Food and Chemical Toxicology 75 (2015) 139–145
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Selenium deficiency sensitizes the skin for UVB-induced oxidative damage and inflammation which involved the activation of p38 MAPK signaling Xiaokang Zhu a, Mingdong Jiang b,*, Erqun Song a, Xiaoji Jiang c, Yang Song a,** a Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China b Department of Radiation Oncology, The Ninth People’s Hospital of Chongqing, Chongqing 400700, China c Department of Rehabilitation and Physiotherapy, No. 324 Hospital of PLA, Chongqing 400020, China
A R T I C L E
I N F O
Article history: Received 26 August 2014 Accepted 18 November 2014 Available online 25 November 2014 Keywords: Selenium deficiency UV radiation Skin Oxidative damage Inflammation p38
A B S T R A C T
Ultraviolet B (UVB) radiation causes oxidative damage and inflammation, and ultimately increases the risk of skin carcinogenesis. Selenium is an essential trace element, previous studies indicated selenium deficiency impairs tissue antioxidant capacity in different experimental models. However, the synergistic effect of selenium deficiency and UVB radiation on skin damage is not clear. In the current study, our data revealed selenium deficiency resulted in further increases of reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS) and phosphorylated H2AX levels, decreases of GSH level and antioxidant enzyme activities in UVB-irradiated mice. Selenium deficiency also exacerbated UVBinduced cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 mRNA expressions. Mechanism studies indicated that UVBinduced p38 signaling was further elevated in the skin of mice maintained with selenium deficiency diet, compared with those maintained with selenium adequate diet. Our investigation suggested that selenium deficiency diet weakens the antioxidant capacity of UVB-irradiated mice skin, which sensitizes to UV radiation-induced oxidative damage and inflammation. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The ozone layer has been usually considered as an important shield to earth, which protects plants and animals on the ground against ultraviolet (UV) radiation. However, human-made chemicals are destroying this shield by depletion of stratospheric ozone. Accumulating evidence demonstrated that UV radiation causes organism damage, including the inhibition of cell growth, inflammation, immunosuppression and cancer (Sime and Reeve, 2004). In particular, UVB (a wavelength range of 280–320 nm) causes acute
Abbreviations: CAT, catalase; COX-2, cyclooxygenase-2; DCFH-DA, 2′, 7′dichlorodihydrofluorescein diacetate; ERK, extracellular signal regulated kinase; GPx, glutathione peroxidase; GSH, glutathione; GST, glutathione S-transferase; IL-1β, interleukin-1beta; iNOS, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TNF-α, tumor necrosis factor-alpha; UV, ultraviolet. * Corresponding author. Department of Radiation Oncology, The Ninth People’s Hospital of Chongqing, Chongqing 400700, China. Tel.: +86 13908375127; fax: +86 23 68251225. E-mail address: [email protected] (M. Jiang). ** Corresponding author. College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China. Tel.: +86 23 68251503; fax: +86 23 68251225. E-mail address: [email protected] (Y. Song). http://dx.doi.org/10.1016/j.fct.2014.11.017 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
skin damages including erythema and the up-regulation of proinflammatory mediators (Tsoyi et al., 2008). People who spend more time outdoors in physical, recreational and social activities are associated with higher chances of having skin cancer (Astell-Burt et al., 2014). New diagnosed skin cancer cases are more than a million in the United States each year, which is the most prevalent form of cancer (Siegel et al., 2013). A mechanism study indicated that UVB radiation induces damage directly and indirectly via the generation of reactive oxygen species (ROS) (Afanas’ev, 2010). Selenium is an essential trace element, which has often been regarded as an antioxidant against ROS attack (Battin and Brumaghim, 2009). Selenium constitutes selenium-containing proteins, including selenoenzymes such as glutathione peroxidase (GPx), selenoprotein-P, and thioredoxin reductase, in the form of selenocysteine. There are at least 25 selenoproteins that have been identified in human genome, and half of them encode enzymes with antioxidant activities (Kryukov et al., 2003). Since selenium is an essential component for the representative selenoprotein GPx, researchers suggested that the saturation of GPx activity in the platelets (or plasma) is considered selenium repletion (Rayman, 2000). The amount of selenium in drinking water is not significant for most people, therefore, the major sources of selenium intake are meat, poultry, fish, and eggs (Fairweather-Tait et al., 2011). On the other
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hand, the amount of selenium in a plant-based food largely depends on the amount of selenium in the soil, and how the plant takes up selenium (Rayman, 2008). Epidemiological data indicated that some population in suboptimal selenium areas, such as China, Europe and New Zealand, are at risk of selenium deficiency because the average intake of selenium is insufficient to meet the daily requirement (Rayman, 2012; Reszka et al., 2012; Xia et al., 2005). Several groups of healthy individuals, including breast-fed neonates, pregnant women and elderly people, may be especially prone to selenium deficiency (Bellinger et al., 2009). Unfortunately, the addition of selenium to oral, enteral and parenteral infant formulas for those selenium deficient populations is not a routine practice in all countries. Previous study indicated that selenium supplementation reduced UV-induced micronucleus frequency through the prevention or repair of DNA damage (Baliga et al., 2007). However, the effect of dietary selenium deficiency on UV-induced mice skin damage remains unclear. In this study, we reported that selenium deficiency sensitizes UVB radiation-induced oxidative damage and inflammation.
2.6. Determination of lipid peroxidation Lipid peroxidation was detected by monitoring the fluorescence of TBARS at 532 nm (F7000, HITACHI). TBARS concentrations of the samples were calculated using the extinction co-efficient of 156,000 M−1 cm−1. 2.7. Assays for antioxidant enzymes The levels of SOD, CAT, GST and GPx activities were measured following previous study (Xu et al., 2014). 2.8. Western blot analysis and densitometry Proteins (25–50 μg) were electrophoresed on 10% SDS-PAGE, transferred onto a nitrocellulose membrane. After blocking non-specific binding sites for 3 h with blocking buffer (5% dried skim milk, 1% Tween 20 in 10 mM TBS), membranes were incubated overnight with primary antibodies. The blot was washed twice and incubated again with HRP-conjugated secondary antibody. Target proteins were developed with DAB and images were visualized with Infinity-3026 (Vilber Lourmat, France). Representative images were presented. 2.9. RNA extraction and real-time PCR analysis
2. Materials and methods
Diagnostic kits used for superoxide dismutase (SOD), determination of catalase (CAT), glutathione S-transferase (GST), GPx and thiobarbituric acid reactive substances (TBARS) content were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). 2-nitro-5-thiobenzoic acid and 2′, 7′dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Rabbit γ-H2AX, c-jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), p38 and β-actin polyclonal primary antibodies, goat anti-rabbit IgG-HRP-conjugated secondary antibody and total protein extraction kit were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals used were of highest commercial grade.
RNA was isolated using an innuPREP Micro RNA Kit according to the manufacturer’s instruction and treated with RNase-free DNase (Ambion, Austin, TX, USA) to remove DNA contamination. The concentration and quality of the samples were verified on a spectrophotometer (UV-2450, SHIMADZU) by determining the ratio A260/A280 nm and all samples had ratios above 1.8. RNA was reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Gibco/BRL). Quantitative realtime PCR was performed with SYBR green PCR reagent using a LightCycler 480 (Roche, Basel, Switzerland) and SYBR green detection system (Bio-Rad Laboratories) with 0.5 μg of cDNA. Primers were designed as shown in Table 1. After denaturation at 95 °C for 2 min followed a 45-cycle amplification at 95 °C for 10 s, annealing at 60 °C for 30 s. Relative quantification was calculated using the comparative Ct method (2−ΔΔCT). Relative mRNA expressions were calibrated using expression of GAPDH gene. SAD group was normalized to 100%. Statistical analyses were carried out for six replicates.
2.2. Animals
2.10. Statistical analysis
All experiments were approved by the Animal Care Committee of Southwest University. Forty-eight female BALB/c hairless mice (4 weeks old, 22–24 g) were purchased from Third Military Medical University (Chongqing, China). Animals were maintained in specific pathogen free laboratory under standard conditions of humidity (50 ± 5%), temperature (25 ± 2 °C) in a 12 h light/12 h dark cycle. They had free access to water, and were acclimatized for at least one week prior to use. Mice were randomly divided into four groups (12 in each group). The first group was used as control and maintained with Se-adequate diet which contains 0.25 mg/kg Se, SAD group. The second group was maintained with Se-deficient diet, which contains 0.01 mg/ kg Se, SDD group. The third group was irradiated and maintained with Se-adequate diet, UVB+SAD group. The fourth group was irradiated and maintained with Sedeficient diet, UVB+SDD group. It is important to point out that there has no concerted criterion on “adequate” selenium supplementation, therefore, the design of selenium concentration on diets was based on previous publications (Barrett et al., 2013; Wei et al., 2014).
Data were presented as the means ± S.D. Statistical significance was determined by one-way analysis of variance (ANOVA) using SPSS 18.0 software and p < 0.05 was considered to be significant.
2.1. Materials
2.3. UVB irradiation and sample collection After fed with Se-adequate or Se-deficient diet for 4 continuous weeks, mice in the third and fourth groups were irradiated three times per week for 4 weeks. The UV irradiation source was a Philips TL/12RS 40W lamp (Medical-Holand) which emits mainly in the range of 270–320 nm, resulting in an irradiation of 0.25 J/cm2 for each treatment. Mice were exposed to UV irradiation with a distance of ~20 cm. After the end of experiment, mice were deep anesthetized with ether and sacrificed with minimum suffering. Skin samples were collected for further analysis. Dorsal skins were shaved with shavers for 2.5 × 3 cm2.
3. Results 3.1. Selenium deficiency enhanced UVB-induced oxidative stress and damage In order to determine the effect of selenium deficiency on UVBinduced oxidative stress, we measured ROS level in skin samples. In UVB-treated mice skin, the levels of ROS were significantly increased (p < 0.01, UVB+SAD vs. SAD group). However, ROS level was further increased in those mice that received irradiation and maintained with Se-deficient diet (p < 0.05, UVB+SDD vs. UVB+SAD group), Fig. 1A. Interestingly, there was no significant difference on ROS level between the SAD group and SDD group. UVB radiation decreased
Table 1 The real-time PCR primer sequences. Gene
Sequences
COX-2
Forward primer, 5′-CCA GAT GCT ATC TTT GGG GAG AC-3′ Reverse primer, 5′-CTT GCA TTG ATG GTG GCT G-3′ Forward primer, 5′-ACA ACA GGA ACC TAC CAG CTC A-3′ Reverse primer, 5′-GAT GTT GTA GCG CTG TGT GTC A-3′ Forward primer, 5′-CCA GAC CCT CAC ACT CAG AT-3′ Reverse primer, 5′-AAC ACC CAT TCC CTT CAC AG-3′ Forward primer, 5′-ATG GCA ACT GTT CCT GAA CTC AAC T-3′ Reverse primer, 5′-CAG GAC AGG TAT AGA TTC TTT CCT TT-3′ Forward primer, 5′-GCT ATG AAG TTC CTC TCT GC-3′ Reverse primer, 5′-CTA GGT TTG CCG AGT AGA TC-3′ Forward primer, 5′-ACC ACA GTC CAT GCC ATC AC-3′ Reverse primer, 5′-TCC ACC ACC CTG TTG CTG TA-3′
2.4. Measurement of ROS level iNOS Intracellular ROS production was measured by DCFH-DA. The fluorescence was recorded with an excitation wavelength of 488 nm and an emission wavelength of 526 nm (F7000, HITACHI). The fluorescence of SAD group was normalized to 100%.
TNF-α IL-1β
2.5. Determination of reduced GSH content Reduced GSH level was measured by monitoring the absorbance of 2-nitro-5thiobenzoic acid (at 405 nm) using a UV–Vis spectrophotometer (UV-2450, SHIMADZU) (Liu et al., 2012).
IL-6 GAPDH
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Fig. 1. Selenium deficiency diet sensitizes UVB-induced oxidative damage in mice skin. (A) ROS, (B) GSH and (C) TBARS. Values are expressed as means ± S.D. of 12 animals in each group. Statistical difference of p < 0.05 was considered significant.
cellular GSH level by 36% in Se-adequate mice (p < 0.001, UVB+SAD vs. SAD group). Consistently, we observed a further decrease in GSH level in Se-deficient mice (p < 0.05, UVB+SDD vs. UVB+SAD group), Fig. 1B. We were surprised that significant difference has also been found in SDD and SAD groups (p < 0.001). In skin sample obtained UVB exposure, lipid peroxidation was significantly elevated as the evidence of TBARS formation (p < 0.001, UVB+SAD vs. SAD group), Fig. 1C. Mice maintained on Se-deficient diet and also received UVBirradiation (UVB+SDD group) have marked increased TBARS level when compared to UVB+SAD group. 3.2. Selenium deficiency enhanced UVB-induced loss of antioxidant enzyme activities To study the synergistic effect of selenium-deficiency and UVB exposure on antioxidant enzyme defense system, the activities of SOD, CAT, GST and GPx were tested. As shown in Fig. 2, SOD, CAT, GST and GPx activities were significantly decreased in UVBirradiated mice by 33% (p < 0.001), 31% (p < 0.01), 24% (p < 0.01) and 21% (p < 0.01), respectively. In addition, mice irradiated and maintained on Se-deficient diet have even lower antioxidant enzyme activities in skin. Interestingly, we found that CAT and GPx activities dramatically dropped in SDD group when compared to SAD group, p < 0.01 and p < 0.05 respectively. 3.3. Selenium deficiency enhanced UVB-induced phosphorylation of H2AX Under the situation of DNA double-strand breaks, H2AX becomes phosphorylated on serine 139 (called γ-H2AX). Therefore, the expression of phosphorylated H2AX has often been used as a biomarker of DNA damage. As shown in Fig. 3, a significant increase in γ-H2AX level was observed in UVB+SAD group. Moreover, UVB+SDD group showed the highest γ-H2AX level. 3.4. Selenium deficiency enhanced UVB-induced COX-2, iNOS and proinflammatory cytokines mRNA gene expressions mRNA levels of COX-2 and iNOS were measured by real-time PCR. Our result indicated that exposure of skin to UVB resulted in elevated mRNA expression of COX-2 (~8 fold) and iNOS (~14 fold). In the skin of mice fed with selenium deficient diet, mRNA levels of COX-2 (~18 fold compared with SAD group) and iNOS (~18 fold com-
pared with SAD group) were further elevated after UVB-exposure, Fig. 4A and B. TNF-α, IL-1β and IL-6 are the representative pro-inflammatory cytokines, which are associated with UVB-induced inflammatory responses. In the present study, we detected the mRNA gene expressions of TNF-α, IL-1β and IL-6, Fig. 4C–E. After UVB radiation, there were significant increases of TNF-α (~15 fold), IL-1β (~14 fold) and IL-6 (~13 fold) mRNA expression, compared with SAD group (p < 0.001). Interestingly, further increases of TNF-α (~20 fold compared with SAD group), IL-1β (~19 fold compared with SAD group) and IL-6 (~28 fold compared with SAD group) mRNA expression were found in UVB+SDD groups, respectively. Also, we found that IL-6 mRNA expression increased in SDD group when compare to SAD group, p < 0.001. 3.5. Selenium deficiency enhanced UVB-induced mitogen-activated protein kinases (MAPK) signaling Previous studies have shown that UVB-radiation induces the phosphorylation of ERK, JNK, and p38 MAPK, which implicated skin carcinogenesis (Vayalil et al., 2003). In order to investigate the synergistic effect of selenium deficiency and UVB-radiation, we determined their effect on MAPK phosphorylation. The data showed that phosphorylations of JNK and p38 MAPK, but not ERK were significantly increased with UVB radiation, Fig. 5. It is interesting to notice that phosphorylation of p38 MAPK was further elevated in UVB+SDD group. 4. Discussion Exposure of UVB is a well-known etiologic factor for the development of nonmelanoma skin cancers (Chen et al., 2013; Khan et al., 2012; Matsumura and Ananthaswamy, 2004). To overcome this issue, some novel agents have been developed for preventing UVBinduced skin damage (Pratheeshkumar et al., 2014). It is widely acknowledged that taking supplementations, such as antioxidants, are effective in protecting against UVB-induced damage. However, there is little information regarding how unhealthy lifestyle, especially diet, affects UVB-induced skin damage. Previous studies showed that administration of a high-fat diet to mice exacerbates their response to short-term UVB-induced inflammation (Meeran et al., 2009) and malignant progression of papillomas to carcinoma (Vaid et al., 2014). Obesity also increases the risk of UVinduced oxidative stress and activation of MAPK and NF-κB signaling
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Fig. 2. Selenium deficiency diet sensitizes UVB-induced loss of antioxidant enzyme activities in mice skin. (A) SOD, (B) CAT, (C) GST and (D) GPx. Values are expressed as means ± S.D. of 12 animals in each group. Statistical difference of p < 0.05 was considered significant.
(Katiyar and Meeran, 2007). To the best of our knowledge, the consequence of taking selenium deficiency diet toward UVB exposure has not been investigated. In the present study, we found that those mice maintained with selenium deficiency diet were more vulnerable to UVB exposure, which were significantly associated with oxidative stress, DNA damage and inflammation. Recent work showed direct evidence that selenium deficiency decreased most of the selenoprotein mRNA expressions in layer chicken liver (Liu et al., 2014). Previous study also found that UVinduced micronuclei formation was higher in genetically modified mice which express reduced selenoprotein levels, compared with wild control (Baliga et al., 2008). Another work reported that selenium deficiency potentiates methamphetamine-induced dopaminergic toxicity by reducing tyrosine hydroxylase-like immunoreactivity, dopamine and dopamine metabolites (Kim et al., 2000). Sanchez et al. showed that selenium deficiency exacer-
Fig. 3. Selenium deficiency diet sensitizes UVB-induced phosphorylation of H2AX. The protein expressions of γ-H2AX were determined in skin tissue samples using western blot analysis, as described in the Materials and methods section. Equal loading of protein samples was confirmed using β-actin.
bates 3,4-methylenedioxymethamphetamine-induced dopaminergic toxicity through impairing the cellular antioxidant status of the mouse brain (Sanchez et al., 2003). Giray and Hincal reported that fenvalerate-exposure induced prooxidant/antioxidant imbalance was aggravated in selenium deficiency status (Giray and Hincal, 2011). Wei et al. demonstrated that lipopolysaccharide-induced inflammatory response was exacerbated in mouse mastitis models (Wei et al., 2014). Taken together, these reports fully illustrated that selenium deficiency experimental models encountered more severe oxidative stress and inflammatory response than those in selenium adequate group. Extensive studies indicated the benefit effect of selenium supplementation, e.g., our recent study showed that selenium supplementation shows protective effects against patulin-induced brain damage in mice (Song et al., 2014). However, some other studies have shown inconsistent associations between selenium intake and health effects. For example, a randomized trial indicated that selenium supplementation is ineffective at preventing basal cell carcinoma and that it increases the risk of squamous cell carcinoma and total nonmelanoma skin cancer (Duffield-Lillico et al., 2003). Another study demonstrated that selenium supplementation did not benefit men with low selenium status but increased the risk of high-grade prostate cancer among men with high selenium status (Kristal et al., 2014). Moreover, several review articles concluded that there has been no convincing evidence regarding the benefit effect of selenium supplements on preventing cancer (Vinceti et al., 2014), Hashimoto’s thyroiditis (van Zuuren et al., 2013) or cardiovascular disease (Rees et al., 2013). Since UVB-radiation induces oxidative damage through increased ROS and lipid
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Fig. 4. Selenium deficiency diet sensitizes mRNA expression of (A) COX-2, (B) iNOS, (C) TNF-α, (D) IL-1β and (E) IL-6 in UVB-irradiated mice skin. The mRNA expressions were determined using real-time PCR as described in the Materials and methods section and presented after normalization to GAPDH using the Ct method. Data were presented as the means ± S.D. of 12 mice. Statistical difference of p < 0.05 was considered significant.
peroxidation (Pratheeshkumar et al., 2014), we start with the examination of the effect of selenium deficiency on UVB-induced oxidative status imbalance. Our result demonstrated that mice maintained with selenium deficiency diet have lower antioxidant capacity compared with those supplied with selenium adequate diet. In turn, these changes build up the status of oxidative stress. Interestingly,
Fig. 5. Selenium deficienct diet sensitizes UVB-induced MAPK signaling. The protein expressions of MAPK were determined in skin tissue samples using western blot analysis, as described in the Materials and methods section. Equal loading of protein samples was confirmed using β-actin.
our previous study showed that selenium supplementation with diet showed protective role on patulin-induced brain damage in mice via increases in GSH-related enzyme activities and expressions (Song et al., 2014). Our current results, combined with previous data indicated that selenium plays an important role for antioxidant enzyme activities and expressions, on the contrary, selenium deficiency diet results in the further depletion of antioxidant defense capabilities upon UVB radiation. The phosphorylation of histone H2AX at Ser139 was identified as a reliable biomarker of different stages of DNA double strand breaks, including the collision of replication and transcription forks and the DNA repair process (Sedelnikova and Bonner, 2006). Previous study indicated that selenium supplementation stimulates the repair of genotoxic DNA damage (Seo et al., 2002) and attenuates phosphorylation of H2AX (Jerome-Morais et al., 2013). In this study, we showed for the first time that selenium deficiency has synergistic effect on UVB-induced formation of γ-H2AX, which further supported the protective role of selenium. Interestingly, Zhao et al. reported AgNO3 argument UVB-induced generation of γ-H2AX in HaCaT cell line via the increased formation of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts (Zhao et al., 2014). COX-2 is a rate-limiting enzyme involved in the synthesis of prostaglandins from arachidonic acid. Overexpression of COX-2 increases the production of inflammatory cytokines under UVB exposure, which was linked to the pathophysiology of inflammation and cancer (Chapple et al., 2000). The inhibition of COX-2 expression has been shown to suppress the development of skin cancer (Tsai et al., 2012). iNOS is a key enzyme which is generating nitric oxide from the amino acid l-arginine. Overexpressed iNOS has also been detected in tumors
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(Chiang et al., 2005). In parallel with previous studies, our study showed increased COX-2 and iNOS mRNA expressions in UVBexposed mouse skin, which were further elevated with selenium deficiency condition. Increased pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, were associated with the pathophysiology of various skin disorders and cancers (Scott et al., 2003). Our data indicated that mice maintained with selenium deficient diet have the highest levels of these proinflammatory cytokines, which may contribute a risk of tumor promotion and development. MAPK signaling is critical to the transduction of a variety of extracellular stimuli into intracellular events, which control the activities of various downstream transcription factors implicated in tumor promotion. The expressions of COX-2 and IL-6 were shown to be affected by intracellular signaling MAPK, such as JNK, ERK and p38 (Ashida et al., 2003). Although the activation of MAPK signaling plays a central role in the responses of UVB irradiation, the MAPK signaling pathway under selenium deficiency condition has not yet been investigated. Our results demonstrate that UVB-induced phosphorylations of JNK and p38 were sensitized with the status of selenium deficiency, which indicated that selenium deficiency may lead to the progression of UVB-induced carcinogenesis. Although JNK, ERK and p38 all belong to MAPK superfamily, they were activated by different signals. JNK and p38 respond strongly to stress signals, such as TNF, IL-1, ionizing, ultraviolet radiation and chemotherapeutic drugs. The phosphorylations of JNK and p38 MAPK induce important cellular event, like apoptotic cell death. However, ERK activates growth factors and cytokines, and plays a major role in regulating cell growth, survival, and differentiation (Munshi and Ramesh, 2013). Previous studies suggested that treatment with antioxidants prevents UVB-induced phosphorylation of MAPK by inhibiting UVB-induced oxidative stress (Mantena and Katiyar, 2006). Other studies also indicated the association with p38 and selenium status. For example, p38 mRNA and protein expression were induced in peripheral blood mononuclear cells from patients with fluorosis, and antagonized by high selenium (Chen et al., 2010). Consistently, our result suggested that the boost of UVB-induced oxidative stress under selenium deficiency condition increased phosphorylation of JNK and p38 MAPK proteins. However, selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells via the generation of ROS (Rudolf et al., 2008), which indicated that selenium might function as a prooxidant rather than an antioxidant, reviewed by Drake (2006). Furthermore, Maraldi et al. showed that selenium compounds are toxic for a human neuron cell line through ROS/RNS increase, with the evidence of increasing iNOS content, suggesting the presence of a higher NO level (Maraldi et al., 2011). Overall, due to the conflicting results of the relationship between selenium and the risk of cancer from different epidemiologic, clinical and laboratory studies, the health effects of selenium need further investigation (Vinceti et al., 2013). In conclusion, our data suggested that selenium deficient diet promotes UVB-induced skin oxidative damage and inflammation through the increased oxidative stress and the stimulation of inflammatory signaling in the UVB-irradiated mice skin. From these data, we speculate that maintaining with selenium deficient diet may sensitize UVB radiation-induced skin cancer risks. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version.
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Selenium deficiency sensitizes the skin for UVB-induced oxidative damage and inflammation which involved the activation of p38 MAPK signaling Xiaokang Zhu a, Mingdong Jiang b,*, Erqun Song a, Xiaoji Jiang c, Yang Song a,** a Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China b Department of Radiation Oncology, The Ninth People’s Hospital of Chongqing, Chongqing 400700, China c Department of Rehabilitation and Physiotherapy, No. 324 Hospital of PLA, Chongqing 400020, China
A R T I C L E
I N F O
Article history: Received 26 August 2014 Accepted 18 November 2014 Available online 25 November 2014 Keywords: Selenium deficiency UV radiation Skin Oxidative damage Inflammation p38
A B S T R A C T
Ultraviolet B (UVB) radiation causes oxidative damage and inflammation, and ultimately increases the risk of skin carcinogenesis. Selenium is an essential trace element, previous studies indicated selenium deficiency impairs tissue antioxidant capacity in different experimental models. However, the synergistic effect of selenium deficiency and UVB radiation on skin damage is not clear. In the current study, our data revealed selenium deficiency resulted in further increases of reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS) and phosphorylated H2AX levels, decreases of GSH level and antioxidant enzyme activities in UVB-irradiated mice. Selenium deficiency also exacerbated UVBinduced cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 mRNA expressions. Mechanism studies indicated that UVBinduced p38 signaling was further elevated in the skin of mice maintained with selenium deficiency diet, compared with those maintained with selenium adequate diet. Our investigation suggested that selenium deficiency diet weakens the antioxidant capacity of UVB-irradiated mice skin, which sensitizes to UV radiation-induced oxidative damage and inflammation. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The ozone layer has been usually considered as an important shield to earth, which protects plants and animals on the ground against ultraviolet (UV) radiation. However, human-made chemicals are destroying this shield by depletion of stratospheric ozone. Accumulating evidence demonstrated that UV radiation causes organism damage, including the inhibition of cell growth, inflammation, immunosuppression and cancer (Sime and Reeve, 2004). In particular, UVB (a wavelength range of 280–320 nm) causes acute
Abbreviations: CAT, catalase; COX-2, cyclooxygenase-2; DCFH-DA, 2′, 7′dichlorodihydrofluorescein diacetate; ERK, extracellular signal regulated kinase; GPx, glutathione peroxidase; GSH, glutathione; GST, glutathione S-transferase; IL-1β, interleukin-1beta; iNOS, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TNF-α, tumor necrosis factor-alpha; UV, ultraviolet. * Corresponding author. Department of Radiation Oncology, The Ninth People’s Hospital of Chongqing, Chongqing 400700, China. Tel.: +86 13908375127; fax: +86 23 68251225. E-mail address: [email protected] (M. Jiang). ** Corresponding author. College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China. Tel.: +86 23 68251503; fax: +86 23 68251225. E-mail address: [email protected] (Y. Song). http://dx.doi.org/10.1016/j.fct.2014.11.017 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
skin damages including erythema and the up-regulation of proinflammatory mediators (Tsoyi et al., 2008). People who spend more time outdoors in physical, recreational and social activities are associated with higher chances of having skin cancer (Astell-Burt et al., 2014). New diagnosed skin cancer cases are more than a million in the United States each year, which is the most prevalent form of cancer (Siegel et al., 2013). A mechanism study indicated that UVB radiation induces damage directly and indirectly via the generation of reactive oxygen species (ROS) (Afanas’ev, 2010). Selenium is an essential trace element, which has often been regarded as an antioxidant against ROS attack (Battin and Brumaghim, 2009). Selenium constitutes selenium-containing proteins, including selenoenzymes such as glutathione peroxidase (GPx), selenoprotein-P, and thioredoxin reductase, in the form of selenocysteine. There are at least 25 selenoproteins that have been identified in human genome, and half of them encode enzymes with antioxidant activities (Kryukov et al., 2003). Since selenium is an essential component for the representative selenoprotein GPx, researchers suggested that the saturation of GPx activity in the platelets (or plasma) is considered selenium repletion (Rayman, 2000). The amount of selenium in drinking water is not significant for most people, therefore, the major sources of selenium intake are meat, poultry, fish, and eggs (Fairweather-Tait et al., 2011). On the other
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hand, the amount of selenium in a plant-based food largely depends on the amount of selenium in the soil, and how the plant takes up selenium (Rayman, 2008). Epidemiological data indicated that some population in suboptimal selenium areas, such as China, Europe and New Zealand, are at risk of selenium deficiency because the average intake of selenium is insufficient to meet the daily requirement (Rayman, 2012; Reszka et al., 2012; Xia et al., 2005). Several groups of healthy individuals, including breast-fed neonates, pregnant women and elderly people, may be especially prone to selenium deficiency (Bellinger et al., 2009). Unfortunately, the addition of selenium to oral, enteral and parenteral infant formulas for those selenium deficient populations is not a routine practice in all countries. Previous study indicated that selenium supplementation reduced UV-induced micronucleus frequency through the prevention or repair of DNA damage (Baliga et al., 2007). However, the effect of dietary selenium deficiency on UV-induced mice skin damage remains unclear. In this study, we reported that selenium deficiency sensitizes UVB radiation-induced oxidative damage and inflammation.
2.6. Determination of lipid peroxidation Lipid peroxidation was detected by monitoring the fluorescence of TBARS at 532 nm (F7000, HITACHI). TBARS concentrations of the samples were calculated using the extinction co-efficient of 156,000 M−1 cm−1. 2.7. Assays for antioxidant enzymes The levels of SOD, CAT, GST and GPx activities were measured following previous study (Xu et al., 2014). 2.8. Western blot analysis and densitometry Proteins (25–50 μg) were electrophoresed on 10% SDS-PAGE, transferred onto a nitrocellulose membrane. After blocking non-specific binding sites for 3 h with blocking buffer (5% dried skim milk, 1% Tween 20 in 10 mM TBS), membranes were incubated overnight with primary antibodies. The blot was washed twice and incubated again with HRP-conjugated secondary antibody. Target proteins were developed with DAB and images were visualized with Infinity-3026 (Vilber Lourmat, France). Representative images were presented. 2.9. RNA extraction and real-time PCR analysis
2. Materials and methods
Diagnostic kits used for superoxide dismutase (SOD), determination of catalase (CAT), glutathione S-transferase (GST), GPx and thiobarbituric acid reactive substances (TBARS) content were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). 2-nitro-5-thiobenzoic acid and 2′, 7′dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Rabbit γ-H2AX, c-jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), p38 and β-actin polyclonal primary antibodies, goat anti-rabbit IgG-HRP-conjugated secondary antibody and total protein extraction kit were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals used were of highest commercial grade.
RNA was isolated using an innuPREP Micro RNA Kit according to the manufacturer’s instruction and treated with RNase-free DNase (Ambion, Austin, TX, USA) to remove DNA contamination. The concentration and quality of the samples were verified on a spectrophotometer (UV-2450, SHIMADZU) by determining the ratio A260/A280 nm and all samples had ratios above 1.8. RNA was reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Gibco/BRL). Quantitative realtime PCR was performed with SYBR green PCR reagent using a LightCycler 480 (Roche, Basel, Switzerland) and SYBR green detection system (Bio-Rad Laboratories) with 0.5 μg of cDNA. Primers were designed as shown in Table 1. After denaturation at 95 °C for 2 min followed a 45-cycle amplification at 95 °C for 10 s, annealing at 60 °C for 30 s. Relative quantification was calculated using the comparative Ct method (2−ΔΔCT). Relative mRNA expressions were calibrated using expression of GAPDH gene. SAD group was normalized to 100%. Statistical analyses were carried out for six replicates.
2.2. Animals
2.10. Statistical analysis
All experiments were approved by the Animal Care Committee of Southwest University. Forty-eight female BALB/c hairless mice (4 weeks old, 22–24 g) were purchased from Third Military Medical University (Chongqing, China). Animals were maintained in specific pathogen free laboratory under standard conditions of humidity (50 ± 5%), temperature (25 ± 2 °C) in a 12 h light/12 h dark cycle. They had free access to water, and were acclimatized for at least one week prior to use. Mice were randomly divided into four groups (12 in each group). The first group was used as control and maintained with Se-adequate diet which contains 0.25 mg/kg Se, SAD group. The second group was maintained with Se-deficient diet, which contains 0.01 mg/ kg Se, SDD group. The third group was irradiated and maintained with Se-adequate diet, UVB+SAD group. The fourth group was irradiated and maintained with Sedeficient diet, UVB+SDD group. It is important to point out that there has no concerted criterion on “adequate” selenium supplementation, therefore, the design of selenium concentration on diets was based on previous publications (Barrett et al., 2013; Wei et al., 2014).
Data were presented as the means ± S.D. Statistical significance was determined by one-way analysis of variance (ANOVA) using SPSS 18.0 software and p < 0.05 was considered to be significant.
2.1. Materials
2.3. UVB irradiation and sample collection After fed with Se-adequate or Se-deficient diet for 4 continuous weeks, mice in the third and fourth groups were irradiated three times per week for 4 weeks. The UV irradiation source was a Philips TL/12RS 40W lamp (Medical-Holand) which emits mainly in the range of 270–320 nm, resulting in an irradiation of 0.25 J/cm2 for each treatment. Mice were exposed to UV irradiation with a distance of ~20 cm. After the end of experiment, mice were deep anesthetized with ether and sacrificed with minimum suffering. Skin samples were collected for further analysis. Dorsal skins were shaved with shavers for 2.5 × 3 cm2.
3. Results 3.1. Selenium deficiency enhanced UVB-induced oxidative stress and damage In order to determine the effect of selenium deficiency on UVBinduced oxidative stress, we measured ROS level in skin samples. In UVB-treated mice skin, the levels of ROS were significantly increased (p < 0.01, UVB+SAD vs. SAD group). However, ROS level was further increased in those mice that received irradiation and maintained with Se-deficient diet (p < 0.05, UVB+SDD vs. UVB+SAD group), Fig. 1A. Interestingly, there was no significant difference on ROS level between the SAD group and SDD group. UVB radiation decreased
Table 1 The real-time PCR primer sequences. Gene
Sequences
COX-2
Forward primer, 5′-CCA GAT GCT ATC TTT GGG GAG AC-3′ Reverse primer, 5′-CTT GCA TTG ATG GTG GCT G-3′ Forward primer, 5′-ACA ACA GGA ACC TAC CAG CTC A-3′ Reverse primer, 5′-GAT GTT GTA GCG CTG TGT GTC A-3′ Forward primer, 5′-CCA GAC CCT CAC ACT CAG AT-3′ Reverse primer, 5′-AAC ACC CAT TCC CTT CAC AG-3′ Forward primer, 5′-ATG GCA ACT GTT CCT GAA CTC AAC T-3′ Reverse primer, 5′-CAG GAC AGG TAT AGA TTC TTT CCT TT-3′ Forward primer, 5′-GCT ATG AAG TTC CTC TCT GC-3′ Reverse primer, 5′-CTA GGT TTG CCG AGT AGA TC-3′ Forward primer, 5′-ACC ACA GTC CAT GCC ATC AC-3′ Reverse primer, 5′-TCC ACC ACC CTG TTG CTG TA-3′
2.4. Measurement of ROS level iNOS Intracellular ROS production was measured by DCFH-DA. The fluorescence was recorded with an excitation wavelength of 488 nm and an emission wavelength of 526 nm (F7000, HITACHI). The fluorescence of SAD group was normalized to 100%.
TNF-α IL-1β
2.5. Determination of reduced GSH content Reduced GSH level was measured by monitoring the absorbance of 2-nitro-5thiobenzoic acid (at 405 nm) using a UV–Vis spectrophotometer (UV-2450, SHIMADZU) (Liu et al., 2012).
IL-6 GAPDH
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Fig. 1. Selenium deficiency diet sensitizes UVB-induced oxidative damage in mice skin. (A) ROS, (B) GSH and (C) TBARS. Values are expressed as means ± S.D. of 12 animals in each group. Statistical difference of p < 0.05 was considered significant.
cellular GSH level by 36% in Se-adequate mice (p < 0.001, UVB+SAD vs. SAD group). Consistently, we observed a further decrease in GSH level in Se-deficient mice (p < 0.05, UVB+SDD vs. UVB+SAD group), Fig. 1B. We were surprised that significant difference has also been found in SDD and SAD groups (p < 0.001). In skin sample obtained UVB exposure, lipid peroxidation was significantly elevated as the evidence of TBARS formation (p < 0.001, UVB+SAD vs. SAD group), Fig. 1C. Mice maintained on Se-deficient diet and also received UVBirradiation (UVB+SDD group) have marked increased TBARS level when compared to UVB+SAD group. 3.2. Selenium deficiency enhanced UVB-induced loss of antioxidant enzyme activities To study the synergistic effect of selenium-deficiency and UVB exposure on antioxidant enzyme defense system, the activities of SOD, CAT, GST and GPx were tested. As shown in Fig. 2, SOD, CAT, GST and GPx activities were significantly decreased in UVBirradiated mice by 33% (p < 0.001), 31% (p < 0.01), 24% (p < 0.01) and 21% (p < 0.01), respectively. In addition, mice irradiated and maintained on Se-deficient diet have even lower antioxidant enzyme activities in skin. Interestingly, we found that CAT and GPx activities dramatically dropped in SDD group when compared to SAD group, p < 0.01 and p < 0.05 respectively. 3.3. Selenium deficiency enhanced UVB-induced phosphorylation of H2AX Under the situation of DNA double-strand breaks, H2AX becomes phosphorylated on serine 139 (called γ-H2AX). Therefore, the expression of phosphorylated H2AX has often been used as a biomarker of DNA damage. As shown in Fig. 3, a significant increase in γ-H2AX level was observed in UVB+SAD group. Moreover, UVB+SDD group showed the highest γ-H2AX level. 3.4. Selenium deficiency enhanced UVB-induced COX-2, iNOS and proinflammatory cytokines mRNA gene expressions mRNA levels of COX-2 and iNOS were measured by real-time PCR. Our result indicated that exposure of skin to UVB resulted in elevated mRNA expression of COX-2 (~8 fold) and iNOS (~14 fold). In the skin of mice fed with selenium deficient diet, mRNA levels of COX-2 (~18 fold compared with SAD group) and iNOS (~18 fold com-
pared with SAD group) were further elevated after UVB-exposure, Fig. 4A and B. TNF-α, IL-1β and IL-6 are the representative pro-inflammatory cytokines, which are associated with UVB-induced inflammatory responses. In the present study, we detected the mRNA gene expressions of TNF-α, IL-1β and IL-6, Fig. 4C–E. After UVB radiation, there were significant increases of TNF-α (~15 fold), IL-1β (~14 fold) and IL-6 (~13 fold) mRNA expression, compared with SAD group (p < 0.001). Interestingly, further increases of TNF-α (~20 fold compared with SAD group), IL-1β (~19 fold compared with SAD group) and IL-6 (~28 fold compared with SAD group) mRNA expression were found in UVB+SDD groups, respectively. Also, we found that IL-6 mRNA expression increased in SDD group when compare to SAD group, p < 0.001. 3.5. Selenium deficiency enhanced UVB-induced mitogen-activated protein kinases (MAPK) signaling Previous studies have shown that UVB-radiation induces the phosphorylation of ERK, JNK, and p38 MAPK, which implicated skin carcinogenesis (Vayalil et al., 2003). In order to investigate the synergistic effect of selenium deficiency and UVB-radiation, we determined their effect on MAPK phosphorylation. The data showed that phosphorylations of JNK and p38 MAPK, but not ERK were significantly increased with UVB radiation, Fig. 5. It is interesting to notice that phosphorylation of p38 MAPK was further elevated in UVB+SDD group. 4. Discussion Exposure of UVB is a well-known etiologic factor for the development of nonmelanoma skin cancers (Chen et al., 2013; Khan et al., 2012; Matsumura and Ananthaswamy, 2004). To overcome this issue, some novel agents have been developed for preventing UVBinduced skin damage (Pratheeshkumar et al., 2014). It is widely acknowledged that taking supplementations, such as antioxidants, are effective in protecting against UVB-induced damage. However, there is little information regarding how unhealthy lifestyle, especially diet, affects UVB-induced skin damage. Previous studies showed that administration of a high-fat diet to mice exacerbates their response to short-term UVB-induced inflammation (Meeran et al., 2009) and malignant progression of papillomas to carcinoma (Vaid et al., 2014). Obesity also increases the risk of UVinduced oxidative stress and activation of MAPK and NF-κB signaling
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Fig. 2. Selenium deficiency diet sensitizes UVB-induced loss of antioxidant enzyme activities in mice skin. (A) SOD, (B) CAT, (C) GST and (D) GPx. Values are expressed as means ± S.D. of 12 animals in each group. Statistical difference of p < 0.05 was considered significant.
(Katiyar and Meeran, 2007). To the best of our knowledge, the consequence of taking selenium deficiency diet toward UVB exposure has not been investigated. In the present study, we found that those mice maintained with selenium deficiency diet were more vulnerable to UVB exposure, which were significantly associated with oxidative stress, DNA damage and inflammation. Recent work showed direct evidence that selenium deficiency decreased most of the selenoprotein mRNA expressions in layer chicken liver (Liu et al., 2014). Previous study also found that UVinduced micronuclei formation was higher in genetically modified mice which express reduced selenoprotein levels, compared with wild control (Baliga et al., 2008). Another work reported that selenium deficiency potentiates methamphetamine-induced dopaminergic toxicity by reducing tyrosine hydroxylase-like immunoreactivity, dopamine and dopamine metabolites (Kim et al., 2000). Sanchez et al. showed that selenium deficiency exacer-
Fig. 3. Selenium deficiency diet sensitizes UVB-induced phosphorylation of H2AX. The protein expressions of γ-H2AX were determined in skin tissue samples using western blot analysis, as described in the Materials and methods section. Equal loading of protein samples was confirmed using β-actin.
bates 3,4-methylenedioxymethamphetamine-induced dopaminergic toxicity through impairing the cellular antioxidant status of the mouse brain (Sanchez et al., 2003). Giray and Hincal reported that fenvalerate-exposure induced prooxidant/antioxidant imbalance was aggravated in selenium deficiency status (Giray and Hincal, 2011). Wei et al. demonstrated that lipopolysaccharide-induced inflammatory response was exacerbated in mouse mastitis models (Wei et al., 2014). Taken together, these reports fully illustrated that selenium deficiency experimental models encountered more severe oxidative stress and inflammatory response than those in selenium adequate group. Extensive studies indicated the benefit effect of selenium supplementation, e.g., our recent study showed that selenium supplementation shows protective effects against patulin-induced brain damage in mice (Song et al., 2014). However, some other studies have shown inconsistent associations between selenium intake and health effects. For example, a randomized trial indicated that selenium supplementation is ineffective at preventing basal cell carcinoma and that it increases the risk of squamous cell carcinoma and total nonmelanoma skin cancer (Duffield-Lillico et al., 2003). Another study demonstrated that selenium supplementation did not benefit men with low selenium status but increased the risk of high-grade prostate cancer among men with high selenium status (Kristal et al., 2014). Moreover, several review articles concluded that there has been no convincing evidence regarding the benefit effect of selenium supplements on preventing cancer (Vinceti et al., 2014), Hashimoto’s thyroiditis (van Zuuren et al., 2013) or cardiovascular disease (Rees et al., 2013). Since UVB-radiation induces oxidative damage through increased ROS and lipid
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Fig. 4. Selenium deficiency diet sensitizes mRNA expression of (A) COX-2, (B) iNOS, (C) TNF-α, (D) IL-1β and (E) IL-6 in UVB-irradiated mice skin. The mRNA expressions were determined using real-time PCR as described in the Materials and methods section and presented after normalization to GAPDH using the Ct method. Data were presented as the means ± S.D. of 12 mice. Statistical difference of p < 0.05 was considered significant.
peroxidation (Pratheeshkumar et al., 2014), we start with the examination of the effect of selenium deficiency on UVB-induced oxidative status imbalance. Our result demonstrated that mice maintained with selenium deficiency diet have lower antioxidant capacity compared with those supplied with selenium adequate diet. In turn, these changes build up the status of oxidative stress. Interestingly,
Fig. 5. Selenium deficienct diet sensitizes UVB-induced MAPK signaling. The protein expressions of MAPK were determined in skin tissue samples using western blot analysis, as described in the Materials and methods section. Equal loading of protein samples was confirmed using β-actin.
our previous study showed that selenium supplementation with diet showed protective role on patulin-induced brain damage in mice via increases in GSH-related enzyme activities and expressions (Song et al., 2014). Our current results, combined with previous data indicated that selenium plays an important role for antioxidant enzyme activities and expressions, on the contrary, selenium deficiency diet results in the further depletion of antioxidant defense capabilities upon UVB radiation. The phosphorylation of histone H2AX at Ser139 was identified as a reliable biomarker of different stages of DNA double strand breaks, including the collision of replication and transcription forks and the DNA repair process (Sedelnikova and Bonner, 2006). Previous study indicated that selenium supplementation stimulates the repair of genotoxic DNA damage (Seo et al., 2002) and attenuates phosphorylation of H2AX (Jerome-Morais et al., 2013). In this study, we showed for the first time that selenium deficiency has synergistic effect on UVB-induced formation of γ-H2AX, which further supported the protective role of selenium. Interestingly, Zhao et al. reported AgNO3 argument UVB-induced generation of γ-H2AX in HaCaT cell line via the increased formation of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts (Zhao et al., 2014). COX-2 is a rate-limiting enzyme involved in the synthesis of prostaglandins from arachidonic acid. Overexpression of COX-2 increases the production of inflammatory cytokines under UVB exposure, which was linked to the pathophysiology of inflammation and cancer (Chapple et al., 2000). The inhibition of COX-2 expression has been shown to suppress the development of skin cancer (Tsai et al., 2012). iNOS is a key enzyme which is generating nitric oxide from the amino acid l-arginine. Overexpressed iNOS has also been detected in tumors
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(Chiang et al., 2005). In parallel with previous studies, our study showed increased COX-2 and iNOS mRNA expressions in UVBexposed mouse skin, which were further elevated with selenium deficiency condition. Increased pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, were associated with the pathophysiology of various skin disorders and cancers (Scott et al., 2003). Our data indicated that mice maintained with selenium deficient diet have the highest levels of these proinflammatory cytokines, which may contribute a risk of tumor promotion and development. MAPK signaling is critical to the transduction of a variety of extracellular stimuli into intracellular events, which control the activities of various downstream transcription factors implicated in tumor promotion. The expressions of COX-2 and IL-6 were shown to be affected by intracellular signaling MAPK, such as JNK, ERK and p38 (Ashida et al., 2003). Although the activation of MAPK signaling plays a central role in the responses of UVB irradiation, the MAPK signaling pathway under selenium deficiency condition has not yet been investigated. Our results demonstrate that UVB-induced phosphorylations of JNK and p38 were sensitized with the status of selenium deficiency, which indicated that selenium deficiency may lead to the progression of UVB-induced carcinogenesis. Although JNK, ERK and p38 all belong to MAPK superfamily, they were activated by different signals. JNK and p38 respond strongly to stress signals, such as TNF, IL-1, ionizing, ultraviolet radiation and chemotherapeutic drugs. The phosphorylations of JNK and p38 MAPK induce important cellular event, like apoptotic cell death. However, ERK activates growth factors and cytokines, and plays a major role in regulating cell growth, survival, and differentiation (Munshi and Ramesh, 2013). Previous studies suggested that treatment with antioxidants prevents UVB-induced phosphorylation of MAPK by inhibiting UVB-induced oxidative stress (Mantena and Katiyar, 2006). Other studies also indicated the association with p38 and selenium status. For example, p38 mRNA and protein expression were induced in peripheral blood mononuclear cells from patients with fluorosis, and antagonized by high selenium (Chen et al., 2010). Consistently, our result suggested that the boost of UVB-induced oxidative stress under selenium deficiency condition increased phosphorylation of JNK and p38 MAPK proteins. However, selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells via the generation of ROS (Rudolf et al., 2008), which indicated that selenium might function as a prooxidant rather than an antioxidant, reviewed by Drake (2006). Furthermore, Maraldi et al. showed that selenium compounds are toxic for a human neuron cell line through ROS/RNS increase, with the evidence of increasing iNOS content, suggesting the presence of a higher NO level (Maraldi et al., 2011). Overall, due to the conflicting results of the relationship between selenium and the risk of cancer from different epidemiologic, clinical and laboratory studies, the health effects of selenium need further investigation (Vinceti et al., 2013). In conclusion, our data suggested that selenium deficient diet promotes UVB-induced skin oxidative damage and inflammation through the increased oxidative stress and the stimulation of inflammatory signaling in the UVB-irradiated mice skin. From these data, we speculate that maintaining with selenium deficient diet may sensitize UVB radiation-induced skin cancer risks. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version.
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