Application of PEG-CdSe@ZnS quantum dots for ROS imaging and evaluation of deoxynivalenol-mediated oxidative stress in living cells
Graphical abstract
Introduction
Mycotoxins, which are fungal secondary metabolites, can contaminate various dietary materials that are consumed by humans and animals (Font and Ruiz, 2019). Deoxynivalenol (DON, also known as vomitoxin), which is a type B trichothecene, is one of the most important mycotoxins and is produced mainly by Fusarium species (Alassane-Kpembi et al., 2013; Zhou et al., 2019). DON most frequently contaminates cereals and their products (Palacios et al., 2017; Simsek et al., 2012). The recent studies suggest that the incidences of DON in globally harvested cereals were 59% approximately in the past ten years, and maximum levels were reported up to 41,157 μg/kg in wheat from China (Lee and Ryu, 2017; Pedroso Pereira et al., 2019). Humans and animals can be exposed through the ingestion of foods that are contaminated with DON (Franco et al., 2019). According to the report, an estimated 500 million people from the developing countries are exposed to mycotoxins including DON, of which 160 million are children younger than five years (Mishra et al., 2019). This exposure can cause a spectrum of symptoms, such as nausea, vomiting, gastroenteritis, growth retardation, and immunosuppression (Fang et al., 2018; Wu et al., 2020; Yang et al., 2019). DON is a potent inhibitor of DNA, RNA, and protein synthesis at the cellular level (Xiong et al., 2019) and has sufficient cytotoxicity for the induction of apoptosis in various types of cells (Lan et al., 2018; Mishra et al., 2014; Peng et al., 2017). Typically, DON can enter the cell membrane, bind with the 60S subunit of the eukaryotic ribosome, and disrupt the activity of the peptidyl transferase center (Garreau de Loubresse et al., 2014). The binding of DON to the ribosome induces a process known as the “ribotoxic stress response” (Payros et al., 2016). Furthermore, DON can interfere with the normal functioning of mitochondria and destroy the mitochondrial electron-transport chain (Pestka, 2008; Ren et al., 2020a). These effects can result in the overproduction of reactive oxygen species (ROS), the induction of oxidative stress, and the rapid activation of MAPK signaling pathways (Liao et al., 2020; Wu et al., 2014). Moreover, they can contribute to the prevention of cell proliferation and the suppression of cell activity and can eventually lead to apoptosis and related diseases.
Intracellular ROS, which constitute a class of highly reactive chemicals (Nishikawa, 2008; Zhou et al., 2015), are the key indicator of oxidative stress that is induced by mycotoxins such as DON and can directly reflect the cellular redox balance (Ren et al., 2020b). ROS include hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide (O2−), hypochlorite (ClO−), hydroxyl radical (·OH), and peroxynitrite (ONOO−) (Wang, 2016). In biological systems, ROS are essential regulatory molecules and play important roles in various pathological and physiological processes of the organism (Wen et al., 2013). The overexpression of ROS results in oxidative stress and disruption of the redox balance (Lee et al., 2009). Due to their high reactivity, the overproduction of ROS can lead to DNA damage, protein oxidation, and lipid peroxidation and can eventually result in mutations and multiple pathologies (Roberts et al., 2009). Thus, the degrees of oxidative stress and toxicity that are induced by DON can be evaluated using the detected ROS level.
Antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione peroxidase (GPX), as well as non-enzymatic antioxidants such as glutathione (GSH) and malondialdehyde (MDA) constitute the antioxidative system of the cell, which are important traditional indicators reflecting the level of intracellular oxidative stress (Peng et al., 2020). Incubation with DON for 6 h could significantly reduce the activities of SOD, CAT and POD in HepG2 cells (Zhou et al., 2020). Yu et al. (2017) found that DON pretreatment downregulated the activity of SOD and the level of GSH and upregulated the level of MDA in placental BeWo cells. After 48 h of DON and zearalenone (ZEN) exposure, the GPX activity and GSH level of porcine splenic lymphocytes were significantly reduced, the antioxidant function is weakened (Ren et al., 2017). In order to achieve the detection purpose as accurately as possible, cells need to be completely stripped from the culture dish in these traditional methods. Then fully lyse them with lysis buffer on ice, centrifuge and remove the cell debris. The collected supernatant is further disrupted by ultrasound, and the total protein concentration needs to be quantified. Therefore, the traditional detection methods are complicated and time consuming usually. Moreover, they are prone to cause cell content target loss and reduce the accuracy of detection results. In addition, these traditional methods cannot achieve in situ monitoring of ROS in living cells.
2′,7′-Dichlorofluorescin diacetate (DCFH-DA) and dihydroethidium (DHE) are two traditional commercial organic fluorescent dyes that are widely used for the detection of ROS in vitro (Ashoka et al., 2020; Das et al., 2019; Liu et al., 2018a). The utilization of commercially organic fluorescent dyes induces higher production of ROS, and this approach was used to evaluate the oxidative statuses of cells (Rota et al., 1999). Kang et al. confirmed that DON increased the intracellular ROS content of IPEC-J2 cells by utilizing DCFH-DA, which decreased the anti-oxidative statuses of cells (Kang et al., 2019). However, organic probes are harmful to human health, and their fluorescence is easily quenched in a short time and cannot satisfy the requirements for continuous monitoring. Recently, with the advancement of nanotechnology, a variety of fluorescent nanocomposite probes have been developed (Chen et al., 2015; Guo et al., 2017; Liu et al., 2017; Zhou et al., 2015). A Förster resonance energy transfer (FRET) nanoassembly of upconversion nanoparticles (UCNPs)-MoS2 nanoflakes was synthesized for the bioimaging of ROS in living cells and zebrafish, which, upon the disassembly of the nanoarchitecture, displayed an excellent fluorescent response toward ROS (Wang et al., 2018). However, most nanosensor systems have intricate structures, require complex premodification and self-assembly processes, are selective toward limited ROS types and cannot be used for total ROS evaluation. Therefore, the construction of a simple, convenient and in situ method for monitoring ROS and evaluating oxidative stress is meaningful for the early screening of mycotoxins such as DON in in vitro toxicology.
In recent years, quantum dots (QDs), which constitute a class of nanoparticles, have attracted widespread attention and have been widely used in optical devices, biomarker technology, biosensing, and bioimaging. Moreover, CdSe@ZnS QDs can be directly oxidized by ROS with significant fluorescence quenching due to their reducing properties. Herein, polyethylene glycol (PEG)-modified CdSe@ZnS QDs (PEG-CdSe@ZnS) were employed as a nanosensor for monitoring intracellular ROS levels and to assess the DON-mediated cellular oxidative stress injury. As a simple and convenient sensor, PEG-CdSe@ZnS QDs can be easily loaded into cells via endocytosis. The intracellular ROS levels increase when cells are treated with DON. The DON-mediated ROS oxidized the reduced PEG-CdSe@ZnS QDs, thereby instigating destruction of the structure, and quenched the FI. Thus, the quenched FI can directly reflect the intracellular ROS levels. This simple and convenient method can be effectively used to evaluate DON-mediated oxidative stress injury in vitro.
Section snippets
Materials and chemicals
DON (purity ≥ 99%), N-acetyl-L-cysteine (NAC) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Commercial CdSe@ZnS QDs (CdSe@ZnS, 5 mg/mL) were purchased from Xingzi New Material Technology Development Co., Ltd. (Shanghai, China). Hydrogen peroxide (30%), iron dichloride, potassium superoxide, sodium hypochlorite and PEG were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco's modified Eagle medium (DMEM), fetal bovine
Principle and verification of the PEG-CdSe@ZnS QDs probe
As illustrated in Scheme 1, PEG-CdSe@ZnS QDs nanoprobe are taken up by cells via endocytosis after incubation in the cell incubator. The simulation of a cell by DON facilitates its penetration into the cell membrane to bind with the 60S subunit of a ribosome and disrupts the normal function of mitochondria, which increases the intracellular ROS levels. The generation of ROS induces the direct oxidization of PEG-CdSe@ZnS QDs probe, resulting in quenched FI. As a result, the ROS levels in living
Conclusions
We have developed a simple, convenient and in situ method for sensing the ROS level in living cells by employing PEG-CdSe@ZnS QDs, which was further implemented to evaluate the degree of DON-mediated oxidative stress. The experimental results demonstrated that the QDs can be directly oxidized by multiple ROS in solution, such as O2−, ONOO−, ·OH, ClO− and H2O2, thereby resulting in green fluorescence quenching. The proposed probe exhibited excellent sensitivity and broad-spectrum adaptability to
CRediT authorship contribution statement
You Zhou: Conceptualization, Methodology, Investigation, Data curation, Writing - original draft, Writing - review & editing. Yan Lv: Formal analysis, Software. Chuxian He: Data curation, Validation. Xianfeng Lin: Visualization, Resources. Cong Li: Writing - review & editing. Wei Xu: Project administration, Resources. Nuo Duan: Project administration, Validation. Shijia Wu: Conceptualization, Funding acquisition, Supervision, Writing - review & editing. Zhouping Wang: Conceptualization, Funding
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 has been supported by National Natural Science Fundation of China (NSFC, 31772086, 32072310), Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF) (CX(18)2025), Project of Hubei Key Laboratory for Processing and Transformation of Agricultural Products (2019HBSQGDKFA04), Fundamental Research Funds for the Central Universities (JUSRP21826), the National First-class Discipline Program of Food Science and Technology (JUFSTR20180303) and the Distinguished Professor Program
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