Cyanotoxins, biosynthetic gene clusters, and factors modulating cyanotoxin biosynthesis.

Cyanobacterial harmful algal blooms (CHABs) are a global environmental concern that encompasses public health issues, water availability, and water quality owing to the production of various secondary metabolites (SMs), including cyanotoxins in freshwater, brackish water, and marine ecosystems. The frequency, extent, magnitude, and duration of CHABs are increasing globally. Cyanobacterial species traits and changing environmental conditions, including anthropogenic pressure, eutrophication, and global climate change, together allow cyanobacteria to thrive. The cyanotoxins include a diverse range of low molecular weight compounds with varying biochemical properties and modes of action. With the application of modern molecular biology techniques, many important aspects of cyanobacteria are being elucidated, including aspects of their diversity, gene-environment interactions, and genes that express cyanotoxins. The toxicological, environmental, and economic impacts of CHABs strongly advocate the need for continuing, extensive efforts to monitor cyanobacterial growth and to understand the mechanisms regulating species composition and cyanotoxin biosynthesis. In this review, we critically examined the genomic organization of some cyanobacterial species that lead to the production of cyanotoxins and their characteristic properties discovered to date.

Variation in cyanobacterial hepatotoxin (microcystin) content of water samples and two species of fishes collected from a shallow lake in Algeria.

Microcystins (MCs) produced from cyanobacteria can accumulate in freshwater fish tissues. In this study, variations in these toxins content were examined monthly in water samples and two species of fish in Lake Oubeira, Algeria, from April 2010 to March 2011. During the study period, MCs were analyzed using protein phosphatase type 2A (PP2A) inhibition assay. In lake water, total (dissolved and intracellular toxins) MC concentrations by PP2A ranged from 0.028 to 13.4 µg equivalent MC-LR/l, with a peak in September 2010. MC-LR was the dominant variant (90 % of the total) in water samples, followed by MC-YR and MC-(H4)YR. The highest MC concentration in the omnivorous common carp (Cyprinus carpio) was found in the order intestine > hepatopancreas > muscle; however, in the carnivorous European eel (Anguilla anguilla) the order was liver > intestine > muscle. Highest MC concentrations in the intestine tissue of the common carp were found between August and November 2010 where high MC concentrations were detected in water samples, whereas high levels of MCs in the liver of the European eel were found later between January and February 2011. During the entire period of study, the World Health Organization (WHO) lifetime limit for tolerable daily intake was exceeded only in common carp muscle.

Studying the impact of phycoerythrin on antioxidant and antimicrobial activity of the fresh rainbow trout fillets.

Marine cyanobacteria present a significant potential source of new bioactive compounds with vast structural diversity and relevant antimicrobial and antioxidant activities. Phycobiliproteins (PBPs) like phycocyanin (PC), phycoerythrin (PE), and water-soluble cyanobacterial photosynthetic pigments, have exhibited strong pharmacological activities and been used as natural food additives. In this study, phycoerythrin (PE) isolated from a marine strain of cyanobacterium Nostoc sp. Ft salt, was applied for the first time as a natural antimicrobial as well as an antioxidant to increase the shelf life of fresh rainbow trout i.e., (Oncorhynchus mykiss) fillets. Fresh trout fillets were marinated in analytical grade PE (3.9 µg/mL) prepared in citric acid (4 mg/mL), and stored at 4 °C and 8 °C for 21 days. Microbiological analysis, antioxidant activity and organoleptic evaluation of both control and treated fish fillets were then statistically compared. The results demonstrated noticeable (P < 0.05) differences in the microbial counts, antioxidant activity, and organoleptic characteristic values between PE-treated and non-treated groups. In addition, we observed that treating fresh fish fillets with a PE solution leads to a significant increase in shelf life by at least 14 days. Consequently, PE could be an alternative to synthetic chemical additives since it does not contain the potentially dangerous residues of the synthetic chemical additives and is thus healthier to the consumers.

Massive fish death associated with the toxic cyanobacterial Planktothrix sp. bloom in the Béni-Haroun Reservoir (Algeria).

In July 2017, a massive bloom of the potentially toxic cyanobacterial species Planktothrix sp. was observed in the Béni-Haroun Reservoir (Algeria), which was followed by a massive fish death. Many questions were raised in association with the role of cyanotoxins and the fish massive mortality. The objective of this paper is twofold: (1) to investigate the variability of physicochemical and cyanobacterial parameters (chlorophyll-a, phycocyanin, allophycocyanin, and microcystins) throughout the period of July 2017 to June 2018; and (2) to determine the free and total MC levels in viscera and muscle tissues of the common carp (Cyprinus carpio), which are found dead in the considered reservoir in October 2017. Our results showed microcystin (MC) concentrations in water samples (by the protein phosphatase PP2A assay) had reached 651.2 ng MC-LR equiv./L. Total MC levels (free + bound) in the viscera and muscle tissues of sampled dead fish were at 960.24 and 438.54 µg MC-LR equiv./kg dw, respectively. It is assumed that high concentrations of MC observed in the tissues of common carp induced a strong degradation of the visceral contents resulting in the complete lysis of the hepatopancreas, and presumably the massive fish death.

Plant-cyanobacteria interactions: Beneficial and harmful effects of cyanobacterial bioactive compounds on soil-plant systems and subsequent risk to animal and human health.

Plant-cyanobacteria interactions occur in different ways and at many different levels, both beneficial and harmful. Plant-cyanobacteria interactions, as a beneficial symbiosis, have long been demonstrated in rice-growing areas (Poaceae) where the most efficient nitrogen-fixing cyanobacteria are present in paddies. Moreover, cyanobacteria may in turn produce and/or secrete numerous bioactive compounds that have plant growth-promoting abilities or that may make the plant more resistant to abiotic or biotic stress. In recent years, there has been a growing worldwide interest in the use of cyanobacterial biomass as biofertilizers to replace chemical fertilizers, in part to overcome increasing organic-farming demands. However, the potential presence of harmful cyanotoxins has delayed the use of such cyanobacterial biomass, which can be found in large quantities in freshwater ecosystems around the world. In this review, we describe the existing evidence for the positive benefit of plant-cyanobacteria interactions and discuss the use of cyanobacterial biomass as biofertilizers and its growing worldwide interest. Although mass cyanobacterial blooms and scums are a current and emerging threat to the degradation of ecosystems and to animal and human health, they may serve as a source of numerous bioactive compounds with multiple positive effects that could be of use as an alternative to chemical fertilizers in the context of sustainable development.

First evidence of okadaic acid in Mytilus galloprovincialis mussels, collected in a Mediterranean Lagoon, Tunisia.

Evidence of the presence of okadaic acid (OA) and its monthly fluctuations are reported for the first time in Mytilus galloprovincialis samples collected from June 2005 to May 2006 in the Bizerte Lagoon. All of the samples with the exception of those taken in August 2005 were found to be contaminated. The level of OA in mussels exceeded the regulatory limit of DSP toxins (16 microg OA/100 g mussel meat) within the European Union (2002) in only two cases: in January and February, 2006 with 31.85 +/- 3.06, and 18.86 +/- 1.88 microg OA/100 g mussel meat respectively. During the investigative period, potentially toxic dinoflagellates were observed. The results reported here, although based on limited sampling, demonstrate for the first time the existence of a risk to public health from diarrheic shellfish poisoning in cultivated bivalve mollusc in Tunisia.

Structural Diversity, Characterization and Toxicology of Microcystins.

Hepatotoxic microcystins (MCs) are the most widespread class of cyanotoxins and the one that has most often been implicated in cyanobacterial toxicosis. One of the main challenges in studying and monitoring MCs is the great structural diversity within the class. The full chemical structure of the first MC was elucidated in the early 1980s and since then, the number of reported structural analogues has grown steadily and continues to do so, thanks largely to advances in analytical methodology. The structures of some of these analogues have been definitively elucidated after chemical isolation using a combination of techniques including nuclear magnetic resonance, amino acid analysis, and tandem mass spectrometry (MS/MS). Others have only been tentatively identified using liquid chromatography-MS/MS without chemical isolation. An understanding of the structural diversity of MCs, the genetic and environmental controls for this diversity and the impact of structure on toxicity are all essential to the ongoing study of MCs across several scientific disciplines. However, because of the diversity of MCs and the range of approaches that have been taken for characterizing them, comprehensive information on the state of knowledge in each of these areas can be challenging to gather. We have conducted an in-depth review of the literature surrounding the identification and toxicity of known MCs and present here a concise review of these topics. At present, at least 279 MCs have been reported and are tabulated here. Among these, about 20% (55 of 279) appear to be the result of chemical or biochemical transformations of MCs that can occur in the environment or during sample handling and extraction of cyanobacteria, including oxidation products, methyl esters, or post-biosynthetic metabolites. The toxicity of many MCs has also been studied using a range of different approaches and a great deal of variability can be observed between reported toxicities, even for the same congener. This review will help clarify the current state of knowledge on the structural diversity of MCs as a class and the impacts of structure on toxicity, as well as to identify gaps in knowledge that should be addressed in future research.

Seasonal occurrence and toxicity of Microcystis spp. and Oscillatoria tenuis in the Lebna Dam, Tunisia.

Physicochemical and biological water quality, including the total microcystin concentrations, was investigated for the first time from January to December 2005 in the Lebna Dam, Tunisia. Microcystin levels and characterization of the different microcystin variants present were measured by protein phosphatase (PP2A) inhibition assays and by LC/MS/MS, respectively. Nutrient values were high, with total inorganic nitrogen and phosphorus concentrations ranging from 0.05 to 8.4 mg L(-1) and from 0.03 to 1.37 mg L(-1), respectively. However, the chlorophyll-a concentrations were very low with a peak (5.32 microg L(-1)) on 20 September 2005 at 9 m depth water samples. Microscopic examination of the phytoplankton samples showed the dominance in the autumn of three morphospecies of the genus Microcystis and the species Oscillatoria tenuis. The total (particulate and dissolved) microcystin concentrations at the surface and at 9 m depth water samples ranged between 0.008 and 1.73, and 0.005 and 5.57 microg microcystin (MC)-LR equivalent L(-1), respectively, with a peak on 20 September. The presence of the microcystin synthetase genes (mcyA, -B, and -C) in the lysates of the three morphospecies of the genus Microcystis and the species O. tenuis indicated that these species were responsible for the microcystin production in this system. The analysis of the field cyanobacterial sample extract containing these species by LC/MS/MS revealed the presence of two microcystin variants: microcystin-LR (MC-LR) and microcystin-YR (MC-YR).

First reported case of turtle deaths during a toxic Microcystis spp. bloom in Lake Oubeira, Algeria.

Microcystins analysis was conducted in field cyanobacterial bloom samples and dead terrapin tissues from Lake Oubeira (Algeria) with an aim of studying the cause of the mortality of the freshwater terrapin species Emys orbicularis and Mauremys leprosa during October 2005. The deaths of these two terrapin species were observed during a bloom of Microcystis spp. The total microcystin content per phytoplankton biomass evaluated with the methanol extraction-protein phosphatase methodology was 1.12 mg MCYST-LR equivalents/g dried bloom material. The analysis of this bloom extract by the LC/MS technique demonstrated the presence of three microcystin variants: microcystin-LR (MCYST-LR), microcystin-YR (MCYST-YR), and microcystin-RR (MCYST-RR). Microcystins were also detected in fresh carcasses of terrapin liver, viscera and muscle tissues using the GC/MS after Lemieux oxidation method and the PP2A inhibition assay. The high level of microcystins detected using the Lemieux oxidation-GC/MS method in the liver tissue (1192.8 microg MCYST-LR equivalent/g dw) and in the viscera tissue (37.19 microg MCYST-LR equivalent/g dw) of the species M. leprosa and E. orbicularis, respectively, and the liver crumbling observed after the necropsy examination of the fresh carcass of M. leprosa support the possibility that cyanobacterial microcystins contribute to the turtle mortalities.

Evaluation of the transfer and the accumulation of microcystins in tomato (Solanum lycopersicum cultivar MicroTom) tissues using a cyanobacterial extract containing microcystins and the radiolabeled microcystin-LR ((14)C-MC-LR).

Microcystins are the most common cyanotoxins and may be expected wherever blooms of cyanobacteria occur in surface waters. Their persistence both in the irrigation water and in the soil can lead to their transfer and bioaccumulation into agricultural plants. The aim of this work was to investigate microcystin accumulation in Solanum lycopersicum cultivar MicroTom. The plant was exposed to either Microcystis aeruginosa crude extracts containing up to 100 µg eq.MC-LRL(-1) in a soil-plant system for 90 days or pure radiolabeled (14)C-MC-LR in a hydroponic condition for 48 h. Toxin bioaccumulation in the soil and different plant tissues was assessed both by the PP2A inhibition assay and by liquid chromatography-mass spectrometry (LC/MS/MS). After 90 days of exposure, microcystins persisted in the soil and their free extractable concentrations accumulated were very low varying between 1.6 and 3.9 µg eq.MC-LR kg(-1) DW. Free MC-LR was detected only in roots and leaves with concentrations varying between 4.5 and 8.1 µg kg(-1) DW and between 0.29 and 0.55 µg kg(-1) DW, respectively. By using radioactivity ((14)C-MC-LR), the results have reported a growing accumulation of toxins within the organs roots>leaves>stems and allowed them to confirm the absence of MC-LR in fruits after 48 h of exposure. The bioconcentration factor (BCF) was 13.6 in roots, 4.5 in leaves, and 1.4 in stems. On the other hand, the results highlight the presence of two radioactive fractions in different plant tissues. The non-extractable fraction of radioactivity, corresponding to the covalently bound MC-LR, was higher than that of the extractable fraction only in roots and leaves reaching 56% and 71% of the total accumulated toxin, respectively. Therefore, results raise that monitoring programs must monitor the presence of MCs in the irrigation water to avoid the transfer and accumulation of these toxins in crops.

High diversity of microcystins in a Microcystis bloom from an Algerian lake.

Microcystins (MCs) are cyanobacterial heptapeptides, produced by several genera and species of cyanobacteria, which have been involved in poisoning of animals throughout the world and have also been implicated in human health problems. They are regarded as the most frequently occurring and widespread of the cyanotoxins, with more than 100 MC variants reported to date including the present study. The lake des Oiseaux is a shallow permanent freshwater lake located in north-eastern Algeria. It is an important natural reserve playing a major role for the migratory birds after the crossing of the Mediterranean Sea and from the Sahara desert. In recent years, possibly related to increased eutrophication of the lake, massive blooms of cyanobacteria identified as Microcystis spp. have been observed. A bloom sample collected in September 2013 was analyzed by the serine/threonine phosphatase PP2A inhibition assay and liquid chromatography-mass spectrometry to determine respectively, the total concentration of MCs and the different variants of these toxins present. The results revealed that the Microcystis spp. bloom sample contained microcystins of which 21 putatively congeners were detected. Among these, 12 known microcystins (MC-RR, MC-LR, MC-FR, MC-WR, MC-YR, MC-LA, MC-(H4)YR, MC-HilR, [Asp(3)]MC-RAba, and [Glu(OCH3)(6)]MC-LR) and two new congeners ([Asp(3)]MC-HarAba and [Glu(OCH3)(6)]MC-FR) were characterized, considering their molecular mass and the fragment ions produced by collision-induced dissociation of the [M+H](+) ions. MC-RR was the major (43.4%) in the bloom sample.

Evaluation of phytotoxicity and ecotoxicity potentials of a cyanobacterial extract containing microcystins under realistic environmental concentrations and in a soil-plant system.

The impact of a crude extract of Microcystis aeruginosa (PCC7820) containing 14 microcystin variants was investigated on seeds germination and radicles development of four agricultural plants: two tomato varieties Solanum lycopersicum (MicroTom and Saint-Pierre), the wheat Triticum aestivum and the lettuce Lactuca sativa. In addition, the effect of 14 d-exposure to irrigation water containing realistic concentrations of microcystins (0-0.1 mg eq. microcystin-LRL(-1)) on the tomato MicroTom seedling growth was further evaluated on roots and aerial part biomasses. Impacts on soil bacterial parameters, as such extracellular enzymatic activities, nitrification activity and abundances of ammonia-oxidizing microorganisms were also investigated. In germination-test, the cyanobacterial extract inhibited only the germination of the wheat seeds, with an EC50 of 11 mg eq. microcystin-LRL(-1); which is 13 times lower than that of the cadmium chloride (EC50 of 145 mg L(-1)). Moreover, the cyanobacterial extract containing low concentrations of microcystins increased the growth of primary roots; however, high concentrations decreased it for all plants except for the wheat. In the soil-plant approach, only aerial part biomass of the tomato MicroTom was enhanced significantly. In addition, only soil nitrification potential and ammonia-oxidizing bacterial abundances were consistently impacted. A significant positive correlation (r=0.56) was found between the increase of nitrification potential and abundances of ammonia-oxidizing bacteria. This work suggested, that exposure to a cyanobacterial extract containing realistic environmental microcystins concentrations could affect seed germination, depending plant species. It was also highlighted, for the first time, disturbances in soil bacteria functioning, evidences on soil nitrification process.

Microcystin-LR and nodularin induce intracellular glutathione alteration, reactive oxygen species production and lipid peroxidation in primary cultured rat hepatocytes.

Microcystin-LR (MCYST-LR) and nodularin (NOD) produced by cyanobacteria are potent specific hepatotoxins. However, the mechanisms of their hepatotoxicity have not been fully elucidated. In the present study the effect of non cytotoxic low concentrations of MCYST-LR and NOD on intracellular reduced glutathione (GSH) alteration, reactive oxygen species (ROS) production and lipid peroxidation was investigated in primary cultured rat hepatocytes. Cell viability was determined by the methylthiazoltetrazolium (MTT) dye assay, reduced GSH was evaluated by enzymatic methods, ROS were evaluated by the dichlorofluorescein diacetate (H2DCF-DA) fluorescent probe and lipid peroxidation by dosing malondialdehyde (MDA) by the thiobarbituric acid method. The 24 h LC50 values of MCYST-LR and NOD were 48 and 62 ng/ml, respectively. Exposure of freshly isolated rat hepatocytes to MCYST-LR or NOD at non cytotoxic low concentrations (2, 10 ng/ml) for 3, 24 and 48 h periods resulted in a significant rise of GSH levels and production of ROS. NOD significantly induced in a time- and concentration-dependent lipid peroxidation. However, MCYST-LR treatment did result in a significant decrease in MDA levels compared with controls. Although MCYST-LR and NOD are closely related in terms of structure and inhibition of protein phosphatases, they induce differently the oxidative stress at non cytotoxic low concentrations. Therefore, the results indicate that oxidative stress mediated by reactive intermediates may be a mechanism by which these cyanotoxins induce their hepatotoxic effect.

Detection by 32P-postlabelling of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA as biomarker of microcystin-LR- and nodularin-induced DNA damage in vitro in primary cultured rat hepatocytes and in vivo in rat liver.

Microcystin-LR (MCYST-LR) and nodularin (NOD) produced by cyanobacteria are potent and specific hepatotoxins. The induction of free-radical formation, reduction of glutathione levels and induction of DNA damage are three major events found in rat hepatocytes treated with these hepatotoxins. However, the mechanism of MCYST-LR- and NOD-mediated induction of oxidative DNA damage has not been fully elucidated. The objective of this study was to determine whether MCYST-LR and NOD increase the formation of a DNA oxidative damage marker such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in vitro in primary rat hepatocytes and in vivo in rat liver cells. Rat hepatocytes were exposed to MCYST-LR or NOD at low doses (2 and 10 ng/ml), at which there is no evidence of morphologically apparent cytotoxic effects, as well as an induced dose- and time-dependent formation of 8-oxo-dG. Moreover, MCYST-LR treatment of rats (50 microg/kg, ip) resulted in a significant increase of 8-oxo-dG in liver DNA, at 24 h after treatment before decreasing at 48 h. However, NOD-induced DNA damage was increased both at 24 and 48 h, in contrast to the MCYST-LR-induced effect. The effects on this oxidative DNA damage marker indicates that MCYST-LR and NOD do evoke oxidative stress, which may contribute, at least in part, to their liver toxicity and carcinogenicity during long-term exposure.

Cyanobacterial toxins: modes of actions, fate in aquatic and soil ecosystems, phytotoxicity and bioaccumulation in agricultural crops.

The occurrence of harmful cyanobacterial blooms in surface waters is often accompanied by the production of a variety of cyanotoxins. These toxins are designed to target in humans and animals specific organs on which they act: hepatotoxins (liver), neurotoxins (nervous system), cytotoxic alkaloids, and dermatotoxins (skin), but they often have important side effects too. When introduced into the soil ecosystem by spray irrigation of crops they may affect the same molecular pathways in plants having identical or similar target organs, tissues, cells or biomolecules. There are also several indications that terrestrial plants, including food crop plants, can bioaccumulate cyanotoxins and present, therefore, potential health hazards for human and animals. The number of publications concerned with phytotoxic effects of cyanotoxins on agricultural plants has increased recently. In this review, we first examine different cyanotoxins and their modes of actions in humans and mammals and occurrence of target biomolecules in vegetable organisms. Then we present environmental concentrations of cyanotoxins in freshwaters and their fate in aquatic and soil ecosystems. Finally, we highlight bioaccumulation of cyanotoxins in plants used for feed and food and its consequences on animals and human health. Overall, our review shows that the information on the effects of cyanotoxins on non-target organisms in the terrestrial environment is particularly scarce, and that there are still serious gaps in the knowledge about the fate in the soil ecosystems and phytotoxicity of these toxins.

First evidence of estrogenic potential of the cyanobacterial heptotoxins the nodularin-R and the microcystin-LR in cultured mammalian cells.

The estrogenic activity of cyanobacterial hepatotoxins microcystin-LR (MC-LR) and nodularin-R (NOD-R) was for the first time investigated invitro in a stably transfect cell line with an estrogen-regulated luciferase gene. Treatment of cells with NOD-R caused a dose-dependent increase in the luciferase activity. NOD-R gave rise to an induction of luciferase activity with an EC(50) value of 66.4 nM, whereas the positive control, 17beta-estradiol (E2) had an EC(50) of 9.6 pM. This indicates that NOD-R is a 6900-fold weaker inducer of luciferase than E2. In contrast, only a slight but significant activation of the luciferase gene was observed with MC-LR between 2.01 and 60.1 nM, and a maximal-induced response was observed with 10.1 nM, approximately 25% of the maximal effect obtained with 1 nM E2. The decrease in the luciferase activity at high MC-LR concentrations can be explained by a cytotoxic effect. No synergistic estrogenic effect was observed when each toxin was co-administrated with E2. However, the induction of the luciferase activity by NOD-R and MC-LR was inhibited by co-treatment with 1 microM of the pure estrogenic receptor (ER) antagonist ICI 182,780, thus proving the ER-dependency of the estrogenic effect.

A new morphospecies of Microcystis sp. forming bloom in the Cheffia dam (Algeria): seasonal variation of microcystin concentrations in raw water and their removal in a full-scale treatment plant.

Toxic cyanobacterial blooms are an increasing problem in Algeria. The production of cyanotoxins (microcystins) and their presence in drinking water represent growing hazards to human health. In this study, seasonal variations in the concentrations of total microcystins and physicochemical parameters (pH, temperature, dissolved oxygen, nitrate, orthophosphate, and chlorophyll-a) were analyzed in the Cheffia dam (Algeria), mainly used to supply drinking water. The removal of cyanobacterial cells and microcystins was also evaluated in full-scale plant associated with the Cheffia reservoir. The levels of microcystins (MCYSTs) in both raw and drinking water were evaluated using the protein phosphatase type 2A (PP2A) inhibition test as MCYST-LR equivalents. Identification of microcystin variants was achieved by LC/MS/MS. During the period of study (March-December 2004), microscopic observation showed the dominance in the autumn months (September-November) of a new morphospecies of Microcystis sp. The MCYST-LR equivalent concentrations in raw water varied between 50.8 and 28,886 ng L(-1). The highest level of toxins was observed in October 2004 and was significantly correlated with the chlorophyll-a. Three variants of microcystins assigned as microcystin-YR (MCYST-YR), microcystin-LR (MCYST-LR), and 6Z-Adda stereoisomer of MCYST-LR were observed in the crude extract of the Microcystis sp. bloom sample. During the bloom period, total elimination of Microcystis sp. and toxins were achieved through a classical treatment plant comprised of coagulation and flocculation, powdered activated carbon at 15 mg L(-1), slow sand filtration and chlorination before storage.

Genotoxic potential of Microcystin-LR and nodularin in vitro in primary cultured rat hepatocytes and in vivo in rat liver.

Microcystin-LR (MCYST-LR) and nodularin (NOD) are known as tumor promoters in experimental animals and so present potential health threats for humans. Although their hepatotoxic mechanisms have been very well documented, many other effects of these toxins are relatively undescribed, indeed controversial, notably those related to their genotoxicity. In the present investigation, we examined how these toxins could induce DNA damage using a combination of in vitro and in vivo approaches. We first used the (32)P-postlabeling assay to test hydrophobic adduct formation on DNA from primary cultured rat hepatocytes treated with noncytotoxic concentrations of MCYST-LR and NOD (2 and 10 ng/mL). Analysis of the autoradiograms of DNA digests isolated from the hepatocytes did not show any hydrophobic DNA adduct formation. However, these toxins significantly decreased the amount of hydrophobic endogenous adducts, termed I compounds. We next investigated oxidative DNA damage by using the (32)P-postlabeling assay to analyze 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) content as a biomarker of possible DNA lesions. Both MCYST-LR and NOD significantly enhanced 8-oxo-dG in time- and dose-dependent manner in vitro in primary cultured hepatocytes and in vivo in rat liver cells. Thus, it appears that the depletion of endogenous DNA adducts (I compounds) and/or the increase of 8-oxo-dG levels by MCYST-LR and NOD could be involved in the formation of hepatic tumors during long-term exposure to these cyanobacterial hepatotoxins.

First report of cyanobacterium Cylindrospermopsis raciborskii from Algerian freshwaters.

This study investigated the first report of the cyanobacterium Cylindrospermopsis raciborskii in Algerian freshwaters. The morphological characteristics of the two morphotypes observed in Lake Oubeira (Algeria) conformed to those of natural populations of C. raciborskii species described in the literature. The two morphotypes produced only straight trichomes. During the study (February 2000-September 2001), this species occurred as a codominant with Microcystis spp., and a peak was observed during the autumn (November 2000) with a remarkable density estimated to be 43 x 10(5) trichomes/L. However, difficulty in isolating and culturing these two morphotypes limited the evaluation of their toxic potential.

First report of a microcystin-containing bloom of the cyanobacteria Microcystis spp. in Lake Oubeira, eastern Algeria.

Lake Oubeira has been used as the main source of drinking water for many communities in the East of Algeria. In this lake, nutrient loading coupled with year-round warm weather favors the growth of cyanobacteria, several of which can produce cyanotoxins, especially the potent liver toxins called microcystins (MCYSTs). The present study evaluated microcystin levels and characterized the different microcystin variants present in the raw water during a 17-month period (April 2000-September 2001), as measured by protein phosphatase inhibition assays and by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, respectively. The results showed that microcystin concentrations in the lake water varied between 3 and 29,163 microg microcystin-LR equivalent per liter. The microscopic examination of the phytoplankton samples showed the dominance of the Microcystis genus in the cyanobacterial bloom. The highest MCYST concentration was observed in August 2001, at 29,163 microg/l. Therefore, the highest total MCYST content per phytoplankton biomass was found in August 2001, with 4,590 microg MCYST-LR equivalents/g dried bloom material. Analysis of the field bloom extract by MALDI-TOF mass spectrometry demonstrated the presence of four variants of microcystins: microcystin-LR (MCYST-LR), microcystin-YR (MCYST-YR), microcystin-RR (MCYST-RR), and a demethylated variant of MCYST-LR (D-MCYST-LR).