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Immunomodulatory potency of microcystin, an important water polluting cyanobacterial toxin Ondrej Adamovsky, Zdena Moosova, Michaela Pekarova, Amrita Basu, Pavel Babica, Lenka Svihalkova Sindlerova, Lukas Kubala, and Ludek Blaha Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02049 • Publication Date (Web): 18 Sep 2015 Downloaded from http://pubs.acs.org on September 20, 2015
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Environmental Science & Technology
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Immunomodulatory potency of microcystin, an important water polluting
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cyanobacterial toxin
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Ondrej Adamovskya*, Zdena Moosovaa, Michaela Pekarovab, Amrita Basua, Pavel Babicaa,
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Lenka Svihalkova Sindlerovab, Lukas Kubalab, Ludek Blahaa
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a
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Republic
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b
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Republic
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Masaryk University, Faculty of Science, RECETOX, Kamenice 753/5, 62500, Brno, Czech
Institute of biophysics, Academy of Sciences, Královopolská 135, 612 65 Brno, Czech
* Corresponding author: e-mail:
[email protected], phone: +420549493449
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Abstract Art
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Abstract Microcystins (MCs) are primarily hepatotoxins produced by cyanobacteria and are
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responsible for intoxication in humans and animals. There are many incidents of chronic
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exposure to MCs which have been attributed to inappropriate treatment of water supplies or
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contaminated food. Using RAW 264.7 macrophages, we showed the potency of microcystin-
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LR (MC-LR) to stimulate production of pro-inflammatory cytokines (tumor necrosis factor α
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and interleukin-6) as a consequence of fast nuclear factor-κB and mitogen-activated protein
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kinase activation. In contrast to other studies, the observed effects were not attributed to
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intracellular inhibition of protein phosphatases 1/2A due to lack of specific trans-membrane
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transporters for MCs. On the other hand, the MC-LR-induced activation of macrophages was
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effectively inhibited by a specific peptide that blocks signaling of receptors, which play a
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pivotal role in the innate immune responses. Taken together, we showed for the first time that
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MC-LR could interfere with macrophage receptors, responsible for triggering the above-
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mentioned signaling pathways. These findings provide an interesting mechanistic explanation
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of some adverse health outcomes associated with toxic cyanobacteria and MCs.
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1. Introduction
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Over the last decades, cyanobacterial blooms have become a serious problem for the
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aquatic environment as well as for water quality and human environmental health.1 Intensive
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human activities, such as releasing of fertilizers or sewage, lead to an increase in the amount
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of phytoplankton in bodies of water as a response to high levels of nutrients. In addition to
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other deleterious effects on ecosystems, such as alteration of the water chemistry and
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formation of dead zones,2 cyanobacteria produce a wide range of biologically active
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compounds, many of them highly toxic. However, only a few of them have been
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toxicologically characterized in detail.3 Hepatotoxic microcystins (MCs) are very stable in the
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environment and rank among the most abundant cyanobacterial toxins identified in reservoirs
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worldwide, and they pose serious risks to the quality of drinking water. The problem is
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relevant not only for developing countries in Asia4 or Africa,5 but also in Europe and North
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America, where drinking water treatment technologies are broadly used and are of much
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higher quality.6,7 In addition, MCs effectively accumulate in fish and other aquatic biota,
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which can represent another exposure pathway to humans.8 Due to the high toxicity and
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apparent risks to human health, the WHO has established a provisional guideline of 1 µg/L for
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microcystin-LR (MC-LR) in drinking water,9 which has been applied in a number of
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countries.10
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The hepatotoxic and tumor-promotion potential of MCs has been investigated
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extensively, but other potential chronic impacts of MCs, including modulation of immune
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responses, have been studied to a much lesser extent, despite the existing evidence of toxicity.
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For example, some studies with complex cyanobacterial bloom extracts revealed their
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immunotoxic potential, as well as significant effects on proliferation, phagocytosis, and
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cytokine production in immune cells.11,12 Apart from whole cyanobacterial cell extracts, a
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study with purified toxin (MC) indicated its high potency to downregulate the numbers of
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human lymphocytes by induction of apoptosis and necrosis, as well as to modulate production
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of numerous cytokines crucial for the signaling network.12,13 With respect to mammalian
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innate immunity, which is the first barrier against invading pathogens, information on MC
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effects is very limited.13,14
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The present study aimed to investigate in detail the effects of MC-LR on macrophages,
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which represent one of the key effector cells within the innate immune responses. The
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selected murine macrophage model RAW 254.7 is a broadly accepted cell line for a range of
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pharmacological15 as well as environmental toxicological studies.16
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Specifically, our study includes investigation of MC-LR effects on macrophage
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activation, associated with production of cytotoxic and cytostatic products such as nitric oxide
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(NO), as well as proinflammatory mediators (e.g. tumor necrosis factor α, TNFα and
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interleukin 6, IL-6). The lipopolysaccharide isolated from gram-negative bacteria (LPS) was
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used as a standard macrophage stimulator. Mechanisms beyond the observed effects of MC-
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LR were explored by studying uptake of toxins into cells (expression of organic anion-
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transporting polypeptide transporters, OATPs) and impacts on intracellular signaling (protein
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phosphatases, PPs; mitogen activated protein kinases, MAPKs; and nuclear factor κB, NF-
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κB). This study brings new mechanistic information on MC effects on innate immunity.
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Interestingly, we observed that macrophages showed a high sensitivity toward MC-LR, which
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– for the first time – suggests that MC-LR acts via Toll-like receptors (TLRs). The study
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provides evidence of new modes of action beyond the toxicity and chronic health impacts of
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highly relevant water polluting natural toxins – MCs.
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2. Material and methods
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The paragraphs within this section contain basic information on the methods used;
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experimental details of the protocols are available in the supporting information (abbreviated
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as Supp).
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Reagents and cell culture MC-LR is commercially available from Enzo Life Sciences, New York with purity
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≥95% (HPLC). The stock solution (1 mg/ml) was dissolved in methanol (50%). For cell
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exposure, MC-LR, bromosulfophthalein (BSP) (Santa Cruz Biotechnology, USA) along with
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lyophilized powder of LPS from Escherichia coli 026:B6 (Sigma-Aldrich, USA) and MyD88
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inhibitory peptide (MYD, InvivoGen, USA) were dissolved in sterile phosphate-buffered
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saline (PBS). An inhibitor of PPs, okadaic acid (OA) (Sigma-Aldrich, USA), and the substrate
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for determination of PP activity 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, Life
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Technologies, Czech Republic) were dissolved in DMSO. All reagents were kept frozen at -
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20 °C.
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RAW 264.7 cell line was obtained from the European Collection of Cell Cultures
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(ECACC, USA) and cultured using standard methods (described in Supp). For all
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experiments, cells were seeded on 12-well tissue culture plates (TPP, Switzerland, 1.66 x 105
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cell/cm2) and left to adhere for 4 h when the exposures were initiated. Various combinations
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of treatments including activation of cells by LPS (10 ng/mL) and/or (co)exposures with MC-
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LR (1–1000 nM), BSP (100 and 300 µM), OA (100 and 500 nM), as well as MYD (10 µM)
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were performed at different time points (30 min to 24 h). Concentrations of vehicles were
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