W. A. Wickramasinghe

The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor

Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non‐luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY.

Genotoxicity investigation of a cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN), a potent cyanobacterial hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies in many parts of the world, and has been associated with the intoxication of humans and livestock. In this study, Balb/c mice were injected via the intraperitoneal (IP) route with a single dose of 0.2 mg/kg CYN. Animals were sacrificed at 6, 12, 24, 48 and 72 h. DNA was isolated from the mouse livers, and examined for strand breakage by alkaline gel electrophoresis (pH 12). Significant DNA strand breakage was observed in the mouse liver exposed to CYN, suggesting that induction of DNA strand breakage is probably one of the key mechanisms for CYN genotoxicity.

X-ray absorption spectroscopy of cadmium phytochelatin and model systems

Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides. We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly. We have used the synthetic ions [Cd(SPh)4]2-, [Cd4(SPh)10]2- and [S4Cd10(SPh)16]4-as crystallographically defined models for the cadmium site. The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes. In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species. The Cd-S distance in the phytochelatin complex is 2.54 A. The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation. Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS. The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for [S4Cd10(SPh)16]4-. This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.

Protective Efficacy of the Antioxidants Vitamin E and Trolox Against Microcystis aeruginosa and Microcystin-LR in Artemia franciscana Nauplii

This study was undertaken to evaluate the protective efficacy of the antioxidants vitamin E and Trolox (a water-soluble vitamin E derivative) against the toxicity of microcystin-LR (MC-LR), Microcystis aeruginosa aqueous extract (CE), and a reference toxin, menadione sodium bisulfite (MSB), in Artemia franciscana nauplii. This was achieved by using the well-established brine shrimp bioassay. The experiment was conducted in 2 stages, with (1) 12-h mortality time course and (2) LC50 determination for 12- and 24-h exposures. Treatments consisted of MC-LR, CE, and MSB alone and with 4-h pretreatments of either vitamin E or Trolox. Sensitivity of A. franciscana nauplii with 24-h LC50 values of 11 (10.1–12.1) μg/ml for MSB and 9.5 (8.8–10.4) μg/ml for MC-LR were in general agreement with values reported for Artemia sp. Both antioxidant pretreatments resulted in significant reductions in mortality of approximately 50% at 9 h postexposure when challenged by either 40 μg/ml MC-LR or 20 μg/ml MSB. In contrast, the antioxidant pretreatments offered little to no protection from CE, suggesting that other uncharacterized bioactive compounds contributed to overall toxicity. The described bioassay is easily accessible, inexpensive, rapid, and complies with animal ethics guidelines of many countries, and thus provides a potential alternative to the mouse bioassay for the initial screening for chemoprotectants against MC-LR toxicity.

Establishing a public health analytical service based on chemical methods for detecting and quantifying Pacific ciguatoxin in fish samples

A referee analysis method for the detection and quantification of Pacific ciguatoxins in fish flesh has recently been established by the public health analytical laboratory for the State of Queensland, Australia. Fifty-six fish samples were analysed, which included 10 fillets purchased as negative controls. P-CTX-1 was identified in 27 samples, and P-CTX-2 and P-CTX-3 were found in 26 of those samples. The range of P-CTX-1 concentrations was 0.04-11.4 microg/kg fish flesh; coefficient of variation from 90 replicate analyses was 7.4%. A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method utilising a rapid methanol extraction and clean-up is reliable and reproducible, with the detection limit at 0.03 microg/kg fish flesh. Some matrix effects are evident, with fish oil content a likely signal suppression factor. Species identification of samples by DNA sequence analysis revealed some evidence of fish substitution or inadvertent misidentification, which may have implications for the management and prevention of ciguatera poisoning. Blinded inspection of case notes from suspect ciguatera poisoning cases showed that reporting of ciguatera-related paraesthesias was highly predictable for the presence of ciguatoxins in analysed fish, with 13 of 14 expected cases having consumed fish that contained P-CTX-1 (p<0.001, Fishers Exact Test).

Chlorination for degrading saxitoxins (paralytic shellfish poisons) in water

Abstract Chlorination was investigated as a treatment option for degrading and thus removing saxitoxins (paralytic shellfish poisons, PSPs) produced by cyanobacteria (blue‐green algae) from water. It was found to be effective with the order of ease of degradation of the saxitoxins being GTX5 (B1) ‐ dcSTX > STX > GTX3 ‐ C2 > C1 > GTX2. However the effectiveness of chlorine was pH dependent. Degradation as a function of pH was not linear with the degree of degradation increasing rapidly at around pH 7.5. At pH 9 > 90% removal was possible provided a residual of 0.5 mg 1−1 free chlorine was present after 30 min contact time. The more effective degradation at higher pH was unexpected as chlorine is known to be a weaker oxidant under these conditions. The more effective degradation, then, must be due to the toxins, which are ionisable molecules, being present in a form at higher pH which is more susceptible to oxidation. The feasibility of using chlorine to remove saxitoxins during water treatment will therefore depend strongly on the pH of the water being chlorinated. Degradation may be improved by pH adjustment but may not be a practical solution. Although saxitoxins were degraded in that the parent compounds were not detected by chemical analysis, there is no indication as to the nature of the degradation products. However, acute toxicity as determined by the mouse bioassay was eliminated.

Occupational and environmental hazard assessments for the isolation, purification and toxicity testing of cyanobacterial toxins

Cyanobacteria can produce groups of structurally and functionally unrelated but highly potent toxins. Cyanotoxins are used in multiple research endeavours, either for direct investigation of their toxicologic properties, or as functional analogues for various biochemical and physiological processes. This paper presents occupational safety guidelines and recommendations for personnel working in field, laboratory or industrial settings to produce and use purified cyanotoxins and toxic cyanobacteria, from bulk harvesting of bloom material, mass culture of laboratory isolates, through routine extraction, isolation and purification. Oral, inhalational, dermal and parenteral routes are all potential occupational exposure pathways during the various stages of cyanotoxin production and application. Investigation of toxicologic or pharmacologic properties using in vivo models may present specific risks if radiolabelled cyanotoxins are employed, and the potential for occupational exposure via the dermal route is heightened with the use of organic solvents as vehicles. Inter- and intra-national transport of living cyanobacteria for research purposes risks establishing feral microalgal populations, so disinfection of culture equipment and destruction of cells by autoclaving, incineration and/or chlorination is recommended in order to prevent viable cyanobacteria from escaping research or production facilities.

First Report of a Toxic Nodularia spumigena (Nostocales/ Cyanobacteria) Bloom in Sub-Tropical Australia. II. Bioaccumulation of Nodularin in Isolated Populations of Mullet (Mugilidae)

Fish collected after a mass mortality at an artificial lake in south-east Queensland, Australia, were examined for the presence of nodularin as the lake had earlier been affected by a Nodularia bloom. Methanol extracts of muscle, liver, peritoneal and stomach contents were analysed by HPLC and tandem mass spectrometry; histological examination was conducted on livers from captured mullet. Livers of sea mullet (Mugil cephalus) involved in the fish kill contained high concentrations of nodularin (median 43.6 mg/kg, range 40.8–47.8 mg/kg dry weight; n = 3) and the toxin was also present in muscle tissue (median 44.0 μg/kg, range 32.3–56.8 μg/kg dry weight). Livers of fish occupying higher trophic levels accumulated much lower concentrations. Mullet captured from the lake 10 months later were also found to have high hepatic nodularin levels. DNA sequencing of mullet specimens revealed two species inhabiting the study lake: M. cephalus and an unidentified mugilid. The two mullet species appear to differ in their exposure and/or uptake of nodularin, with M. cephalus demonstrating higher tissue concentrations. The feeding ecology of mullet would appear to explain the unusual capacity of these fish to concentrate nodularin in their livers; these findings may have public health implications for mullet fisheries and aquaculture production where toxic cyanobacteria blooms affect source waters. This report incorporates a systematic review of the literature on nodularin measured in edible fish, shellfish and crustaceans.

BENZYLMERCAPTAN (BENZYLTHIOL) AND DIBENZYLDISULPHIDE FROM THE MARINE SPONGE CRELLA SPINULATA, (HENTSCHEL) (POECILOSCLERIDA: CRELLIDAE)

Benzylmercaptan (benzylthiol) and its oxidised product dibenzyldisulphide have been isolated and characterised from the marine sponge Crella spinulata (Hentschel) found in the Great Barrier Reef, northern Australia. The structures of the two compounds were determined on the basis of their spectral and chemical properties. These two compounds were found only in gravid females collected during the reproductive season. The biochemical interest of these sulphur-containing compounds in the gravid females is briefly discussed in relation to the role of sulphydryl groups in biological systems.

Interaction of Copper-Binding Proteins from Enterococcus hirae

Copper is imported into prokaryotic cells by CPx-type ATPases. CPx-type ATPases have the transmembrane characteristics typical of P-type ATPases involved in the translocation of many ions. A conserved Cys-Pro-X (X = C or H) sequence within the transmembrane channel and a variable number of distinct amino-terminal domains define the CPx classification (1). The cytoplasmic subdomains of the CPx-ATPases have a MxCxxC or M/HxxMDHS/GxM metal-binding site (x = any amino acid). Intracellular copper is utilized in the activation of enzymes, such as cytochrome-c oxidase, superoxide dismutase, and lysyl oxidase. Copper also has the potential to cause cellular damage because of its redox properties. To overcome this dichotomy, the cell regulates copper levels and prevents toxicity with overlapping mechanisms: sequestration, export, and inhibition of entry.