Cylindrospermopsin: A Decade of Progress on Bioaccumulation Research

Cylindrospermopsin (CYN) is rapidly being recognised as one of the most globally important of the freshwater algal toxins. The ever-expanding distribution of CYN producers into temperate zones is heightening concern that this toxin will represent serious human, as well as environmental, health risks across many countries. Since 1999, a number of studies have demonstrated the ability for CYN to bioaccumulate in freshwater organisms. This paper synthesizes the most current information on CYN accumulation, including notes on the global distribution of CYN producers, and a précis of CYN’s ecological and human effects. Studies on the bioaccumulation of CYN are systematically reviewed, together with an analysis of patterns of accumulation. A discussion on the factors influencing bioaccumulation rates and potential is also provided, along with notes on detection, monitoring and risk assessments. Finally, key gaps in the existing research are identified for future study

On the Chemistry, Toxicology and Genetics of the Cyanobacterial Toxins, Microcystin, Nodularin, Saxitoxin and Cylindrospermopsin

The cyanobacteria or “blue-green algae”, as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that inhabit a wide range of aquatic and terrestrial environments, and display incredible morphological diversity. Many aquatic, bloom-forming species of cyanobacteria are capable of producing biologically active secondary metabolites, which are highly toxic to humans and other animals. From a toxicological viewpoint, the cyanotoxins span four major classes: the neurotoxins, hepatotoxins, cytotoxins, and dermatoxins (irritant toxins). However, structurally they are quite diverse. Over the past decade, the biosynthesis pathways of the four major cyanotoxins: microcystin, nodularin, saxitoxin and cylindrospermopsin, have been genetically and biochemically elucidated. This review provides an overview of these biosynthesis pathways and additionally summarizes the chemistry and toxicology of these remarkable secondary metabolites

Investigation of the mechanisms involved in cylindrospermopsin toxicity : hepatocyte culture and reticulocyte lysate studies / Suzanne M. Froscio.

Copy of author's previously published work inserted.Bibliography: leaves 121-139.xvii, 139 leaves : ill. (some col.) ; 30 cm.The aim of this study was to determine the extent to which protein synthesis inhibition, lowered glutathione (GSH) levels and toxin metabolism contribute to the toxicity of cyclindrospermopsin. Both hepatocyte cultures and reticulocyte lysates were utilized as in vitro tools of investigation. The findings imply that the inhibition of protein synthesis by direct action of the toxin cannot be considered a primary cause of hepatocyte cell death over an acute time frame. Cytochrome P450-derived metabolites may play a crucial role in cytotoxicity, and the toxicity process does not appear to involve oxidative damage.Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 2002

An examination of the antibiotic effects of cylindrospermopsin on common gram-positive and gram-negative bacteria and the protozoan Naegleria lovaniensis

Copyright © 2008 Wiley Periodicals, Inc., A Wiley CompanyThe importance of the toxin cylindrospermopsin to the function and fitness of the cyanobacteria that produce it remains a matter of conjecture. Given that the structure of cylindrospermopsin has commonalities with other antibacterial protein synthesis inhibitors, such as streptomycin, authors tested the possibility that the toxin might act as an antibacterial compound that can kill competing microbes. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were tested by the minimal inhibitory concentration method and significant antibacterial activity was only observed at a cylindrospermopsin concentration of 300 microg mL(-1) after exposure for 5 days. No effect on log phase growth of E. coli was observed for this same toxin concentration. Protein synthesis was inhibited by cylindrospermopsin in E. coli 70S extracts, reduced by 25% compared with controls when treated with 41.5 microg mL(-1) of the toxin; however, a much greater reduction of 97% was observed for chloramphenicol in the same experiment. Naegleria lovaniensis, a phagotrophic protozoan, was more susceptible to cylindrospermopsin, with a decrease in the number of N. lovaniensis plaques after 24-h treatment with 5-50 microg mL(-1) of toxin and an LC(50) of approximately 60 microg mL(-1). Given these results, cylindrospermopsin is clearly not antibacterial at concentrations found in environmental waters, nor will it adversely affect N. lovaniensis at these concentrations. For organisms that are able to ingest cylindrospermopsin-producing cells, the response of N. lovaniensis to the toxin suggests that only a few ingested cells would be enough to kill predatory organisms with similar susceptibility.J. Paul Rasmussen, Michael Cursaro, Suzanne M. Froscio and Christopher P. Sain

Cytotoxicity screening for the cyanobacterial Toxin cylindrospermopsin

The cell lines C3A, HepG2, NCI-87, HCT-8, HuTu-80, Caco-2, and Vero were screened for sensitivity to the cyanobacterial toxin cylindrospermopsin (CYN), with the aim of determining the most sensitive cells to be used in cytotoxicity tests. Cell lines were chosen to be representative of the organs targeted by the toxin; liver, kidney, intestine, and were expected to have different metabolic activities and uptake capabilities. Over the range of cell lines tested, IC₅₀ determinations at 24 h (MTT assay) ranged fourfold, from 1.5 μM for hepatocyte-derived cell lines (C3A IC₅₀ = 1.5 ± 0.54; HepG2 IC₅₀ = 1.5 ± 0.87) to 6.5 ± 3.3μM for the colon-derived Caco-2 cell line. The cell-line sensitivity seemed to decrease in cell lines derived from progressively more distal regions of the gastrointestinal tract: gastric > duodenal > ileal > colonic. The greater sensitivity of the hepatic cell lines to CYN was also apparent in 7-d exposure studies, with low toxin concentrations exerting cytotoxic effects that were not seen in other cell lines. Short-term exposure of C3A cells to CYN (1-6 h) was shown to induce cytotoxicity at 24 h despite a washout and recovery incubation, demonstrating the protracted and apparently irreversible nature of CYN's toxic effects.Suzanne M. Froscio, Stella Fanok and Andrew R. Humpag

Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells

Copyright © 2009 Elsevier Ltd. All rights reserved.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.S.M. Froscio, E. Cannon, H.M. Lau and A.R. Humpag

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

Copyright © 2008 Elsevier B.V.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.S. Laingama, S.M. Froscioa and A.R. Humpag