Oxidative damage mechanism in Saccharomyces cerevisiae cells exposed to tetrachlorobisphenol A

https://doi.org/10.1016/j.etap.2020.103507Get rights and content

Highlights

  • Three pathways contributed to ROS accumulation in yeast while being exposed to TCBPA.

  • TCBPA activated NADPH oxidase through regulation of three pathways.

  • TCBPA promoted expressions of genes which are responsible for ROS accumulation.

  • These results clarified that how TCBPA induced ROS accumulation in S. cerevisiae.

Abstract

Tetrachlorobisphenol A (TCBPA) can promote intracellular reactive oxygen species (ROS) accumulation. However, limited attention has been given to mechanisms underlying TCBPA exposure-associated ROS accumulation. Here, such mechanisms were explored in the simple eukaryotic model organism Saccharomyces cerevisiae exposed to multiple concentrations of TCBPA. Addition of diphenyleneiodonium, a specific inhibitor of NADPH oxidase, blocked TCBPA treatment-associated intracellular ROS accumulation. NADPH oxidase can be activated by calcineurin, mitogen-activated protein kinase (MAPK), and tyrosine kinase. Therefore, corresponding specific inhibition respectively on these three kinases was performed and results suggested that the Ca2+ signaling pathway, MAPK pathway, and tyrosine kinase pathway all contributed to the TCBPA exposure-associated intracellular ROS accumulation. In addition, TCBPA exposure-associated up-regulation of genes involved in ROS production and down-regulation of catalase promoted ROS accumulation in S. cerevisiae. To sum up, our current results provide insights into the understanding of TCBPA exposure-associated ROS accumulation.

Introduction

Today, flame retardants, which have been intensively applied in various fields of industry, are widely added into industrial and daily supplies to fit flammability criterion for building products, furnishings, and electronics (Lorber, 2008). However, widespread use of flame retardants promotes its accumulation in organisms through exposure, thereby causing significant threats to biological health (Shaw et al., 2010; Thomsen et al., 2001a). Tetrachlorobisphenol A (TCBPA), a widely used flame retardant, can cause serious physiological damage through accumulation in humans and other animals. (Riu et al., 2011; Song et al., 2014) TCBPA concentration is mostly maintained at the μg/L level in the environment and 4–200 pg/g in human plasma (Ji et al., 2018; Thomsen et al., 2001b). TCBPA acts as a thyroid system disruptor by competing with thyroxine for human transthyretin (Sun et al., 2009) and disturbing the endocrine system (Mccormick et al., 2010). Moreover, our previous study also indicated that TCBPA could induce oxidative stress via increasing intracellular levels of reactive oxygen species (ROS) in yeast (Ji et al., 2018).

Accumulated intracellular ROS damages cellular structures and disturbs the intracellular redox equilibrium, thereby further aggravating damage to DNA, RNA or amino acid residues in proteins (Brooker and Minnesota, 2005; Devasagayam et al., 2004). The oxidation of DNA secondary to ROS accumulation generates mutations through several types of DNA damage (Waris and Ahsan, 2006). ROS can also cause apoptosis (Nakagawa et al., 2007; Reistad and Mariussen, 2005). According to the free-radical theory, oxidative damage secondary to ROS accumulation is a key factor involved in the hypofunction that is characteristic of aging (Van Raamsdonk and Hekimi, 2009). Although the ROS accumulation-associated damage to cells has been characterized, the exact mechanisms underlying ROS accumulation secondary to TCBPA exposure still remain poorly understood. Therefore, to better understand the biological toxicity caused by TCBPA, further studies are required to clarify how TCBPA induces the intracellular ROS accumulation.

Saccharomyces cerevisiae, which is widely used in genetics and cell biology, has some experimental superiorities in comparison to higher eukaryotic cells, such as its simple cell structures, fully interpreted genetic background, and easy manipulation (Goffeau et al., 1996; Nickoloff and Haber, 2001). Because of these advantages, S. cerevisiae was used as an ideal eukaryotic model for clarifying mechanisms underlying TCBPA exposure-associated intracellular ROS accumulation. In S. cerevisiae, there are three signaling pathways primarily involved in ROS accumulation (Levin et al., 1994; Neet and Hunter, 2010); the calcium signaling pathway, mitogen-activated protein kinase (MAPK) pathway, and tyrosine kinase pathway (Fig. 1). Ca2+ acts as a universal secondary signal transducer in many kind of cells (Matheos et al., 1997) and can promote ROS generation (Zheng et al., 2007). Previous studies suggest a possible link between ROS accumulation and the MAPK pathway (Blumer and Johnson, 1994; Eaton et al., 2011). Tyrosine kinase could enhance ROS accumulation through regulating the MAPK pathway or by directly activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Dikic and Blaukat, 1999; Toone and Jones, 1998). In light of the above, all of these three pathways could activate NADPH oxidase to promote the ROS accumulation (Choksi et al., 2004; Porras et al., 1981). Given what we know, the next worthy issue is to determine which of those pathway(s) mainly accounts for TCBPA exposure-associated intracellular ROS accumulation in S. cerevisiae cells.

In this study, to clarify the concrete pathway(s) that are involved in TCBPA exposure-associated intracellular ROS accumulation respective specific inhibitors or gene knockouts were adopted to inhibit the corresponding key enzymes in the above mentioned three pathways, and the subsequent intracellular ROS level in yeast cells was determined. To further evaluate the regulation of these three pathways on intracellular ROS accumulation in the presence of TCBPA, TCBPA exposure-associated expression changes of key genes involved in ROS production and elimination were determined by quantitative real-time PCR (qRT-PCR). This work helps to clarify the mechanisms of how TCBPA induces the ROS accumulation in S. cerevisiae and may hint at similar effects in humans and other animals.

Section snippets

Strains, media and culture conditions

S. cerevisiae S288c strain (ATCC number 204508) was purchased from China General Microbiological Culture Collection Center (CGMCC) (Beijing, China). Single-gene deletion strains BY4741Δynl307c (Y01137) and BY4741Δyjl095w (Y01328) with knock outs of MCK1 and BCK1, respectively, used in the current work were purchased from Euroscarf (Oberursel, Germany). The detailed information of these strains is listed in Table 1. Yeast strains were cultured in YPD (2% glucose, 2% peptone, 1% yeast extract)

Effect of inhibition of certain pathways on induced ROS accumulation secondary to TCBPA exposure

Under aerobic culture conditions, S. cerevisiae cells mainly rely on respiration pathways to generate energy, which terminates in the electron transfer chain (ETC). As a byproduct of reducing oxygen to water, the ETC produces a low amount of endogenous ROS. Environmental stimulation can increase intracellular ROS through activation of NADPH oxidase, which is specifically inhibited by DPI (Lesuisse et al., 1996). Consistent with this, DPI did effectively reverse the intracellular ROS

Discussion

TCBPA exposure induces cell damage through promotion of intracellular ROS accumulation. Although numerous publications have indicated the TCBPA exposure-associated ROS accumulation and subsequent related cytotoxicity, the mechanisms underlying such ROS accumulation needed to be uncovered.

Ca2+ plays a very important role in cell signal regulation and combines with the regulatory subunit of calcineurin to regulate its activity (Matheos et al., 1997). Upon activation by Ca2+, calcineurin acts on

CRediT authorship contribution statement

Xiaoru Zhang: Methodology, Investigation, Writing - original draft, Writing - review & editing. Yaxian Zhang: Conceptualization, Investigation, Writing - original draft. Zhihua Ji: Investigation, Writing - review & editing. Fengbang Wang: Investigation. Lei Zhang: Investigation. Maoyong Song: Conceptualization, Methodology, Supervision, Funding acquisition. Hao Li: Conceptualization, Methodology, Writing - review & editing, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB14010301), the National Natural Science Foundation of China (No.21677170) and the Higher Education and High-quality and World-class Universities (No. PY201617).

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      Moreover, in a two-hybrid assay in yeast on a rat liver S9 preparation (Kitamura et al., 2002; Terasaki et al., 2011), Cl3BPA and Cl4BPA were shown to increase thyroid hormone activities but inhibit triiodothyronine (T3) binding compared to Cl2BPA, ClBPA and BPA. Additionally, Cl4BPA exposure induces cell damage through promotion of intracellular reactive oxygen species (ROS) accumulation using Saccharomyces cerevisiae as a model and in terms of subsequent related cytotoxicity (Zhang et al., 2020). Cl4BPA has been shown to induce lipid accumulation in vitro (Riu et al., 2011a; Riu et al., 2011b).

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    These authors contributed equally to this work.

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