Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors

Okadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.

A novel toxicity fingerprinting method for pollutant identification with lux-marked biosensors

A novel technique is described for the identification and quantification of environmental pollutants based on toxicity fingerprinting with a metabolic lux-marked bacterial biosensor. This method involved characterizing the toxicity-based responses of the biosensor to seven calibration pollutants as acute temporal-dose response fingerprints. An algorithm is described to allow comparisons of responses of an unknown pollutant to be made against the calibration data. This is based on predicting pollutant concentration at each of six different time points over the course of a 5-min assay. If the prediction is consistent between the unknown pollutant and a calibration pollutant at the 95% test level, this is considered to be a positive identification. All seven calibration pollutants could be successfully distinguished from each other with this technique. Environmental samples, individually spiked with single concentrations of pollutants, were compared in this way against the calibration pollutants. An 83% identification success was achieved, with no false positives at the 95% test level. This is a simple and rapid technique that potentially can be applied to monitoring of industrial wastewater or as a screening tool for regulators.

Ecotoxicological screening of Kenyan tannery dust using a luminescent-based bacterial biosensor

Ecotoxicological screening of dust sampled throughout a Kenyan tannery was conducted using a luminescence (lux)-based bacterial biosensor for both solid and liquid assays. This was complemented by chemical analysis in an attempt to identify possible causative toxic components. The biosensor results showed a highly significant (p <0.001) difference in both solid and liquid phase toxicity in samples collected from various identified sampling points in the tannery. A positive correlation was observed between results of the solid and liquid phase techniques, for most of the sampling points indicating that the toxic contaminants were bioavailable both in the solid and liquid state. However, the results generally indicated toxicity associated with liquid phase except certain areas in solid phase such as chemical handling, buffing area and weighing. The most toxic tannery area identified was the weighing area (p <0.001), showing the lowest bioluminescence for both the solid (0.38 +/- 2.21) and liquid phases (0.01 +/- 0.001). Chromium was the metal present in the highest concentration indicating levels higher than the stipulated regulatory requirement of 0.5 mg Cr/m3 for total Cr (highest Cr concentration was at chemical handling at 209.24 mg l(-1)) in all dust samples. The weighing area had the highest Ni concentration (1.87 mg l(-1)) and the chemical handling area showed the highest Zn concentration (31.9 mg l(-1)). These results raise environmental health concerns, as occupational exposure to dust samples from this site has been shown to give rise to elevated concentrations (above the stipulated levels) of chromium in blood, urine and some body tissues, with inhalation being the main route. Health and Safety Executive (HSE), UK, and American Conference of Governmental Industrial Hygienist (ACGIH) and National Institute for Occupational Safety and Health (NIOSH), USA stipulates an occupational exposure limit of 0.5 mg Cr/m3 (8 h TWA) for total chromium. However, schedule 1 of Controls of substances hazardous to health (COSHH) regulations developed by HSE, indicate 0.05 mg m3 (8 h TWA reference periods) to be the limit for Cr (VI) exposure. The exposure limit for individual (e.g., Cr, Zn, Ni etc.) contaminants (homogeneity) was not exceeded, but potential impact of heterogeneity (multi-element synergistic effect) on toxicity requires application of the precautionary principle.

Toxicity assessment of xenobiotic contaminated groundwater using lux modified Pseudomonas fluorescens

A bacterial bioassay, suitable for rapid screening to assess the relative toxicity of xenobiotic contaminated groundwater has been developed. The quantitative bioassay utilizes a decline in luminescence of the lux marked soil bacterium Pseudomonas fluorescens on exposure to contaminated groundwaters from which effective concentration (EC) values can be assessed and compared. P. fluorescens was most sensitive to semi-volatile organics in groundwaters but there was no correlation between EC value and chemical content. The sensitivity and reproducibility of the P. fluorescens bioassay was compared with that of Microtox and results showed that mean EC₅₀ values for diluted ground water replicate samples were 20% and 18% respectively. This suggested that the P. fluorescens bioassay was as applicable to groundwater screening as the widely used Microtox bioassay.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Inhibition and fibril formation and toxicity of a fragment of alpha-synuclein by an N-methylated peptide analogue

Alpha-synuclein has been linked to amyloidogenesis in Parkinson's disease and other neurodegenerative disorders. We have previously shown that a peptide comprising residues 68-78 of alpha-synuclein is the minimum fragment that, like alpha-synuclein itself, forms amyloid fibrils and exhibits toxicity towards cells in culture. Hughes et al. [J. Biol. Chem. 275 (2000) 25109] showed that an N-methylated derivative of Abeta(25-35) inhibited the formation of fibrils by Abeta(25-35) and reduced its toxicity. We have now extended this concept to an amyloidogenic alpha-synuclein-based peptide. Alpha-synuclein(68-78), N-methylated at G1y73, was compared to non-methylated peptide. Whereas alpha-synuclein(68-78) formed fibrils and was toxic to cells, the N-methylated analogue had neither of these properties. Moreover, an equimolar mixture of the non-methylated and methylated peptides formed very few fibrils and toxicity was markedly reduced.

Identification of the region of non-A beta component (NAC) of alzheimer's disease amyloid responsible for its aggregation and toxicity

The non-beta-amyloid (Aß) component of Alzheimer's disease amyloid (NAC) and its precursor a-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and a-synuclein both form ß-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3±18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8±18) and NAC(8±16) are toxic, whereas NAC(12±18), NAC(9±16) and NAC(8±15) are not. Circular dichroism indicates that none of the peptides displays ß-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce ß sheet, fragments NAC(8±18) and NAC(8±16) both form ß-sheet structure. Only NAC(8±18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8±16 of NAC, equivalent to residues 68±76 in a-synuclein, comprise the region crucial for toxicity.

Inhibitors of alpha-synuclein oligomerization and toxicity: a future therapeutic strategy for Parkinson's disease and related disorders.

An abundance of genetic, histopathological, and biochemical evidence has implicated the neuronal protein, alpha-synuclein (alpha-syn) as a key player in the development of several neurodegenerative diseases, the so-called synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. Development of disease appears to be linked to events that increase the intracellular concentration of alpha-syn or cause its chemical modification, either of which can accelerate the rate at which it forms aggregates. Examples of such events include increased copy number of genes, decreased rate of degradation via the proteasome or other proteases, or altered forms of alpha-syn, such as truncations, missense mutations, or chemical modifications by oxidative reactions. Aggregated forms of the protein, especially newly formed soluble aggregates, are toxic to cells, so that one therapeutic strategy would be to reduce the rate at which such oligomerization occurs. We have therefore designed several peptides and also identified small molecules that can inhibit alpha-syn oligomerization and toxicity in vitro. These compounds could serve as lead compounds for the design of new drugs for the treatment of PD and related disorders in the future.

Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

Synucleins are small proteins that are highly expressed in brain tissue and are localised at presynaptic terminals in neurons. alpha-Synuclein has been identified as a component of intracellular fibrillar protein deposits in several neurodegenerative diseases, and two mutant forms of alpha-synuclein have been associated with autosomal-dominant Parkinson's Disease. A fragment of alpha-synuclein has also been identified as the non-Abeta component of Alzheimer's Disease amyloid. In this review we describe some structural properties of alpha-synuclein and the two mutant forms, as well as alpha-synuclein fragments, with particular emphasis on their ability to form beta-sheet on ageing and aggregate to form amyloid-like fibrils. Differences in the rates of aggregation and morphologies of the fibrils formed by alpha-synuclein and the two mutant proteins are highlighted. Interactions between alpha-synuclein and other proteins, especially those that are components of amyloid or Lewy bodies, are considered. The toxicity of alpha-synuclein and related peptides towards neurons is also discussing in relation to the aetiology of neurodegenerative diseases.