# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 1 Introduction  
Cyanobacteria are photosynthetic microorganisms that appeared on Earth 3.5 billion years ago and can be observed in various freshwater, brackish and marine ecosystems [1]. In freshwater ecosystems, cyanobacteria have significant proliferative capacities [2] favored by different natural factors (e.g., temperature above 25 \({}^{\circ}\)C, alkaline pH) but also anthropogenic factors (e.g., high nitrate and phosphorus inputs) [3; 4; 5]. Many toxins can be produced by cyanobacteria during blooms, such as neurotoxic anatorus and hepatotoxic microcystins (MCs). The hepatotoxin MCs are the most frequently detected cyanotoxins in cyanobacterial blooms [2]. There is a great diversity of MCs-producing strains of cyanobacteria. MC producers can be found in several different orders, including _Chroococcales_, _Oscillatoriales_, _Nostocales_, and _Stigonematales_. Within these orders, it is important to note that not all genera of an order produce MCs (for example, in the order _Nostocales_, some species of the genus _Anabaena_ are producers while other genera of this order, such as _Aphanizomenon_, have never been described as MC-producers). Moreover, a species can be both producing and non-producing depending on the strain, and a producing strain depends on environmentalfactors [6]. Among the genera known to include MC-producing (and non-producing) species: _Microcytsis_, _Planktothrix_, _Anabena_, and _Hapalosiphon_ are the main ones. However, there are many other producing species (Table S1, [6; 7]). MCs are monocyclic heptapeptides containing two atypical amino acids called Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), which is a fragment common to all MCs variant and nodularins and Mdah (N-methyl-dehydroalanine) groups, which plays a role in the binding of MCs to proteins. MCs present with two variable amino acids, characteristic of the identity of the MC variant (Figure 1). For example, MC-LR, the first chemically-identified MC, is a variant where L and R represent the amino-acid leucine and arginine, respectively [8]. Thus, the identification of the MCs is mainly due to the variations in the AAs in positions X and Z (Figure 1) but also due to the variations of the other sites of the MCs, which can also be subject to substitutions. To date, up to 279 MCs congeners have been described [9]. MCs are mainly intracellular during cyanobacteria proliferation and are released in media, becoming extracellular (dissolved in water or adsorbed on particles) once cell death occurs after bloom collapse. After the ingestion of cyanobacterial cells or the ingestion of contaminated water, MCs can accumulate in various organisms, such as zooplankton, zoobenthos, or fish, with a possible transfer between trophic levels [10; 11; 12; 13]. MCs can accumulate in two forms in organisms: a free, available cellular fraction and a bound form (_id._ intracellular for the cyanobacterial matrices or covalently bound to proteins in biota tissues). These covalently-bound forms are particularly bound to the serine/threonine residues of enzyme protein phosphatases 1 (PP1) and 2A (PP2A) [14]. The mechanism of the binding of MCs to PP is a two-step mechanism, first with the formation of a weakly-interacting bond of a hydrophobic and reversible nature between the Mdah group of MCs and the proteins and, after a few hours, the Adda group can form a strong covalent thioether bond with the cysteine residues present at the catalytic subunits of PP [15; 16; 17; 18]. The binding of MCs to proteins results in the deregulation of the PP by disrupting the phosphorylation mechanisms [19; 20]. This deregulation by MCs may induce a cascade of negative side effects, in particular, tumor promotion and apoptosis, but also the production of reactive oxygen species, resulting in oxidative stress (reviewed in [20; 21]). MC toxicity can occur in different organs but was initially discovered in the liver, resulting in the classification of MCs as hepatoxins.