Microcystin Toxicokinetics, Molecular Toxicology, and Pathophysiology in Preclinical Rodent Models and Humans.

Microcystins are ubiquitous toxins produced by photoautotrophic cyanobacteria. Human exposures to microcystins occur through the consumption of contaminated drinking water, fish and shellfish, vegetables, and algal dietary supplements and through recreational activities. Microcystin-leucine-arginine (MCLR) is the prototypical microcystin because it is reported to be the most common and toxic variant and is the only microcystin with an established tolerable daily intake of 0.04 µg/kg. Microcystin toxicokinetics is characterized by low intestinal absorption, rapid and specific distribution to the liver, moderate metabolism to glutathione and cysteinyl conjugates, and low urinary and fecal excretion. Molecular toxicology involves covalent binding to and inhibition of protein phosphatases, oxidative stress, cell death (autophagy, apoptosis, necrosis), and cytoskeleton disruption. These molecular and cellular effects are interconnected and are commonly observed together. The main target organs for microcystin toxicity are the intestine, liver, and kidney. Preclinical data indicate microcystins may also have nervous, pulmonary, cardiac, and reproductive system toxicities. Recent evidence suggests that exposure to other hepatotoxic insults could potentiate microcystin toxicity and increase the risk for chronic diseases. This review summarizes the current knowledge for microcystin toxicokinetics, molecular toxicology, and pathophysiology in preclinical rodent models and humans. More research is needed to better understand human toxicokinetics and how multifactorial exposures contribute to disease pathogenesis and progression.

Kinetics of Microcystin-LR Removal in a Real Lake Water by UV/H2O2 Treatment and Analysis of Specific Energy Consumption.

The hepatotoxin microcystin-LR (MC-LR) represents one of the most toxic cyanotoxins for human health. Considering its harmful effect, the World Health Organization recommended a limit in drinking water (DW) of 1 µg L-1. Due to the ineffectiveness of conventional treatments present in DW treatment plants against MC-LR, advanced oxidation processes (AOPs) are gaining interest due to the high redox potential of the OH• radicals. In this work UV/H2O2 was applied to a real lake water to remove MC-LR. The kinetics of the UV/H2O2 were compared with those of UV and H2O2 showing the following result: UV/H2O2 > UV > H2O2. Within the range of H2O2 tested (0-0.9 mM), the results showed that H2O2 concentration and the removal kinetics followed an increasing quadratic relation. By increasing the initial concentration of H2O2, the consumption of oxidant also increased but, in terms of MC-LR degraded for H2O2 dosed, the removal efficiency decreased. As the initial MC-LR initial concentration increased, the removal kinetics increased up to a limit concentration (80 µg L-1) in which the presence of high amounts of the toxin slowed down the process. Operating with UV fluence lower than 950 mJ cm-2, UV alone minimized the specific energy consumption required. UV/H2O2 (0.3 mM) and UV/H2O2 (0.9 mM) were the most advantageous combination when operating with UV fluence of 950-1400 mJ cm-2 and higher than 1400 mJ cm-2, respectively.

In vitro assessment of cyanotoxins bioaccessibility in raw and cooked mussels.

The oral route by ingestion of water and food contaminated with cyanotoxins is the main route of exposure to these toxins. This study addresses for the first time the bioaccessibility of some of the most common Microcystins (MC-LR, MC-RR and MC-YR) and Cylindrospermopsin (CYN) simultaneously in raw and steamed mussels spiked at 250 ng/g fresh weight of each cyanotoxin, after an in vitro digestion, including the salivary (incubation with artificial saliva, 30s), gastric (with pepsin, 2h, pH 2), duodenal (with pancreatin and bile salts, 2h, pH 6.5) and colonic phases (with lactic-acid bacteria, 48h, pH 7.2). The results obtained suggest that the potential absorption of these cyanotoxins by consumption of contaminated mussels is lower than expected. After the total effect of cooking and digestion, the mean bioaccessibility levels recorded were 24.65% (CYN), 31.51% (MC-RR), 17.51% (MC-YR) and 13.20% (MC-LR). Moreover, toxins were transferred to the steaming waters at 3.77 ± 0.24 µg L-1 CYN, 2.29 ± 0.13 µg L-1 MC-LR, 6.60 ± 0.25 µg L-1 MC-RR and 3.83 ± 0.22 µg L-1 MC-YR. These bioaccessibility results should be considered for a more accurate risk assessment related to these cyanotoxins in mussels, including the fact that the steaming waters could also represent a risk after human consumption.

Development and Application of Extraction Methods for LC-MS Quantification of Microcystins in Liver Tissue.

A method was developed to extract and quantify microcystins (MCs) from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using HPLC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) from spiked liver tissue and the MC-LR cysteine adduct (MC-LR-Cys) created by the glutathione detoxification pathway. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ≥ 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 µg or 100 µg of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (experiment) mice were used as a model to study non-alcoholic fatty liver disease. The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest.

Machine learning prediction of cyanobacterial toxin (microcystin) toxicodynamics in humans.

Microcystins (MC) represent a family of cyclic peptides with approx. 250 congeners presumed harmful to human health due to their ability to inhibit ser/thr-proteinphosphatases (PPP), albeit all hazard and risk assessments (RA) are based on data of one MC-congener (MC-LR) only. MC congener structural diversity is a challenge for the risk assessment of these toxins, especially as several different PPPs have to be included in the RA. Consequently, the inhibition of PPP1, PPP2A and PPP5 was determined with 18 structurally different MC and demonstrated MC congener dependent inhibition activity and a lower susceptibility of PPP5 to inhibition than PPP1 and PPP2A. The latter data were employed to train a machine learning algorithm that should allow prediction of PPP inhibition (toxicity) based on MCs 2D chemical structure. IC50 values were classified in toxicity classes and three machine learning models were used to predict the toxicity class, resulting in 80-90% correct predictions.

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards.

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC.

Bioavailability of bound microcystins in mice orally fed with contaminated tilapia edible tissues: Implications to human health.

This study investigates for the first time in vivo potential release of bound microcystins (MCs) from edible fish tissues. Bound MCs were released in the mouse digestive system and reached the liver (2.1-14.3 ng g-1) and blood (1-5 ng ml-1). The released MCs were active and negatively affected liver functions of treated mice. The estimated daily intake (EDI) for bound MCs (93.5 µg kg-1 day-1) is about 2300 times higher than the WHO safe EDI (0.04 µg kg-1 day-1), compared to lower EDI for free MCs (0.002 µg kg-1 day-1). Therefore, bound MCs in fish tissues should be considered when fish are monitored for human consumption.

Hepatotoxicity induced by paclitaxel interaction with turmeric in association with a microcystin from a contaminated dietary supplement.

A 67-year-old Caucasian male with lung cancer was presented to the Emergency Department with asthenia, anorexia, jaundice and choluria. The patient's lung cancer was being treated medically by a combination of paclitaxel/carboplatin with bi-monthly frequency. The patient was also self-medicating with several natural products, including Chlorella (520 mg/day), Silybum marianum (total of 13.5 mg silymarin/day), zinc sulphate (5.5 mg), selenium (50 µg) and 15 g/day of Curcuma longa. In first chemotherapy cycle no toxicity was observed even he was taking other medications as budesonide and sitagliptin. The toxic events started only after the introduction of the dietary products. Chlorella had contamination with cyanobacteria (Oscillatoriales) and 1.08 µg of cyanotoxin Microcystin-LR (MC-LR) per gram of biomass was found. Patient was consuming ca 0.01 µg MC-LR/kg/day. This case report describes the first known case of paclitaxel toxicity probably related to pharmacokinetic interaction with Turmeric and a contaminated Chlorella supplement resulting in an acute toxic hepatitis and the impact on oncologic patient health.

Chronic exposure of nanomolar MC-LR caused oxidative stress and inflammatory responses in HepG2 cells.

Low dose but long-term exposure of microcystin-LR (MC-LR) could induce human hepatitis and promote liver cancer according to epidemiological investigation results, but the exact mechanism has not been completely elucidated. In the present study, a chronic toxicity test of MC-LR exposure on HepG2 cells at 0.1-30 nM for 83 d was conducted under laboratory conditions. The western blot assay result revealed that MC-LR entered HepG2 cells, even at the concentration of 0.1 nM, after 83 d of exposure, but no cytotoxicity was observed in the HepG2 cells, as determined by the CCK-8 and LDH tests. However, the results of the DCF fluorescence assay showed that the intracellular ROS level in the 30 nM MC-LR-treated cells was significantly higher than that of the control cells, and 5 and 10 nM of MC-LR exposure totally increased the activity of SOD in HepG2 cells. These results indicate that MC-LR exposure at low concentration also induced excessive ROS in HepG2 cells. Additionally, long-term exposure of MC-LR at low concentration remarkably promoted the expression of NF-κB p65, COX-2, iNOS, TNF-α, IL-1ß, and IL-6 in the cells, suggesting that long-term MC-LR exposure at low concentration can induce inflammatory reaction to HepG2 cells, which might account for MC-induced human hepatitis. Thus, we hypothesized that the pathogenesis of human hepatitis and hepatocarcinoma caused by MCs might be closely associated with oxidative stress and inflammation.

Lettuce facing microcystins-rich irrigation water at different developmental stages: Effects on plant performance and microcystins bioaccumulation.

This study investigated the microcystins (MCs)-rich irrigation water effect on lettuce of different developmental stages, i.e. during a two months period, covering the whole period from seed germination to harvest at marketable size of the plant. We followed four lettuce plant groups receiving MCs-rich water (1.81µgl-1 of dissolved MCs), originating from the Karla Reservoir, central Greece: 1) from seeds, 2) the cotyledon, 3) two true leaves and 4) four true leaves stages, all of which were compared to control plants that received tap water. Lettuce growth, photosynthetic performance, biochemical and mineral characteristics, as well as MCs accumulation in leaves, roots and soil were measured. The overall performance of lettuce at various developmental stages pointed to increased tolerance since growth showed minor alterations and non-enzymatic antioxidants remained unaffected. Plants receiving MCs-rich water from the seed stage exhibited higher photosynthetic capacity, chlorophylls and leaf nitrogen content. Nevertheless, considerable MCs accumulation in various plant tissues occurred. The earlier in their development lettuce plants started receiving MCs-rich water, the more MCs they accumulated: roots and leaves of plants exposed to MCs-rich water from seeds and cotyledons stage exhibited doubled MCs concentrations compared to respective tissues of the 4 Leaves group. Furthermore, roots accumulated significantly higher MCs amounts than leaves of the same plant group. Concerning human health risk, the Estimated Daily Intake values (EDI) of Seed and Cotyledon groups leaves exceeded Tolerable Daily Intake (TDI) by a factor of 6, while 2 Leaves and 4 Leaves groups exceeded TDI by a factor of 4.4 and 2.4 respectively. Our results indicate that irrigation of lettuce with MCs-rich water may constitute a serious public health risk, especially when contaminated water is received from the very early developmental stages (seed and cotyledon). Finally, results obtained for the tolerant lettuce indicate that MCs bioaccumulation in edible tissues is not necessarily coupled with phytotoxic effects.

Microcystin-LR bioconcentration induces antioxidant responses in the digestive gland of two marine bivalves Crassostrea gigas and Mytilus edulis.

Microcystins (MCs) are a major group of potent cyanobacterial toxins found in freshwater and even brackish waterbodies. To understand the putative correlation between bioconcentration of MCs and antioxidant responses of the digestive gland of bivalves, Pacific oyster Crassostrea gigas and blue mussel Mytilus edulis were exposed to different concentrations (0.1, 1, 10 and 20µgL-1) of MC-Leucine-Arginine (LR) for seven days. MC-LR bioconcentrated in the digestive glands of both bivalves during exposure period. The levels were slightly reduced when the bivalves were exposed to seawater during depuration (7days), while approximately 0.1µgL-1 of MC-LR was observed in the 10 and 20µgL-1 exposed bivalves at the end of depuration. Intracellular malondialdehyde (MDA) and glutathione (GSH) levels were significantly elevated in the 10 and 20µgL-1 exposed bivalves at 7day, and the levels were maintained during depuration in both bivalves. Overall, significant higher levels of enzymatic activities of antioxidant defense systems such as glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed in the 10 and 20µgL-1 exposed bivalves. Interestingly, most of higher levels of Pacific oyster were detected at exposure period, while blue mussel showed higher levels at depuration phase, suggesting a species-specific sensitivity upon MC-LR. These patterns were correlated with the bioconcentration patterns of MC-LR as Pacific oyster was highly accumulated by MC-LR during exposure period, but blue mussel showed prolonged high levels of MC-LR for depuration phase. Our results will be useful to understand species-specific bioconcentration of MC-LR in bivalves and their effects on intracellular oxidative status via accumulation.

Effects of microcystin-LR and cylindrospermopsin on plant-soil systems: A review of their relevance for agricultural plant quality and public health.

Toxic cyanobacterial blooms are recognized as an emerging environmental threat worldwide. Although microcystin-LR is the most frequently documented cyanotoxin, studies on cylindrospermopsin have been increasing due to the invasive nature of cylindrospermopsin-producing cyanobacteria. The number of studies regarding the effects of cyanotoxins on agricultural plants has increased in recent years, and it has been suggested that the presence of microcystin-LR and cylindrospermopsin in irrigation water may cause toxic effects in edible plants. The uptake of these cyanotoxins by agricultural plants has been shown to induce morphological and physiological changes that lead to a potential loss of productivity. There is also evidence that edible terrestrial plants can bioaccumulate cyanotoxins in their tissues in a concentration dependent-manner. Moreover, the number of consecutive cycles of watering and planting in addition to the potential persistence of microcystin-LR and cylindrospermopsin in the environment are likely to result in groundwater contamination. The use of cyanotoxin-contaminated water for agricultural purposes may therefore represent a threat to both food security and food safety. However, the deleterious effects of cyanotoxins on agricultural plants and public health seem to be dependent on the concentrations studied, which in most cases are non-environmentally relevant. Interestingly, at ecologically relevant concentrations, the productivity and nutritional quality of some agricultural plants seem not to be impaired and may even be enhanced. However, studies assessing if the potential tolerance of agricultural plants to these concentrations can result in cyanotoxin and allergen accumulation in the edible tissues are lacking. This review combines the most current information available regarding this topic with a realistic assessment of the impact of cyanobacterial toxins on agricultural plants, groundwater quality and public health.

A review of neurotoxicity of microcystins.

Cyanobacterial blooms-produced microcystins are secondary metabolites which can accumulate in the food chain and contaminate water, thus posing a potential threat to the health of aquatic animals and even humans. Microcystin toxicity affects not only the liver but also the other organs, i.e., the brain. The serious neurotoxicity effects caused by microcystins then lead to various symptoms. This review focuses on the neurotoxicity of microcystins. Microcystins can cross blood-brain barrier with the transport of Oatps/OATPs, causing neurostructural, functional, and behavioral changes. In this review, potential uptake mechanisms and neurotoxicity mechanisms are summarized, including neurotransmissions, neurochannels, signal transduction, oxidative stress, and cytoskeleton disruption. However, further researches are needed for detailed studies on signaling pathways and the downstream pathways of neurotoxicity of microcystins.

Combined effects of toxic cyanobacteria Microcystis aeruginosa and hypoxia on the physiological responses of triangle sail mussel Hyriopsis cumingii.

The single and combined effects of toxic cyanobacteria Microcystis aeruginosa and hypoxia on the energy budget of triangle sail mussel Hyriopsis cumingii were determined in terms of scope for growth (SfG). Mussels were exposed to different combinations of toxic M. aeruginosa (0%, 50%, and 100% of total dietary dry weight) and dissolved oxygen concentrations (1, 3, and 6.0mg O2l(-1)) with a 3×3 factorial design for 14 days, followed by a recovery period with normal conditions for 7 days. Microcystin contents in mussel tissues increased with the increase in the exposed M. aeruginosa concentration at each sampling time. Adverse physiological responses of H. cumingii under toxic M. aeruginosa and hypoxic exposure were found in terms of clearance rate, absorption efficiency, respiration rate, excretion rate, and SfG. Results emphasized the importance of combined effects of hypoxia and toxic cyanobacteria on H. cumingii bioenergetic parameters, highlighted the interactive effects of toxic algae and hypoxia, and implied that the two stressors affected H. cumingii during the exposure period and showed carryover effects later. Thus, if H. cumingii is used as a bioremediation tool to eliminate M. aeruginosa, the waters should be oxygenated.

Influence of captopril on the cellular uptake and toxic potential of microcystin-LR in non-hepatic adhesive cell lines.

Microcystin-LR (MC-LR) is a toxin produced by various cyanobacterial strains. Its cytotoxicity is due to inhibition of the protein phosphatases PP1 and PP2A, resulting in hyperphosphorylation of a number of functional and cytoskeletal proteins. To penetrate through the plasma membrane, MC-LR needs specific transporters such the organic anion-transporting polypeptides (OATP) that are highly expressed on the hepatocytes. Hence, our goal was to investigate the role of the membrane transport proteins for the cytotoxic effect of MC-LR on adhesive cell lines different from hepatocytes. We have used three cell lines--A549 (human lung carcinoma), SK-Hep-1 (human liver adenocarcinoma), FL (human amniotic normal cells), and two inhibitors of the OATP (cyclosporine A and captopril). To examine the cytotoxic effect of MC-LR we applied MTT and Neutral Red assays. In addition, a fluorescent staining of the mitochondria by JC-1 was performed. A dose-dependent cytotoxic effect was observed for the three cell lines, as this effect was most pronounced in A549. No cytotoxicity was detected when the captopril was added 2 h before treatment of the cells with MC-LR. Addition of captopril to the cells 2 h after treatment with MC-LR leads to enhancement of the cytotoxic effect. Reduced mitochondrial membrane potential after treatment with MC-LR was detected in the three cell lines, compared to untreated control cells. Results from the NR-cytotoxicity assay indicated that MC-LR does not affect the lysosomes. Captopril is an effective inhibitor of both OATP influx membrane transport proteins and the P-gp efflux pumps involved in the transport of MC-LR. It protects the cells from toxic effects of the cyanotoxin MC-LR.

Quantitatively evaluating detoxification of the hepatotoxic microcystin-LR through the glutathione (GSH) pathway in SD rats.

Glutathione (GSH) plays crucial roles in antioxidant defense and detoxification metabolism of microcystin-LR (MC-LR). However, the detoxification process of MC-LR in mammals remains largely unknown. This paper, for the first time, quantitatively analyzes MC-LR and its GSH pathway metabolites (MC-LR-GSH and MC-LR-Cys) in the liver of Sprague-Dawley (SD) rat after MC-LR exposure. Rats received intraperitoneal (i.p.) injection of 0.25 and 0.5 lethal dose 50 (LD50) of MC-LR with or without pretreatment of buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis. The contents of MC-LR-GSH were relatively low during the experiment; however, the ratio of MC-LR-Cys to MC-LR reached as high as 6.65 in 0.5 LD50 group. These results demonstrated that MC-LR-GSH could be converted to MC-LR-Cys efficiently, and this metabolic rule was in agreement with the data of aquatic animals previously reported. MC-LR contents were much higher in BSO + MC-LR-treated groups than in the single MC-LR-treated groups. Moreover, the ratio of MC-LR-Cys to MC-LR decreased significantly after BSO pretreatment, suggesting that the depletion of GSH induced by BSO reduced the detoxification of MCs. Moreover, MC-LR remarkably induced liver damage, and the effects were more pronounced in BSO pretreatment groups. In conclusion, this study verifies the role of GSH in the detoxification of MC-LR and furthers our understanding of the biochemical mechanism for SD rats to counteract toxic cyanobacteria.

Time dependent uptake, bioaccumulation and biotransformation of cell free crude extract microcystins from Lake Amatitlán, Guatemala by Ceratophyllum demersum, Egeria densa and Hydrilla verticillata.

Recent studies evidence that macrophytes can uptake and bioaccumulate microcystins (MC) from contaminated environments, suggesting their use in phytoremediation. In the present study Ceratophyllum demersum, Egeria densa and Hydrilla verticillata were exposed to cell free crude extracts (CE) containing three MC congeners MC-LR, MC-RR and MC-YR at a total MC concentration of 104.4 ± 7.6 µg/L from Lake Amatitlán, Guatemala. Time dependent total glutathione (tGSH), glutathione disulfide (GSSG), disappearance of MC from exposure medium and macrophyte uptake as well as calculated uptake and biotransformation rates and bioconcentration factors (BCF) were monitored after 1, 4, 8 hours (h) and 1, 3, 7 and 14 days (d). Results showed that tGSH concentrations in all exposed macrophytes were enhanced by CE. Disappearance of 62.1 ± 13, 40.8 ± 3.1 and 37.8 ± 3.5 µg/L total MCs from exposure mediums with E. densa, H. verticillata and C. demersum were observed after 1 h. Followed by the total elimination of MCs in exposure medium from H. verticillata after 14 d. Highest MC bioaccumulation capacity (BCF), was observed in E. densa followed by C. demersum and H. verticillata. The here presented results imply the strong MC phytoremediation potential of the evaluated macrophytes.

Oxidative effects and toxin bioaccumulation after dietary microcystin intoxication in the hepatopancreas of the crab Neohelice (Chasmagnathus) granulata.

We studied the accumulation and depuration of microcystin-LR (MCLR) in the hepatopancreas of the crab Neohelice granulata fed twice weekly with either non toxic or MCLR-producing Microcystis aeruginosa (strain NPDC1 or NPJB, respectively) during seven weeks. We also analyzed MCLR effects on the oxidative stress- and detoxification-related variables, superoxide dismutase and glutathione-S-transferase activities, and the levels of reduced glutathione and lipid peroxidation (as thiobarbituric acid reactive substances, TBARS). Hepatopancreas MCLR content slightly increased during the first three weeks, up to 8.81±1.84ngg(-1) wet tissue mass (WTM) and then started to decrease to a minimum of 1.57±0.74ngg(-1) WTM at the seventh week (p<0.05 with respect to that in the first week). TBARS levels were about 55% higher in treated than in control N. granulata (p<0.001 and p<0.05) during the first three weeks of the experimental period. GSH content became 50% lower than in control individuals (p<0.01) during weeks 6 and 7. SOD activity was increased by about 2-fold (p<0.05 or p<0.001) from week 3 to 7 in treated crabs with respect to control ones, while GST activity was about 70% higher in treated than in control crabs from week 4 to week 7 (p<0.05). Our data suggest that in the hepatopancreas of N. granulata MCLR accumulation and oxidative damage are limited and reversed by detoxification-excretion and antioxidant mechanisms. The activation of these defensive mechanisms becomes evident at 3-4 weeks after the start of the intoxication.

Low in vitro permeability of the cyanotoxin microcystin-LR across a Caco-2 monolayer: with identification of the limiting factors using modelling.

Microcystins (MCs) are toxins produced by several cyanobacteria species found worldwide. MC-LR is the most frequent. Here, we used the human Caco-2 cell line grown on semi-permeable filter supports as an in vitro model for determining MC-LR intestinal bidirectional transport. In this study, there was very low and time-dependent apparent permeability of MC-LR. To identify the limiting factors involved in the low permeability of MC-LR, a mathematical model was constructed to get physiologically relevant and informative parameters. The apical-to-basolateral transport was characterised by a rapid and substantial decrease in apical MC-LR concentrations (24-40% of the initial amount). In the basolateral compartment, the concentrations increased slowly after a lag time, but represented only a small fraction of the loaded concentrations (0.3-1.3%) after 24 h. This weak permeability was mainly due to a low clearance of efflux (from the cellular to the basolateral compartment) and effective secretion (from the cellular to the apical compartment). During the basolateral-to-apical transport, we observed a slow decrease in basolateral concentrations and a rapid increase in apical concentrations. In conclusion, modelling has the potential to highlight the key mechanisms involved in the complex kinetics of toxin transport.

Reproductive toxicity on female mice induced by microcystin-LR.

There are increasing concerns about health risk of human exposure to microcystin-LR (MC-LR) over the past few decades. Nevertheless, its female mammalian reproductive toxicity has not been addressed yet. In the present study, we firstly reported MC-LR could impact female reproductive function of mammals. After 28 days MC-LR exposure, relative ovary weight significantly reduced in 20 µg/kg MC-LR group and this reduction may be relative with pathomorphological changes of ovary. The result of histological evaluation of follicles showed that primordial follicles decreased roughly in half at high dose level compared with control. Since serum hormone assay indicated that MC-LR induced decrease of progesterone but not FSH or LH, disturbance of estrus cycle was seemed to result from direct impact of ovary rather than indirectly from hypothalamus or pituitary. As expected, MC-LR was detected in the ovaries of MC-LR exposure mice by immunoblot analysis.