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.
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 (EGSH), 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 EGSH 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.
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 contaminants1,2 and pharmaceutical agents3,4 have been documented to induce oxidative stress by either direct action of the xenobiotic compound itself5,6 or secondarily by production of oxidant species as part of a cellular response7,8,9,10. It is therefore of great interest in toxicology to accurately observe and characterize the oxidative processes leading to adverse outcomes. Conventional methods of measuring oxidative stress involve identification of oxidized biomolecules11,12,13,14,15 or antioxidants16,17,18,19,20, or direct measurement of reactive species themselves21,22,23,24. However, these methods typically require cellular disruption, which often consumes the sample, eliminates spatial resolution, and potentially introduces artifacts25. The development of more sensitive and specific methods for the detection of oxidant species and markers of oxidative stress is broadly applicable to the investigation of the adverse effects of xenobiotic exposure.
Live-cell microscopy using a new generation of genetically-encoded fluorogenic sensors has emerged as a powerful tool to monitor the intracellular redox status of living cells. These sensors are typically expressed using a vector under the control of a viral promoter that is introduced via transfection or transduction methodologies. High expression efficiencies are not necessary, since cells expressing the fluorescent sensor can be easily identified visually. For toxicological assessments, cells expressing these sensors can be observed using fluorescence microscopy as they are exposed to xenobiotic compounds in real-time. This experimental design permits repeated measurements in the same cell, allowing each cell's established baseline to act as its own control. The high temporal resolution afforded by live-cell imaging is well-suited for the detection of oxidative events, particularly those that are modest in magnitude or transient in nature. In addition to being both sensitive and specific to their target molecules, the fluorescence of some of these sensors can be excited using two wavelengths of light. This phenomenon allows the fluorescent emission to be expressed as a ratio, which permits the discernment of signal changes associated with authentic sensor responses from those caused by artefacts such as variations in sensor expression, cell thickness, lamp intensity, photobleaching, and sensitivity of the fluorescence detector26. Another advantage of the use of fluorogenic sensors is that they can be targeted to specific cellular compartments, creating a level of spatial resolution that is unmatched by conventional methods25,26,27.
A large family of genetically-encoded sensors based on green fluorescent protein (GFP) have been developed and characterized to report on a broad variety of physiological markers, including pH, temperature, calcium concentrations, and the ATP/ADP ratio25,28,29,30,31. Included among these are sensors of the glutathione redox potential (EGSH) and hydrogen peroxide (H2O2). While these sensors were developed for applications in redox biology and physiology, they have also been adapted to study xenobiotic-induced oxidative stress. Specifically, the protocol outlined here describes the use of the EGSH sensor roGFP2 and the H2O2 sensor HyPer.
roGFP2 reports on the redox potential of intracellular reduced and oxidized glutathione (GSH/GSSG) through a redox relay involving glutathione peroxidase (GPx), glutaredoxin (Grx), and glutathione reductase (GR) (Figure 1)25,32,33. Glutathione is the predominant cellular antioxidant molecule and is present primarily in its reduced form (GSH) in millimolar concentrations in the cytosol25,34. While EGSH has not been linked to any functional outcome, it is recognized as an important indicator of intracellular oxidative status34. A relatively small increase in the concentration of GSSG results in an increase in EGSH that is detectable by roGFP2. Equally important, monitoring of EGSH using roGFP2 during xenobiotic exposures can potentially reveal much about the mechanism of action at several points in the redox relay and associated pathways, such as the pentose phosphate shunt (Figure 1)35. The second sensor discussed here, HyPer, is an intracellular H2O2 probe derived from the insertion of yellow fluorescent protein (YFP) into the regulatory domain of bacterial H2O2-sensitive transcription factor OxyR136. Although it has previously been considered a damaging reactive oxygen intermediate, H2O2 is increasingly being recognized as an important intracellular signaling molecule under physiological conditions37,38, suggesting that unrecognized roles for H2O2 exist in toxicology as well. For instance, excess H2O2 induced by a xenobiotic exposure could be a precursor to dysregulation in cellular signaling or a shift in bioenergetics.
Both genetically-encoded fluorogenic sensors have been expressed in several established cell lines, including the human epidermoid carcinoma cell line A431 and the human bronchial epithelial cell line BEAS-2B, to observe changes in EGSH and H2O2 in response to a variety of toxicological exposures. These include gaseous pollutants (ozone35), soluble components of particulate matter (1,2 naphthoquinone39,40 and zinc41), and nickel nanoparticles (unpublished data). These studies represent only a small subset of the possible applications of these two sensors. Theoretically, any cell type that is capable of receiving and expressing the DNA of these sensors through conventional molecular biology techniques can be utilized to assess the effects of xenobiotics suspected to alter the cellular oxidative state. To date, one or more of these sensors has been expressed in various prokaryotes and eukaryotes, including several mammalian, plant, bacterial, and yeast cell types25,26,36,42. The readout for both EGSH and H2O2 sensors is a change in the intensity of fluorescence emitted at 510 nm upon excitation with 488 and 404 nm light. This method is widely adaptable across fluorometric platforms, including various types of microscopy (confocal and wide-field) and plate readers. The method presented here allows for sensitive and specific observation of intracellular EGSH and H2O2 de in vitro toxicological systems.
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 presented to the microscope). The steps described below provide details for preparing cell transductions to be performed in a 6-well cell culture plate. If this format is not an appropriate size for the desired application, other vessels can be substituted.
2. Microscopy Set-up
NOTE: The protocol described below is performed using a confocal microscope equipped with laser lines at 404 and 488 nm. Other means of making fluorometric assessments of the sensors described within this protocol at their prescribed excitations/emission should also yield viable data. Importantly, equipment settings can vary greatly depending on the type, age, and condition of the instrument being used; thus, any instrument values mentioned may not be specific to the equipment used in other laboratories.
3. Data Acquisition
4. Data Analysis
The use of roGFP2 and HyPer in detecting changes in EGSH and intracellular H2O2 has been well described previously25,36,42,43 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 images collected throughout the experiment were then exported and analyzed as described in section 4 of the protocol. As shown, the addition of exogenous H2O2 as a positive control produces reproducible responses when the data are normalized to their respective baseline and a defined dynamic range is established through addition of known exogenous oxidants/reductants (i.e., H2O2 and DTT) (Figures 3, 4, and 5).
Responses to toxicological stimuli are typically more variable than those induced by controls. This is to be expected, as xenobiotic compounds may have pleiotropic effects on cells, ranging from redox cycling to adduction of intracellular proteins. Examples of roGFP2 and HyPer responses induced by exposure to the toxicant 9,10-PQ, an oxidizing air pollutant formed by atmospheric reactions of phenanthrene44, are presented in Figures 6 and 7. The graphs in these figures depict the progression of each step involved in the data analysis outlined in section 4 of the protocol. As shown, normalization of each cell to its own baseline and positive control (Figure 6B), reduces intercellular variability in the magnitude of the sensor response from the raw data (Figure 6A). In cases where the variance in the cellular response is of interest, normalization of the data can be performed up to this point, with individual cells represented by their own lines on the graph. Variation in the magnitude or kinetics of the responses among cells in the same dish can reveal important mechanistic insights that reflect differences in the cell cycle, for instance. Alternatively, the averaged response of the cells in the dish can be presented along with the standard deviation (Figure 6C). Otherwise, if replicates of separate experiments are being presented, the data may be presented as the normalized relative fluorescence intensity values or a percentage of the maximum response induced by the control oxidant (Figures 3, 4, 5, and 6D), showing the average of all replicates accompanied by the standard error of the mean. The ambient air contaminant 9,10-PQ is known to be a potent redox cycler, which we hypothesize is responsible for the cyclical peaks of hydrogen peroxide production detected by HyPer (Figure 7) and stepwise EGSH increases detected by roGFP2 (Figure 6D).
Figure 1. The roGFP2 glutathione redox relay. Glutathione peroxidases (GPx) oxidize reduced glutathione (GSH) to GSSG in response to hydrogen peroxide (H2O2), thus increasing the glutathione redox potential (EGSH). The fluorogenic sensor roGFP2 equilibrates with intracellular EGSH when oxidized by glutaredoxin (Grx). In the reductive pathway, Grx catalyzes the reduction of roGFP2 through deglutathionylation as GSSG levels decrease and normal levels of GSH are reestablished by glutathione reductase (GR) at the expense of NADPH. NADPH is supplied by glucose through the pentose phosphate pathway (PPP). Adapted from Gibbs-Flournoy et al35. Please click here to view a larger version of this figure.
Figure 2. Confocal microscopy images of the human bronchial epithelial cell line BEAS-2B expressing cytosolic roGFP2. Cells were illuminated at 488 nm (A-D), followed by 404 nm (E-H). Images show fluorescence emitted at 510 nm for the following conditions: baseline (A, E), 100 µM H2O2(B, F), 1 mM H2O2 (C, G), and 5 mM DTT (D, H). 60X Plan Apo VC objective. Scale bars represent 25 µm. Please click here to view a larger version of this figure.
Figure 3. Dose dependent changes in roGFP2 induced by H2O2 in BEAS-2B cells. Cells were equilibrated for 30 min in phenol-red free keratinocyte basal media (KBM) prior to exposures. All additions of H2O2 occurred after a baseline period of 5 min. roGFP2 fluorescence emitted at 510 nm was collected using a 525/30 nm band pass filter following laser excitation at 404 and 488 nm. Cellular responses were normalized to their respective baseline and maximum sensor response following the addition of 1 mM H2O2. Values are presented as mean ± standard error, n=3, where n consists of an average of 5 – 10 independent cells. Please click here to view a larger version of this figure.
Figure 4. Response of glucose starved BEAS-2B cells expressing roGFP2 to various doses of H2O2. Cells were equilibrated for 2 hours in a minimal salt medium that does not contain glucose prior to exposures. All additions of H2O2 occurred after a baseline period of 5 min. roGFP2 fluorescence emitted at 510 nm was collected using a 525/30 nm band pass filter following laser excitation at 404 and 488 nm. Cellular responses were normalized to their respective baseline and maximum sensor response following the addition of 1 mM H2O2. Values are presented as the mean ± standard error, n = 3, where n consists of an average of 5 – 10 distinct cells. Please click here to view a larger version of this figure.
Figure 5. Response of BEAS-2B cells expressing HyPer to various doses of H2O2. Cells were equilibrated for 30 min in phenol-red free KBM prior to exposures. All additions of H2O2 occurred after a baseline period of 5 min. Fluorescence emitted at 510 nm was collected using a 525/30 nm band pass filter following laser excitation at 404 and 488 nm. Cellular responses were normalized to their respective baseline and maximum sensor response following the addition of 1 mM H2O2. Values are presented as the mean ± standard error, n = 3, where n consists of an average of 5 – 10 distinct cells. Please click here to view a larger version of this figure.
Figure 6. 9,10-PQ-induced responses of BEAS-2B cells expressing roGFP2. Cells were equilibrated for 30 min in phenol-red free KBM prior to exposure. Addition of 25 µM 9,10-PQ with mixing occurred at 5 min, followed by addition of 1 mM H2O2 at 20 min and 5 mM DTT at 24 min. Panel A depicts the raw roGFP2 ratio of individual cells in the dish, each cell represented by a separate line. Panel B shows normalization of values from each individual cell to its baseline sensor fluorescence, and the average and standard deviation of this data is superimposed on the individual normalized ratios in Panel C. The percentage of maximal sensor response averaged across all cells is depicted in Panel D. Please click here to view a larger version of this figure.
Figure 7. 9,10-PQ-induced responses of BEAS-2B cells expressing HyPer. Cells were equilibrated for 30 min in phenol-red free KBM prior to exposure. Addition of 25 µM 9,10-PQ with mixing occurred at 5 min, followed by addition of 1 mM H2O2 at 20 min and 5 mM DTT at 25 min. Fluorescence emitted at 510 nm was collected using a 525/30 nm band pass filter following laser excitation at 404 and 488 nm. Cellular responses were normalized to their respective baseline and maximum sensor response following the addition of 1 mM H2O2. Values are presented as the mean of 5 – 10 distinct cells. Please click here to view a larger version of this figure.
The use of roGFP2 and HyPer in detecting changes in intracellular EGSH 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 reports an effective way to implement these genetically-encoded redox sensors in pointed toxicological studies designed to assess xenobiotic-induced perturbations of intracellular redox homeostasis. Most importantly, the proper use of these sensors links any changes observed to a specific redox pair or ROS (EGSH or H2O2), ultimately clarifying the lack of specificity with many conventional oxidative stress approaches. In assuring the quality of the data generated from these sensors, a few aspects must be taken into consideration when designing experiments and analyzing/interpreting data. They include: 1) the use of proper controls, 2) verification of appropriate sensor responses, and 3) manipulation of experimental parameters to elucidate mechanisms. Of these, the addition of exogenous H2O2 and DTT as controls at the end of the observation period serves multiple purposes. First, it affords an opportunity to assess the magnitude of the response to the toxic exposure relative to the maximal and minimal signals achievable from the sensor. This is most useful in normalizing responses for comparisons between experiments. In addition, ending the experiment with the addition of strong exogenous oxidant and reductive stimuli can evaluate the effect of the preceding exposure on sensor/relay integrity. Furthermore, aldrithiol can be added at the end of the run to bypass the relay and test the functionality of the sensor directly for potential damage that the exposure may have inflicted to the fluorophore itself.
Just as with other techniques, when applying this methodology for the specific examination of toxicological outcomes, it is important to make qualitative assessments regarding the accuracy of the observations made. When utilizing roGFP2 or HyPer with any new xenobiotic it is important to first test a range of doses, while ensuring that the fluorescence intensity (510 nm emission) from each excitation wavelength (404 nm and 488 nm) changes appropriately (i.e. increase or decrease) for the redox senor being used. The goal is to identify a dose at which a sensor response is detectable, but is not overwhelmed by overt manipulation of the sensor or other non-specific effects of the compound. Direct adduction or damage to the sensor may manifest as atypical alterations in fluorescence at either excitation wavelength (488 or 404 nm). In situations where either sensor has been observed to consistently respond inappropriately to the addition of a toxicant across exposures, it is recommended that the investigator consider examining experimental parameters in relation to dosimetry, cell viability, sensor expression, and/or optical/chromatic abnormalities of the instrumentation being used to observe the responses.
As with any toxicological readout, the data acquired are usually more meaningful when the observed responses are examined or validated using a mechanistic approach, and the experimental limitations are considered. To this end, several experimental parameters combined with the inherent properties of the sensor can be utilized to generate robust redox assessments. Investigations specific to the use of the roGFP2 sensor ultimately concern the redox relay through which roGFP2 senses EGSH (Figure 1). The relay involves intracellular enzymes, including glutaredoxin (Grx), glutathione reductase (GR), and glutathione peroxidase (GPx). Differences in endogenous levels of Grx across both cellular compartments and cell types can influence measurements of EGSH and make comparisons between experiments difficult43. To address this problem, a series of "fusion probes" have been synthesized, which link the relevant enzyme directly to the roGFP2 sensor. Most recently, human Grx-1 was linked to roGFP2 by Gutscher and colleagues32 to form Grx1-roGFP2. In addition to having a greater dynamic range than roGFP1 and being pH insensitive, Grx1-roGFP2 can monitor EGSH in cell types or compartments in which Grx activity would otherwise be limiting. Differences in NADPH levels across cell types or even among cells in the same dish can also contribute to variability in roGFP2 responses. NADPH is produced from glucose through the pentose-phosphate pathway, and can be utilized by glutathione reductase (GR) to reduce GSSG back to GSH (Figure 1). If cells in the same field begin the experiment with different reducing capacities, their EGSH changes and resulting roGFP2 responses to a stimulus may not be uniform. This problem can be surmounted through a period of glucose starvation prior to the experiment, which depletes cellular NADPH stocks and harmonizes roGFP2 responses. A decreased capacity for EGSH recovery can also be seen in the 25 µM H2O2 dose in cells that were starved of glucose as compared to cells supplemented with normal glucose concentrations (Figures 3 and 4). Thusly, manipulation of NADPH levels through glucose starvation can serve as a means to check the involvement of multiple relay components in transducing the effect of a xenobiotic exposure to the sensor. Another concern when using roGFP2 in a toxicological context is the potential for the xenobiotic to interact directly in oxidizing the sensor, rather than acting through the redox relay to alter EGSH. Direct oxidation of roGFP2 can be determined by probing each point of the redox relay and assessing its effect on the roGFP2 signal. An example of this technique using ozone as a stimulus is demonstrated in the study by Gibbs-Flournoy et al 35.
For experiments with the H2O2 sensor, the above concerns are not as relevant, as the OxyR1 domain of HyPer senses H2O2 directly rather than through a redox relay. However, unlike roGFP2, H2O2 sensor is sensitive to changes in pH, which can cause changes in fluorescence not attributable to H2O2 during an experiment. For this reason, the use of properly buffered media, an environmental chamber to maintain the desired temperature, and the use of vehicle controls are vital to any experiment utilizing the H2O2 sensor. Additionally, fluorogenic sensors have been developed to specifically monitor pH, such as pHRed, which emits fluorescence in the red channel and can be co-expressed in cells also expressing HyPer45. The ideal pH control for the H2O2 sensor is SypHer, which is a version of the H2O2 sensor that has a single point mutation rendering it unable to sense H2O2, yet preserves responsiveness to pH46. It has also been observed that cells expressing H2O2 sensor require a longer baseline period to equilibrate, particularly after being transported from an incubator to the site of analysis, during which they experience slight changes in media temperature and pH. It is recommended to acquire at least 5 data points after the baseline has stabilized before beginning any exposure.
The sensors discussed in this method, roGFP2 and HyPer, are two of many fluorogenic sensors that have a variety of emerging applications in the field of toxicology. This includes sensors designed to detect not only EGSH and H2O2, but peroxynitrite47, nitric oxide48,49, and even ozone50. These sensors can be utilized in live-cell imaging studies to specifically and sensitively monitor the oxidative species of interest. With advances in techniques such as spectral unmixing, it is also possible to co-express two sensors with similar spectral characteristics (within 5 nm of each other) in the same cell, and separate the proportion of the fluorescence emitted by each sensor51. This technique is of particular interest for roGFP2 and HyPer, as they involve excitations and emission at the same wavelengths. As briefly mentioned here, certain sensors can be targeted to specific intracellular compartments, such as the mitochondria, to observe oxidative events of interest. While these emerging sensors and techniques are beyond the scope of this method, the reader is referred to several recent publications on the topic of intracellular redox sensors, such as the review by Wages and colleagues52. The use of genetically-encoded fluorogenic sensors with live-cell imaging is a robust tool for the monitoring of oxidative responses during toxicological assessments.
The authors have nothing to disclose.
The authors would like to thank Katelyn Lavrich for assistance with experimental design and manuscript edits.
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 |