Microcystin-LR activates the ERK1/2 kinases and stimulates the proliferation of the monkey kidney-derived cell line Vero-E6.

Microcystin-LR (MCLR) is a peptide produced by freshwater cyanobacteria that induces severe hepatotoxicity in humans and animals. MCLR is also a potent tumour promoter and it has been proposed that this activity is mediated by the inhibition of protein phosphatases PP1/PP2A, possibly through the activation of proto-oncogenes c-jun, c-fos and c-myc. However, the mechanisms underlying MCLR-induced tumour promotion are still largely unknown, particularly in non-liver cells. In previous studies we have demonstrated that micromolar concentrations of MCLR induce cytotoxic effects in the kidney Vero-E6 cell line. The purpose of the present work was to evaluate whether the exposure to subcytotoxic concentrations of MCLR was sufficient to induce the proliferation of Vero-E6 cells. Through BrdU incorporation assay we show that at nanomolar concentrations MCLR stimulates cell cycle progression in Vero-E6 kidney cell line. Moreover, the analysis of mitogen-activated protein kinases p38, JNK and ERK1/2 activity revealed that the proliferative effect of MCLR is associated with the activation of the pro-proliferative ERK1/2 pathway. These results emphasise the importance to confirm in vivo the impact of MCLR on tumour promotion at kidney level.

Comparative study of the cytotoxic effect of microcistin-LR and purified extracts from Microcystis aeruginosa on a kidney cell line.

Microcystin-LR (MCLR) is a potent hepatotoxin, but increasing evidences suggest that it might also induce kidney injury. The aim of this work was to evaluate the cytotoxicity of MCLR on a kidney cell line (Vero-E6). Cells were exposed for up to 72 h either to Microcystis aeruginosa extracts from both MCLR-producer and non-MCLR-producer isolates or to pure MCLR (1.5-200 microM). The cytotoxic effects were evaluated by several cell viability assays (MTT, Neutral Red and LDH). Pure MCLR, the extract from MCLR-producer and the mixture of the non-MCLR-producer with pure MCLR, induced cell viability decrease in a similar dose/time-dependent manner. Conversely, no effects were induced by the extract of non-MCLR-producer. These results suggest that the cytotoxic effects of M. aeruginosa extract were due to MCLR and excluded the eventual toxicity of other cyanobacteria bioactive compounds. The lowest cytotoxic MCLR concentration varied between 11 and 100 microM depending on the employed cell viability assay and is within the range of MCLR dosage reported to affect other mammalian cell lines. The NR assay was the most sensitive to evaluate the MCLR-induced cytotoxicity. Our results suggest that Vero-E6 cell line may constitute a cell model to evaluate the nephrotoxicity of microcystins.

Morphological and ultrastructural effects of microcystin-LR from Microcystis aeruginosa extract on a kidney cell line.

The aim of this study was to examine the toxic effects of a microcystin-LR (MCLR)-containing cyanobacteria extract on the subcellular organization of a kidney cell line (Vero-E6). Cells were exposed to different MCLR concentrations (1.3-150 microM) for 24, 48 and 72h and two cytotoxicity assays were performed. This information was combined with the analysis of lysosomal, mitochondrial and cytoskeleton integrity and with an ultrastructural study. Biochemical and microscopic data revealed a good agreement and demonstrated that cellular response to MCLR is dependent on the dose/exposure time. Cell viability decayed markedly after 24h of exposure to toxin concentrations greater than 30 microM. Furthermore, it was demonstrated that lysosome destabilization precedes mitochondria dysfunction. The ultrastructural analysis showed that mild toxin incubation conditions induce endoplasmic reticulum (ER) vacuolization and assembly of large autophagic vacuoles, suggesting that autophagy is an early cellular response to the toxin. After exposure to higher MCLR doses, the number of apoptotic cells increased, as identified by microscopic observations and confirmed with TUNEL assay. Additionally, drastic exposure conditions induced the increase of necrotic cells. These results suggest that the ER is the primary microcystin target in Vero cells and that autophagy, apoptosis and necrosis are induced in a dose- and time-dependent manner.

High-fish consumption and risk prevention: assessment of exposure to methylmercury in Portugal.

The aim of this study was to evaluate the exposure to methylmercury (MeHg) of potential populations at risk living in Portugal. To ascertain youth exposure, a questionnaire was distributed to 300 students of a middle secondary school in Sesimbra and to 429 students studying in Canecas, selected as the control population. The average number of fish meals consumed by person was 4.1 and 3 per week in Sesimbra and Canecas, respectively. The subpopulations of high intake (PHI) corresponding to those ingesting 7 or more fish meals per week were also analyzed separately, with 17% of the students belonging to the PHI of Sesimbra versus 6.1% in Canecas. Socioeconomic aspects such as relative's professional involvement with fisheries correlated with the higher intakes in Sesimbra. Fish samples were collected in the dock of Sesimbra and total mercury (Hg) was determined by flow injection cold vapor atomic fluorescence spectroscopy (FI-CV-AFS). The mean value found for nonpredators was 0.035 microg/g. Dogfish specimens surpassed the legislated limit for predator species and increased the predators mean to 1 microg/g. The cross-sectional data were integrated with the fish analysis results to estimate the population exposure to MeHg. The indices of risk calculated for youth reached values of 4.5, demonstrating the existence of risk to a part of the population exceeding the provisional tolerable weekly intake (PTWI) level mandated by WHO (1.6 microg/kg bw). The results indicate that monitoring of Hg levels in fish is mandatory and counseling should be provided to populations at risk, encouraging them to prevent the risk.

Risk of human exposure to paralytic toxins of algal origin.

The most significant neurotoxins produced by harmful algal blooms (HABs) are paralytic shellfish toxins (PSTs) found in shellfish and freshwater. Human exposure to neurotoxins through the food consumption represents a severe hazard to human health and the exposure through contaminated water represents an added risk often difficult to recognize. Furthermore, there is an insufficient knowledge of toxicokinetics of these complex toxins produced by HABs. If human acute exposure occurs, the diagnosis of intoxication is typically based upon symptomatology and analysis of shellfish tissue by mouse bioassay, HPLC-FLD analysis and mouse neuroblastoma assay. However, the health risks due to chronic exposure should also be considered and its prevention could be reached with a better understanding of sub-lethal doses of these toxins. In this context, information required for development of a diagnostic protocol should include knowledge about toxicokinetics and toxicodynamics of these neurotoxins. We emphasise the importance of research on biomarkers to prevent, predict and diagnose acute and chronic human exposure to PST.