A novel role of topical iodine in skin: Activation of the Nrf2 pathway
Introduction
Iodine is a chemical element which has been obtained from seaweeds, fish, and seawater since early 19th century [1], [2]. It is sparingly soluble in water (0.33 g/l at 25 °C) and its solubility can be raised in the presence of iodide ions, such as potassium iodide, to form the tri-iodide ion [1], [3]. In humans, it was used in the treatment of thyroid disorders since it is vital for the biosynthesis of the thyroid hormones, triiodothyronine and thyroxine, which ensure proper metabolism [1], [2], [4]. However, the use of iodine was rapidly extended over time, and is now being used as an active dermal agent in the treatment of inflammatory, immune-mediated and infectious diseases (e.g. psoriasis, eczema, lupus vulgaris, syphilis) [2]. In fact, the use of iodine as a bactericidal, antiseptic agent in wound care has been reported as being effective for more than 150 years [1], [2], [5]. Iodine offers a wide spectrum of antimicrobial activity and is capable of rapidly inhibiting yeasts, molds, protozoa and viruses [1], [6], [7], [8]. Invaded microorganisms are prone to be attacked simultaneously in multiple sites, following iodine treatment, without developing any resistance [1]. The combined activity of iodine on microorganisms' cell wall, membrane and cytoplasm may be detrimental, as iodine binds to proteins, react with C-C bonds in fatty acids and may oxidize disulfide bonds in amino acids (e.g. cysteine, methionine). In addition, it may prevent hydrogen bonding by reacting with N-H groups in arginine, histidine and lysine and preclude hydrogen bonding in nucleic acids (e.g. adenine, cytosine) [1], [7].
Topical iodine application has been reported to provide protection against sulfur-mustard-induced skin lesions, heat-induced and acid-induced skin burns [9], [10] in both haired guinea-pigs and mouse ear swelling models [11]. Surprisingly, the mechanism of action by which iodine provides relief in these cases, as well as for skin immune-mediated and/or inflammatory diseases, is not fully understood and further comprehensive research is required. Although many suggestions have made, including iodine's ability to inhibit apoptosis and/or proteinase activity [10], reduce collagenolytic activity [12], reduce inducible nitric oxide synthase expression [13], depress oxidative-burst effect, and scavenge hydroxyl suppressing the induction of TNF-α and, perhaps, other inflammatory mediators as well [14], additional studies are needed.
Iodine is an oxidizing agent (standard reduction potential at 25 °C, E°=0.536 V) [15] and therefore may affect cellular pathways. Here, we suggest a novel mode of action by which iodine provides skin relief and protects against stress-mediated skin injuries. We hypothesize that iodine is capable of activating the NF-E2-related factor 2 (Nrf2) pathway, due to its electrophilic properties. To the best of our knowledge, it has never been suggested that iodine can activate the Nrf2 pathway and induce phase-II enzymes in skin cells. This is a cytoprotective signaling pathway, which serves as a major intersection for many other signaling pathways that cross-talk with each other and determine cell functionality and fate [16], [17], [18]. Nrf2 is capable of coordinating the cellular response to endogenous as well as exogenous stressors by tight regulation of phase-II protective enzymes [16], [17], [18] which are responsible for the antioxidant response, xenobiotic disposition, inflammatory response, metabolic programming and cell proliferation and survival [19]. The involvement of the Nrf2 pathway in skin is of high importance; playing a role in skin homeostasis and skin renovation and a variety of other skin diseases, e.g. allergic skin inflammation atopic dermatitis, psoriasis, epidermal blistering disease, and vitiligo vulgaris [20]. Pharmacological activation of Nrf2 was demonstrated to be efficient in the prevention of skin carcinogenesis in a variety of animal studies [20]. Moreover, protection against UV-induced cytotoxicity and photo-aging was shown [20].
The classic activation of the Nrf2 pathway is regulated by two cysteine-rich proteins called Keap1 (Kelch-like ECH associated protein1) [21], [22], [23], [24], [25]. Under basal conditions, Nrf2 is anchored to the cytoplasm via the Keap1 actin cytoskeleton binding protein and degraded via the ubiquitin proteasome pathway resulting in Nrf2 half-life of approximately 10–20 min [21], [24], [25], [26], [27]. However, upon oxidative/electrophilic stress conditions, i.e. exogenous or endogenous electrophile or reactive oxygen species, the Keap1 protein is modified, resulting in the suppression of Keap1-mediated proteasomal degradation of Nrf2, thereby causing Nrf2 stabilization with half-life extending to approximately 100–200 min [28], [29], translocation and accumulation in the nucleus [30]. As Nrf2 accumulates in the nucleus, it heterodimerizes with additional proteins (e.g. small Maf proteins), and binds to the antioxidant-response element (ARE), also known as the electrophile-response element (EpRE) in the regulatory sequences of its target genes [16], [31]. As a result, large networks of gene-encoding enzymes and detoxifying proteins, are expressed [32], [33], [34], [35], [36]. In addition, the involvement of many kinases and phosphatases contributes to pathway complications [37], [38], [39], [40]. Additional mechanisms of action were suggested for Nrf2 activation including inhibition of Keap1-Nrf2 interaction by blocking the Keap1 domain [41], methylation/demethylation of CpGs in the promoter regions, acetylation/deacetylation and methylation/demethylation of histones, or targeting of mRNAs by miRNAs [42]. Moreover, certain non-coding RNAs are also capable of influencing Nrf2 [42].
The aim of this study is to investigate whether iodine may obtain its beneficial effects, described above as a dermal protecting agent, at least partially, via activation of the Nrf2 pathway in the skin. In addition, the activation of Nrf2 pathway by iodine was elucidated. Moreover, iodide's (I-) ability to activate the Nrf2 pathway was also reported and discussed.
Section snippets
Materials
All chemicals were of analytical grade and were used as received. Water for preparation of the solutions was purified using a Milli-Q purification system. Iodine was purchased from MERCK Chemicals, Switzerland, potassium iodide was purchased from J.T.Baker Chemicals, USA. Diethylenetriaminepentaacetic acid (DETAPAC) and Tert-Butyl hydroperoxide solution were purchased from Sigma-Aldrich, Israel. NaOH was purchased from FRUTAROM, Israel. Strong iodine solution was prepared by dissolving 5 g of
Skin-cell viability is not affected by iodine and iodide treatments
Keratinocyte viability following 24-h treatment with strong iodine solution (USP) and potassium iodide solution was evaluated by MTT assay. Fig. 1 shows cell viability (%) for treatments ranging from 0 to 750 µM. As can be seen there is no significant difference between the control group (untreated cells), strong iodine solution and potassium iodide, indicating that the concentrations used are nontoxic.
Iodine and iodide demonstrate different potential in activation of Nrf2 pathway in skin
Nrf2 is a redox "master switch" that can turn on the cellular signaling involved in the
Discussion
Activation of the Nrf2 pathway in the skin is a major strategy for maintaining skin redox balance and is highly important for skin redox homeostasis, functionality and repair processes [20]. Moreover, this strategy was proven to be effective under stress conditions, in different skin models and pathologies [20]. The strength of this strategy derived from a few different contributions. First, there is the obvious rationale of utilizing the self-protection mechanisms that skin is equipped with,
Conclusions
This work revealed a novel mode of action by which iodine and iodide operate in human keratinocytes and the skin. We demonstrated that iodine and iodide are capable of activating the Nrf2 pathway, thus providing, protection against UVB-induced damage and a relief under inflamed skin condition. The enhanced potency of potassium iodide vs. strong iodine in activating the system was shown. A possible underlying mechanism of activity for Nrf2 activation was suggested. Further studies are needed to
Conflict of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
Acknowledgments
We thank Dr. Sabine Werner for kindly providing us with Nrf2 knock-out and Nrf2 dominant- negative cell lines and for her generous assistance. Dr. Ron Kohen is affiliated with the Bloom Center of Pharmacy. He is the incumbent of the Richard and Jean Zarbin Chair in Medical Studies at the Hebrew University of Jerusalem. This work was funded by the David and Ines Myers Fund of Cleveland, Ohio, USA; Yissum, the technology transfer company of the Hebrew University (Grant no. 0344809); the Hebrew
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Current address: Institute of Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology (ETH) Zurich, CH 8093 Zurich, Switzerland.