Preclinical Characterization of Antioxidant Quinolyl Nitrone QN23 as a New Candidate for the Treatment of Ischemic Stroke.

Nitrones are encouraging drug candidates for the treatment of oxidative stress-driven diseases such as acute ischemic stroke (AIS). In a previous study, we found a promising quinolylnitrone, QN23, which exerted a neuroprotective effect in neuronal cell cultures subjected to oxygen-glucose deprivation and in experimental models of cerebral ischemia. In this paper, we update the biological and pharmacological characterization of QN23. We describe the suitability of intravenous administration of QN23 to induce neuroprotection in transitory four-vessel occlusion (4VO) and middle cerebral artery occlusion (tMCAO) experimental models of brain ischemia by assessing neuronal death, apoptosis induction, and infarct area, as well as neurofunctional outcomes. QN23 significantly decreased the neuronal death and apoptosis induced by the ischemic episode in a dose-dependent manner and showed a therapeutic effect when administered up to 3 h after post-ischemic reperfusion onset, effects that remained 11 weeks after the ischemic episode. In addition, QN23 significantly reduced infarct volume, thus recovering the motor function in a tMCAO model. Remarkably, we assessed the antioxidant activity of QN23 in vivo using dihydroethidium as a molecular probe for radical species. Finally, we describe QN23 pharmacokinetic parameters. All these results pointing to QN23 as an interesting and promising preclinical candidate for the treatment of AIS.

The first detection of potentially toxic Microcystis strains in two Middle Atlas Mountains natural lakes (Morocco).

Aguelmam Azizgza (LAZ) and Dayet Afourgah (DAF) are two Moroccan natural lakes located in a humid hydrographic basin of the Middle Atlas Mountains. Both are considered important reservoirs of plant and animal biodiversity. In addition, they are extensively used for recreational and fishing activities and as a water source for irrigation of agricultural crops. Recurrent cyanobacteria scum episodes in the two water bodies have been reported, Microcystis being the main genus in the scums. Here, we report on the toxic potential of three Microcystis aeruginosa strains isolated from those lakes: Mic LAZ and Mic B7 from LAZ and Mic DAF isolated from DAF. The toxic potential was checked by their microcystin (MC) content and the presence of mcy genes involved in MC synthesis. The identification and quantification of MC variants were performed by high-performance liquid chromatography-photo-diode array. The detection of mcy genes was achieved by whole-cell multiplex PCR that allowed the simultaneous amplification of DNA sequences corresponding to specific mcy regions. MC content of cultured cells, as MC-LR equivalents per gram cell biomass, was slightly higher in Mic LAZ (ca. 860) than in Mic B7 (ca. 700) and Mic DAF (ca. 690). Four MC variants were identified in the three isolates: MC-WR, MC-RR, MC-DM-WR, and MC-YR. The presence of toxic Microcystis strains in the two studied lakes may be regarded as an environmental and health hazard, especially during periods of bloom proliferation. It would be recommended the use of two complementary techniques, as those utilized herein (HPLC and mcy detection) to alert on highly probable toxicity of such lakes.

Assessment of microcystins in lake water and fish (Mugilidae, Liza sp.) in the largest Spanish coastal lake.

Cyanobacteria dominance and cyanotoxin production can become major threats to humans and aquatic life, especially in warm shallow lakes, which are often dominated by cyanobacteria. This study investigates the occurrence and distribution of microcystins (MCYST) in water, cell-bound and in the tissues of the commercial mugilid Liza sp. in the largest, coastal, Spanish Mediterranean lake (Albufera of Valencia). This is the first report concerning microcystin accumulation in tissues of mugilid fish species. Considerable amounts of microcystins were found in the water and seston, which correlated with development of Microcystis aeruginosa populations in the lake. The MCYST concentrations found in Lake Albufera (mean 1.7 and 17 µg/L and maximum 16 and 120 µg/L in water and seston, respectively) exceeded by one to two orders of magnitude the guideline levels proposed by the World Health Organization and were higher than that reported in other lakes of the Mediterranean zone. The presence of MCYST was found in all the fishes studied and accumulated differently among tissues of the commercial species Liza sp. Toxin accumulation in fish tissues showed that although the target organ for MCYST was the liver, high concentrations of microcystins were also found in other analysed tissues (liver>intestine>gills>muscle). Human tolerable daily intake for microcystins is assessed relative to the WHO guidelines, and potential toxicological risks for humans, wildlife and related ecosystems of the lake are discussed.

Detection of potentially producing cylindrospermopsin and microcystin strains in mixed populations of cyanobacteria by simultaneous amplification of cylindrospermopsin and microcystin gene regions.

Cyanobacterial blooms are frequently formed by heterogeneous populations of toxin-producing and non-producing strains. Microcystins (MC) and cylindrospermopsin (CYN) are the most representative cyanobacterial toxins. We have developed a multiplex PCR assay that allows simultaneous detection of MC(+) and/or CYN(+) strains in mixed populations of cyanobacteria. Various primer sets were designed using mcy and aoa gene sequences related with MC and CYN synthesis respectively, to amplify at the same time aoa and mcy sequences. Purified DNA, cultured cell mixtures and field samples with MC and CYN producing strains were used as DNA template. The results show: (i) the expected amplicons were only observed with toxic strains; (ii) cells were suitable as a source of purified DNA for the multiplex PCR; (iii) the assay could detect simultaneously 3 aoa and 3 mcy gene regions with mixed CYN(+) and MC(+) cyanobacteria cells. The method could be applied to environmental samples, allowing in a rapid, economical and easy way to detect simultaneously the presence of CYN(+) and MC(+) cyanobacteria in sestonic fractions of water samples.

Identification of microcystins from three collection strains of Microcystis aeruginosa.

Microcystins (MCs) are toxic cyclic heptapeptides produced by various cyanobacteria genera, especially Microcystis. We identified 10 out of 12 MCs produced by three Microcystis aeruginosa strains from cyanobacteria collections, UTEX 2666, UTEX 2670 and UAM 1303, by using two analytical methods: Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) and HPLC Photodiode Array Detector coupled to a hybrid Quadrupole Time of Flight Mass Spectrometry (HPLC-PDA-QTOF/MS). MALDI-TOF/MS failed to detect non-polar MCs, such as MC-LY and MC-LW. HPLC-QTOF/MS permitted the accurate identification of most MCs present in methanolic extracts. Besides, three new MCs, namely: [D-Glu(OCH3)6, D-Asp3] MC-LAba, MC-YL and MC-YM were detected by HPLC-QTOF/MS.

Effect of different microcystin profiles on toxin bioaccumulation in common carp (Cyprinus carpio) larvae via Artemia nauplii.

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin (MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B (100 microg L(-1)EqMC-LR) for 2h. The MC concentration in the nauplii was 73.60+/-7.88ngEqMC-LRg(-1)FW (n=4, mean+/-SE) for bloom A and 87.04+/-10.31ngEqMC-LRg(-1)FW for bloom B. These contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval weight and length from day 9 were significantly different between groups A and B, and in both cases lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely correlated with the growth performance, was also significantly different between groups A and B (37.43+/-2.61 and 54.55+/-3.01ngEqMC-LRg(-1) FW, respectively, at the end of the experimental period). These results indicate that MC profile of a bloom may have differential effects on toxin accumulation/transfer and toxicity.

Typing of toxinogenic Microcystis from environmental samples by multiplex PCR.

Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.

Physiological changes in Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars, caused by irrigation with water contaminated with microcystins: a laboratory experimental approach.

The aim of the present study was to investigate the effect of exposure to a microcystin (MC)-containing extract from a cyanobacteria bloom on growth, development, mineral nutrient accumulation, and photosynthetic activity of Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars. The MCs in the extract, identified by HPLC and/or mass spectrometry (MS) were: MC-RR, -LR, -YR, -(H4)YR, -WR, and -FR. Plant growth and development was tested along 30 exposure days. After this period, MC-extract caused a clear reduction in plant growth and productivity, as well as deleterious effects on development and Photosystem II activity, measured by Fv/Fm fluorescence. However, the chlorophyll (a + b) content hardly varied, and the accumulation of Na+, K+, Ca2+, P and N was enhanced. All the effects observed were plant species, MC concentration, and exposure-time dependent. Relative accumulation of each MC variant greatly varied among plant species and plant organ. The data obtained supports the idea that the use of surface water containing MCs for crop irrigation can affect both plant yield and quality, and secondly, that MC accumulation in edible plants might pose a potential risk for human and animal health, if the MC intake exceeded the recommended tolerable limits.

Compensatory growth induced in zebrafish larvae after pre-exposure to a Microcystis aeruginosa natural bloom extract containing microcystins.

Early life stage tests with zebrafish (Danio rerio) were used to detect toxic effects of compounds from a Microcystis aeruginosa natural bloom extract on their embryolarval development. We carried out the exposure of developing stages of fish to complex cyanobacterial blooms containing hepatotoxic molecules - microcystins. Fish embryo tests performed with the bloom extract containing 3 mg.L(-1) Eq microcystin-LR showed that after 24 h of exposure all fish embryos died. The same tests performed with other diluted extracts (containing 0.3, 0.1 and 0.03 mg.L(-1) Eq microcystin-LR) were shown to have an influence on zebrafish development and a large number of embryos showed malformation signs (edema, bent and curving tail). After hatching the larvae were transferred to a medium without toxins to follow the larval development under the new conditions. The specific growth of the pre-exposed larvae was significantly more important than that of the control larvae. This may represent a compensatory growth used to reduce the difference in size with the control fish noted after hatching.

Phytotoxic effects of cyanobacteria extract on the aquatic plant Lemna gibba: microcystin accumulation, detoxication and oxidative stress induction.

The occurrence of toxic cyanobacteria in the aquatic environment constitutes a serious risk for the ecological balance and the functioning of ecosystems. The presence of cyanotoxins in ecosystems could have eventual adverse effects on aquatic plants, which play an important biological role as primary producers. The original aim of this study was to investigate microcystin (MC) accumulation, detoxication and oxidative stress induction in the free-floating aquatic vascular plant Lemna gibba (Duckweed, Lemnaceae). Experiments were carried out with a range of MC levels, obtained from toxic Microcystis culture extracts (0.075, 0.15, 0.22 and 0.3 microg equiv.MC-LR mL(-1)). During chronic exposure of the plant to MC, we examined the growth, photosynthetic pigment contents and also the physiological behavior related to toxin accumulation, possible biodegradation and stress oxidative processes of L. gibba. For the last reason, changes in peroxidase activity and phenol compound content were determined. This is a first report using phenol compounds as indicators of biotic stress induced by MC contamination in aquatic plants. Following MC exposure, a significant decrease of plant growth and chlorophyll content was observed. Also, it was demonstrated that L. gibba could take up and bio-transform microcystins. A suspected MC degradation metabolite was detected in treated Lemna cells. In response to chronic contamination with MCs, changes in the peroxidase activity and qualitative and quantitative changes in phenolic compounds were observed after 24h of plant exposure. The physiological effects induced by chronic exposure to microcystins confirm that in aquatic ecosystems plants coexisting with toxic cyanobacterial blooms may suffer an important negative ecological impact. This may represent a sanitary risk due to toxin bioaccumulation and biotransfer through the food chain.

Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions.

Reliable cyanotoxin monitoring in water reservoirs is difficult because of, among other reasons, unpredictable changes in cyanobacteria biomass, toxin production, and inadequate sampling frequency. Therefore, it would be useful to identify potentially microcystin-producing strains of cyanobacterial populations in field samples. With this aim, we developed a methodology to distinguish microcystin-producing from non-producing Microcystis strains by amplifying six characteristic segments of the microcystin synthetase mcy cluster, three corresponding to the nonribosomal peptide synthetase, genes mcyA, mcyB, and mcyC, and three to the polyketide synthase, genes mcyD, mcyE, and mcyG. For this purpose five new primer sets were designed and tested using purified DNA, cultured cells, and field colonies as DNA sources. Simultaneous amplification of several genes in multipex PCR reactions was performed in this study. The results obtained showed that: (i) the expected specific amplicons were obtained with all microcystin-producing strains but not with nonproducing strains; (ii) cells could be directly used as DNA templates, 2000 cells being a sufficient number in most cases; (iii) simultaneous amplification of several gene regions is feasible both with cultured cells and with field colonies. Our data support the idea that the presence of various mcy genes in Microcystis could be used as a criterion for ascribing potential toxigenicity to field strains, and the possibility of applying whole-cell assays for the simultaneous amplification of various genes may contribute significantly to simplifying toxigenicity testing.