Water H2O2 Levels as Factor in Swimmers Melanoma

Introduction: This study proposes afoundation which could demonstrate a published H2O2 hypothesis of cancerogenesis. A mechanism explaining the high incidence of melanoma in swimmers is described. Methods: Humanhairs immersed in drops of pure water with resistivity of 18.2 MΩ × cm (million ohms) were in indirect contact with adjacent drops of 35% H2O2. Digital microphotographs and video-recordings were obtained for further analysis. Results: This approach allows visualization of details that could not be analyzed previously.The findings show that H2O2 penetrates hair follicles either at the sites of injury to external structures or at the intact shaft/skin junction. Conclusions: In hairs immersed in pure water mixed with a low concentration of H2O2, sebum and contiguous dermal sheaths blocked exogenous H2O2. Conversely, in injured hair follicles and at the intact shaft/skin junction, H2O2 penetrated the tissue and was subsequently decomposed by catalase. This mechanism is proposed for the high incidence of Swimmers Melanoma.


Introduction
Analytical measurement of the effects of hydrogen peroxide (H 2 O 2 ) on tissues has been difficult [1] . The objectives of the present study are to introduce an experimental method to reduce H 2 O 2 substrate concentration in solutions in contact with human hair follicles; thus mimicking surface fresh and saltwater H 2 O 2 levels. A second objective is to demonstrate the protective role of the external skin layers from penetration of reactive oxygen species (ROS), namely H 2 O 2 .
One factor impeding optical microscopy studies of the effect of H 2 O 2 decomposition on tissue is the rapid decomposition caused by the enzyme catalase. Invariably, multiple layers of gas bubbles rapidly form, obstructing the viewing field ( Figure  1). Reports of "swimmers melanomas" in adults and children, as well as reports of H 2 O 2 formation resulting principally from the excitation of humic substances in fresh and sea water by the ultraviolet (UV) portion of sunlight, have been published [2,3] . These studies raise the question of whether H 2 O 2 present in surface waters is a risk factor for the development of melanoma in swimmers.
Successful attempts to suppress the rate of H 2 O 2 decomposition reaction utilizing activated carbons have been described [4] . The hair follicle has been described as a dynamic miniorgan [5] with independent cell division and differentiation and adjacent sebaceous gland, as well as dermal sheaths [6] .H 2 O 2 vapors are air-bound and used for bio-decontamination through deposition on surfaces via micro-condensation [7] . This manuscript introduces a simple method to use the human hair follicle as sentinel following immersion in pure water to slow down the explosive repetitive decomposition of H 2 O 2 . www.ommegaonline.org

Materials and Methods
Glass slides (25×75×1 mm, #1301) were purchased from Globe Scientific Inc. Certified food grade H 2 O 2 (35%) and bovine liver liquid catalase (purified, thymol-free) was purchased from Sigma-Aldrich (StockC-40, one gram 11000 units). Very pure water with resistivity of 18.2 MΩ × cm (million 112 ohms) supplied by The University of Oklahoma, Health Sciences Center.was used. The equipment included a model OS425-LS non-contact infrared thermometer, Celestron model 44348 digital video microscope, 117 Acurite model 01538CDI remote weather station, Apple MacBook computer, and Apple Inc. iPhoto 8.1.2 application.

Harvesting and mounting of hair samples
Sixteen hairs were plucked from the scalp of the author using tweezers. Care was taken to select hairs with large visible roots. Using a fresh double-edge razor blade, ten of the hair follicles were gently injured after placing on a dry slide. This maneuver produced four partially transected follicles with different follicular injury patterns. The individual hairs (12 intact and 4 injured) were individually placed on the center of clean glass slides then covered by two drops of very pure water with a resistivity of 18.2 MΩ × cm. Two to three drops of 35% H 2 O 2 were delivered via a micropipette to the same slide 4 to 5 mm distant from the pure water drops. Care was taken not to mix the drops. Each preparation was mounted on a digital video-microscope platform and observed for any changes of the hair follicles. Gas bubbles emanating from the hair follicles appeared slowly on the hair tissue. Once the slow bubbling started after approximately 8 ± 3 minutes ( Figure 1A), microphotographs and video-recordings were obtained and the data were stored for subsequent analysis. Ancillary testing demonstrated the gradual transfer of H 2 O 2 into the pure water drops.

Results
All intact follicles showed bubbling activity; it occurred near the shaft/skin boundary in 12 follicles (     Concerning the cause(s) of the observed slow H 2 O 2 decomposition by the hair follicles, it could be theorized that H 2 O 2 molecules gradually penetrated the pure water adjacent to the 35% drops of H 2 O 2 adjacently placed on the same glass slide as shown in Figure 1A.

Discussion
Oxidative stress due to tissue metabolism in hair follicles has been previously demonstrated [11,12] . Hair follicles produce catalase in order to decompose ROS and achieve homeostasis. The present findings were possibly due to the very slow bubbling observed during the endogenous catalase-mediated decomposition of H 2 O 2 that penetrated the hair follicles.
The first thought was that the presence of catalase in the scalp [13] could be the cause of the intense bubbling observed at the shaft/skin junction site. High magnification views showed that the bubbling originated away from the shaft/skin boundary surface. Since the shaft/skin junction area was uninjured, the observations prompted the consideration of whether the shaft/skin junction area in the skin was a point of spontaneous entry for H 2 O 2 .

Sebum/Dermal sheaths as barriers
Observed was that exogenous H 2 O 2 penetrated deep into hair follicles. In seeking a mechanism for this observation, it was noted that when hair follicles were injured, the protective sebum coat and dermal sheaths were compromised ( Figure. 6). This would allow the exogenous H 2 O 2 to penetrate the internal tissue layers where it would be decomposed by the ubiquitous enzyme catalase.
The present study also presents an association of sebum combined with intact dermal sheaths acting as a barrier to ROS external toxicity.

Swimmers melanoma
Two decades ago, it was postulated that the recreational exposure to sunlight did not fully explain the current trends In melanoma Incidence [14] . The authors suggested that the "positive association between a history of swimming and melanoma risk suggests that carcinogenic agents in water, possibly chlorination by products, play a role in melanoma aetiology". Additionally, considering the skin dryness of swimmers, the same authors stated that this is "caused by a combination of the dilution of natural sebum and by the osmotic gradient produced when the body is immersed in water, drawing hydration from the outer skin layers." Another study documented several dermatological problems related to prolonged or repetitive water immersion, including skin denuding of the protective sebum coat [15] . The protective barrier function of the skin against ROS has been identified previously [16] .
The experimental documentation presented in this manuscript supports the view that H 2 O 2 fuels aging, inflammation, cancer metabolism, and metastasis [17] . Furthermore, the present observations provide a basis for testing the hypothesis that ROS reactions are associated with cancerogenesis [18] . We demonstrated that in hair follicles, compromised sebum/dermal sheath layers and uncompromised shaft/skin junction allow H 2 O 2 penetra-tion, resulting in repetitive ROS reactions and possible initiation of diseases. This could include melanoma.

Addendum
The data presented in this manuscript also supports that in fresh or seawater, the penetration of ROS into the submerged hairs occurs at the hair shaft/skin interface, as well as through the injured external structures. The risk for "swimmers melanoma" could be explained as follows: "The formation of hydrogen peroxide results principally from the UV portion of sunlight exciting humic substances in the water and thereby leads to the formation of superoxide ion, which reacts with itself to form H 2 O 2 . Because this production is limited to the depth of UV light penetration, its vertical distribution provides a sensitive tracer for mixing processes" [19] .