Raquel M. Soares

Microcystin contamination in fish from the Jacarepaguá Lagoon (Rio de Janeiro, Brazil): ecological implication and human health risk.

Chronic and subchronic toxicity from exposure to microcystins, cyclic peptide liver toxins from certain cyanobacteria, poses an important hazard, which has received little study. No in vivo information exists on accumulation and transfer of microcystin from the food chain to humans. This paper present results of a 3-year study that demonstrates bioaccumulation of microcystins by fish and potential rates of microcystin ingestion by humans. The study was carried out in a shallow coastal lagoon in the city of Rio de Janeiro (Jacarepaguá Lagoon). Fish (Tilapia rendalli) were collected every 2 weeks from August 1996 to November 1999. Microcystins were analyzed by HPLC in phytoplankton, fish liver and viscera while fish muscle tissue was analyzed by enzyme linked immunosorbant assay (ELISA). Phytoplankton samples, dominated by the genus Microcystis, were confirmed to contain microcystins as were fish livers, viscera and muscle tissue. During the entire study period, including times of low water bloom densities, fish muscle tissue contained concentrations of microcystins close to or above the recommended limit for human consumption (0.04 microg x kg(-1) day). Our findings demonstrate that microcystins can accumulate in fish tissue used for human consumption. Rates of ingestion routinely exceed the TDI guidelines as set by the WHO for drinking water. Appropriate epidemiology and risk assessment should be undertaken so that an acceptable TDI and appropriate risk management decisions can be made for human consumption of fish which are harvested from cyanobacterial blooms that contain cyanotoxins.

Microcystins in South American aquatic ecosystems: occurrence, toxicity and toxicological assays.

The acute poisoning of chronic renal patients during hemodialysis sessions in 1996 in Caruaru City (Pernambuco State, Brazil) stimulated an intensive search for the cause of this severe complication. This search culminated in the identification of microcystins (MC), hepatotoxic cyclic heptapeptides produced by cyanobacteria, as the causative agents. More than ten years later, additional research data provides us with a better understanding of the factors related to cyanobacterial bloom occurrence and production of MC in Brazil and other South American countries. The contamination of water bodies and formation of toxic blooms remains a very serious concern, especially in countries in which surface water is used as the main source for human consumption. The purpose of this review is to highlight the discoveries of the past 15 years that have brought South American researchers to their current level of understanding of toxic cyanobacteria species and that have contributed to their knowledge of factors related to MC production, mechanisms of action and consequences for human health and the environment.

Sublethal Microcystin Exposure and Biochemical Outcomes among Hemodialysis Patients

Cyanobacteria are commonly-occurring contaminants of surface waters worldwide. Microcystins, potent hepatotoxins, are among the best characterized cyanotoxins. During November, 2001, a group of 44 hemodialysis patients were exposed to microcystins via contaminated dialysate. Serum microcystin concentrations were quantified with enzyme-linked immunosorbent assay which measures free serum microcystin LR equivalents (ME). We describe serum ME concentrations and biochemical outcomes among a subset of patients during 8 weeks following exposure. Thirteen patients were included; 6 were males, patients’ median age was 45 years (range 16–80), one was seropositive for hepatitis B surface antigen. The median serum ME concentration was 0.33 ng/mL (range: <0.16–0.96). One hundred thirty nine blood samples were collected following exposure. Patients’ biochemical outcomes varied, but overall indicated a mixed liver injury. Linear regression evaluated each patient’s weekly mean biochemical outcome with their maximum serum ME concentration; a measure of the extrinsic pathway of clotting function, prothrombin time, was negatively and significantly associated with serum ME concentrations. This group of exposed patients’ biochemical outcomes display evidence of a mixed liver injury temporally associated with microcystin exposure. Interpretation of biochemical outcomes are complicated by the study population’s underlying chronic disease status. It is clear that dialysis patients are a distinct ‘at risk’ group for cyanotoxin exposures due to direct intravenous exposure to dialysate prepared from surface drinking water supplies. Careful monitoring and treatment of water supplies used to prepare dialysate is required to prevent future cyanotoxin exposure events.

LASSBio 596 per os avoids pulmonary and hepatic inflammation induced by microcystin-LR

Cyanobacterial blooms that generate microcystins (MCYSTs) are increasingly recognized as an important health problem in aquatic ecosystems. We have previously reported the impairment of pulmonary structure and function by microcystin-LR (MCYST-LR) exposure as well as the pulmonary improvement by intraperitoneally injected (i.p.) LASSBio 596. In the present study, we aimed to evaluate the usefulness of LASSBio 596 per os on the treatment of pulmonary and hepatic injuries induced by MCYST-LR. Swiss mice received an intraperitoneal injection of 40 μl of saline (CTRL) or a sub-lethal dose of MCYST-LR (40 μg/kg). After 6 h the animals received either saline (TOX and CTRL groups) or LASSBio 596 (50 mg/kg, LASS group) by gavage. Eight hours after the first instillation, lung impedance (static elastance, elastic component of viscoelasticity and resistive, viscoelastic and total pressures) was determined by the end-inflation occlusion method. Left lung and liver were prepared for histology. In lung and hepatic homogenates MCYST-LR, TNF-α, IL-1β and IL-6 were determined by ELISA. LASSBio 596 per os (LASS mice) kept all lung mechanical parameters, polymorphonuclear (PMN) cells, pro-inflammatory mediators, and alveolar collapse similar to control mice (CTRL), whereas in TOX these findings were higher than CTRL. Likewise, liver structural deterioration (hepatocytes inflammation, necrosis and steatosis) and inflammatory process (high levels of pro-inflammatory mediators) were less evident in the LASS than TOX group. LASS and CTRL did not differ in any parameters studied. In conclusion, orally administered LASSBio 596 prevented lung and hepatic inflammation and completely blocked pulmonary functional and morphological changes induced by MCYST-LR.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 microl, ip) and group Ci received a sublethal dose of microcystins (48.2 microg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262%) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161%). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.

Can LASSBio 596 and dexamethasone treat acute lung and liver inflammation induced by microcystin-LR?

The treatment of microcystin-LR (MCYST-LR)-induced lung inflammation has never been reported. Hence, LASSBio 596, an anti-inflammatory drug candidate, designed as symbiotic agent that modulates TNF-alpha levels and inhibits phosphodiesterase types 4 and 5, or dexamethasone were tested in this condition. Swiss mice were intraperitoneally (i.p.) injected with 60 microl of saline (CTRL) or a sub-lethal dose of MCYST-LR (40 micrg/kg). 6 h later they were treated (i.p.) with saline (TOX), LASSBio 596 (10 mg/kg, L596), or dexamethasone (1 mg/kg, 0.1 mL, DEXA). 8 h after MCYST-LR injection, pulmonary mechanics were determined, and lungs and livers prepared for histopathology, biochemical analysis and quantification of MCYST-LR. TOX showed significantly higher lung impedance than CTRL and L596, which were similar. DEXA could only partially block the mechanical alterations. In both TOX and DEXA alveolar collapse and inflammatory cell influx were higher than in CTRL and L596, being LASSBio 596 more effective than dexamethasone. TOX showed oxidative stress that was not present in CTRL and L596, while DEXA was partially efficient. MCYST-LR was detected in the livers of all mice receiving MCYST-LR and no recovery was apparent. In conclusion, LASSBio 596 was more efficient than dexamethasone in reducing the pulmonary functional impairment induced by MCYST-LR.

Single sublethal dose of microcystin-LR is responsible for different alterations in biochemical, histological and physiological renal parameters

Microcystins (MCYSTs) are very stable cyclic peptidic toxins produced by cyanobacteria. Their effects on hepatic tissue have been studied extensively, and they are considered to be a potent hepatotoxin. However, several effects of MCYST on other organs have also been described, but generally in studies using higher doses of MCYST. In the present work, we investigated the effect of a single sublethal dose of MCYST-LR (55 μg/kg) in Wistar rats and analyzed different aspects that influenced renal physiology, including toxin accumulation, excretion, histological morphology, biochemical responses and oxidative damage in the kidney. After 24 h of exposure to MCYST-LR, it was possible to observe an increased glomerular filtration rate (6.28 ± 1.56 vs 2.16 ± 0.48 μl/min per cm(2)) compared with the control group. Increase of interstitial space and collagen deposition corresponded to a fibrotic response to the increased production of reactive oxygen species. The observed decrease of Na(+) reabsorption was due to inhibition of the activity of both Na(+) pumps in proximal tubules cells. We suggested that this modulation is mediated by the effect of MCYST as a phosphatase protein inhibitor that maintains the sustained kinase-mediated regulatory phosphorylation of the ATPases. The observed alteration of Na(+) active transporters lead to damage of renal function, since are involved in regulation of water and solute reabsorption in proximal tubules. The results of this report reinforce the importance of understanding the molecular effects of a single sublethal dose of MCYST-LR, which, in this study, was responsible for macro-alterations found in the renal parenchyma and renal physiology in rats.

Pulmonary and hepatic injury after sub-chronic exposure to sublethal doses of microcystin-LR

We had previously shown that microcystin-LR (MCLR) could induce lung and liver inflammation after acute exposure. The biological outcomes following prolonged exposure to MCLR, although more frequent, are still poorly understood. Thus, we aimed to verify whether repeated doses of MCLR could damage lung and liver and evaluate the dose-dependence of the results. Male Swiss mice received 10 intraperitoneal injections (i.p.) of distilled water (60 μL, CTRL) or different doses of MCLR (5 μg/kg, TOX5), 10 μg/kg (TOX10), 15 μg/kg (TOX15) and 20 μg/kg (TOX20) every other day. On the tenth injection respiratory mechanics (lung resistive and viscoelastic/inhomogeneous pressures, static elastance, and viscoelastic component of elastance) was measured. Lungs and liver were prepared for histology (morphometry and cellularity) and inflammatory mediators (KC and MIP-2) determination. All mechanical parameters and alveolar collapse were significantly higher in TOX5, 10, 15 and 20 than CTRL, but did not differ among them. Lung inflammatory cell content increased dose-dependently in all TOX groups in relation to CTRL, being TOX20 the largest. The production of KC was increased in lung and liver homogenates. MIP-2 increased in the liver of all TOX groups, but in lung homogenates it was significantly higher only in TOX20 group. All TOX mice livers showed steatosis, necrosis, inflammatory foci and a high degree of binucleated hepatocytes. In conclusion, sub-chronic exposure to MCLR damaged lung and liver in all doses, with a more important lung inflammation in TOX20 group.

Repeated intranasal exposure to microcystin-LR affects lungs but not nasal epithelium in mice

Microcystin-LR (MC-LR) is a harmful cyanotoxin able to induce adverse outcomes in the respiratory system. We aimed to examine the lungs and nasal epithelium of mice following a sub-chronic exposure to MC-LR. Swiss mice were intranasally instilled with 10 μL of distilled water (CTRL, n = 10) or 6.7 ng/kg of MC-LR diluted in 10 μL of distilled water (TOX, n = 8) during 30 consecutive days. Respiratory mechanics was measured in vivo and histology measurements (morphology and inflammation) were assessed in lungs and nasal epithelium samples 24 h after the last intranasal instillation. Despite the lack of changes in the nasal epithelium, TOX mice displayed an increased amount of PMN cells in the lungs (× 10(-3)/μm(2)), higher lung static elastance (cmH2O/mL), resistive and viscoelastic/inhomogeneous pressures (cmH2O) (7.87 ± 3.78, 33.96 ± 2.64, 1.03 ± 0.12, 1.01 ± 0.08, respectively) than CTRL (5.37 ± 4.02, 26.65 ± 1.24, 0.78 ± 0.06, 0.72 ± 0.05, respectively). Overall, our findings suggest that the nasal epithelium appears more resistant than lungs in this model of MC-LR intoxication.

Dualistic evolution of liver damage in mice triggered by a single sublethal exposure to Microcystin-LR.

Microcystins (MCYST) are the most frequently reported cyanotoxins in human poisoning incidents. Despite the well-described mechanism of acute and lethal injury, the sublethal effects of this toxin require further investigation. The aim of this study was to contribute to the knowledge of the variant MCYST-LR effects at sublethal doses by investigating biochemical changes and tissue damage in a murine model. For this purpose, mice were intraperitoneally injected with 45 μg of MCYST-LR/kg body weight. Their organs were collected at 2, 8, 24, 48 or 96 h after injection. Control animals received saline solution. We detected oxidative imbalance in the liver, particularly at 8 h after exposure. Furthermore, biomarkers of liver injury were detected in high concentration in the serum of the exposed animals. Stereological analyses of the liver indicated two different phases in the intoxication process: an initial phase characterized by an increase in steatosis was followed by a second, later phase characterized by increased inflammation and hepatocyte binucleation. Formation of areas of necrosis and increased blood vessel diameter were observed throughout the experimental period. The number of hepatocytes per area unit also decreased. However, these parameters recovered over the period of exposure. MCYST accumulated in liver and was detectable until the end of the monitoring period. These results confirm the necessity for further studies of processes involved in sublethal exposure to MCYST.