Topical hypochlorous acid (HOCl) blocks inflammatory gene expression and tumorigenic progression in UV-exposed SKH-1 high risk mouse skin

Jana Jandova ^a,b , Jeremy Snell ^a,b , Anh Hua ^a,b , Sally Dickinson ^b , Jocelyn Fimbres ^a,b , Georg T. Wondrak ^a,b,*

^aDepartment of Pharmacology and Toxicology, College of Pharmacy and UA Cancer Center, University of Arizona, Tucson, AZ, USA

^bUA Cancer Center, University of Arizona, Tucson, AZ, USA

ABSTRACT

Hypochlorous acid (HOCl) is the active oxidizing principle underlying drinking water disinfection, also delivered by numerous skin disinfectants and released by standard swimming pool chemicals used on a global scale, a topic of particular relevance in the context of the ongoing COVID-19 pandemic. However, the cutaneous consequences of human exposure to HOCl remain largely unknown, posing a major public health concern. Here, for the first time, we have profiled the HOCl-induced stress response in reconstructed human epidermis and SKH-1 hairless mouse skin. In addition, we have investigated the molecular consequences of solar simulated ultraviolet (UV) radiation and HOCl combinations, a procedure mimicking co-exposure experienced for example by recreational swimmers exposed to both HOCl (pool disinfectant) and UV (solar radiation). First, gene expression elicited by acute topical HOCl exposure was profiled in organotypic human reconstructed epidermis. Next, co-exposure studies (combining topical HOCl and UV) performed in SKH-1 hairless mouse skin revealed that the HOCl-induced cutaneous stress response blocks redox and inflammatory gene expression elicited by subsequent acute UV exposure (Nos2, Ptgs2, Hmox1, Srxn1), a finding consistent with emerging clinical evidence in support of a therapeutic role of topical HOCl formulations for the suppression of inflammatory skin conditions (e.g. atopic dermatitis, psoriasis). Likewise, in AP-1 transgenic SKH-1 luciferase-reporter mice, topical HOCl suppressed UV-induced inflammatory signaling assessed by bioluminescent imaging and gene expression analysis. In the SKH-1 high-risk mouse model of UV-induced human keratinocytic skin cancer, topical HOCl blocked tumorigenic progression and inflammatory gene expression (Ptgs2, Il19, Tlr4), confirmed by immunohisto-chemical analysis including 3-chloro-tyrosine-epitopes. These data illuminate the molecular consequences of HOCl-exposure in cutaneous organotypic and murine models assessing inflammatory gene expression and modulation of UV-induced carcinogenesis. If translatable to human skin these observations provide novel insights on molecular consequences of chlorination stress relevant to environmental exposure and therapeutic.

Keywords:

Pathway-focused gene expression profiling Hypochlorous acid Skin cancer Solar UV radiation Inflammation Oxidative stress

Antimicrobial Hypochlorous Wound Irrigation Solutions Demonstrate Lower Anti-biofilm Efficacy Against Bacterial Biofilm in a Complex in-vitro Human Plasma Biofilm Model (hpBIOM) Than Common Wound Antimicrobials

Julian-Dario Rembe^{1 *†} , Lioba Huelsboemer^2† , Isabell Plattfaut² , Manuela Besser² and Ewa K. Stuermer³

¹Department of Vascular and Endovascular Surgery, University Hospital Düsseldorf, Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany, ²Chair for Translational Wound Research, Centre for Biomedical Education and Research, Witten/Herdecke University, Witten, Germany, ³ Department of Vascular Medicine, University Heart and Vascular Center Hamburg, Translational Wound Research, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany

Biofilms pose a relevant factor for wound healing impairment in chronic wounds. With 78% of all chronic wounds being affected by biofilms, research in this area is of high priority, especially since data for evidence-based selection of appropriate antimicrobials and antiseptics is scarce. Therefore, the objective of this study was to evaluate the anti-biofilm efficacy of commercially available hypochlorous wound irrigation solutions compared to established antimicrobials. Using an innovative complex in-vitro human plasma biofilm model (hpBIOM), quantitative reduction of Pseudomonas aeruginosa, Staphylococcus aureus, and Methicillin-resistant S. aureus (MRSA) biofilms by three hypochlorous irrigation solutions [two <0.08% and one 0.2% sodium hypochlorite (NaClO)] was compared to a 0.04% polyhexanide (PHMB) irrigation solution and 0.1% octenidine-dihydrochloride/phenoxyethanol (OCT/PE). Efficacy was compared to a non-challenged planktonic approach, as well as with increased substance volume over a prolonged exposure (up to 72 h). Qualitative visualization of biofilms was performed by scanning electron microscopy (SEM). Both reference agents (OCT/PE and PHMB) induced significant biofilm reductions within 72 h, whereby high volume OCT/PE even managed complete eradication of P. aeruginosa and MRSA biofilms after 72 h. The tested hypochlorous wound irrigation solutions achieved no relevant penetration and eradication of biofilms despite increased volume and exposure. Only 0.2% NaClO managed a low reduction under prolonged exposure. The results demonstrate that low-dosed hypochlorous wound irrigation solutions are significantly less effective than PHMB-based irrigation solution and OCT/PE, thus unsuitable for biofilm eradication on their own. The used complex hpBIOM thereby mimics the highly challenging clinical wound micro-environment, providing a more profound base for future clinical translation.

Keywords: wound infection, biofilm, antimicrobial, antiseptic, hypochlorous acid, human plasma biofilm model, wound irrigation, chronic wound

Antiseptic Effects and Biosafety of a Controlled-Flow Electrolyzed Acid Solution Involve Electrochemical Properties, Rather than Free Radical Presence

Alejandro Cabrera-Wrooman ^1,†, Silvestre Ortega-Peña ^1,† , Rosa M. Salgado ¹ , Belinda Sandoval-Cuevas ² and Edgar Krötzsch ^1,*

¹Laboratory of Connective Tissue, Centro Nacional de Investigación y Atención de Quemados, Instituto Nacional de Rehabilitación “Luis Guillermo Ibarra Ibarra”, Calzada México-Xochimilco 289, Colonia Arenal de Guadalupe, Tlalpan, Mexico City 14389, Mexico; 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。 (A.C.-W.); 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。 (S.O.-P.); 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。 (R.M.S.)

²Wound Care Clinic, Hospital General Regional Number 2, Instituto Mexicano del Seguro Social, Calzada de las Bombas 117, Coapa, Girasoles I, Tlalpan, Mexico City 14310, Mexico; 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

^*Correspondence: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。; Tel.: +52-1-552106-6140 † These authors contributed equally to this work.

Abstract: Electrolyzed acid solutions produced by different methods have antiseptic properties due to the presence of chlorine and reactive oxygen species. Our aim was to determine whether a controlled-flow electrolyzed acid solution (CFEAS) has the ability to improve wound healing due to its antiseptic and antibiofilm properties. First, we demonstrated in vitro that Gram-negative and Gram-positive bacteria were susceptible to CFEAS, and the effect was partially sustained for 24 h, evidencing antibiofilm activity (p < 0.05, CFEAS-treated vs. controls). The partial cytotoxicity of CFEAS was mainly observed in macrophages after 6 h of treatment; meanwhile, fibroblasts resisted short-lived free radicals (p < 0.05, CFEAS treated vs. controls), perhaps through redox-regulating mechanisms. In addition, we observed that a single 24 h CFEAS treatment of subacute and chronic human wounds diminished the CFU/g of tissue by ten times (p < 0.05, before vs. after) and removed the biofilm that was adhered to the wound, as we observed via histology from transversal sections of biopsies obtained before and after CFEAS treatment. In conclusion, the electrolyzed acid solution, produced by a novel method that involves a controlled flow, preserves the antiseptic and antibiofilm properties observed in other, similar formulas, with the advantage of being safe for eukaryotic cells; meanwhile, the antibiofilm activity is sustained for 24 h, both in vitro and in vivo.

Keywords: biofilm; cytotoxicity; oxidation-reduction; wound

HOCl-producing electrochemical bandage is active in murine polymicrobial wound infection

Derek Fleming,¹ Ibrahim Bozyel,² Christina A. Koscianski,¹ Dilara Ozdemir,² Melissa J. Karau,¹ Luz Cuello,¹ Md Monzurul Islam Anoy,² Suzanne Gelston,² Audrey N. Schuetz,¹ Kerryl E. Greenwood-Quaintance,¹ Jayawant N. Mandrekar,³ Haluk Beyenal,² Robin Patel_^1,4 AUTHOR AFFILIATIONS See affiliation list on p. 13.

ABSTRACT Wound infections, exacerbated by the prevalence of antibiotic-resistant bacterial pathogens, necessitate innovative antimicrobial approaches. Polymicrobial infections, often involving Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), present challenges due to biofilm formation and antibiotic resistance. Hypochlorous acid (HOCl), a potent antimicrobial agent, holds promise as an alternative therapy. An electrochemical bandage (e-bandage) that generates HOCl in situ via precise polarization controlled by a miniaturized potentiostat was evaluated for the treatment of murine wound biofilm infections containing both P. aeruginosa with “difficult-to-treat” resistance and MRSA. Previously, HOCl-producing e-bandage was shown to reduce murine wound biofilms containing P. aeruginosa alone. Here, in 5-mm excisional skin wounds containing 48-h biofilms comprising MRSA and P. aeruginosa combined, polarized e-bandage treatment reduced MRSA by 1.1 log10 CFU/g (P = 0.026) vs non-polarized e-bandage treatment (no HOCl production), and 1.4 log10 CFU/g (0.0015) vs Tegaderm only controls; P. aeruginosa was similarly reduced by 1.6 log10 CFU/g (P = 0.0032) and 1.6 log10 CFU/g (P = 0.0015), respectively. For wounds infected with MRSA alone, polarized e-bandage treatment reduced bacterial load by 1.1 log10 CFU/g (P = 0.0048) and 1.3 log10 CFU/g (P = 0.0048) compared with non-polarized e-bandage and Tegaderm only, respectively. The e-bandage treatment did not negatively impact wound healing or cause tissue toxicity. The addition of systemic antibiotics did not enhance the antimicrobial efficacy of e-bandages. This study provides additional evidence for the HOCl-producing e-bandage as a novel antimicrobial strategy for managing wound infections, including in the context of antibiotic resistance and polymicrobial infections.

IMPORTANCE New approaches are needed to combat the rise of antimicrobial-resistant infections. The HOCl-producing electrochemical bandage (e-bandage) leverages in situ generation of HOCl, a natural biocide, for broad-spectrum killing of wound pathogens. Unlike traditional therapies that may exhibit limited activity against biofilms and antimicrobial-resistant organisms, the e-bandage offers a potent, standalone solution that does not contribute to further resistance or require adjunctive antibiotic ther apy. Here, we show the ability of the e-bandage to address polymicrobial infection by antimicrobial resistant clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa, two commonly isolated, co-infecting wound pathogens. Effectiveness of the HOCl-producing e-bandage in reducing pathogen load while minimizing tissue toxicity and avoiding the need for systemic antibiotics underscores its potential as a tool in managing complex wound infections.

KEYWORDS electrochemical bandage, hypochlorous acid, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, anti-biofilm, in vivo wound infection

Hypochlorous Acid: From Innate Immune Factor and Environmental Toxicant to Chemopreventive Agent Targeting Solar UV-Induced Skin Cancer

Jeremy A. Snell, Jana Jandova and Georg T. Wondrak*

Department of Pharmacology and Toxicology, R.K. Coit College of Pharmacy & UA Cancer Center, University of Arizona, Tucson, AZ, United States

A multitude of extrinsic environmental factors (referred to in their entirety as the ‘skin exposome’) impact structure and function of skin and its corresponding cellular components. The complex (i.e. additive, antagonistic, or synergistic) interactions between multiple extrinsic (exposome) and intrinsic (biological) factors are important determinants of skin health outcomes. Here, we review the role of hypochlorous acid (HOCl) as an emerging component of the skin exposome serving molecular functions as an innate immune factor, environmental toxicant, and topical chemopreventive agent targeting solar UV-induced skin cancer. HOCl [and its corresponding anion (OCl- ; hypochlorite)], a weak halogen-based acid and powerful oxidant, serves two seemingly unrelated molecular roles: (i) as an innate immune factor [acting as a myeloperoxidase (MPO)-derived microbicidal factor] and (ii) as a chemical disinfectant used in freshwater processing on a global scale, both in the context of drinking water safety and recreational freshwater use. Physicochemical properties (including redox potential and photon absorptivity) determine chemical reactivity of HOCl towards select biochemical targets [i.e. proteins (e.g. IKK, GRP78, HSA, Keap1/NRF2), lipids, and nucleic acids], essential to its role in innate immunity, antimicrobial disinfection, and therapeutic anti-inflammatory use. Recent studies have explored the interaction between solar UV and HOCl-related environmental co-exposures identifying a heretofore unrecognized photo-chemopreventive activity of topical HOCl and chlorination stress that blocks tumorigenic inflammatory progression in UV-induced high-risk SKH-1 mouse skin, a finding with potential implications for the prevention of human nonmelanoma skin photocarcinogenesis.

Keywords: hypochlorous acid, chlorination stress, environmental exposure, skin exposome, solar ultraviolet radiation, inflammation, skin cancer

The Immediate and Delayed Post-Debridement Effects on Tissue Bacterial Wound Counts of Hypochlorous Acid Versus Saline Irrigation in Chronic Wounds

John M. Hiebert, MD, FACN, FACS, and Martin C. Robson, MD, FACS, Hon. FRCS, Hon. FRACS

Truman Medical Center, University of Missouri–Kansas City; and Division of Plastic Surgery, University of South Florida, Tampa

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Keywords: ultrasonic debridement, bioburden, irrigation, hypochlorous acid, wound closure Published December 1, 2016

Introduction: Wound debridement is considered essential in chronic wound management. Hypochlorous acid has been shown to be an effective agent in reducing wound bacterial counts in open wounds. Ultrasound-enabled wound debridement is an effective and efficient method of debridement. This study compared ultrasound irrigation with hypochlorous acid versus saline irrigation for wound debridement on pre- and postoperative wounds and determined regrowth of bacteria over 1 week period of time. Finally, the outcome of definitive wound closure of the clinically clean-appearing wounds was

Methods: Seventeen consenting adult patients with chronic open wounds were randomly selected for study. The patients were randomly divided into the hypochlorous acid irrigation or saline irrigation group. All patients provided pre- and postoperative tissue samples for qualitative and quantitative bacteriology. For the time (7 days) between the debridement procedure and the definitive closure procedure, the wounds were dressed with a silver-impregnated dressing and a hydroconductive dressing. Results: Both types of irrigation in the ultrasonic system initially lowered the bacterial counts by 4 to 6 logs. However, by the time of definitive closure, the saline-irrigated wounds had bacterial counts back up to 105 whereas the hypochlorous acid–irrigated wounds remained at 102 or fewer. More than 80% of patients in the saline group had postoperative closure failure compared with 25% of patients in the hypochlorous acid group.

Conclusions: Hypochlorous acid irrigation with ultrasound debridement reduced bacterial growth in chronic open wounds more efficiently than saline alone. Postoperative wound closure outcomes suggest a remarkable reduction in wound complications after wound debridement using hypochlorous acid irrigation with ultrasound versus saline