New records on toxic cyanobacteria from Brazil: Exploring their occurrence and geography.

Cyanobacterial Harmful Algal Blooms (CyanoHABs) pose a significant threat to communities globally, impacting ecosystems and public health. This study provides an in-depth review of the current state of cyanotoxins and the distribution of CyanoHABs species in Brazil, while also detailing the methods used for their detection. Four hundred and twenty-one incidents were analyzed from 1993 to 2021, compiling cyanotoxin records and toxic CyanoHABs occurrences. The investigation begins with the first detection of microcystins in 1994 and highlights pivotal moments, like the 1996 "Caruaru Syndrome" outbreak. This event encouraged research and updated cyanotoxin-monitoring guidelines. The Brazilian drought period of 2015-2016 exacerbated cyanobacterial growth and saxitoxin levels, coinciding with Zika-related microcephaly. This study delves into methods used for cyanotoxin analysis, including ELISA, bioassays, HPLC, and LC-MS. Additionally, we investigated the toxicity of 37 cyanobacterial strains isolated from various Brazilian environments. Extracts were tested against Artemia salina and analyzed by LC-MS. Results revealed toxicity in extracts from 49 % of cyanobacterial strains. LC-MS results were analyzed using GNPS MS/MS molecular networking for comparing experimental spectra with those of cyanotoxin standards against in-house databases and the existing literature. Our research underscores the variability in cyanotoxin production among species and over time, extending beyond microcystins. LC-MS results, interpreted through the GNPS platform, revealed six cyanotoxin groups in Brazilian strains. Yet, compounds present in 75 % of the toxic extracts remained unidentified. Further research is crucial for fully comprehending the impact of potentially harmful organisms on water quality and public health management strategies. The study highlights the urgent need for continuously monitoring cyanobacteria and the cyanotoxin inclusion of management in public health policies.

Cyanopeptides occurrence and diversity in a Brazilian tropical reservoir: Exploring relationships with water quality.

Microcystins (MCs) are a class of toxic secondary metabolites produced by some cyanobacteria strains that endanger aquatic and terrestrial organisms in various freshwater systems. Although patterns in MC occurrence are being recognized, divergences in the global data still hamper our ability to predict the toxicity of cyanobacterial blooms. This study aimed (i) to determine the dynamics of MCs and other cyanopeptides in a tropical reservoir, (ii) to investigate the correlation between peptides and potential cyanotoxin producers (iii) identifying the possible abiotic factors that influence the peptides. We analyzed, monthly, eight MC variants (MC-RR, -LA, -LF, -LR, -LW, -YR, [D-Asp3]-RR and [D-Asp3]-LR) and other peptides in 47 water samples collected monthly, all season long, from two sampling sites in a tropical eutrophic freshwater reservoir, in southeastern Brazil. The cyanopeptides were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The biomass of potential cyanobacterial producers and water quality variables were measured. MCs were detected in both sampling sites year-round; the total MC concentration varied from 0.21 to 4.04 µg L-1, and three MC variants were identified and quantified (MC-RR, [D-Asp3]-RR, -LR). Additionally, we identified 28 compounds belonging to three other cyanopeptide classes: aeruginosin, microginin, and cyanopeptolin. As potential MC producers, Microcystis spp. and Dolichospermum circinalis were dominant during the study, representing up to 75% of the total phytoplankton. Correlational and redundancy analysis suggested positive effects of dissolved oxygen, nitrate, and total phosphorus on MC and microginins concentration, while water temperature appeared to favor aeruginosins. A comparison between our results and historical data showed a reduction in total phosphorus and cyanobacteria, suggesting increased water quality in the reservoir. However, the current MC concentrations indicate a rise in cyanobacterial toxicity over the last eight years. Moreover, our study underscores the pressing need to explore cyanopeptides other than MCs in tropical aquatic systems.

A systematic review on guanitoxin: General characteristics and ecological risks.

Guanitoxin (GNT) is a potent cyanotoxin, with a relatively low number of publications (n = 51) compared to other cyanotoxins. Among the published studies, 35 % were on the effect of the toxin in animals, mainly in rodents and in vitro testing, followed by studies that identified species of cyanobacteria that produce GNT in aquatic systems and consequently accidental poisoning in wild and domestic animals (27 %). Studies that developed or tested methods for identifying the molecule, based on colorimetric and analytical techniques, represented 14 %, while 8 % were on GNT biosynthesis. Review articles and chemical isolation (6 %) and on the stability of the molecule (4 %) were the topics with the lowest number of publications. The results show the occurrence of GNT was identified mainly in eutrophic environments with a higher incidence in the American continent. Chemical characteristics of the molecule, such as short half-life in the environment, instability in solutions with alkaline pH values, temperature >23 °C, added to the lack of an analytical standard, are factors that make it difficult to identify and quantify it. However, GNT monitoring can be performed using LC-MS-MRM methods or genes specific to the newly discovered molecule.

Oxidation of chlortetracycline and its isomers by Botrytis aclada laccase in the absence of mediators: pH dependence and identification of transformation products by LC-MS.

Tetracyclines are antibiotics considered emerging pollutants and currently, wastewater treatment plants are not able to remove them efficiently. Laccases are promising enzymes for bioremediation because they can oxidize a wide variety of substrates. The aim of this study was to evaluate the Botrytis aclada laccase for the oxidation of chlortetracycline and its isomers in the absence of a mediator molecule, at a pH range between 3.0 to 7.0, and to characterize the transformation products by LC-MS. Chlortetracycline and three isomers were detected in both, controls and reaction mixtures at 0 h and in controls after 48 h of incubation but in different proportions depending on pH. An additional isomer was also detected, but only in the presence of BaLac. Based on the transformation products identified in the enzymatic reactions and information from literature, we assembled a network of transformation pathways starting from chlortetracycline and its isomers. The spectrometric analysis of the products indicated the probable occurrence of oxygen insertion, dehydrogenation, demethylation and deamination reactions. Four new products were identified, and we also described a novel transformation product without the chloro group. We observed that increasing pH led to higher diversity of main products. This is the first study using the laccase from fungi Botrytis aclada to oxidate chlortetracycline and its isomers and it can be considered as an ecological alternative to be used in bioremediation processes such as wastewater.

Metabolomics Applied to Cyanobacterial Toxins and Natural Products.

The biological and chemical diversity of Cyanobacteria is remarkable. These ancient prokaryotes are widespread in nature and can be found in virtually every habitat on Earth where there is light and water. They are producers of an array of secondary metabolites with important ecological roles, toxic effects, and biotechnological applications. The investigation of cyanobacterial metabolites has benefited from advances in analytical tools and bioinformatics that are employed in metabolomic analyses. In this chapter, we review selected articles highlighting the use of targeted and untargeted metabolomics in the analyses of secondary metabolites produced by cyanobacteria. Here, cyanobacterial secondary metabolites have been didactically divided into toxins and natural products according to their relevance to toxicological studies and drug discovery, respectively. This review illustrates how metabolomics has improved the chemical analysis of cyanobacteria in terms of speed, sensitivity, selectivity, and/or coverage, allowing for broader and more complex scientific questions.

Lethal and sublethal effects towards zebrafish larvae of microcystins and other cyanopeptides produced by cyanobacteria.

Cyanobacterial blooms affect aquatic ecosystems across the globe and one major concern relates to their toxins such as microcystins (MC). Yet, the ecotoxicological risks, particularly non-lethal effects, associated with other co-produced secondary metabolites remain mostly unknown. Here, we assessed survival, morphological alterations, swimming behaviour and cardiovascular functions of zebrafish (Danio rerio) upon exposure to cyanobacterial extracts of two Brazilian Microcystis strains. We verified that only MIRS-04 produced MCs and identified other co-produced cyanopeptides also for the MC non-producer NPCD-01 by LC-HRMS/MS analysis. Both cyanobacterial extracts, from the MC-producer and non-producer, caused acute toxicity in zebrafish with LC50 values of 0.49 and 0.98 mgdw_biomass/mL, respectively. After exposure to MC-producer extract, additional decreased locomotor activity was observed. The cyanopeptolin (micropeptin K139) contributed 52% of the overall mortality and caused oedemas of the pericardial region. Oedemas of the pericardial area and prevented hatching were also observed upon exposure to the fraction with high abundance of a microginin (Nostoginin BN741) in the extract of the MC non-producer. Our results further add to the yet sparse understanding of lethal and sublethal effects caused by cyanobacterial metabolites other than MCs and the need to better understand the underlying mechanisms of the toxicity. We emphasize the importance of considering mixture toxicity of co-produced metabolites in the ecotoxicological risk assessment of cyanobacterial bloom events, given the importance for predicting adverse outcomes in fish and other organisms.

A rapid LC-MS/MS method for multi-class identification and quantification of cyanotoxins.

Cyanobacteria can form harmful blooms in specific environmental conditions due to certain species producing toxic metabolites known as cyanotoxins. These toxins pose significant risks to public health and the environment, making it critical to identify and quantify them in food and water sources to avoid contamination. However, current screening methods only focus on a single class of cyanotoxins, limiting their effectiveness. Thus, fast and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed to analyze eighteen cyanotoxins simultaneously. A simplified extraction procedure using lyophilized samples of cyanobacterial biomass was also used, eliminating the need for traditional solid-phase extraction methods. This method uses multiple reaction monitoring and allows accurate determination and quantification of eighteen cyanotoxins, including anatoxin-a, homoanatoxin-a, cylindrospermopsin, deoxy-cylindrospermopsin, nodularin, guanitoxin, seven microcystins (RR, [D-Asp3] RR, LA, LR, LY, LW, and YR), and five saxitoxins (gonyautoxins - GTX-1&4, GTX-2&3, GTX-5), decarbamoylgonyautoxin (dcGTX-2&3), and N-Sulfocarbamoylgonyautoxin (C1&C2), all in a short acquisition time of 8 min. Therefore, this method provides a simple and efficient approach to identify and quantify harmful compounds produced by cyanobacteria. Hence, this represents the first method to detecting guanitoxin among cyanotoxins. By expanding the range of toxins analyzed, this method can help ensure high-quality food and drinking water and protect recreational users from exposure to cyanotoxins.

Ecotoxicological assessment of guanitoxin-producing cyanobacteria in Danio rerio and Daphnia similis.

Anthropogenic activity has dramatically deteriorated aquatic ecosystems in recent years. Such environmental alterations could change the primary producers' composition, exacerbating the proliferation of harmful microorganisms such as cyanobacteria. Cyanobacteria can produce several secondary metabolites, including guanitoxin, a potent neurotoxin and the only naturally occurring anticholinesterase organophosphate ever reported in the literature. Therefore, this study investigated the acute toxicity of guanitoxin-producing cyanobacteria Sphaerospermopsis torques-reginae (ITEP-024 strain) aqueous and 50% methanolic extracts in zebrafish (Danio rerio) hepatocytes (ZF-L cell line), zebrafish embryos (fish embryo toxicity - FET) and specimens of the microcrustacean Daphnia similis. For this, hepatocytes were exposed to 1-500 mg/L of the ITEP-024 extracts for 24 h, the embryos to 31.25-500 mg/L for 96 h, and D. similis to 10-3000 mg/L for 48 h. Non-target metabolomics was also performed to analyze secondary metabolites produced by the ITEP-024 using LC-MS/MS. Metabolomics indicated the guanitoxin presence just in the aqueous extract of the ITEP-024 and the presence of the cyanopeptides namalides, spumigins, and anabaenopeptins in the methanolic extract. The aqueous extract decreased the viability of zebrafish hepatocytes (EC(I)50(24h) = 366.46 mg/L), and the methanolic extract was not toxic. FET showed that the aqueous extract (LC50(96) = 353.55 mg/L) was more toxic than the methanolic extract (LC50(96) = 617.91 mg/L). However, the methanolic extract had more sublethal effects, such as abdominal and cardiac (cardiotoxicity) edema and deformation (spinal curvature of the larvae). Both extracts immobilized daphnids at the highest concentration analyzed. However, the aqueous extract was nine times more lethal (EC(I)50(48h) = 108.2 mg/L) than the methanolic extract (EC(I)50(48h) = 980.65 mg/L). Our results showed an imminent biological risk for aquatic fauna living in an ecosystem surrounded by ITEP-024 metabolites. Our findings thus highlight the urgency of understanding the effects of guanitoxin and cyanopeptides in aquatic animals.

Toxicological effects of cyanobacterial metabolites on zebrafish larval development.

Freshwater cyanobacteria are known worldwide for their potential to produce toxins. However, these organisms are also found in marine, terrestrial and extreme environments and produce unique compounds, other than toxins. Nevertheless, their effects on biological systems are still barely known. This work tested extracts of different cyanobacterial strains against zebrafish (Danio rerio) larvae and analyzed their metabolomic profiles using liquid chromatography combined with mass spectrometry. Strains Desertifilum tharense, Anagnostidinema amphibium, and Nostoc sp. promoted morphological abnormalities such as pericardial edema, edema in the digestive system region, curvature of the tail and spine in zebrafish larvae in vivo. In contrast, Microcystis aeruginosa and Chlorogloeopsis sp. did not promote such changes. Metabolomics revealed unique compounds belonging to the classes of terpenoids, peptides, and linear lipopeptides/microginins in the nontoxic strains. The toxic strains were shown to contain unique compounds belonging to the classes of cyclic peptides, amino acids and other peptides, anabaenopeptins, lipopeptides, terpenoids, and alkaloids and derivatives. Other unknown compounds were also detected, highlighting the rich structural diversity of secondary metabolites produced by cyanobacteria. The effects of cyanobacterial metabolites on living organisms, mainly those related to potential human and ecotoxicological risks, are still poorly known. This work highlights the diverse, complex, and unique metabolomic profiles of cyanobacteria and the biotechnological potential and associated risks of exposure to their metabolites.

Chronic alcohol administration alters metabolomic profile of murine bone marrow.

Introduction: People with hazardous alcohol use are more susceptible to viral, bacterial, and fungal infections due to the effect of alcohol on immune system cell function. Metabolized ethanol reduces NAD+ to NADH, affecting critical metabolic pathways. Here, our aim was to investigate whether alcohol is metabolized by bone marrow cells and if it impacts the metabolic pathways of leukocyte progenitor cells. This is said to lead to a qualitative and quantitative alteration of key metabolites which may be related to the immune response. Methods: We addressed this aim by using C57BL/6 mice under chronic ethanol administration and evaluating the metabolomic profile of bone marrow total cells by gas chromatography-coupled mass spectrometry (GC-MS). Results: We identified 19 metabolites. Our data demonstrated that chronic ethanol administration alters the metabolomic profile in the bone marrow, resulting in a statistically diminished abundance of five metabolites in ethanol-treated animals: uracil, succinate, proline, nicotinamide, and tyrosine. Discussion: Our results demonstrate for the first time in the literature the effects of alcohol consumption on the metabolome content of hematopoietic tissue and open a wide range of further studies to investigate mechanisms by which alcohol compromises the cellular function of the immune system.

Exploring the Relationship between Biosynthetic Gene Clusters and Constitutive Production of Mycosporine-like Amino Acids in Brazilian Cyanobacteria.

Cyanobacteria are oxygenic phototrophic prokaryotes that have evolved to produce ultraviolet-screening mycosporine-like amino acids (MAAs) to lessen harmful effects from obligatory exposure to solar UV radiation. The cyanobacterial MAA biosynthetic cluster is formed by a gene encoding 2-epi-5-epi-valiolone synthase (EVS) located immediately upstream from an O-methyltransferase (OMT) encoding gene, which together biosynthesize the expected MAA precursor 4-deoxygadusol. Accordingly, these genes are typically absent in non-producers. In this study, the relationship between gene cluster architecture and constitutive production of MAAs was evaluated in cyanobacteria isolated from various Brazilian biomes. Constitutive production of MAAs was only detected in strains where genes formed a co-linear cluster. Expectedly, this production was enhanced upon exposure of the strains to UV irradiance and by using distinct culture media. Constitutive production of MAAs was not detected in all other strains and, unexpectedly, production could not be induced by exposure to UV irradiation or changing growth media. Other photoprotection strategies which might be employed by these MAA non-producing strains are discussed. The evolutionary and ecological significance of gene order conservation warrants closer experimentation, which may provide a first insight into regulatory interactions of genes encoding enzymes for MAA biosynthesis.

Evaluation of the Toxicity of Microcyclamide Produced by Microcystis aeruginosa in Danio rerio Embryos.

The genus of cyanobacteria Microcystis is one of the most recurrent in blooms and is associated with the hepatotoxin microcystin production. In addition to cyanotoxins, these bacteria produce a wide range of secondary metabolites with a wide repertoire of activities. The co-occurrence of cyanotoxins and other cyanopeptides during blooming is quite common, and the negative effects are not always limited to one class of toxins, which makes it essential to investigate the toxicity of the other compounds individually. The objective of this study was to isolate the cyanopeptide microcyclamide produced by the strain Microcystis aeruginosa LTPNA 08 by liquid chromatography coupled to high-resolution mass spectrometry with a quadrupole-time-of-flight analyzer (LC-HR-QTOF-MS/MS) and to evaluate its acute toxicity in embryos of Danio rerio through the Fish Embryo Acute Toxicity (FET) assay. The fraction containing microcyclamide (95% purity) caused lethality in 62% of the embryos after 96 h exposure (50 µg mL-1), with evidence of cardiotoxicity (cardiac edema). The calculated LC50 value was 42.98 µg mL-1 (with a concentration range of 37.79-48.89 µg mL-1). The characterization of the secondary metabolites produced by cyanobacteria and the investigation of the toxicity of these compounds individually are essential for the identification of the substances responsible for negative effects on living organisms and on the ecosystem, in addition to assisting in the development of risk management policies.

Cyanotoxins and water quality parameters as risk assessment indicators for aquatic life in reservoirs.

We assessed the extent of pollution in an essential public water supply reservoir (southeastern Brazil). An environmental monitoring study was performed at the Billings Reservoir (at the water catchment site) to assess the water quality in 2017, 2018, and 2019. Physicochemical parameters were analyzed, quantifying the total cyanobacteria and the cyanotoxins microcystins (MCs) and saxitoxins (SXTs), as well as their possible ecological risk to the aquatic environment. We also determined metals and metalloids (As, Ba, Cd, Pb, Cu, Cr, Fe, Mn, Ni, Zn, and Sb) and fecal bacteria (Escherichia coli). Monthly samplings were performed for 2017, 2018, and 2019 (totaling 36 sampling campaigns). Metals, metalloids, and E. coli values were below the maximum limit allowed by the Brazilian legislation. High concentrations of total cyanobacteria (3.07 × 104 - 3.23 × 105 cells/mL), microcystin variants MC-LR (0.67-23.63 µg/L), MC-LA (0.03-8.66 µg/L), MC-RR (0.56-7.92 µg/L), and MC-YR (0.04-1.24 µg/L), as well as the saxitoxins GTX2 (0.18-5.37 µg/L), GTX3 (0.13-4.40 µg/L), and STX (0.12-2.92 µg/L) were detected. From an ecotoxicological point of view, the estimated values for the risk quotient (RQ) for microcystins and saxitoxins were largely greater than 1, indicating a high risk to aquatic life. Therefore, further efforts need to be made to delay the eutrophication of the reservoir.

Ecological risk of imidacloprid on the Brazilian non-target freshwater organisms Chironomus sancticaroli and Poecilia reticulata.

Imidacloprid (IMI) is a neonicotinoid insecticide widely used in agriculture worldwide. This pesticide has been found in freshwater ecosystems, including Brazilian freshwaters. For this reason, studies are being conducted to detect the presence of IMI in freshwater and understand its effects on the aquatic biota. In the present study, the acute toxic effect of the imidacloprid commercial formulation (ICF) Galeão® on the Brazilian non-target aquatic organisms Chironomus sancticaroli and Poecilia reticulata was evaluated. Enzymatic activities (glutathione S-transferase (GST), catalase (CAT), and ascorbate peroxidase (APX)) were also determined. Moreover, we considered 11 studies that detected IMI concentrations up to 3.65 µg.L-1 in 28 different Brazilian freshwaters to evaluate the acute ecological risk of IMI in these environments. From the ecotoxicological assays, we determined the LC50 values for C. sancticaroli (LC50-48 h 1.52 µg.L-1) and P. reticulata (LC50-96 h 122.65 mg.L-1). The high sensitivity of C. sancticaroli demonstrates that this species could be used as a bioindicator in studies investigating the contamination of freshwater by IMI. Enzymatic activity changes were observed in both organisms and offered sublethal responses to the effects of the pollution by IMI on aquatic biota. Our results suggest that the presence of IMI in Brazilian aquatic ecosystems can represent a potential ecological risk for the aquatic insect populations and, consequently, cause an imbalance in these ecosystems. The present study provides relevant and comparable toxicity information that may be useful to develop public policies to protect the Brazilian aquatic ecosystem from IMI contamination.

Reannotation of Fly Amanita l-DOPA Dioxygenase Gene Enables Its Cloning and Heterologous Expression.

The l-DOPA dioxygenase of Amanita muscaria (AmDODA) participates in the biosynthesis of betalain- and hygroaurin-type natural pigments. AmDODA is encoded by the dodA gene, whose DNA sequence was inferred from cDNA and gDNA libraries almost 30 years ago. However, reports on its heterologous expression rely on either the original 5'-truncated cDNA plasmid or artificial gene synthesis. We provide unequivocal evidence that the heterologous expression of AmDODA from A. muscaria specimens is not possible by using the coding sequence previously inferred for dodA. Here, we rectify and reannotate the full-length coding sequence for AmDODA and express a 205-aa His-tagged active enzyme, which was used to produce the l-DOPA hygroaurin, a rare fungal pigment. Moreover, AmDODA and other isozymes from bacteria were submitted to de novo folding using deep learning algorithms, and their putative active sites were inferred and compared. The wide catalytic pocket of AmDODA and the presence of the His-His-His and His-His-Asp motifs can provide insight into the dual cleavage of l-DOPA at positions 2,3 and 4,5 as per the mechanism proposed for nonheme dioxygenases.

Biosynthesis of Guanitoxin Enables Global Environmental Detection in Freshwater Cyanobacteria.

Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin (gnt) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.

Response of Oreochromis niloticus (Teleostei: Cichlidae) exposed to a guanitoxin-producing cyanobacterial strain using multiple biomarkers.

Changes in environmental conditions in aquatic ecosystems caused by anthropic actions can modify the composition of primary producers, promoting the excessive proliferation of cyanobacteria. These organisms can form cyanobacterial blooms, which directly affect aquatic life. The present study investigated the mutagenicity of the cyanobacterium Sphaerospermopsis torques-reginae (strain ITEP-024), guanitoxin-producing (natural organophosphate), and sublethal effects on fish in relevant environment concentrations. For this, the Ames test (Salmonella/microsome) was performed as a mutagenic assay for extracts of the ITEP-024 strain. Specimens of Oreochromis niloticus (Teleostei: Cichlidae) were subjected to acute 96 h exposure to different concentrations of aqueous extract of the strain: C = control group; T1 = 31.25 mg/L; T2 = 62.5 mg/L; T3 = 125 mg/L; and T4 = 250 mg/L. Genotoxic, biochemical, osmoregulatory, and physiologic biomarkers were analyzed. Our results showed that the cyanobacterium had a weak mutagenic response for the TA102 strain of Salmonella with and without metabolic activation by S9. Strains TA98 and TA100 were not affected. Fish from treatments T3 and T4 showed changes in oxidative stress (CAT, SOD, and GST enzymes), inhibition of the enzyme acetylcholinesterase activity, micronucleus formation, and osmoregulatory disorders. No guanitoxin accumulation was detected in the different tissues of O. niloticus by LC-MS/MS. Our results showed unprecedented mutagenicity data of the guanitoxin-producing cyanobacteria by the Ames test and biochemical, osmoregulatory, and genotoxic disorders in fish, providing efficient aquatic contamination biomarkers. Despite the great concern related to the presence of guanitoxin in blooms in freshwater ecosystems, its concentration is not yet regulated, and thus there is no monitoring agenda in current legislation.

Effects of different cultivation conditions on the production of ß-cyclocitral and ß-ionone in Microcystis aeruginosa.

BACKGROUND: Cyanobacteria blooms have become a major environmental problem and concern because of secondary metabolites produced by cyanobacteria released into the water. Cyanobacteria produce volatile organic compounds (VOCs), such as the compounds ß-cyclocitral and ß-ionone, which comprise odors, off-flavors, defense compounds, as well as growth regulators. Therefore, the general objective of this work was to evaluate the VOCs produced by two strains of Microcystis aeruginosa, differing in their ability to produce microcystins (LTPNA 01-non-producing and LTPNA 08-toxin-producing). The analysis of VOC production was carried out in (1) normal culture conditions, (2) under different light intensities (LI), and (3) after the external application of ß-ionone in both cultures. RESULTS: The results showed that ß-cyclocitral and ß-ionone are produced in all growth phases of LTPNA 01 and LTPNA 08. Both strains were producers of ß-cyclocitral and ß-ionone in normal culture conditions. It was observed that the ß-cyclocitral concentration was higher than ß-ionone in all light intensities investigated in this study. Additionally, the strain LTPNA 01 produced more ß-cyclocitral than LTPNA 08 at almost all times and LIs analyzed. However, the strain LTPNA 08 produced more ß-ionone, mainly at the initial times. In addition, the experiment results with the external addition of ß-ionone in the cultures showed that the strain LTPNA 01 produced more ß-cyclocitral in control conditions than in treatment. Nonetheless, ß-ionone production was higher in treatment conditions in LTPNA 08, indicating that the addition of ß-ionone may favor the production of these compounds and inhibit the production of ß-cyclocitral. CONCLUSION: Our results showed that some abiotic factors, such as different light intensities and external application of ß-ionone, can be triggers that lead to the production of VOCs.

Determining the Presence of 5-Fluorouracil in Hamster Saliva by HPLC / Determinação, por CLAE, da Presença do 5-Fluorouracil em Saliva de Hamsters

Abstract Introduction: Various methods of analysis for the assay of chemotherapeutic agent 5-Fluorouracil (5-FU) in human and animal biological fluids have previously been reported. However, there is no standardization for detecting 5-FU in the hamsters' saliva that received the chemotherapeutic agent. Objective: Considering that the administration of 5-FU in some way changes the morphology and function of the salivary glands, and that the presence of the chemotherapeutic agents in the oral mucosa may lead to some oral complications, the aim of this study was to determine the presence of 5-FU in the hamsters' saliva that received the chemotherapeutic agent, by means of the High Performance Liquid Chromatography technique (HPCL) since this animal model is used in studies of 5-FU induced oral mucositis and glandular hypofunction. Methods: Twelve animals were divided into 4 groups: CP and CPI, in which the animals received pilocarpine (CP) or pilocarpine + isoproterenol (CPI) and the chemotherapy vehicle intraperitoneally; and Groups QP and QPI, in which the animals received the same secretagogues listed above, and the chemotherapeutic agent 5-FU, respectively. After the secretagogue administration, saliva was collected from all the animals for a period of 60 mins. Subsequently, the saliva was frozen at -80 ˚C for later determination of the chemotherapeutic agent by HPLC. After the the chromatograms analysis, and based on the results obtained, it was possible to identify the presence of 5-FU in the saliva samples from hamsters that received the chemotherapeutic agent intraperitonally, by the HPLC technique. (AU)

Resumo Vários métodos de análise para o ensaio do quimioterápico 5-Fluorouracil (5-FU) em fluidos biológicos de humanos e animais, foram previamente relatados. No entanto, não há uma padronização para detecção de 5-FU na saliva de hamsters que receberam o quimioterápico. Considerando que a administração do 5-FU altera de alguma maneira a morfologia e função das glândulas salivares, e que a presença do quimioterápico na mucosa oral pode levar a algumas complicações orais, este trabalho teve como objetivo de determinar a presença de 5-FU na saliva de hamsters que receberam o quimioterápico pela técnica de Cromatografia Líquida de Alta Eficiência (CLAE), uma vez que este modelo animal é usado nos estudos com mucosite oral e hipofunção glandular, induzidas por 5-FU. Doze animais foram divididos em 4 grupos: CP e CPI, onde os animais receberam intraperitonealmente pilocarpina (CP) ou pilocarpina + isoproterenol (CPI) e o veículo do quimioterápico, e os grupos QP e QPI, onde os animais receberam, respectivamente, os mesmos secretagogos listados acima e o quimioterápico 5-FU. Após a administração do secretagogo, foi coletada a saliva de todos os animais, por um período de 60 min. Em seguida, a saliva foi congelada a -80 ˚C para posterior determinação do quimioterápico por CLAE. Após análise dos cromatogramas, e com base nos resultados obtidos, foi possível identificar a presença do 5-FU nas amostras de saliva de hamsters que receberam o quimioterápico via intraperitoneal pela técnica da CLAE. (AU)

Permanent occurrence of Raphidiopsis raciborskii and cyanotoxins in a subtropical reservoir polluted by domestic effluents (Itupararanga reservoir, São Paulo, Brazil).

Toxic cyanobacteria blooms are a frequent problem in subtropical reservoirs and freshwater systems. The purpose of this study was to investigate the occurrence of potentially toxic cyanobacteria and the environmental conditions associated with the presence of cyanotoxins in a Brazilian subtropical reservoir. Five collections were carried out at seven sampling locations in the reservoir, during the rainy and dry seasons, between the years 2016 and 2017. There was permanent occurrence of Raphidiopsis raciborskii (Woloszynska) Aguilera, Berrendero Gómez, Kastovsky, Echenique & Salerno (Phycologia 57(2):130-146, 2018), ranging between dominant and abundant, with an average biomass of 38.8 ± 29.9 mg L-1. Also abundant were Dolichospermum solitarium, D. planctonicum, Planktothrix isothrix, and Aphanizomenon gracile. Saxitoxin (STX) was detected in all the collected samples (0.11 ± 0.05 µg L-1). Microcystin (MC) was also detected, but at lower concentrations (0.01 ± 0.0 µg L-1). Low availability of NO3- and phosphorus limitation had significant effects on the R. raciborskii biomass and the levels of STX and MC. It was observed that R. raciborskii was sensitive to thermal stratification, at the same time that STX levels were higher. This suggested that STX was produced under conditions that restricted the growth of R. raciborskii. These are important findings, because they add information about the permanent occurrence of STX and R. raciborskii in an aquatic ecosystem limited by phosphorus, vulnerable to climatic variations, and polluted by domestic effluents.