Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa.

Cannabidiol (CBD), one of the main Cannabis sativa bioactive compounds, is utilized in the treatment of major epileptic syndromes. Its efficacy can be attributed to a multimodal mechanism of action that includes, as potential targets, several types of ion channels. In the brain, CBD reduces the firing frequency in rat hippocampal neurons, partly prolonging the duration of action potentials, suggesting a potential blockade of voltage-operated K+ channels. We postulate that this effect might involve the inhibition of the large-conductance voltage- and Ca2+-operated K+ channel (BK channel), which plays a role in the neuronal action potential's repolarization. Thus, we assessed the impact of CBD on the BK channel activity, heterologously expressed in HEK293 cells. Our findings, using the patch-clamp technique, revealed that CBD inhibits BK channel currents in a concentration-dependent manner with an IC50 of 280 nM. The inhibition is through a direct interaction, reducing both the unitary conductance and voltage-dependent activation of the channel. Additionally, the cannabinoid significantly delays channel activation kinetics, indicating stabilization of the closed state. These effects could explain the changes induced by CBD in action potential shape and duration, and they may contribute to the observed anticonvulsant activity of this cannabinoid.

Effects of Full-Spectrum Cannabis Oil with a Cannabidiol:Tetrahydrocannabinol 2:1 Ratio on the Mechanisms Involved in Hepatic Steatosis and Oxidative Stress in Rats Fed a Sucrose-Rich Diet.

Introduction: This study aimed to analyze the effects of cannabis oil (cannabidiol:tetrahydrocannabinol [CBD:THC], 2:1 ratio) on the mechanisms involved in hepatic steatosis and oxidative stress in an experimental model of metabolic syndrome (MS) induced by a sucrose-rich diet (SRD). We hypothesized that noninvasive oral cannabis oil administration improves hepatic steatosis through a lower activity of lipogenic enzymes and an increase in carnitine palmitoyltransferase-1 (CPT-1) enzyme activity involved in the mitochondrial oxidation of fatty acids. Furthermore, cannabis oil ameliorates liver oxidative stress through the regulation of the main regulatory factors involved, nuclear factor erythroid 2 (NrF2) and nuclear factor-kB (NF-κB) p65. For testing this hypothesize, a relevant experimental model of MS was induced by feeding rats with a SRD for 3 weeks. Methods: Male Wistar rats were fed the following diets for 3 weeks: reference diet: standard commercial laboratory diet, SRD, and SRD + cannabis oil: noninvasive oral administration of 1 mg/kg body weight cannabis oil daily. The full-spectrum cannabis oil presents a total cannabinoid CBD:THC 2:1 ratio. Serum glucose, triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol, uric acid, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase (AP), N-arachidonoylethanolamine or anandamide and 2-arachidonoylglycerol endocannabinoids levels, thiobarbituric acid reactive substance (TBARS) levels, and non-enzymatic antioxidant capacity (ferric ion-reducing antioxidant power [FRAP]) were evaluated. In the liver tissue: histology, nonalcoholic fatty liver disease activity score (NAS), triglycerides and cholesterol content, lipogenic enzyme activities (fatty acid synthase, acetyl-CoA carboxylase, malic enzyme, and glucose-6-phosphate dehydrogenase), enzyme related to mitochondrial fatty acid oxidation (CPT-1), reactive oxygen species, TBARS, FRAP, glutathione, catalase, glutathione peroxidase, and glutathione reductase enzyme activities. 4-hydroxynonenal, NrF2, and NF-κB p65 levels were analyzed by immunohistochemistry. Results: The results showed that SRD-fed rats developed dyslipidemia, liver damage, hepatic steatosis (increase of key enzymes related to the novo fatty acid synthesis and decrease of key enzyme related to mitochondrial fatty acid oxidation), lipid peroxidation, and oxidative stress. Hepatic NrF2 expression was significantly decreased and NF-κB p65 expression was increased. Cannabis oil administration improved dyslipidemia, liver damage, hepatic steatosis, lipid peroxidation (improving enzymes involved in lipid metabolism), and oxidative stress. In the liver tissue, NrF2 expression increased, and NF-κB p65 expression was reduced. Conclusion: The present study revealed new aspects of liver damage and steatosis, lipid peroxidation, and oxidative stress in dyslipidemic insulin-resistant SRD-fed rats. We demonstrated new properties and molecular mechanisms of cannabis oil (CBD:THC, 2:1 ratio) on lipotoxicity and hepatic oxidative stress in an experimental model of MS.

Cannabinoid Content in Cannabis Flowers and Homemade Cannabis-Based Products Used for Therapeutic Purposes in Argentina.

Introduction: A recent law (DCTO-2020-883-APN-PTE-Law No. 27,350. Regulation) passed in Argentina put an end to the ban imposed for the last 60 years on cannabis cultivation within the country. The law permits restricted access to cannabis derivatives for medicinal, therapeutic, and palliative use by individuals and communities, allowing self- and community-based cannabis production. This is cause for concern in view of the lack of quality controls for cannabis derivatives. The several varieties of cannabis grown in Argentina have different chemical profiles and are processed in a variety of ways-mostly by alcohol extraction or maceration at different temperatures and for different amounts of times-making the cannabinoid content of these preparations highly variable. Determining the characteristics of home- and community-grown cannabis products will facilitate the implementation of public policies conducive to their safety and improvement. Objective: The aim of this study was to determine the cannabinoid chemotypes used for therapeutic purposes in Argentina and evaluate whether the cannabinoids present in homemade derivatives are comparable to those in commercially available products. Materials and Methods: High performance liquid chromatography with ultraviolet and diode array detector (HPLC/UV-DAD) analysis of 436 samples (oils, resins, and inflorescences) was carried out to determine the identity and concentration of five cannabinoids: tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), and cannabinol (CBN). From three different sources, the samples represent the type of medical cannabis preparations to which patients have access. Results: The results indicate that the medium-to-low cannabinoid concentration in a significant number of homemade oil samples is similar to that found in commercial products. Most of the samples have a THC/CBD ratio >1 or only contain THC. Acidic cannabinoids were detected in homemade preparations, but were not reported in package inserts of commercial products. Conclusions: Our results indicate that despite their considerable variability, homemade preparations as a whole show cannabinoid levels and profiles equivalent to the commercially available products commonly used for medicinal, therapeutic, and palliative purposes in Argentina.

Determination of variability of terpenes and terpenoids in Cannabis sativa by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry.

Absolute content of terpenes in inflorescences of two strains of Cannabis sativa L., CAT 1 and CAT 3, has been determined. Twenty terpenes commonly present in these samples were quantified by solid phase microextraction combined with gas chromatography and flame ionization detection (SPME/GC-FID). High amounts of ß-myrcene, α-pinene, ß-pinene, limonene, (E)-ß-ocimene, ß-caryophyllene, α-humulene, (E)-nerolidol, and linalool, were found in both strains. Lower concentrations (< 20 µg·g-1) of other terpenes were also determined. Only (E)-ß-ocimene was detected at 50 µg·g-1 in CAT 3 whereas it was below the LOD in CAT 1. Concentrations of other compounds for which standards were not available, were estimated based on a response factor obtained from the calibration curves of compounds with similar chemical structures. Fingerprints of both CAT strains were obtained and the identities of most volatile compounds were assigned using gas chromatography coupled to mass spectrometer detector (GC-MS). Additionally, an assessment of variability of terpenes was achieved by analyzing ten plants of each strain grown under controlled conditions and harvested at the same time. This variability was about 20%, considering terpenes at concentration above 20 µg·g-1.

[D-Leu1]MC-LR Has Lower PP1 Inhibitory Capability and Greater Toxic Potency than MC-LR in Animal and Plant Tissues.

Two microcystins, MC-LR and [D-Leu1]MC-LR, present in La Plata Basin blooms, are differentiated by substitution of D-Alanine for D-Leucine at position 1. Our objective was to evaluate acute toxicity of [D-Leu1]MC-LR and MC-LR in mice (N:NIH Swiss) and beans (Phaseolus vulgaris). We observed variations in [D-Leu1]MC-LR lethal doses with respect to those reported for MC-LR (100 µg/kg), with an increased liver/body weight ratio and intrahepatic hemorrhages in mice exposed to 50-200 µg [D-Leu1]MC-LR/kg and slight steatosis after a single 25 µg [D-Leu1]MC-LR/kg i.p. dose. Our study in the plant model showed alterations in germination, development, morphology and TBARs levels after a single contact with the toxins during imbibition (3.5 and 15 µg/mL), those treated with [D-Leu1]MC-LR being more affected than those treated with the same concentration of MC-LR. Protein phosphatase 1 (PP1) IC50 values were 40.6 nM and 5.3 nM for [D-Leu1]MC-LR and MC-LR, respectively. However, the total phosphatase activity test in root homogenate showed 60% inhibition for [D-Leu1]MC-LR and 12% for MC-LR. In mouse liver homogenate, 50% inhibition was observed for [D-Leu1]MC-LR and 40% for MC-LR. Our findings indicate the need for further research into [D-Leu1]MC-LR toxicity since together with oxidative stress, the possible inhibition of other phosphatases could explain the differences detected in the potency of the two toxins.

[D-Leu1]MC-LR and MC-LR: A Small-Large Difference: Significantly Different Effects on Phaseolus vulgaris L. (Fabaceae) Growth and Phototropic Response after Single Contact during Imbibition with Each of These Microcystin Variants.

[D-Leu1]MC-LR and MC-LR, two microcystins differing in one amino acid, constitute a sanitary and environmental problem owing to their frequent and concomitant presence in water bodies of the Americas and their association with human intoxication during recreational exposure to cyanobacterial bloom. Present in reservoirs used for irrigation as well, they can generate problems in the development of crops such as Phaseolus vulgaris, of nutritional and economic interest to the region. Although numerous works address the toxic effects of MC-LR, information on the toxicity of [D-Leu1]MC-LR is limited. Our objective was to study the toxic effects of [D-Leu1]MC-LR and MC-LR (3.5 µg/ml) on P. vulgaris after a single contact at the imbibition stage. Our findings indicate that 10 days post treatment, [D-Leu1]MC-LR generates morphological and physiological alterations more pronounced than those caused by MC-LR. In addition to the alterations produced by [D-Leu1]MC-LR in the development of seedlings and the structure of the leaves, roots and stems, we also found alterations in leaf stomatal density and conductivity, a longer delay in the phototropic response and a decrease in the maximum curvature angles achieved with respect to that observed for MC-LR. Our findings indicate that these alterations are linked to the greater inhibition of phosphatase activity generated by [D-Leu1]MC-LR, rather than to oxidative damage. We observed that 30 days after treatment with MC-LR, plants presented better development and recovery than those treated with [D-Leu1]MC-LR. Further studies are required on [D-Leu1]MC-LR and MC-LR toxicity and their underlying mechanisms of action.

Effect of temperature on microcystin-LR removal and lysis activity on Microcystis aeruginosa (cyanobacteria) by an indigenous bacterium belonging to the genus Achromobacter.

Microcystis is a frequent cyanobacterium bloom-forming with cosmopolitan distribution which can produce a hepatotoxin group called microcystins (MCs). These MCs are resistant to the traditional processes employed in the water treatment plants and they are often detected after conventional treatments. Because of this, the bio-removal studies have obtained a great interest in the last decades. In this work, a bacterial strain namely LG1 with the ability to remove microcystin-LR (MC-LR) under laboratory conditions was isolated from Rio de la Plata River and it was identified as Achromobacter spp. This ubiquitous bacterium was able to remove 79.5% MC-LR in 7 days with average removal time of 3.33 ± 0.08, 3.06 ± 0.05, and 2.77 ± 0.05 days at 28, 32, and 36 ± 1 °C, being higher at high temperature (36 °C) with an activation energy = 16.79 ± 1.99 kJ mol-1. LG1 grew better at higher temperature (from 28 to 36 ± 1 °C) increasing the specific growth rate (µ) and reducing 2-fold the lag phase duration (LPD) without significant differences (p > 0.05) between maximum population density (MPD). In addition, LG1 showed a lysis activity on two M. aeruginosa native strains in 7 days measured as chlorophyll a (Chl-a) concentration. The lysis activity increased around 2-fold when increasing the temperature from 28 to 36 ± 1 °C. This is the first report of an indigenous bacterium belonging to the genus Achromobacter spp. isolated from the Rio de la Plata River with the capacity to remove MC-LR and lysis activity on M. aeruginosa.

Monitoring human genotoxicity risk associated to urban and industrial Buenos Aires air pollution exposure.

The quality of life in large megacities is directly affected by its air quality. In urban environments, suspended particles from anthropogenic origin is one of the main air contaminants identified as highly genotoxic, mutagenic, or carcinogenic. Atmospheric monitoring is therefore imperative, and bioassays to detect the effects of genotoxic agents give usually excellent results. Analysis of micronucleus (MN) in exfoliated oral mucosa cells is a sensitive non-invasive method for monitoring genetic damage in human populations. The first aim of this study was to analyze and characterize levels of volatile organic compounds (VOCs), particulate matter (PM), and polycyclic aromatic hydrocarbons (PAHs) in two areas from Buenos Aires: La Plata city, an urban (U) area and Ensenada, an industrial (I) area. Secondly, we evaluated the possible health risk of its inhabitants through a simple genotoxic assay on exfoliated oral mucosa cells. Whole blood cell count and nuclear abnormalities frequencies were evaluated in the exfoliated oral mucosa cells from urban and industrial inhabitants. Smoking habit represented a significant factor increasing MN percentage while, age did not increase the production of any of the nuclear aberrations assayed (micronuclei, binucleated, karyorrhexis) when the inhabitants from the urban and the industrial areas were compared. In addition, changes in MN and binucleated cell percentages in males and females were found to be area-dependent. We suggest that regardless PM concentration, PM-specific characteristics (size, shape, chemical elements, etc.) and VOCs levels could be responsible for the different harmful genotoxic effects seen in the two areas. Although this is a preliminary study, our results allowed to recognize that individuals living in both the urban and the industrial areas could be considered susceptible groups and should periodically undergo biological monitoring and appropriate care.

Relationship between Ocular Surface Alterations and Concentrations of Aerial Particulate Matter.

PURPOSE: To evaluate ocular surface alterations in two populations at different exposure levels to particulate matter (PM) in their living and work environments. METHODS: A cross-sectional study was conducted, including 78 volunteers from Argentina who lived and worked under different pollution levels in an urban (U; n = 44) or industrial zone (I; n = 34). Mean exposure level to PM was evaluated. Responses to the Ocular Symptom Disease Index and McMonnies questionnaire were obtained from all subjects. Subsequently, an assessment through the Schirmer I test (ST), slit lamp microscopy, vital staining, and tear breakup time was conducted. Statistical analyses with Chi-square and Bartlett's tests, as well as Student's t-tests and principal component analysis (PCA), were performed. RESULTS: Particles of size < 2.5 µm (PM 2 . 5 ) level was significantly higher in the I group than the U group (P = 0.04). Ocular surface parameters including bulbar redness, eyelid redness, and the degree of vital staining with fluorescein (SF) and lissamine green (SLG) exhibited difference between the groups. With regards to the tear film, statistically significant differences in the ST value and meibomian gland dysfunction between the groups were detected (P = 0.003 and P = 0.02, respectively). Conjunctival SF and SLG, and ST values were identified as factors which could distinguish groups exposed to different PM levels. CONCLUSION: Subjects exposed to higher levels of PM in the outdoor air presented greater ocular surface alterations. Thus, ST, SF, and SLG values could be used as convenient indicators of adverse health effects due to exposure to air pollution.

Inhalation of marijuana affects Drosophila heart function.

We investigated the effect of inhalation of vaporized marijuana on cardiac function in Drosophila melanogaster, a suitable genetic model for studying human diseases. Adult flies were exposed to marijuana for variable time periods and the effects on cardiac function were studied. Short treatment protocol incremented heart-rate variability. Contractility was augmented only under prolonged exposure to cannabis and it was associated with incremented calcium transient within cardiomyocytes. Neither the activity of the major proteins responsible for calcium handling nor the calcium load of the sarcoplasmic reticulum were affected by the cannabis treatment. The observed changes manifested in the cardiomyocytes even in the absence of the canonical cannabinoid receptors described in mammals. Our results are the first evidence of the in vivo impact of phytocannabinoids in D. melanogaster. By providing a simple and affordable platform prior to mammalian models, this characterization of cardiac function under marijuana exposure opens new paths for conducting genetic screenings using vaporized compounds.This article has an associated First Person interview with the first author of the paper.

Comparative study of tear lipid composition in two human populations with different exposure to particulate matter in La Plata, Argentina.

To identify the changes in the lipid profile of the tear film in two human populations exposed to different levels of particulate material, and its relationship with dry eye, by gas chromatography with mass spectrometry (GC-MS) detection. A panel study involving 78 volunteers, who live and work in two locations in Argentina with different pollution levels: urban zone (n = 44) and industrial zone (n = 34). We measured the mean levels of particulate matter (PM) exposure. The tear samples were analyze by gas GC-MS detection and the dry eye was diagnose using Schirmer test, fluorescein breakup time, vital staining with fluorescein and lissamine green, and lid parallel conjunctival folds (LIPCOF). Statistical analysis was performed using Chi-Square, Bartlett's, Mann-Whitney tests, and Multiple Correspondence Analysis. PM10 level was significantly higher in industrial zone than in urban area (p < 0.05). Subjects exposed to higher levels of PM10 in outdoor air presented more presence of fatty acids (FA) of long chain, a higher proportion of saturated fatty acids (SFA), and lower unsaturated fatty acids (UFA), showing a differentiated profile, which may be associated with a PM level. The incidence of dry eye was greater in the industrial zone (p < 0.001), showing in both populations for this pathology higher FA ω-6 levels, which are responsible for the inflammation process. The lipid profile in populations exposed to higher levels of PM10, like the industrial zone, shows a differentiated profile of FA and more incidence of dry eye with higher FA ω-6 levels, which are responsible for the inflammation process.

Physiological responses and toxin production of Microcystis aeruginosa in short-term exposure to solar UV radiation.

The aim of this study was to evaluate the effects of short-term (hours) exposure to solar UV radiation (UVR, 280-400 nm) on the physiology of Microcystis aeruginosa. Three solar radiation treatments were implemented: (i) PAR (PAR, 400-700 nm), (ii) TUVA (PAR + UVAR, 315-700 nm) and (iii) TUVR (PAR + UVAR + UVBR, 280-700 nm). Differential responses of antioxidant enzymes and the reactive oxygen species (ROS) production to UVR were observed. Antioxidant enzymes were more active at high UVR doses. However, different responses were observed depending on the exposure to UVAR or UVBR and the dose level. No effects were observed on the biomass, ROS production or increased activity of superoxide dismutase (SOD) and catalase (CAT) compared to the control when UVR + PAR doses were lower than 9875 kJ m-2. For intermediate doses, UVR + PAR doses between 9875 and 10 275 kJ m-2, oxidative stress increased while resistance was imparted through SOD and CAT in the cells exposed to UVAR. Despite the increased antioxidant activity, biomass decrease and photosynthesis inhibition were observed, but no effects were observed with added exposure to UVBR. At the highest doses (UVR + PAR higher than 10 275 kJ m-2), the solar UVR caused decreased photosynthesis and biomass with only activation of CAT by UVBR and SOD and CAT by UVAR. In addition, for such doses, a significant decrease of microcystins (MCs, measured as MC-LR equivalents) was observed as a consequence of UVAR. This study facilitates our understanding of the SOD and CAT protection according to UVAR and UVBR doses and cellular damage and reinforces the importance of UVR as an environmental stressor. In addition, our results support the hypothesized antioxidant function of MCs.

Recreational Exposure during Algal Bloom in Carrasco Beach, Uruguay: A Liver Failure Case Report.

In January 2015, a 20-month-old child and her family took part in recreational activities at Carrasco and Malvín beaches (Montevideo, Uruguay). An intense harmful algae bloom (HAB) was developing along the coast at that time. A few hours after the last recreational exposure episode, the family suffered gastrointestinal symptoms which were self-limited except in the child's case, who was admitted to hospital in Uruguay with diarrhea, vomiting, fatigue, and jaundice. The patient had increased serum levels of liver enzymes and bilirubin and five days later presented acute liver failure. She was referred to the Italian Hospital in Buenos Aires, being admitted with grade II-III encephalopathy and hepatomegaly and requiring mechanical respiratory assistance. Serology tests for hepatitis A, B, and C, Epstein-Barr virus, and cytomegalovirus were negative. Laboratory features showed anemia, coagulopathy, and increased serum levels of ammonium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin. Autoimmune Hepatitis Type-II (AH-II) was the initial diagnosis based on a liver kidney microsomal type 1 antibodies (LKM-1) positive result, and twenty days later a liver transplant was performed. The liver histopathology had indicated hemorrhagic necrosis in zone 3, and cholestasis and nodular regeneration, which were not characteristic of AH-II. LC/ESI-HRMS (liquid chromatography electrospray ionization high-resolution mass spectrometry) analysis of MCs in the explanted liver revealed the presence of Microsytin-LR (MC-LR) (2.4 ng·gr-1 tissue) and [D-Leu¹]MC-LR (75.4 ng·gr-1 tissue), which constitute a toxicological nexus and indicate a preponderant role of microcystins in the development of fulminant hepatitis.

Expresión del síndrome metabólico frente a factores ambientales / Expression of metabolic syndrome against environmental factors / Expressão da síndrome metabólica frente a fatores ambientais

El Síndrome Metabólico (SM) se define como la asociación de alteraciones metabólicas e inflamatorias a nivel molecular, celular o hemodinámico, que pueden presentarse en forma simultánea o secuencial en un mismo individuo. Esto imprime un mayor riesgo de desarrollar diabetes y enfermedades cardiovasculares, teniendo como base la resistencia insulínica. Su diagnóstico se presenta cuando existe obesidad abdominal y dos o más componentes adicionales: triglicéridos elevados, lipoproteína de alta densidad (HDL) baja, alteración en la regulación de la glucemia y presión arterial alta. En este contexto, y dada su relación con los factores ambientales, el objetivo del presente trabajo fue evaluar la relación del SM en poblaciones expuestas a diferentes niveles de contaminación atmosférica, determinando dicha asociación mediante las respuestas obtenidas de una encuesta socioeconómica, de antecedentes de salud, y contrastándolas con análisis sanguíneos. Finalmente, los resultados obtenidos evidencian intercurrencias entre el grado de contaminación atmosférica y el SM.

The metabolic syndrome (MS) is defined as the association of metabolic disorders and inflammatory diseases at molecular, cellular or hemodynamic levels, which may occur simultaneously or sequentially in the same individual. This adds an increased risk of developing diabetes and cardiovascular disease, based on insulin resistance. MS diagnosis is made when there are two or more additional components and abdominal obesity: elevated triglycerides, high density lipoprotein (HDL) low, altered regulation of blood glucose and high blood pressure. In this context, and given its relationship with environmental factors, the objective of this study was to evaluate the relationship of MS in populations exposed to different levels of air pollution,determining the association with the responses obtained from a socio-economic survey and health history, and contrasting them with a blood test. Finally, the results show intercurrences between the degree of air pollution and SM.

A síndrome metabólica (SM) é definida como a associação de alterações metabólicas e inflamatórias em nível molecular, celular ou hemodinâmico, que podem ocorrer em forma simultânea ou sequencial num mesmo indivíduo. Isto adiciona um maior risco de desenvolver diabetes e doenças cardiovasculares, tendo como base a resistência à insulina. Seu diagnóstico ocorre quando há obesidade abdominal e dois ou mais componentes adicionais: aumento dos triglicerídeos, lipoproteína de alta densidade (HDL) baixa, alteração na regulação da glicemia e pressão arterial elevada. Neste contexto, e devido a sua relação com os fatores ambientais, o objetivo desse estudo foi avaliar a relação da SM em populações expostas a diferentes níveis de poluição do ar, determinando tal associação através das respostas obtidasa em um levantamento socioeconômico, histórico de saúde e em contraste com análises de sangue. Por fim, os resultados mostram intercorrências entre o grau de poluição do ar e a SM.

Growth, toxin production, active oxygen species and catalase activity of Microcystis aeruginosa (Cyanophyceae) exposed to temperature stress.

Microcystis are known for their potential ability to synthesize toxins, mainly microcystins (MCs). In order to evaluate the effects of temperature on chlorophyll a (Chl a), growth, physiological responses and toxin production of a native Microcystis aeruginosa, we exposed the cells to low (23°C) and high (29°C) temperature in addition to a 26°C control treatment. Exponential growth rate was significantly higher at 29°C compared to 23°C and control, reaching 0.43, 0.32 and 0.33day(-)(1) respectively. In addition, there was a delay of the start of exponential growth at 23°C. However, the intracellular concentration of Chl a decreased significantly due to temperature change. A significant increase in intracellular ROS was observed in coincidence with the activation of enzymatic antioxidant catalase (CAT) during the first two days of exposure to 23° and 29°C in comparison to the control experiment, decreasing thereafter to nearly initial values. Five MCs were determined by LC-MS/MS analysis. In the experiments, the highest MC concentration, 205fg [Leu(1)] MC-LR.cell(-1) expressed as MC-LR equivalent was measured in the beginning of the experiment and subsequently declined to 160fg.cell(-1) on day 2 and 70fg.cell(-1) on day 4 in cells exposed to 29°C. The same trend was observed for all other MCs except for the least abundant MC-LR which showed a continuous increase during exposure time. Our results suggest a high ability of M. aeruginosa to perceive ROS and to rapidly initiate antioxidant defenses with a differential response on MC production.

Occurrence and toxicity of Microcystis aeruginosa (Cyanobacteria) in the Paraná River, downstream of the Yacyretá dam (Argentina) / Presencia y toxicidad de Microcystis aeruginosa (Cianobacteria) en el río Paraná, aguas abajo de la represa Yacyretá (Argentina)

AbstractCyanobacteria constitute the main toxin producers in inland water ecosystems and have extensive global distribution. The presence of hepatotoxins in aquatic environments is hazardous to human and animal health; even though the presence and identification of hepatotoxic microcystins in rivers and reservoirs of the world have been confirmed by several studies in the last few years. Herein, we studied the abundance and toxicity of Microcystis aeruginosa in the Argentine section of the Paraná River at the beginning of the Middle Paraná (Corrientes Hydrometer), near Corrientes city (27º28´ S - 58º51´ W) and approximately 220 km downstream of the Yacyretá dam (High Paraná). The Paraná River basin, with a drainage area of 3.1 x 106 km2 and 3 965 km in length, is the second largest catchment of South America, after that of the Amazon. The Paraná River is the main source of drinking water supply for the Northeastern Argentine region. Phytoplankton samples were collected and environmental variables were measured in a monthly basis (exceptionally fortnightly), from March 2004 to June 2008. Fifty-eight samples were analyzed for phytoplankton density and biomass. Five samples were used for toxicity testing; the latter were obtained during the cyanobacteria blooms from 2005 to 2008. Phytoplankton counts were performed with an inverted microscope, and biomass was expressed as biovolume. Bioassays with mice and high-performance liquid chromatography (HPLC) analysis were performed to evaluate the presence of cyanotoxins. Phytoplankton mainly consisted of Cryptophyta, Chlorophyta and Bacillariophyta. Microcystis aeruginosa was identified during the warmer months each year (November to March). Density varied between 189 and 25 027 cells/mL (1-10 colonies/mL) and biomass from 0.34 to 44 mm3/L. Taking into account the number of cells, the highest abundance occurred in April 2004 (25 027 cells/mL), coinciding with the largest biovolume (44 mm3/L). All mice subjected to intraperitoneal injections with samples obtained during bloom episodes showed positive results for the presence of hepatotoxins. Three microcystins variants: LR, RR and [D-Leu1] Mcyst-LR were detected by analysis with semi-preparative high-performance liquid chromatography with diode array detector system (HPLC-PDA). This constitutes the first report of microcystins recorded during M. aeruginosa blooms in the Argentine stretch of the Paraná River at the beginning of the Middle Paraná (Corrientes Hydrometer), approximately 220 km downstream of the Yacyretá dam (High Paraná).

ResumenLas Cyanobacterias constituyen el principal productor de toxinas en ecosistemas acuáticos y tienen una amplia distribución mundial. La presencia e identificación de microcistinas hepatotóxicas en ríos y embalses de todo el mundo fue confirmada por diferentes estudios durante los últimos años. La presencia de hepatotoxinas en cuerpos de agua son riesgosas para la salud humana y animal. Se estudió la abundancia y toxicidad de Microcystis aeruginosa (Kütz.) Kütz. en el río Paraná (Argentina), cerca de la ciudad de Corrientes (27°28' S - 58°51' W), aproximadamente a 220 km aguas abajo de la represa Yacyretá. La cuenca del río Paraná, con un área de drenaje de 3.1 x 106 km2 y 3 965 km de longitud, es la segunda mayor cuenca de Sudamérica, después del Amazonas. El río Paraná es la principal fuente de abastecimiento de agua potable para el Nordeste de la República Argentina. Los muestreos se realizaron mensualmente (excepcionalmente fueron quincenales) con medición de variables ambientales, entre Marzo 2004 y Junio 2008. Se tomaron un total de 58 muestras para analizar la densidad y biomasa del fitoplancton; mientras que cinco muestras fueron utilizadas en ensayos de toxicidad, estas últimas fueron obtenidas durante floraciones de cianobacterias entre 2005 y 2008. Los recuentos de fitoplancton fueron realizados con un microscopio invertido y la biomasa fue expresada como biovolumen. Para determinar la presencia de cianotoxinas se utilizaron bioensayos con ratones y análisis con Cromatografia líquida de alta resolución (HPLC). El fitoplancton estuvo representado principalmente por Cryptophyta, Chlorophyta y Bacillariophyta. Cyanobacteria fue dominante durante los meses cálidos de cada año (Noviembre a Marzo), con alta densidad de Microcystis aeruginosa. La densidad de M. aeruginosa varió entre 189 y 25 027 cells/mL (1-10 colonies/mL) y la biomasa entre 0.34 y 44 mm3/L. Teniendo en cuenta el número de células, la mayor abundancia ocurrió en abril 2004 (25 027 cells/ mL), coincidiendo con el gran biovolumen (44 mm3/L). Todos los ratones inyectados intraperitonealmente presentaron síntomas correspondientes a hepatotoxicidad. Tres variantes de microcystinas: LR, RR y [D-Leu1] Mcyst-LR, fueron detectadas por análisis de cromatografía líquida de alta resolución con detector de diodos (HPLC-PDA). Este es el primer trabajo de microcistinas registradas durante las floraciones de M. aeruginosa en el tramo argentino del río Paraná en los inicios del Paraná Medio (Hidrómetro Corrientes), aproximadamente a 220 km aguas abajo de la represa de Yacyretá (Alto Paraná).

Occurrence and toxicity of Microcystis aeruginosa (Cyanobacteria) in the Paraná River, downstream of the Yacyretá dam (Argentina).

Cyanobacteria constitute the main toxin producers in inland water ecosystems and have extensive global distribution. The presence of hepatotoxins in aquatic environments is hazardous to human and animal health; even though the presence and identification of hepatotoxic microcystins in rivers and reservoirs of the world have been confirmed by several studies in the last few years. Herein, we studied the abundance and toxicity of Microcystis aeruginosa in the Argentine section of the Paraná River at the beginning of the Middle Paraná (Corrientes Hydrometer), near Corrientes city (27º28´ S - 58º51´ W) and approximately 220 km downstream of the Yacyretá dam (High Paraná). The Paraná River basin, with a drainage area of 3.1 x 10(6) km(2) and 3 965 km in length, is the second largest catchment of South America, after that of the Amazon. The Paraná River is the main source of drinking water supply for the Northeastern Argentine region. Phytoplankton samples were collected and environmental variables were measured in a monthly basis (exceptionally fortnightly), from March 2004 to June 2008. Fifty-eight samples were analyzed for phytoplankton density and biomass. Five samples were used for toxicity testing; the latter were obtained during the cyanobacteria blooms from 2005 to 2008. Phytoplankton counts were performed with an inverted microscope, and biomass was expressed as biovolume. Bioassays with mice and high-performance liquid chromatography (HPLC) analysis were performed to evaluate the presence of cyanotoxins. Phytoplankton mainly consisted of Cryptophyta, Chlorophyta and Bacillariophyta. Microcystis aeruginosa was identified during the warmer months each year (November to March). Density varied between 189 and 25 027 cells/mL (1-10 colonies/mL) and biomass from 0.34 to 44 mm(3)/L. Taking into account the number of cells, the highest abundance occurred in April 2004 (25 027 cells/mL), coinciding with the largest biovolume (44 mm(3)/L). All mice subjected to intraperitoneal injections with samples obtained during bloom episodes showed positive results for the presence of hepatotoxins. Three microcystins variants: LR, RR and [D-Leu(1)] Mcyst-LR were detected by analysis with semi-preparative high-performance liquid chromatography with diode array detector system (HPLC-PDA). This constitutes the first report of microcystins recorded during M. aeruginosa blooms in the Argentine stretch of the Paraná River at the beginning of the Middle Paraná (Corrientes Hydrometer), approximately 220 km downstream of the Yacyretá dam (High Paraná).

Hepatic and intestine alterations in mice after prolonged exposure to low oral doses of Microcystin-LR.

Oral intake of Microcystin-LR (MC-LR) is the principal route of exposure to this toxin, with prolonged exposure leading to liver damage of unspecific symptomatology. The aim of the present paper was therefore to investigate the liver and intestine damage generated by prolonged oral exposure to low MC-LR doses (50 and 100 µg MC-LR/kg body weight, administrated every 48 h during a month) in a murine model. We found alterations in TBARS, SOD activity and glutathione content in liver and intestine of mice exposed to both doses of MC-LR. Furthermore, the presence of MC-LR was detected in both organs. We also found hepatic steatosis (3.6 ± 0.6% and 15.3 ± 1.6%) and a decrease in intraepithelial lymphocytes (28.7 ± 5.0% and 44.2 ± 8.7%) in intestine of 50- and 100-µg MC-LR/kg treated animals, respectively. This result could have important implications for mucosal immunity, since intraepithelial lymphocytes are the principal effectors of this system. Our results indicate that prolonged oral exposure at 50 µg MC-LR/kg every 48 h generates significant damage not only in liver but also in intestine. This finding calls for a re-appraisal of the currently accepted NOAEL (No Observed Adverse Effect Level), 40 µg MC-LR/kg body weight, used to derive the guideline value for MC-LR in drinking water.

Seven new microcystin variants discovered from a native Microcystis aeruginosa strain--unambiguous assignment of product ions by tandem mass spectrometry.

RATIONALE: High-resolution mass spectrometry was applied to the study of a Microcystis aeruginosa strain previously reported as a [D-Leu(1)]MC-LR producer. Detailed analysis revealed new microcystin (MC) variants produced from the strain, and seven of these were previously unreported variants. This work shows the importance of mass accuracy for the identification of unknown MCs. METHODS: The M. aeruginosa strain was isolated from a bloom sample collected from Argentina and acclimated to lab conditions. The MC variants in the strain were separated by UV/Vis detection-guided high-performance liquid chromatography, and their structures were unambiguous determined by tandem mass spectrometry (MS/MS). RESULTS: A simple strategy was developed for quickly locating the low-abundance MC precursors from complex samples. MS/MS anlysis revealed ten MC variants produced from the strain, of which seven have never been reported before. CONCLUSIONS: This work shows the interference of isobarics and isomers in the study of unknown MCs, and, therefore, high mass accuracy is important to avoid false assignments. Moreover, the peak list provided here (30-50 fragments unambiguously assigned for ten MCs) can be used as a reference for the discovery of MCs from environmental samples.

Application of hydrogen peroxide to the control of eutrophic lake systems in laboratory assays.

We exposed water samples from a recreational lake dominated by the cyanobacterium Planktothrix agardhii to different concentrations of hydrogen peroxide (H2O2). An addition of 0.33 mg·L-1 of H2O2 was the lowest effective dose for the decay of chlorophyll-a concentration to half of the original in 14 h with light and 17 h in experiments without light. With 3.33 mg·L-1 of H2O2, the values of the chemical oxygen demand (COD) decreased to half at 36 and 126 h in experiments performed with and without light, respectively. With increasing H2O2, there is a decrease in the total and faecal coliform, and this effect was made more pronounced by light. Total and faecal coliform were inhibited completely 48 h after addition of 3.33 mg·L-1 H2O2. Although the densities of cyanobacterial cells exposed to H2O2 did not decrease, transmission electron microscope observation of the trichomes showed several stages of degeneration, and the cells were collapsed after 48 h of 3.33 mg·L-1 of H2O2 addition in the presence of light. Our results demonstrate that H2O2 could be potentially used in hypertrophic systems because it not only collapses cyanobacterial cells and coliform bacteria but may also reduce chlorophyll-a content and chemical oxygen demand.