In vitro assessment of the combination of cylindrospermopsin and the organophosphate chlorpyrifos on the human neuroblastoma SH-SY5Y cell line
Introduction
Cyanobacterial blooms are a global environmental concern due to their increased occurrence in terrestrial, marine and freshwater ecosystems, caused by climate change and human influence. Among their detrimental effects, they produce unpleasant organoleptic properties where they proliferate (Manning and Nobles, 2017). However, the main hazard is their capacity to produce cyanotoxins, toxic secondary metabolites that threaten water safety and aquatic life (Brooks et al., 2017; Buratti et al., 2017). There are different exposure routes to them, standing out the oral one as one of the most common and dangerous. Nonetheless, other relevant routes such as dermal contact or inhalation are worth to be mentioned as well (Buratti et al., 2017).
Cylindrospermopsin (CYN) is one of the most common aquatic cyanotoxicants as a consequence of the increasing occurrence of Cylindrospermopsis raciborskii blooms, the most relevant producer species (Antunes et al., 2015). However, other cyanobacterial species such as Chrysosporum ovalisporum, Aphanizomenon flos-aquae, Anabaena bergii, or Raphidiopsis curvata are able to produce this compound (Manning and Nobles, 2017). Cylindrospermopsin consists of an alkaloid with a tricyclic guanidine linked to a hydroxymethyl uracil group (Ohtani et al., 1992). The main episode of human intoxication by CYN occurred in the outbreak of 1979 in Palm Island (Australia) when 146 people were hospitalized in a local clinic with malaise, vomits, anorexia and hepatomegaly after drinking from a water supply containing CYN-producing C. raciborskii (Bourke et al., 1983; Griffiths and Saker, 2003). However, despite intoxication by drinking contaminated water, it is also feasible to be exposed by ingestion of contaminated food. In fact, the bioaccumulation of these cyanotoxicants by plants irrigated with contaminated water (Testai et al., 2016) or in exposed aquatic organisms has been demonstrated, enhancing the risk of human intoxication by the food chain (Cordeiro-Araújo et al., 2017; Gutiérrez-Praena et al., 2013; Machado et al., 2017).
Although its target organ is the liver, CYN has widely demonstrated its capacity to damage other organs, including the nervous system (Guzmán-Guillén et al., 2015; Falconer, 1999; Hawkins et al., 1985; Humpage et al., 2005; Terao et al., 1994). Its main mechanism of action is the irreversible inhibition of protein and glutathione (GSH) synthesis (Froscio et al., 2003). Moreover, CYN has demonstrated its ability to increase reactive oxygen species (ROS) production, linked to apoptosis and DNA damage (Guzmán-Guillén et al., 2013; Puerto et al., 2011), and progenotoxic effects in several cell lines (Puerto et al., 2018; Žegura et al., 2011). In addition, several studies have shown the capacity of CYN to cause neuronal damage in vitro (Hinojosa et al., 2019; Takser et al., 2016) and in vivo (Da Silva et al., 2018; Guzmán-Guillén et al., 2015), by inducing changes in the acetylcholinesterase activity (AChE) and oxidative stress (Hinojosa et al., 2019).
Furthermore, the pollution of water by pesticides and their metabolites has been of great concern due to the increasing number of pesticides used in agricultural practices and detected in water, as well as to their persistence, mobility, and toxicity (Carvalho, 2012). Among all types of pesticides, organophosphates (OP) are about 38% of the global pesticides used, due to their high effectiveness against target pests and relatively low toxicity to non-target organisms (Koly and Khan, 2018). One of the most common OP used in agriculture and residential pest is chlorpyrifos (CPF) [O,O-diethyl 0-(3, 5, 6- trichloro-2-pyridinol) phosphorothionate] (Mehta et al., 2009). This OP has been detected in many samples from all over the world (Koly and Khan, 2018).
The cases of human intoxication with CPF are numerous, including symptoms of headache, dizziness, nausea, sweating, salivation, muscle twitching, unconsciousness, convulsion, and death (Eaton et al., 2008). The main mechanism of action for CPF is the inhibition of the acetylcholinesterase activity, which leads to accumulation of acetylcholine in the synaptic space, causing an excessive stimulation of postsynaptic neuronal receptors and consequent signs of toxicity (Al-Badrany and Mohammad, 2007; Mehta et al., 2009; Zheng et al., 2000). Moreover, CPF induces changes on macromolecule synthesis (DNA, RNA, proteins), on neurotransmitter receptors, and in signal transduction pathways, neuronal differentiation, and neurochemical effects. Furthermore, it is also capable of producing oxidative stress by lipid peroxidation and increasing ROS (Uchendu et al., 2012).
Recently, the European Food Safety Authority has established the need of studying the toxicity of the mixtures of cyanotoxins and other chemicals, as some OP pesticides were reported to potentiate the anatoxin-induced toxicity, for instance (Cook et al., 1988; Testai et al., 2016). In this sense, due to the possible coexistence of CYN and CPF in water or their bioaccumulation in aquatic organisms or crops, the aim of the present work aimed to study, for the first time, the potential interaction and effects of the combination of CYN and the pesticide CPF in the human neuroblastoma cell line SH-SY5Y, including cytotoxicity, oxidative stress, AChE inhibition, and morphological changes.
Section snippets
Supplies and chemicals
Cylindrospermopsin (purity > 95% by HPLC) was purchased from Enzo Life Sciences. Nutrient Mixture F-12 Ham and CPF were purchased from Sigma-Aldrich (Madrid, Spain). Minimum essential medium (MEM), fetal bovine serum (FBS) and cell culture reagents were obtained from Gibco (Biomol, Sevilla, Spain).
The Bradford reagent was purchased from Sigma-Aldrich (Madrid, Spain). The supplier of MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulphophenyl)-2H-tetrazolium salt) Cell Titer
Cytotoxicity assays
A concentration-dependent decrease in the viability was observed in the SH-SY5Y cells after being exposed to 0–200 μg/mL CPF for 24 and 48 h (Fig. 1). The EC50 values obtained were 83.98 ± 2.74 μg/mL and 85.31 ± 7.67 μg/mL after 24 h of exposure for the MTS assay and the PC assay, respectively. In the case of the cells exposed to CPF for 48 h, the EC50 values were 69.78 ± 6.02 μg/mL and >200 μg/mL for the MTS and PC assays, respectively. Thus, the MTS assay demonstrated to be the most sensitive
Discussion
The interactive effects of chemicals and natural stressors have been reviewed (Holmstrup et al., 2010; Laskowski et al., 2010). Synergistic interactions were reported in more than 50% of the available studies of interactions between chemicals with abiotic stressors. The coexistence of multiple cyanotoxins or with some other active compounds has been described (Al-Sammak et al., 2014; Chen et al., 2017; Martínez-Ruiz and Martínez-Jerónimo, 2016; Pathmalal, 2019; Tatters et al., 2017). However,
Conclusions
Our findings indicate that the combination of CYN and CPF induces GSH depletion, AChE activity inhibition, and cell death by apoptosis in the human neuroblastoma SH-SY5Y cell line. In comparison to CYN and CPF alone, an intensification of these effects was observed in this cell line after exposure to the mixture. However, these observations were less severe than expected, which was corroborated by the isobologram method. Thus, a mainly antagonistic response was established between both
CRediT authorship contribution statement
M.G. Hinojosa: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. A.I. Prieto: Conceptualization, Validation, Writing - review & editing, Supervision. D. Gutiérrez-Praena: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft, Writing - review & editing, Visualization, Supervision. F.J. Moreno: Validation, Investigation. A.M. Cameán: Conceptualization, Validation, Writing - review &
Acknowledgements
The authors wish to thank the Ministerio de Economía y Competitividad of Spain (AGL 2015-64558-R, MINECO/FEDER, UE) for the financial support, the Junta de Andalucía for the contact of María Gracia Hinojosa (USE-16667), and the Biology Service and Microscopy Service of Centro de Investigación, Tecnología e Innovación from Universidad de Sevilla (CITIUS), for providing technical assistance.
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