Changes in the neurotransmitter profile in the central nervous system of marine medaka (Oryzias melastigma) after exposure to brevetoxin PbTx-1 - A multivariate approach to establish exposure biomarkers.

A strategy to construct multivariate biomarkers for exposure to algal neurotoxins via correlating changes to the profiles of a series of neurotransmitters and their metabolites in the central nervous system (CNS) of exposed test organism is reported. 3-Month-old marine medaka (Oryzais melastigma) were exposed to waterborne brevetoxin PbTx-1 at two sub-lethal dose levels (0.5 and 2.5 µg-PbTx-1 L-1) for a duration of 12 h before quantification of 43 selected neurotransmitters and metabolites in their CNS were measured via dansyl chloride derivatization and LC-MS/MS determination. The profiling data were analyzed by multivariate statistical analyses, including principle component analysis (PCA), projection on latent structure-discriminate analysis (PLS-DA) and orthogonal projection on latent structure-discriminate analysis (OPLS-DA). Neurotransmitters and metabolites related to activation of voltage-gated sodium channels (VGSCs), N-methyl-D-aspartic acid receptors (NMDARs) and cholinergic neurotransmission were found to contribute significantly to class separation in the corresponding OPLS-DA models. Those models obtained from different exposure dosages were correlated by the Shared and Unique Structures Plot (SUS-plot) to identify appropriate variables for the construction of exposure biomarkers in the form of multivariate predictive scores. The established biomarkers for male and female medaka fish were able to predict acute sub-lethal exposure to PbTx-1 with good sensitivity and specificity (male fish: sensitivity 94.7%, specificity 80.0%; female fish: sensitivity 91.4%, specificity 83.3%). Neurotransmitter profiles in the CNS of medaka fish that should have recovered from exposure to PbTx-1 have also been determined to reveal long-term impacts to the CNS of the affected organism even after the exposure has been interrupted.

Profiling of Selected Functional Metabolites in the Central Nervous System of Marine Medaka (Oryzias melastigma) for Environmental Neurotoxicological Assessments.

The simultaneous profiling of 43 functional metabolites in the brain of the small model vertebrate organism, marine medaka (Oryzais melastigma), has been accomplished via dansyl chloride derivatization and LC-MS/MS quantification. This technique was applied to examine effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), one of the most abundant polybrominated diphenyl ether flame retardants in the natural environment, on the central nervous system (CNS) of vertebrates. The model teleosts were fed with bioencapsulated Artemia nauplii for up to 21 days. Multivariate statistical analysis has demonstrated that levels of numerous classical neurotransmitters and their metabolites in the CNS of the fish were perturbed even at the early phase of dietary exposure. Subsequent metabolic pathway analysis further implied potential impairment of the arginine and proline metabolism; glycine, serine and threonine metabolism; D-glutamine and D-glutamate metabolism; alanine, aspartate, and glutamate metabolism; valine, leucine, and isoleucine biosynthesis, and the cysteine and methionine metabolism in the brain of the test organism. Our results demonstrate that targeted profiling of functional metabolites in the CNS may shed light on how the various neurological pathways of vertebrates, including humans, are affected by toxicant/stress exposure.

Acute Exposure to Pacific Ciguatoxin Reduces Electroencephalogram Activity and Disrupts Neurotransmitter Metabolic Pathways in Motor Cortex.

Ciguatera fish poisoning (CFP) is a common human food poisoning caused by consumption of ciguatoxin (CTX)-contaminated fish affecting over 50,000 people worldwide each year. CTXs are classified depending on their origin from the Pacific (P-CTXs), Indian Ocean (I-CTXs), and Caribbean (C-CTXs). P-CTX-1 is the most toxic CTX known and the major source of CFP causing an array of neurological symptoms. Neurological symptoms in some CFP patients last for several months or years; however, the underlying electrophysiological properties of acute exposure to CTXs remain unknown. Here, we used CTX purified from ciguatera fish sourced in the Pacific Ocean (P-CTX-1). Delta and theta electroencephalography (EEG) activity was reduced remarkably in 2 h and returned to normal in 6 h after a single exposure. However, second exposure to P-CTX-1 induced not only a further reduction in EEG activities but also a 2-week delay in returning to baseline EEG values. Ciguatoxicity was detected in the brain hours after the first and second exposure by mouse neuroblastoma assay. The spontaneous firing rate of single motor cortex neuron was reduced significantly measured by single-unit recording with high spatial resolution. Expression profile study of neurotransmitters using targeted profiling approach based on liquid chromatography-tandem mass spectrometry revealed an imbalance between excitatory and inhibitory neurotransmitters in the motor cortex. Our study provides a possible link between the brain oscillations and neurotransmitter release after acute exposure to P-CTX-1. Identification of EEG signatures and major metabolic pathways affected by P-CTX-1 provides new insight into potential biomarker development and therapeutic interventions.