See related commentary on pg 1496 ORIGINAL ARTICLE

Real-Time Monitoring of Oxidative Stress in Live Mouse Skin Alexander M. Wolf1, Kiyomi Nishimaki1, Naomi Kamimura1 and Shigeo Ohta1

Oxidative stress is involved in many age-associated diseases, as well as in the aging process itself. The development of interventions to reduce oxidative stress is hampered by the absence of sensitive detection methods that can be used in live animals. We generated transgenic mice expressing ratiometric redox-sensitive green fluorescent protein (roGFP) in the cytosol or mitochondria of several tissues, including skin epidermal keratinocytes. Crossbreeding into hairless albino mice allowed noninvasive optical measurement of skin oxidative state. Topical application of hydrogen peroxide emulsion shifted the keratinocyte redox state toward oxidation within minutes and could be observed in real time by fluorescence ratio imaging. Exposing skin to 365 nm UVA radiation oxidized roGFP localized in keratinocyte mitochondria, but not when roGFP was localized in the cytosol. This suggests that significant amounts of the endogenous photosensitizers that mediate UVA– induced oxidative stress are located in the mitochondria. UVR is the major environmental cause of skin aging and UVA–mediated oxidative stress has been associated with the development of wrinkles in humans. Direct measurements of redox state in defined cell compartments of live animals should be a powerful and convenient tool for evaluating treatments that aim to modulate oxidative stress. Journal of Investigative Dermatology (2014) 134, 1701–1709; doi:10.1038/jid.2013.428; published online 14 November 2013

INTRODUCTION of assessing oxidative state in vivo remains a big hurdle in the Oxidative stress, most often defined as an excess in the development of effective antioxidants, and preselecting production of reactive oxygen species (ROS) over their substances that can actually affect oxidative stress in vivo elimination by endogenous antioxidants, is involved in the will be crucial to avoid costly failures (Vickers, 2007). pathogenesis of cancer (Serrano and Blasco, 2007), Oxidative stress is usually assessed by an increase in down- inflammation and atherosclerosis (Lusis, 2000; Rocha and stream biomarkers or fluorogenic ROS indicators, invasive Libby, 2009), diabetes (Brownlee, 2001; Houstis et al., 2006), single-point indicators of oxidative stress with a variety of and neurodegenerative (Esposito et al., 2002; Beal, 2003; drawbacks (Wardman, 2007; Chen et al., 2010; Murphy et al., Dawson and Dawson, 2003) and other diseases (James et al., 2011). Questions like how much of which ROS is produced 2012). During aging, depletion of endogenous antioxidants how, where,andwhen remain mostly unanswered. Here, we has a critical role in disease progression (Chinta and Andersen, report the generation of transgenic mice expressing redox- 2006) and oxidative stress is often assumed to be the main sensitive green fluorescent protein (roGFP) in the skin. RoGFP factor driving the aging process (Balaban et al., 2005; Beal, is a ratiometric redox sensor with two cysteine residues on its 2005; Dickinson and Chang, 2011). Even though clinical trials surface. Oxidation by hydrogen peroxide (H2O2)causesthe using antioxidants have been very disappointing (Thomson formation of a disulfide bond, changing its absorption spectrum et al., 2007; Bjelakovic et al., 2012), treatments that can (Dooley et al., 2004; Hanson et al., 2004). RoGFP is reduced by effectively modulate oxidative stress continue to be highly the thioredoxin and glutathione systems, which also eliminate sought after (Finkel, 2005; Ohsawa et al., 2007). The difficulty the majority of H2O2 (Cox et al., 2010), resulting in continuous formation and release of the roGFP disulfide bridge depending on the local redox potential (Dooley et al., 2004; Meyer and 1Department of Biochemistry and Cell Biology, Institute of Development and Dick, 2010). Although defining oxidative stress as an excess of Aging Sciences, Nippon Medical School, Kawasaki, Kanagawa, Japan ROS is overly simplistic, any change in roGFP redox state is a Correspondence: Alexander M. Wolf, Department of Biochemistry and Cell reliable indicator of oxidative stress as perturbed redox signaling Biology, Institute of Development and Aging Sciences, Nippon Medical School, 1-396 Kosugi, Nakahara-ku, Kawasaki, Kanagawa 211-8533 Japan. (Jones, 2006, 2008). RoGFP was targeted to two subcellular E-mail: [email protected] locations, cytosol and mitochondria. Expressed in a defined cell Abbreviations: CCD, charge coupled device; GSH, glutathione in reduced type, it allowed the real-time visualization of skin redox state. form; GSSG, glutathione in oxidized form; H2O2, hydrogen peroxide; LED, light-emitting diode; roGFP, redox-sensitive green fluorescent protein; ROS, RESULTS reactive oxygen species Transgenic mice expressing roGFP1 under the control of the Received 26 April 2013; revised 10 September 2013; accepted 16 September 2013; accepted article preview online 15 October 2013; published online elongation factor 1a promoter were generated using nuclear 14 November 2013 injection. We chose roGFP1 over its close cousin roGFP2

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because roGFP2 fluorescence is also sensitive to variations in 1.5 kb 0.7 kb 0.7 kb pH (Hanson et al., 2004), whereas roGFP1 is not Cytosol (Supplementary Figure S1 online). The constructs for cytosolic EF-1α promoter roGFP1 Poly (A) and mitochondrial expression are shown in Figure 1a. For 1.5kb 1.0 kb 0.7 kb cytosolic roGFP, 17 founder transgenic mice were obtained, Mitochondria of which 11 yielded viable offspring containing the transgene. EF-1α promoter PDHL roGFP1 Poly (A) Of these, five lines showed appreciable roGFP fluorescence. For mitochondrially targeted roGFP, 10 of 22 founder mice passed on the transgene, with 6 lines exhibiting detectable roGFP fluorescence. For each type, two lines with the strongest roGFP expression were kept for experiments. RoGFP fluorescence was recorded using an imaging device specially built for this purpose (Figure 1b). The object is sequentially illuminated with excitation light from CCD camera three light-emitting diodes (LEDs) emitting light of around 405, 450, and 470 nm (Figure 1c). Fluorescence emission from the animal is collected synchronized to excitation with a cooled charge coupled device (CCD) camera. The fluorescence emission spectrum (Supplementary Figure S1a online) is independent of excitation wavelength, and the 1 5 2 F roGFP redox state is determined from the ratios of 4 3 3 intensities at the different excitation wavelengths. The redox 2 4 state of pure roGFP can be determined from the ratio 1 5 1 of fluorescence at 405 versus 470 nm illumination 2 (Supplementary Figure S1b online). In founder mice, roGFP expression could be observed in the 3 skin, eyes (Figure 2a and Supplementary Figure S4 online), and several internal organs including the brain, pancreas, LED LED LED

thymus, and intestine (Supplementary Figure S2 online). For 405 nm 450 nm 470 nm analysis of redox state, the ratio of intensities at the excitation wavelengths of 405 and 470 nm was converted to color using a scale between adjustable minimum and maximum ratios, and brightness adjusted to absolute fluorescence intensity Oxidized roGFP (Figure 2b). To compare the fluorescence properties of Reduced roGFP reduced and oxidized roGFP among transgenic and wild-type animals, front paws from a single litter (11 pups, 7 roGFP positive) were either oxidized with H2O2 (right paw, upper row) or reduced with dithiothreitol (left paw, lower row) (Figure 2c). Although fluorescence at 405 nm excitation was much higher in transgenic animals, the contribution of mouse Intensity/absorption autofluorescence to total mouse fluorescence was not negli- gible. When roGFP fluorescence dominates, the ratio of fluorescence indicates the extent of oxidation and thus the roGFP redox state, while canceling out the amount of 450 470 indicator and absolute fluorescence intensity (Dooley et al., 405 2004; Hanson et al., 2004). However, the amount of RoGFP 360 380 400 420 440 460 480 500 expression could vary between transgenic animals. This led to Wavelength (nm) an expression-dependent difference in roGFP ratio even if Figure 1. In vivo redox state recording system. (a) Representation roGFP was fully oxidized or reduced (Figure 2c). In non- of the construct used to express redox-sensitive green fluorescent protein transgenic animals, the 405/470 nm fluorescence ratio was (roGFP1) in the cytosol and mitochondria of transgenic mice. (b) Scheme much lower and unaffected by oxidation state (Figure 2c and of the setup used to record roGFP fluorescence. Light from three light-emitting Supplementary Figure S3 online). We included a third illumi- diodes (LEDs) turned on sequentially synchronized to the charge coupled nation LED emitting around 450 nm light in the hope of using device (CCD) camera is combined with a randomized fiber bundle. spectral unmixing to distinguish between roGFP and auto- (c) Absorption spectra of the oxidized and reduced forms of roGFP and emission spectra of the LEDs used for excitation ratio imaging. High fluorescence, but the 450 to 470 nm excitation fluorescence 405/470 nm fluorescence ratios indicate oxidation, whereas low ratios indicate ratios of autofluorescence and roGFP were very close, making reduction of roGFP. spectral unmixing impossible with the currently available wavelengths (data not shown).

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405 nm Excitation 450 nm Excitation 470 nm Excitation

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DTT 405/470 ratio Color scale 405/470 ratio Color scale

Figure 2. Redox-sensitive green fluorescent protein (roGFP) expression and function. (a) Raw fluorescence image of a transgenic mouse head expressing roGFP in the cytosol at the three available excitation wavelengths of the setup. (b) Color-coded image representing roGFP redox state formed from the 405/470 nm fluorescence ratio. (c) Color-coded fluorescence ratio of the front paws from a 3-day-old litter of mice. Pairs of paws from each pup are oxidized with hydrogen peroxide (H2O2) (upper row) or reduced with dithiothreitol (DTT) (lower row). Numbers 1, 4, 5, 6, 7, 10, and 11 are paws from pups expressing roGFP in the mitochondria. Numbers 2, 3, 8, and 9 are from wild-type pups.

Although roGFP fluorescence was detected in the skin, fur freely moving animal. Applying the oxidizing emulsion did and melanin absorption interfere with roGFP ratio recording in not seem to cause any discomfort to the mouse. Within the C57BL/6J background. Crossing with the albino hairless 5 minutes, roGFP ratio in the area where microemulsion was Hos:HR-1 strain yielded mice with high fluorescence obser- applied increased from an average of about 2.3 to 3.5 at vable over the whole skin. Confocal imaging of frozen skin peak (Figure 3c). RoGFP redox state reverted to baseline sections revealed roGFP expression to be restricted to kerati- within approximately 30 minutes, after which a slight over- nocytes, with no roGFP fluorescence detectable in the dermis shoot to reduction was observed in the area of application. (Figure 3a). In interfollicular epidermis, roGFP fluorescence A similar response could be observed when roGFP was was restricted to the basal cell layer and a single layer of expressed in the cytosol (Supplementary Figure S6f online), viable prickle cells directly above the basal cell layer and the response increased when skin glutathione content (Supplementary Figure S5 online). No fluorescence could be was reduced by exposing mice to 5 g l 1 buthionine sulfox- detected in the stratum corneum. When a large piece of skin imine in drinking water for 1 week (Supplementary Figure was removed from the back of a mouse that was killed, the S6g online). A variety of substances could induce keratino- underlying tissue composed of muscle and bone showed cyte roGFP oxidation. Supplementary Figure S6a shows negligible fluorescence (Figure 3b). Inserting black aluminum the in vivo time course for application of diallyl disulfide, foil under the skin also had little effect on the fluorescence a component of garlic. Compared with ex vivo skin ratio (data not shown). The fluorescence signal from albino (Supplementary Figure S6b þ d online), the redox state hairless mice expressing roGFP therefore originates from quickly reverted to baseline in vivo (Figure 3c and epidermal keratinocytes. Supplementary Figure S6c þ g online), most probably To confirm the functionality of the roGFP construct in live because of superior nutrient and oxygen supply in vivo. animals, an emulsion containing 10% H2O2 was applied RoGFP fluorescence was higher when roGFP was expressed 2 thinly (B2mgcm ,a20mM layer) to the back of a mouse in the mitochondria. As a consequence, fluorescence and expressing roGFP in keratinocyte mitochondria. After appli- fluorescence ratio changes in response to chemical oxidants cation, the mouse was put in a small chamber covered with were larger when roGFP was localized in the mitochondria. a glass plate and roGFP fluorescence was monitored in the Qualitatively, however, the responses in the cytosol and

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FL TL FL+TL

4.00 3.50 3.00 2.50 2.00 1.50 1.00 Baseline +13 min +19 min +30 min ~ 2 h ~ 4 h

Figure 3. Redox-sensitive green fluorescent protein (roGFP) fluorescence in the skin and in vivo response to oxidative stress. (a) Confocal fluorescence (FL), transmitted light (TL), and overlay image of roGFP in the skin (bar ¼ 50 mm). RoGFP expression is restricted to viable keratinocytes. (b) Fluorescence image of a hairless mouse expressing roGFP in the mitochondria at 405 nm excitation. Cutting away a large piece of skin in the center reveals negligible fluorescence from underlying tissues. (c) Time series of color-coded ratio images of a freely moving mouse expressing roGFP in the mitochondria. At time zero, 10–15 mg of microemulsion containing 10% hydrogen peroxide is applied to the back of the mouse.

mitochondria were similar, as might be expected from an (Supplementary Movie S1 online). When UVA exposure external oxidant overwhelming cellular redox defenses. was stopped after 100 minutes, roGFP fluorescence was We also tested the stability of roGFP during intense almost eliminated in the center, while still being present in illumination. RoGFP fluorescence could be bleached with an oxidized state in the surrounding area receiving lower very high intensity blue light (405 nm; B320 mW cm 2), but intensity UVA radiation (Figure 4c). When roGFP was located wasalmoststableatamediumintensity(B50 mW cm 2, in the cytosol, however (Figure 4d), no oxidation could be data not shown). The excitation light intensities used to record detected even after 1 hour (Figure 4e), and roGFP fluores- roGFP fluorescence were below 1 mW cm 2. cence was only slightly reduced (Supplementary Movie S2 These transgenic mice are hence especially suitable for online). This suggests that the photosensitizer responsible for the monitoring of oxidative stress in the skin and eye, as the production of singlet oxygen under 365 nm UVA light is fluorescence from these organs is optically accessible without located mostly in the mitochondria. harming the animal. Owing to the involvement of oxidative When anesthesia was stopped and the mouse allowed to stress in UV light–induced skin aging, we decided to wake up, roGFP in the mitochondria of viable keratinocytes investigate whether we could detect UV–induced oxidative recovered from oxidation (75 minutes after the end of UVA stress. In contrast to UVB (o320 nm light), which damages exposure; Supplementary Figure S7a online). The next day, DNA directly, longer wavelength UVA (320–400 nm) induces roGFP was again fully reduced (Supplementary Figue S7b oxidative stress via photosensitized production of singlet online) and the epidermis gradually regrew over the course of oxygen. UVA radiation (emission spectrum: Supplementary several weeks (Supplementary Figure S7c–f online). Figure S8b online) from an LED was focused onto the back of Preventing UVA from reaching the epidermis also prevented a hairless mouse expressing roGFP in keratinocyte mitochon- the oxidative response (Supplementary Figure S8 online). dria (Figure 4a) with an average intensity of 250 W m 2 Two locations on the same mouse were exposed to the (about five times higher than sunlight UVA and about a same amount of UVA radiation previously confirmed to quarter of total sunlight intensity). After about 10 minutes, cause oxidation of roGFP in the mitochondria. Oxidation oxidation of roGFP could be detected (Figure 4b). Oxidation (Supplementary Figure S8a online) and epidermal damage progressed up to until 30 minutes, after which roGFP fluores- apparent 1 week later (Supplementary Figure S8d online) were cence gradually disappeared in the center, suggesting damage prevented if the illuminated area was protected by a UVA– to mitochondrial proteins after prolonged UVA exposure absorbing sunscreen (Supplementary Figure S8c online).

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roGFP in the mitochondria

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Figure 4. UVA induces oxidative stress in keratinocyte mitochondria, but not the cytosol. (a) Photo of setup exposing anesthetized mouse skin to UVA radiation (B365 nm, mean intensity 250 W m 2). (b) Color-coded ratio image of a hairless mouse expressing redox-sensitive green fluorescent protein (roGFP) in keratinocyte mitochondria 15 minutes after the start of UVA irradiation in the area marked by the yellow polygon. (c) Time series of color-coded ratio images at the indicated times after irradiation start around the irradiated area (marked by the thin white square in b; color scale as in b). (d and e)Asinb and c, except that roGFP expression is in the cytosol instead of the mitochondria. Color scale of e as in d.

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DISCUSSION (by the thioredoxin and glutathione antioxidant systems) of This transgenic mouse provides a highly convenient method for oxidizing species. Besides a lower concentration of photo- noninvasive monitoring of oxidative stress in mice. Previously, sensitizer, roGFP oxidation in the cytosol could be prevented Guzman et al. (2010) impressively used roGFP to show why by better cytosolic antioxidant defenses. The keratinocyte dopaminergic neurons selectively experience oxidative stress. cytosol might well become oxidized when using other RoGFP was also used to map redox state in Drosophila wavelengths or higher light intensities. Although roGFP has (Albrecht et al., 2011) and Caenorhabditis elegans (Back been demonstrated to equilibrate with the GSH/GSSG redox et al., 2012; Knoefler et al., 2012). Alternative techniques to pool, this equilibration might be imperfect (Go and Jones, assess oxidative stress in vivo include chemoselective 2008; Gutscher et al., 2008; Meyer and Dick, 2010). The bioluminescent reporters (Van de Bittner et al., 2010) or lower concentration of roGFP in the cytosol might also cause redox-sensitive transcription factor activation (Oikawa et al., a lower sensitivity to localized redox changes. The absence of 2012). RoGFP is a reversible sensor (Dooley et al., 2004; bulk cytosol GSH/GSSG changes does not exclude activation Hanson et al., 2004) permitting time-course measurements of redox-sensitive transcription factors if their equilibrium previously impossible or very labor-intensive. The redox state potential lies below that of GSH/GSSG (or that of roGFP) of the glutathione pool, which is usually measured by (Meyer and Dick, 2010) or if activation can occur bypassing determining GSH and GSSG (glutathione in both reduced redox changes (McMahon et al., 2010). As the lifetime of and oxidized forms) concentrations from tissue samples, can singlet oxygen is probably below 1 ms in vivo, the diffusion of now be followed in real time in the live animal. Targeting singlet oxygen is almost negligible (Baker and Kanofsky, 1992). roGFP to subcellular structures also allows the observation of We assume that besides proteins, singlet oxygen reacts with oxidative stress confined to cellular compartments (Hansen and intracellular ascorbate to produce H2O2 directly (Kramarenko Go, 2006), such as seen here in keratinocyte mitochondria. et al., 2006) or with NAD(P)H (Petrat et al., 2003), producing One drawback of the current method is that fluorescence superoxide, which is in turn converted to H2O2 by manganese recording systems need to have light sources suitable for the superoxide dismutase and detected by roGFP. selective excitation of the roGFP absorption maxima at around RoGFP fluorescence from transgenic mice was sufficiently 400 and 480 nm, which might necessitate the exchange or bright to record fluorescence images significantly below addition of light sources or filters in existing in vivo fluores- 100 ms exposure time. This should allow sufficiently short cence recording apparatuses. Another drawback is the vari- exposure times to record redox state continuously in freely able roGFP expression. In cultured cells, roGFP is in high moving animals and not only intermittently when the animal excess over autofluorescence and the roGFP fluorescence keeps still by chance, as in Figure 3c. However, sensitive ratio is a quantitative indicator of what percentage of roGFP measurement of redox state changes in moving animals would is in oxidized versus reduced state. In our mice, the contribu- necessitate precise tracking of the animal. We think that tion of intrinsic autofluorescence to total fluorescence is not measurements in anesthetized animals will remain preferable negligible. The fluorescence ratio of autofluorescence, which for most applications. is lower than the fluorescence ratio of reduced roGFP, will Oxidative stress has an important role not only as a affect the whole animal fluorescence ratio depending on the signaling factor but also in disease, where it is often seen as amount of roGFP (Supplementary Figure S3 online). Local the causative factor of a detrimental process associated with variations in skin baseline fluorescence ratio are due to aging. Despite the lack of success for several decades (Jones, variations in roGFP expression level rather than because of 2008; Bjelakovic et al., 2012), antioxidants are investigated as variations in baseline redox state. A fluorescence ratio from a a serious treatment strategy. The major difficulty in developing certain point on the whole animal cannot be assigned to a effective antioxidants is the absence of reliable in vivo certain redox state when using epifluorescence, but might be oxidative stress markers. Many antioxidant studies remain possible using confocal microscopy. For the same reasons, any inconclusive as they fail to demonstrate a reduction in change in autofluorescence (from treatments) or the amount of oxidative stress biomarkers. The antioxidant in question roGFP (from changes in transcription or photobleaching) will might simply fail to affect the oxidative damage responsible affect the apparent fluorescence ratio in the absence of any for the particular pathology (Murphy et al., 2011). redox state change. Mice expressing roGFP should be a powerful method to Previous research clearly demonstrates that UVA light can evaluate antioxidant treatments, especially in skin. Skin aging is activate redox-sensitive transcription factors located in the caused by intrinsic aging (also called chronologic or ‘‘normal’’ cytosol (Gao and Talalay, 2004), as well as shift the total cell aging) and extrinsic aging (or photoaging), mainly caused by GSH/GSSG balance in keratinocytes in vitro (He et al., 2003; UVR (Yaar and Gilchrest, 2007). Short wavelength UVB Banach-Latapy et al., 2013). Several factors could explain radiation (280–320 nm) directly damages DNA. More than why roGFP in the cytoplasm did not respond to UVA 95% of UVR, however, is UVA, which does not irradiation. The subcellular location and amount of damage DNA directly and is alone roughly 10,000 times less oxidizing species produced will depend on the absorbing carcinogenic than UVB (Diffey, 1991). UVA has therefore photosensitizer, and therefore on the illumination wave- received less attention as a skin aging factor. Nevertheless, length. In addition, the redox state of the GSH/GSSG redox UVA has been associated with the formation of wrinkles (Mac- pool is determined by the balance between photosensitized Mary et al., 2010). The number of skin care products containing production (dependent on illumination intensity) and removal antioxidants is overwhelming. The mitochondrial free radical

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theory of aging (Gruber et al., 2008) proposes ROS–induced sequence, for the expression in the mitochondria) (both kind gifts mutations in mtDNA as a major cause of aging, and previous from Prof. SJ Remington, University of Oregon, Eugene, OR) was research showed that high-intensity UVA radiation could digested with NheIandNotI restriction enzymes to remove the induce mtDNA damage (Berneburg et al.,1999).Many cytomegalovirus promoter. The mammalian expression vector pEF- aspects of photoaging resemble chronological aging (Yaar BOS (Mizushima and Nagata, 1990) carrying the human polypeptide and Gilchrest, 2007), and it is tempting to speculate chain elongation factor 1a promoter was cut with XbaI. RoGFP1 was that this might be due to the significant mitochondrial inserted and used to transform competent high DH5a Escherichia coli location of UVA–induced oxidative stress discovered here. The (Toyobo, Osaka, Japan). DNA was prepared using an endotoxin-free chemical identity of the photosensitizer(s) responsible for UVA– plasmid kit (Qiagen, Tokyo, Japan) and digested with PvuII. Fragments mediated singlet oxygen formation remains to be determined were recovered from gels using disposable chips (TaKaRa RECOCHIP, (Wondrak et al., 2006), but their location is important for the TaKaRa, Otsu, Japan) and further purified (Wizard DNA Cleanup development of antioxidants against UVA–mediated skin aging. System; Promega, Tokyo, Japan). Transfection into HeLa cells was An obvious area of application of the current method is in used to confirm correct function of the construct. Transgenic mice the evaluation of antioxidant treatments. As intracellular redox were created by nuclear injection into B400 eggs each at the Center state is highly controlled, we do not expect it to change much for Animal Resources and Development (Kumamoto University, at baseline in a live animal. The comparison of responses to a Kumamoto, Japan). The medical ethical committee of Nippon well-defined short-term stress (such as H2O2 or UVA) will be Medical School approved all described studies (approval reference the preferred assay to measure the capability of cellular numbers 22–114, 23–175, and 24–048). antioxidative enzymes and for comparison with genetically manipulated or pharmacologically treated animals. An alter- RoGFP ratiometric imaging system native to delivering antioxidant substances is electrophilic To excite roGFP fluorescence, the light of three high-power LEDs substances like sulforaphane, which can increase cellular emitting around 405 (M405L1, Thorlabs, Newton, NJ), 450 (Cree defenses by causing transient oxidative stress that induces XLamp XR-E Royal Blue), and 470 nm (Cree XLamp XR-E Royal Blue, redox-sensitive transcription factors such as NF-E2–related Cree, Durham, NC) light was focused into the split ends of a comb factor-2 (Martin-Montalvo et al., 2011). Topical application randomized trifurcated fiber optic bundle (Oriel Instruments, Strat- of sulforaphane could induce such phase 2-detoxifying ford, CT). LED light was collimated with an f ¼ 20 mm antireflection- enzymes in mice, and reduce UV–induced tumor formation coated aspheric condenser lens (Thorlabs) and passed through (Dinkova-Kostova et al., 2006). Detecting short-term oxidative cleanup filters to remove green light overlapping with roGFP stress in live skin will clarify which substances can induce fluorescence. To ensure a homogeneous distribution of excitation this protective adaptation. The redox response to topical light, a frosted glass plate was placed in front of the common end of application of an exogenous oxidant will also depend on the the fiber bundle. LED operating current was controlled with Buck- formulation of the delivery vehicle, which can be used to Puck LED power modules (LuxDrive, LEDdynamics, Randolph, VT), determine the effect of penetration enhancers on the diffusion which in turn were controlled by an USB-1208FS data acquisition of substances through the stratum corneum. device (Measurement Computing, Norton, MA). Fluorescence passed Photodynamic therapy is a promising noninvasive treatment through an emission filter (HQ535/50x; Chroma Technologies, Rock- for keratinocyte tumors, including basal cell carcinoma, the ingham, VT) and was recorded with a cooled CCD camera (Spot RT; most common human cancer. In photodynamic therapy, an Diagnostic Instruments, Sterling Heights, MI) using the Kodak KAI- exogenous photosensitizer is used for the selective elimination 2001 monochrome CCD chip (1600 1200 7.4 mmsquarepixels). of cancerous cells via light-mediated induction of oxidative LEDs were turned on and off synchronized to individual frames of stress, and this transgenic mouse would provide a convenient CCD camera exposure. The whole setup was controlled using method to monitor photosensitizer-mediated oxidative stress software written in LabView (National Instruments, Austin, TX). induced by light treatment. Applying substances or light to the back of an anesthetized RoGFP excitation and emission spectra mouse needs little experimental skill. Fluorescence from Suspensions of PC12 cells expressing roGFP in the mitochondria roGFP is bright enough to provide high signal-to-noise ratios (described in Wolf et al., 2010) were suspended in phosphate-buffered and color-coded movies reflecting skin redox state are saline containing 50 mM HEPES and either 50 mM dithiothreitol for intuitively understandable. These mice should be a valuable reduced or 50 mM H2O2 for oxidized roGFP1. Excitation spectra were tool to understand the dynamics of oxidative stress in vivo obtained at 520 nM emission, and emission spectra were taken with and test the efficacy of treatments aimed at reducing 400 nM excitation with a Shimadzu RF-5300PC spectrophotometer oxidative stress. (Shimadzu, Kyoto, Japan). To correct for cell autofluorescence, the fluorescence of an equal protein amount of PC12 cells not expressing MATERIALS AND METHODS roGFP1 reduced or oxidized in the same way was subtracted. The Transgene construction and generation of mice amount of protein was determined using the BCA protein assay Two transgenes for the expression of roGFP1 in the cytosol and (Pierce, Thermo Fisher Scientific, Rockford, IL). mitochondria were made. The mammalian expression vector pEGFP- N1 (Clontech, Takara Bio, Mountain View, CA) containing roGFP1 LED emission spectra and illumination intensities (for the expression in the cytosol) or roGFP1 with a mitochondrial The emission spectra of the LED light sources were measured using an targeting sequence (pyruvate dehydrogenase E1 subunit leader USB4000-FL spectrometer (Ocean Optics, Dunedin, FL) coupled to a

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