Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals.

Cycads are known to host symbiotic cyanobacteria, including Nostocales species, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition to Nostoc spp., symbiotic cyanobacteria of the genus Aulosira were identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includes Aulosira spp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

On the trail of iron uptake in ancestral Cyanobacteria on early Earth.

Cyanobacteria oxygenated Earth's atmosphere ~2.4 billion years ago, during the Great Oxygenation Event (GOE), through oxygenic photosynthesis. Their high iron requirement was presumably met by high levels of Fe(II) in the anoxic Archean environment. We found that many deeply branching Cyanobacteria, including two Gloeobacter and four Pseudanabaena spp., cannot synthesize the Fe(II) specific transporter, FeoB. Phylogenetic and relaxed molecular clock analyses find evidence that FeoB and the Fe(III) transporters, cFTR1 and FutB, were present in Proterozoic, but not earlier Archaean lineages of Cyanobacteria. Furthermore Pseudanabaena sp. PCC7367, an early diverging marine, benthic strain grown under simulated Archean conditions, constitutively expressed cftr1, even after the addition of Fe(II). Our genetic profiling suggests that, prior to the GOE, ancestral Cyanobacteria may have utilized alternative metal iron transporters such as ZIP, NRAMP, or FicI, and possibly also scavenged exogenous siderophore bound Fe(III), as they only acquired the necessary Fe(II) and Fe(III) transporters during the Proterozoic. Given that Cyanobacteria arose 3.3-3.6 billion years ago, it is possible that limitations in iron uptake may have contributed to the delay in their expansion during the Archean, and hence the oxygenation of the early Earth.

Metagenomic Insights Into the Microbial Iron Cycle of Subseafloor Habitats.

Microbial iron cycling influences the flux of major nutrients in the environment (e.g., through the adsorptive capacity of iron oxides) and includes biotically induced iron oxidation and reduction processes. The ecological extent of microbial iron cycling is not well understood, even with increased sequencing efforts, in part due to limitations in gene annotation pipelines and limitations in experimental studies linking phenotype to genotype. This is particularly true for the marine subseafloor, which remains undersampled, but represents the largest contiguous habitat on Earth. To address this limitation, we used FeGenie, a database and bioinformatics tool that identifies microbial iron cycling genes and enables the development of testable hypotheses on the biogeochemical cycling of iron. Herein, we survey the microbial iron cycle in diverse subseafloor habitats, including sediment-buried crustal aquifers, as well as surficial and deep sediments. We inferred the genetic potential for iron redox cycling in 32 of the 46 metagenomes included in our analysis, demonstrating the prevalence of these activities across underexplored subseafloor ecosystems. We show that while some processes (e.g., iron uptake and storage, siderophore transport potential, and iron gene regulation) are near-universal, others (e.g., iron reduction/oxidation, siderophore synthesis, and magnetosome formation) are dependent on local redox and nutrient status. Additionally, we detected niche-specific differences in strategies used for dissimilatory iron reduction, suggesting that geochemical constraints likely play an important role in dictating the dominant mechanisms for iron cycling. Overall, our survey advances the known distribution, magnitude, and potential ecological impact of microbe-mediated iron cycling and utilization in sub-benthic ecosystems.

A low-cost automized anaerobic chamber for long-term growth experiments and sample handling.

The handling of oxygen sensitive samples and growth of obligate anaerobic organisms requires the stringent exclusion of oxygen, which is omnipresent in our normal atmospheric environment. Anaerobic workstations (aka. Glove boxes) enable the handling of oxygen sensitive samples during complex procedures, or the long-term incubation of anaerobic organisms. Depending on the application requirements, commercial workstations can cost up to 60.000 €. Here we present the complete build instructions for a highly adaptive, Arduino based, anaerobic workstation for microbial cultivation and sample handling, with features normally found only in high cost commercial solutions. This build can automatically regulate humidity, H2 levels (as oxygen reductant), log the environmental data and purge the airlock. It is built as compact as possible to allow it to fit into regular growth chambers for full environmental control. In our experiments, oxygen levels during the continuous growth of oxygen producing cyanobacteria, stayed under 0.03 % for 21 days without needing user intervention. The modular Arduino controller allows for the easy incorporation of additional regulation parameters, such as CO2 concentration or air pressure. This paper provides researchers with a low cost, entry level workstation for anaerobic sample handling with the flexibility to match their specific experimental needs.

Exploring cycad foliage as an archive of the isotopic composition of atmospheric nitrogen.

Molecular nitrogen (N2 ) constitutes the majority of Earth's modern atmosphere, contributing ~0.79 bar of partial pressure (pN2 ). However, fluctuations in pN2 may have occurred on 107 -109  year timescales in Earth's past, perhaps altering the isotopic composition of atmospheric nitrogen. Here, we explore an archive that may record the isotopic composition of atmospheric N2 in deep time: the foliage of cycads. Cycads are ancient gymnosperms that host symbiotic N2 -fixing cyanobacteria in modified root structures known as coralloid roots. All extant species of cycads are known to host symbionts, suggesting that this N2 -fixing capacity is perhaps ancestral, reaching back to the early history of cycads in the late Paleozoic. Therefore, if the process of microbial N2 fixation records the δ15 N value of atmospheric N2 in cycad foliage, the fossil record of cycads may provide an archive of atmospheric δ15 N values. To explore this potential proxy, we conducted a survey of wild cycads growing in a range of modern environments to determine whether cycad foliage reliably records the isotopic composition of atmospheric N2 . We find that neither biological nor environmental factors significantly influence the δ15 N values of cycad foliage, suggesting that they provide a reasonably robust record of the δ15 N of atmospheric N2 . Application of this proxy to the record of carbonaceous cycad fossils may not only help to constrain changes in atmospheric nitrogen isotope ratios since the late Paleozoic, but also could shed light on the antiquity of the N2 -fixing symbiosis between cycads and cyanobacteria.

An investigation into the effects of increasing salinity on photosynthesis in freshwater unicellular cyanobacteria during the late Archaean.

The oldest species of bacteria capable of oxygenic photosynthesis today are the freshwater Cyanobacteria Gloeobacter spp., belonging to the class Oxyphotobacteria. Several modern molecular evolutionary studies support the freshwater origin of cyanobacteria during the Archaean and their subsequent acquisition of salt tolerance mechanisms necessary for their expansion into the marine environment. This study investigated the effect of a sudden washout event from a freshwater location into either a brackish or marine environment on the photosynthetic efficiency of two unicellular freshwater cyanobacteria: the salt-tolerant Chroococcidiopsis thermalis PCC7203 and the cyanobacterial phylogenetic root species, Gloeobacter violaceus PCC7421. Strains were cultured under present atmospheric levels (PAL) of CO2 or an atmosphere containing elevated levels of CO2 and reduced O2 (eCO2 rO2 ) in simulated shallow water or terrestrial environmental conditions. Both strains exhibited a reduction in growth rates and gross photosynthesis, accompanied by significant reductions in chlorophyll a content, in brackish water, with only C. thermalis able to grow at marine salinity levels. While the experimental atmosphere caused a significant increase in gross photosynthesis rates in both strains, it did not increase their growth rates, nor the amount of O2 released. The differences in growth responses to increasing salinities could be attributed to genetic differences, with C. thermalis carrying additional genes for trehalose synthesis. This study demonstrates that, if cyanobacteria did evolve in a freshwater environment, they would have been capable of withstanding a sudden washout into increasingly saline environments. Both C. thermalis and G. violaceus continued to grow and photosynthesise, albeit at diminished rates, in brackish water, thereby providing a route for the evolution of open ocean-dwelling strains, necessary for the oxygenation of the Earth's atmosphere.

Global cellular responses to ß-methyl-amino-L-alanine (BMAA) by olfactory ensheathing glial cells (OEC).

This study utilised a proteomics approach to identify any differential protein expression in a glial cell line, rat olfactory ensheathing cells (OECs), treated with the cyanotoxin ß-methylamino-l-alanine (BMAA). Five proteins of interest were identified, namely Rho GDP-dissociation inhibitor 1 (RhoGDP1), Nck-associated protein 1 (NCKAP1), voltage-dependent anion-selective channel protein 1 (VDAC1), 3-hydroxyacyl-CoA dehydrogenase type-2 (3hCoAdh2), and ubiquilin-4 (UBQLN4). Four of these candidates, nuclear receptor subfamily 4 group A member 1 (Nur77), cyclophilin A (CyPA), RhoGDP1 and VDAC1, have been reported to be involved in cell growth. A microarray identified UBQLN4, palladin and CyPA, which have been implicated to have roles in excitotoxicity. Moreover, the NCKAP1, UBQLN4, CyPA and 3hCoAdh2 genes have been associated with abnormal protein aggregation. Differential expression of genes involved in mitochondrial activity, Nur77, 3hCoAdh2, VDAC1 and UBQLN4, were also identified. Confirmatory reverse transcription quantitative PCR (RT-qPCR) analysis of transcripts generated from the genes of interest corroborated the differential expression trends identified in the global protein analysis. BMAA induced cell cycle arrest in the G2/M phase of OEC and apoptosis after 48 h at concentrations of 250 µM and 500 µM. Collectively, this work advances our understanding of the mechanism of BMAA-mediated glial-toxicity in vitro.

Climate change and regulation of hepatotoxin production in Cyanobacteria.

Harmful, bloom-forming cyanobacteria (CyanoHABs) are occurring with increasing regularity in freshwater and marine ecosystems. The most commonly occurring cyanobacterial toxins are the hepatotoxic microcystin and nodularin. These cyclic hepta- and pentapeptides are synthesised nonribosomally by the gene products of the toxin gene clusters mcy and nda, respectively. Understanding of the regulation of hepatotoxin production is incomplete, although there is strong evidence supporting the roles of iron, light, higher nitrate availability and inorganic carbon in modulating microcystin levels. The majority of these studies have focused on the unicellular freshwater, microcystin-producing strain of Microcystis aeruginosa, with little attention being paid to terrestrial or marine toxin producers. This review intends to investigate the regulation of microcystin and nodularin production in unicellular and filamentous diazotrophic cyanobacteria against the background of changing climate conditions. Special focus is given to diazotrophic filamentous cyanobacteria, for example Nodularia spumigena, capable of regulating their nitrogen levels by actively fixing dinitrogen. By combining data from significant studies, an overall scheme of the regulation of toxin production is presented, focussing specifically on nodularin production in diazotrophs against the background of increasing carbon dioxide concentrations and temperatures envisaged under current climate change models. Furthermore, the risk of sustaining and spreading CyanoHABs in the future ocean is evaluated.

Gliotoxicity of the cyanotoxin, ß-methyl-amino-L-alanine (BMAA).

The amino acid variant ß-methyl-amino-L-alanine (BMAA) has long been associated with the increased incidence and progression of the amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). Previous studies have indicated that BMAA damages neurons via excitotoxic mechanisms. We have challenged rat olfactory ensheathing cells (OECs) with exogenous BMAA and found it to be cytotoxic. BMAA also induces a significant increase in Ca2+ influx, enhanced production of reactive oxygen species (ROS), and disrupts mitochondrial activity in OECs. This is the first study investigating BMAA toxicity using pure glial cells. These findings align BMAA with the three proposed mechanisms of degeneration in ALS, those being non-cell autonomous death, excitotoxicity and mitochondrial dysfunction.

Excitotoxic potential of the cyanotoxin ß-methyl-amino-L-alanine (BMAA) in primary human neurons.

The toxicity of the cyanobacterial modified amino acid, BMAA, has been described in rat, mouse and leech neurons. Particular emphasis has been placed on the potential ability of BMAA to induce neuronal damage via excitotoxic mechanisms. Here we present data indicating that the effects observed on lower organisms are also evident in a human model. Our data indicates that BMAA induces increased intracellular Ca²âº influx, DNA damage, mitochondrial activity, lactate dehydrogenase (LDH) release and generation of reactive oxygen species (ROS). The amelioration of LDH release in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 indicates that the neurotoxic effects of BMAA are mediated via NMDA receptor activation. Additionally, we have shown that BMAA induces the expression of neuronal nitric oxide synthase (nNOS) and caspase-3 indicating that it can stimulate apoptosis in human neurons, presumably via activation of NMDA receptors.

Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp.

The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. 'Macrozamia riedlei 65.1' and Nostoc sp. 'Macrozamia serpentina 73.1' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng µg-1 chlorophyll a in Nostoc sp. 'Macrozamia riedlei 65.1' and 12.5±8.4 ng µg-1 Chl a in Nostoc sp. 'Macrozamia serpentina 73.1' extracts. Further scans indicated the presence of the rare isoform [L-Har(2)] nodularin, which contains L-homoarginine instead of L-arginine. Nodularin was also present at 1.34±0.74 ng ml(-1) (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har(2)] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har(2)] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils.

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths > 10 cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.

Does α-amino-ß-methylaminopropionic acid (BMAA) play a role in neurodegeneration?

The association of α-amino-ß-methylaminopropionic acid (BMAA) with elevated incidence of amyotrophic lateral sclerosis/Parkinson's disease complex (ALS/PDC) was first identified on the island of Guam. BMAA has been shown to be produced across the cyanobacterial order and its detection has been reported in a variety of aquatic and terrestrial environments worldwide, suggesting that it is ubiquitous. Various in vivo studies on rats, mice, chicks and monkeys have shown that it can cause neurodegenerative symptoms such as ataxia and convulsions. Zebrafish research has also shown disruption to neural development after BMAA exposure. In vitro studies on mice, rats and leeches have shown that BMAA acts predominantly on motor neurons. Observed increases in the generation of reactive oxygen species (ROS) and Ca(2+) influx, coupled with disruption to mitochondrial activity and general neuronal death, indicate that the main mode of activity is via excitotoxic mechanisms. The current review pertaining to the neurotoxicity of BMAA clearly demonstrates its ability to adversely affect neural tissues, and implicates it as a potentially significant compound in the aetiology of neurodegenerative disease. When considering the potential adverse health effects upon exposure to this compound, further research to better understand the modes of toxicity of BMAA and the environmental exposure limits is essential.

How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection?

Mycobacteria have thwarted detection by scientists for centuries. Mycobacterium paratuberculosis is one of the most fastidious of the Mycobacteriaceae, and has been implicated in both animal and human diseases. In domestic livestock, M. paratuberculosis has been associated with Johne's disease, which given its increasing incidence, is currently a cause for concern, due to the potential for M. paratuberculosis to enter our food chain. In addition, a tenuous link has been reported between M. paratuberculosis and Crohn's disease, however evidence to support this link is hampered by the lack of accurate methodologies for detection of M. paratuberculosis in humans. This review compares the sensitivity and specificity of traditional and more recent techniques to the culture and molecular detection of M. paratuberculosis. While serology and culture are popular choices for the livestock industry they have not produced useful data for human infection. Although the advent of molecular biology has enabled faster diagnosis of M. paratuberculosis in human infection, there is currently no gold standard such as culture on which to validate these findings. Even with DNA/RNA detection methods, there is the ever present issue of the genetic relatedness of M. paratuberculosis to other mycobacteria of the Mycobacterium avium complex, some of which also infect humans with very different pathological outcomes. Recent developments in this field include more rapid methods of M. paratuberculosis culture as well as the development of more accurate and sensitive PCR assays. The application of these techniques should offer a greater insight as to the role of M. paratuberculosis in human gastrointestinal diseases.

Host selection of symbiotic cyanobacteria in 31 species of the Australian cycad genus: Macrozamia (Zamiaceae).

The nitrogen-fixing cyanobacterium Nostoc is a commonly occurring terrestrial and aquatic cyanobacterium often found in symbiosis with a wide range of plant, algal, and fungal species. We investigated the diversity of cyanobacterial species occurring within the coralloid roots of different Macrozamia cycad species at diverse locations throughout Australia. In all, 74 coralloid root samples were processed and 56 endosymbiotic cyanobacteria were cultured. DNA was isolated from unialgal cultures and a segment of the 16S rRNA gene was amplified and sequenced. Microscopic analysis was performed on representative isolates. Twenty-two cyanobacterial species were identified, comprising mostly Nostoc spp. and a Calothrix sp. No correlation was observed between a cycad species and its resident cyanobiont species. The predominant cyanobacterium isolated from 18 root samples occurred over a diverse range of environmental conditions and within 14 different Macrozamia spp. Phylogenetic analysis indicated that endosymbionts were not restricted to previously described terrestrial species. An isolate clustering with Nostoc PCC7120, an aquatic strain, was identified. This is the first comprehensive study to identify the endosymbionts within a cycad genus using samples obtained from their natural habitats. These results indicate that there is negligible host specialization of cyanobacterial endosymbionts within the cycad genus Macrozamia in the wild.

Genetic variation of the bloom-forming Cyanobacterium Microcystis aeruginosa within and among lakes: implications for harmful algal blooms.

To measure genetic variation within and among populations of the bloom-forming cyanobacterium Microcystis aeruginosa, we surveyed a suite of lakes in the southern peninsula of Michigan that vary in productivity (total phosphorus concentrations of approximately 10 to 100 microg liter(-1)). Survival of M. aeruginosa isolates from lakes was relatively low (i.e., mean of 7% and maximum of 30%) and positively related to lake total phosphorus concentration (P = 0.014, r2 = 0.407, n = 14). In another study (D. F. Raikow, O. Sarnelle, A. E. Wilson, and S. K. Hamilton, Limnol. Oceanogr. 49:482-487, 2004), survival rates of M. aeruginosa isolates collected from an oligotrophic lake (total phosphorus of approximately 10 mug liter(-1) and dissolved inorganic nitrogen:total phosphorus ratio of 12.75) differed among five different medium types (G test, P of <0.001), with higher survival (P = 0.003) in low-nutrient media (28 to 37% survival) than in high-nutrient media. Even with the relatively low isolate survivorship that could select against detecting the full range of genetic variation, populations of M. aeruginosa were genetically diverse within and among lakes (by analysis of molecular variance, Phi(sc) = 0.412 [Phi(sc) is an F-statistic derivative which evaluates the correlation of haplotypic diversity within populations relative to the haplotypic diversity among all sampled populations], P = 0.001), with most clones being distantly related to clones collected from lakes directly attached to Lake Michigan (a Laurentian Great Lake) and culture collection strains collected from Canada, Scotland, and South Africa. Ninety-one percent of the 53 genetically unique M. aeruginosa clones contained the microcystin toxin gene (mcyA). Genotypes with the toxin gene were found in all lakes, while four lakes harbored both genotypes possessing and genotypes lacking the toxin gene.

Comparison of the structure of key variants of microcystin to vasopressin.

Microcystins (MCs) are cyclic heptapeptide compounds [where X(2) (position 2) and Z(4) (position 4) are variable l-aminoacids] produced by cyanobacteria and responsible for severe liver damage in animals ingesting acute doses of the toxic compounds. Certain variants of microcystins are more toxic than others, the differences being commonly ascribed to the hydrophobic nature of the variant. Microcystin-LR (MCLR) [X = l-leucine (L); Z = l-arginine (R); R1 = R2 = CH(3)] is the most toxic of all the microcystins investigated to date. This study investigates the similarity of the structures of MCLR and selected MC variants to the liver specific hormone vasopressin. Structures were compiled in HyperChem(®) (professional version 5.1). Initial comparisons of the MCLR and vasopressin indicated comparable volumes, surface areas and masses. Further studies using RMS overlays show that the microcystin derivative MCLR(Dha(7)) is comparably similar to vasopressin in terms of tertiary structure.

The effect of intraperitoneally administered microcystin-LR on the gastrointestinal tract of Balb/c mice.

Microcystin-LR (MCLR) has been associated with the development of gastrointestinal complaints in people ingesting cyanobacterial bloom contaminated water. The symptoms usually present a day or two after exposure raising questions as to the toxic effects of MCLR on the gastrointestinal tract. This study investigated the apoptotic effect of ip administered MCLR over time on the duodenum, jejenum and ileum of mice receiving a single 75% LD(50) dose. The apoptotic index was significantly raised in all sections at 8 h post exposure (pe) and continued to rise for the 16, 24 and 32 h pe groups, while the glycogen levels were normal at 24 h pe. The duodenum exhibited the most significant increase in apoptotic index overall, followed by the jejenum and ileum. Immunohistochemistry indicated the presence of MCLR in the lamina propria suggesting a role for MCLR in the induction of apoptosis in the GIT of mice exposed to a single sublethal dose of MCLR.

The role of microcystin-LR in the induction of apoptosis and oxidative stress in CaCo2 cells.

The increasing presence of toxic cyanobacteria in drinking and recreational water bodies, and their potential to impact on human and animal health is cause for concern. Recent work suggests that apoptosis plays a major role in the toxic effects induced by microcystin-LR (MCLR) in the gastrointestinal tract; however, the biochemical pathway remains elusive. Exposure of CaCo2, a human colon carcinoma cell line, and MCF-7, a cell line deficient in pro-caspase-3, cells to 50 microM MCLR induced similar reductions in cell viability as measured by MTT and LDH leakage. The role of MCLR induced oxidative stress in the initiation of apoptosis was investigated over a 2-hr period, and it was found that there was an increase in the release of H(2)O(2) in the first 30 min of exposure for both cell lines. Both cell lines exhibited a dose-dependent increase in both micro- and millicalpain after 24 h exposure to MCLR suggesting a role for protease activation in MCLR-induced apoptosis in non-hepatic human derived cell lines.

An investigation into the detoxification of microcystin-LR by the glutathione pathway in Balb/c mice.

Toxin-producing cyanobacteria pose a world-wide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. The predominant cyanotoxin, microcystin-LR (MCLR), targets the liver and its toxicity depends on the uptake and removal rates in the liver. The role of the glutathione detoxification pathway in protecting the liver from the effects of MCLR was investigated. Mice exposed to a single 75% LD(50) dose of pure MCLR were sacrificed at 8, 16, 24 and 32 h post-exposure (pe). Toxin induced liver damage was observed 8 and 16 h pe as evidenced by raised serum ALT and LDH levels, reduced glycogen levels and liver histology. A significant increase in lipid peroxidation was seen at 16 h pe that decreased after 24 and 32 h pe, the time-points which showed significant increases in GPX activity. An increase in soluble GST activity was noted between 8 and 16 h pe, levels of total GSH increased at 24 h while oxidised glutathione increased throughout the investigation. The increase in activity of both GPX and GST corresponded with increased transcription of these enzymes, as well as the rate-limiting enzyme in GSH synthesis, gamma-glutamyl transferase. In conclusion, this study confirms that an increase in GST activity is critical for the detoxification of MCLR, that this is regulated at the transcriptional level, and that exposure to MCLR induces the de novo synthesis of GSH. Finally, we report the involvement of GPX in the removal of MCLR-induced lipid hydroperoxides.