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Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial pulmonary disease characterized with radiographically evident pulmonary infiltrates and extracellular matrix deposition with limited treatment options. We previously described that microcystin-LR (MC-LR) reduces transforming growth factor (TGF)-β1/Smad signaling and ameliorates pulmonary fibrosis in bleomycin (BLM)-induced rat models. In the present study, we further demonstrate that microcystin-RR (MC-RR), an MC congener with lower toxicity than MC-LR, exerted an anti-fibrotic effect on BLM-induced pulmonary fibrosis rodent models and compared it with MC-LR. Our data show that MC-RR treatment attenuated BLM-associated pulmonary inflammation and collagen deposition in both therapeutic and preventive models. MC-RR reduced the expression of fibrotic markers, including vimentin, α-smooth muscle actin, collagen 1α1, and fibronectin, in rat pulmonary tissues. Furthermore, the core features of BLM-induced pulmonary fibrotic lesions were better alleviated by MC-RR than by MC-LR. MC-RR treatment substantially decreased the number of pulmonary M2 macrophages. In vitro, MC-RR attenuated the epithelial-mesenchymal transition and fibroblast-myofibroblast transition triggered by M2 macrophages. Therefore, we highlight MC-RR as a promising molecule for developing therapeutic and prophylactic strategies against IPF, a refractory lung disease.

The effects of microcystin-RR (MC-RR) on water metabolism were studied on Sprague–Dawley (SD) rats and KunMing (KM) mice. In the single dose toxicity test, polydipsia, polyuria, hematuria and proteinuria were found in group of rats receiving a MC-RR dose of 574.7 μg/kg, and could be relieved by dexamethasone (DXM). Gradient damage was observed in kidney and liver in rats with gradient MC-RR doses of 574.7, 287.3, and 143.7 μg/kg. No significant water metabolic changes or kidney injuries were observed in mice treated with MC-RR doses of 210.0, 105.0, and 52.5 μg/kg. In the continuous exposure test, in which mice were administrated with 140.0, 70.0, and 35.0 μg/kg MC-RR for 28 days, mice in the 140.0 μg/kg group presented increasing polydipsia, polyuria, and liver damage. However, no anatomic or histological changes, including related serological and urinary indices, were found in the kidney. In summary, abnormal water metabolism can be induced by MC-RR in rats through kidney injury in single dose exposure, the kidney of SD rats is more sensitive to MC-RR than that of KM mouse, and polydipsia and polyuria in mice exposed to MC-RR for 28 days occurred but could not be attributed to kidney damage.

Microcystins (MCs) are toxins produced by several cyanobacterial species found worldwide. While MCs have a common structure, the variation of two amino acids in their structure affects their toxicity. As toxicodynamics are very similar between the MC variants, their differential toxicity could rather be explained by toxicokinetic parameters. Microcystin-RR (MC-RR) is the second most abundant congener and induces toxicity through oral exposure. As intestinal permeability is a key parameter of oral toxicokinetics, the apparent permeability of MC-RR across a differentiated intestinal Caco-2 cell monolayer was investigated. We observed a rapid and large decrease of MC-RR levels in the donor compartment. However, irrespective of the loaded concentration and exposure time, the permeabilities were very low from apical to basolateral compartments (from 4 to 15 × 10−8 cm·s−1) and from basolateral to apical compartments (from 2 to 37 × 10−8 cm·s−1). Our results suggested that MC-RR would be poorly absorbed orally. As similar low permeability was reported for the most abundant congener microcystin-LR, and this variant presented a greater acute oral toxicity than MC-RR, we concluded that the intestinal permeability was probably not involved in the differential toxicity between them, in contrast to the hepatic uptake and metabolism.

The canonical mode of action (MOA) of microcystins (MC) is the inhibition of protein phosphatases, but complete characterization of toxicity pathways is lacking. The existence of over 200 MC congeners complicates risk estimates worldwide. This work employed RNA-seq to provide an unbiased and comprehensive characterization of cellular targets and impacted cellular processes of hepatocytes exposed to either MC-LR or MC-RR congeners. The human hepatocyte cell line, HepaRG, was treated with three concentrations of MC-LR or -RR for 2 h. Significant reduction in cell survival was observed in LR1000 and LR100 treatments whereas no acute toxicity was observed in any MR-RR treatment. RNA-seq was performed on all treatments of MC-LR and -RR. Differentially expressed genes and pathways associated with oxidative and endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) were highly enriched by both congeners as were inflammatory pathways. Genes associated with both apoptotic and inflammatory pathways were enriched in LR1000. We present a model of MC toxicity that immediately causes oxidative stress and leads to ER stress and the activation of the UPR. Differential activation of the three arms of the UPR and the kinetics of JNK activation ultimately determine whether cell survival or apoptosis is favored. Extracellular exosomes were enrichment of by both congeners, suggesting a previously unidentified mechanism for MC-dependent extracellular signaling. The complement system was enriched only in MC-RR treatments, suggesting congener-specific differences in cellular effects. This study provided an unbiased snapshot of the early systemic hepatocyte response to MC-LR and MC-RR congeners and may explain differences in toxicity among MC congeners., Highlights • Microcystin-LR and microcystin-RR have similar transcriptional responses. • Genes associated with oxidative stress and the unfolded protein response were enriched by congeners. • Genes associated with extracellular exosomes were enriched, suggesting a potential new mechanism for cell signaling. • Complement associated genes were strongly enriched only by microcystin-RR. • Identified a potential molecular mechanism underlying the cellular fate of hepatocyte.

The spatio-temporal distribution of environmental factors, chlorophyll-a (Chl-a), and microcystins (MCs) in a shallow lake, Lake Taihu (China), were investigated from 2009 to 2011 on a monthly basis at nine sampling stations. The annual mean concentration ranges of total nitrogen (TN), total phosphorus (TP), Chl-a, MC-LR and MC-RR were 0.17–10.53 mg/L, 0.027–0.581 mg/L, 0.10–129.75 µg/L, 0.013–2.019 µg/L and 0.002–0.794 µg/L, respectively. The average TN, ammonium (NH4+) and TP concentrations in Meiliang Bay decreased from 3.54 to 2.26 mg/L, 0.63 to 0.31 mg/L and 0.150 to 0.124 mg/L, respectively, when compared with values from 2006–2008, indicating that water quality has improved in severe cyanobacterial bloom areas in recent years. Additionally, the distribution of MCs was northern lake areas > western lake areas > central lake areas > macrophyte-dominated areas. Correlation analysis revealed that nutrients were the most important variable accounting for the variation of extracellular MC-LR concentration in heavy cyanobacterial bloom areas of Lake Taihu. During the study period, the maximum MCs concentration reached 2.75 ± 0.27 μg/L in the bloom period in the northern lake areas, which is more than two times the safety limit of 1 μg/L MCs required for drinking water. However, microcystins decreased gradually as the water quality improved from 2009 to 2011, indicating that the risk of MCs exposure was slightly decreased in Lake Taihu.

The Hartbeespoort (HBP) Dam is a reservoir used for agricultural, domestic supply of raw potable water and recreational activities in South Africa’s North-West Province. Eutrophication and cyanobacterial blooms have long been a cause of water-quality problems in this reservoir. The most prevalent bloom-forming species is Microcystis aeruginosa, often producing the toxin microcystin, a hepatotoxin which can negatively impact aquatic animal and human health, and poses a problem for potable water supply. Algal samples were collected monthly from four pre-determined sites in the dam during the summer months (December 2010–March 2011). Intracellular microcystins (MCs) were extracted using SPE C18 cartridges, followed by separation, identification and quantification using LC-ESI-MS techniques. Quantitative variation studies of MCs were conducted with respect to MC congener isolated, sampling site and month. Three main MC congeners (MC-RR, -LR and-YR) were isolated, identified and quantified. In addition, three minor MCs (MC-WR, MC-(H4)YR and (D-Asp3, Dha7)MC-RR were also identified, but were not quantified. The MC dominance followed the order MC-RR>MC-LR>MC-YR across all sites and time. The maximum and minimum concentrations were 268 µg/g and 0.14 µg/g DW for MC-RR and MC-YR, respectively, of the total MCs quantified from this study. One-way ANOVA showed that there were no significant differences between average MC concentrations recorded across months (P = 0.62), there was, however, a marginally-significant difference in concentrations among MC congeners (P = 0.06). ANCOVA revealed a highly significant interaction between sites and MC congeners on MC concentration (P < 0.001).

Publicaciones del III Congreso Ibérico de Cianotoxinas (Blanes, 2013) Microcystins (MCs) are hepatotoxins that are abundantly produced by cyanobacteria. Studies have shown that these toxins affect many multicellular organisms that inhabit aquatic ecosystems; however, their impact on bacteria that co-occur with freshwater cyanobacteria is still unclear. In this study, the impact of the three most common variants of MCs (MCLR, MCRR, and MCYR) on the growth of heterotrophic bacteria isolated from four Portuguese reservoirs was evaluated. Some isolates were derived from freshwater sources where blooms of cyanobacteria that often contain microcystin-producing strains are frequently observed and from a reservoir where these phenomena do not occur. Morphological and molecular characterisation of the bacterial isolates was performed, and these bacteria were exposed to three different concentrations of each MC variant. The effect of MCs on bacterial growth was then evaluated. This study showed that MCLR, MCRR and MCYR can reduce the growth of some heterotrophic bacteria isolated from freshwater sources. To our knowledge, this is the first study in which the impact of several variants of MCs was evaluated in a diverse group of freshwater heterotrophic bacteria. E. Valério is the recipient of grant SFRH/BPD/75922/2011 from Fundação para a Ciência e a Tecnologia.

A bacterial strain EMS with the capability of degrading microcystins (MCs) was isolated from Lake Taihu, China. The bacterium was tentatively identified as a Stenotrophomonas sp. The bacterium could completely consume MC-LR and MC-RR within 24 hours at a concentration of 0.7 microg/mL and 1.7 microg/mL, respectively. The degradation of MC-LR and MC-RR by EMS occurred preferentially in an alkaline environment. In addition, mlrA gene involved in the degradation of MC-LR and MC-RR was detected in EMS. Due to the limited literature this gene has rare homologues. Sequencing analysis of the translated protein from mlrA suggested that MlrA might be a transmembrane protein, which suggests a possible new protease family having unique function.