Trion：号称百分百抵抗uva与氧化应激的抗氧化物 faseb-1402-Comparing-140211

The FASEB Journal • Research Communication Comparing the effects of mitochondrial targeted and localized antioxidants with cellular antioxidants in human skin cells exposed to UVA and hydrogen peroxide Anne O. Oyewole,* Marie-Claire Wilmot,* Mark Fowler,† and Mark A. Birch-Machin*,1 *Department of Dermatological Sciences, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle Upon Tyne, UK; and †Unilever Research and Development Colworth, Sharnbrook, UK ABSTRACT Skin cancer and aging are linked to increased cellular reactive oxygen species (ROS), par- ticularly following exposure to ultraviolet A (UVA) in sunlight. As mitochondria are the main source of cellular ROS, this study compared the protective ef- fects of mitochondria-targeted and -localized antioxi- dants (MitoQ and tiron, respectively) with cellular antioxidants against oxidative stress-induced [UVA and hydrogen peroxide (H2O2)] mitochondrial DNA (mtDNA) damage in human dermal fibroblasts. With the use of a long quantitative PCR assay, tiron (EC50 10 mM) was found to confer complete (100%) protection (P<0.001) against both UVA- and H2O2-induced mtDNA damage, whereas MitoQ (EC50 750 nM) pro- vided less protection (17 and 32%, respectively; P<0.05). This particular protective effect of tiron was greater than a range of cellular antioxidants investi- gated. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway provides cellular protection against oxidative stress. An ELISA assay for the Nrf2 target gene heme oxygenase-1 (HO-1) and studies using Nrf2 small interfering RNA both indicated that tiron’s mode of action was Nrf2 independent. The comet assay showed that tiron’s protective effect against H2O2- induced nuclear DNA damage was greater than the cellular antioxidants and MitoQ (P<0.001). This study provides a platform to investigate molecules with sim- ilar structure to tiron as potent and clinically relevant antioxidants.—Oyewole, A. O., Wilmot, M.-C., Fowler, M., Birch-Machin, M. A. Comparing the effects of mitochondrial targeted and localized antioxidants with cellular antioxidants in human skin cells ex- posed to UVA and hydrogen peroxide. FASEB J. 28, 485–494 (2014). www.fasebj.org Key Words: DNA damage � mitochondria � ultraviolet radia- tion � reactive oxygen species � ROS � Nrf2 Exposure to solar ultraviolet radiation (UVR) plays a key role in the development of skin cancers and aging (1). There is evidence that ultraviolet A (UVA) may have a causative role in skin cancer (2, 3), but its mechanism of action is thought to be different from that of higher energy UVB occurring via an indirect mechanism involving reactive oxygen species (ROS), including hydrogen peroxide (H2O2; ref. 4). This oxi- dative stress can subsequently exert downstream effects, including lipid peroxidation and generation of DNA damage. As electrons leak from the electron transport chain (ETC), mitochondria are the major cellular generator of superoxide (1, 5), which can be readily converted into other ROS. Mitochondrial DNA (mtDNA) is close to the site of superoxide production, making it highly vulnerable to oxidative damage. The accumulation of mtDNA mutations, through oxidative stress mecha- nisms, has been proposed as an underlying cause of the carcinogenic and aging process in many tissues, includ- ing skin (1, 4, 5). We and others have shown that mtDNA damage accumulates in skin due to UVR irra- diation, photoaging, and skin cancer (6–10), thereby pioneering the use of mtDNA damage as a biomarker of UV exposure (7, 10, 11). 1 Correspondence: Dermatological Sciences, Institute of Cellular Medicine, Medical School, Framlington Pl., New- castle University, Newcastle upon Tyne, NE2 4HH, UK. E-mail: m.a.birch-machin@ncl.ac.uk doi: 10.1096/fj.13-237008 Abbreviations: EC50, half-maximal effective concentration; ETC, electron transport chain; HDFn, human dermal fibroblast neonatal; HO-1, heme oxygenase 1; Keap1, kelch-like ECH- associated protein 1; mtDNA, mitochondrial DNA; MTS, 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt; NAC, N-acetyl cysteine; nDNA, nuclear DNA; NF104, neonatal fibroblast 104; Nrf2, nuclear factor erythroid 2-related factor 2; qPCR, quantitative real-time PCR; RNAi, RNA interference; ROS, reactive oxygen species; SED, standard erythemal dose; SF, sulforaphane; tiron, 4,5-dihydroxy-1,3-benzenedisulfonic; siRNA, short interfering RNA; UVA, ultraviolet A; UVB, ultraviolet B; UVR, ultraviolet radiation 4850892-6638/14/0028-0485 © FASEB Antioxidants have been shown to provide protection to the skin against oxidative stress. For example, caro- tenoids protect against UVR-induced erythema, possibly by acting as a ROS scavenger (12), and with collaborators we have shown that UVR-induced mtDNA damage in human skin is protected by a diet rich in tomato paste (high in the antioxidant lycopene; ref. 13). However, the ability of antioxidants to abrogate oxidative stress in skin and indeed other tissues has been the subject of much discussion in the face of little comparative data for the different antioxidants and their targets, partic- ularly at the level of mitochondria (4). As mitochondria are a major source of ROS, this has led to the develop- ment of several antioxidant compounds with the ubiquinone structure and designed to target the mito- chondria. A widely used compound is MitoQ, a ubiqui- none derivative conjugated to triphenylphosphonium that enables this molecule to enter the mitochondria (14–16). An unrelated compound, 4,5-dihydroxy-1,3-ben- zenedisulfonic (tiron), which chelates iron and other metals (17) and exhibits ROS scavenging properties (17– 20), is also able to permeabilize the mitochondrial mem- brane and thereby be localized rather than targeted to the mitochondrion from the cytoplasm (18, 20). The comparative effects of a mitochondria-targeted antioxidant, MitoQ; an antioxidant preferentially local- ized to the mitochondria, tiron; and cellular antioxi- dants have not been previously investigated, particu- larly in relation to protection against oxidative stress- induced DNA (mitochondrial and nuclear) damage under the same experimental conditions. Therefore, in a novel approach, we have compared the protective effects of mitochondria-targeted and localized antioxi- dants (MitoQ and tiron, respectively) with each other and with cellular antioxidants [including N-acetyl cys- teine (NAC), resveratrol, curcumin, and sulforaphane (SF)] against oxidative stress (UVA and H2O2)-induced mtDNA damage in human dermal fibroblasts utilizing a long quantitative PCR (qPCR) assay to amplify the majority of the genome. We have investigated the wider effects of these compounds through comparison of their protective effects against H2O2-induced nuclear DNA (nDNA) damage using the comet assay. The nuclear factor erythroid 2-related factor 2 (NrF2) sig- naling pathway significantly contributes to cellular protection against oxidative stress in the skin (21– 24), and it may provide an “intramitochondrial anti- oxidant network” (25, 26). Therefore, experiments were performed [including an ELISA assay for the Nrf2 target gene heme oxygenase 1 (HO-1) and studies using Nrf2 short interfering RNA (siRNA)] to determine whether the mode of action of tiron (the most potent antioxidant observed in our study) was Nrf2 pathway dependent. Prevention of excessive ROS in a physiolog- ical manner based on medicinal chemical studies of the tiron structure may provide a tractable therapeutic platform beyond skin to other tissues where the process of aging and disease is associated with increased levels of oxidative stress. MATERIALS AND METHODS Cell viability and half-maximal effective concentration (EC50) determination The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (Pro- mega, Southampton, UK) measures the ability of viable cells to metabolize the tetrozolium salt MTT by succinate dehydroge- nase. The absorbance of nontreated (control) cells on each 96-well plate was set as reference of 100% cell viability, and the viabilities of cultured human dermal fibroblast cells treated in the presence of various compounds (after 24 h in culture medium) were calculated relative to this value. All compounds were tested in at least 3 independent experiments, and for each experiment the results for all concentrations assayed are the means of 8 replicates. The EC50 values (concentration inducing 50% of cell death) were calculated using dose-response curves from �3 independent experiments. Statistical significance was performed using a 1-way ANOVA with Dunnett’s correction, where P � 0.05 was considered significantly different from control (untreated). UVA/H2O2 treatment Human dermal fibroblast neonatal (HDFn) cells were grown in DMEM containing 10% fetal bovine serum, 5 IU/ml penicillin, and 5 g/ml streptomycin. The cells were treated in advance with the optimum, sublethal dose (determined by the MTS assay of the antioxidant for 24 h in phenol red-free DMEM) and washed before treatment with 200 �M H2O2 (Sigma, UK) or irradiated with a sublethal dose [9.2 J/cm2, equivalent to 0.5 standard erythemal dose (SED)] of UVR using a TL-09 lamp (315– 400 nM, peak emission at 360 nM; Philips, Amsterdam, The Netherlands). Exposure to this physiologically relevant dose of UVA equated to �45 min exposure time. Total cellular DNA was extracted using the Qiagen DNeasy tissue extraction kit (Qiagen, Man- chester, UK). qPCR MtDNA damage was measured as amplification efficiency for a large (11,095 bp) amplicon normalized to a short (83 bp) amplicon as described previously (27) and performed using the real-time PCR assay as described previously (28). At least 3 independent PCR analyses on �3 biological replicates were performed. DNA primer sequences for the 11,059-bp amplicon were as follows: D1B (282-255), 5=-ATGATGTCTGTGTGGAAAGTG- GCTGTGC-3=; and OLA (5756–5781), 5=-GGGAGAAGC- CCCGGCAGGTTTGAAGC-3=. DNA primer sequences for the 83-bp amplicon were as follows: IS1 (16,042-16,066), 5=- GATTTGGGTACCACCCAAGTATTG-3=; and IS2 (16,101- 16,124), 5=-AATATTCATGGTGGCTGGCAGTA-3=. Quantification of the Nrf2 target HO-1 using ELISA Fibroblast cells (3�104/well) were seeded and treated with the optimum, sublethal dose of each antioxidant for 24 h. This experimental work was conducted at Unilever Colworth, where the standard operating protocol for the HO-1 ELISA assay used human neonatal fibroblast 104 (NF104) cells. These cells can be considered as interchangeable with the HDFn cells used throughout the rest of the study, as both cells are human neonatal fibroblast cells isolated from foreskin. In addition, previous proof-of-concept studies between New- 486 Vol. 28 January 2014 OYEWOLE ET AL.The FASEB Journal � www.fasebj.org castle University and Unilever have shown that both cell types behave in the same way in the oxidative stress-induced mtDNA strand-break assays. Cell lysates from the NF104 cells were analyzed for HO-1 content using the DuoSet IC ELISA kit (R&D Systems, Abingdon, UK) and a microplate reader at 450 nM [results expressed as picograms per microgram protein using the Pierce bicinchoninic acid (BCA) assay; Thermo Fisher Scientific, Waltham, MA, USA). Comet assay The comet assay was performed as described previously by Atherton et al. (29). Nucleoids were selected (100/slide), and the tail length (measure of DNA migration from head of comet) for each treatment was measured and normalized against the control (untreated). According to the established conditions in our laboratory and reports from collaborators, a lower concentration (i.e., 10 �M) of H2O2 was required in the comet assay compared with the concentration (i.e., 200 �M) used in the long qPCR assay to detect mtDNA damage. As a result, it was observed that the maximal concentration of antioxidant required to provide the greatest protective effect against H2O2-induced nDNA damage was much lower than that required in the long qPCR mtDNA damage assay (with the exception of tiron). siRNA and Nrf2 studies At 24 h after transfection (Lipofectamine RNAiMAX; Invitro- gen, Paisley, UK) with siRNA (20 nM) against Nrf2 (or a negative control using On-TargetPlus ontargeting pool; Thermo Scientific), cells were treated with medium contain- ing the compound for 24 h before UVA irradiation. Nrf2 mRNA levels were determined 48 h post-transcription follow- ing cDNA synthesis (Applied Biosystems, Warrington, UK) from isolated RNA (RNeasy; Qiagen) and quantification of the Nrf2 mRNA transcripts (TaqMan Gene Expression Assay; Applied Biosystems). Primer/probe sets for human Nrf2 and �-actin (endogenous control) were from Applied Biosystems. Nrf2 results were normalized to �-actin using the ��Ct method (30, 31). Statistical analyses ANOVAs with correction for multiple groups were performed using commercially available software (GraphPad Prism 5; GraphPad, San Diego, CA, USA). RESULTS Tiron confers greater protection than MitoQ against both UVA- and H2O2-induced mtDNA damage in dermal fibroblasts The first aim of the study was to determine and compare the relative effects of MitoQ and tiron in terms of providing protection against both UVA- and H2O2-induced mtDNA damage in cultured HDFn cells. First, it was crucial to derive a sublethal experimental concentration of tiron and MitoQ by performing an extensive range of cell viability dose-response experi- ments (MTS assay). The derived maximum concentra- tion of MitoQ and tiron without inducing significant (not �5%) death in the HDFn cells was 150 and 3 mM, respectively (1-way ANOVA with Dunnett’s correction; treatments were not significantly different when com- pared with untreated control, P�0.001; n�3). These concentrations were then selected for the subsequent experiments. The EC50 for cell viability for MitoQ and tiron in the HDFn cells was derived from the experi- mental series as 750 and 10 mM, respectively. There- fore, the derived sublethal concentrations of 150 nM MitoQ and 3 mM tiron are not only well below the EC50 values but do not cause any significantly observable cell death (i.e., �5%). The protective effect of these sublethal concentra- tions of MitoQ (150 nM) and tiron (3 mM) against UVA-induced mtDNA damage was determined in HDFn cells utilizing the previously validated real-time long qPCR assay (28, 32). The long qPCR assay mea- sures DNA lesions in the majority (�70%) of the mitochondrial genome. The cells were exposed to a physiological dose of UVA (0.5 SED) that we have previously shown to be well tolerated by the cultured skin cells (28). First, the results in Fig. 1 show that exposure to physiologically relevant levels of UVA re- sulted in a statistically significant increase in the levels of mtDNA damage (P�0.001; Fig. 1). This is expressed as an increase in Ct value due to the decrease in the relevant proportions of undamaged:damaged circular mtDNA molecules as the UVA-induced DNA damage blocks the progression of the DNA polymerase. The data are expressed as a fold difference in C(t), with each treatment corrected for fold difference in relation to the value obtained from the nonirradiated control sample (which is normalized as 1). For example, the level of UVA-induced damage in Fig. 1B is calculated as a fold increase of 1.72, which approximates to a 128- fold increase in mtDNA damage. The second and most important observation in Fig. 1 is that tiron appears to provide a clear and complete (100%) protection against the UVA-induced mtDNA damage. This obser- vation is in sharp contrast to MitoQ, which provided only a 17% protective effect. UVA has been postulated as a stimulator of oxidative stress in skin (1), and the data from Fig. 1 suggest, therefore, that tiron may be affecting this pathway. To address this issue, the experimental protocol repre- sented in Fig. 1 was repeated with the only exception of replacing UVA with H2O2 (200 �M as used in our previous study; ref. 28) as a direct oxidative inducer of mtDNA damage in the HDFn cells (Fig. 2). There are 2 key observations from the data presented in Fig. 2. First, in a similar fashion to the results observed in Fig. 1 (UVA induction), the data in Fig. 2 show that 200 �M H2O2 induced a statistically significant induction in mtDNA damage, as represented by an increase in Ct value. Second, and more important, tiron appears to provide a clear and complete (100%) protection against the H2O2-induced mtDNA damage (P�0.001). Once again, this observation is in sharp contrast to the results in Fig. 2B, where MitoQ provided a 32% protec- tive effect (P�0.05). 487EFFECT OF MITOCHONDRIAL AND CELLULAR ANTIOXIDANTS Protective ability of a range of cellular antioxidants against UVA and H2O2-induced mtDNA damage is lower than that exhibited by tiron The next question to be addressed is how this greater protection of mtDNA damage exhibited by tiron com- pares to a range of cellular antioxidants that do not penetrate the mitochondrial membrane with the same experimental protocol used in Figs. 1 and 2. In the same manner as the experiment described in Fig. 1A, sublethal experimental concentration for each cellular A Co ntr ol Tir on Tir on + UV A UV A 0.0 0.5 1.0 1.5 2.0 *** Fo ld D iff er en ce C (t) Co ntr ol Mi toQ Mi toQ + UV A UV A 0.0 0.5 1.0 1.5 2.0 * Fo ld D iff er en ce C (t) B Figure 1. Protective effect of MitoQ and tiron against UVA-induced mtDNA damage in HDFn cells. Effects of tiron (A) and MitoQ (B) against UVA-induced mtDNA damage in HDFn skin cells using the lesion-specific real-time PCR assay. Statistical significance was assessed by performing a 1-way ANOVA with Newman-Keuls multiple comparison test. Bars represent means sem (n�3). A) Tiron (3 mM) appears to completely prevent UVA-induced mtDNA damage (i.e., 100% protection). ***P� 0.001 vs. UVA treatment alone. B) In contrast MitoQ (150 nM) was able to offer less (17%) protection against UVA-induced mtDNA damage. *P � 0.05 vs. UVA treatment alone. A Co ntr ol Tir on Tir on + H 2 O 2 0.0 0.5 1.0 1.5 *** Fo ld D iff er en ce C (t) 0.0 0.5 1.0 1.5 * Fo ld D iff er en ce C (t) H 2 O 2 Co ntr ol Mi toQ Mi toQ + H 2 O 2 H 2 O 2 B Figure 2. Protective effect of MitoQ and tiron against H2O2-induced mtDNA damage in HDFn cells. Effects of tiron (A) and MitoQ (B) against H2O2-induced mtDNA damage in HDFn skin cells using the lesion-specific real-time PCR assay. Statistical significance was assessed by a 1-way ANOVA with Newman-Keuls multiple comparison test. Bars represent means sem (n�3). A) Tiron (3 mM) appears to completely (i.e., 100%) prevent H2O2-induced mtDNA damage. ***P � 0.001 vs. H2O2 treatment alone. B) In contrast, MitoQ (150 nM) was able to offer less (32%) protection against H2O2-induced mtDNA damage. *P� 0.05 vs. H2O2 treatment alone. 488 Vol. 28 January 2014 OYEWOLE ET AL.The FASEB Journal � www.fasebj.org antioxidant was determined by performing an exten- sive range of cell viability dose-response experiments (MTS assay). The derived maximum concentration (without inducing �5% cell death) was 100 �M for resveratrol, 8 mM for NAC, and 8 �M for curcumin (1-way ANOVA with Dunnett’s correction; treatments were not significantly different when compared with untreated control, P�0.001; n�3). The protective ef- fect of these concentrations of antioxidants against UVA and H2O2-induced mtDNA damage was deter- mined in HDFn cells using the long qPCR strand-break assay (Table 1). As the aim of the experiment was to compare the protective effect of tiron with that of known cytosolic antioxidants against UVA- and H2O2- induced mtDNA damage, the degree of protection offered by each compound was expressed as a percent- age of the protection offered by tiron, which appeared to be complete (