Dietary l-carnitine prevents histopathological changes in tilapia (Oreochromis Niloticus) exposed to cylindrospermopsin.

Cylindrospermopsin (CYN) is a cytotoxin highly water-soluble, which is easily taken up by several aquatic organisms. CYN acts as a potent protein and glutathione synthesis inhibitor, as well as inducing genotoxicity, oxidative stress, and histopathological alterations. This is the first study reporting the protective effect of a l-carnitine (LC) pretreatment (400 or 880 mg LC/kg bw fish/day, for 21 days) on the histopathological alterations induced by pure CYN or Aphanizomenon ovalisporum lyophilized cells (400 µg CYN/kg bw fish) in liver, kidney, heart, intestines, and gills of tilapia (Oreochromis niloticus) acutely exposed to the toxin by oral route. The main histopathological changes induced by CYN were disorganized parenchyma with presence of glycogen and lipids in the cytoplasm (liver), glomerulonephritis, glomerular atrophy, and dilatation of Bowman's capsule (kidney), myofibrolysis, loss of myofibrils, with edema and hemorrhage (heart), intestinal villi with necrotic enterocytes and partial loss of microvilli (gastrointestinal tract), and hyperemia and hemorrhage (gills). LC pretreatment was able to totally prevent those CYN-induced alterations from 400 mg LC/kg bw fish/day in almost all organs, except in the heart, where 880 mg LC/kg bw fish/day were needed. In addition, the morphometric study indicated that LC managed to recover totally the affectation in the cross sections of the proximal and distal convoluted tubules in CYN-exposed fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 241-254, 2017.

Cyanobacterium producing cylindrospermopsin cause histopathological changes at environmentally relevant concentrations in subchronically exposed tilapia (Oreochromis niloticus).

The acute toxicity of cylindrospermopsin (CYN) has been established in rodents, based on diverse intraperitoneal an oral exposure studies and more recently in fish. But no data have been reported in fish after subchronic exposure to cyanobacterial cells containing this cyanotoxin, so far. In this work, tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels of CYN (10 or 100 µg CYN L(-1)) and deoxy-cylindrospermopsin (deoxy-CYN) (0.46 or 4.6 µg deoxy-CYN L(-1)), during two different exposure times: 7 or 14 d. This is the first study showing damage in the liver, kidney, hearth, intestines, and gills of tilapia after subchronic exposure to cyanobacterial cells at environmental relevant concentrations. The major histological changes observed were degenerative processes and steatosis in the liver, membranous glomerulopathy in the kidney, myofibrolysis and edema in the heart, necrotic enteritis in the gastrointestinal tract, and hyperemic processes in gill lamellae and microhemorrhages. Moreover, these histopathological findings confirm that the extent of damage is related to the CYN concentration and length of exposure. Results from the morphometric study indicated that the average of nuclear diameter of hepatocytes and cross-sections of proximal and distal convoluted tubules are useful to evaluate the damage induced by CYN in the main targets of toxicity.

Polymethoxy-1-alkenes from Aphanizomenon ovalisporum inhibit vertebrate development in the zebrafish (Danio rerio) embryo model.

Cyanobacteria are recognized producers of a wide array of toxic or otherwise bioactive secondary metabolites. The present study utilized the zebrafish (Danio rerio) embryo as an aquatic animal model of vertebrate development to identify, purify and characterize lipophilic inhibitors of development (i.e., developmental toxins) from an isolate of the freshwater cyanobacterial species, Aphanizomenon ovalisporum.Bioassay-guided fractionation led to the purification, and subsequent chemical characterization, of an apparent homologous series of isotactic polymethoxy-1-alkenes (1-6), including three congeners (4-6) previously identified from the strain, and two variants previously identified from other species (2 and 3), as well as one apparently novel member of the series (1). Five of the PMAs in the series (1-5) were purified in sufficient quantity for comparative toxicological characterization, and toxicity in the zebrafish embryo model was found to generally correlate with relative chain length and/or methoxylation. Moreover, exposure of embryos to a combination of variants indicates an apparent synergistic interaction between the congeners. Although PMAs have been identified previously in cyanobacteria, this is the first report of their apparent toxicity. These results, along with the previously reported presence of the PMAs from several cyanobacterial species, suggest a possibly widespread distribution of the PMAs as toxic secondary metabolites and warrants further chemical and toxicological investigation.

Influence of Glycine and Arginine on Cylindrospermopsin Production and aoa Gene Expression in Aphanizomenon ovalisporum.

Arginine (Arg) and glycine (Gly) seem to be the only substrates accepted by the amidinotransferase that catalyze the first step of the synthesis pathway of the cyanotoxin cylindrospermopsin (CYN), leading to guanidinoacetate (GAA). Here, the effect of these amino acids on the production of CYN in cultures of the cylindrospermopsin-producing strain, Aphanizomenon ovalisporum UAM-MAO, has been studied. Arg clearly increased CYN content, the increment appearing triphasic along the culture. On the contrary, Gly caused a decrease of CYN, observable from the first day on. Interestingly, the transcript of the gene ntcA, key in nitrogen metabolism control, was also enhanced in the presence of Arg and/or Gly, the trend of the transcript oscillations being like that of aoa/cyr. The inhibitory effect of Gly in CYN production seems not to result from diminishing the activity of genes considered involved in CYN synthesis, since Gly, as Arg, enhance the transcription of genes aoaA-C and cyrJ. On the other hand, culture growth is affected by Arg and Gly in a similar way to CYN production, with Arg stimulating and Gly impairing it. Taken together, our data show that the influence of both Arg and Gly on CYN changes seems not to be due to a specific effect on the first step of CYN synthesis; it rather appears to be the result of changes in the physiological cell status.

First evidence of accumulation in cyanobacteria of guanidinoacetate, a precursor of the toxin cylindrospermopsin.

Guanidinoacetate (GAA) is one of the most extensively studied toxic guanidine compounds. Changes in GAA can affect the nervous system and induce hyperhomocysteinemia, representing a risk factor for cardiovascular diseases. In cyanobacteria, GAA is thought to be an intermediate in the synthesis of the toxin cylindrospermopsin (CYN), one of the most common known cyanotoxins that affects multiple organs and functions in animals and plants. In spite of the evidence supporting GAA toxicity and its role in CYN synthesis, no data have been reported on the accumulation of GAA in any cyanobacterium. We have analyzed and compared the content of GAA in cultures of diverse cyanobacteria types, both cylindrospermopsin producing (CYN(+)) and not producing (CYN(-)). The results obtained show that GAA accumulates in the majority of the strains tested, although the highest content was found in one of the CYN(+) strain, Aphanizomenon ovalisporum UAM-MAO. In this strain, both GAA and CYN can be located within and out the cells. In conclusion, GAA appears to be a general cyanobacterial metabolite that due to its proven toxic should be considered when studying and managing cyanobacteria toxicity.

Detection of cylindrospermopsin toxin markers in cyanobacterial algal blooms using analytical pyrolysis (Py-GC/MS) and thermally-assisted hydrolysis and methylation (TCh-GC/MS).

The hepatotoxin cylindrospermopsin (CYN) is produced by freshwater cyanobacteria becoming an emerging threat for human health. Methods for the rapid determination of CYN in environmental samples are needed. Conventional analytical pyrolysis (Py-GC/MS) and thermally-assisted hydrolysis and methylation (TCh-GC/MS) were used to study a CYN standard, two Aphanizomenon ovalisporum cultures (CYN+) and one culture of Cylindrospermopsis raciborskii (CYN-). A micro-furnace pyrolyzer was used directly attached to a GC/MS system fitted with a 30 m × 250 µm × 0.25 µm film thickness column (14% cyanopropyl phenyl, 86% dimethyl polysiloxane pahase composition). Oven temperature was held at 50 °C for 1 min and increased to 100 °C at 30 °C min(-1), from 100 °C to 300 °C at 10 °C min(-1), and stabilized at 300 °C for 10 min using helium (1 mL min(-1)) as carrier gas. Pyrolysis at 500 °C yield over 70 compounds with 20 specific for CYN+ samples. Two peaks containing a diagnostic fragment (m/z 194) were found at 25.0 and 28.9 min only in CYN+ samples. Fewer peaks with limited diagnostic value were released after TCh-GC/MS, including breakdown products and TMAH adducts. A compound was detected that may correspond to the CYN molecule (MW 415 Da) thermoevaporation product after the loss of SO3 (MW 80 Da). This TCh-GC/MS peak (m/z 336) together with the fragments obtained by conventional Py-GC/MS (m/z 194) are diagnostic ions with potential use for the direct detection of CYN toxin in environmental samples at last with an estimated 5 ppm detection threshold.

Effects of depuration on oxidative biomarkers in tilapia (Oreochromis niloticus) after subchronic exposure to cyanobacterium producing cylindrospermopsin.

Cylindrospermopsin (CYN) is a cytotoxic polyketide-derived alkaloid produced by several freshwater cyanobacterial species. It is now considered the second most studied cyanotoxin worldwide. Among the toxic mechanisms suggested for CYN pathogenicity are inhibition of protein and glutathione synthesis, genotoxicity by DNA fragmentation, and oxidative stress. The study of depuration of cyanobacterial toxins by aquatic organisms, particularly by fish, is important for fish economy and public health, but in the case of CYN is practically nonexistent. In this work, we investigated the efficiency of two distinct depuration periods, 3 or 7d, in a clean environment, as a mean of restoring the levels of several oxidative stress biomarkers in tilapia (Oreochromis niloticus) subchronically exposed to CYN by immersion in an Aphanizomenon ovalisporum culture (by adding 10 µg CYN/L every two days during 14 d). Lipid peroxidation (LPO) and DNA oxidation returned to normal values after 7d of depuration, whereas the time needed for restoring of the oxidatively damaged proteins was longer. Superoxide dismutase (SOD) and gamma-glutamyl-cysteine-synthetase (γ-GCS) activities recovered after just 3d of depuration, while catalase (CAT) activity needed up to 7d to return to control values. Ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) returned to control levels after 7d of depuration in both organs. These results validate the depuration process as a very effective practice for detoxification in fish contaminated with these toxins.