Stress In Human Dermal Fibroblasts Research Articles

Assessment of the Antioxidant Properties of Horned Turban (Turbo cornutus) Viscera, Marine By-Products with Potential Application in Nutraceuticals

Horned turban (Turbo cornutus) is an edible gastropod that occurs along the intertidal zone and basalt coastline, and is an important marine resource in Jeju, Korea. However, T. cornutus viscera are mostly discarded following processing. In this study, the antioxidant activity of viscera and muscle extracts was compared. In addition, the protective effect of T. cornutus viscera ethanol extract (TVEE) against H2O2-induced oxidative stress in human dermal fibroblasts (HDFs) was investigated. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydrogen peroxide scavenging activity of the viscera (IC50 = 0.64 ± 0.64 and 0.76 ± 0.01 mg/mL, respectively) showed higher activity than that in muscle. TVEE increased the reactive oxygen species (ROS) scavenging effect and cell viability in H2O2-induced HDFs without cytotoxicity. Furthermore, the fluorescence intensity of HDFs was high in those from H2O2-induced intracellular ROS production, but TVEE treatment decreased ROS production. H2O2 activated the mitogen-activated protein kinase (MAPK) signaling and matrix metalloproteinase 2 (MMP-2) in HDFs. However, MMP2 expression was confirmed to be reduced via MAPK (ERK, JNK, and p38) signaling phosphorylation. In conclusion, various antioxidant effects were confirmed in T. cornutus viscera, which instead of being discarded as marine by-products, can be applied as nutraceuticals in various industries.

Human hair proteins as natural reactive oxygen species scavengers for in vitro applications.

Human hair proteins are recognized for their intrinsically high cysteine content. They can be solubilized while preserving their highly reductive thiol groups for free radical scavenging applications. The presence of aromatic and nucleophilic amino acids such as methionine, serine, phenylalanine, and threonine further contribute to the antioxidative potential of this material. Herein, utilizing the DPPH (2,2-diphenyl-1-picrylhydrazyl) and acellular 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) assays, keratins are demonstrated to possess the highest radical scavenging activity among the studied hair proteins. Consequently, protection against hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs) cultured in human hair keratin supplemented media is demonstrated. Quenching of reactive oxygen species in the HDF is observed using the CellROX Green dye and the expression levels of antioxidant (HMOX1, SOD2, GPX1) and tumor suppressor (TP53) genes is analyzed using qPCR. Collectively, this study presents further evidence and demonstrates the in vitro application potential of hair proteins, especially keratins, as an antioxidizing supplement.

Antiaging effect of inotodiol on oxidative stress in human dermal fibroblasts

Oxidative damage is one of the major causes of human skin aging. Inotodiol is a lanostane triterpenoid that demonstrates antiviral, anticancer, and anti-inflammatory activities. Previous studies have reported that inotodiol also has antiallergic effects. However, whether inotodiol inhibits oxidative stress-induced human skin aging is not known. Stimulation of human dermal fibroblast cells with hydrogen peroxide is related to skin aging. Inotodiol inhibited the expression of mitogen-activated protein kinase (MAPK) and NADPH Oxidase 5 (NOX5). Moreover, inotodiol effectively decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), as well as nitric oxide (NO), reactive oxygen species (ROS), cyclooxygenase-2 (COX-2), and cytokines such as IL-1β, IL-6, and TNF-α. Based on our results, inotodiol protects human dermal fibroblast by preventing MAPK-NOX5 and NF-κB activation and attenuates the expression of aging genes. Inotodiol may therefore be considered a potential candidate for developing natural antiaging products, because it protects the human skin from oxidative stress-induced skin aging by inhibiting the MAPK-NOX5 and NF-κB signaling pathways.

9-Hydroxy-6,7-dimethoxydalbergiquinol suppresses hydrogen peroxide-induced senescence in human dermal fibroblasts through induction of sirtuin-1 expression

Objective: To investigate the potential anti-aging mechanism of 9-hydroxy-6,7-dimethoxydalbergiquinol (HDDQ) on hydrogen peroxide (H2O2)-induced oxidative stress in human dermal fibroblasts (HDFs). Methods: The effect of HDDQ on cell viability was assessed by MTT assay, and the effects of HDDQ on senescence-like phenotypes were determined by senescence-associated β-galactosidase (SA-β-gal) staining, Western blotting analysis, and a cell proliferation assay. The expression level and activity of sirtuin-1 (SIRT1) induced by HDDQ were also measured. Results: HDDQ reversed senescence-like phenotypes in the oxidant-challenged model, through reducing SA-β-gal activity and promoting cell growth. Meanwhile, decreases in ac-p53, p21Cip1/WAF1, and p16Ink4a and an increase in pRb were observed. HDDQ induced the expression of SIRT1 in a concentration- and time-dependent manner. Moreover, HDDQ inhibited H2O2-induced phosphorylation of Akt by SIRT1 up-regulation and reduced SA-β-gal staining. Conclusions: HDDQ inhibits H2O2-induced premature senescence and upregulation of SIRT1 expression plays a vital role in the inhibition of the senescence phenotype in HDFs.

Pueraria montana var. lobata root extract inhibits photoaging on skin through Nrf2 pathway.

Pueraria montana var. lobata is a bioactive substance, in possession of a variety of beneficial health effects, which has long been extensively used as a traditional medication for the treatment of fever, acute dysentery, diabetes, and cardiovascular diseases in North-East Asian countries. The purpose of this study was to evaluate the cytoprotective activity of Pueraria montana var. lobata ethanol extract (PLE) for ultraviolet B (UVB) induced oxidative stress in human dermal fibroblasts (HDF). It was hypothesized that PLE treatment (25-100 μg/mL) would reduce intracellular reactive oxygen species (ROS) levels as well as increase collagen production in UVB-irradiated HDF. The results confirmed this theory, with collagen production increasing in the PLE treatment group in a dose-dependent manner. In addition, regulators of cellular ROS accumulation, including HO-1 and NOQ-1, were activated by Nrf2, which was mediated by PLE. Hence, intracellular levels of ROS were also reduced in the PLE treatment group in a dose-dependent manner. In conclusion, PLE increases collagen production and maintains hyaluronic acid (HA) levels in human dermal fibroblasts exposed to UVB-irradiation, thereby inhibiting photoaging.

Protective effect of flavonoids from Foeniculum vulgare against ultraviolet-B-induced oxidative stress in human dermal fibroblasts

Background: Traditionally, Foeniculum vulgare (fennel seeds) has been used for its antimicrobial, analgesic, antipyretic, antiflatulence, antispasmodic, and antiandrogenic activities. Materials and Methods: In the present study, the protective effect of flavonoids from fennel seeds was investigated against ultraviolet (UV)-B radiation-induced cell damage and oxidative stress in human dermal fibroblast (HDF) cells. Results: Flavonoid-enriched fraction (FEF) of fennel seeds showed high flavonoid content and antioxidant potential as well as the presence of a marker compound rutin. Pretreatment of HDF cells with the FEF (15–45 μg/ml) significantly protected against UV-B-induced cytotoxicity, endogenous enzymatic antioxidant depletion, oxidative DNA damage, intracellular reactive oxygen species generation, and apoptotic morphological changes. Conclusion: The current study proved for the first time that the FEF of fennel seeds reduced oxidative stress through the nuclear factor E2-related factor 2-antioxidant response elements pathway. Flavonoids from fennel seeds have a potential as UV-B protectants and can be explored against diseases, in which oxidative stress is closely implicated.

Combined extract of Moringa oleifera and Centella asiatica modulates oxidative stress and senescence in hydrogen peroxide-induced human dermal fibroblasts.

Moringa oleifera Lam. and Centella asiatica (L.) Urb. leaves have been previously reported to exhibit antioxidant activity. The objective of the present study is to determine the in vitro antioxidant activity of the combined extracts of M. oleifera and C. asiatica (TGT-PRIMAAGE) and its effect on hydrogen peroxide (H 2O2)-induced oxidative stress in human dermal fibroblasts. TGTPRIMAAGE acted on the mechanism of hydrogen transfer as it showed scavenging activity in the DPPH assay. This is due to the presence of phenolics and flavonoids in TGT-PRIMAAGE. TGT-PRIMAAGE effectively reduced cellular generation of reactive oxygen species induced by H O2. The activities of superoxide dismutase and catalase were also increased in cells treated with TGT-PRIMAAGE. 2 Treatment with TGT-PRIMAAGE showed significant reduction (P < 0.05) in the number of senescent cells. Significant reduction (P < 0.05) of malondialdehyde was also seen in cells treated with TGT-PRIMAAGE. The p53 protein level was reduced in TGT-PRIMAAGEtreated cells, which indicates its potential in protecting the cells from oxidative stress induced by H2O2.

Protective properties of geniposide against UV-B-induced photooxidative stress in human dermal fibroblasts

Context: Geniposide (genipin-1-O-β-d-glucoside) is a major bioactive ingredient in the fruits of gardenia [Gardenia jasminoides J. Ellis (Rubiaceae)], a traditional herbal medicine in Asian countries.Objective: This work assesses the skin anti-photoaging potential of geniposide in human dermal fibroblasts under UV-B irradiation.Materials and methods: The anti-photoaging property of geniposide, at varying concentrations (5, 12 and 30 μM) treated for 30 min prior to UV-B irradiation, was evaluated by analysing reactive oxygen species (ROS), promatrix metalloproteinase-2 (proMMP-2), glutathione (GSH), superoxide dismutase (SOD), nuclear factor erythroid 2-related factor 2 (Nrf2) and cellular viability.Results: Geniposide suppressed the ROS elevation under UV-B irradiation, which was revealed using three ROS-sensitive fluorescent dyes. The use of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE) and dihydrorhodamine 123 (DHR-123) elicited the IC50 values of 10.5, 9.8 and 21.0 μM, respectively. Geniposide attenuated proMMP-2 at activity and protein levels that were elevated under UV-B-irradiation. Geniposide at 5, 12 and 30 μM augmented the UV-B-reduced total GSH content to 1.9 ± 0.1-, 2.2 ± 0.2- and 4.1 ± 0.2-fold, respectively. Geniposide at 5, 12 and 30 μM upregulated total SOD activity to 2.3 ± 0.1-, 2.5 ± 0.3- and 3.3 ± 0.3-fold, respectively, under UV-B irradiation. The UV-B-reduced Nrf2 levels were also upregulated by geniposide treatment. Geniposide, at the concentrations used, was unable to interfere with cellular viabilities under UV-B irradiation.Discussion and conclusions: After the skin anti-photoaging potential of geniposide may be further verified, it can be utilized as a safer resource in the manufacture of effective anti-aging cosmetics.

Photoprotective Effects of Cycloheterophyllin against UVA-Induced Damage and Oxidative Stress in Human Dermal Fibroblasts.

Ultraviolet (UV) radiation, particularly ultraviolet A (UVA), is known to play a major role in photoaging of the human skin. Many studies have demonstrated that UV exposure causes the skin cells to generate reactive oxygen species and activates the mitogen-activated protein kinase (MAPK) pathway. Previous studies have also demonstrated that cycloheterophyllin has an antioxidant effect and can effectively scavenge free radicals. Extending the aforementioned investigations, in this study, human dermal fibroblasts were used to investigate the protective effect of cycloheterophyllin against UV-induced damage. We found that cycloheterophyllin not only significantly increased cell viability, but also attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce hydrogen peroxide (H2O2) generation and down-regulate H2O2-induced MAPK phosphorylation. In the in vivo studies, the topical application of cycloheterophyllin before UVA irradiation significantly decreased trans-epidermal water loss (TEWL), erythema, and blood flow rate. These results indicate that cycloheterophyllin is a photoprotective agent that inhibits UVA-induced oxidative stress and damage, and could be used in the research on and prevention of skin photoaging.

149 The protective effects of stem cell-derived epidermal progenitor cell-conditioned media against oxidative stress in human dermal fibroblasts

149 The protective effects of stem cell-derived epidermal progenitor cell-conditioned media against oxidative stress in human dermal fibroblasts

Antioxidative properties of ginsenoside Ro against UV-B-induced oxidative stress in human dermal fibroblasts.

Ginsenoside Ro (Ro), an oleanolic acid-type ginsenoside, exhibited suppressive activities on reactive oxygen species (ROS) and matrix metalloproteinase-2 (MMP-2) elevation in UV-B-irradiated fibroblasts. Ro could overcome the reduction of the total glutathione (GSH) contents in UV-B-irradiated fibroblasts. Ro could not interfere with cell viabilities in UV-B-irradiated fibroblasts. Collectively, Ro possesses a potential skin anti-photoaging property against UV-B radiation in fibroblasts.

Age-associated reduction of cell spreading induces mitochondrial DNA common deletion by oxidative stress in human skin dermal fibroblasts: implication for human skin connective tissue aging.

BackgroundReduced cell spreading is a prominent feature of aged dermal fibroblasts in human skin in vivo. Mitochondrial DNA (mtDNA) common deletion has been reported to play a role in the human aging process, however the relationship between age-related reduced cell spreading and mtDNA common deletion has not yet been reported.ResultsTo examine mtDNA common deletion in the dermis of aged human skin, the epidermis was removed from full-thickness human skin samples using cryostat. mtDNA common deletion was significantly elevated in the dermis of both naturally aged and photoaged human skin in vivo. To examine the relationship between age-related reduced cell spreading and mtDNA common deletion, we modulated the shape of dermal fibroblasts by disrupting the actin cytoskeleton. Reduced cell spreading was associated with a higher level of mtDNA common deletion and was also accompanied by elevated levels of endogenous reactive oxygen species (ROS). Boosting cellular antioxidant capacity by using antioxidants was found to be protective against mtDNA common deletion associated with reduced cell spreading.ConclusionmtDNA common deletion is highly prevalent in the dermis of both naturally aged and photoaged human skin in vivo. mtDNA common deletion in response to reduced cell spreading is mediated, at least in part, by elevated oxidative stress in human dermal fibroblasts. These data extend current understanding of the mitochondrial theory of aging by identifying the connection between mtDNA common deletion and age-related reduction of cell spreading.

Ginsenoside Rb2 Attenuates UV-B Radiation-Induced Reactive Oxygen Species and Matrix Metalloproteinase-2 through Upregulation of Antioxidant Components in Human Dermal Fibroblasts

Aims: The antiphotoaging activities of ginsenoside Rb2 on the skin, one of the predominant protopanaxadiol-type ginsenosides, were evaluated in cultured human dermal fibroblasts. Methods: The antiphotoaging activity was examined by analyzing the levels of reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP-2), total glutathione (GSH) and superoxide dismutase (SOD) activity as well as cell viability for fibroblasts under UV-B irradiation. Results: When cultured fibroblasts were exposed to Rb2 prior to UV-B irradiation, Rb2 displayed suppressive activities on UV-B-induced ROS elevation and MMP-2 on both activity and protein levels, while it exhibited an enhancing activity on total GSH level and SOD activity diminished by UV-B irradiation. However, Rb2 could not interfere with cell viabilities in UV-B-irradiated fibroblasts. Conclusion: Ginsenoside Rb2 plays a photoprotective role against UV-B-induced oxidative stress in human dermal fibroblasts, which implies its skin antiphotoaging potential.

Cordyceps militaris extract protects human dermal fibroblasts against oxidative stress-induced apoptosis and premature senescence.

Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS.

Inhibitory effect of kohlrabi juices with antioxidant activity on oxidative stress in human dermal fibroblasts (LB394)

Inhibitory effect of kohlrabi juices with antioxidant activity on oxidative stress in human dermal fibroblasts Dan‐Bi Kim1, Ji‐Won Oh1, Gi‐Hae Shin1, Young‐Hyun Kim1, Jong Seok Lee1, In‐Jae Park2 and Ju Hyun Cho2, Ok‐Hwan Lee1,* 1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200‐701, Korea 2Hurum Central Research Institute, Ochang 363‐880, Korea This study was to investigate the antioxidant activity of green kohlrabi juice (GKJ) and purple kohlrabi juice (PKJ) using different in vitro methods. GKJ exhibited the higher total phenol content (153.74 GAE mg/mL), and antioxidant activities than that of PKJ. However, PKJ exhibited the higher ORAC value (664.32 μM TE/mL) than that of GKJ. Human dermal fibroblasts were co‐incubated with various concentrations (10~25 μg/mL) of GKJ and PKJ for 24 h, and then exposed to 1 mM hydrogen peroxide for 3 h. Hydrogen peroxide treatment significantly decreased cell viability (p&lt;0.05), whereas, GKJ and PKJ protected against hydrogen peroxide‐induced cell death in a dose‐dependent manner. Also, pre‐treatment with GKJ and PKJ suppressed intracellular reactive oxygen species (ROS) production in hydrogen peroxide‐treated human dermal fibroblasts. These results demonstrate that both kohlrabi juices have strong antioxidative activity, which could be a good candidate for natural antioxidant and anti‐aging agents.

Antioxidant Properties of Cerium Oxide Nanocrystals as a Function of Nanocrystal Diameter and Surface Coating

This work examines the effect of nanocrystal diameter and surface coating on the reactivity of cerium oxide nanocrystals with H2O2 both in chemical solutions and in cells. Monodisperse nanocrystals were formed in organic solvents from the decomposition of cerium precursors, and subsequently phase transferred into water using amphiphiles as nanoparticle coatings. Quantitative analysis of the antioxidant capacity of CeO2-x using gas chromatography and a luminol test revealed that 2 mol of H2O2 reacted with every mole of cerium(III), suggesting that the reaction proceeds via a Fenton-type mechanism. Smaller diameter nanocrystals containing more cerium(III) were found to be more reactive toward H2O2. Additionally, the presence of a surface coating did not preclude the reaction between the nanocrystal surface cerium(III) and hydrogen peroxide. Taken together, the most reactive nanoparticles were the smallest (e.g., 3.8 nm diameter) with the thinnest surface coating (e.g., oleic acid). Moreover, a benchmark test of their antioxidant capacity revealed these materials were 9 times more reactive than commercial antioxidants such as Trolox. A unique feature of these antioxidant nanocrystals is that they can be applied multiple times: over weeks, cerium(IV) rich particles slowly return to their starting cerium(III) content. In nearly all cases, the particles remain colloidally stable (e.g., nonaggregated) and could be applied multiple times as antioxidants. These chemical properties were also observed in cell culture, where the materials were able to reduce oxidative stress in human dermal fibroblasts exposed to H2O2 with efficiency comparable to their solution phase reactivity. These data suggest that organic coatings on cerium oxide nanocrystals do not limit the antioxidant behavior of the nanocrystals, and that their redox cycling behavior can be preserved even when stabilized.

Photoprotective Effects of Cycloheterophyllin Isolated from Artocarpus heterophyllus against UVA‐Induced Damage and Oxidative Stress in Human Dermal Fibroblasts

Ultraviolet (UV) radiation is considered a cause of skin aging and may result in pathological changes in the skin. Particularly, ultraviolet A (UVA), a long known aging ray and a significant source of oxidative stress, plays a major role in the photoaging of human skin. In the past, many studies have documented that skin cells produce reactive oxygen species after UV exposure. The mitogen‐activated protein kinase (MAPK) pathway would be activated, resulting in phosphorylation of JNK, ERK, and p38 kinase in human skin cells. Previous studies also demonstrated that cycloheterophyllin, a prenylflavone isolated from the root bark of Artocarpus Heterophyllus, has an antioxidant effect and can effectively scavenge free radicals. Although cycloheterophyllin has been studied, the molecular mechanisms involved in the protection of human skin cells from photoaging have not been investigated. Therefore, we used human dermal fibroblasts to investigate protective effect of cycloheterophyllin on UV‐induced damage. We found that cycloheterophyllin significantly increased cell viability and attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce H2O2 generation. In the in vivo studies, we found that the topical application of cycloheterophyllin before UVA irradiation significantly decreased the value of trans‐epidermal water loss (TEWL), erythema, corneometer and the speed of blood flow. These results indicate that photoprotective effects of cycloheterophyllin inhibited UVA‐induced oxidative stress and damage, and may be beneficial in the prevention of skin photoaging.

Ethanol Extract of Peanut Sprout Induces Nrf2 Activation and Expression of Antioxidant and Detoxifying Enzymes in Human Dermal Fibroblasts: Implication for its Protection Against UVB‐irradiated Oxidative Stress

A peanut sprout is known to contain a significant level of resveratrol, which was reported to have beneficial effects in our body due to its antioxidant activities. The purpose of this study was to evaluate the cytoprotective activity of ethanol extract of peanut sprout (EPS) from ultraviolet B (UVB)-induced oxidative stress in human dermal fibroblasts (HDF). EPS was revealed to contain 54.2μgg(-1) of trans-resveratrol. The DCF-DA-positive reactive oxygen species level was increased by 50mJcm(-2) of UVB irradiation (2150±450% of nonirradiated control), which was markedly suppressed by EPS treatment (180±42% of control). Annexin V-positive apoptotic cell death induced by UVB irradiation (16.4±4.5%) was also significantly inhibited by EPS treatment (6.7±2.5%). EPS induced up-regulation and nuclear translocation of Nrf2, a transcription factor for antioxidant and detoxifying enzymes, in HDF as a dose-dependent manner. UVB irradiation up-regulated Nrf2-dependent enzymes of heme oxygenase-1, NAD(P)H:quinine oxidoreductase-1 and glutathione-S-transferase pi, and they were further stimulated by EPS treatment. Taken together, EPS is an efficient cytoprotective agent against UVB-induced oxidative stress by activation of Nrf2 and upregulation of Nrf2-relating antioxidant and detoxifying enzymes in HDF.

Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli

Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H 2O 2 stimulation.

Effect of UVA Fluence Rate on Indicators of Oxidative Stress in Human Dermal Fibroblasts

During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.