Toxicological evaluation of the neurotoxic properties of cylindrospermopsin and its potential interactions with cyanotoxins and chemical pollutants present in food

UNIVERSIDAD DE SEVILLA FACULTAD DE FARMACIA DEPARTAMENTO DE NUTRICIÓN Y BROMATOLOGÍA, TOXICOLOGÍA Y MEDICINA LEGAL “TOXICOLOGICAL EVALUATION OF THE NEUROTOXIC PROPERTIES OF CYLINDROSPERMOPSIN AND ITS POTENTIAL INTERACTIONS WITH CYANOTOXINS AND CHEMICAL POLLUTANTS PRESENT IN FOOD” Memoria que presenta la Graduada MARIA GRACIA HINOJOSA HIDALGO para optar al título de Doctor por la Universidad de Sevilla con la mención de “Doctorado Europeo” Sevilla, 2021 INDEX INDEX I. Summary II. Introduction 1. Cyanobacteria 1.1. Cyanotoxins 1.1.1. Exposure routes 1.1.2. Intoxications after exposure 1.1.3. Microcystins 1.1.3.1. Geographical distribution 1.1.3.2. Bioaccumulation of MCs 1.1.3.3. Cases of intoxication 1.1.3.4. Toxicity of MCs 1.1.3.4.1. Toxicokynetics 1.1.3.4.2. Mechanisms of action 1.1.4. Cylindrospermopsin 1.1.4.1. Geographical distribution 1.1.4.2. Bioaccumulation 1.1.4.3. Cases of intoxication 1.1.4.4. Toxicity of CYN 1.1.4.4.1. Toxicokynetics 1.1.4.4.2. Mechanisms of action 2. Pesticides 2.1. Organophosphates 2.1.1. Mechanisms of action 2.1.2. Chlorpyrifos 2.1.2.1. Toxicity of CPF 3. Importance of the combinational studies 4. Neurotoxicity induced by cyanotoxins and pesticides III. Results IV. General discussion V. Conclusions VI. References I. SUMMARY SUMMARY Cyanobacteria are prokaryotic microorganism present in many aquatic and terrestrial environmental samples worldwide able to produce some toxic compounds known as cyanotoxins. Among them, microcystins, in special microcystin-LR (MC-LR) and CYN are some of the most common ones. Both are produced by several species of cyanobacteria. Cylindrospermopsin (CYN) is a cyanotoxin produced by several species of cyanobacteria. Their incidence is increasing in the last decades due to anthropogenic activities and climate change. These hepatotoxin and cytotoxin, respectively, are able to cause intoxication cases in the food chain from fish to humans, as they can also be bioaccumulated. The main mechanisms of action for MC-LR are the the inhibition of protein phosphatases, oxidative stress generation and effects on DNA repair, among others. Furthermore, MC-LR has demonstrated to exert neurotoxicity by irreversible inhibition of Serine/Threonine protein phosphatases 1 and 2A (PP1 and PP2A), Tau hyperphosphorylation, oxidative stress and modifications in neurotransmitters such as ACh, DA and GABA. Concerning the mechanisms of action for CYN, the most important ones are the proteinsynthesis inhibition, oxidative stress generation and apoptosis. Furthermore, its metabolite has demonstrated also to be able to cause genotoxicity. Despite being the liver and the kidneys its main targets, the nervous system has recently been studied as a possible target as well. The studies performed in this regard point out a possible effect on the production of oxidative stress and alterations in the acetylcholinesterase activity both in vitro and in vivo. However, the studies in this aspect are scarce. Nonetheless, these cyanotoxins are not usually found in nature as a pure compound, but in combination with other chemicals such as other cyanotoxins. Taking into account all this information, a state of the art of the neurotoxicity induced by these cyanotoxins was perfomed, which led to the publication: NEUROTOXICITY INDUCED BY MICROCYSTINS AND CYLINDROSPERMOPSIN: A REVIEW (Hinojosa et al., 2019. Science of the Total Environment 668, 547-565) According to EFSA, it is important to study their toxicity not only isolated but also in combination, as they could be found in nature, due to their possible interaction, which could lead to changes in their effect. In order to assess the toxicological properties that these cyanotoxins would exert in a human cell line, both isolated and in combination, some studies concerning cytotoxicity, oxidative stress, acetylcholinesterase activity, and morphological analysis were Figure 1. Cyanobloms of Raphidiopsis raciborskii and Microcystis aeruginosa (from https://freshwaterecology.wordpress.com/2018/03/30/cylindrospermopsis-raciborskii-and-microcystis-aeruginosa- competing-under-different-conditions-of-ph-and-inorganic-carbon/) This feature is potentiated by hydrological changes in aquatic ecosystems, global warming, and climate change, causing an increase in their incidence (Codd et al., 2017). In the last decades, climate change and anthropogenic activities have led to an increase in the incidence of algal blooms with species that are more toxic and with larger duration and geographical extent, causing major problems for water quality (Heisler et al., 2008; Trevino-Garrison et al., 2015). This overgrowth forms a slime, which causes the reduction of light penetration and oxygenation of water, inducing hypoxia and anoxia for fish, benthic invertebrates, and submerged aquatic vegetation (Huisman et al., 2018). In addition, bad organoleptic properties can be also generated (Khan and Ansari, 2005), which interfere with the use of reservoirs for drinking water or recreational activities in freshwater bodies (Huisman et al., 2018). The phenomenon by which an excessive growth of aquatic flora such as cyanobacteria occurs due to nutrient enrichment is known as eutrophication (Yang et al., 2008). The main cause for eutrophication is the unloading of high amounts of nutrients such as nitrogen, phosphorus, or carbon into the water bodies, mainly derived from intensified agriculture (Khan and Ansari, 2005). Lately, several studies have pointed out that the increase of CO2 levels, global warming, and eutrophication increase the frequency, intensity, and duration of cyanobacterial blooms in many aquatic ecosystems across the globe (Huisman et al., 2018). Temperature and pH levels can also increase the rate of cyanobacterial growth. More than 150 genera and about 2000 different species of cyanobacteria have been identified so far, presenting a wide range of cellular morphologies, physiological capacities, and adaptations (Machado et al., 2017). Many bloom-forming cyanobacteria reach their maximal growth rates above 25°C, faster than eukaryotic phytoplankton, contributing to a longer duration and range expansion of cyanobacterial blooms (Huisman et al., 2018). Consequently, they dwell in both terrestrial and aquatic ecosystems including extreme habitats (Bownik, 2016; Buratti et al., 2017). Due to their ability to withstand adverse and extreme environmental conditions, together with their capacity for photosynthesis and for fixing gaseous nitrogen into ammonia and amino acids, cyanobacteria are primary colonisers. In fact, cyanobacteria can store essential nutrients such as phosphorus or nitrogen when abundant to be able to grow under nutrient-limiting conditions (Codd et al., 2017). Their adaptative ability together with their high nutrient content and their small requirement of volumes of water for growth and development, make cyanobacteria an attractive substitute for sustainable food production. In this sense, cyanobacteria can be used as a potential food supplement and provide nutritional, therapeutic and beneficial values. Due to their high content of minerals and vitamins, some species of cyanobacteria, such as Spirulina, Anabaena, or Nostoc, are consumed as a food supplement in many countries in different pharmaceutical forms like capsules, tablets, and liquids (Chittora et al., 2020). Furthermore, these prokaryotic organisms can have the potential to fix atmosphere nitrogen, which could be used as a biofertilizer for the cultivation of economically important crops. In addition, these organisms are producers of metabolites with pharmaceutical properties, such as antimicrobial, antibacterial, antialgal, antifungal, antiviral, antiprotozoal, antitumoural, cytotoxic, antiinflammatory, antioxidant, antimalarials, antimycotics, herbicides, insecticides, etc. (Demay et al., 2019; Chittora et al., 2020). However, some other compounds like aldehydes, terpenes, ketones, etc., constitute human and animal health hazards (Zanchett and Oliveira-Filho, 2013; Demay et al., 2019). Among these compounds, cyanotoxins are the main concern. 1.1. Cyanotoxins Cyanotoxins are secondary metabolites produced by several species of cyanobacteria able to cause harm in many different organisms (Wood, 2016). These chemicals are of special interest due to their presence in many human and animal intoxication cases (Codd et al., 2017). The production of these toxins has significant adverse effects on aquatic and terrestrial ecosystems, which leads to a reduction of diversity, and also being a potential health hazard to other living organisms, including humans (Wood, 2016; Lee et al., 2017). These toxic metabolites are the main reason for the classification of cyanobacteria as emerging pathogens by the Organization for Economic Cooperation and Development (OECD), in spite of not having the ability to colonize or invade hosts (OECD, 2005; Machado et al., 2017). Cyanotoxins are usually stored inside the cyanobacteria and their release occurs at the end of the natural lifecycle of the bloom or the cyanobacteria themselves, being the cause of their presence in the water even after the dissipation of the bloom and thus, constituting still a health risk (Wood, 2016). In addition to the producing species itself, the total production of cyanotoxins concerning their quantity and their profile of cyanotoxin variants relies on a combination of different environmental factors such as temperature, light, and nutrients, among others (Manganelli et al., 2012). Cyanotoxins have been considered in scientific literature as ‘secondary metabolites’. However, this label might not be accurate, as secondary metabolites are defined as “compounds not used by organisms for primary metabolism”, and there might be some evidence of their current or past potential role in this process (Holland and Kinnear, 2013). The reason for the cyanobacterial production of cyanotoxins has not been elucidated yet. However, they might be a possible evolutionary adaptation for defensive functions against grazing, to reduce competition for resources, cell signaling, iron scavenging, nutrient uptake, homeostasis, or protection against oxidative stress (Holland and Kinnear, 2013; Huisman et al., 2018). In fact, some studies report that the presence of predators has an impact on the level of cyanotoxinsproduction, varying between toxins even when the same producer is involved (Zanchett and Oliveira-Filho, 2013). The production of these toxicants implicates risks to animal and human health, being able to cause severe poisoning in a short- and long-term. Cyanotoxins are thus important chemical compounds, from an ecotoxicological, toxicological, and environmental points of view (Zanchett and Oliveira-Filho, 2013). Cyanotoxins vary in their toxicology and they are typically classified according to their main organ target as hepatotoxins, cytotoxins, neurotoxins, or dermatotoxins (Wood, 2016): Table 1: summary of cyanotoxins based on Codd et al. (2019) 1.1.1. Exposure routes Humans can be exposed to cyanotoxins by several routes (Fig. 2). The main habitat where cyanotoxins can be found is water, so oral exposure through drinking water or contaminated food, or even accidental ingestion during recreational activities, are usually the most accessible route for human exposure to cyanotoxins (Metcalf and Codd, 2012). Classification Cyanotoxin Structure Mechanisms of action Hepatotoxins Microcystins Hepatotoxic, tumour promoting, oxidative stress, inhibition of the protein phosphatases PP1 and PP2A Nodularin Similar to MCs Cylindrospermopsin Multiple organ toxicity, neurotoxic, genotoxic, protein synthesis inhibitor Neurotoxins Anatoxins Inhibition on the acetylcholinesterase Saxitoxins Blockage of voltage-gated sodium channels β-N-methylamino-L- alanine Erroneuos insertion into proteins causing misfolding Dermatotoxins Aplysiatoxins and lyngbiatoxins Inflammation and tumour promotion Lipopolysaccharides Inflammatory, promotion of cytokine secretion Figure 2. Routes of exposure for cyanotoxins. Drinking contaminated water is of special concern in arid and developing countries, where inhabitants depend on stored water in reservoirs (Buratti et al., 2017). There are several methods to treat water for cyanotoxin removal, although no single procedure can be recommended as the nature and characteristics of each cyanotoxin are different (Metcalf and Codd, 2012). However, with adequate monitoring and prevention, the risk of exposure can be minimised (Metcalf and Codd, 2012). Ingestion of contaminated aquatic organisms is another important source of intoxaction (Guzmán-Guillén et al., 2011). Cyanotoxins are of special concern as they may accumulate by their ingestion in edible aquatic organisms such as fish, mussels, or crustaceans, whose safety has not been routinely checked for consumers (Buratti et al., 2017). In this sense, it is known that bivalves are able to concentrate all kinds of toxics present in the water and among them, different types of cyanotoxins (Metcalf and Codd, 2012). Consumption of vegetables irrigated with contaminated water could also be an additional source of exposure, as the soil can also retain the toxins (Lee et al., 2017). Thus, although plants are rarely killed by environmental concentrations of cyanotoxins, their growth and crop yields can be affected, depending their bioaccumulation on the frequency and time of exposure (Prieto et al., 2011; Gutiérrez-Praena et al., 2014; Guzmán- Guillén et al., 2017; Lee et al., 2017). Cyanotoxins can also be present in health-food supplements based on Spirulina or Chrysosporum flos-aquae grown in artificial ponds or collected from open environments such as natural lakes (Gutiérrez-Praena et al., 2013; Lee et al., 2017). Dermal and inhalatory routes are also important ways of exposure for cyanotoxins. In this regard, water-based recreational activities such as swimming, paddling, kayaking, or even showering at lakes with presence of cyanobacteria and cyanotoxins have implicated some adverse health outcomes as well (Metcalf and Codd, 2012). Lastly, the parenteral route has probably been underestimated over the world, as the quality of water used for hemodyalisis is not regulated in most countries despite demonstrating to be the main cause for some cyanotoxin intoxications, among others (Manganelli et al., 2012). 1.1.2. Intoxication reports Reports of animal poisoning due to cyanotoxins have been documented worldwide for more than a century (Wood, 2016; Díez-Quijada et al., 2019a,b). The first record of a possible animal intoxication by cyanobacterial blooms was carried out by Francis (1878), after the death of livestock and domestic dogs at Lake Alexandrina in South Australia, probably due to nodularin from an extensive bloom of Nodularia spumigena had occurred (Metcalf and Codd, 2012). From then, it is known that a wide range of animal species have succumbed to cyanotoxins, including fish, birds, bats, sheep, cattle, horses, pigs, and primates (Wood, 2016). Some reports point out several cases of dog poisoning by anatoxin-a(s) (ATXs) after drinking and bathing in contaminated waters in Scotland, France, Ireland, and USA (Manganelli et al., 2012). Furthermore, there are some cattle death attributed to microcystins (MCs), ATXs, and cylindrospermopsin (CYN) (Mez et al., 1997; Saker et al., 1999; Metcalf and Codd, 2012). Anatoxin-a(s) have been frequently associated with massive mortality of birds (Manganelli et al., 2012). The documented cases of human poisoning with cyanotoxins have been less frequent but also relevant (Metcalf and Codd, 2012). Most of these cases have been studied retrospectively, with little useful data for the exposure assessment. Nonetheless, epidemiological evidence is highly valuable in elucidating direct connections between cyanotoxins and health effects (Svircěv et al., 2017). In this sense, the main cyanotoxins present in short-term human intoxications were MCs in Brazil, England, and Argentina; CYN in Brazil and Australia, and cyanobacterial lipopolysaccharides (LPS) in USA and Brazil, causing mostly gastrointestinal, respiratory, and neuronal symptoms (Metcalf and Codd, 2012). In addition, there are some other cases where the role of cyanotoxins was not confirmed as the main cause of intoxication, but they could be a possible explanation. Even so, increasing evidence indicates that these secondary metabolites also have long-term human health implications, as MCs proved in China or β-N-methylamino-L- alanine (BMAA) in the USA, causing an increase in tumour promotion and neurodegenerative diseases (Metcalf and Codd, 2012). Among all the cyanotoxins, MCs and CYN are the most frequently detected and responsible for many intoxication cases (Wood, 2016). For this reason, they are the main object of this doctoral thesis. 1.1.3. Microcystins According to Catherine et al. (2017), about 23 cyanobacterial genera and 47 species are suspected to be MCs-producers. The main genera involved are Microcystis, Anabaena, Planktothrix/Oscillatoria, Anabaenopsis, Nostoc, and Chrysosporum (De Figueiredo et al., 2004). The general structure of MCs consists of a cyclo-(D-Ala-X-D-MeAsp-Z-Adda-Arg-D- Glu-Mdha) heptapeptide, where X and Z are variable L-amino acids in positions 2 and 4. D- MeAsp is D-erythro-b-methylaspartic acid, Mdha is methyldehydroalanine and Adda, which is essential for their toxic action, is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Fig. 3) (Chorus and Bartram, 1999; Machado et al., 2017). The overall structure of these toxins is quite hydrophilic due to the carboxylic acids at positions 3 and 6 and the presence of arginine in either 2, 4, or both positions (Catherine et al., 2017). More than 240 MC congeners have been reported to date, with molecular weight varying between 900 and 1100 Da, originating by different amino acid combinations and chemical variations such as methylation/desmethylation of some functional groups (Catherine et al., 2017). The major congeners are MC-LR, MC-RR, and MC-YR, with different combinations of leucine (L), arginine (R), or tyrosine (Y), being MC-LR the most studied due to its ubiquity, prevalence, and toxicity (Campos and Vasconcelos, 2010). Figure 3. Structure of MC-LR Microcystins are chemically stable molecules and relatively resistant to degradation, although biodegradation may occur (Metcalf and Codd, 2012). In this line, some species/strains of bacteria reported to degrade MCs in a matter of hours to days are Sphingomonas, Sphingocinicella, Arthrobacter, Brevibacterium, Rhodococcus, and Burkholderia (Kato et al., 2009; Mazur-Marzec and Plínski 2009). The degradation process starts by attacking the cyclic ring, degradation of the linear peptide to a tetrapeptide, and breakdown of Adda, causing a depletion of its toxicity (Metcalf and Codd, 2012). The MCs production is highly variable, depending on space and time, making it hard to predict from species composition and cyanobacterial abundance (Huisman et al., 2018). Regarding this, high light intensities and warm temperatures are related to increases in the transcription of the responsible genes for toxin production (Holland and Kinnear, 2013). For instance, MC-RR production seems to be influenced by high temperatures, while lower ones favour MC-LR synthesis (Rapala et al., 1997; Rapala and Sivonen, 1998; de Figueiredo et al., 2004). The effects of nitrogen and phosphorus concentrations are controversial on MCs content of cyanobacteria, but its production by Microcystis strains is influenced by variations in the ratio N:P, with different responses according to the strain (Vézie et al., 2002; de Figueiredo et al., 2004). Microcystins are mostly intracellular, constituting about 70% of the total MCs concentration in raw water (Graham et al., 2010; Buratti et al., 2017). However, high extracellular concentrations can be found because of blooms lysis (Rohrlack and Hyenstrand, 2007; Díez- Quijada et al., 2019b). These concentrations vary considerably both from one bloom to another and during the course of a single bloom (Buratti et al., 2017). In addition, genotype diversity between strains seems to be the main factor affecting toxicity levels in blooms of the same species (Rohrlack et al., 2001; Kurmayer et al., 2002; de Figueiredo et al., 2004). In this respect, the genotypes can differ in growth strategy, interaction with zooplankton, plasmids, MCs synthetase genes cluster, and MCs content, leading to different MCs variants with different toxic properties (Hesse and Kohl, 2001; Mikalsen et al., 2003; de Figueiredo et al., 2004). Concerning the purpose of MCs synthesis, there exist different explanations (de Figueiredo et al., 2004). They could act as a chemical defense against grazing (Kurmayer and Jüttner, 1999; Henning et al., 2001), have an allelopathic effect over algal competitors (Kearns and Hunter, 2001), as a nitrogen reserve or as a regulator of endogenous protein phosphatases (de Figueiredo et al., 2004). However, this remains still unclear (Humbert and Fastner, 2017). To avoid human toxicity, the World Health Organization (WHO) released a provisional drinking water guideline of 1 µg/L for MC-LR (WHO, 2020a). This value has been adopted as a guideline in many countries. However, some countries such as Canada and Australia proposed 1.5 mg/L and 1.3-10 mg/L, respectively (USEPA, 2001). Furthermore, Canada also proposed for short-term exposure the value of 10 mg/L (Fitzgerald, 2001; de Figueiredo et al., 2004). Also, WHO set the TDI value of 0.04 μg/kg bw/ day. Based on updated data, the Environmental Protection Agency (EPA, 2006) in USA proposed acute and chronic TDI guidelines (0.006 and 0.003 MC‐LR μg/ kg bw/day, respectively). 1.1.3.1. Geographical distribution The distribution and concentration in waterbodies of MCs depend on the biomass of the producer cyanobacteria, the amount and congeners in the cells, the share of toxigenic genotypes in a population, and the release of extracellular MCs into the water (Humbert and Fastner, 2017). Different MC congeners are frequently found at the same time in the same bloom (Catherine et al., 2017). Blooms of MC-producing cyanobacteria have been reported in 80 countries, being more relevant in Europe and North America (Fig. 4) (Buratti et al., 2017; Catherine et al., 2017). Figure 4. Blooms of MCs producers - cyanobacterial strains. In general, MCs are the most common cyanotoxins, with levels up to 126.42 μg/L in Asia, 11400 μg/L in Europe, and 10000 μg/L in the USA (Buratti et al., 2017). 1.1.3.2. Bioaccumulation of MCs In general, bioaccumulation patterns have been demonstrated to depend on the species, probably due to interspecific differences in selective ingestion and depuration rates (Kim et al., 2017; Díez-Quijada et al., 2019a). Among all the organisms able to bioaccumulate MCs, bivalves are very important due to their filtering capacity, providing a relevant human health threat since they are consumed on a regular basis in many countries (Ferrão-Filho and Kozlowsky-Suzuki, 2011; Gutiérrez-Praena et al., 2013). Thus, the edible mussel Mytilus galloprovincialis has been shown to accumulate higher quantities of MCs than the tolerable daily intake (TDI) established by WHO, which is of 0.04 µg/kg/day, with the consequent risks to humans (WHO, 1998; 2020a). In addition, crustaceans feed on phytoplankton, including cyanobacteria, so their metabolites can accumulate in their tissues. There are several studies of bioaccumulation in different species of crustaceans reporting higher values of MCs than those recommended as TDI by WHO (WHO, 1998; Chen and Xie, 2005; García et al., 2010). Normally, bivalves and crustaceans would be safer for humans if the target organs of MCs were removed before consuming them, but these organisms are usually fully eaten, providing risky levels of MCs for humans (Gutiérrez-Praena et al., 2013). Additionally, some of the most important aquatic organisms consumed worldwide are fish. Fish can be exposed to cyanotoxins by directly feeding on phytoplankton or food web, or by uptaking dissolved toxins via epithelium (Ibelings and Chorus, 2007; Gutiérrez-Praena et al., 2013). Fish are able to bioaccumulate toxins mainly in liver and muscle (Magalhães et al., 2001; Mohamed et al., 2003; Gutiérrez-Praena et al., 2013). In this respect, different studies on tilapia (Oreochromis niloticus) and carps (Cyprinus carpio) exposed to MCs-producing blooms in different reservoirs found that the TDI value was exceeded in most of the samples analyzed. The higher MCs concentrations were found in guts > liver > kidney > muscle, changing the concentrations according to the different depuration abilities of the fish (Mohamed et al., 2003; Chen 2006, 2007; Deblois et al., 2008; Guzmán-Guillén et al., 2017). Although is not the main organ containing higher concentrations of MCs, muscle has demonstrated to bioaccumulate higher concentrations than the TDI, constituting a potential human health risk (Gutiérrez-Praena et al., 2013). In addition, MCs bioaccumulation by aquatic animals is a multifactorial process depending on their feeding guild, physiology, length, time of exposure, accumulation, depuration, and metabolic rates of the fish species (Jia et al., 2014; Díez-Quijada et al., 2019a). Furthermore, it has also been demonstrated that different cooking procedures (microwave oven, boiling, steaming etc.) can affect the level of different MC-congeners reaching humans (Zhang et al., 2010; Guzmán-Guillén et al., 2011; Buratti et al., 2017). Although aquatic ecosystems are an important source of cyanotoxins-bioaccumulation, cyanobacterial blooms can also appear in water reservoirs for plant irrigation. In this sense, plants can also be exposed to MCs and accumulate them (Gutiérrez-Praena et al., 2013). Several studies performed in different plant species such as mustard, broccoli, clover, rape, parsley, rice, apple, spinach, or lettuce among others, demonstrated their capacity for MCs-bioaccumulation and the effects of these cyanotoxins in their physiology (Kós et al., 1995; Crush et al., 2008; Mohamed and Al Shehri, 2009; Chen et al., 2010; 2012; Prieto et al., 2011; Gutiérrez-Praena et al., 2013; Freitas et al., 2015; Buratti et al., 2017). Crush et al. (2008) also concluded that plants with big leaves retain more contaminated water than the ones with small leaves. However, edible plants accumulate MCs not only on the leaves but also in the roots and sprouts (Díez-Quijada et al., 2019a). In general, the results obtained predict the exposure of humans to higher concentrations than the TDI for MCs (Gutiérrez-Praena et al., 2013, WHO, 2020a). recognize their blooms for the naked eye (Yang et al., 2021). Nonetheless, this cyanotoxin has been also detected in different species, causing an increase in its presence around the globe (Fig. 7). In general, CYN has demonstrated to be present in higher concentrations in freshwaters influenced by human activities (WHO, 2020). Figure 7. Blooms of CYN producerscyanobacterial strains Different studies have been performed in order to elucidate the effects of environmental factors on CYN production, such as temperature, nitrogen and phosphate sources, and light intensity, which have been demonstrated to vary among the different CYN-producers (Kokociński et al., 2019). In general, these producers-organisms can be found in lakes, reservoirs, rivers, ponds, and dams (Kokociński et al., 2019). In regard, their adaptability together with climate change and increasing eutrophication is allowing these species to expand into habitats that were not suitable before, making it likely to lead to an ever-increasing incidence of CYN in the future (Scarlett et al., 2020). Furthermore, the CYN-congeners deoxy-CYN and 7-epi-CYN have been detected in cyanobacterial blooms of R. raciborskii, R. curvata, R. mediterranea, A. ovalisporum, and Oscillatoria spp. (Mazmouz et al., 2010; Mc Gregor et al., 2011; Li et al., 2001; Norris et al., 1999; Banker et al., 2000; Kokociński et al., 2019). 1.1.4.2. Bioaccumulation of CYN The bioaccumulation studies available are scarce and dependent on the species and the CYN conditions (purified, in a cell extract, in a producer species, etc.), although the results are worth mentioning. In this sense, CYN has demonstrated its ability to be bioaccumulated by a range of organisms from different parts of the food chain, including mollusks, fish, toads, and plants (Kinnear, 2010). In mollusks, Saker and Eaglesham (1999) exposed Anodonta cygnea to 14-90 µg/L CYN for 16 days, obtaining bioaccumulation in haemolymph, viscera, mantle, foot, and gonads (Kinnear, 2010). Furthermore, after 14 days of depuration, almost 50% of the toxin remained in the tissues (Saker and Eaglesham, 1999; Kinnear, 2010). Likewise, Anderson et al. (2003) reported CYN accumulation levels of 130-560 µg / kg in Alathyria pertexta after being exposed to less than 0.8 µg/L CYN (Kinnear, 2010). Concerning the study performed by Berry and Lind (2010) in Tegogolo snails (Pomecea patula catemacensis), their field CYN exposure gave a BCF value of 157, while the environmental concentrations measured were of 20 ng/L CYN, demonstrating their bioaccumulation even at low CYN concentrations (Berry and Lind, 2010; Kinnear, 2010). In addition, White et al. (2006) studied the CYN-bioaccumulation in Melanoides tuberculata exposed over seven and fourteen days (Kinnear, 2010). They also found much higher bioaccumulation when exposed to live cultures of R. raciborskii than to cell extracts containing the aqueous toxin, in agreement with the results obtained by Seifert (2007) in Lemna puntata (White et al., 2007; Kinnear, 2010). Moreover, White et al. (2007) reported bigger concentrations of deoxy-CYN than of CYN (Kinnear, 2010). This is in agreement with the results obtained by Seifert (2007) who observed a much bigger bioaccumulation value for muscle and viscera in Corbiculina australis mussels for deoxy-CYN than for CYN when exposed to a known CYN-producing bloom (Kinnear, 2010). The studies concerning CYN-bioaccumulation in crustaceans also point out its ability to stay in the organism. In this sense, Saker and Eaglesham (1999) detected CYN values of 900 and 4300 µg/kg freeze-dried tissue from muscle and hepatopancreas, respectively, in crayfish Cherax quadricarinatus from an aquaculture pond containing 589 µg/L CYN (Kinnear, 2010). Seifert (2007) confirmed these results in Cherax crayfish (Kinnear, 2010). However, the study performed in Daphnia magna exposed to C. raciborskii lead to a non-significant CYN-bioaccumulation after 24 and 48 hours (Nogueira et al., 2004). In the bioaccumulation studies using fish, the results are also species-dependent. The first authors reporting CYN bioaccumulation were Saker and Eaglesham (1999), in rainbow fish Melanotaenia eachamensis, with values of 1200 μg/kg freeze-dried tissue. Furthermore, Seifert (2007) reported the bioaccumulation of both CYN and deoxy-CYN in eel-tailed catfish (Tandanus tandanus), but not in Australian bass (Macquaria novemaculeata), golden perch (M. ambigua), or silver perch (Bidyanus bidyanus) (Kinnear, 2010). In this line, Berry et al. (2012) detected CYN in muscle tissues from different fish species such as Bramocharax caballeroi, Cichlasoma uropthalmus, C. helleri, Dorosoma mexicana, Heterandria jonesii, Oreochromis aureus, Rhamidia sp., Vieja sp. and V. finestrata (Berry et al., 2012; Scarlett et al., 2020). In addition, Guzmán-Guillén et al. (2015) reported bioaccumulation of CYN after 14 days of exposure in the brain of tilapia, reducing the levels after 3 and 7 days of depuration (Guzmán-Guillén et al., 2015). This is in agreement with the detection of CYN in muscle, liver and brain of Hoplias malabaricus exposed for 7 and 14 days to both purified CYN and CYN-producing cyanobacterial extract (da Silva et al., 2018). Moreover, there is only one study concerning the CYN-bioaccumulation in tadpoles of the cane toad Bufo marinus. In this experimental model, White et al. (2007) reported BCF of 19.27 after exposure to live C. raciborskii cultures containing CYN, although no bioaccumulation was detected after their exposure to whole-cell extracts (White et al., 2007; Kinnear, 2010). Regarding the studies concerning CYN-bioaccumulation by plants, White et al. (2005) and Kinnear et al. (2007) reported little concentrations of CYN after exposure to Hydrilla verticillata and Lemna punctata, respectively (Kinnear, 2010). In this aspect, Seifert (2007) also reported in Lemna punctata a low bioconcentration factor (BCF). However, when cell extracts were used instead of the purified toxin, the BCF values were up to 86.67 (Kinnear, 2010). Furthermore, the BCF decreased with increasing CYN exposure concentrations in arugula (Eruca sativa Mill.) and lettuce (Lactuca sativa L.) after 3, 5, and 10 mg/L (Cordeiro-Araújo et al., 2017). For all of this, a provisional TDI of 0.03 µ/kg bw has been established for CYN (WHO, 2020). 1.1.4.3. Intoxication reports The most famous case of human intoxication due to CYN was through contaminated drinking water, causing an outbreak of hepatic enteritis on Palm Island, Queensland, Australia, known as “Palm Island Mystery Disease” in 1979. In this incident, over 139 children and 10 adults showed various symptoms of gastroenteritis, such as anorexia, vomiting, headache, hepatomegaly, initial constipation followed by bloody diarrhea and dehydration (Byth, 1980; Hawkins et al., 1985; Griffiths and Saker, 2003; Codd et al., 2016). This case occurred after a few days of the treatment with copper sulfate of the Solomon Dam reservoir to control an algal bloom containing R. raciborskii, from where all the patients reported the intake of drinking water. This water treatment caused cellular lysis, and thus, the release of CYN (Metcalf and Codd, 2012; Zanchett and Oliveira-Filho, 2013). Furthermore, CYN was also postulated to be implied in the Caruaru syndrome. However, as no CYN quantification was reported, its contribution to the toxic effects observed cannot be evaluated (Carmichael et al., 2001; Buratti et al., 2017). In addition, there are cases of animal intoxication by contaminated water intake, causing the death of fish, birds, and cattle (Yang et al., 2021). 1.1.4.4. Toxicity of CYN 1.1.4.4.1. Toxicokynetics Due to its hydrophilic properties, CYN is unlikely to cross the lipidic bilayer-membrane cell, requiring the mediation of the bile acid transport system (Runnegar et al., 2002; Froscio et al., 2009; Pichardo et al., 2017b). However, passive diffusion through biological membranes has also been demonstrated because of its small molecular weight (Chong et al., 2002; Buratti et al., 2017). Cylindrospermopsin uptake varies among different types of cells, as it seems to be fast in primary mouse hepatocytes (Froscio et al., 2003) and slow in kidney or colon cells (Froscio et al., 2009a,b; Fernandez et al., 2014). In this sense, the main pathway involved in CYN uptake in intestinal cells seems to be the paracellular route, with a minor carrier-mediated transcellular transport H+ and GSH-dependent (Pichardo et al., 2017a,b). However, this uptake has not been reported so far in other cell lines and in Caco-2 cells is very limited (Pichardo et al., 2017a,b). In general, the differences in the toxicity exerted by CYN in different experimental models could be linked to their metabolic capacity (Pichardo et al., 2017a). Regarding this, although no studies have demonstrated direct CYN biotransformation, the cytochrome P450 (CYP450) enzymatic system seems to be involved in the bioactivation of CYN, as some reports have demonstrated with both inducers and inhibitors of CYP450 on CYN toxicity (Norris et al., 2002; Froscio et al., 2003; Humpage et al., 2005; Bazin et al., 2010b). Accordingly, some authors have reported that S9 fraction-inducing metabolism led to an increase in the cytotoxic and genotoxic effects of CYN (Runnegar et al., 1994; Lankoff et al., 2008; Froscio et al., 2003; Puerto et al., 2018; Pichardo et al., 2017a). Furthermore, it has been demonstrated the ability of CYN to upregulate genes coding for phase I enzymes (CYP1A1, CYP1B1, ALDH1A2, and CES2) and phase II enzymes (UGT1A6, UGT1A1, NAT1, and GSTM3) in HepG2 cells (Štraser et al., 2013). However, Ließel et al. (2015) reported no increase in CYN toxicity after pre-inducing the expression of CYPs. In addition, Kittler et al. (2016) reported no phase I biotransformation after exposure of HepaRG cells, a metabolically competent human cell line, to CYN after 24 hours. Nonetheless, when exposed to the CYP3A4 inhibitor ketoconazole, the cytotoxicity was significantly decreased (Kittler et al., 2016). Besides, a substantial GSH depletion was observed after oral CYN administration to rats, meaning a possible CYN conjugation with GSH or a GSH synthesis-inhibition by CYN (Runnegar et al., 1995; Buratti et al., 2017). Concerning its excretion, both urinary and fecal pathways have demonstrated to be quite rapid, requiring 0-12 hours for a complete excretion, not being almost observable after 24 hours (Norris et al., 2001). However, the studies regarding its elimination are still limited (WHO, 2020). 1.1.4.4.2. Mechanisms of action The mechanisms of action for CYN are not well elucidated yet (WHO, 2020). However, it is known that they depend on different factors such as the magnitude and frequency of dose, exposure duration, and the characteristics of the exposed organism, such as life stage, age or sex (WHO, 2020). In this regard, CYN seems to act through different mechanisms (Fig. 8): Figure 8. Mecanisms CYN toxicity (based on Yang et al., 2021) - Complete and irreversible concentration-dependently protein synthesis-inhibition, both in vitro and in vivo, which leads to hepatic and renal (cyto)toxicity (Froscio et al., 2003; Terao et al., 1994; Buratti et al., 2017). In addition, stomach, lungs, small intestine, thymus, and spleen can also be affected by histopathological lesions and cellular necrosis (Codd et al., 2019). In this regard, Terao et al. (1994) described the liver as the main target for CYN, which leads to protein synthesis inhibition, membrane proliferation, fat droplet accumulation, and cell death. This blockage, caused by the uracil moiety and the hydroxyl at C7 position, occurs at ribosomic level during the peptide chain elongation (Banker et al., 2001; Froscio et al., 2003; Machado et al., 2017). However, when CYP450 inhibitors were used, CYN toxicity decreased but no protection against the effect on protein synthesis was observed (Froscio et al., 2003; Buratti et al., 2017). Thus, the possible metabolites could exert toxicity through different mechanisms (Buratti et al., 2017). - Apoptosis and morphological alterations were observed in HUVEC and Caco-2 cells (Gutiérrez-Praena et al., 2012a,b; Buratti et al., 2017). - Increase of the oxidative stress biomarkers which, as a consequence, activate the p53 transcription factor, as described in cultured human dermal fibroblasts and HepG2 (Bain et al., 2007; Buratti et al., 2017). Cylindrospermopsin is thought to be the native toxin, as those cell lines do not present a significant metabolic activity (Buratti et al., 2017). Furthermore, intracellular formation of ROS was reported in HepG2 and human umbilical vein endothelial cell line (HUVEC), detecting in the last one an increase of c-glutamylcysteine synthetase and GSH content as well (Štraser et al., 2013c; Gutiérrez-Praena et al., 2012, Buratti et al., 2017). In this sense, Runnegar et al. (1994; 2002) found a decrease of GSH in rat hepatocytes, which was attributed to the inhibition of GSH synthesis (Codd et al., 2016). Besides, a synergistic effect was observed after exposure to both CYN and a GSH inhibitor in the same experimental model (Soldatow et al., 2013). However, the results concerning the effects of CYN in GSH are not consistent. In this line, Ließel et al. (2011) did not find any alterations in the primary hepatocytes from the fish Prochilodus lineatus, while Gutiérrez-Praena et al. (2011) found a significant increase at low CYN concentrations but a reduction after exposure to high in fish PLHC-1 cells (Pichardo et al., 2017a). The depletion of GSH could be directly correlated to the increase in ROS levels and, these, to other toxic mechanisms, such as genotoxicity (Humpage et al., 2005; Pichardo et al., 2017a). However, this decrease on GSH levels could also be due to the protein-synthesis inhibition (WHO, 2020). The studies concerning other oxidative stress biomarkers such as lipoperoxidation or CAT are also varied (Pichardo et al., 2017a). Nonetheless, GSH reduction is not considered to contribute meaningfully to CYN acute toxicity in vivo (Norris et al., 2002; Pichardo et al., 2017a). In fact, when a toxicogenomic study was performed by Štraser et al. (2013c) on HegG2, only catalase (CAT) and thioredoxin reductase (TXNRD1) were upregulated, while NOS2, SOD1, or glutathione enzymes were not affected (Štraser et al., 2013c; Buratti et al., 2017). - Connected with the oxidative stress damage, CYN has also been described as progenotoxic (Moreira et al., 2012; Pichardo et al., 2017b). This is based on its potentially reactive guanidine and sulphate groups (Shen et al., 2002; Humpage et al., 2000), favouring the interaction with nucleic acids (Bain et al., 2007; Pichardo et al., 2017b). The pro-genotoxic properties of CYN have been widely demonstrated in eukaryotic cells in vitro (Žegura et al., 2011; Puerto et al., 2018). In this sense, Humpage et al. (2005) and Bazin et al. (2010) demonstrated that CYP450 inhibitors such as omeprazole, SKF25A, or ketoconazole inhibited the genotoxicity induced by CYN alone, which indicates that the metabolism by CYP450 is needed for its genotoxicity (Humpage et al., 2005; Bazin et al., 2010). Furthermore, DNA damage was induced by CYN in HepG2 cells and human peripheral blood lymphocytes cells at non-cytotoxic concentrations, together with an upregulation of some CYP450 genes (Štraser et al., 2011; Žegura et al., 2011). However, when the Ames test was performed, no changes were observed after exposure to CYN with or without metabolic activation (Sierolawska, 2013; Buratti et al., 2017), which is in agreement with the results obtained in CHO-K1 cells by Fessard and Bernard (2003) and Lankoff et al. (2007). It is also relevant that CYN also led to an overexpression of the proteins involved in DNA repair, together with modifications of nucleosomal histones in Caco-2 cells (Huguet et al., 2014; Buratti et al., 2017). In agreement, Štraser et al. (2013a,b) observed DNA damage oxidative stress-independent after exposure of HepG2 cells to non-cytotoxic concentrations of CYN. In vivo, CYN induces DNA-strand breakage in colon and bone marrow after oral administration to 1-4 mg/kg in mice after 24 hours (Bazin et al. 2012). However, the doses tested were quite high, which could induce cytotoxicity and thus, DNA damage (Buratti et al., 2017). Therefore, the genotoxicity potencial of CYN is still controversial (Buratti et al., 2017; Díez-Quijada et al., 2019c, 2020). 2. Pesticides In addition to cyanotoxins, freshwater reservoirs can contain some pollutants able to cause environmental toxicity. In fact, the occurrence of harmful chemicals in the environment has become an issue of great concern in the last decades, being pesticides one of the most important groups (Bao et al., 2015; Damalas and Koutroubas, 2016). Pesticides are chemical compounds designed to protect agricultural products from insects, undesirable plants, or fungal diseases, which led to a noticeable increase in crop yields and food production (WHO, 1996; Alexandratos and Bruisnma, 2012; Carvalho, 2017). These chemicals are applied annually in millions of tons of yields throughout the world (EPA 2017b; Dar et al., 2020), out of which 45% are applied in Europe and 25% in the USA (De et al., 2014; 2020). Despite being an agricultural advantage, their overuse leads to high toxicity, affecting soil, water, and natural resources where can be present, which leads to several environmental, animal, and human health issues (Toni et al., 2006; Bhardwaj et al., 2020). In this regard, a considerable fraction of the pesticides applied is dispersed into the environment, being found in every habitat, and are routinely detected in both aquatic and terrestrial animals (Vorley and Keeney, 1998; Law, 2014; Choudhary et al., 2018). Due to their perdurability, these compounds can be found after a long period, being able to exert toxic effects and disturb ecological balance by harming non-target organisms or causing genetic modifications that may lead to resistance (Andersson et al., 2014; Asghar et al., 2016). Based on their chemical structure, pesticides can be divided into organochlorines, organophosphorus, carbamates, pyrethroids, and neonicotinoids (Hakeem et al., 2017). From them, organophosphates (OPs) account for more than one third of the total pesticides globally consumed (Singh and Walker, 2006; Dar et al., 2020). 2.1. Organophosphates These pesticides need to be metabolically activated via CYP450 through a desulfuration reaction to form an oxon (Hodgson and Rose, 2007; Richardson et al., 2019). These chemicals are extensively applied in agriculture, horticulture, veterinary medicine, domestic purposes, and control of disease vectors (Dar et al., 2020). Their massive use is due to their high effectiveness against target pests and their relatively low toxicity to non-target organisms compared to other pesticides (Singh and Walker, 2006; Vijayalakshmi and Usha, 2012). Due to their widespread availability and their high-intensity use, OPs are associated with morbidity and mortality through occupational and accidental exposure or via suicidal attempts (Rohlman et al., 2011; Roman et al., 2019). General population may be exposed to pesticide residues in food and drinking water on a daily basis or to pesticide drift in residences close to spraying areas (Damalas and Eleftherohorinos, 2011; Damalas and Koutroubas, 2016). In this sense, according to the WHO, about 3 million people are exposed to OPs worldwide, leading to 300000 deaths each year (WHO, 2001; Roman et al., 2019). On the other hand, the treatment with OPs of agricultural products can lead to their discharge into the soil, leaching into groundwater, contaminating drinking water, and getting drift and thus, causing also air pollution (Dar et al., 2020). In addition, OPs are mainly used due to their degradable organic nature and less persistence in the environment compared to other pests (Yang et al., 2005; Dar et al., 2020). However, despite their expected rapid degradation in aquatic systems, residual concentrations have been detected in water, soil, food, and human fluids, demonstrating their persistence for days/weeks and their bioaccumulation (Maurya and Malik, 2016; Carvalho et al., 1992; 2017). The bioaccumulation process usually is the consequence of the runoff from pesticide-contaminated agricultural land areas into the waterbodies such as streams, rivers, and oceans (Dar et al., 2020). Thus, these contaminants are ingested by fish and accumulate into the food chain, reaching humans (Maurya and Malik, 2016; Dar et al., 2020). Thus, OPs have been found in blood, urine, semen, breast milk, amniotic fluid, adipose tissue, and umbilical cord blood from humans (CSE report 2005; Kumar et al., 2016). The main routes of exposure for OPs are the dermal route (by direct chemical contact), the oral route (ingestion of contaminated food or water), and inhalation (Sacramento, 2008; Dar et al., 2020). 2.1.1. Mechanisms of action In general, all OPs present similar mechanisms of action, mainly affecting the synapsis: - Irreversible inhibition of acetylcholinesterase (AChE): this inhibition occurs after phosphorylation of the hydroxyl group of the Serine 203 at the active site of the enzyme AChE (covalent bond), creating an AChE-OP complex. This leads to the accumulation of acetylcholine (Ach) at cholinergic synapses and neuromuscular junctions, causing a disruption of the nervous impulse transmission at a synaptic level, leading to overstimulation (Kwong, 2002; Carvalho, 2017; Jokanovic, 2018). Death usually occurs as a consequence of respiratory failure from a combination of central and peripheral effects, paralysis of the respiratory muscles, and depression of the brain respiratory center (Karchmar, 2007; WHO, 1986; IPCS, 1998; Clark, 2002; Eyer, 2003; Marrs and Vale, 2006; Jokanović et al., 2011; 2018). The hazardous effects include impairment in normal body functioning, neurotoxicity, carcinogenicity, reproductive, and several other problems (Singh et al. 2007; Bhardwaj et al., 2020). - Overstimulation of nicotinic and muscarinic acetylcholine receptors (AChRs and mAChRs): is a consequence derived from the AChE inhibition (Jokanović, 2018). The high stimulation of mAChR and nAChR disrupts the balance of glutamatergic, GABAergic, and cholinergic activities and changes the concentration of Ca2+ both inside and outside the cells (Aluigi et al., 2005; Grasshoff et al., 2003; Firozjaei et al., 2015). This change in Ca2+ concentrations affects ion concentration, hyperosmolarity, and protein function in the endoplasmic reticulum membrane, indicating possible prolonged effects on the structure and function of the brain (Angelini et al., 2004; Wade et al., 1987; McDonough and Shih, 1997; Piekut and Phipps, 1999; Firozjaei et al., 2015). For 50 years, organophosphorus (OP) pesticides have been the most heavily and ubiquitously used worldwide, accounting for more than 50% of the total insecticide use worldwide being chlorpyrifos (CPF) on of special relevance (Burke et al., 2017; Rahman et al., 2021). 2.1.2. Chlorpyrifos Chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) is a broadspectrum chlorinated organophosphate insecticide (Fig. 9) used for pest control on crops such as corn, citrus fruits, cotton, nuts, beets, legumes, potatoes, etc. (Alizadeh et al., 2018), lawns and ornamental plants, being detected in most of the countries worldwide (Lai, 2020; John and Shaike, 2015). Its overuse is due to its low persistence, cost-efficiency, wide-ranging applicability, and low range of toxicity (Rahman et al., 2021). In this regard, CPF is registered for its use in nearly 100 countries and is annually applied to 8.5 million crops acres (Mukti et al., 2018; Koly and Khan, 2018). Furthermore, this pesticide is the most used one in the European Union (1226 tons, Eurostat) and one the most common in the United States (8-11 million pounds applied in 2007 (US EPA, 2016; Lai, 2020). This excessive and continuous use in agriculture led to the persistence of CPF residues in various environmental compartments, such as soil, water, and air, being detected even at 24 kilometers from the application site (Vijayalakshmi and Usha, 2012; Koly and Khan, 2018). However, this OP has been recently removed from the list of allowed chemicals in EU (EFSA, 2019). Figure 9. CPF chemical structure. In general, CPF enters the soil environment by direct pesticide application, leading to its detection in edibles, sediments, streams, sumps, reservoirs, rivers, sea, urban storm drains, freshwater lakes, groundwater, ice, snow, fog, rain and air in many countries, including the artic regions (Pengphol et al., 2012; Watts, 2012). However, its absortion depends on many environmental factors (John and Shaike, 2015). The half-life of CPF in soil generally depends on many factors such as soil microorganisms, application rate, pH, moisture content, pesticide formulation, organic carbon content, and climatic conditions (John and Shaike, 2015). However, its persistence has been shown to vary from a few days up to 4 years (Rahman et al., 2021). Chlorpyrifos has been demonstrated to exert acute toxicity in insects, birds, mammals, and aquatic life, by the affectation of the CNS as well as cardiovascular, endocrine, and respiratory systems in non-target organisms (Kavitha and Rao, 2008; Shafiq-Ur-Rehman and Waliullah, 2012). In general, in target and non-target organisms, acute exposure to CPF can lead to tingling sensation, numbness, dizziness, vomiting, sweating, nausea, stomach cramps, headache, incoordination, muscle twitching, vision disturbances, slurred speech, drowsiness, confusion, anxiety, depression and, in worst cases, respiratory arrest, unconsciousness, convulsions, and even death (Rayu et al., 2017). The main routes of exposure for CPF in humans are through contact, ingestion, and inhalation, as 70% of the absorption is by oral route and less than 3% through dermal contact (Alizadeh et al., 2018). In this sense, the exposure to CPF occurs mainly through contaminated environment and dietary intake (Saunders et al., 2012; Todd, 2017). 2.1.2.1. Toxicity of CPF The liver is the main responsible for CPF metabolism, being usually excreted without bioaccumulation through urine within 24 hours (Griffin et al., 1999; Wagner et al., 1999; Lai, 2020; Alizadeh et al., 2018). However, animals convert CPF to its oxidized forms “oxon”, which 1. Significance Taking into account the previously mentioned, it would be of interest to study the effects that the combinations of CYN + MC-LR and CYN + CPF could exert in the human nervous system, together with the neurotoxic properties of CYN, using for this purpose the human neuronal cell line SH-SY5Y and murine primary neuron cultures. The SH-SY5Y cell line is one of the most commonly used experimental models among all the available for neurotoxicity studies (Todd, 2017). This cell line is a subclone of SK-SN-SH neuroblastoma cell line isolated from a bone marrow of a 4-year-old female patient, which possesses a stable karyotype of 47 chromosomes (Shipley et al., 2016, 2017). One of the most important characteristics of this immortal cell line is its capacity to differentiate, resembling, chemically and morphologically, different mature neuronal populations (Påhlman et al., 1995; Shipley et al., 2016; Todd, 2017). Furthermore, the SH-SY5Y cell line presents a basal expression of neurotransmitters, essential for dopaminergic and cholinergic signaling, making this cell line a really interesting experimental model for the study of the Parkinson’s or Alzheimer’s diseases, among others (Agholme et al., 2010; Kovalevich et al., 2013; Xicoy et a., 2017; Xie et al., 2010; Todd, 2017). Undifferentiated SH-SY5Y cells proliferate rapidly and are morphologically characterized as neuroblast-like non-polarized cells, expressing immature neuronal markers (Kovalevich and Langford, 2013; Shipley et al., 2016; Todd, 2017). In this regard, these cells are considered most reminiscent of immature catecholaminergic neurons (Kovalevich and Langford, 2013). However, during differentiation, the proliferation rate decreases, and cells start to express mature neuronal protein markers (Shipley et al., 2016). In addition, the differentiation process leads to the formation and extension of neuritic processes, electrical excitability of the plasma membrane, formation of synaptophysin-positive functional synapses, expression of various neurotransmitters, and neuron-specific enzymes (Kovalevich and Langford, 2013). When fully differentiated, some of the markers of mature neurons appear in this cell line: neuronal nuclei (NeuN), synaptophysin (SYN), growth-associated protein (GAP-43), neuron-specific enolase (NSE), synaptic vesicle protein II (SV2), and microtubule-associated protein (MAP), among others (Gimenez-Cassina et al., 2006; Påhlman et al., 1984; Xie et al., 2010; Cheung et al., 2009; Shipley et al., 2016). Furthermore, the removal of the trophic factor BDNF leads to cellular apoptosis, as occurs in mature neurons (Encinas et al., 2000; Shipley et al., 2016). There are different procedures to differentiate cells according to the subtype of interest according to the specific neuronal subtype population desired. In this respect, RA is a vitamin A derivative known for causing inhibition of cellular growth and differentiation-promoting properties (Kovalevich and Langford, 2013). This differentiation leads to a cholinergic neuron phenotype, with is evidenced by the increase in the choline acetyltransferase (ChAT) activity expression and vesicular monoamine transporter (VMAT) expression (Lopes et al., 2010; Presgraves et al., 2004; Kovalevich and Langford, 2013). Furthermore, the expression of both mAChRs and nAChRs have been reported in SH-SY5Y cells differentiated with RA (Kovalevich and Langford, 2013). The murine neuronal primary cultures are also an important experimental model for neurotoxicity and developmental neurotoxicity studies, as they are real neurons. Thus, these cultures allow the examination of the activity and properties of neurons at cellular and synaptic levels (Nunez, 2008). In addition, the main difference between cell lines and primary cultures is that the primary cultures do not derive from tumours, which makes of these cells a more interesting cell model in terms of neuronal properties in vivo. However, this type of culture is hard to maintain, as mature neurons do not proliferate. Furthermore, it is required to separate the cell type of interest from the rest, such as glial cells (Gordon et al., 2013). Both differentiated SH-SY5Y and primary cultures express biomarkers of interest. In this sense, the NeuN protein is a commonly used marker to identify post-mitotic neurons, as is expressed exclusively in nervous mature cells (Todd, 2017). In addition, the MAP-2 is a protein that stabilizes dendrites in post-mitotic neurons (Soltani et al., 2005). The expression of this protein depends on growth factors and neurotransmitters, and also appears during cell proliferation and differentiation, although not only in neuronal tissues (Johnson and Jope, 1992; Todd, 2017). Concerning the synapsis, synaptophysin and postsynaptic density protein 95 (PSD95) are pre- and postsynaptic proteins, respectively. Synaptophysin is an integral membrane protein of small synaptic vesicles and is found in all nervous tissue, while PSD95 is a synaptic protein that interferes in the synaptic maturation by interacting with several receptors of the postsynaptic membrane (Glantz et al., 2008; Coley and Gao, 2018). Both proteins are of great interest for investigating the neuronal synaptic function. 2. Purposes Therefore, the specific aims established in the present Doctoral Thesis have been: 1. To perform a comprehensive literature review of the studies performed in vitro and in vivo concerning the neurotoxic effects of MCs and CYN. 2. To assess the effects of MC-LR, the most abundant MCs variant, and CYN in cell viability, oxidative stress generation, AChE activity, and morphological alterations, alone and in combination, in undifferentiated and differentiated SH-SY5Y cells. 3. To study the effects of CPF isolated and in combination with CYN, in the undifferentiated and differentiated SH-SY5Y cell line concerning viability, oxidative stress parameters, AChE activity and morphological alterations. 4. To evaluate the effects of a CYN-producer and non-producer cyanobacterial extracts in the SH-SY5Y cell line using viability and oxidative stress as biomarkers. 5. To study the effects of pure CYN in murine primary cultures in terms of viability and synapsis by immunocytochemistry. 6. To study the effects of CYN, CPF and their combination in the nAChRs in order to assess a possible mechanism of action acute toxicity in 3 days-differentiated SH-SY5Y. Furthermore, studies concerning the effect on developmental neurotoxicity of these toxics in SH-SY5Y during 6 days of differentiation by assessing their effect on cell viability and neurite outgrowth. The experimental work for aims 1-6 h was performed at the Area of Toxicology of the Faculty of Pharmacy of the University of Sevilla, using the infrastructures from the Biology and Microscopy Services of the Centro de Investigación, Tecnología e Innovación of the University of Sevilla (CITIUS) as support. The morphological analysis was performed in collaboration with Dr. Francisco Javier Moreno, from the Ultrastructural Cytochemistry group of the Department of Cellular biology from the Faculty of Biology of the University of Sevilla. The experimental work for aim 7 was performed at the Department of Biochemistry and Biophysics of the Stockholm University, under the supervision of Dr. Anna Forsby. Furthermore, an internship for 7 months that led to a contract for 10 more months was performed in Anna Forsby’s group. In this sense, one research work has been published in collaboration with the EU-Tox Risk project. In addition, according to the regulations from the University of Sevilla, this thesis has been completely written in English to fulfill the requirements to aim the PhD with International Mention. III. RESULTS CHAPTER 1 María Gracia Hinojosa, Ana Isabel Prieto, Daniel Gutiérrez Praena, Francisco Javier Moreno, Ana María Cameán, Ángeles Jos NEUROTOXIC ASSESSMENT OF MICROCYSTIN-LR, CYLINDROSPERMOPSIN AND THEIR COMBINATION ON THE HUMAN NEUROBLASTOMA SH-SY5Y CELL LINE Chemosphere. 2019;224:751-764. UNCORRECTED PROOF Chemosphere xxx (xxxx) xxx-xxx Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com Neurotoxic assessment of Microcystin-LR, cylindrospermopsin and their combination on the human neuroblastoma SH-SY5Y cell line M.G. Hinojosa a, A.I. Prieto a, D. Gutiérrez-Praena a, ∗, F.J. Moreno b, A.M. Cameán a, A. Jos a aÁrea de Toxicología, Facultad de Farmacia, Universidad de Sevilla, C/Profesor García González 2, 41012, Sevilla, Spain bÁrea de Biología Celular, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes s/n, 41012, Sevilla, Spain ARTICLE INFO Article history: Received 29 October 2018 Received in revised form 12 February 2019 Accepted 24 February 2019 Available online xxx Handling Editor: A. Gies Keywords: MC-LR CYN MC-LR+CYN combination SH-SY5Y cells Neurotoxicity ABSTRACT Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN) are produced by cyanobacteria. Although being considered as a hepatotoxin and a cytotoxin, respectively, different studies have revealed neurotoxic properties for both of them. The aim of the present work was to study their cytotoxic effects, alone and in combination, in the SH-SY5Y cell line. In addition, toxicity mechanisms such as oxidative stress and acetylcholinesterase (AChE) activity, and morphological studies were carried out. Results showed a cytotoxic response of the cells after their exposure to 0–100μg/mL of MC-LR or 0–10μg/mL CYN in both differentiated and undifferentiated cells. Thus, CYN resulted to be more toxic than MC-LR. Respect to their combination, a higher cytotoxic effect than the toxins alone in the case of undifferentiated cells, and almost a similar response to the presented by MC-LR in differentiated cells were observed. However, after analyzing this data with the isobolograms method, an antagonistic effect was mainly obtained. The oxidative stress study only showed an affectation of glutathione levels at the highest concentrations assayed of MC-LR and the combination in the undifferentiated cells. A significant increase in the AChE activity was observed after exposure to MC-LR in undifferentiated cells, and after exposure to the combination of both cyanotoxins on differentiated cells. However, CYN decreased the AChE activity only on differentiated cultures. Finally, the morphological study revealed different signs of cellular affectation, with apoptotic processes at all the concentrations assayed. Therefore, both cyanotoxins isolated and in combination, have demonstrated to cause neurotoxic effects in the SH-SY5Y cell line. © 2019. 1. Introduction Cyanobacteria are present in a variety of aquatic and terrestrial ecosystems due to their adaptive ability, even in extreme conditions (Svirčev et al., 2014). Under favorable conditions of light, pH, nutrients (nitrogen and phosphorus) and interaction with other organisms, they present the capability of forming blooms and producing secondary metabolites called cyanotoxins, whose occurrence is increasing due to long term climate change (Buratti et al., 2017). According to their target organ, these toxins can be classified as hepatotoxins (e.g. microcystins, nodularins), dermatotoxins (e.g. lungbyatoxin), neurotoxins (e.g. anatoxin-a, homoanatoxin, saxitoxins), irritant toxins (e.g. lipopolysaccharides) and cytotoxins (e.g. cylindrospermopsin) (Testai et al., 2016). The exposure to these metabolites can occur by different paths such as the oral route, dermal contact or inhalation, although the oral route is the most significant one, since intoxication may take place by the intake of contaminated water, food or dietary supplements based on algae (Buratti et al., 2017). Among all cyanotoxins, microcystins (MCs) and cylindrospermopsin (CYN) have focused great interest, since they have been involved in the death of different animal species and humans (Azevedo et al., 2002; ∗Corresponding author. Email address: [email protected] (D. Gutiérrez-Praena) Bourke et al., 1983; Carmichael et al., 2001; Malbrouck and Kestemont, 2006). Microcystins are cyclic heptapeptides synthetized by several cyanobacterial species such as Mycrocistis aeruginosa, Oscillatoria agardhii, Plankthotrix agardii, and Planktothris rubescens, etc. (Sivonen and Jones, 1999). Up to date, more than 246 congeners of MCs are known, being MC-LR the most potent congener and frequently identified (Spoof and Catherine, 2017). Mainly considered as a hepatotoxin, MC-LR can also affect other organs such as kidneys, heart or brain (Li et al., 2012; Qiu et al., 2009; Zeng et al., 2014, Zhang et al., 2018). One of the main MC-LR-mechanisms of action is the inhibition of protein serine/threonine phosphatases, causing a cascade of effects such as the deregulation of phosphoproteins, which lead to tumor promotion and apoptosis (MacKintosh et al., 1990). In addition, many studies have also demonstrated its in vitro cytotoxic potential in different cell lines from fish, mammals and humans (Ding et al., 2017; Feurstein et al., 2009; Gutiérrez-Praena et al., 2012; Meng et al., 2011, 2013; Pichardo et al., 2005; Rozman et al., 2017). Moreover, several authors have also described that this toxin induces oxidative stress by increasing reactive oxygen species (ROS) and reducing glutathione (GSH) levels, leading to cell apoptosis (Li et al., 2015; Liu et al., 2016; Puerto et al., 2011; Qian et al., 2018), although these effects have not been studied using human neuronal cell lines yet. https://doi.org/10.1016/j.chemosphere.2019.02.173 0045-6535/ © 2019. UNCORRECTED PROOF 2 Chemosphere xxx (xxxx) xxx-xxx Cylindrospermopsin is an alkaloid consisting in a tricyclic guanidine combined to a hydroxylmethyl uracil group. This toxin presents a highly water-soluble structure, being commonly found out of the cells (Falconer and Humpage, 2006). Several cyanobacterial species are able to produce this toxin, such as Cylindrospermopsis raciborskii, Umezakia natans,Chrysosporum ovalisporum, Anabaena bergii, etc. (Banker et al., 1997; Harada et al., 1994; Schembri et al., 2001; Shaw et al., 1999). The main target of this cytotoxin is the liver, although kidneys, lungs, thymus, marrow bone, adrenal gland, gastrointestinal tract, immune, heart and nervous system have been also described as potential targets (Falconer, 1999; Guzmán-Guillén et al., 2015; Hawkins et al., 1985; Humpage et al., 2005; Terao et al., 1994). The most well-known mechanism of action of CYN is the inhibition of protein and GSH synthesis (Froscio et al., 2003; Runnegar et al., 1995; Terao et al., 1994). This cyanotoxin also enhances ROS production, which could lead to apoptosis or DNA damage (Gutiérrez-Praena et al., 2011, 2012; Guzmán-Guillén et al., 2013; Puerto et al., 2011). In addition, some studies have indicated the pro-genotoxic properties of CYN, being essential its previous metabolic activation by the enzymatic cytochrome P-450 complex (CYP450) (Humpage et al., 2005; Puerto et al., 2018; Žegura et al., 2011). Both cyanotoxins have evidenced to induce neurotoxic effects in different experimental models (Florzyk et al., 2014). Thus, MCs have shown to cause neuronal damage in vitro in different rodent cell lines such as primary murine cerebellar granule neurons (CGNs) and primary rat astrocytes (Feurstein et al., 2011; Rozman et al., 2017). Furthermore, many in vivo studies have manifested a clear neurotoxic potential, mostly of MC-LR, in different animal species such as rodents, fish and nematodes, affecting to their behavior, enhancing ROS levels, and modifying proteins related to neurodegenerative diseases (Baganz et al., 2004; Wang et al., 2013; Wu et al., 2017). In fact, MC-LR has produced pathological damage in hippocampus, neuronal degenerative changes, inflammation in memory-related brain regions and apoptosis in rats, suggesting that this toxin can be related to Alzheimer's disease in humans (Li et al., 2012; 2014). Actually, some studies confirm its transport through the blood-brain-barrier using OATP1A2, a variant of the organic anion transport system (OATP), because of its relatively large hydrophilic structure (Feurstein et al., 2009; Fischer et al., 2005; Menezes et al., 2013). On the contrary, neurotoxic effects of CYN are still not well elucidated. In this sense, there is only one report studying its effects in different in vitro murine cell lines (Takser et al., 2016). Meanwhile, some in vivo reports have showed in vivo neurotoxic effects in snails, tadpoles and fish, such as behavioral alterations or histopathological changes (Da Silva et al., 2018; Guzmán-Guillén et al., 2015; Kinnear et al., 2007; Kiss et al., 2002; White et al., 2007). Moreover, it is worthy to point out that the majority of studies concerning cyanotoxins toxicity are focused on single purified toxins, setting apart the fact that organisms are exposed simultaneously to a wide variety of cyanotoxins when they are present in aquatic systems. In fact, several studies have described the concomitant occurrence of MCs- and CYN-producing cyanobacteria, as well as the presence of both cyanotoxins at the same time (Bittencourt-Oliveira et al., 2014; Bogialli et al., 2006; Oehrle et al., 2010; Vasas et al., 2004). In the case of neurotoxicity, only a study conducted by Takser et al. (2016) showed the effects of a combination of MCs and CYN, although they also included the neurotoxin anatoxin-a in the combination (1:1:1). The SH-SY5Y cell line is a commonly used neuronal model due to its biochemical and functional properties, being very appropriate for neurotoxicity studies. Moreover, the differentiation of this cell line provides functional, biochemical and morphologically mature neurons, which are more similar to those present in the human brain (Xie et al., 2010). For this reason, both types of SH-SY5Y cells are a very interesting experimental model to assess the possible damage induced by MC-LR and CYN in human neural cells. Thus, considering all this, the aim of the present study was to assess the neurotoxic potential of MC-LR, CYN and their combination in vitro using the human neuroblastoma SH-SY5Y cell line, by exploring the cell viability, oxidative stress (ROS and GSH levels), acetylcholinesterase (AChE) activity and morphological changes after the exposure to these cyanotoxins. 2. Materials & methods 2.1. Supplies and chemicals MC-LR and CYN (both purity>95% by HPLC) were purchased from Enzo Life Sciences. Minimum essential medium (MEM), cell culture reagents, and fetal bovine serum (FBS) were obtained from Gibco (Biomol, Sevilla, Spain). Nutrient Mixture F-12 Ham, retinoic acid (RA), and brain-derived neurotrophic factor human (BDNF) were purchased in Sigma-Aldrich (Madrid, Spain). The MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-car- boxymethoxyphenyl)-2-(4- sulphophenyl)-2H-tetrazolium salt) Cell Titer 96®AQueous One Solution Cell Proliferation Assay was purchased in Promega (Biotech Iberica, Madrid, Spain). The Bradford Reagent and the neutral red (NR) were purchased from Sigma-Aldrich (Madrid, Spain). 2.2. Model system SH-SY5Y cells derived from a human neuroblastoma were obtained from ATCC (CRL-2266). They were maintained at 37°C in an atmosphere containing 5% CO2at 95% relative humidity (CO2in- cubator, NuAire®, Spain) in a medium consisting in MEM and F-12 (1:1) supplemented with 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, 1% L-glutamine 200mM, and 1% penicillin/streptomycin solution. Cells were grown near confluence in 75-cm2plas- tic flasks and harvested weekly with 0.25% trypsin-EDTA (1X). Cells were quantified in a Neubauer chamber. SH-SY5Y cells were plated at density of 2·105cells/mL to perform all the experiments. 2.3. Cell differentiation SH-SY5Y cells were differentiated using the protocol provided by Encinas et al. (2000) with some modifications. Cells were plated at density of 5·102cells/mL in plates of 48 wells, changing the medium every 48h with 1% of FBS, 10μM RA and 50ng/mL BDNF, for a week. The differentiation process was evaluated by morphological analysis. 2.4. Toxin test solutions Stock solutions of 4mg/mL MC-LR and 1mg/mL CYN were prepared in absolute ethanol and sterilized milliQ water, respectively. Both solutions were maintained at −20°C until their use. 2.5. Cytotoxicity assays Undifferentiated SH-SY5Y cells were seeded in 96-well tissue-culture plates for basal cytotoxicity tests and incubated at 37°C for 24h prior to exposure. Differentiated cells were exposed after a week from the start of the differentiation process in the same 48-well plates where the differentiation process took place. From the stock solution UNCORRECTED PROOF Chemosphere xxx (xxxx) xxx-xxx 9 Fig. 12. Morphology of differentiated SH-SY5Y cells after 24h of exposure to MC-LR. Phase-contrast microscopy of cells exposed to 45μg/mL MC-LR. Rounded cells (arrows) with clear signs of cell death. Bar=100μm (A). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 45μg/mL MC-LR. Presence of numerous apoptotic bodies (arrows) as clear sign of cell death. Presence of dilated endoplasmic reticulum (arrowheads). Bar=25μm (B). Transmission electronic microscopy of differentiated SH-SY5Y cells exposed to 45μg/mL MC-LR. Chromatin condensation (Chr), presence of numerous mitochondria (Mit), and nucleolar segregation of apoptotic nuclei (N). Nucleolus (n).Bar=2.5μm (C, D). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 22.5μg/mL MC-LR. Enlarged cells with cytoplasmic elongations as lamellipodiums (arrows) and presence of apoptotic cellular debris (arrowheads). Bar=25μm (E, F). Transmission electronic microscopy of differentiated SH-SY5Y cells exposed to 22.5μg/mL MC-LR. Cells with irregular shape presenting nuclei (N) with chromatin condensation (Chr). Bar=2μm (G). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 11.25μg/ mL MC-LR. Numerous dilated endoplasmic reticuli (arrows) with protein condensation (arowheads). Bar=25μm (H). Transmission electronic microscopy of differentiated SH-SY5Y cells exposed to 11.25μg/mL MC-LR. Presence of mitochondria (Mit), protein condensation (arrowheads) and lipidic vacuoles (Lp). Bar=2μm (I, J). caspases protein family, well-known apoptotic proteins. Thus, Feurstein et al. (2011) and Rozman et al. (2017) demonstrated that MC-LR induced cell death by apoptosis through the activation of caspase proteins in primary murine CGN cells and primary rat astrocytes, respectively. These findings support our results, since our most sensitive cytotoxicity biomarker was the MTS assay, which assesses the mitochondrial health and its activity, which is related with cell death (Tait and Green, 2013). Zhang et al. (2018) demonstrated that 10μM MC-LR with endoporter caused cell death in SH-SY5Y cells by using the lactate dehydrogenase (LDH) release assay. In this experimental model, the authors indicated that MC-LR induced phosphorylation of protein Tau, promoting dissociation of Tau from microtubules and aggregation of phospho-paired helical filaments-Tau, and consequently, neuronal degeneration and cell death. Cytoskeleton disruption is considered to be one of the cytotoxicity triggering caused by MC-LR (Hu et al., 2016) based on the alterations induced in vivo by the toxin in diverse cytosqueletal proteins in brains of rats (Zhao et al., 2015), and in vitro experiments (Meng et al., 2011). Cellular death can be also corroborated through morphological studies. In this regard, the present work shows how MC-LR induced the most common characteristics of cellular death such as cytoplasm fragmentation, chromatin condensation, and nucleolar segregation, endoplasmic reticulum dilatation, lipidic vacuoles, and presence of heterophagosomes, both in the undifferentiated and differentiated Fig. 13. Morphology of undifferentiated SH-SY5Y cells after 24h of exposure to CYN. Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 1μg/mL CYN. Detention of cellular growth and decrease of cell number caused by cellular death by apoptosis. Apoptotic bodies (arrows). Heterochromatin in apoptotic nucleus (arrowhead). Bar=20μm (A, B). Transmission electronic microscopy of SH-SY5Y cells exposed to 1μg/mL CYN. Fractionated cytoplasm and formation of apoptotic bodies (arrows). Condensed chromatin in the inner face of the nucleolar membrane (asterisks). Fibrillar component of the nucleolus (n). Nucleus (N). Bar=2μm (C). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.5μg/mL CYN. No evidence of morphological alterations. Bar=20μm (D). Transmission electronic microscopy of SH-SY5Y cells exposed to 0.5 and 0.25μg/mL CYN. Euchromatic nuclei with irregular shape (N). Cytoplasm with numerous free ribosomes (asterisk). Mitochondria (arrows). Bar=2μm (E). SH-SY5Y cells. In this sense, Feurstein et al. (2011) found that MC-LR induced a slight impairment of the neurite network in primary murine CGN cells. In addition, Meng et al. (2011) described some apoptotic effects such as the reorganization of cytoskeletal architectures in differentiated PC12cells exposed to 10μM MC-LR, which was also observable in the differentiated SH-SY5Y cells from our study. Moreover, Zhang et al. (2018) described neurites degeneration and cell death in SH-SY5Y cells exposed to 10μM MC-LR. Two other toxic mechanisms studied in the present work are the oxidative stress generation and the AChE disruption. For the oxidative stress evaluation two related parameters were studied, ROS generation and GSH depletion, since it has been stated that free-radical damage is one of the toxic mechanisms of MC-LR (Meng et al., 2013). In the present work, ROS levels did not suffer any alteration in undifferentiated cells exposed to pure MC-LR. However, Meng et al. (2013) found that concentrations up to 10μM MC-LR induced significant enhancement of ROS levels at early times, reaching the highest levels at 3h after the exposure. Nonetheless, these authors also found that after this time, ROS levels started to decline to basal levels due to a rapid response of cells (differentiated PC12cells) against MC-LR. Moreover, in the present study, GSH levels decreased after 24h of exposure at all the concentrations assayed. This could confirm UNCORRECTED PROOF 10 Chemosphere xxx (xxxx) xxx-xxx Fig. 14. Morphology of differentiated SH-SY5Y cells after 24h of exposure to CYN. Phase-contrast microscopy of cells exposed to 0.3μg/mL CYN. Rounded cells (arrows) with clear signs of cell death. Bar=100μm (A). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.3μg/mL CYN. Presence of numerous apoptotic bodies (arrows) as clear sign of cell death. Presence of dilated endoplasmic reticulum (arrowheads). Bar=25μm (B). Transmission electronic microscopy of SH-SY5Y cells exposed to 0.3μg/mL CYN. Condensed chromatin in the inner face of the nucleolar membrane (Chr). Bar=2μm (C). Phase-contrast microscopy of cells exposed to 0.15μg/mL CYN. Rounded cells (arrows) with clear signs of cell death. Bar=100μm (D). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.15μg/mL CYN. Presence of dilated endoplasmic reticulum (arrowheads) and lipidic vacuoles (arrows). Bar=25μm (E). Transmission electronic microscopy of SH-SY5Y cells exposed to 0.15μg/mL CYN. Condensed chromatin (Chr) in apoptotic nucleus (N). Bar=2μm (F). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.075μg/mL CYN. Presence of numerous apoptotic bodies (arrows) as clear sign of cell death. Bar=25μm (G). Transmission electronic microscopy of SH-SY5Y cells exposed to 0.075μg/mL CYN. Euchromatic nuclei with irregular shape (N). Dilate endoplasmic reticulum (ER) and lipidic vacuoles (Lp). Bar=2μm (H). the fact that ROS levels appeared unaffected since GSH could act directly against ROS (Circu and Aw, 2010). These oxidative stress parameters were not evaluated in differentiated SH-SY5Y cells since several authors have highlighted that these cells are more resistant to oxidative stressors due to changes in mitochondrial metabolism and antioxidant defenses (Cecchi et al., 2008; Cheung et al., 2009; Schneider et al., 2011). In contrast, diverse in vivo studies have reported the implication of oxidative stress in the neurotoxicity induced by MC-LR (Li et al., 2014 Mello et al., 2018; Wang et al., 2010; Zhao et al., 2015). Concerning the AChE activity, this enzyme is a well-known biomarker of neuronal damage, as the affectation of the cholinergic system could lead to a malfunctioning of the locomotor system, behavior and cognitive processes (Kist et al., 2012). MCs may influence brain AChE indirectly via the inhibition of serine/threonine phosphatases (Hu et al., 2016). In our study, a significant increase of AChE activity was observed when undifferentiated SH-SY5Y cells were exposed to the highest concentration of MC-LR (37μg/mL). The increased AChE activity could lead to a reduction of the cholinergic neurotransmission efficiency because of the lack of acetylcholine in the synaptic space, possibly contributing to a progressive cognitive impairment (Teodorak et al., 2015). Despite this, some other authors have demonstrated that AChE plays a role by promoting or suppressing cell death. An enhanced AChE activity take part in apoptosis, participating in the formation of apoptosomes or influencing the expression of Fig. 15. Morphology of undifferentiated SH-SY5Y cells after 24 h of exposure to MC-LR + CYN. Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 37 μg/mL MC-LR + 1 μg/mL CYN. Intense cellular death by apoptosis. Heterochromatin accumulations in apoptotic nuclei (arrows). Cellular debris as apoptotic bodies (arrowheads). Bar = 20 μm (A). Transmission electronic microscopy of SH-SY5Y cells exposed to 37 μg/mL MC-LR + 1 μg/mL CYN. Heterochromatin condensation in the inner face of the nuclear membrane (asterisks). Nucleolar segregation with the fibrillary component (n). Apoptotic bodies formation (arrows). Autophagy vacuoles in the cytoplasm (arrowheads). Nucleus (N). Bar = 2 μm (B). Semithin sections of SH-SY5Y cells stained with toluidine blue. Cells exposed to 18.5 μg/ mL MC-LR + 0.5 μg/mL CYN. Blister formation in cellular surface (arrows). Apoptotic bodies (arrowheads). Bar = 20 μm (C, D). apoptotic genes (Park et al., 2004; Ben-Ari et al., 2006; Zhu et al., 2007). Zhu et al. (2007) also stated that AChE expression during apoptosis is associated with calcium mobilization. These facts are in agreement with our findings, since it has been shown that MC-LR induced cell death by apoptosis at the highest concentration assayed, correlating this effect with the AChE activity enhancement. On the contrary, when differentiated SH-SY5Y cells were exposed to 45μg/ mL MC-LR, no significant changes in the AChE activity were detected compared to the control group. This could be due to structural and functional modifications of the cells after the differentiation process, which could impede the effects of MC-LR over the AChE activity. However, cell death by apoptosis was also observed in these cells, which implies that not only the rise of the AChE could be involved in the cellular death, but also other potential factors such as intracellular calcium levels etc., that should be further investigated. Other authors have also evaluated the AChE activity disruption induced by MC-LR, but their studies have been carried out in vivo systems (Kist et al., 2012; Qian et al., 2018; Wu et al., 2016, 2017), obtaining contradictory and not conclusive results, depending on the administration route, or the experimental model assayed, etc. Conse UNCORRECTED PROOF Chemosphere xxx (xxxx) xxx-xxx 11 Fig. 16. Morphology of differentiated SH-SY5Y cells after 24 h of exposure to MC-LR + CYN. Transmission electronic microscopy of SH-SY5Y cells exposed to 45 μg/mL MC-LR + 0.3 μg/mL CYN. Apoptotic nucleus (N) with heterochromatin condensation (Chr). Lipidic degeneration (Lp). Dilated endoplasmic reticulum (ER) with protein condensation (arrowheads). Pre-apoptotic bodies (arrows). Heterophagosome (Het). Nucleoulus (n) with the presence of a nuclear body (NB). Bar = 2 μm (A, B, C, D). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 45 μg/mL MC-LR + 0.3 μg/mL CYN. Clear formation of apoptotic bodies (arrowheads). Bar = 25 μm (E). Cells exposed to 22.5 μg/mL MC-LR + 0.15 μg/mL CYN and 11.25 μg/mL MC-LR + 0.075 μg/mL CYN. No significant alterations observed. Bar = 25 μm (F, G). quently, further research should be carried out in order to clarify the effects of MCs on this key enzyme in the nervous system. Concerning CYN, a very scarce number of studies have been carried out in order to elucidate its neurotoxicity. However, due to its zwitterionic behavior and its small size, CYN is likely to be taken by the cells through diffusion, being able the crossing through the blood brain barrier (Florczyk et al., 2014; Valério et al., 2010). In the present study, CYN showed EC50 values even lower than MC-LR both in undifferentiated and differentiated cells. In agreement, Takser et al. (2016) found that pure CYN reached an EC50 value between 0.1 and 10μM CYN in N2a cells after 24, 48 and 72h of exposure. These authors also found that CYN induced almost a total cellular death at a concentration of 10μM in RAW264.7 and BV-2cells. These findings are also in agreement with the morphological results, which showed clear signs of cellular death by apoptosis in both types of SH-SY5Y cells exposed to 1μg/mL (2.4μM) and 0.3μg/mL (0.7μM) CYN. Relative to oxidative stress, although numerous works demonstrate in vitro the oxidative stress induction by CYN in different cell lines (reviewed by Pichardo et al., 2017), this work investigated the potential effects on ROS and GSH levels in the undifferentiated SH-SY5Y cell line. However, no effects were found, which is in disagreement with some in vivo studies, where oxidative stress was a main component of the cellular damaged observed (Guzmán-Guillén et al., 2015; Da Silva et al., 2018). These discrepancies could be due to the different experimental model or the concentrations used. In relation to the AChE activity disruption, to our knowledge, no other papers have been published concerning the effects of CYN on AChE activity in vitro. In the present work, our results did not indicate any alteration at all CYN concentrations assayed (up to 1μg/ mL) in undifferentiated cells. This is in agreement with Da Silva et al. (2018), who described no significant differences in the AChE activity in brain of fish (Hoplias malabaricus) exposed to pure CYN after 7- and 14-days post treatment. In addition, these authors also reported increased AChE activity by 44% in brain at 7 days of exposure to aqueous CYN-producing cyanobacteria extracts, returning to control levels after 14 days. However, when differentiated SH-SY5Y cells were exposed to 0–0.3μg/mL CYN, cultures showed a significant decrease of AChE activity at all CYN concentrations. These observations are in agreement with the findings reported by Guzmán-Guillén et al. (2015), who found a significant inhibition of 35% of AChE ac tivity in the brain of tilapia subchronically exposed to CYN (10μg/L CYN) by immersion in an A. ovalisporum culture for 14 days has been reported. In this study, after a depuration process (7 days) a recovery of the enzyme was found. These contrary responses could be due to differences in the experimental conditions, highlighting the need to perform further studies. Once the neurotoxic effects of pure MC-LR and CYN were studied separately, it was interesting to study their effects in combination, since these toxins can appear together in the nature (Bittencourt-Oliveira et al., 2014; Oehrle et al., 2010; Vasas et al., 2004). In this sense, in the present work the MC-LR + CYN combination resulted more cytotoxic than each individual toxin after 24 and 48 h of exposure in undifferentiated SH-SY5Y cells. However, the differentiation process resulted in a lower cytotoxicity of the toxin combination, as the effect of MC-LR + CYN was similar to that obtained for MC-LR. The combination interaction was analyzed by the isobolograms method described by Chou and Talalay (1984), which establish the foundations for assessing whether cytotoxicity induced by a combination of cyanotoxins is more or less harmful than the expected for individual cyanotoxins. This method is independent of the mode of action of the compounds and considers both the potency (EC50,Dm) and the shape (m) of the dose-effect curve for each toxin (Ruiz et al., 2011a, 2011b). The method allows a prediction of synergism/antagonism at all effect levels (fa) for a combination of a different number of cyanotoxins. Depending on the equipotency level of MC-LR and CYN, it is feasible the cyanotoxin interaction can vary between antagonism and additivity. To explore this, the IC values at 50% inhibition (IC50) and 90% inhibition (IC90) were determined. The combined effect of the combination observed on undifferentiated SH-SY5Y cells is of antagonistic nature, with a slightly tendency to additivity at higher concentrations. In differentiated cells, only an antagonistic behavior was observed. However, experimental cytotoxicity and histopathological changes obtained by the combination appeared to be more related with additivity than antagonism in both cell types, although the antagonistic effect seems to be probable since the effect of the combination was not as intense as it could be expected. Similar results were obtained by Gutiérrez-Praena et al. (2018) in the hepatic cell line HepG2, where the combination of both cyanotoxins also presented an antagonistic response in the cells. This response could be due to complex dissimilar actions of these different cyanotoxins, although the mechanisms of interaction remain unknown. However, it is difficult to give an explanation to this phenomenon because of the isobolograms method only allows quantitative determination of synergism or antagonism, and the elucidation of the mechanism by which these relations occur is a separate issue that requires a different kind of approach (Lu et al., 2013). Similar to our results, Takser et al. (2016) found that a combination of MC-LR, CYN and anatoxin-a in an equimolar proportion (3.33μM) induced a significant reduction of cell viability in N2a cells after 24, 48 and 72h of exposure. Moreover, these authors also found that this combination induced a total cell death in the RAW264.7 and BV-2cell lines. They also suggested that the combination was more toxic compared with the individual compounds. Regarding cell death, Takser et al. (2016) showed that the combination induced clear signs of cell apoptosis in all the cell lines used. This is in agreement with our morphological results, where an intense cell death was observed at the highest concentrations of the combination assayed for both SH-SY5Y cell types (37 μg/mL MC-LR + 1 μg/mL CYN and 45 μg/mL MC-LR + 0.3 μg/mL CYN for undifferentiated and differentiated cultures, respectively), mainly by apoptosis. Concerning oxidative stress and the AChE activity, no significant changes were observed respect to the control group at any of the combination concentrations assayed in the UNCORRECTED PROOF 12 Chemosphere xxx (xxxx) xxx-xxx undifferentiated cells. However, in the differentiated SH-SY5Y cells a significant increase of AChE activity was observed at the highest combination concentration (45 μg/mL MC-LR + 0.3 μg/mL CYN), which could lead to the consequences previously described. To our knowledge, this is the first report concerning these toxicological parameters, obtaining different responses after the exposure to the combination and to the isolated toxins, highlighting the importance of considering more realistic exposure-scenarios. Therefore, further investigations would be needed to clarify the effects of the MC-LR-CYN-com- bination on neuronal cells, and in different experimental models. 5. Conclusions Our results showed a cytotoxic effect caused by the exposure to MC-LR and CYN individually and in combination in both undifferentiated and differentiated SH-SY5Y cells. CYN resulted more cytotoxic than MC-LR, but the combination presented the highest cytotoxicity. However, the isobolograms method establishes that these toxins together induce, mainly, an antagonistic response. Concerning oxidative stress biomarkers, only MC-LR and the combination decreased GHS levels at the highest concentration assayed. Moreover, AChE activity also showed different results for individual toxins and their combination. The morphology study corroborated those results observed with the cytotoxicity assays, since cell death by apoptosis was observed at almost all the concentrations assayed of both toxins and the combination. Taking all this into account, both cyanotoxins seem to present neurotoxic effects in the SH-SY5Y cell line. Thus, as the potential neurotoxicity induced by MC-LR and CYN is of interest, more studies concerning the different mechanisms by which both cyanotoxins can cross the blood-brain barrier (diffusion, transporters, direct affectation of the barrier, etc.) would be required. In addition, the affinity of these two cyanotoxins by the different cells composing the nervous system would be also of interest, together with the study of the toxic mechanisms these cyanotoxins can exert and their changes when single and combination toxin exposure are considered. 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Sci. 162, 475–487. https://doi.org/ 10.1093/toxsci/kfx271. Zhao, S., Li, G., Chen, J., 2015. A proteomic analysis of prenatal transfer of microcystin-LR induced neurotoxicity in rat offspring. J. Proteonomics 114, 197–213. https://doi.org/10.1016/j.jprot.2014.11.015. Zhu, H., Gao, W., Jiang, H., Jin, Q.H., Shi, Y.F., Tsim, K.W., Zhang, X.J., 2007. Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargin-induced apoptosis. Int. J. Biochem. Cell Biol. 39, 93–108. https://doi.org/10.1016/j.biocel.2006.06.012. CHAPTER 2 María Gracia Hinojosa, Daniel Gutiérrez Praena, Ana Isabel Prieto, Remedios Guzmán Guillén, Ángeles Jos, Ana María Cameán NEUROTOXICITY INDUCED BY MICROCYSTINS AND CYLINDROSPERMOPSIN: A REVIEW Science of the Total Environment. 2019;668:547-565. UNCORRECTED PROOF Science of the Total Environment xxx (xxxx) xxx-xxx Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com Review Neurotoxicity induced by microcystins and cylindrospermopsin: A review M.G. Hinojosa , D. Gutiérrez-Praena , A.I. Prieto ⁎, R. Guzmán-Guillén , A. Jos , A.M. Cameán Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, C/Profesor García González 2, 41012 Seville, Spain ARTICLE INFO Article history: Received 14 December 2018 Received in revised form 25 February 2019 Accepted 27 February 2019 Available online xxx Editor: Yolanda Picó Keywords: Cyanotoxins MCs CYN Nervous system Ecotoxicology Environmental risk ABSTRACT Microcystins (MCs) and cylindrospermopsin (CYN) are among the most frequent toxins produced by cyanobacteria. These toxic secondary metabolites are classified as hepatotoxins and cytotoxin, respectively. Furthermore, both may present the ability to induce damage to the nervous system. In this sense, there are many studies manifesting the potential of MCs to cause neurotoxicity both in vitro and in vivo, due to their probable capacity to cross the blood-brain-barrier through organic anion transporting polypeptides. Moreover, the presence of MCs has been detected in brain of several experimental models. Among the neurological effects, histopathological brain changes, deregulation of biochemical parameters in brain (production of oxidative stress and inhibition of protein phosphatases) and behavioral alterations have been described. It is noteworthy that minority variants such as MC-LF and -LW have demonstrated to exert higher neurotoxic effects compared to the most studied congener, MC-LR. By contrast, the available studies concerning CYN-neuro- toxic effects are very scarce, mostly showing inflammation and apoptosis in neural murine cell lines, oxidative stress, and alteration of the acetylcholinesterase activity in vivo. However, more studies are required in order to clarify the neurotoxic potential of both toxins, as well as their possible contribution to neurodegenerative diseases. © 2018. 1. Introduction Cyanobacteria are a group of Gram-negative prokaryotes capable of growing under almost every environmental condition (Chorus and Bartram, 1999). Due to climate change and anthropogenic activities, their presence is increasing (Davis and Gobler, 2016). As a consequence, there is an enhancement of the production of toxic secondary metabolites of great importance for the ecotoxicology known as cyanotoxins (Duy et al., 2000). These toxins are classified as hepatotoxins (e.g. microcystins, nodularins), cytotoxins (e.g. cylindrospermopsin), neurotoxins (e.g. anatoxin-a, homoanatoxin, saxitoxins), dermatotoxins (e.g. lungbyatoxin) or irritant toxins (e.g. lipopolysaccharides) (Testai et al., 2016). There are different exposure routes for cyanotoxins, being the most important the oral route. In fact, many aquatic organisms are able to live in presence of cyanotoxins, and some of them have proved to bioaccumulate these secondary metabolites, acting as a reservoir for animals higher up the trophic chain, and also for humans (Berry and Lind, 2010; Gutiérrez-Praena et al., 2013). However, dermal, inhaling or even parenteral exposures are also possible (Buratti et al., 2017). Thus, the variety of targets and exposure routes together with the rise of cyanobacterial proliferations make of cyanotoxins a serious concern for animal livestock, human activities and public health (Testai et al., 2016). ⁎Corresponding author. Email address: [email protected] (A.I. Prieto) In the last decades, the toxic effects of cyanotoxins on the nervous system have been widely studied, not only those caused by the so-called neurotoxins with well-defined mechanisms of action in this system such as anatoxins (ATX) and saxitoxins (STX), but also, by other cyanotoxins with different target organs (Florczyk et al., 2014). Neurotoxicity could be described as ‘any adverse effect on the central or peripheral nervous system caused by chemical, biological or physical agents’(Costa et al., 2008). The keys to the brief communication within the nervous system are the generation of a potential of action as a quick response of dendrites to the neurotransmitters released from contiguous neurons, and its fast travelling for the neuronal axon for its release afterwards (Kem, 2000). In this sense, among all the cyanotoxins, microcystins (MCs) and cylindrospermopsin (CYN) have proven to exert damage in the nervous system as well, in spite of not being considered as neurotoxins per sé. These are very common cyanotoxins (Table 1) able to put health at risk due to their ubiquity (Gutiérrez-Praena et al., 2013), as previously demonstrated in different human poisoning cases. The most serious episode associated with human exposure to MCs occurred when 126 people were intoxicated at a haemodialysis clinic in Caruaru (Brazil), causing the death of almost half of them. All patients presented malaise, weakness, dizziness, vertigo, tinnitus, mild deafness and, in severe cases, visual disturbance and blindness, grand mal convulsions, and gastrointestinal and hepatic symptoms (Pouria et al., 1998; Carmichael et al., 2001). Most of these symptoms have a neuronal origin, standing out the possible MCs-crossing the blood-brain barrier (BBB) as several authors have reported (Feurstein et al., 2009, 2010, 2011; Zhao et al., 2015a), causing their toxic effects. https://doi.org/10.1016/j.scitotenv.2019.02.426 0048-9697/ © 2018. UNCORRECTED PROOF 2 Science of the Total Environment xxx (xxxx) xxx-xxx Table 1 Properties and environmental concentrations of some MCs congeners and CYN. Toxin Chemical structure Molecular properties Environmental concentrations Molecular weight [M+H]+Molecular composition Kow BCF Plants In surface waters (μg/L) In mollusks and fish samples (ng/g d.w) MCLR Cyclo(−D-Ala-L-Leu-D-erythro-β-methylAsp(isolinkage)-L-Arg-Adda-D- Glu(isolinkage)-N-methyldehydro-Ala 995.5561 C49H75N10O12 2.16 (Ward and Codd, 1999) Up to 680.05±40.88 (Romero- Oliva et al., 2014) Up to 2100 Faassen and Lürling (2013) Up to 130 in fish muscle (Roy- Lachapelle et al., 2015) MC-LF Cyclo(−D-Ala-L- Leu -D-erythro-β-methylAsp(isolinkage)-L-Fe-Adda-D- Glu(isolinkage)-N-methyldehydro-Ala 986.5234 C52H72N7O12 3.56 (Ward and Codd, 1999) nf Up to 51 (Graham et al., 2010) Up to 300 in common carp (Gurbuz et al., 2016) MCLW Cyclo(−D-Ala-L- Leu -D-erythro-β-methylAsp(isolinkage)-L-Trp-Adda-D- Glu(isolinkage)-N-methyldehydro-Ala 1025.5343 C54H73N8O12 3.46 (Ward and Codd, 1999) nf Up to 260 (Faasen and Lurling 2013) Up to 15.5 in bivalves (Preece et al., 2015) MCRR Cyclo(−D-Ala-L- Arg -D-erythro-β-methylAsp(isolinkage)-L- Arg -Adda-D- Glu(isolinkage)-N-methyldehydro-Ala 1038.5731 C49H76N13O12 1.54 (Liang et al., 2011) Up to 54.09±17.01 (Romero- Oliva et al., 2014) Up to 16,000 (Graham et al., 2010) Up to 463,000 in silver carp (Xie et al., 2007) MCYR Cyclo(−D-Ala-L-Tir-D-erythro-β-methylAsp(isolinkage)-L- Arg -Adda-D- Glu(isolinkage)-N-methyldehydro-Ala 1045.5353 C52H73N10O13 nf nf Up to 343 (Simiyu et al., 2018) Up to 20,000 in bivalves (Kim et al., 2017) CYN 2,4(1H,3H)-Pyrimidinedione, 6-[(R)- hydroxy[2aR,3S,4R,5aR,7S)-2,2a,3,4,5,5a,6,7-octahydro-3-methyl-4-(sulfooxy)-1H-1,8,8b- triazaacenaphthylen-7-yl]methyl]-, rel-(−)- (9CI) 416.1234 C15H21N5O7S Highly watersoluble Up to 3.88±0.33 (Cordero- Araújo et al., 2017) Up to 800 (Shaw et al., 2000) Up to 200 in crayfish (Saker and Eaglesham, 1999) Abbreviations: BCF: bioconcentration factor; d.w: dry weight; Kow: octanol/water partition coefficients; nf: not found; In the case of CYN, the most important outbreak occurred in Palm Island (Australia) in 1979, when 146 people were hospitalized with symptoms of malaise, vomits, anorexia, and hepatomegaly after drinking from a water supply that contained a CYN-producing Cylindrospermopsin raciborskii strain (Bourke et al., 1983; Griffiths and Saker, 2003). However, it is important to mention that CYN was also present in the Caruaru outbreak, possibly contributing to the neurological affectation reported (Bláha et al., 2009) although it is hard to differentiate the effects caused for each toxin in the symptoms observed, as both toxins are often present together in nature (Gkelis and Zaoutsos, 2014; Trainer and Hardy, 2015; Loftin et al., 2016; Buratti et al., 2017). Due to the low molecular weight of CYN, it might be able to cross the BBB. In fact, CYN was detected in brains of two fish species (Guzmán-Guillén et al., 2015; da Silva et al., 2018). Thus, although not being considered as neurotoxins, both cyanotoxins have demonstrated its neurotoxic potential in different in vitro and in vivo experimental models, increasing the interest of the scientific community in this matter. Taking into account all these facts, the aim of the present work was to gather the existent knowledge about the potential to exert neurotoxic effects of both toxins from 1998 to 2018. 2. Microcystins Microcystins (MCs) are cyclic heptapeptides molecules containing a hydrophobic C20 D-amino acid commonly known as ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid), crucial for the toxicity of these cyanotoxins due to their interaction with protein phosphatases (Song et al., 2006) (Fig. 1). >246 isoforms of MCs have been detected (Spoof and Catherine, 2017), mainly differing in the L-amino acids at positions 2 and 4, causing differences in toxicokinetic and toxicodynamic properties (Rinehart et al., 1994). These compounds are the most widespread cyanobacterial toxins detected in freshwaters (Spoof and Catherine, 2017), being many the cyanobacteria genera capable of synthesize them: Microcystis, Plankthotrix, Anabaena, Nostoc, Aphanizomenon, Anabaenopsis,Rivularia and Fisherella, among others (Sivonen and Jones, 1999; Rao et al., 2002; Carey et al., 2007; Bittencourt-Oliveira et al., 2014; Cirés et al., 2014). The most known mechanism of action of MCs is the protein serine/threonine phosphatases inhibition, able to cause phosphoprotein-deregulation, which leads to tumor promotion and apoptosis (MacKintosh et al., 1990; Vichi et al., 2016). Furthermore, the potential of MCs to increase reactive oxygen species (ROS) and to reduce glutathione (GSH) levels, causing oxidative stress and, therefore, apoptosis, has already been demonstrated (Puerto et al., 2011; Wang et al., 2013; Li et al., 2015a, 2015b; Liu et al., 2016; Qian et al., 2018). Although being considered as hepatotoxins, MCs can damage other organs such as intestines, heart or kidneys (Moreno et al., 2003; Atencio et al., 2008; Qiu et al., 2009; Li et al., 2011a; Zeng et al., 2014). In this sense, it has been demonstrated that MCs require organic anion transporting polypeptides (OATPs for humans/Oatps for rodents) in order to cross cell membranes (Chen and Xie, 2016). The OATP1B1 and OATP1B3 are common in liver cells, while OATP1A2 is thought to be the responsible for the transport of MC-LR, across the BBB and the kidneys, for example (Fischer et al., 2005; Feurstein et al., 2009). This means that significant amounts of MCs could reach UNCORRECTED PROOF Science of the Total Environment xxx (xxxx) xxx-xxx 3 Fig. 1. Structure of MCs. the brain across the BBB and induce brain pathology, depending on the type and expression of OATPs/Oatps at the BBB, the blood-cere- brospinal fluid barrier, and the neuronal cell membrane (Bronger et al., 2005; Huber et al., 2007; Westholm et al., 2009). Most of the existent studies have been carried out using MC-LR, due to its major presence and its wide demonstration of causing neurotoxic effects in several experimental models, although other more toxic congeners such as MC-LW and MC-LF have also been studied (Feurstein et al., 2009, 2010, 2011; Rozman et al., 2017). This can be due to the hydrophobicity of MC-LF and MC-LW. Structure variations and differences in molecular properties such as hydrophilicity/hydrophobicity can lead to a modification on molecular interactions with lipid membranes (Vesterkvist and Meriluoto, 2003) modifying PP-inhibitory activity (Díez-Quijada et al., 2018). Concerning to their effects in the nervous system, Florczyk et al. (2014), Hu et al. (2016) and Mello et al. (2018) have reviewed the main mechanisms of neurotoxicity of MCs at different levels. Firstly, neurotransmission, by causing effects on GABAergic neurons. Secondly, neurochannels, by affecting the ionic concentrations in and outside the cells. Linked to this, signal transduction, as a consequence of the deregulation of Ca2+, which, by activating calcineurin leads to apoptosis. Moreover, the production of oxidative stress, by deregulating several antioxidant enzymes such as catalase or superoxide dismutase (SOD). And finally, cytoskeleton disruption, by alteration of structural brain proteins such as Tau. However, important contributions have been made lately, confirming these mechanisms using mostly in vivo experimental models. In this sense, the studies carried out using different animal models (mice, fish) revealed an important effect on the neurotransmission induced by MC-LR (Wu et al., 2016; Qian et al., 2018; Shin et al., 2018; Wang et al., 2018), together with an enhancement in oxidative stress in mice (Shin et al., 2018; Wang et al., 2018), and cytoskeleton disruption in the case of rats (Zhang et al., 2018) (Fig. 2). 2.1. Neurotoxicological in vitro studies performed with microcystins Table 2 shows the different in vitro assays performed with MC-LR and some other congeners in different neuronal cell lines and primary cultures. The in vitro studies are relatively recent, comprising a range of ten years (2009–2018) (Table 2). This fact demonstrates the importance that MCs have lately acquired concerning their neurotoxicity nowadays. Thus, it is possible to find different studies carried out in permanent cell lines (PC12, BV-2, N2a, GT1–7, and SH-SY5Y) and in several primary cell cultures. It is also important to remark that all the toxins used in these studies are commercial standards with a purity >95%, which guarantees that the results reported are due to the MC itself and not to other potential bioactive compounds that can be present in cyanobacterial extracts (Falconer, 2007). Furthermore, it is Fig. 2. Main mechanisms of neurotoxic action of MCs. UNCORRECTED PROOF 10 Science of the Total Environment xxx (xxxx) xxx-xxx Table 4 In vivo neurotoxicity studies in different terrestrial models exposed to MCs. Microcystin congener/ cyanobacteria Experimental model Experimental conditions Assays performed Relevant Results References Nematodes Pure MC-LR Caenorhabditis elegans 1, 10, 20, 40, 80 or 160μg/ L for 24h in sterile culture plates Behavioral study and gene expression Decrease of chemotaxis to NaCl and diacetyl from 40μg/L Decrease of thermotaxis from 20μg/L Decrease of expression patterns of sensory neurons (ASE, AWA, AFD and AIY) Li et al. (2009a) Pure MC-LR Caenorhabditis elegans 1, 10, 20, 40 or 80μg/L in different periods of time, in sterile culture plates Behavioral study, morphological changes, gene expression and lifecycle indices Decrease of head thrash, body bends Decrease of body size Enhance of gfp gene expression Decrease of life span, brood size, generation time Li et al. (2009b) Pure MC-LR Caenorhabditis elegans 0.1, 1, 10 or 100μg/L for 8 or 24h, in 12-well sterile culture plates Behavioral studies, morphologic changes and gene expression Decrease of locomotion behavior Enhance of neuronal loss of GABAergic neurons, presenting aberrant neuronal morphology at 10–100μg/L No changes in cholinergic, serotonergic, dopaminergic and glutamatergic neurons Decrease of Gene expression affecting GABAergic neurons Ju et al. (2013) Pure MC-LR Microcystis aeruginosa culture containing MC-LR, MC-RR and MC-YR Caenorhabditis elegans 0.1, 1, 10 or 100μg/L for 24 or 72h, in sterile culture plates 300μg/L for 24 or 72h, in sterile culture plates Behavioral study Decrease of body bends, move length, pharyngeal pumping frequency and touch response Alteration of the thermotactic behavior after 72h of exposure to 100μg/L Enhance of motile and pumping activity Ju et al. (2014) Pure MC-LR Pure MC-LF Caenorhabditis elegans 1, 10, 40, 80, 160, 320, 500, or 1000μg/L for 24h, in sterile culture plates 1, 10, 100, 160 or 320μg/L for 24h, in sterile culture plates Function of sensory neurons Affectation of AWA sensory neurons, but not of AWC sensory neurons: MC-LF>MC-LR Moore et al. (2014) Pure MC-LR Caenorhabditis elegans 1, 50 or 100μg/L in different periods for life cycle, in sterile culture plates Life-cycle indices and gene expression Decrease of lifespan, body length and brood size after 100μg/L of exposure Alteration of genes expression after 100μg/L of exposure Saul et al. (2014) Birds Cyanobacterial biomass containing MC-LR, MC-RR, MC-YR and MCs similar compounds Japanese quail (Coturnix coturnix japonica) 0.045, 0.459, 4.605 or 46.044μg/day for 10 or 30days, by oral route Oxidative stress parameters Alteration in brain, after acute exposure: Decrease of GSH, Enhance of TBARS, Enhance of EROD. After subchronic exposure: Enhance of GSH, Enhance of GPx Enhance of TBARS, Enhance of EROD Pašková et al. (2008) Mammals MC raw extracts containing mainly [D-Leu1]MC-LR Rats 1μL of extracts containing 0.01 or 20μg/L (equivalent to 0.045x10E-6 and 9.1x10E-5μg/kg) by intrahippocampal injection Behavioral study, oxidative stress parameters and DNA damage Enhance of latency of long-term memory in rats exposed to 20μg/L Decrease of latency of memory retrieval Enhance of working and reference memory errors after 8days of exposure Enhance of GST activity in brain rats exposed to 0.01μg/L Enhance of LPO content in brain rats exposed to 20μg/L DNA damage in brain of both MCs doses treated rats Maidana et al. (2006) Extracted and purified MC-LR and MC-RR from blooms Rats 80μg MC-LReq/kg b.w. injected i.v. The analysis was performed 1, 2, 4, 6, 12 and 24h post-injection Determination of MCs content in different tissues by LC-MS MCs contents in brain (0.2%): 2>24>1>12>6≈4h post-injection kidney>lung>stomach> liver> small intestine> gonad> spleen> muscle> heart>brain Wang et al. (2008) Pure MC-LR Rats 1μL containing 1 or 10μg/ L MC-LR (equivalent to 5x10E-6 or 5x10E-5μg/ kg), bilaterally injected into hippocampal. Parameters were measured 15days post-injection Behavioral study, histopathological study and oxidative stress parameters Enhance of latencies to find the platform Decrease of swimming distance in the target zone Swimming speed did not change Decrease of total hipoccampal neurons Highest MC-LR dose: Enhance of LPO, Enhance of CAT, Enhance of GPx, Enhance of SOD Lowest MC-LR dose: Enhance of LPO, Enhance of CAT Li et al. (2012a) UNCORRECTED PROOF Science of the Total Environment xxx (xxxx) xxx-xxx 11 Table 4 (Continued) Microcystin congener/ cyanobacteria Experimental model Experimental conditions Assays performed Relevant Results References Pure MC-LR Rats 1 or 10μg/kgdayi.p. injected for 50days Behavioral study, histopathological study, protein expression, MC-LR content analysis Enhance of latencies to find the platform Decrease of swimming distance in the target zone Enhance of degeneration and apoptosis of hippocampal cells Hyperphosphorylation of tau 41.6±8.45ng/g d.w. of MC-LR was detected in brain of rats exposed to 10μg/kgday Li et al. (2012b) Pure MC-LR Rats 10μL containing 5 or 25μg/L (equivalent to 2.5x10E-4 or 1.25x10E-3μg/kg), by i.c.v. injection MC-LR+LiCl and SB216763 inhibitors of GSK-3β Electrophysiologycal studies Enhance of PPs activity Decrease of phosphorilated GSK-3β Decrease of LTP Inhibitors avoid effects produced by MC-LR Wang et al. (2013) Pure MC-LR Rats 0.2, 1 or 5μg/kg every 2days for 8weeks, by intragastric route Behavioral study, histopathological study and immunohistochemistry staining Enhance of escape latencies in 5μg/kg MC-LR- treated rats Decrease of frequencies entering the enlarged platform in 1 and 5μg/kg MC-LR-treated rats No significant differences in the number of damaged neurons Enhance of astrocyte density and NO concentration in hippocampus exposed to 5.0μg/ kg Li et al. (2014) Pure MC-LR Rats 1, 5 or 20μg/kg every 2days for 8weeks, by intragastric route. Later the rats became pregnant of a non-exposed male Maternal toxicity and reproductive outcome, simple motor and locomotor activities, behavioral study and oxidative stress parameters Decrease of mean body weight gain in maternal rats. Decrease of number of pregnant rats Alteration of behavior and neurodevelopment in rat offsprings Enhance of MDA and SOD in hippocampus of offsprings Li et al. (2015b) Extracted and purified MC-LR from blooms Pregnant rats and pups 10μg/kg daily from day 8 to postnatal day 15 Oxidative stress parameters, determination of MC-LR, histopathological study and protein expression Enhance of MDA, Decrease of GSH andAChE activity No significant PPs changes 3.75±0.94ng/g d.w. were detected in brain of pup rats Morphological changes Alteration of proteins involved in neuronal processes in pup rats Zhao et al. (2015) Extracted and purified MC-LR from blooms Mice 1μL containing 1–20ng/μL, by i.c.v. route. All parameters measured 3h, 1day, 3day and 7day after exposure Behavioral study, histopathological study and oxidative stress parameters Decrease of memory impairment Morphological changes in hippocampal neurons from 10ng/μL Enhance of protein oxidation, LPO, ROS, SOD, GPx and Nrf2 Decrease of GSH/GSSG Shin et al. (2018) Pure MC-LR Mice 1, 5, 10, 20 or 40μg/L 12weeks, by oral route Histopathological study and protein expression Pathological changes in hippocampus and cortical cells in a dose-dependent way. Differences between hippocampus and cerebral cortex in the affectation of mRNA and proteins expression: ATP6, COX3, CYTB, POLG, mtSSB and TFAM Wang et al. (2018) Pure MC-LR Rats 3μL of 0.1μg MC-LR/μL (equivalent to 1.5μg/kg) via hippocampal injection. All parameters measured 24h, before and after exposure Protein expression and behavioral study Enhance of desmethylation of PP2Ac, phosphorylation of GSK-3βand tau, spatial memory deficit. Zhang et al. (2018) Abbreviations: AChE: acetylcholinesterase; ATP6: adenosine triphosphate-6; b.w.: body weight; CAT: catalase; COX3: cyclooxygenase-3; CYTB: Cytochrome B; EROD: cytochrome P-450-dependent 7- ethoxyresorufin O deethylase; GLU: glucose; GPx: glutathione peroxidase; GSH: reduced glutathione; GSK-3β: Glycogen synthase kinase 3 beta; GSSG: oxidized gluthatione; GST: Glutathione-S-transferase; i.c.v.: intracerebroventricular; LPO: lipid peroxidation; LTP: long term period; MDA: malondialdehyde; mtSSB: mitochondrial single-stranded DNA binding protein; NO: nitric oxide; POLG: DNA polymerase g; PP: protein phosphatase; PP2Ac: catalytic subunit of protein phosphatase 2A; ROS: reactive oxygen species; SOD: superoxide dismutase; TBARS: total thiobarbituric acid reactive species; TFAM: mitochondrial transcription factor A. lighting the fact that AWC and AWA neurons act as independent targets. Moreover, these effects were compared to the ones caused by the exposure to MC-LF, suggesting a more potent effect by MC-LF than MC-LR. Up to date, only this neurotoxicity study has been carried out with this congener in nematodes, despite MC-LF is transported more efficiently into the neurons (Feurstein et al., 2010). Furthermore, Saul et al. (2014) obtained a significant decrease in all life trait variables, measured at different periods of the nematode life cycle, only at the highest concentration of MC-LR assayed. They inves tigated widely the variation in the gene expression, reporting an enhancement of 125, among which was unc-30, related to the GABAergic response, and a decrease of 76. Although these results may seem contradictory to the ones obtained by Ju et al. (2013), as they described a diminish of unc-30 gene expression, it is important to highlight that the duration of the stress exposure is essential for their regulation, being the possible cause for their discordance (Nadal et al., 2011). Moreover, Saul et al. (2014) also reported a down-regulation in let-7 expression, which could play a role in the development and UNCORRECTED PROOF 12 Science of the Total Environment xxx (xxxx) xxx-xxx the reproductive processes, contributing, therefore, to the effects observed in the brood size and growth. Their results manifested that many of the affected genes by MC-LR are involved in neurogenesis, signaling or neurological behavior processes, reinforcing those results previously obtained by Li et al. (2009a) and Ju et al. (2013), where MC-LR played an important role in the neuromodulating action. In general, the different behavioral studies agree that MC-LR produced a decrease in autonomic (body bend, head thrash, move length, pharyngeal pumping, touch response) and sensory (chemical, thermal) functions reflecting an alteration in the nervous system functions to generate appropriate behaviors from sensory signals in nematodes (Li et al., 2009a, 2009b; Ju et al., 2013, 2014; Moore et al., 2014). Therefore, MC-LR at environmentally relevant concentrations, could affect the nervous system regulation to receive, process, integrate and interpret sensory signals, as suggested by the gene expression results (Li et al., 2009a, 2009b; Ju et al., 2013; Saul et al., 2014; Hu et al., 2016). It is important to point out that not always a variation in the gene expression can be translated to a change in protein levels, being required complementary studies in order to assure the neurotoxic role of this toxin (Saul et al., 2014). Although previous studies confirmed the suitability of the C. elegans test as a neurotoxicity screening test for MCs (Ju et al., 2014), it should be taken into account that this experimental model is much simpler than the mammals-nervous system. The only neurotoxicity study performed in birds was carried out in Japanese quail exposed to Microcystis biomass containing MC-LR, MC-RR, MC-YR and MCs-similar compounds (Pašková et al., 2008). This study focused on the determination of oxidative stress, where a significant enhancement was reported in cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase (EROD) levels in the brain after acute and sub-chronic exposure at medium concentrations of MCs. The LPO levels were also enhanced after acute and sub-chronic exposure, so did the GSH levels, decreasing, nonetheless, after acute exposure. Howbeit, no significant changes were observed in GST activity in this organ. In general, a rise in the oxidative stress parameters was described by these authors in brain (Pašková et al., 2008). Oxidative stress as a mechanism of toxic action of MCs has been widely studied in other organs such as liver or kidney in different species (Li et al., 2003; Jos et al., 2005; Skocovska et al., 2007; Weng et al., 2007; Prieto et al., 2009); however, these investigations are very scarce in brain. The increase of ROS could be involved in the mitochondrial dysfunction and activation of calpain and Ca2+/calmodulin-dependent protein kinase II (Ding and Nam, 2003), generating damage in the brain structure and neurological functions. Mice exposed to pure MC-LR showed differences in the effects on hippocampus and cortex after oral exposure by drinking water with 1–40μg/L MC-LR for a year (Wang et al., 2018). Histopathological changes were observed in the hippocampus (bright eosinophil-like angular shape and nuclear fragments) and in the cortex (shrunken bodies and pyknotic nuclei) dose-dependently. Likewise, MC-LR produced different impacts on mRNA transcription genes and in their protein expression (ATP6, COX3, CYTB, DNA polymerase ɣ(POLG), mitochondrial single-stranded DNA-binding protein (mtSSB) and mitochondrial transcription factor A (TFAM)), mainly affecting the hippocampus. In accordance with these results, Shin et al. (2018) described a dose-dependent neuronal loss in the same hippocampal cells due to several morphological changes, but in this case, after exposure to a cyanobacterial extract containing MC-LR. Moreover, several behavioral studies demonstrated memory impairment after Morris water maze (MWM) and passive avoidance tests. However, these effects were only observed after exposure to 4μg/mL MC-LR, suggesting that the neuronal loss is not the main cause for these toxic effects in mice. After exposure to the same doses, no effects on spatial working and visual recognition memory were detected by Y-maze and novel object recognition tests, respectively. These effects were patent only in non-transgenic (non-Tg) mice compared to those overexpressing glutathione peroxidase (GPx Tg). Besides, in non-Tg group, these authors observed significant changes in oxidative stress biomarkers such as increased protein oxidation, LPO and ROS, together with a decrease of the GSH/Glutathione disulfide (GSSG) ratio. Moreover, the rise in SOD enzyme activity was more evident in non-Tg compared to the increase observed in GPx-1 Tg, while the enhancement of GPx enzyme activity was more visible in this last group of mice. Furthermore, no proinflammatory tumor necrosis factor-α(TNFα) and allograft inflammatory factor-1 (Iba1) levels were affected after the MC-LR exposure. All of the results obtained in this study suggest that memory impairments in mice exposed were due to oxidative stress in spite of by neuroinflammation process, which could be confirmed by the enhancement of nuclear factor erythroid-derived 2 (Nrf2) observed in both exposed groups. In addition, the reduced responses of GPx-1 Tg compared to non-Tg mice suggest a possible prevention of memory impairment by compounds implied in antioxidant activity (Shin et al., 2018). Furthermore, it is important to highlight that, although not being neurotoxicological studies per sé, some studies exposing mice to pure MC-LR have proven its capacity to cause effects in the HPI axis at hypothalamic level, altering the neurohormonal control of reproduction (Wang et al., 2012; Xiong et al., 2014; Chen et al., 2016). In rats exposed to MC-LR after infusion into hippocampus presented longer periods of time searching the platform and shorter swimming distance in the target zone, but no significant differences in swimming speed were appreciated compared to the control group (Li et al., 2012a). This would point out the spatial learning and memory impairment caused by the exposure to the toxin. Furthermore, some neuronal injury was observed by shrunk nuclei and cellular edema or dissolved cell organelles, diminishing significantly the number of CA1 pyramidal cells in the hippocampus. Nonetheless, after exposure to the lowest dose, some morphological changes were appreciated in the neurons, like swollen and degranulated endoplasmic reticulum or puffed periplast. In fact, a more significant rise in oxidative stress parameters was reported after exposure to the highest concentration (LPO, CAT, GPx, and SOD) versus the lowest (LPO and CAT). In agreement with these results, Li et al. (2012b) reported that chronic exposure produced neither changes in intake, body weight and overall mobility, nor visual and locomotor deficits, although they demonstrated the presence of this toxin in brain. However, treated rats did take longer to find the platform, mainly over the late days, spending less time in the target zone, which implies effects on spatial learning and memory as well. This impairment was also confirmed by the degeneration and apoptosis of hippocampal cells in rats exposed to MC-LR for 50days. Furthermore, the authors observed the presence of proteins involved in neurodegenerative diseases such as septin 5, a-in- ternexin and a-synuclein, and a PPs inhibition after exposure to 10μg/ kg MC-LR, which may lead to Tau hyperphosphorylation, implied in the generation of Alzheimer's disease. This is the first scientific study correlating MC-LR exposure to an age-associated neurodegenerative disorder. Additionally, Wang et al. (2013) reported a significant PPs activity enhancement after exposure to pure MC-LR in rats, in disagreement with the results obtained by Li et al. (2012b). This effect could be the cause for the reduction, at all concentrations, of the phosphorylation of GSK-3βin the hippocampus and, consequently, for the described long-term potential concentration-dependent effects, leading to a loss UNCORRECTED PROOF Science of the Total Environment xxx (xxxx) xxx-xxx 13 of neuronal plasticity. Moreover, this study showed, for the first time, the prevention of the neurotoxic effects caused by MC-LR by simultaneous treatment with a GSK-3βinhibitor. In agreement with Li et al. (2012a, 2012b), an investigation of the effects of MC-LR on learning and memory ability in rats was performed by Li et al. (2014), obtaining that the rats exposed to the highest dose presented prolonged escape latencies on the third day of training, while those exposed to lower doses had shorter frequencies entering the enlarged platform. Despite no significant differences in the number of damaged neurons were observed, an increase of astrocyte cells density in the hippocampus was reported after the exposure to the highest dose. This could be related to the increase of nitrogen reactive species, an inflammatory indicator, reported in the hippocampus at the same dose, playing a role in the central neuron system inflammatory reactions and affecting spatial memory impairment. The only study evaluating the transmission of the toxic effects of MC-LR in female rats to offspring was performed by Li et al. (2015b). In maternal rats, a decrease in the mean body weight gain was significant only at the highest dose of exposure. Respecting the behavior of the offspring, a significant reduction of the ability in the cliff avoidance test was observed, although no differences were perceived after the surface righting reflex and the negative geotaxis tests. However, no significant alterations in the locomotor activity were observed. In the MWM test, the frequencies in reaching the platform zone decreased dose-dependently in male offspring at all exposure doses, while in the case of female offspring, the diminishment of frequency was produced only after exposure to the highest doses, together with the effects on the swimming speed. Furthermore, although no evident pathological alterations in the hippocampus were observed, a significant increase of LPO and SOD levels were reported in male and female offspring after exposure to the highest dose, and an increase of LPO levels after 5μg/kg MC-LR exposures only in male subjects. Recently, Zhang et al. (2018) indicated an accumulation of MC-LR in the hippocampus after 24h of injection, causing demethylation of PP2Ac (inhibition of PP2Ac) and phosphorylation of GSK-3β(activation of GSK-3β). This could lead to the hyperphosphorylation of Tau, being in agreement with Li et al. (2012b) and Wang et al. (2013). These results confirm the effects obtained, as mentioned above, in the SH-SY5Y in vitro model in the same study (Zhang et al., 2018). Moreover, going along with the results obtained in the MWM test by Li et al. (2012a, 2012b, 2014, 2015b), a reduction of the swimming distance spent in the target zone was observed as well, in this case, after day 8 compared to day 6, producing, consequently, memory impairments. Although they represent a more realistic scenario, only two neurotoxicity studies have been carried out with MCs contained in extracts of cyanoblooms. In this sense, Maidana et al. (2006) used the step-down inhibitory avoidance test by injection of MC-LR containing raw extract. They reported a significant effect on long-term memory and the impairment of its retrieval at both doses assayed, while no significant changes were produced in short-term memory at any dose. Furthermore, using the radial arm maze to test the spatial memory, the number of working and reference memory errors increased only at day 8 of exposure at both concentrations, being probably caused by the accumulation of previous extracts-administrations. Surprisingly, an increase of the time spent to consume all the baits was reported in the same test only at the lowest dose. Moreover, these authors also studied different oxidative stress parameters, obtaining higher GST activity after exposure to the lowest dose compared to the highest. In the case of LPO levels, higher levels were obtained after exposure to the highest MCs dose, although the lower dose also caused lipid peroxidation. These parameters could be the cause for the increase of DNA damage observed after exposure to MCs in the comet assay, corroborating the role of oxidative stress in the neurotoxic effects produced by MC-containing extracts, as was previously demonstrated with pure MCs (Li et al., 2012a, 2015b). In agreement with these oxidative stress results, Zhao et al. (2015a) obtained an increase in the LPO levels in the brain of the pups after maternal exposure to MCs-ex- tract, together with a decrease in the GSH levels and in AChE activity in the cerebral cortex. Moreover, although these authors verified the presence of MC-LR in the offspring brains, no changes were obtained in the PP activity after maternal exposure, which would be in disagreement with the PP activity enhancement reported by Wang et al. (2013) and its decrease reported by Li et al. (2012b) and Zhang et al. (2018). This could be due to the experimental subjects since in both cases the parameter was measured in a direct object, the adult rats, versus an indirect object, their pups; or the discordance in the administration route, being, in this case, subcutaneous. Furthermore, similar ultrastructural changes were obtained in brain offspring by Li et al. (2012a). Likewise, an alteration of proteins involved in neurodevelopment was detected as well, in agreement with Li et al. (2012b). Taken together, all the experiments conclude that MCs both pure and contained in cyanoblooms extracts produced important neurotoxic effects in several species by different exposure routes. Mostly, MCs caused oxidative stress and alteration of biochemical chains that ended up leading to huge effects such as hyperphosphorylation of Tau. In fact, most of them demonstrate the spatial learning and memory impairment by several behavioral tests. Howbeit, very few studies have been performed using other MC congeners besides MC-LR, isolated and contained in the mixture in a cyanobacterial extract, although some of them have demonstrated to exert more severe neurotoxic effects, being the case of MC-LF for instance. 3. Cylindrospermopsin Cylindrospermopsin consists of a tricyclic guanidine group combined with a hydroxylmethyl uracil group (Ohtani et al., 1992). Its structure presents a zwitterionic nature and a low molecular weight (415Da) (Falconer and Humpage, 2006). This cyanotoxin is produced by several cyanobacterial genera such as Cylindrospermopsis, Aphanizomenon, Umezakia, Chrysosporum, and Anabaena, among others (Harada et al., 1994; Banker et al., 1997; Shaw et al., 1999; Schembri et al., 2001) (Fig. 3). (Fig. 4.) Fig. 3. Structure of CYN. UNCORRECTED PROOF 14 Science of the Total Environment xxx (xxxx) xxx-xxx Fig. 4. Main mechanisms of neurotoxic action of CYN. Despite being the liver its main target, many other organs such as kidneys, lungs, thymus, marrow bone, adrenal gland, gastrointestinal tract, immune and nervous systems, and heart have been described as potential targets as well (Hawkins et al., 1985; Terao et al., 1994; Falconer et al., 1999; Humpage et al., 2000; Guzmán-Guillén et al., 2015). The most well-known mechanism of action for CYN is the protein and GSH synthesis-inhibition (Terao et al., 1994; Runnegar et al., 1995; Froscio et al., 2003). In addition, due to its ability to enhance ROS production, this toxin can lead to DNA damage, causing cell death by apoptosis (Roos and Kaina, 2006; Gutiérrez-Praena et al., 2011; Puerto et al., 2011; Gutiérrez-Praena et al., 2012; Guzmán-Guillén et al., 2013) (Fig. 4). Moreover, some studies have demonstrated the importance of its previous metabolic activation by the enzymatic complex cytochrome P-450, since it is able to exert genotoxic potential (Runnegar et al., 1995; Norris et al., 2001; Froscio et al., 2003; Humpage et al., 2005; Žegura et al., 2011; Puerto et al., 2018). As a cytotoxin, these effects could be also caused in the nervous system. Besides, it is important to notice that the chemical structure of CYN is more alike to neurotoxins than to hepatotoxins, as it was classified at first, not being unexpected for this cyanotoxin to also cause neurological disorders (Kiss et al., 2002). Furthermore, although it is not likely for CYN to cross the BBB by passive diffusion due to its hydrophilic properties (Banks, 2009), its low molecular weight might play a role in its entrance to the nervous system. There are some studies pointing out its neurotoxicity in different in vitro and in vivo models, although the mechanisms for which CYN could exert neurotoxic effects in the brain remain unknown. 3.1. Neurotoxicological in vitro studies performed with cylindrospermopsin Up to date, in comparison with MCs, very few studies have brought to light the potential neurotoxicity CYN can exert (Table 5). Furthermore, most of them have been performed using extracts or cultures of Cylindrospermopsis raciborskii or Aphanizomenon ovalisporum. In this sense, the first study suggesting its neurotoxic effect was performed by Kiss et al. (2002), who exposed CNS neurons of two species of snail, Helix pomatia L. and Lymnaea stagnalis L., to a C. raciborskii purified fraction and ATX-a. They suggested that the purified fraction could be CYN, and although it had no direct effect on the membrane of the neurons, it decreased the ACh-induced membrane response, suggesting a neuroactive effect on the cell membrane for the first time. On the contrary, Vehovszky et al. (2013) reported that application of a CYN-producing strain to CNS preparations of H. pomatia (at 20mg/mL) did not display the same cholinergic inhibitory effects, although these were observed after exposure to a non-CYN-produc- ing C. raciborskii bloom, which authors attribute to some ATX-a like compound. In the case of CYN, contrary to MCs, there is only one work with pure toxin, performed by Takser et al. (2016). These authors evaluated in vitro the individual and combined effects of CYN, MC-LR and ATX-a, at environmentally relevant low concentrations (10μM alone and 3.3μM in mixture), in brain cell lines. Their findings revealed that CYN individually and the mixture containing CYN were 3–15 times more potent than the individual toxins, inducing apoptosis and inflammation in murine BV-2 microglia cells and N2a murine neuroblasts cells. Besides, the latest were more sensitive to the mixture than BV-2 cells, causing a meaningful pro-inflammatory response to CYN and the mixture, demonstrating that low concentrations of CYN are highly relevant for neurodegeneration. These outcomes could have potential implications in future research on neurodegenerative diseases. Nevertheless, care should be taken in the extrapolation of these in vitro results to in vivo circumstances, including human health effects, mainly concerning the developing brain where there is no BBB yet. 3.2. Neurotoxicological in vivo studies performed with cylindrospermopsin Studies concerning CYN neurotoxicity in vivo are scarce (Table 5), although they provide interesting results. In this regard, White et al. (2007) reported that 7day-exposure of Bufo marinus tadpoles to whole cell extracts or live cultures of C. raciborskii at 400 or 232μg/L, respectively, appeared to decrease their activity levels, mostly swimming behavior, which could make them more vulnerable to prey, but also be used as an avoidance strategy from visually-oriented hunters. This effect, however, might have been caused by damage in some other organs. It is worth to mention that live C. raciborskii cultures contained a mixture of intra- and extracellular CYN, whereas the cell extracts only had extracellular CYN, and they also reported the presence of deoxy-CYN. This work by White et al. (2007) was the first one using amphibians as experimental model, whose changes in behavior gain relevance as they are usually the first indication of sublethal exposure (Henry, 2000), being a possible indicator of CYN neurotoxicity. In agreement with these results, Kinnear et al. (2007), using the same model and conditions, but nearly half the concentrations (200 and 107μg/L, for the cell extracts and the live cultures, respectively), reported a reduction in the swimming ability and un-coordination in tadpoles of B. marinus. They suggested that it could be due to the disintegration of the brain, as the encephalon had a loosely arranged matrix and brain cells were disintegrated and sometimes necrotic, showing a mix of the outer matrix and inner cells, together with general organ failure. Besides, authors also hypothesized that degeneration of the gill epithelia could have led to suffocation, and finally to the consequently reduced activity. To our knowledge, there are only two studies concerning the neurotoxicity of CYN in fish. CYN was detected by ELISA in the brain of all tilapia fish (Oreochromis niloticus) exposed subchronically (14days) by immersion to repeated concentrations (10μg/L) of an A. ovalisporum culture containing CYN and deoxy-CYN (Guzmán-Guillén et al., 2015). As a result, a marked increase in LPO levels, and a reduction in AChE activity in tilapia brains was observed, al UNCORRECTED PROOF Science of the Total Environment xxx (xxxx) xxx-xxx 15 Table 5 Neurotoxicity studies performed with CYN. CYN/Cyanobacteria Experimental model Experimental conditions Assays performed Relevant Results References In vitro Crude extracts of C. raciborskii Neurons of Helix pomatia Extracts of both the bloom sample and the laboratory isolate of the bloom were diluted in physiological Helix saline and applied by perfusion at a constant flow rate Electrophysiological experiments No cholinergic alteration was observed with the CYN-producing strain Vehovszky et al. (2013) Pure CYN N2a murine neuroblastoma derived cells 0.001, 0.1 and 10μM for 24, 48 and 72h MTT assay Apoptotic cell death TNF-α measurement Concentration and time-dependent decrease of cell viability after all time exposures to both 0.1 and 10μM. Significant rise in proapoptotic caspases after exposure to 10μM Takser et al. (2016) BV-2 microglia murine cells Concentration-dependent decrease of cell viability after all exposure times to both 0.1 and 10μM. Significant rise in proapoptotic caspases after exposure to 10μM In vivo Whole cell extracts of C. raciborskii and live cultures of C. raciborskii Bufo marinus tadpoles 0–200 and 0-107μg/L, respectively, for 7days, by transdermal route Histopathological study No mortality observed. Several histopathological changes in the encephalon Kinnear et al. (2007) Whole cell extracts of C. raciborskii Bufo marinus tadpoles 0–400μg/L for 7days, by transdermal route Behavioral studies Toxin analysis Decrease in behavior scores Neither mortality nor growth rates were affected White et al. (2007) Live cultures of C. raciborskii 0–232μg/L for 7days, by transdermal route Decrease in behavior scores Time-dependent increase in mortality Negative growth rates A. ovalisporum culture containing CYN Tilapia fish (Oreochromis nicotilus) 10μg/L for 14days, by transdermal and oral route AChE activity, LPO, histopathological study and ELISA Inhibition of the AChE activity Rise in LPO levels Necrosis, hyperemia, haemorrhagia and edema CYN detection in all brain samples Guzmán- Guillén et al. (2015) Purified CYN (CYNp) and extract of C. raciborskii containing CYN (CYNex) Trahira (Hoplias malabaricus) Single dose of 50μg/kg b.w. for 7 and 14days by intraperitoneal injection AChE activity, GST activity, LPO and ELISA Increase of AChE activity after 7days of exposure to CYNex, decreasing after 14days. Decrease of GST after 7days of exposure to CYNex and increase after 7days of exposure to CYNex and after 14days of exposure to CYNp and CYNex. Rise in LPO levels after 7 and 14days of exposure to CYNp and CYNex. Detection of CYN in brain da Silva et al. (2018) Abbreviations: A. ovalisporum:Aphanizomenon ovalisporum; AChE: Acetylcholinesterase; BV-2: cellosaurus cell line; b.w.: body weight; C. raciborskii:Cylindrospermopsis raciborskii; CYN: cylindrospermopsin; CYNp: purified cylindrospermopsin; CYNex: extract containing cylindrospermopsin; GST: glutathione-S-transferase; LPO: lipid peroxidation; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N2a: fast-growing mouse neuroblastoma cells. though the inhibition of AChE activity was too low to induce neurological symptoms. In addition, signs of necrosis, vacuolization, chromatin condensation, cytoplasmic edema and mitochondrial swelling were reported as well. Recently, detection of CYN has also been reported in brains of the fish Hoplias malabaricus exposed by a single i.p. injection (50μg/kg b.w.) to purified CYN or to extracts of a CYN-producing strain of C. raciborskii, even 7 and 14days after exposure (Da Silva et al., 2018). In addition, detected CYN levels were higher after exposure to the extracts, which could point out the importance of other compounds in the extract (i.e. lipopolysaccharides) that might also affect CYN crossing of the BBB. Nonetheless, no significant effects were noticed on AChE activity after CYN exposure in any form tested, contrary to the results obtained by Guzmán-Guillén et al. (2015), which could be due to differences in the exposure concentrations and times (subchronic versus acute exposure) or in the fish species, although both studies agree on the rise of LPO levels. Moreover, GSH levels did not vary in H. malabaricus after exposure to CYN, but different responses were obtained for GST activity for extracts and pure CYN. To exert neurotoxic effects, toxins must be transported into cells or interact with channels or receptors of the cell membrane (Stillwell, 2013), suggesting the interference of other compounds present in the extract (Da Silva et al., 2018). Some neurological symptoms after exposure of alligators (Schoeb et al., 2002) and mice (Saker et al., 2003; Zagatto et al., 2012) to C. raciborskii strains have been attributed to CYN (Poniedziałek et al., 2012). However, it is important to clarify that neither of these studies proved the presence of CYN in those strains, so the reported effects might be due to different compounds present in the extracts or different secondary metabolites, such as STX. 4. Conclusions This review summarizes, as far as we know, the reports available on the scientific literature dealing with the neurotoxicity assays performed in vitro and in vivo to elucidate the toxic effects that MCs and CYN can exert in the nervous system. In the case of MCs, they have proven to cause neurotoxicity by their crossing using the OATPs, which are present in the BBB and in most neural cells, leading to a rise in the [Ca2+]ilevels and, therefore, apoptosis. These cyanotoxins have demonstrated to exert neurotoxic effects mostly in the limbic system. In fact, some histopathological studies have described impor UNCORRECTED PROOF 16 Science of the Total Environment xxx (xxxx) xxx-xxx tant damages in the hippocampus and in the cortex, together with global biochemical alterations, being especially relevant Tau hyperphosphorylation, characteristic of some neurodegenerative diseases such as Alzheimer's disease. On the other hand, these toxins have proven to cause damage in the hypothalamus as well, having an impact in other systems of the organism such as the reproductive or the endocrine. Furthermore, MCs have exerted a rise in oxidative stress and lipid peroxidation, together with neurotransmission alterations (DA, ACh and GABA levels), leading to autonomic and sensory responses. Thus, MCs not only cause effects in the CNS but also in the peripheral nervous system. Furthermore, some other minor variants such as MC-LF or MC-LW require attention as well, since both have demonstrated to be even more toxic in neural cells, in spite of being less environmentally abundant. Special attention should be paid to the fact that very little studies have been carried out in vivo using one of the major congeners in nature, MC-RR. In the case of CYN, the number of studies performed is even scarcer, reporting deregulation of some oxidative stress parameters was observed together with alteration of AChE activity, which could be linked to the histological changes observed. Thus, although neurotoxicity mechanisms for CYN are still unknown, it seems to be caused by damage in the CNS. For all mentioned above, further research is required in order to clarify the neurotoxic potential of several MC congeners and CYN, as well as their possible contribution in neurodegenerative diseases. 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Supplies and chemicals 125 CylindrospermopsinYN (purity > 95% by HPLC) was purchased from Enzo Life 126 Sciences. Nutrient Mixture F-12 Ham and CPF were purchased from Sigma-Aldrich 127 (Madrid, Spain). Minimum essential medium (MEM), fetal bovine serum (FBS) and 128 cell culture reagents were obtained from Gibco (Biomol, Sevilla, Spain). 129 The Bradford reagent was purchased from Sigma-Aldrich (Madrid, Spain). The 130 supplier of MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- 131 sulphophenyl)-2H-tetrazolium salt) Cell Titer 96® AQueous One Solution Cell 132 Proliferation Assay was Promega (Biotech Iberica, Madrid, Spain). 133 2.2. Model system 134 SH-SY5Y cells, derived from a human neuroblastoma, were obtained from the 135 American Type Culture Collection (CRL-2266). These cells were maintained in a MEM 136 and F-12 (1:1) medium supplemented with 10% FBS, 1% L-glutamine 200 mM, 1% 137 sodium pyruvate, 1% non-essential amino acids, and 1% penicillin/streptomycin 138 solution, in an atmosphere containing 5% CO2 at 95% relative humidity at 37ºC (CO2 139 incubator, NuAire®, Spain). Cells were grown 80% confluent in 75-cm2 plastic flasks 140 and harvested twice a week with 0.25% trypsin-EDTA (1X). The quantification of the 141 cells was performed in a Neubauer chamber. SH-SY5Y cells were plated at density of 142 1·105 cells/mL to perform all the experiments. 143 2.3. Toxin test solutions 144 Stock solution of 1 mg/mL CYN was prepared in sterilized milliQ water, and 145 maintained at -20˚C until its use. Stock solution of 50 mg/mL CPF was prepared in 146 absolute ethanol and maintained at -20˚C until its use. 147 7 2.4. Cytotoxicity assays 148 SH-SY5Y cells were seeded for basal toxicity tests in 96-well tissue-culture 149 plates and, after being incubated at 37˚C for 24 hours, the exposure took place. From 150 the CYN-stock solution, serial dilutions in medium without serum were prepared (0-1 151 µg/mL CYN) based on previous studies (Hinojosa et al., 2019a). From the stock 152 solution of CPF, serial dilutions in medium without serum were prepared (0-200 µg/mL 153 CPF). This concentration was selected based oin previous studies (Amani et al., 2016; 154 Fu et al., 2019). Non-treated cells were used as a negative control. A solvent control 155 was also evaluated to ensure its innocuous role (data not shown). After replacing the 156 medium, the addition of the exposure solutions to the plates and incubation for 24 and 157 48 hours at 37°C took place. The endpoints assayed were protein content (PC) and 158 tetrazolium salt reduction (MTS). All the assays were performed by triplicate. All the 159 endpoints assayed are well described in Hinojosa et al. (2019a). 160 2.5. Assessment of the effect of CYN-CPF combination by the isobolograms method 161 Concentrations used to evaluate the toxic potential of the combination CYN-162 CPF were chosen based on the cytotoxicity results of CYN and CPF previously 163 obtained (Hinojosa et al., 2019a). The highest exposure concentrations for the 164 combination studies were those obtained as the mean effective concentration (EC50) of 165 the most sensitive endpoint, in this case, the MTS assay at 24 hours, along with the 166 fractions EC50/2 and EC50/4. Thus, SH-SY5Y cells were exposed for 24 and 48 hours to 167 binary pure pollutants combinations: EC50 CYN + EC50 CPF, EC50/2 CYN + EC50/2 168 CPF and EC50/4 CYN + EC50/4 CPF. 169 The isobologram method was used according to Tatay et al. (2014) to determine 170 the type of interaction occurring when CYN and CPF are combined. This method 171 consists of plotting the concentration-effects curves for each compound and their 172 8 combinations in multiple diluted concentrations using the median-effect equation. The 173 combination index (CI) quantifies the synergism (CI < 1), the additivity (CI = 1), or the 174 antagonism (CI > 1) of the combinations. The CalcuSyn software calculates these CI 175 values automatically (Biosoft, Cambridge, UK). 176 2.6. Oxidative stress assays 177 2.6.1. Reactive Oxygen Species (ROS) generation 178 The ROS production was assessed in 96-well plates using the 179 dichlorofluorescein (DCF) assay, previously optimized by Medrano-Padial et al. (2019). 180 Fluorescence was measured at 535 nm (emission) and 485 nm (excitation) after 181 incubation for 4, 8, 12 and 24 hours. 182 2.6.2. Glutathione (GSH) content 183 The GSH content was measured according to Maisanaba et al. (2018). Cells 184 were exposed and incubated for 4, 8, 12 or 24 hours. Buthionine sulfoximine (BSO) 1µM 185 was used as positive control. Fluorescence was measured 460 nm (emission) and 380 186 nm (excitation). 187 2.7. Acetylcholinesterase (AChE) activity determination 188 Acetylcholinesterase activity was measured following the method of Ellman et 189 al. (1961) with modifications (Santillo et al., 2015). Viable SH-SY5Y cells were 190 exposed to the toxins and incubated at 37ºC for 24 hours. A solution of parathion 50 nM 191 was used as a positive control. Then, the medium was discarded and replaced by 200 µL 192 of a reaction mixture containing 0.5 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) 193 and 100 µM acetyltiocholine (ATCh) per well. The resulting product of the reaction, 5-194 thio-2-nitrobenzoate (TNB), was measured at 410 nm every 90 seconds up to 1 hour. 195 2.8. Morphology 196 9 The concentrations used for the morphological study were the ones obtained in 197 the cytotoxicity assays, being the EC50 values those obtained after 24 hours of exposure. 198 These values were chosen as the highest concentrations of exposure along with the 199 fractions EC50/2 and EC50/4. SH-SY5Y cells were exposed for 24 hours. The procedures 200 for the contrast, light and electron microscopies were described by Hinojosa et al. 201 (2019a). 202 2.10. Calculations and statistical analysis 203 Data for the cytotoxicity assays, oxidative stress biomarkers and AChE were 204 presented as mean ± standard deviation (SD) in relation to control. Analysis of variance 205 (ANOVA) followed by Dunnett’s multiple comparison was carried out to perform the 206 statistical analysis, using GraphPad InStat software (GraphPad Software Inc., La Jolla, 207 USA). Differences were considered significant from P<0.05. EC50 values were the 208 result of the linear regression in the concentration-response curves. 209 3. Results 210 3.1. Cytotoxicity assays 211 A concentrationdependent decrease in the viability was observed in the SH-212 SY5Y cells after being exposed to 0-200 µg/mL CPF for 24 and 48 hours (Fig. 1). The 213 EC50 values obtained were 83.98 ± 2.74 µg/mL and 85.31 ± 7.67 µg/mL after 24 hours 214 of exposure for the MTS assay and the PC assay, respectively. In the case of the cells 215 exposed to CPF for 48 hours, the EC50 values were 69.78 ± 6.02 µg/mL and >200 216 µg/mL for the MTS and PC assays, respectively. Thus, the MTS assay demonstrated to 217 be the most sensitive biomarker. 218 3.2. Assessment of the effect of CYN-CPF combination by the isobolograms method 219 10 Using the EC50 value previously indicated in Hinojosa et al. (2019a), the 220 exposure concentrations of CYN were 0.25, 0.5 and 1 µg/mL, and while for CPF were 221 21, 42 and 84 µg/mL of CPF, being both combined in a proportion of 1:84 for 24 or 48 222 hours (Fig. 2). The experiments performed with the combination presented a CI>1 in the 223 case of the cells exposed for 24 hours, confirming an antagonistic response (Fig. 3A). 224 However, in those cells exposed for 48 hours, the response obtained was mainly 225 synergic at low concentrations (CI<1) and antagonistic at higher ones (CI>1) (Fig. 3B). 226 3.3. Oxidative stress assays 227 The exposure to 21, 42 or 84 µg/mL CPF led to no significant changes in the 228 ROS levels compared to the control group in SH-SY5Y cells after any of the times of 229 exposure (4, 8, 12 or 24 hours) (Fig. 4A). However, it caused a significant decrease of 230 the GSH levels after all the times of exposure to the three concentrations assayed (Fig. 231 4B). 232 Similarly, the combination of the cyanotoxin and the organophosphate OP (1:84) 233 caused a reduction of the GSH levels in all the concentrations and times of exposure 234 assayed (Fig. 4D), despite the ROS levels were not altered compared to the control 235 group (Fig. 4C). 236 3.4. Acetylcholinesterase activity 237 All the concentrations of CPF (21, 42 and 84 µg/mL) reduced the AChE activity 238 in SH-SY5Y cells. Concerning the combination, all the concentrations assayed induced 239 a reduction of the AChE activity (Fig. 5). 240 3.5. Morphology study 241 Unexposed SH-SY5Y cells observed under phase-contrast microscopye showed 242 a star-shape morphology with neurites contacting adjacent cells. The presence of mitotic 243 11 processes evidenced the active proliferation of these cells (Fig. 6A). The light 244 microscope revealed clear euchromatic nuclei with obvious nucleoli (Fig. 6B). Under 245 electronic microscope, cells showed a big number of mitochondria in the cytoplasm, 246 which also presented vacuole with a dense matrix (Fig. 6C). 247 3.5.1. Microscope observations of cells exposed to CPF 248 Cells exposed to 42.5 and 84 µg/mL CPF presented, under phase-contrast and 249 light microcopies, cell death signs. Cytoplasmic projections diminished with a low 250 proliferation rate. At the lowest concentration, a vacuolization of the cytoplasm was 251 observed (Fig. 6D-E). Ultrastructurally, mitochondrial organelles increased their size 252 due to the entrance of water. In addition, an alteration of the pattern of the 253 mitochondrial crests was observed. These cellular processes drove to the presence of 254 heterophagosomes and to condensation of the chromatin, a clear sign of apoptosis (Fig. 255 6F). 256 3.5.2. Microscope observations of cells exposed to the combination CYN+CPF 257 Concerning the combination, an intense vacuolization of the cytoplasm, cellular 258 cycle detention, loss of cytoplasmic projections, and cell deathd were observed at the 259 three concentrations assayed (Fig. 6G-H). Under the transmission electronic 260 microscopye, small lipid droplets associated with mitochondrial organelles were 261 observed. Moreover, at the highest dose assayed (1 µg/mL CYN + 84 µg/mL CPF), 262 rough endoplasmic reticulum (RER) cisternae appeared dilated with dense content, and 263 an intense presence of heterophagosomes was observed (Fig. 6I). 264 265 4. Discussion 266 12 The interactive effects of chemicals and natural stressors have been reviewed 267 (Holmstrup et al., 2010; Laskowski et al., 2010). Synergistic interactions were reported 268 in more than 50% of the available studies of interactions between chemicals with abiotic 269 stressors. The coexistence of multiple cyanotoxins or with some other active 270 compounds has been described (Al-Sammak et al., 2014; Chen et al., 2017; Martínez-271 Ruiz and Martínez-Jerónimo, 2016; Pathmalal, 2019; Tatters et al., 2017). However, 272 although pesticides are candidates to appear together with cyanotoxins, literature 273 regarding this subject is almost non-existent (Asselman et al., 2014; Daam et al., 2011; 274 Moe et al., 2012). In this sense, EFSA has highlighted the importance of addressing the 275 concomitant exposure of other chemicals, such as pesticides, along with cyanotoxins in 276 order to have information on mixture toxicity and on the environmental factors affecting 277 toxicity of a bloom (Testai et al., 2016). 278 The present work focused on the potential effects induced by the combination of 279 the cyanotoxin CYN and the organophosphate CPF in the neuronal SH-SY5Y cell line. 280 To address this research, a cytotoxicity study of CYN and CPF in the cell model was 281 required in order to establish the EC50 values of the test items to be applied in the 282 combination study. In the case of CYN, the EC50 value of 1 µg/mL CYN was taken 283 from a previous work, where SH-SY5Y cells were exposed to 0-10 µg/mL CYN for 24 284 and 48 hours (Hinojosa et al., 2019a). Regarding to CPF our study confirms its 285 damaging properties in neuronal cells, showing a time- and concentration-dependent 286 cytotoxicity. In agreement with our results, Park et al. (2013) demonstrated that CPF 287 induced cytotoxic effects in a concentration-dependent manner in SH-SY5Y cells 288 exposed to 0-70 µM CPF, establishing an EC50 value of ~32 µg/mL CPF after 24 hours 289 of exposure. Similarly, Raszewski et al. (2015) also found a time- and concentration-290 dependent cytotoxic behavior of CPF in SH-SY5Y cells exposed during 24, 48, and 72 291 13 hours to 0-175 µg/mL CPF, obtaining an EC50 value for 24 hours of ~of ~100 µg/mL 292 CPF. 293 The potential mechanisms by which CPF induces its neurotoxic effects are 294 oxidative stress and acetylcholinesterase inhibition, among others (Park et al., 2013; 295 2015). In our study, CPF did not show any alteration of ROS levels at any of the 296 concentrations assayed. However, GSH levels presented a concentration-dependent 297 decrease, which could indicate that GSH is scavenging ROS. In this sense, Park et al. 298 (2015) and Xu et al. (2017) found an increment in ROS levels when SH-SY5Y cells 299 were exposed to CPF. However, they did not evaluate the GSH levels, which could give 300 an idea about the antioxidant activity of the neuroblastoma cell line. On the other hand, 301 Giordano et al. (2007) evaluated GSH levels of SH-SY5Y cells exposed to CPF. The 302 authors did not find alterations in the tripeptide levels, although they observed and 303 increment in oxidized GSH levels, which indicated that an antioxidant response was 304 taking place, suggesting that cytotoxicity may be mediated by oxidative stress. In 305 addition, Park et al. (2013, 2015) suggested that CPF-oxidative stress induction was 306 related towith AChE inhibition, together with its usual mechanism of action. In 307 agreement with this, CPF induced a clear inhibition of the AChE activity at all the 308 concentrations assayed in this case. Other authors have also pointed out that a depletion 309 of GSH levels is linked to with a mitochondrial dysfunction, which could lead to 310 apoptotic processes (de Oliveira et al., 2016; Park et al., 2013). This is in concordance 311 with our morphological findings, where the highest concentrations of CPF induced 312 mitochondrial alterations, leading to cell death by apoptosis. This process is related to 313 the activation of caspase-9 and -3 and an increment of cytosolic cytochrome c, as Park 314 et al. (2015) reported for SH-SY5Y cells exposed to CPF. Considering this, Raszewski 315 et al. (2015) proposed that CPF induces neurotoxicity through apoptotic mechanisms. 316 14 Once the toxic effects of CYN (Hinojosa et al., 2019a) and CPF alone on the 317 neuronal SH-SY5Y cell line were elucidated, the behavior of their combination on the 318 same cell line was evaluated. Our results showed that the combination was more 319 cytotoxic than both toxicants alone after 24 and 48 hours. However, in order to establish 320 the type of interaction between both compounds, the isobologram method was used. 321 Thus, after 24 hours of exposure, an antagonistic response was observed. This pattern 322 changed aAfter 48 hours, this pattern changed, showing a synergistic response at low 323 concentrations, and turning to an antagonistic response at higher concentrations. A 324 similar pattern was found when the combined toxicity of the cyanotoxin Microcystin 325 (MC)-LR and Cu exposure was investigated in the aquatic plant Vallisneria natans, 326 producing synergistic effects when combined at low concentrations (Wang et al., 2017). 327 Considering this, it is noteworthy to mention that CYN usually appears at low 328 concentrations in nature (de la Cruz et al., 2013), making a synergistic toxic response 329 very likely to appear. 330 To our knowledge, no studies concerning the effects of the combination of 331 cyanotoxins and pesticides using neuronal cell lines have been carried out. However, in 332 vivo, the first combination of cyanobacteria and pesticides was performed by Cerbin et 333 al. (2010), using a strain of Microcystis aeruginosa and the carbamate carbaryl. In this 334 study, the combination demonstrated a mostly additive interaction in Daphnia pulicaria 335 after 24 hours of exposure, while their effect in body deformations of newborns was 336 even synergistic. Ondracek et al. (2012) exposed for 10 days Japanese quails for 10 337 days to a combination of a cyanobacterial biomass with MCs (61.62 µg/day MCs), the 338 organophosphate OP paraoxon (2 doses of 250 µg/Kg paraoxon in that period), and an 339 anticoagulant (2 doses of 500 mg/Kg bromadiolone). They observed that the 340 combination induced a more severe damages in the birds than the isolated compounds. 341 15 This brings to light that the combination of cyanotoxins and pesticides usually drives to 342 a synergistic response, which is in agreement with our results at low concentrations 343 after 48 hours of exposure. In contrast, Asselman et al. (2013) observed non-interactive 344 and antagonistic effects for the combination of carbaryl with four species of 345 cyanobacteria (some of them CYN-producers) on Daphnia pulex. Moreover, when these 346 same authors studied the effects of the combination of four different insecticides 347 (including CPF) and M. aeruginosa on Daphnia pulex after 21 days of exposure, a 348 different response was found (Asselman et al., 2014). They observed an additive 349 responsehus, for CPF and the cyanobacterial strain they observed an additive response, 350 different to the antagonistic response that they found using carbaryl, which presents the 351 same mechanism of action (Asselman et al., 2013). For this reason, they postulated that 352 interactive effects could not be generalized for compounds targeting the same pathway 353 (Asselman et al., 2014). 354 Regarding the oxidative stress induction, the combination of CYN and CPF did 355 not induce a significant variation in ROS levels compared to the control group, 356 following the trend showed by CYN in Hinojosa et al. (2019a) and CPF described in the 357 present paper. Regarding the GSH levels, the combination of both compounds led to a 358 reduction of its levels. However, the decline was lower than the one caused by CPF 359 alone. This could be due to the fact that CYN alone did not induce any alteration on 360 GSH levels in SH-SY5Y cells (Hinojosa et al., 2019a). This decrease, as mentioned 361 before, could be the consequence of athe compensating mechanism for the oxidative 362 stress, explaining the non-altered ROS levels shown by the mixture. 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Toxicol Sci. 2000;55:124-132. 639 Doi: 10.1093/toxsci/55.1.124 640 1 In vitro assessment of the combination of Cylindrospermopsin and the 1 organophosphate chlorpyrifos on the human neuroblastoma SH-SY5Y cell line 2 Hinojosa MG1, Prieto AI1, Gutiérrez-Praena D1*, Moreno FJ2, Cameán AM1, Jos A1 3 1 Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla, C/Profesor García 4 González 2, 41012 Sevilla, Spain. 5 2 Área de Biología Celular, Facultad de Biología, Universidad de Sevilla, Avda. Reina 6 Mercedes s/n, 41012 Sevilla, Spain 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 * Corresponding author: Daniel Gutiérrez Praena. Área de Toxicología, Facultad de 26 Farmacia, Universidad de Sevilla. C/Profesor García González 2, 41012 Sevilla, Spain. 27 Mail: [email protected] 28 *Manuscript Click here to download Manuscript: Hinojosa et al., 2020_revised version_withoutcontrol.docxClick here to view linked References 2 Abstract 29 Cylindrospermopsin (CYN) is a cyanotoxin which occurrence is increasing due 30 to climate change. This toxin is able to exert damage in the organism at several levels, 31 among them, in the nervous system. Moreover, it is important to take into account that 32 this toxin is not usually present isolated in nature, but in combination with some other 33 pollutants, being the case of the pesticide chlorpyrifos (CPF). Thus, the aim of the 34 present work was to assess the effects of the interaction of CYN in combination with 35 CPF in the human neuroblastoma cell line SH-SY5Y by evaluating cytotoxicity and 36 mechanistic endpoints. The mixtures 0.25+21, 0.5+42, 1+84 µg/mL of CYN+CPF 37 based on cytotoxicity results, were evaluated, and the isobologram method detected an 38 antagonistic effect after 24 and 48 hours of exposure. Moreover, although no alterations 39 of reactive oxygen species were detected, a significant decrease of glutathione levels 40 was observed after exposure to both, CPF alone and the combination, at all the 41 concentrations and times of exposure assayed. In addition, CYN + CPF caused a 42 marked decrease in the acetylcholinesterase activity, providing similar values to CPF 43 alone. However, these effects were less severe than expected. All these findings, 44 together with the morphological study results obtained, point out that it is important to 45 take into account the interaction of CYN with other pollutants. Further research is 46 required to contribute to the risk assessment of CYN and other contaminants 47 considering more realistic exposure scenarios. 48 49 50 Keywords: Cylindrospermopsin, chlorpyrifos, SH-SY5Y cells, chemicals combination, 51 pollutants 52 3 1. Introduction 53 Cyanobacterial blooms are a global environmental concern due to their increased 54 occurrence in terrestrial, marine and freshwater ecosystems, caused by climate change 55 and human influence. Among their detrimental effects, they produce unpleasant 56 organoleptic properties where they proliferate (Manning and Nobles, 2017). However, 57 the main hazard is their capacity to produce cyanotoxins, toxic secondary metabolites 58 that threaten water safety and aquatic life (Brooks et al., 2017; Buratti et al., 2017). 59 There are different exposure routes to them, standing out the oral one as one of the most 60 common and dangerous. Nonetheless, other relevant routes such as dermal contact or 61 inhalation are worth to be mentioned as well (Buratti et al., 2017). 62 Cylindrospermopsin (CYN) is one of the most common aquatic cyanotoxins as a 63 consequence of the increasing occurrence of Cylindrospermopsis raciborskii blooms, 64 the most relevant producer species (Antunes et al., 2015). However, other 65 cyanobacterial species such as Chrysosporum ovalisporum, Aphanizomenon flos-aquae, 66 Anabaena bergii, or Raphidiopsis curvata are able to produce this toxin as well 67 (Manning and Nobles, 2017). Cylindrospermopsin consists of an alkaloid with a 68 tricyclic guanidine linked to a hydroxymethyl uracil group (Ohtani et al., 1992). The 69 main episode of human intoxication by CYN occurred in the outbreak of 1979 in Palm 70 Island (Australia) when 146 people were hospitalized in a local clinic with malaise, 71 vomits, anorexia and hepatomegaly after drinking from a water supply containing CYN-72 producing C. raciborskii (Bourke et al., 1983; Griffiths and Saker, 2003). However, 73 despite intoxication by drinking contaminated water, it is also feasible to be exposed by 74 ingestion of contaminated food. In fact, the bioaccumulation of these cyanotoxins by 75 plants irrigated with contaminated water (Testai et al., 2016) or in exposed aquatic 76 organisms has been demonstrated, enhancing the risk of human intoxication by the food 77 4 chain (Cordeiro-Araújo et al., 2017; Gutiérrez-Praena et al., 2013; Machado et al., 78 2017). 79 Although its target organ is the liver, CYN has widely demonstrated its capacity 80 to damage other organs, including the nervous system (Guzmán-Guillén et al., 2015; 81 Falconer, 1999; Hawkins et al., 1985; Humpage et al., 2005; Terao et al., 1994). Its 82 main mechanism of action is the irreversible inhibition of protein and glutathione 83 (GSH) synthesis (Froscio et al., 2003). Moreover, CYN has demonstrated its ability to 84 increase reactive oxygen species (ROS) production, linked to apoptosis and DNA 85 damage (Guzmán-Guillén et al., 2013; Puerto et al., 2011), and progenotoxic effects in 86 several cell lines (Puerto et al., 2018; Žegura et al., 2011). In addition, several studies 87 have shown the capacity of CYN to cause neuronal damage in vitro (Hinojosa et al., 88 2019a; Takser et al., 2016) and in vivo (da Silva et al., 2018; Guzmán-Guillén et al., 89 2015), by inducing changes in the acetylcholinesterase activity (AChE) and oxidative 90 stress (Hinojosa et al., 2019b). 91 Furthermore, the pollution of water by pesticides and their metabolites has been 92 of great concern due to the increasing number of pesticides used in agricultural practices 93 and detected in water, as well to their persistence, mobility, and toxicity (Carvalho, 94 2012). Among all types of pesticides, organophosphates (OP) are about 38% of the 95 global pesticides used, due to their high effectiveness against target pests and relatively 96 low toxicity to non-target organisms (Koly and Khan, 2018). One of the most common 97 OP used in agriculture and residential pest is chlorpyrifos (CPF) [O,O-diethyl 0-(3, 5, 6- 98 trichloro-2-pyridinol) phosphorothionate] (Mehta et al., 2009). This OP has been 99 detected in many samples from all over the world (Koly and Khan, 2018). 100 The cases of human intoxication with CPF are numerous, including symptoms of 101 headache, dizziness, nausea, sweating, salivation, muscle twitching, unconsciousness, 102 11 Cells exposed to 42.5 and 84 µg/mL CPF presented, under phase-contrast and 247 light microcopies, cell death signs. Cytoplasmic projections diminished with a low 248 proliferation rate. At the lowest concentration, a vacuolization of the cytoplasm was 249 observed (Fig. 6D-E). Ultrastructurally, mitochondrial organelles increased their size 250 due to the entrance of water. In addition, an alteration of the pattern of the 251 mitochondrial crests was observed. These cellular processes drove to the presence of 252 heterophagosomes and condensation of the chromatin, a clear sign of apoptosis (Fig. 253 6F). 254 3.5.2. Microscope observations of cells exposed to the combination CYN+CPF 255 Concerning the combination, intense vacuolization of the cytoplasm, cellular 256 cycle detention, loss of cytoplasmic projections, and cell death were observed at the 257 three concentrations assayed (Fig. 6G-H). Under transmission electronic microscopy, 258 small lipid droplets associated with mitochondrial organelles were observed. Moreover, 259 at the highest dose assayed (1 µg/mL CYN + 84 µg/mL CPF), rough endoplasmic 260 reticulum (RER) cisternae appeared dilated with dense content, and an intense presence 261 of heterophagosomes was observed (Fig. 6I). 262 263 4. Discussion 264 The interactive effects of chemicals and natural stressors have been reviewed 265 (Holmstrup et al., 2010; Laskowski et al., 2010). Synergistic interactions were reported 266 in more than 50% of the available studies of interactions between chemicals with abiotic 267 stressors. The coexistence of multiple cyanotoxins or with some other active 268 compounds has been described (Al-Sammak et al., 2014; Chen et al., 2017; Martínez-269 Ruiz and Martínez-Jerónimo, 2016; Pathmalal, 2019; Tatters et al., 2017). However, 270 12 although pesticides are candidates to appear together with cyanotoxins, literature 271 regarding this subject is almost non-existent (Asselman et al., 2014; Daam et al., 2011; 272 Moe et al., 2012). In this sense, EFSA has highlighted the importance of addressing the 273 concomitant exposure of other chemicals, such as pesticides, along with cyanotoxins in 274 order to have information on mixture toxicity and on the environmental factors affecting 275 toxicity of a bloom (Testai et al., 2016). 276 The present work focused on the potential effects induced by the combination of 277 the cyanotoxin CYN and the organophosphate CPF in the neuronal SH-SY5Y cell line. 278 To address this research, a cytotoxicity study of CYN and CPF in the cell model was 279 required to establish the EC50 values of the test items to be applied in the combination 280 study. In the case of CYN, the EC50 value of 1 µg/mL CYN was taken from a previous 281 work, where SH-SY5Y cells were exposed to 0-10 µg/mL CYN for 24 and 48 hours 282 (Hinojosa et al., 2019a). Regarding CPF our study confirms its damaging properties in 283 neuronal cells, showing a time- and concentration-dependent cytotoxicity. In agreement 284 with our results, Park et al. (2013) demonstrated that CPF induced cytotoxic effects in a 285 concentration-dependent manner in SH-SY5Y cells exposed to 0-70 µM CPF, 286 establishing an EC50 value of ~32 µg/mL CPF after 24 hours of exposure. Similarly, 287 Raszewski et al. (2015) also found a time- and concentration-dependent cytotoxic 288 behavior of CPF in SH-SY5Y cells exposed during 24, 48, and 72 hours to 0-175 289 µg/mL CPF, obtaining an EC50 value for 24 hours of ~100 µg/mL CPF. 290 The potential mechanisms by which CPF induces its neurotoxic effects are 291 oxidative stress and acetylcholinesterase inhibition, among others (Park et al., 2013; 292 2015). In our study, CPF did not show any alteration of ROS levels at any of the 293 concentrations assayed. However, GSH levels presented a concentration-dependent 294 decrease, which could indicate that GSH is scavenging ROS. In this sense, Park et al. 295 13 (2015) and Xu et al. (2017) found an increment in ROS levels when SH-SY5Y cells 296 were exposed to CPF. However, they did not evaluate the GSH levels, which could give 297 an idea about the antioxidant activity of the neuroblastoma cell line. On the other hand, 298 Giordano et al. (2007) evaluated GSH levels of SH-SY5Y cells exposed to CPF. The 299 authors did not find alterations in the tripeptide levels, although they observed and 300 increment in oxidized GSH levels, which indicated that an antioxidant response was 301 taking place, suggesting that cytotoxicity may be mediated by oxidative stress. In 302 addition, Park et al. (2013, 2015) suggested that CPF-oxidative stress induction was 303 related to AChE inhibition, together with its usual mechanism of action. In agreement 304 with this, CPF induced a clear inhibition of the AChE activity at all the concentrations 305 assayed in this case. Other authors have also pointed out that a depletion of GSH levels 306 is linked to mitochondrial dysfunction, which could lead to apoptotic processes (de 307 Oliveira et al., 2016; Park et al., 2013). This is in concordance with our morphological 308 findings, where the highest concentrations of CPF induced mitochondrial alterations, 309 leading to cell death by apoptosis. This process is related to the activation of caspase-9 310 and -3 and an increment of cytosolic cytochrome c, as Park et al. (2015) reported for 311 SH-SY5Y cells exposed to CPF. Considering this, Raszewski et al. (2015) proposed that 312 CPF induces neurotoxicity through apoptotic mechanisms. 313 Once the toxic effects of CYN (Hinojosa et al., 2019a) and CPF alone on the 314 neuronal SH-SY5Y cell line were elucidated, the behavior of their combination on the 315 same cell line was evaluated. Our results showed that the combination was more 316 cytotoxic than both toxicants alone after 24 and 48 hours. However, in order to establish 317 the type of interaction between both compounds, the isobologram method was used. 318 Thus, after 24 hours of exposure, an antagonistic response was observed. This pattern 319 changed after 48 hours, showing a synergistic response at low concentrations, and 320 14 turning to an antagonistic response at higher concentrations. A similar pattern was 321 found when the combined toxicity of the cyanotoxin Microcystin (MC)-LR and Cu 322 exposure was investigated in the aquatic plant Vallisneria natans, producing synergistic 323 effects when combined at low concentrations (Wang et al., 2017). Considering this, it is 324 worth to mention that CYN usually appears at low concentrations in nature (de la Cruz 325 et al., 2013), making a synergistic toxic response very likely to appear. 326 To our knowledge, no studies concerning the effects of the combination of 327 cyanotoxins and pesticides using neuronal cell lines have been carried out. However, in 328 vivo, the first combination of cyanobacteria and pesticides was performed by Cerbin et 329 al. (2010), using a strain of Microcystis aeruginosa and the carbamate carbaryl. In this 330 study, the combination demonstrated a mostly additive interaction in Daphnia pulicaria 331 after 24 hours of exposure, while their effect in body deformations of newborns was 332 even synergistic. Ondracek et al. (2012) exposed Japanese quails for 10 days to a 333 combination of a cyanobacterial biomass with MCs (61.62 µg/day MCs), the OP 334 paraoxon (2 doses of 250 µg/Kg paraoxon in that period), and an anticoagulant (2 doses 335 of 500 mg/Kg bromadiolone). They observed that the combination induced more severe 336 damages in the birds than the isolated compounds. This brings to light that the 337 combination of cyanotoxins and pesticides usually drives to a synergistic response, 338 which is in agreement with our results at low concentrations after 48 hours of exposure. 339 In contrast, Asselman et al. (2013) observed non-interactive and antagonistic effects for 340 the combination of carbaryl with four species of cyanobacteria (some of them CYN-341 producers) on Daphnia pulex. Moreover, when these same authors studied the effects of 342 the combination of four different insecticides (including CPF) and M. aeruginosa on 343 Daphnia pulex after 21 days of exposure, a different response was found (Asselman et 344 al., 2014). They observed an additive response for CPF and the cyanobacterial strain, 345 15 different to the antagonistic response that they found using carbaryl, which presents the 346 same mechanism of action (Asselman et al., 2013). For this reason, they postulated that 347 interactive effects could not be generalized for compounds targeting the same pathway 348 (Asselman et al., 2014). 349 Regarding the oxidative stress induction, the combination of CYN and CPF did 350 not induce a significant variation in ROS levels compared to the control group, 351 following the trend showed by CYN in Hinojosa et al. (2019a) and CPF described in the 352 present paper. Regarding the GSH levels, the combination of both compounds led to a 353 reduction of its levels. However, the decline was lower than the one caused by CPF 354 alone. This could be due to the fact that CYN alone did not induce any alteration on 355 GSH levels in SH-SY5Y cells (Hinojosa et al., 2019a). This decrease, as mentioned 356 before, could be the consequence of a compensating mechanism for the oxidative stress, 357 explaining the non-altered ROS levels shown by the mixture. Up to date, no more data 358 concerning the oxidative stress induction of the combination of CYN and CPF in vitro 359 is available. However, the oxidative stress induction of both toxicants has been assayed 360 combined with other chemicals. The scientific literature is very scarce concerning to 361 CYN. Our research group demonstrated that the combination of CYN with MC-LR did 362 not induce an alteration of ROS levels but a slight reduction of GSH levels in SH-SY5Y 363 cells (Hinojosa et al., 2019). This is in agreement with the results presented in this 364 paper, being worthy to point out that CYN ameliorates GSH levels in the mixture 365 compared to CPF alone. Regarding CPF, its combination with other chemicals using 366 SH-SY5Y cells as in vitro model has resulted in different outcomes. Thus, Raszewski et 367 al. (2015) found that the combination of CPF-cypermethrin, a synthetic pyrethroid, 368 induced a synergistic response in the MTT assay, whereas a mixture of CPF and Cd 369 presented an antagonistic response, not showing a deterioration of the oxidative stress 370 16 status previously induced by CPF alone (Xu et al., 2017). These results support the 371 theory that CYN could be the limiting factor of the combination. 372 Concerning the AChE status, the combination of CYN and CPF showed a 373 similar pattern than CPF alone, thus is, an intense inhibition of the AChE activity. This 374 same fact was described by Xu et al. (2017) in SH-SY5Y cells, who found that 375 cadmium had no effect on the inhibitory AChE activity in combination with CPF. This 376 result reveals, for the first time, that in a CYN-CPF mixture, CPF is the one that mainly 377 determines the inhibition of the AChE enzyme. 378 Finally, regarding the morphological findings, Hinojosa et al. (2019a) showed 379 that CYN induced clear morphological alterations leading to cell death (apoptotic 380 bodies, heterochromatin condensation, nuclei with an irregular shape, segregated 381 nucleolus, and numerous mitochondria in the cytoplasm). These observations were very 382 similar to those observed for CPF. Thus, the induction of mitochondrial dysfunction 383 observed after CPF exposure could be explained not only by its intrinsic pro-oxidant 384 capacity but also by altering mitochondrial architecture and dynamics (de Oliveira et al., 385 2016). The combination of CYN and CPF showed a more intense cell death by 386 apoptosis than the individual exposure to CYN and CPF. This result is in agreement 387 with those reported by Raszewski et al. (2015) and Xu et al. (2017) in SH-SY5Y cells 388 exposed to a combination of CPF and cypermethrin, and CPF and Cd, respectively. In 389 the last case, the observations at mitochondrial levels were very similar to those 390 described in the present paper, with swelling and fragmentation. 391 392 5. Conclusions 393 17 Our findings indicate that the combination of CYN and CPF induces GSH 394 depletion, AChE activity inhibition, and cell death by apoptosis in the human 395 neuroblastoma SH-SY5Y cell line. In comparison to CYN and CPF alone, an 396 intensification of these effects was observed in this cell line after exposure to the 397 mixture. However, these observations were less severe than expected, which was 398 corroborated by the isobologram method. Thus, a mainly antagonistic response was 399 established between both compounds. Nevertheless, a synergistic effect between both 400 substances was observed at low concentrations after 48 hours, which requires special 401 attention in order to not underestimate the exposure concentrations. Hence, from a 402 toxicological point of view, it is important to consider the exposure pattern (single or 403 mixtures) as results can differ. Further research is required to contribute to the risk 404 assessment of CYN and other contaminants considering more realistic exposure 405 scenarios. 406 407 Acknowledgements 408 The authors wish to thank the Ministerio de Economía y Competitividad of 409 Spain (AGL2015-64558-R, MINECO/FEDER, UE) for the financial support, the Junta 410 de Andalucía for the contact of María Gracia Hinojosa (USE-16667), and the Biology 411 Service and Microscopy Service of Centro de Investigación, Tecnología e Innovación 412 from Universidad de Sevilla (CITIUS), for providing technical assistance. 413 414 415 416 417 18 References 418 Al-Badrany YMA, Mohammad FK. Effects of acute and repeated oral exposure to the 419 organophosphate insecticide chlorpyrifos on open-field activity in chicks. Toxicol 420 Lett. 2007;174:110-116. 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Toxicol Sci. 2000;55:124-132. 633 Doi: 10.1093/toxsci/55.1.124 634 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure captions Figure 1. Reduction of tetrazolium salt (MTS) and protein content (PC) on SH-SY5Y cells after 24 h (A) and 48 h (B) of exposure to 0-200 µg/mL CPF. All values are expressed as mean ± s.d. ** MTS significantly different from control group (p<0.01), §§ all parameters significantly different from control group (p<0.01). Figure 2. Reduction of tetrazolium salt (MTS) on SH-SY5Y cells after 24 h (A) and 48 h (B) of exposure to different concentrations of CYN+CPF combinations at a ratio of EC50 CYN / EC50 CPF (1:84). All values are expressed as mean ± s.d. ** significantly different from control group (p<0.01) for CPF and the combination, εε significantly different from control group (p<0.01) for CYN and the combination, and δδ significantly different from control group (p<0.01) for CYN, CPF and the combination. Figure 3. Combination index (CI)/fraction affected (Fa) curve in SH-SY5Y cells exposed to CYN-CPF combination after 24 h (A) and 48 h (B) of exposure. Each point represents the CI ± s.d. at a fractional effect. The dotted line (CI = 1) indicates additivity, the area under the dotted line points out a synergist effect, and the area above the dotted line signifies antagonism. Figure 4. Reactive oxygen species (ROS) levels on SH-SY5Y cells after 4, 8, 12 and 24 hours of exposure to 0-84 µg/mL CPF (A) and a CYN-CPF combination at a ratio 1:84 of EC50 CYN / EC50 CPF (C). Reduced glutathione (GSH) levels on SH-SY5Y cells after 4, 8, 12 and 24 hours of exposure to 0-84 µg/mL CPF (B) and a CYN-CPF combination at a ratio 1:84 of EC50 CYN / EC50 CPF (D). All values are expressed as mean ± s.d. The significance levels observed are ** p<0.01 significantly different from control group. Figure 5. In vitro effects of SH-SY5Y cells to their exposure to CYN (0-1 µg/mL), CPF (0-84 µg/mL) or CYN-CPF combination on the acetylcholinesterase activity (AChE) after 24 hours. All values are expressed as mean ± s.d. ** Indicates significant difference from control group value (p<0.01). Figure 6. Morphology of control SH-SY5Y cells after 24 h of exposure to culture medium without serum. Contrast-phase microscopy of a SH-SY5Y cell culture in normal neuronal growth. Cells present cytoplasmic projections contacting with other cells (arrows) Bar=100µm (A). Semithin sections of cells culture were stained with toluidine blue. Cells in mitosis processes (arrowheads) with big nucleoli in the nucleus Figure Captions Click here to download Supplementary Material: Figure captions.docx (arrows). Bar=20µm (B). Transmission electronic microscopy of SH-SY5Y cells with numerous mitochondria (m). Bar=1 µm (C). Morphology of SH-SY5Y cells after 24 h of exposure to CPF. Phase-contrast microscopy of cells exposed to 42.5 µg/mL CPF presented rounded cells (arrows) with clear signs of cell death. Bar=100 µm (D). Semithin sections of cells exposed to 85 µg/mL CPF presented nuclei with condensed chromatin (arrowheads) as clear sign of cell death. Bar=20 µm (E). Electronic microscopy of cells exposed to 85 µg/mL CPF showed irregular nuclei with chromatin condensation (Ch), appearance of blisters in the cellular membrane (arrows), dilated RER with protein content (RER) and presence of heterophagosomes (H). Bar=1 µm (F). Phase-contrast microscopy of SH-SY5Y cells exposed to the combination of CYN and CPF showed cell death signs at all the concentrations assayed. Bar=100 µm (G). Semithin sections of cells culture exposed to 0.5 + 42 µg/mL CYN + CPF presented lipid droplets (arrows) and big nucleoli (arrowheads). Bar=20 µm (H). The TEM of cells exposed to the highest concentration of the combination showed altered dilated mitochondria (arrows) surrounded by lipidic droplets (arrowheads). The endoplasmic reticulum cisternae appeared dilated (RER) and lipidic vacuoles were formed (Lip). Bar=1 µm (I). Credit Author Statement - María Gracia Hinojosa: Conceptualization, Methodology, Formal Analysis, Investigation, Writing-Original Draft, Writing-Review & Editing, Visualization - Ana Isabel Prieto: Conceptualization, Validation, Writing-Review & Editing, Supervision - Daniel Gutiérrez-Praena: Conceptualization, Methodology, Validation, Formal Analysis, Writing-Original Draft, Writing-Review & Editing, Visualization, Supervision - Francisco Javier Moreno: Validation, Investigation. - Ana María Cameán: Conceptualization, Validation, Writing-Review & Editing, Supervision, Project administration, Funding adquisition. - Ángeles Jos: Conceptualization, Validation, Writing-Review & Editing, Supervision, Project administration, Funding adquisition. *Credit Author Statement