Nitrogen forms and concentration influence the impact of titanium dioxide nanoparticles on the biomass and antioxidant enzyme activities of Microcystis aeruginosa.

Nanoparticles (NPs) are becoming more widely produced, used, and released into the aquatic environment. In aquatic ecosystems, these NPs affect different populations of photosynthesizing organisms, such as cyanobacteria. This study aimed to evaluate the effects of titanium dioxide (TiO2) NPs (48 mg l-1) combined with low (0.04 mM) and high (9 mM) concentrations of urea and nitrate on Microcystis aeruginosa. Microcystins (MCs) production and release were monitored in the cyanobacterium. The results showed that high urea concentration (9 mM) combined with TiO2 NPs inhibited growth, pigment, and malondialdehyde (MDA) content by 82%, 63%, and 47%, respectively. The treatment also increased the reactive oxygen species (ROS) and glutathione S-transferase (GST) activity by 40.7% and 67.7%, respectively. Similarly, low nitrate (0.04 mM) combined with TiO2 NPs inhibited growth by 40.3% and GST activity by 36.3% but stimulated pigment production and ROS concentration in M. aeruginosa. These responses suggest that high urea combined with TiO2.NPs and high nitrate combined with TiO2 NPs induced oxidative stress in cyanobacteria. The peroxidase (POD) activity of M. aeruginosa decreased by 17.7% with increasing urea concentrations. Our findings suggest that TiO2 NPs combined with changing nutrient (urea and nitrate) concentrations may adversely affect cyanobacterial development and antioxidant defense enzymes.

Microcystin leucine arginine induces human sperm damage: Involvement of the Ca2+/CaMKKß/AMPK pathway.

As a common pollutant in the water environment, microcystin leucine arginine (MC-LR) can enter semen and damage the sperm in animals. However, the mechanism by which MC-LR damages human sperm is unclear. Therefore, human sperm samples were obtained from the Henan Provincial Sperm Bank and exposed to different concentrations (0, 1, 10, and 100 µg/L) of MC-LR for 1, 2, 4, and 6 h, to invegest the effects and potential mechanism of MC-LR on sperm. The results showed that MC-LR mainly accumulated in the neck and flagellum of human sperm. Compared to the control group, the sperm capacitation rate and motility were significantly decreased in the 100 µg/L group. After exposure of 100 µg/L of MC-LR, the central microtubule and microtubule doublet of sperm flagellum were blurred, asymmetrical, or even lost. Furthermore, the expression levels of flagellin DNAH17, SPEF2, SPAG16, SPAG6, and CFAP44 in human sperm were reduced. Also, the phosphorylation levels of CaMKKß and AMPK can be inhibited by MC-LR. These findings revealed that MC-LR can induce functional and structural damage in human sperm, and the Ca2+/CaMKKß/AMPK pathway may be involved in this process. This study will provide a basis for prevention and treatment of male fertility declines caused by MC-LR.

Compounds to mitigate cyanobacterial blooms affect growth and toxicity of Microcystis aeruginosa.

Numerous products and techniques are used to combat harmful cyanobacterial blooms in lakes. In this study, we tested nine products, the phosphate binders Phoslock® and Aqual-PTM, the coagulant chitosan, the phosphorus binder and coagulant aluminum salts (aluminum sulphate and sodium aluminate), the copper-based algicides SeClear, Captain® XTR and CuSO4·5H2O, the antibiotic Streptomycin and the oxidant hydrogen peroxide (H2O2) on their efficiency to manage the cyanobacterium Microcystis aeruginosa (M. aeruginosa). To this end, 7 days of laboratory experiments were conducted and effects were determined on chlorophyll-a, photosystem II efficiency (PSII), soluble reactive phosphorus (SRP) and intracellular and extracellular microcystin (MC) concentrations. The algicides, chitosan and H2O2 were the most powerful in reducing cyanobacteria biomass. Biomass reductions compared to the controls yielded: Chitosan (99.8%) > Hydrogen peroxide (99.6%) > Captain XTR (98.2%) > SeClear (98.1%) > CuSO4·5H2O (97.8%) > Streptomycin (86.6%) > Phoslock® (42.6%) > Aqual-PTM (28.4%) > alum (5.5%). Compounds that caused the largest reductions in biomass also strongly lowered photosystem II efficiency, while the other compounds (Phoslock®, Aqual-PTM, aluminum salts) had no effect on PSII, but strongly reduced SRP. Intracellular MC concentration followed the biomass patterns, extracellular MC was generally lower at higher doses of algicides, chitosan and H2O2 after one week. Recovery of PSII was observed in most algicides and chitosan, but not at the highest doses of SeClear and in all streptomycin treatments. Our results revealed that M. aeruginosa can be killed rapidly using several compounds, that in some treatments already signs of recovery occurred within one week. P fixatives are efficient in reducing SRP, and thus acting via resource suppression, which potentially may provide an addition to fast-acting algicides that kill most of the cells, but allow rapid regrowth as sufficient nutrients remain.

A new identity of microcystins: Environmental endocrine disruptors? An evidence-based review.

Microcystins (MCs) are widely distributed cyanobacterial toxins in eutrophic waters. At present, the endocrine-disrupting effects of MCs have been extensively studied, but whether MCs can be classified as environmental endocrine disruptors (EDCs) is still unclear. This review is aimed to evaluate the rationality for MCs as to be classified as EDCs based on the available evidence. It has been identified that MCs meet eight of ten key characteristics of chemicals that can be classified as EDCs. MCs interfere with the six processes, including synthesis, release, circulation, metabolism, binding and action of natural hormones in the body. Also, they are fit two other characteristics of EDC: altering the fate of producing/responding cells and epigenetic modification. Further evidence indicates that the endocrine-disrupting effect of MCs may be an important cause of adverse health outcomes such as metabolic disorders, reproductive disorders and effects on the growth and development of offspring. Generally, MCs have endocrine-disrupting properties, suggesting that it is reasonable for them to be considered EDCs. This is of great importance in understanding and evaluating the harm done by MCs on humans.

Adverse role of colonial morphology and favorable function of microcystins for Microcystis to compete with Scenedesmus.

In eutrophic freshwaters, Microcystis usually becomes dominant in phytoplankton communities due to the synergistic effects of its special eco-physiological traits and environmental factors. Colonial morphology can protect Microcystis from zooplankton grazing, which indirectly favors Microcystis to outcompete other phytoplankton, although the colonial form is not conducive to the absorption of nutrients. Moreover, unicellular Microcystis usually has competitive advantages over other phytoplankton due to its efficient absorption capacity for nutrients and releasing microcystins. However, the consequence of direct competition between toxic colonial Microcystis and green algae without external grazing pressure still remained unknown. In this study, the competition between toxic colonial Microcystis aeruginosa and a common green alga Scenedesmus obliquus was explored. Results showed that: (1) colonial M. aeruginosa had a higher requirement for key macro-nutrient phosphorus than S. obliquus, and thus its population declined and was replaced by S. obliquus eventually; (2) microcystins released by colonial M. aeruginosa inhibited the photosynthetic activity and growth of S. obliquus at early stage of the competition; (3) the photosynthetic potential of colonial M. aeruginosa was stimulated in response to the competitive stress from S. obliquus, although the population of colonial M. aeruginosa declined eventually; (4) microcystin production of colonial M. aeruginosa was enhanced by phosphorus limitation due to S. obliquus competition and was positively related to photosynthetic potential of colonial M. aeruginosa. These results indicated that, in the absence of complex natural environment, colonial Microcystis cannot outcompete Scenedesmus in a pure competition, although microcystins can play a favorable role in the competition, which clarified the opposite role of colonies and microcystins in the competition of colonial Microcystis against other phytoplankton.

Higher content of microcystin-leucine-arginine promotes the survival of intrahepatic cholangiocarcinoma cells via regulating SET resulting in the poorer prognosis of patients.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) has over the last 10 years become the focus of increasing concern largely due to its rising incidence and high mortality rates worldwide. Microcystin-leucine-arginine (MC-LR) has been reported to be carcinogenic, but there are no data on the linkage between MC-LR and ICC. This study aimed to explore whether the content levels of MC-LR in the tumour tissues of ICC patients be associated with the prognosis and if so, to characterize the mechanism in ICC cells. METHODS: We conducted a retrospective study to evaluate the prognostic value of MC-LR in ICC after resection. All patients were divided into two groups according to the content of MC-LR in tumour via immunohistochemistry: low-MC-LR group (n = 28) and high-MC-LR group (n = 30). RESULTS: Multivariate analysis showed high-MC-LR level was the prognostic factor for OS and RFS after hepatectomy (P = .011 and .044). We demonstrated that MC-LR could promote the survival of human ICC cell lines and SET was identified as an important mRNA in the progression via RNA array. CONCLUSIONS: We provide evidence that MC-LR was an independent prognostic factor for ICC in humans by modulating the expression of SET in human ICC cells.

Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB-. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3ß-hydroxysteroid dehydrogenase (3ß-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3ß-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- had an opposite effect on 3ß-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11ß1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.

Microcystin-LR ameliorates pulmonary fibrosis via modulating CD206+ M2-like macrophage polarization.

Idiopathic pulmonary fibrosis (IPF) is a group of chronic interstitial pulmonary diseases characterized by myofibroblast proliferation and extracellular matrix deposition with limited treatment options. Based on our previous observation, we hypothesized microcystin-leucine arginine (LR), an environmental cyanobacterial toxin, could potentially suppress pulmonary fibrosis. In this study, we first demonstrated that chronic exposure of microcystin-LR by oral for weeks indeed attenuated the pulmonary fibrosis both on bleomycin-induced rat and fluorescein isothiocyanate-induced mouse models. Our data further indicated that treatment with microcystin-LR substantially reduced TGF-ß1/Smad signaling in rat pulmonary tissues. The experiments in vitro found that microcystin-LR was capable of blocking epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT) through suppressing the differentiation of CD206+ macrophages. Mechanically, microcystin-LR was found to bind to glucose-regulated protein 78 kDa (GRP78) and suppress endoplasmic reticulum unfolded protein response (UPRER) signaling pathways. These events led to the modulation of M2 polarization of macrophages, which eventually contributed to the alleviation of pulmonary fibrosis. Our results revealed a novel mechanism that may account for therapeutic effect of microcystin-LR on IPF.

MC-LR induced overproduction of progesterone via inhibiting miR-3473g: in vitro and in vivo evidence.

Health risk of human exposure to microcystin-leucine arginine (MC-LR) has drawn more and more attention in recent years. In the present study, MC-LR inhibited miR-3473g expression of mouse granulosa cells both in vitro and in vivo. By dual-luciferase reporter assay, we confirmed miR-3473g is able to bind the 3'-UTR of StAR protein (StAR) mRNA and suppress StAR expression. Thus, downregulation of miR-3473g after MC-LR exposure led to StAR overexpression. Excessive StAR probably transported much more cholesterol into the inner membrane of the mitochondria and finally resulted in overproduction of progesterone. Our results revealed that MC-LR exposure was associated with premature luteinization of granulosa cells and may adversely affect women's fertility.

Effects of Chronic Exposure to Microcystin-LR on Kidney in Mice.

Microcystin-LR (MC-LR) is a potent hepatotoxin, but a few studies suggested that it might also induce nephrotoxicity. However, nephrotoxicity induced by prolonged oral exposure to MC-LR is unknown. The aim of this study was to evaluate the potential influence of MC-LR on the kidney in mice following chronic exposure to MC-LR. In this study, we evaluated the nephrotoxicity of MC-LR in mice drinking water at different concentrations (1, 30, 60, 90, and 120 µg/L) for 6 months for the first time. The results showed that the kidney weights and the kidney indexes of mice were not altered in the MC-LR treated mice, compared with the control group. In addition, the renal function indicators revealed that the serum creatinine (SCr) levels were not significant changes after exposure to MC-LR. The blood urea nitrogen (BUN) levels were markedly decreased after exposure to 90 and 120µg/L MC-LR for 3 months. The BUN levels were lower than that of the control group after exposure to 120µg/L MC-LR for 6 months. The histopathological investigation revealed enlarged renal corpuscles, widened of kidney tubules, and lymphocyte infiltration in the interstitial tissue and the renal pelvis after exposure to 60, 90, and 120 µg/L MC-LR. Consequently, our results suggested that long-term exposure to MC-LR might be one important risk of kidney injury, which will provide important clues for the prevention of renal impairment.

The effects of temperature on Bosmina longirostris susceptibility to microcystin-LR acute toxicity.

Harmful algal blooms are an ongoing threat to many aquatic systems throughout the world. In the Chowan River, North Carolina, the frequency of toxin producing Microcystis aeruginosa blooms has increased since 1975 along with an average 0.71°C rise in water temperature. The combined effect of microcystin-LR toxin and rising temperatures on a dominant zooplankter in the system, Bosmina longirostris, was the focus of this study. Laboratory studies were conducted to determine how microcystin-LR, produced from M. aeruginosa blooms, affected B. longirostris mortality under different temperature regimes. At 25°C, the LC50 for B. longirostris was 26.3 µg L-1 suggesting that B. longirostris can survive typical current bloom microcystin-LR concentrations ranging 0.1µg L-1 to 2.0 µg L-1, but would be susceptible to higher concentrations they may be periodically exposed to. Mortality was assessed at a constant microcystin-LR concentration of 26.3 µg L-1 over 15-35°C, and it was found that B. longirostris mortality increased at higher temperatures. B. longirostris mortality increased approximately 18% due to microcystin-LR alone over 2°C between 25°C and 27°C when exposed to the LC50 concentration. The increased prevalence of toxic M. aeruginosa blooms and increasing temperatures due to climate change may reduce B. longirostris populations, potentially affecting larval fish and fisheries in the Chowan River, North Carolina.

Environmental microcystin targets the microbiome and increases the risk of intestinal inflammatory pathology via NOX2 in underlying murine model of Nonalcoholic Fatty Liver Disease.

With increased climate change pressures likely to influence harmful algal blooms, exposure to microcystin, a known hepatotoxin and a byproduct of cyanobacterial blooms can be a risk factor for NAFLD associated comorbidities. Using both in vivo and in vitro experiments we show that microcystin exposure in NAFLD mice cause rapid alteration of gut microbiome, rise in bacterial genus known for mediating gut inflammation and lactate production. Changes in the microbiome were strongly associated with inflammatory pathology in the intestine, gut leaching, tight junction protein alterations and increased oxidative tyrosyl radicals. Increased lactate producing bacteria from the altered microbiome was associated with increased NOX-2, an NADPH oxidase isoform. Activationof NOX2 caused inflammasome activation as shown by NLRP3/ASCII and NLRP3/Casp-1 colocalizations in these cells while use of mice lacking a crucial NOX2 component attenuated inflammatory pathology and redox changes. Mechanistically, NOX2 mediated peroxynitrite species were primary to inflammasome activation and release of inflammatory mediators. Thus, in conclusion, microcystin exposure in NAFLD could significantly alter intestinal pathology especially by the effects on microbiome and resultant redox status thus advancing our understanding of the co-existence of NAFLD-linked inflammatory bowel disease phenotypes in the clinic.

Crude microcystins extracted from Microcystis aeruginosa exert anti-obesity effects by downregulating angiogenesis and adipogenesis related signaling molecules in HUVEC and 3 T3-L1 cells.

BACKGROUND: Obesity is a risk factor for many diseases including diabetes, cancer, arthritis, and cardiovascular diseases. Angiogenesis nourishes adipose tissues and contributes to obesity; it can be prevented by suppressing the expression of associated signaling molecules. Natural products have garnered attention owing to their safety and efficacy in treating several diseases, including obesity. METHODS: Crude Microcystins were extracted from the blooming Microcystis aeruginosa under stress conditions, by ultrasonication following by solvent extraction. The microcystin extract was evaluated for its potential of inhibiting angiogenesis and adipogenesis. The antiangiogenic activity of the microcystins extract was investigated using human umbilical vein endothelial cells (HUVECs), and its anti-obesity activity was determined in vitro by quantification of the accumulated lipids in mouse 3 T3-L1 cells via Oil Red O staining method. RESULTS: The microcystin extract suppressed HUVECs proliferation and tubes formation in Matrigel in a dose-dependent manner. RT-PCR analysis revealed the downregulation of the mRNA expression of angiogenesis-related signaling molecules, such as PI3K, ß-catenin, vascular endothelial growth factor receptor-2 (VEGFR-2), vascular endothelial-cadherin, Akt1, and NF-κB. Additionally, it inhibited the differentiation of premature 3 T3 cells and lipid accumulation in a dose-dependent manner. It suppressed adipogenesis and lipogenesis by reducing the expression level of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and sterol regulatory element-binding protein. CONCLUSIONS: Crude microcystin exerts anti-angiogenic and anti-obesity effects due to the inhibitory effects on the genes expression of associated signaling molecules and transcriptional factors.

Direct and indirect effects of a common cyanobacterial toxin on amphibian-trematode dynamics.

Wildlife diseases are emerging at unprecedented rates. While there are likely several factors at play, human-mediated environmental alterations may play a significant role. Of growing interest is the effect that microcystin-LR (MC-LR), a cyanotoxin, may have on disease outcomes. In this study, using an amphibian-trematode model we examined (1) the lethal effects of MC-LR on cercariae of trematodes; (2) the sublethal effects of MC-LR exposure on the ability for trematodes to infect green frog tadpoles; and (3) the sublethal effects of MC-LR on green frog tadpole susceptibility to trematodes. We found that environmentally-relevant concentrations of MC-LR at 50, 100, and 500 µg L-1 increased cercariae rate of mortality (LC50-14h = 134.24 µg L-1). However, sublethal exposure of trematodes to 2 and 10 µg L-1 MC-LR did not alter their infectivity. Conversely, sublethal exposure of tadpoles to 2 µg L-1 increased their susceptibility to trematodes by 147%. However, 10 µg L-1 of MC-LR did not affect tadpole susceptibility to trematodes, indicating a non-linear response to sublethal MC-LR exposure. Overall, our findings suggest that high concentrations of MC-LR (≥50 µg L-1) have the potential to limit trematode transmission to amphibian hosts through MC-LR-induced mortality. However, at lower concentrations (<10 µg L-1) MC-LR's effect on tadpole-cercariae disease outcome is likely driven by its effect on the tadpole host. Collectively, this work highlights the need to consider how toxicants influence both host and parasite at multiple concentrations to better understand the impacts of cyanotoxins on disease dynamics.

Effects of cylindrospermopsin on cultured immortalized human airway epithelial cells.

Anthropogenic eutrophication of freshwater bodies increases the occurrence of toxic cyanobacterial blooms. The cyanobacterial toxin cylindrospermopsin (CYN) is detected in the environment with increasing frequency, driving the scientific effort to assess emerging health risks from CYN-producing blooms. Oral exposure to CYN results primarily in hepatotoxicity. Nevertheless, extrahepatic manifestations of CYN toxicity have been reported. Furthermore, cyanotoxins have been detected in aerosols and dust particles, suggesting potential toxic effects in the respiratory tract. To assess the susceptibility of airway epithelia towards cyanotoxins, monolayers of immortalized human bronchial epithelial cells HBE1 and 16HBE14o- were exposed to a concentration range of 0.1-10 µM CYN. Cytotoxic endpoints were assessed as morphologic alterations, resazurin reduction capacity, esterase activity, neutral red uptake, and by impedimetric real-time cell analysis. Depending on the endpoint assessed, EC50 values ranged between 0.7 and 1.8 µM (HBE1) and 1.6-4.8 µM (16HBE14o-). To evaluate alterations of other cellular events by subcytotoxic concentration of CYN (1 µM), phosphorylation of mitogen-activated protein kinases ERK and p38 was determined. Only a slight increase in p38 phosphorylation was induced by CYN in HBE1 cell line after 48 h, while activities of both ERK1/2 and p38 gradually and significantly increased in 16HBE14o- cells during 8-48 h exposure. This study suggests possible hazards of inhalation CYN exposures, which may severely impact the integrity of airway epithelia and epithelial cell signaling. Further research of CYN-induced toxicity and underlying mechanisms is needed, as well as more data on environmental concentrations of cyanotoxins in aerosols for exposure assessment.

Microcystin-LR promotes migration via the cooperation between microRNA-221/PTEN and STAT3 signal pathway in colon cancer cell line DLD-1.

Previous researches have reported that microcystin-LR (MC-LR) contributes to the progression of multiple types of carcinomas including colon cancer; however, the underlying molecular mechanisms remain unclear and require in-depth investigation. Here, the colon cell line DLD-1 was arranged for the analysis by the microRNA microarray which was associated with the cancer metastasis after MC-LR exposure. 31 human microRNAs were differentially expressed, including miR-221, which targeted 3'-UTR of PTEN mRNA and PTEN level was down-regulated by MC-LR treatment. Besides, MC-LR also induced the phosphorylation of STAT3, which can be reversed by adding miR-221 inhibitor and PTEN expression plasmid. Furthermore, miR-221 inhibitor, STAT3 siRNA and PTEN expression plasmid could reverse the effects of MC-LR induced migration with the accumulation of ß-catenin in nuclei. In conclusion, our study suggested that MC-LR promoted the progression of colon carcinoma, at least in part, by regulating the expression miR-221, PTEN and STAT3 phosphorylation, which offers a novel perspective to understand the connection between MC-LR and colon cancer.

Assessment of the cytotoxicity, antioxidant activity and chemical composition of extracts from the cyanobacterium Fischerella major Gomont.

Cyanoprokaryotes (Cyanobacteria/Cyanophyta) are ancient photosynthetic prokaryotic organisms with cosmopolitan distribution. They are producers of a number of biologically active substances with antitumor and antifungal activity, vitamins, antibiotics, algaecides, insecticides, repellents, hormones, immunosuppressants and toxins. So far, the cyanobacterium Fischerella major Gomont has not been studied regarding its impact on the environment and human health. In this study, the cytotoxic, antioxidant and antitumor activities of four extracts prepared from Fischerella major were evaluated in vitro. In addition, the total phenolic content and the potential for production of cyanotoxins were also analyzed. The conducted GC/MS analysis identified 45 compounds with different chemical nature and biological activity. Presence of microcystins and saxitoxins was detected in all Fischerella major extracts. In vitro testing on cell cultures showed a significant concentration- and time-dependent cytotoxic effect on all cell lines (HeLa, SK-Hep-1 and FL) treated at three exposure times (24, 48 and 72 h) with four extracts. A selective antitumor effect was not observed. This is the first study demonstrating biological activity of extracts from Fischerella major, which makes it an interesting subject for further research, including environmental risk assessments (as producer of cyanotoxins) or as a potential source of pharmaceuticals.

Microcystin-LR regulates circadian clock and antioxidant gene expression in cultured rat cardiomyocytes.

BACKGROUND: Microcystins are waterborne environmental toxins that induce oxidative stress and cause injuries in the heart. On the other hand, many physiological processes, including antioxidant defense, are under precise control by the mammalian circadian clock. RESULTS: In the present study, we evaluated the effect of microcystin-LR (MC-LR) on the rhythmic expression patterns of circadian and antioxidant genes in rat cardiomyocytes using the serum shock technique. We found that a non-toxic dose (10 µm) of MC-LR decreased the amplitudes of rhythmic patterns of clock genes, while it increased the expression levels of antioxidant genes. CONCLUSIONS: Our results indicate an influence of MC-LR on the circadian clock system and clock-controlled antioxidant genes, which will shed some light on the explanation of heart toxicity induced by MC-LR from the viewpoint of chronobiology.

A Quantitative Chemical Proteomic Strategy for Profiling Phosphoprotein Phosphatases from Yeast to Humans.

A "tug-of-war" between kinases and phosphatases establishes the phosphorylation states of proteins. While serine and threonine phosphorylation can be catalyzed by more than 400 protein kinases, the majority of serine and threonine dephosphorylation is carried out by seven phosphoprotein phosphatases (PPPs). The PPP family consists of protein phosphatases 1 (PP1), 2A (PP2A), 2B (PP2B), 4 (PP4), 5 (PP5), 6 (PP6), and 7 (PP7). The imbalance in numbers between serine- and threonine-directed kinases and phosphatases led to the early belief that PPPs are unspecific and that kinases are the primary determinants of protein phosphorylation. However, it is now clear that PPPs achieve specificity through association with noncatalytic subunits to form multimeric holoenzymes, which expands the number of functionally distinct signaling entities to several hundred. Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases.We have developed a chemical proteomic strategy for the systematic interrogation of endogenous PPP catalytic subunits and their interacting proteins, including regulatory and scaffolding subunits (the "PPPome"). PP1, PP2A, PP4, PP5, and PP6 were captured using an immobilized, specific but nonselective PPP inhibitor microcystin-LR (MCLR), followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a single analysis. Here, we combine this approach of phosphatase inhibitor bead profiling and mass spectrometry (PIB-MS) with label-free and tandem mass tag (TMT) quantification to map the PPPome in human cancer cell lines, mouse tissues, and yeast species, through which we identify cell- and tissue-type-specific PPP expression patterns and discover new PPP interacting proteins.

Validation of Housekeeping Genes as Internal Controls for the Study of the Effects of Microcystin-LR in Zebrafish by Real-Time PCR.

Microcystin-leucine arginine (MC-LR) is a natural toxin produced by cyanobacterial blooms that causes severe liver damage in fish. It is crucial to investigate if housekeeping genes are affected by MC-LR in zebrafish, to permit the adequate evaluation of gene expression by RT-qPCR. We evaluated the gene expression stability (M value) and regulation by chemical treatment (using E-Ct and E-ΔCt) to validate the use of eight housekeeping genes in fish exposed to 0, 0.31, and 6.10 µg L-1 MC-LR for 24 h. We suggest the use of the combination of ß-actin1, b2m, and arnt2 in the liver and ß-actin1, 18S rRNA, and arnt2 in gills as housekeeping genes. The evaluation of gene regulation following MC-LR exposure denoted a strong repression of 18S rRNA (17- and 10-fold decrease) and tbp (10- and 2-fold decrease) and induction of ef-1α (8- and 14-fold increase) in the liver of zebrafish exposed to 0.31 and 6.10 µg L-1 MC-LR, respectively. This is the first study showing that housekeeping genes commonly used in gene expression could be affected in the liver by environmentally relevant concentrations of MC-LR. The study validates adequate housekeeping genes that could be used in toxicological studies with MC-LR in zebrafish.