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Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

ABSTRACT: Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O2•-) and H2O2 in the presence of NADH. Generation of the free radical O2•- and H2O2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1? stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.

SUBMITTER: Ganini D

PROVIDER: S-EPMC5362137 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

Ganini Douglas D Leinisch Fabian F Kumar Ashutosh A Jiang JinJie J Tokar Erik J EJ Malone Christine C CC Petrovich Robert M RM Mason Ronald P RP

Redox biology 20170310

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as ...[more]

PMID: 28334681

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Project description:Lead (Pb) is a heavy metal that can have harmful effects on the environment, which has severe cytotoxicity in many animal tissues. N-acetylcysteine (NAC) has antioxidant activity, reducing lead-induced oxidative stress and apoptosis, but its role in chicken cells is unknown. The current study explored the antagonistic effect of NAC on lead-induced apoptosis and oxidative stress in chicken embryo fibroblast (CEF). In this study, CEF was used as a model to measure the cytotoxic effects of lead nitrate at different concentrations, demonstrating a dose-dependent effect on CEF activity. Employing inverted microscopy, the investigation of morphological alterations in CEF cells was conducted. Fluorescence staining methodology enabled the assessment of reactive oxygen species (ROS) levels within CEF cells. Moreover, an enzyme-linked immunosorbent assay was utilized to detect the presence of oxidative damage indicators encompassing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activity, malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC) within CEF cells. Furthermore, the determination of the apoptosis rate of CEF cells was accomplished through the utilization of the Hoechst 33258 staining method in combination with the Annexin V-FITC dual staining method. By using RT-qPCR for detection, lead treatment increased expression of pro-apoptotic genes, caspase-3, and caspase-9, and reduced expression of anti-apoptotic genes, Bcl-2, and BI-1. Reduced antioxidant capacity was shown by increased ROS and MDA levels in CEF cells after lead treatment. The results showed that NAC inhibited the expression of caspase-3 and caspase-9 in lead-treated CEF cells, while NAC had a certain inhibitory effect on the relative expression of Bcl-2 and BI-1 mRNA in lead-induced CEF cells. NAC significantly reduced lead-induced oxidative damage and apoptosis. Overall, our results demonstrate a novel protective effect of NAC against lead-induced injury in chicken cells, providing a theoretical basis for future investigations of drugs that are effective in preventing lead poisoning in animals.

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Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

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Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

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