In vitro toxicity and genotoxicity assessment of disinfection by-products, organic N-chloramines.

Disinfection by-products (DBPs) are of concern to both water industries and health authorities. Although several classes of DBPs have been studied, and there are regulated safe levels in disinfected water for some, a large portion of DBPs are not characterized, and need further investigation. Organic N-chloramines are a group of DBPs, which can be formed during common disinfection processes such as chlorination and chloramination, but little is known in terms of their toxicological significance if consumed in drinking water. Only a few in vitro studies using bacterial assays have reported some genotoxic potential of organic N-chloramines, largely in the context of inflammatory processes in the body rather than exposure through drinking water. In this study, we investigated 16 organic N-chloramines produced by chlorination of model amino acids and amines. It was found that within the drinking water-relevant micromolar concentration range, four compounds were both cytotoxic and genotoxic to mammalian cells. A small reduction of cellular GSH was also observed in the treatment with these four compounds, but not of a magnitude to account for the cytotoxicity and genotoxicity. The results presented in this study demonstrate that some organic N-chloramines, at low concentrations that might be present in disinfected water, can be harmful to mammalian cells.

Evaluation of the Abraxis Strip Test for Microcystins™ for use with wastewater effluent and reservoir water.

Rapid tests for the microcystin-type cyanobacterial toxins that are designed to be able to be used in the field have recently become available. The tests provide a semi-quantitative result over a relatively narrow concentration range (10-fold) and are available with detection limits relevant for drinking water and recreational water compliance testing (1 µg/L and 10 µg/L, respectively). The aim of this research was to assess the applicability of these tests for the determination of microcystin-related toxicity in treated effluent from the Western Treatment Plant and potable source water from Tarago Reservoir, both near Melbourne, Australia. Accuracy, precision, cross-reactivity, matrix effects and inter-operator variability were assessed. The claimed mLR concentration response range of the tests was confirmed within reasonable limits, although the false negative and false positive rates were significant for spike concentrations below 2.5 µg/L (Recreational Strip Test). Inter-operator variability was reasonably high (CV=23%) and this was exacerbated by the use of untrained scorers. Contributing to this was significant inter-assay variability in test band intensity (CV=28%). The strip tests responded to all 8 microcystin analogues tested and also to a mixture of another 7 analogues contained in a Certified Bloom Material. Cross-reactivity was always greater than 50%. Matrix effects due to the test waters or to cyanobacterial cell material were also relatively minor, being of the order of 2-fold at the maximum. Overall, these Strip Tests were found to be reliable for relatively rapid detection of microcystins around the upper limits of their response ranges, as recommended by the manufacturer. While the Recreational Water Strip test was less reliable in the lower ranges, it can be used in conjunction with the Drinking Water Strip test to reduce uncertainty around the 1 µg/L concentration. Despite limitations, both strip tests provide near real-time information which can assist with day to day operational decisions. When results indicate microcystin concentrations near compliance limits it is recommended that use of the test kits should be supported by accurate quantitative toxin testing together with traditional algal cell counts, and possibly emerging qPCR methods for species and toxin gene detection.

Comparison of analytical tools and biological assays for detection of paralytic shellfish poisoning toxins.

The paralytic shellfish poisoning toxins (PSTs) were, as their name suggests, discovered as a result of human poisoning after consumption of contaminated shellfish. More recently, however, the same toxins have been found to be produced by freshwater cyanobacteria. These organisms have worldwide distribution and are common in our sources of drinking water, thus presenting another route of potential human exposure. However, the regulatory limits for PSTs in drinking water are considerably lower than in shellfish. This has increased the need to find alternatives to the mouse bioassay, which, apart from being ethically questionable, does not have a limit of detection capable of detecting the PSTs in water at the regulated concentrations. Additionally, the number of naturally occurring PSTs has grown substantially since saxitoxin was first characterised, markedly increasing the analytical challenge of this group of compounds. This paper summarises the development of chromatographic, toxicity, and molecular sensor binding methodologies for detection of the PSTs in shellfish, cyanobacteria, and water contaminated by these toxins. It then summarises the advantages and disadvantages of their use for particular applications. Finally it recommends some future requirements that will contribute to their improvement for these applications.

Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin increasingly found in drinking water sources worldwide. Toxicity studies have shown CYN can induce effects in a range of different cell types with primary hepatocytes consistently shown to be the most sensitive cellular model. How CYN enters the intracellular environment is not clear, although the size and hydrophilic nature of the toxin suggest it would not readily cross a lipid bilayer. In this study, a Vero cell line expressing green fluorescent protein (GFP) was used to monitor for CYN uptake based on the toxin's potent effects on protein synthesis. Effects on the GFP signal were compared with inhibitors cycloheximide (CHEX) and emetine. While CYN potency was demonstrated in a cell-free system (CYN>CHEX>emetine) it was considerably reduced in the Vero-GFP cell model (CHEX, emetine>>CYN). In contrast to other inhibitors, CYN effects on GFP signal increased 6 fold over 4-24 h incubation indicating slow, progressive uptake of the toxin. Confirming that the uptake process is not energy dependent CYN entry also occurred at 4 degrees C, while competition experiments excluded the uracil nucleobase transporter system as potential mechanism for CYN uptake. Dilution of media enhanced CYN uptake by Vero-GFP cells although mechanism by which this occurred is unknown.

Flow-cytometric analysis of in vitro micronucleus formation: comparative studies with WIL2-NS human lymphoblastoid and L5178Y mouse lymphoma cell lines.

The development of a flow cytometry-based micronucleus (FCMMN) assay for measuring the micronucleus (MN) frequency in cells following exposure to test chemicals has potential for improving reproducibility and reducing turn-around time when compared with the traditional microscopy-based micronucleus method. A major drawback of the FCMMN assay is that a false-positive interpretation could result from the presence of large numbers of apoptotic or necrotic bodies in the measured sample. Although several studies have reported ways in which the FCMMN assay could be improved using different staining techniques or electronic gating strategies, to date none of these protocols are suitable for use as a screening assay. To reduce the interference from apoptosis, performing the FCMMN assay with an apoptosis-resistant cell line may be an alternative approach. This study reports the use of p53-mutated cell lines to minimise the interference found in the FCMMN assay. Two commonly used cell lines (WIL2-NS and L5178Y) were investigated by comparison of (1) cytotoxicity and micronucleus induction in the FCMMN assay following treatment with model genotoxicants and (2) apoptotic responses after exposure to inducers of apoptosis. Both cell lines were responsive to all genotoxicants, producing concentration-dependent results with respect to genotoxicity. WIL2-NS cells were found to be more tolerant to apoptosis induction than L5178Y cells. This characteristic could be beneficial to minimise the interference from apoptotic nuclei in the FCMMN genotoxicity-screening assay.

Interaction of the cyanobacterial toxin cylindrospermopsin with the eukaryotic protein synthesis system.

The cyanobacterial toxin cylindrospermospin (CYN) was shown to inhibit the eukaryotic protein synthesis apparatus with similar potency in plant and mammalian cell extracts, IC(50) of 334 nM in wheat germ extract and 110 nM in reticulocyte lysate. [(14)C] CYN binding was assessed in reticulocyte lysates, following separation of ribosomes from free toxin on Sephadex G-50 size exclusion columns. While toxin binding was shown to be concentration-dependent (60-600 nM [(14)C] CYN), it did not correlate with ribosome content. The molar ratio of toxin to ribosomes was 0.02:1 at the binding plateau. Significant binding of [(14)C] CYN was identified in both the ribosome fraction (> 90% total ribosomes) and the first wash fraction eluting from the Sephadex G-50 column, despite the low ribosome content (<10% total ribosomes) in the latter. Bound [(14)C] CYN could be partially displaced by incubation with an excess of unlabelled CYN in both the ribosome and wash fractions, indicating a non-covalent linkage. Molecular weight cut-off filters identified that [(14)C] CYN was associated with high molecular weight material > 100 kDa. These results suggest that CYN's target may not be the ribosome itself, but rather one of the soluble proteins associated with the eukaryotic translation system.

Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin.

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.

Microcystins (cyanobacterial toxins) in drinking water enhance the growth of aberrant crypt foci in the mouse colon.

Microcystis aeruginosa produces toxic cyclic peptides called microcystins, potent hepatotoxins that have been implicated in tumor promotion in skin and liver. The model used in this investigation was the azoxymethane (AOM)-induced aberrant crypt focus (ACF) in the male C57Bl/6J mouse colon. Three intraperitoneal (i.p.) injections of 5 mg/kg AOM were administered at 7-d intervals to mice; 19 d after the last AOM injection, drinking water containing Microcystis extract was commenced and continued for a further 212 d. The content of microcystins in the drinking water was determined by mouse bioassay, high-performance liquid chromatography (HPLC), capillary eletrophoresis, and protein phosphatase inhibition. The doses employed were 0, 382, and 693 micrograms/kg bodyweight/d at the midpoint of the trial. Following postmortem examination blood cells, serum enzymes and organ pathology were investigated. A significant microcystin dose-dependent increase in the area of aberrant crypt foci was observed. There was no marked increase in the number of crypts/colon. Two overt colonic tumors (approximately 30 mm3) were seen in microcystin-treated mice, and one microscopic colonic tumor in an AOM-alone-treated mouse. This investigation provides the first evidence for the stimulation of preneoplastic colon tumor growth by microcystin.