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EFFECT OF DEPLETED-DEUTERIUM WATER ON NORMAL HUMAN SKIN CELLS IN CULTURE AND 3D SKIN EQUIVALENT
Abstract
BACKGROUND
Deuterium-depleted water (DDW) or light water, the opposite of heavy water, is microbiologically pure distilled water characterized by a D/(D+H) deuterium concentration of 20 - 130, i.e. under natural water content which is within 140 - 150 ppm. Variations occur as depending on the geographical zone and altitude. The amount of deuterium varies from 90 ppm in melted Antarctic ice to 180 ppm in underground water below the Sahara Desert. Although mainstream research focused primarily on the effects of deuterated water in organisms, investigations about DDW is scarce. Recent studies unveiled its important role in the regulation of aging by modulating cell growth and other key biochemical processes.
OBJECTIVE
To further investigate this parameter, we examined for the first time the influence of DDW on cutaneous cells proliferation, differentiation, and extracellular matrix synthesis in monolayer cultures and 3D full-thickness skin equivalent model.
METHODOLOGY
To conduct such research, normal human dermal fibroblasts, keratinocytes cultured in 2D and 3D skin equivalents were exposed to DDW at different concentrations (50, 75, 100 ppm) and compared to 150 ppm deuterium ultrapure water (UPW) as a control. The 3D skin model based on a unique collagen glycosaminoglycan-chitosan porous polymer was cultured in DDW for 35 days. Skin equivalent samples were systematically harvested during the course of culture to evaluate by histology and immunohistology the influence of DDW on skin regeneration.
RESULTS / CONCLUSION
Our results in cell monolayer cultures showed that DDW treatments significantly increased the proliferation of fibroblast without a significant effect on keratinocyte proliferation. DDW also promoted the deposit of laminin 332 by epidermal keratinocyte up to 120% in a dose effect manner. Stimulation of collagen I synthesis after 48 hours was only significantly at 50 ppm. Interestingly, DDW significantly promoted the reconstruction of both epidermal and dermal compartments in the 3D skin equivalent models. Treatments dramatically enhanced keratinocyte differentiation, epidermal thickness and extracellular matrix synthesis.
These results support for the first time the importance of deuterium in skin healing process, and provide an insight into DDW utilization in the formulation of dermatological products. The results also show the uses of the full-thickness skin equivalent engineered with a collagen-glycosaminoglycan-chitosan scaffold as a model for evaluation of parameters involved in skin regeneration.
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Work with sub-natural levels of deuterium (D) in animals has demonstrated an anti-cancer effect of low D-concentration in water. Our objective was to investigate whether deuterium-depleted water (DDW) can overturn reverse manganese (Mn)-induced reduction in life span, using the Caenorhabditis elegans (C. elegans) as a model system. DDW per se had no effect on worm's life span 48 h after treatment; however, it reversed the Mn-induced decrease in C. elegans life span. Mn reduced DAF-16 levels, a transcription factor strongly associated with life-span regulation. Low D-concentration (90 ppm) restored the Mn-induced changes in DAF-16 to levels indistinguishable from controls, suggesting DDW can regulate the DAF-16 pathway. We further show that insulin-like receptor DAF-2 levels were unaltered by Mn exposure, tAKT levels increased, whilst superoxide dismutase (SOD-3) levels were decreased by Mn. DDW (90 ppm) restored the levels of tAKT and superoxide dismutase (SOD) to control values without changing DAF-2 levels. Treatment of Mn exposed worms with DDW (90 ppm) restored life-span, DAF-16 and SOD-3 levels to control levels, strongly suggesting that low D concentrations can protect against Mn toxic effects.
Elastic fibers form a complex network that contributes to the elasticity of connective tissues. Alterations in the elastic fiber network are involved in several disease affecting organs in which compliance of the connective tissue is essential: skin, main vasculature, lung, joints, muscle, and ligament. The aim of our work was to study the deposition, maturation, and organization of elastic fiber components in a dermal equivalent model consisting of collagen-GAG-chitosan seeded with fibroblasts. The influence of keratinocytes was studied in parallel, thus constituting a skin equivalent model. These models were examined by transmission electron microscopy (TEM) and by immunohistochemistry to determine the staining patterns of fibrillin-1 and elastin proteins representative of the microfibrillar framework and of the elastic fibers, respectively. After 2 mo of fibroblast culture in the dermal equivalent, elastin was undetectable, whereas fibrillin-1 staining was weak and microfibrils were infrequently observed by TEM. In the skin equivalent, fibrillin-1 and elastin were detected by immunostaining 15 d after epidermization and TEM revealed the typical structure and organization of the elastic network in the dermis, with elastin deposition on the microfibrillar scaffold. This in vitro skin equivalent model is to our knowledge the first in which elastic fibers have been detected, thus demonstrating the influence of keratinocytes on the maturation and organization of the elastic network.Keywords: dermal equivalent, fibrillin, immunohistochemistry, skin model, transmission electron microscopy
To investigate the in vivo and in vitro inhibitory effects of deuterium-depleted water (DDW) on human lung cancer and the possible mechanisms underlying these effects, we cultured and treated human lung carcinoma cell line A549 and human embryonic lung fibroblasts HLF-1 with various concentrations of DDW from 2 to 72 h. Cellular growth inhibition rates were determined using the 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium-bromide) (MTT) proliferation assay. A549 cells were treated with 50±5 ppm DDW, and the morphology and structure of cells were observed by scanning electron microscopy (SEM). We observed alterations in the cellular skeleton by transmission electron microscopy (TEM) and changes in cell cycle by flow cytometry. Our data showed that DDW significantly inhibited the proliferation of A549 cells at a specific time point, and cells demonstrated the characteristic morphological changes of apoptosis under SEM and TEM. The length of the S phase increased significantly in cells treated with 50 ppm DDW, whereas the G0 to G1 phase and G2 to M phase were decreased. We observed DDW-induced cellular apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragment analyses. In addition, we established a tumor transplantion model by injecting H460 tumor cells into subcutaneous tissue of BALB/c mice treated with DDW for 60 days. We determined the tumor inhibition rate of treated and control groups and found that the tumor weight was significantly decreased and the tumor inhibition rate was approximately 30% in the DDW group. We conclude that DDW is a promising new anticancer agent with potential for future clinical application.
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