A novel recombinant cell fluorescence biosensor based on toxicity of pathway for rapid and simple evaluation of DON and ZEN

Jian Ji,Hui Jiang,Jiadi Sun,Wenshu Gu,Yinzhi Zhang,Chao Sun,Xiulan Sun

doi:10.1038/srep31270

Jian Ji, Hui Jiang + Show 5 more

Open Access

https://doi.org/10.1038/srep31270

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Abstract

During an exposure, humans and animals are most often exposed to a mixture rather than individual mycotoxins. In this study, a Human Embryonic Kidney 293 cell (HEK-293) fluorescence sensor was developed to detect and evaluate mycotoxins, deoxynivalenol (DON) and zearalenone (ZEN) compounds, produced by Fusarium culmorum that are common food contaminants. TRE-copGFP (green fluorescent protein) and ERE-TagRFP (red fluorescent protein) plasmids were constructed and cotransfected into HEK-293 cells through a highly efficient, lipid-mediated, DNA-transfection procedure. Results show that fluorescence intensity was proportional to DON and ZEN concentrations, ranging from 2 to 40 ng/mL and 10 to 100 ng/mL respectively, with a detection limit of 0.75 ng/mL and 3.2 ng/mL respectively. The EC50 of DON and ZEN are 30.13 ng/mL and 76.63 ng/mL respectively. Additionally, ZEN may have a synergistic effect on enhancing AP-1 activity of the toxicity pathway of DON. These data indicate the high sensitivity and effectiveness of our biosensor system in the evaluation of the combined toxicity of ZEN, DON and their derivatives. In addition, this approach is suitable for an early warning method for the detection of ZEN and DON family mycotoxins contamination without higher-priced, conventional analytical chemistry methods.

Highlights

Endocrine glands and serum hormone levels[18]
This assay is based on the stable cotransfection of HEK293 cells with two plasmids that separately encode the green fluorescent protein GFP reporter gene under the transcriptional control of the TRE promoter (TRE-GFP) and the red fluorescent protein RFP reporter gene under the transcriptional control of estrogen response element (ERE) promoter (ERE-RFP)
The PCR products of TRE and ERE and the vectors of pcopGFP and pTagRFP were double-digested with Xba I and EcoR I, ligated with T4 DNA ligase respectively, and separately transfected into DH5a competent cells (Fig. 2A,C)

Summary

Introduction

Endocrine glands and serum hormone levels[18] These compounds have a high relative binding affinity for estrogen receptor and exhibit high transactivation activity[19], acting through Ers[20,21,22] to activate the transcription of estrogen-responsive genes both in vivo[23,24,25] and in vitro[26,27]. Biological assays of cell activity and viability evaluation, such as by measuring the mitochondrial reduction of tetrazolium salts into an insoluble dye (the MTT test), the release of the enzyme lactate dehydrogenase (LDH), the measurement of reactive oxygen species (ROS), or mitochondrial apoptosis can be used to evaluate toxicity[31] These methods require specific reagents, significant sample preparation time, and biological expertise.

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