Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
The possible role of microcystin (D-Leu1 MC-LR) as an antioxidant on Microcystis aeruginosa (Cyanophyceae). In vitro and in vivo evidence
Graphical abstract
Introduction
When rivers and lakes become eutrophic, Microcystis spp. is one of the cyanobacteria genus that usually increases in relative abundance forming blooms (D'Angelo et al., 1998). Cyanobacteria may produce different types of secondary metabolites such as cyanotoxins (e.g. the microcystin, MC). Over 90 structural analogues of MC have been identified up to date, but only a few occur frequently at high MCs concentration. It consists of a general cyclic heptapeptide structure with variable amino acids (Qi et al., 2015). Qi et al. (2015) have shown seven new MC variants in the same strain of Microcystis aeruginosa used for the present study, which has been previously reported as a [D-Leu1]MC-LR producer (Rosso et al., 2014). Such structural variants of MCs can be explained by the multispecificity of single domains of the MC biosynthesis complex. At this variable position, [D-Leu1]MC-LR contains the amino acid leucine, whereas MC-LR (also present in our strain) contains alanine (Christiansen et al., 2003).
There is an increasing concern that changes in climate (Stocker et al., 2013) will cause changes in phytoplankton community structure and composition. MCs are predominantly produced by freshwater cyanobacteria of the genera Microcystis, Planktothrix and Anabaena (Dittmann et al., 2013), which would have adverse effects on humans and environment (Babica et al., 2006). Moreover, some studies found a direct influence of environmental factors on the MC production (e.g. Jähnichen et al., 2011; Giannuzzi et al., 2016).
MC is known to irreversibly inhibit eukaryotic protein phosphatases of types 1 and 2A (Chorus and Bartram, 1999). However, the physiological role of MC is still unclear (Pflugmacher, 2002; Yoshida et al., 2007).For example, recent studies examining cyanotoxin production have found close links between these compounds and certain physiological functions that may be considered part of the primary metabolism of the cell, with a close connection to primary metabolism (Neilan et al., 2012). The function of MCs has been postulated as allelopathic or intercellular communication (Schatz et al., 2007). Also, a series of evidence has reinforced the hypothesis of its antioxidant function (Zilliges et al., 2011; Dziallas and Grossart, 2011; Yang et al., 2015; Zhang et al., 2018).
Reactive oxygen species (ROS) are inevitably generated in processes such as respiration and photosynthesis (Apel and Hirt, 2004). Cyanobacteria are often exposed to changing conditions, including drastic fluctuations in light intensity and temperature, and therefore ROS are easily produced. Several environmental conditions may produce increased ROS concentrations. For example, high amounts of ROS in phytoplankton were observed because of increased ultraviolet-B radiation (UVBR) (Chen et al., 2009; Hernando et al., 2011). For M. aeruginosa, Hernando et al. (2018) demonstrated that the highest ultraviolet radiation doses produced an increase in ROS as well as decreased photosynthesis and biomass with only activation of the enzymatic antioxidant catalase (CAT) by UVBR and superoxide dismutase (SOD) and CAT by ultraviolet radiation-A (UVAR). In addition, for such high doses, a significant decrease of MCs was observed because of UVAR exposure (Hernando et al., 2018). High Solar irradiances in addition to increased greenhouse gas emissions may produce an increased temperature. In this century, global temperatures are expected to increase about additional 2–5 °C (Houghton et al., 2001). However, harmful cyanobacteria such as Microcystis have been found to have an optimal temperature for growth and photosynthesis at 25 °C or above (Reynolds, 2006). Giannuzzi et al. (2016) had demonstrated a significant increase in intracellular ROS in coincidence with the activation of CAT during the first two days of 29 °C exposure in M. aeruginosa.
Furthermore, the production of ROS in M. aeruginosa is significantly increased under exogenous H2O2 conditions (Bouchard and Purdie, 2011). It has been shown that ROS are responsible for the degradation of Clorophyll-a (Chla) and the decrease in the activity of photosystem II (PSII) in phytoplankton photosynthetic antenna (Saison et al., 2010). In addition, ROS cause the inhibition of cyanobacterial growth (Dziallas and Grossart, 2011). However, ROS are under the control of an elucidated antioxidant system (Latifi et al., 2009), consisting of small antioxidant molecules such as tocopherols, ascorbate or glutathione, and antioxidant enzymes such as CAT, SOD and peroxidases. Under stress conditions, the balance between the oxidative impact and the antioxidant defense system could be disturbed leading to oxidative stress. The response of the antioxidant system was shown in M. aeruginosa by several studies (Hernando et al., 2018; Giannuzzi et al., 2016).
At present, it is not clear whether the intra- or the extracellular portion of MC has a larger impact on cellular physiology and to what extent the intra- and extracellular roles are connected.
Here, we hypothesize a radical scavenger function of MCs. Recently, several studies have proposed a mechanism on how MCs are beneficial for toxin-producing cells exposed to oxidative stress (Dziallas and Grossart, 2011; Zilliges et al., 2011; Yang et al., 2015; Giannuzzi et al., 2016; Hernando et al., 2018).
The aim of this study was to evaluate the microcystin [D-Leu1]MC-LR antioxidant capacity by in vitro assays. Moreover, the physiological effects of toxin addition will be determined on a long-term exposure (7 days) of M. aeruginosa to high temperature condition (HT) as stress factor.
Section snippets
Chemicals
MC-LR (CAS N° 101043-37-2); Ascorbic acid (CAS N° 50-81-7); Dimethylsulfoxide (DMSO) (CAS N° 67-68-5), 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) (CAS N°3317-61-1); 2,7-Dichlorodihydrofluorescein di-acetate (DCFH-DA) (CAS N° 4091-99-0); 2′,7′-Dichlorofluorescein (DCFH) (CAS N° 76-54-0); Hydrogen Peroxide (H2O2) (CAS N° 7722-84-1); Acetonitrile HPLC grade, ≥99.93% (CAS N° 75-05-8); Formic acid (CAS N° 64-18-6); Methanol HPLC grade, ≥99.9% (CAS N° 67-56-1) were provided by Sigma Chemical Co. (St.
EPR spectra
A typical EPR spectrum of A was recorded (Fig. 1A) and showed the characteristic two lines at g = 2.005 and aH = 1.8 G in accordance with computer spectral simulated signals obtained using the parameters given in the Material and Methods section (Section 2) (Fig. 1Aa). DMSO was examined and no DMSO signal was observed (Fig. 1Ab). Fig. 1B showed generation of A content, assessed by quantification of EPR signals. The addition of [D-Leu1]MC-LR exhibited a maximum scavenging activity, reducing the A
Role of MCs
The physiological role of MC is still unclear (Pflugmacher, 2002; Yoshida et al., 2007). MC is known to irreversibly inhibit eukaryotic protein phosphatases of types 1 and 2A (Chorus and Bartram, 1999). Nevertheless, such defensive function of MC is in doubt. Through evolutionary analysis it was observed that defense genes were already present in the common ancestor of all cyanobacteria, before the appearance of a eukaryotic grazer (Rantala et al., 2004). Other studies suggest that its function
Conclusions
The results presented here are consistent with the hypothesis that indicates that A and OH (hydrosoluble radicals) were efficiently quenched in vitroby [D-Leu1]MC-LR addition, under these experimental conditions. As far as we know, this is the first in vitro record of the role of MC as antioxidant for hydrosoluble radicals. The maximum scavenging activity of both radicals was observed at low concentrations, ensuring a potential antioxidant function considering that cellular concentrations of
Acknowledgements
We would especially like to thank Dr. Dario Andrinolo and Daniela Sedan, from the La Plata University, for providing the isolated [D-Leu1]MC-LR. We also thank Florencia de la Rosa for the CAT activity measurements. The authors are grateful to Dr. Paula Denise Prince for checking the grammar and spelling of the manuscript. We appreciate the comments and corrections of the two anonymous reviewers, which effectively contributed to the improvement of this manuscript. This study was supported by
References (64)
- et al.
Analysis of cyanobacterial hepatotoxins in water samples by microbore reversed phase liquid chromatography–electrospray ionisation mass spectrometry
J. Chromatogr. A
(2002) - et al.
Growth, toxin production, active oxygen species and antioxidants responses of Microcystis aeruginosa (Cyanophyceae) to temperature stress
Comp Biochem Physiol C Toxicol Pharmacol.
(2016) - et al.
The reaction of ascorbic acid with different heme iron redox states of myoglobin
FEBS Lett.
(1993) - et al.
Chemistry of dioxygen
Meth. Enzymol.
(1984) - et al.
Responses of enzymatic antioxidants and non-enzymatic antioxidants in the cyanobacterium Microcystis aeruginosa to the allelochemical ethyl 2-methyl acetoacetate (EMA) isolated from reed (Phragmites communis)
J. Plant Physiol.
(2008) - et al.
Microcystin production by Microcystis aeruginosa: direct regulation by multiple environmental factors
Harmful Algae
(2011) - et al.
Topology and enhanced toxicity of bound microcystins in Microcystis PCC 7806
Toxicon
(2008) - et al.
Continous monitoring of cellular nitric oxide generation by spin trapping with an iron-dithiocarbamate complex
Biochim. Biophys. Acta
(1996) - et al.
ROI-scavenging enzyme activities as toxicity biomarkers in three species of marine microalgae exposed to model contaminants (copper, Irgarol and atrazine)
Ecotoxicol. Environ. Saf.
(2014) - et al.
Effect of core–shell copper oxide nanoparticles on 566 cell culture morphology and photosynthesis (photosystem II energy distribution) in the green alga, 567 Chlamydomonas reinhardtii
Aquat.Toxicol.
(2010)
Relevance of the capacity of phosphorylated fructose to scavenge the hydroxyl radical
Carbohydr. Res.
Intrapopulation clonal variability in allelochemical potency of the toxigenic dinoflagellate Alexandrium tamarense
Harmful Algae
Exploring the interaction of microcystin-LR with proteins and DNA
Toxicol. in Vitro
Effects of UV-B radiation on microcystin production of a toxic strain of Microcystis aeruginosa and its competitiveness against a non-toxic strain. J
Hazard. Mater.
PAHs would alter cyanobacterial blooms by affecting the microcystin production and physiological characteristics of Microcystis aeruginosa
Ecotoxicol. Environ. Saf.
Reactive oxygen species: metabolism, oxidative stress, and signal transduction
Annu. Rev. Plant Biol.
Ascorbate system in plant development
J Bioenerg Biomembr.
Exploring the natural role of microcystins—a review of effects on photoautotrophic organisms
J. Phycol.
Catalase
Effect of elevated temperature, darkness and hydrogen peroxide treatment on oxidative stress and cell death in the bloom-forming toxic cyanobacterium Microcystis aeruginosa
J. Phycol.
Competition between microcystin- and non-microcystin-producing Planktothrix agardhii (cyanobacteria) strains under different environmental conditions
Environ. Microbiol.
UV-B-induced oxidative damage and protective role of exopolysaccharides in desert cyanobacterium Microcoleus vaginatus
J. Integr. Plant Biol.
Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management
Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation
J. Bacteriol.
Advances in spin trapping
Electron Paramagnetic Resonance
UVB radiation as a potential selective factor favoring microcystin producing bloom forming cyanobacteria
PLoS One
Cyanobacterial toxins: biosynthetic routes and evolutionary roots
FEMS Microbiol. Rev.
Increasing oxygen radicals and water temperature select for toxic Microcystis sp
PLoS One
Cellular Oxidant/Antioxidant Network: Update on the Environmental Effects over Marine Organisms
The open mar. biol. j..: Bentham Open
Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life
Plant Physiol.
Non-enzymatic antioxidant photoprotection against potential UVBR-induced damage in an Antarctic diatom (Thalassiosira sp)
Lat. Am. J. Aquat. Res.
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2021, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyCitation Excerpt :However, it was also demonstrated that the incorporation of toxins in molluscs produces an increase in TBARS. Although the quota of MCs were significantly lower in M. aeruginosa exposed to 29 °C compared to 26 °C, probably due to its antioxidant function proposed by Malanga et al. (2019), the presence of MCs could be enough for the lipid damage observed after 15 days of feeding on M. aeruginosa. MCs incorporation per se can be the first event that triggers glutathione depletion and the consequent increase in ROS concentration (Amado and Monserrat, 2010).
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2020, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyCitation Excerpt :A lower ROS concentration and lipid damage has allowed an exponential growth after D1 in both 26 and 29 °C conditions, being significantly higher at high temperature (11%) probably as a result of greater enzymatic antioxidant protection (Giannuzzi et al., 2016). Another mechanism by which M. aeruginosa can protect itself from the physiological consequences of temperature rise is using toxins (microcystins, MCs) as antioxidants (Malanga et al., 2019). However, MCs have different responses to temperature increase.
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Both authors contributed equally to this manuscript.