Name: imaging approaches to assessments of toxicological oxidative stress using genetically-encoded fluorogenic sensors
Uploaded: 2018-02-07T14:00:00
Description: Watch this Scientific Journal Video about Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors at JoVE.com

The authors would like to thank Katelyn Lavrich for assistance with experimental design and manuscript edits.

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 proced...

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 r...

The term &#34;oxidative stress&#34; 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&#39;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 in in vitro toxicological systems.

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			<td height="45" style="height:45px;width:216px;">roGFP2 Plasmid</td>
			<td style="width:123px;">University of Oregon</td>
			<td style="width:243px;">N/A</td>
			<td style="width:167px;">Generous gift of S.J. Remington</td>
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			<td height="40" style="height:40px;width:216px;">HyPer Plasmid</td>
			<td style="width:123px;">Evrogen</td>
			<td>FP941</td>
			<td style="width:167px;"></td>
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			<td height="40" style="height:40px;width:216px;">Hydrogen Peroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:243px;">H1009</td>
			<td style="width:167px;"></td>
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			<td height="40" style="height:40px;width:216px;">Dithiothreitol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:243px;">10708984001</td>
			<td style="width:167px;"></td>
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			<td height="40" style="height:40px;width:216px;">9,10-Phenanthrenequinone</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:243px;">156507</td>
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			<td height="40" style="height:40px;width:216px;">Black Wall Glass Bottomed Dishes</td>
			<td style="width:123px;">Ted Pella</td>
			<td>14029-20</td>
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			<td height="40" style="height:40px;width:216px;">BEAS-2B cell line</td>
			<td style="width:123px;">American Type Culture Collection</td>
			<td style="width:243px;">CRL-9609</td>
			<td></td>
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			<td height="40" style="height:40px;width:216px;">Keratinocyte Basal Medium</td>
			<td style="width:123px;">Lonza</td>
			<td style="width:243px;">192151</td>
			<td></td>
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			<td height="40" style="height:40px;width:216px;">Keratinocyte Growth Medium BulletKit</td>
			<td style="width:123px;">Lonza</td>
			<td style="width:243px;">192060</td>
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			<td height="40" style="height:40px;width:216px;">Excel&#160;</td>
			<td style="width:123px;">Microsoft Office Suite</td>
			<td style="width:243px;">N/A</td>
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			<td height="40" style="height:40px;width:216px;">NIS-Elements AR Imaging Software</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:243px;">N/A</td>
			<td></td>
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			<td height="40" style="height:40px;width:216px;">Nikon C1si Confocal Imaging System</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:243px;">N/A</td>
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imaging approaches to assessments of toxicological oxidative stress using genetically-encoded fluorogenic sensors

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 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 ...

Watch this Scientific Journal Video about Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors at JoVE.com

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

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 ...

The overall goal of this methodology is to establish the use of genetically-encoded redox sensors for the specific assessment of toxicant-induced perturbations in redox homeostasis. By overcoming the significant limitations of conventional approaches used with oxidative stress, this method will help answer key questions pertaining to toxicological oxidative stress. The main advantage of this technique is that it combines a high degree of specificity and sensitivity with unparalleled spacio-temporal resolution to investigate toxicological oxidative stress in living cells.Perform all imaging analysis using a confocal microscope equipped with laser lines at 404 and 488 nanometers, and environmental controls to maintain an appropriate temperature, humidity, and/or gas concentration suitable for the cells throughout the duration of the experiment. If using high values of relative humidity, use an objective heater and/or heating tape to keep any surfaces that may come in contact with the humidified atmosphere at or slightly above the temperature at which the humidity is generated to prevent condensation. Turn on all microscope components, and set up all equipment required for sequential excitation at 488 and 404 nanometers with emission at 510 nanometers.Ensure all components of the optical configuration are set appropriately for real-time acquisition. Set up a stage-top environmental chamber to maintain constant temperature at 37 degrees celsius, 5%CO2 atmosphere, and greater than 95%humidity. Prior to starting image acquisition, equilibrate the environmental chamber for at least 10 minutes after the initial set-up of all environmental controls.Place the dish of cells on the stage top within the environmental chamber. With the desired objective lens, find the focal plane of cells using the eyepiece and white light and ensure normal morphology. A 1.4 numerical aperture 60 X violet corrected oil-immersed objective lens is commonly used, which permits identification of intracellular compartments while maximizing the optical resolution of the confocal system.Check the fluorescence expression of the cells in the field of view while visualizing the cells under wide-field fluorescence illumination with an appropriate filter set such as flourescian-isothioanate or FITC. At this point, using wide-field illumination while looking through the eyepiece is more convenient because it is easier to move the dish to select a field of cells to study. Once a desired field of view is found, close the environmental chamber.Use the focus maintaining feature to facilitate maintenance of a stable focal plane throughout the study. Next, set up acquisition parameters to ensure optimal assessment of the sensor of interest across the desired exposure period. To do so, adjust the laser power for excitation at 488 nanometers and emission at 510 nanometers.Choose a laser power level that allows visualization of the cells and keep this constant between samples or replicate dishes. Use the confocal controls of the acquisition software to ensure that the selected focal plane has been optimized for maximal florescence emission intensity in the center of the cells by scanning at 488 nanometers while adjusting the Z-plane. Use a high gain setting while adjusting for the Z-plane with the most over-exposed cells.Once the appropriate focal plane has been found, return the gain to a setting that is most optimal for the fluorescence of the reporter being used without over-saturating the pixels being observed. Then, use the gain to fine-tune the baseline fluorescence. With roGFP2, establish the baseline near the upper limit of the instrument without over-saturation, as these cells will lose 510 nanometer fluorescence induced by 488 nanometer excitation when the glutathione redox potential increases.Repeat these steps with excitation at 404 nanometers and emission at 510 nanometers. Gain settings for the 404 nanometer excitation wavelength are opposite to those used with 488 nanometer excitation for each sensor. In general, fluorescence of the 404 nanometer excitation will be considerably lower than that obtainable with the 488 nanometer excitation in cells expressing roGFP2, as a 404 nanometer peek is a relatively minor excitation maximum for this sensor.Set up the acquisition software to sequentially excite the two excitation wavelengths and collect emissions for both at 510 nanometers and at a predetermined time interval throughout the desired length of the experiment. For real-time assessment of experimental parameters, choose at least five to 10 sensor-expressing cells in the field, and establish them as regions of interest to monitor their fluorescence changes during the experiment. Next, dissolve the environmental toxicant, 9, 10-PQ in dimethyl sulfoxide to a concentration of 15 millimolar.Dilute the dissolved toxicant in basal-cell media to a 250 micromolar working solution for a final concentration of 25 micromolar. Additionally, prepare a working solution of hydrogen peroxide for later use in this protocol. Once the experimental parameters have been defined, begin the time course acquisition.Establish a baseline period of at least five minutes prior to starting xenobiotic exposures. Expose the cells to the toxicant being investigated using conventional approaches for in-vitro dosing. Monitor changes in fluorescence during the exposure period.Perform subsequent injections as needed. Take great care not to shift the dish during injections so that the same cells are followed throughout the entire time course while imaged in the same focal plane. At the end of the experiment, expose the cells to the appropriate controls to fully oxidize and reduce roGFP2.The addition of control simuli validates sensor responsiveness at the end of the exposure and confirms that the responses observed are not the direct result of damage to the forport. So, it is especially important in the initial evaluation of a toxin. For these studies, use one millimolar hydrogen peroxide to determine the maximum sensor response.Wait at least five minutes to allow the sensor to respond and then inject a reducing agent such as DTT to reduce the sensor and return it to a level of fluorescence at or near its established baseline. Finally, perform data analysis as described in the text protocol. Shown here are representative results illustrating an established dose response generated using the bronchial epithelial cell line Bees 2B expressing roGFP2.Cursor shown to exposure five micromolar, 25 micromolar, or 100 micromolar doses of hydrogen peroxide. Exposure of bees 2B cells to a previously untested environmental toxicant, 9, 10-PQ induces a gradual but potent increase in the intracellular glutathione redox potential as assessed by roGFP2. Here, each line depicts the raw roGFP2 ratio of individual cells in the dish.Although the test compound produces a maximal response comparable to one millimolar hydrogen peroxide, this known redox cycler caused a change in the sensor that was fully reduced with the addition of five millimolar DTT, suggesting that the sensor remains functional following exposure to this redox-active organic compound. This approach will aid toxicologists in investigating key mechanistic events in toxican-induced oxidative stress, paving the way for more focus studies in the emergent field of redox toxicology across a broad spectrum of toxicants and cell types. After watching this video, you should have a good understanding of how to use genetically encoded sensors and live-cell microscopy to monitor pivotal markers of toxicological oxidative stress with high sensitivity and specificity at unmatched spacio-temporal resolution.

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