Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

Summary

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

Abstract

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (E_GSH), while HyPer directly detects hydrogen peroxide (H₂O₂). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular E_GSH and H₂O₂ generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

Introduction

The term "oxidative stress" is frequently cited as a mechanism in toxicology, yet rarely is this term described specifically. Oxidative stress can refer to several intracellular processes, including generation of reactive oxygen species, damage caused by free radicals, the oxidation of antioxidant molecules, and even the activation of specific signaling cascades. A broad range of environmental contaminants^1,2 and pharmaceutical agents^3,4 have been documented to induce oxidative stress by either direct action of the xenobiotic compound itself

Protocol

1. Preparation of Cells

NOTE: This procedure describes the lentiviral transduction of an immortalized cell line (BEAS-2B; ATCC, Manassas, VA) to express the desired reporter (roGFP2 or HyPer). Other cell lines/types and/or methods of gene transfer, including transfection, can be utilized as long as they result in a level of reporter expression adequate to visualize a sufficient number of sensor-expressing cells per field of view (typically 5 - 10 cells). If using transfection methods, the procedure should be performed in the dish that will ultimately be used for the method of analysis (e.g., transfect the cells in the same dish that will be p....

Results

The use of roGFP2 and HyPer in detecting changes in E_GSH and intracellular H₂O₂ has been well described previously^25,36,42,43 and is demonstrated here. Confocal images of cells expressing roGFP2 at baseline and following addition of H₂O₂ and DTT are shown in Figure 2. Data from .......

Discussion

The use of roGFP2 and HyPer in detecting changes in intracellular E_GSH and H₂O₂, respectively, has been well-described in previous physiological and toxicological studies ^{25,35,36,39,40,41,42,43}. The protocol outlined here r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
roGFP2 Plasmid	University of Oregon	N/A	Generous gift of S.J. Remington
HyPer Plasmid	Evrogen	FP941
Hydrogen Peroxide	Sigma Aldrich	H1009
Dithiothreitol	Sigma Aldrich	10708984001
9,10-Phenanthrenequinone	Sigma Aldrich	156507
Black Wall Glass Bottomed Dishes	Ted Pella	14029-20
BEAS-2B cell line	American Type Culture Collection	CRL-9609
Keratinocyte Basal Medium	Lonza	192151
Keratinocyte Growth Medium BulletKit	Lonza	192060
Excel	Microsoft Office Suite	N/A
NIS-Elements AR Imaging Software	Nikon	N/A
Nikon C1si Confocal Imaging System	Nikon	N/A