statistical significance of toxicant challenge and general gender-specific alterations, and multivariate models (principal component analysis [PCA], partial least squares discriminant analysis [PLS-DA], and orthogonal partial least squares discriminant analysis [OPLS-DA]) were generated to fit and interpret the neurometabolomics data.
Chapter 1 gives a general background of metabolomics, the CNS, neurotransmitters and chemometrics. Metabolomics is the study of metabolism at the global level, and is performed by advanced analytical technologies combined with sophisticated statistical methods (chemometrics) for information extraction and data interpretation. Metabolomics gives a snapshot of all molecules produced by cells at one time. With the help of metabolomic profiles, we gain insights to the neural mechanisms underlying the observed effects of the selected external stimuli.
Chapter 2 reports the development of the quantitative analytical protocol for the metabolomic profiling of selected classical neurotransmitters and their metabolite in the brain of marine medaka. The method utilized a dansylation derivatization to improve sensitivity of detection and metabolome coverage, a fast liquid chromatography (LC) for separation and tandem electrospray mass spectrometry (ESI-MS/MS) for detection. Different dansylation conditions were optimized. Operational parameters of ESI-MS/MS and LC conditions were also carefully tuned to obtain the highest signal to noise ratio.
In Chapter 3, we apply the high throughout neurometabolomic platform based on LC-ESI-MS/MS to detect and assess the neurotoxicological impacts of BDE-47, a predominant PBDEs residue in the aquatic ecosystem, on the CNS of vertebrates. PBDEs are a class of 209 structurally related man-made chemicals. They are mainly used as additive flame-retardants. PBDEs are ubiquitous organic contaminants in our global ecosystem. They are toxic to both humans and the environment, and are suspected of causing neurobehavioral effects and endocrine disruption. Their critical endpoint of concern for human health is neurobehavioral effects.
Our results indicate that exposure to BDE-47 is able to induce abnormal expression of a number of neurotransmitters in the CNS of marine medaka. Significant relationship was observed between the neurometabolomic profiles and the concentration of PBDEs, suggesting that marine medaka responded to PBDEs at a metabolic level. The observed metabolic responses to PBDEs via either dietary or waterborne exposure are indicative of activation of N-methyl-D-aspartate (NMDA) receptors. That dysfunctional overactivation of NMDA receptors causes excitotoxic neuronal death and may lead to disturbances of neuronal plasticity, learning and memory.
In Chapter 4, we apply the neurometabolomic platform to detect and assess the neurotoxicological impacts of brevetoxins on CNS of vertebrates. Brevetoxins, unique activators of voltage-gated sodium channels (VGSC), are a series of neurotoxic trans-fused cyclic polyethers produced naturally by Karenia brevis, the dominant marine dinoflagellate of Florida red tide. Brevetoxins adversely impact human and animal health, local economies, and ecosystem function. They share the same binding site with ciguatoxins, another class of globally distributed marine neurotoxins. Our results reveal that exposure to PbTx-1, the most potent and most lipophilic brevetoxins congeners, and PbTx-2, the most abundant congener in nature, are able to induce abnormal expression of a number of neurotransmitters in the CNS of the model organism. Significant relationship was observed between metabolic profiles and the concentration of brevetoxins, suggesting that marine medaka responded to both PbTx-1 and PbTx-2 at a metabolic level. The observed metabolic responses to both PbTx-1 (A-type brevetoxin) and PbTx-2 (B-type brevetoxin) are indicative of activation of sodium channels, which is a known effect of brevetoxins. Both A-type and B-type brevetoxins act on the neurotransmitter systems in similar ways. In addition, numerous changes were observed in the metabolomic profiles of brain samples during the course of, and after cessation of brevetoxin exposure. Response trajectories based on those metabolomic profiles suggest that fish can partially recover from the effects of both A-type and B-type brevetoxins after cessation of exposure. This highlights the usefulness of metabolomics for investigating impacts of sublethal ecological interactions on organism physiology. Furthermore, by inference, our findings may be extended to ciguatoxins, which also bind to site 5 on the α subunit of VGSC and produce neurotoxic syndromes similar to that of brevetoxins.
To the best of our knowledge, this is the first report in the literature of a quantitative metabolomics platform for studying in vivo metabolic profiles of classical neurotransmitters and their metabolites in brain samples. It gives a snapshot of concentrations of these compounds in the brain of the exposed organism. Our results in Chapter 3 and 4 also demonstrated the ability of our neurometabolomics platform for early detection and impact assessment of toxicant on the CNS of vertebrates. With the help of the multiparametric metabolic data generated from this platform, we can gain insight into the neural mechanisms underlying observed effects of those xenobiotic.
| Date of Award | 3 Oct 2014 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Hon Wah Michael LAM (Supervisor) |
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