Oxidative stress and related gene expression effects of cyfluthrin in human neuroblastoma SH-SY5Y cells: Protective effect of melatonin

Highlights

• . Cyfluthrin neurotoxicity in human neuroblastoma SH-SY5Y cells.

• . Cyfluthrin decreased MTT cell viability.

• . Cyfluthrin increased MDA, NO and ROS production and decreased NQO1 activity.

• . Cyfluthrin affected oxidative stress signaling pathways.

• . Melatonin showed effective protection against cyfluthrin-induced oxidative stress.

Abstract

This study was designed to assess oxidative stress induction in human neuroblastoma SH-SY5Y cells in response to cyfluthrin exposure. Cell viability MTT assay was carried out to assess cyfluthrin cytotoxicity; IC₃₀ and IC₅₀ values for cyfluthrin were calculated to be 4.81 ± 0.92 μM and 19.39 ± 3.44 μM, respectively. Cyfluthrin induced a significant increase in ROS generation, lipid peroxides measured as malondialdehyde (MDA) and nitric oxide (NO) production and a significant decrease in NQO1 activity. The antioxidant activity of melatonin (MEL), Trolox, N-acetylcysteine (NAC) and Sylibin against cyfluthrin-induced oxidative stress was examined. Cyfluthrin increased significantly gene expressions of apoptosis, proinflammation and oxidative stress (Bax, Bcl-2, Casp-3, BNIP3, AKT1, p53, APAF1, NFκB1, TNFα and Nrf2) mediators. In the most genes, the mRNA levels induced by cyfluthrin were partially reduced by MEL (1 μM). Cyfluthrin effects on gene expression profiling of oxidative stress pathway by Real-Time PCR array analysis showed that of the 84 genes examined, (fold change > 1.5) changes in mRNA levels were detected in 31 genes: 13 upregulated and 18 down-regulated. A fold change >3.0 fold was observed on upregulated CYBB, DUOX1, DUOX2, AOX1, BNIP3, HSPA1A, NOS2, and NQO1 genes. The greater fold change reversion (2.5 fold) by MEL (1 μM) was observed on cyfluthrin-upregulated CYBB, AOX1, BNIP3 and NOS2 genes. These results demonstrated that oxidative stress is a key element in cyfluthrin induced neurotoxicity as well as MEL may play a role in reducing cyfluthrin-induced oxidative stress.

Introduction

Pyrethroids are a class of synthetic, neurotoxic, insecticides that are structurally based on purified extracts (pyrethrins) of Chrysanthemum sp. flowers. Characteristically, pyrethroids are described as phenoxybenzoic and cyclopropane moieties joined by an ester bond. Pyrethroids are frequently grouped according to the absence (Type I) or presence (Type II) of a cyano group at the α-carbon of the phenoxybenzoic constituent. The principal target site for pyrethroids is defined as the voltage-dependent sodium channel in the neuronal membrane leading to depolarization and hyperexcitation of the nervous system (Narahashi et al., 1998; Soderlund et al., 2002). This group of compounds has also been shown to act on isoforms of voltage sensitive calcium channels (Hildebrand et al., 2004), thereby contributing to the release of neurotransmitters and hence leading to pyrethroid induced toxicity (Symington and Clark, 2005). Pyrethroids are classified as Type I or Type II based on their chemical structure and also based on the toxic syndrome than they produce in rodents. Type I pyrethroids are associated to hyper-excitation and fine tremors (T-syndrome), and Type II pyrethroids are associated to a more complex syndrome, including clonic seizures (choreoathetosis) and salivation (CS syndrome) (Verschoyle and Aldridge, 1980; Aldridge, 1990). Some pyrethroids produce tremors and salivation, classified as the intermediate TS-syndrome (Soderlund et al., 2002; Soderlund, 2012). Types I and II pyrethroids are extensively applied to control pests in residential and agricultural settings, to treat head lice and scabies in humans and fleas in pets, for public health vector control, and for disinfection of commercial aircrafts (Anadón et al., 2009, Anadón et al., 2013a; USEPA, 2013, 2017). Data from in vivo and in vitro studies have shown that pyrethroids undergo extensive metabolism by carboxylesterases and CYP (Godin et al., 2007; Scollon et al., 2009). However, pyrethroids could interact with the normal metabolism of drugs and xenobiotics and some pyrethroids are found to induce CYP enzyme activities (Yang et al., 2009; Anadón et al., 2013b; Martínez et al., 2018). Moreover, there is evidence suggesting that the detoxifying enzymes of pyrethroids are present at lower levels during fetal and early postnatal development than they are later in life (Cantalamessa, 1993; Sheets, 2000). Previous experiments showed that the toxicity of pyrethroids increases as the age of the animal decreases, so that neonates appear highly sensitive to pyrethroid exposure (Cantalamessa, 1993). A comprehensive review summarizing the existing 22 studies of the developmental neurotoxicity of pyrethroids suggests that pyrethroids may exert developmental neurotoxicity (Shafer et al., 2005). Developmental neurotoxicity involves alterations in behavior, neurohistology, neurochemistry and/or dysmorphology of the central nervous system occurring in the adult age, as a result of neonatal exposure to pyrethroids. Learning and memory impairments in pyrethroid-treated rats and neurobehavioral changes could also be attributed to increased oxidative stress (Liu et al., 2003; Ansari et al., 2012a, b; Nasuti et al., 2007, 2013; Gargouri et al., 2018). Induction of oxidative stress is an important mechanism in pesticide induced toxicities, being the major endpoints damage to DNA, proteins and membrane lipids (Banerjee et al., 2001; Rehman et al., 2006; Tiwari et al., 2010; Romero et al., 2012, 2017). Because, the increased use of pyrethroids has made human exposure almost inevitable, further studies are necessary to assess the neurotoxicity of pyrethroids. It is accepted that animal testing should be reduced, refined or replaced as far as it is practicably possible. Alternative in vitro testing strategies are required, in particular for screening, as part of a tired testing scheme, and routine testing (Anadón et al., 2013c).

Cyfluthrin, a commonly used Type II pyrethroid, was selected for the current investigation. Cyfluthrin, a mixture of four diastereoisomeric pairs of enantiomers (I, II, III, IV) was first registered for use in the United States in 1987 (USEPA, 1987), frequently it is used in veterinary medicine, agriculture against grasshoppers and pests, industrial and residential settings, and public health and, in some countries for the protection of stored products (Ritter and Chappel, 1997; FAO, 1999; Surgan et al., 2002). Despite beneficial roles in agricultural and household products, recent studies in Wistar rats showed that oral exposure to cyfluthrin induced hepatic and renal CYP2E, CYP1A and CYP4A subfamilies, and also increased the β-oxidation of palmitoyl-coenzyme A and carnitine acetyltransferase activity, supporting cyfluthrin classification as a peroxisome proliferator with possible implications in oxidative stress (Anadón et al., 2013b). Also, alteration of GPx and AchE activities in liver and kidney of Wistar rats following intraperitoneal treatment of cyfluthrin has been described (Yilmaz et al., 2015). Cyfluthrin is readily absorbed by the oral route, enters the brain, accumulates in significant quantity and therefore it is appropriate that cyfluthrin affects the CNS function of the non-target organisms (Rodríguez et al., 2018). Because cyfluthrin is one of the more used pyrethroid insecticides worldwide (Surgan et al., 2002), and studies describing mechanisms underlying neurotoxicity of this insecticide (Mense et al., 2006; Eraslan et al., 2007; Rodríguez et al., 2016) are limited, in the present study the possible link between oxidative stress pathways and cyfluthrin neurotoxicity, was investigated. This in vitro study was undertaken (i) to characterize the concentration-dependent cytotoxicity of cyfluthrin using cell viability MTT assay and to determine the protective role of selected antioxidant substances (MEL, Trolox, NAC and Sylibin) on MTT reduction, lipid peroxidation, NO and ROS production and NQO1 activity and (ii) to evaluate gene expressions of apoptosis, proinflammation and oxidative stress (Bax, Bcl-2, Casp-3, BNIP3, AKT1, p53, APAF1, NFκB1, TNFα and Nrf2) mediators as well as to examine by Real-Time PCR array the expression of key genes related to oxidative stress after cyfluthrin and cyfluthrin plus MEL exposure. In the present study, human dopaminergic neuroblastoma cell line (SH-SY5Y) was used as an in vitro model to determine cytotoxic mechanisms of cyfluthrin. The human dopaminergic neuroblastoma cell line, SH-SY5Y, is a commonly used cell line in studies related to neurotoxicity, oxidative stress, and neurodegenerative diseases (Pahlman, 1990; Krishna et al., 2014).