Arene-Ruthenium(II) Complexes with Carbothiamidopyrazoles as a Potential Alternative for Antibiotic Resistance in Human.

To meet the demand for alternatives to commonly used antibiotics, this paper evaluates the antimicrobial potential of arene-ruthenium(II) complexes and their salts, which may be of value in antibacterial treatment. Their antimicrobial activity (MIC, MBC/MFC) was examined in vitro against Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa, Proteus vulgaris and Candida albicans and compared with classic antibiotics used as therapeutics. Selected arene-ruthenium(II) complexes were found to have synergistic effects with oxacillin and vancomycin against staphylococci. Their bactericidal effect was found to be associated with cell lysis and the ability to cut microbial DNA. To confirm the safety of the tested arene-ruthenium(II) complexes in vivo, their cytotoxicity was also investigated against normal human foreskin fibroblasts (HFF-1). In addition, the antioxidant and thus pro-health potential of the compounds, i.e., their nonenzymatic antioxidant capacity (NEAC), was determined by two different methods: ferric-TPTZ complex and DPPH assay.

Human Induced Pluripotent Stem Cell-Based Skin for Assessing Transdermal Drug Permeability and Irritancy.

To establish a system for assessing drug permeation and irritation of the skin, the permeation of benzoic acid and isosorbide dinitrate, which are listed in the Pharmacopoeia, and the chemical irritation were evaluated using skin generated from human induced pluripotent stem cells (iPSCs). Multilayer structures and cellular markers (keratin 14 and 10, which are in basal and suprabasal epidermal layers) were clearly detected in our iPSC-based skin. Transepidermal water loss (TEWL) decreased after iPSC-derived keratinocytes were cultured on collagen gels from human primary fibroblasts. These results indicate that the barrier function was partly increased by formation of the living epidermis. The cumulative amount of benzoic acid and isosorbide dinitrate across human iPSC-based skin gradually increased after an initial lag time. Moreover, the irritancy of various chemicals (non-irritants: ultrapure water, allyl phenoxy-acetate, isopropanol, and hexyl salicylate and irritants: 5% sodium dodecyl sulfate (SDS), heptanal, potassium hydroxide (5% aq.) and cyclamen aldehyde) to iPSC-based skin was almost met the irritation criteria of the Organisation for Economic Co-operation and Development (OECD) guideline. The results of our iPSC-based skin evaluation provide useful basic information for developing an assessment system to predict the permeation and safety of new transdermal drugs in human skin.

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis.

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.

Caviar Extract and Its Constituent DHA Inhibits UVB-Irradiated Skin Aging by Inducing Adiponectin Production.

In this study, caviar (sturgeon eggs) was used to elucidate its roles in adiponectin production and skin anti-aging. Recently, caviar has been largely used not only as a nutritional food, but also in cosmetic products. In particular, it has been reported that docosahexaenoic acid (DHA), as one of the main phospholipid components of caviar extract, induces intracellular lipid accumulation and the expression of adiponectin in adipocytes. Although adipocytes are well known to be associated with the skin dermis by secreting various factors (e.g., adiponectin), the effects of caviar extract and DHA on the skin are not well studied. Here, we demonstrate the effects of caviar extract and DHA on adipocyte differentiation and adiponectin production, resulting in a preventive role in UV-irradiated skin aging. Caviar extract and DHA enhanced adipocyte differentiation and promoted the synthesis of transcription factors controlling adipocyte differentiation and adiponectin. In addition, the mRNA expression levels of matrix metalloproteinase-1 (MMP-1) were decreased in UVB-irradiated Hs68 fibroblasts that were cultured in conditioned medium from caviar extract or DHA-treated differentiated adipocytes. Taken together, these results indicate that caviar extract and DHA induce adipocyte differentiation and adiponectin production, thereby inhibiting UVB-induced premature skin aging via the suppression of MMP-1 production.

Celastrol Alleviates Gamma Irradiation-Induced Damage by Modulating Diverse Inflammatory Mediators.

The present study aimed to explore the possible radioprotective effects of celastrol and relevant molecular mechanisms in an in vitro cell and in vivo mouse models exposed to gamma radiation. Human keratinocytes (HaCaT) and foreskin fibroblast (BJ) cells were exposed to gamma radiation of 20Gy, followed by treatment with celastrol for 24 h. Cell viability, reactive oxygen species (ROS), nitric oxide (NO) and glutathione (GSH) production, lipid peroxidation, DNA damage, inflammatory cytokine levels, and NF-κB pathway activation were examined. The survival rate, levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in blood, and p65 and phospho-p65 expression were also evaluated in mice after exposure to gamma radiation and celastrol treatment. The gamma irradiation of HaCaT cells induced decreased cell viability, but treatment with celastrol significantly blocked this cytotoxicity. Gamma irradiation also increased free radical production (e.g., ROS and NO), decreased the level of GSH, and enhanced oxidative DNA damage and lipid peroxidation in cells, which were effectively reversed by celastrol treatment. Moreover, inflammatory responses induced by gamma irradiation, as demonstrated by increased levels of IL-6, TNF-α, and IL-1ß, were also blocked by celastrol. The increased activity of NF-κB DNA binding following gamma radiation was significantly attenuated after celastrol treatment. In the irradiated mice, treatment with celastrol significantly improved overall survival rate, reduced the excessive inflammatory responses, and decreased NF-κB activity. As a NF-κB pathway blocker and antioxidant, celastrol may represent a promising pharmacological agent with protective effects against gamma irradiation-induced injury.

Phenolic and Non-Polar Fractions of the Extracts from Fruits, Leaves, and Twigs of Elaeagnus rhamnoides (L.) A. Nelson-The Implications for Human Barrier Cells.

Biological potential of plant extracts are widely described. Because their oral or topical administration is usually recommended, intestinal mucous and skin are the first surfaces exposed to such preparations. Therefore, we asked the question whether phenolic and non-polar fractions of the extracts from fruits, twigs, and leaves of sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) would be able to modulate the functions of human physiological barrier. The study was carried on caucasian colon epithelial-like Caco-2 cells and human foreskin fibroblasts HFF-1 line. Cell secretory activity (ELISA), the expression of cell surface molecules (flow cytometry), cell migration during wound healing in vitro (scratch assay) were assessed. It was demonstrated for the first time, that sea buckthorn extracts can improve intestinal and skin barrier by increasing of ICAM-1 expression on colon epithelial cells and intensification of IL-8 production by fibroblasts. On the other hand, an inhibition of fibroblasts migration in the presence of those preparations was noted. Therefore, greater attention should be paid on precise description of plant extracts effect depended on target cells and their role to give adequate recommendations for such preparations use.

Activity of Thymus capitatus essential oil components against in vitro cultured Echinococcus multilocularis metacestodes and germinal layer cells.

The essential oil (EO) of Thymus capitatus, seven fractions (F1-F7) obtained from silica gel chromatography, and several pure EO components were evaluated with respect to in vitro activities against Echinococcus multilocularis metacestodes and germinal layer (GL) cells. Attempts to evaluate physical damage in metacestodes by phosphoglucose isomerase (PGI) assay failed because EO and F1-F7 interfered with the PGI-activity measurements. A metacestode viability assay based on Alamar Blue, as well as transmission electron microscopy, demonstrated that exposure to EO, F2 and F4 impaired metacestode viability. F2 and F4 exhibited higher toxicity against metacestodes than against mammalian cells, whereas EO was as toxic to mammalian cells as to the parasite. However, none of these fractions exhibited notable activity against isolated E. multilocularis GL cells. Analysis by gas chromatography-mass spectrometry showed that carvacrol was the major component of the EO (82.4%), as well as of the fractions F3 (94.4%), F4 (98.1%) and F5 (90.7%). Other major components of EO were ß-caryophyllene, limonene, thymol and eugenol. However, exposure of metacestodes to these components was ineffective. Thus, fractions F2 and F4 of T. capitatus EO contain potent anti-echinococcal compounds, but the activities of these two fractions are most likely based on synergistic effects between several major and minor constituents.

Polygonum multiflorum Radix extract protects human foreskin melanocytes from oxidative stress in vitro and potentiates hair follicle pigmentation ex vivo.

OBJECTIVE: To examine the ability of an extract from traditional Chinese medicine, Polygonum multiflorum Radix, to protect melanocyte viability from oxidative stress, a key mechanism in the initiation and progression of hair greying. METHODS: To assess the antioxidant capacity of Polygonum multiflorum Radix extract, primary human foreskin melanocytes were treated with a commercially available Polygonum multiflorum Radix extract added to culture medium and exposed to hydrogen peroxide (H2 O2 ), using intracellular reactive oxygen species concentrations and glutathione/protein ratios as endpoints. To improve solubility for cosmetic uses, a new Polygonum multiflorum Radix extract was derived. As hair greying is the consequence of melanocyte disappearance in an oxidative stress environment, we checked whether the antioxidant capacity of the new Polygonum multiflorum Radix extract could preserve melanocyte viability in response to H2 O2 -induced oxidative stress, and preserve pigmentation within ex vivo human hair follicles. RESULTS: In vitro treatment of primary human foreskin melanocytes with traditional available Polygonum multiflorum Radix extract resulted in decreased intracellular ROS accumulation in response to H2 O2 exposure with a concomitant preservation of glutathione-to-protein ratio, consistent with a protective response against H2 O2 exposure and demonstrating the promise of this extract for protecting melanocytes against oxidative stress. Melanocytes treated with the improved Polygonum multiflorum Radix extract exhibited attenuated H2 O2 -induced cell death, demonstrating a clear cytoprotective effect. Treatment of ex vivo human hair follicles with the improved Polygonum multiflorum Radix extract resulted in a higher level of melanin compared to vehicle-treated controls, demonstrating an ex vivo protective effect on hair pigmentation. CONCLUSION: Polygonum multiflorum Radix extract protects in vitro primary human foreskin melanocytes from the deleterious effects of H2 O2 exposure and improves pigmentation within ex vivo human hair follicles, demonstrating the utility of Polygonum multiflorum Radix extract as a potential active ingredient for the protection of melanocytes against premature death. This data provides in vitro mechanistic evidence consistent with existing in vivo studies for the use of Polygonum multiflorum Radix extract as a strategy for the prevention of oxidative stress-induced hair greying, in line with traditional Polygonum multiflorum Radix uses.

Actomyosin Cortical Mechanical Properties in Nonadherent Cells Determined by Atomic Force Microscopy.

The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface. We extracted an average tension of T ∼ 20.25 nN/µm, which agrees with macroscopic experimental methods. We then measured cortical mechanical properties in nonadherent human foreskin fibroblasts and THP-1 human monocytes before and after pharmacological perturbations of actomyosin activity. Our results show that myosin II activity and actin polymerization increase cortex tension and intracellular pressure, whereas branched actin networks decreased them. Interestingly, myosin II activity stiffens the cortex and branched actin networks soften it, but actin polymerization has no effect on cortex stiffness. Our method is capable of detecting changes in cell mechanical properties in response to perturbations of the cytoskeleton, allowing characterization with physically relevant parameters. Altogether, this simple method should be of broad application for deciphering the molecular regulation of cell cortical mechanical properties.

Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD.

The brighter (and evolutionarily older) face of the metabolic syndrome: evidence from Trypanosoma cruzi infection in CD-1 mice.

BACKGROUND: Infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, results in chronic infection that leads to cardiomyopathy with increased mortality and morbidity in endemic regions. In a companion study, our group found that a high-fat diet (HFD) protected mice from T. cruzi-induced myocardial damage and significantly reduced post-infection mortality during acute T. cruzi infection. METHODS: In the present study metabolic syndrome was induced prior to T. cruzi infection by feeding a high fat diet. Also, mice were treated with anti-diabetic drug metformin. RESULTS: In the present study, the lethality of T. cruzi (Brazil strain) infection in CD-1 mice was reduced from 55% to 20% by an 8-week pre-feeding of an HFD to induce obesity and metabolic syndrome. The addition of metformin reduced mortality to 3%. CONCLUSIONS: It is an interesting observation that both the high fat diet and the metformin, which are known to differentially attenuate host metabolism, effectively modified mortality in T. cruzi-infected mice. In humans, the metabolic syndrome, as presently construed, produces immune activation and metabolic alterations that promote complications of obesity and diseases of later life, such as myocardial infarction, stroke, diabetes, Alzheimer's disease and cancer. Using an evolutionary approach, we hypothesized that for millions of years, the channeling of host resources into immune defences starting early in life ameliorated the effects of infectious diseases, especially chronic infections, such as tuberculosis and Chagas disease. In economically developed countries in recent times, with control of the common devastating infections, epidemic obesity and lengthening of lifespan, the dwindling benefits of the immune activation in the first half of life have been overshadowed by the explosion of the syndrome's negative effects in later life.

Discovery of (E)-5-(benzylideneamino)-1H-benzo[d]imidazol-2(3H)-one derivatives as inhibitors for PTK6.

A lead compound 1, which inhibits the catalytic activity of PTK6, was selected from a chemical library. Derivatives of compound 1 were synthesized and analyzed for inhibitory activity against PTK6 in vitro and at the cellular level. Selected compounds were analyzed for cytotoxicity in human foreskin fibroblasts using MTT assays and for selectivity towards PTK members in HEK 293 cells. Compounds 20 (in vitro IC50=0.12µM) and 21 (in vitro IC50=0.52µM) showed little cytotoxicity, excellent inhibition of PTK6 in vitro and at the cellular level, and selectivity for PTK6. Compounds 20 and 21 inhibited phosphorylation of specific PTK6 substrates in HEK293 cells. Thus, we have identified novel PTK6 inhibitors that may be used as treatments for PTK6-positive carcinomas, including breast cancer.

In vitro effect of bisphosphonates on oral keratinocytes and fibroblasts.

PURPOSE: Osteonecrosis of the jaws is a potential complication of bisphosphonate (BP) therapy. The underlying mechanisms remain unclear. Although most research has concentrated on the effects of BPs on osteoclast and osteoblast functions, the disease is diagnosed and classified based on of mucosal breakdown, suggesting that oral soft tissues may be involved in its pathogenesis. The aim of this study was to determine the effect of 3 different BP drugs (alendronate, zoledronate, and clodronate) on the function of oral keratinocytes and fibroblasts. MATERIALS AND METHODS: Human oral keratinocytes (OKF6) and fetal foreskin fibroblasts (HFFF2) were exposed to each drug at several concentrations and the effect on cell proliferation was assessed by counting the viable cells after different lengths of treatment. The effect on cell migration was examined using Transwell migration assays. An organotypic coculture model using keratinocytes and fibroblasts, which recapitulated the morphology of the oral mucosa, was used to assess the effect of the drugs on epithelial stratification and differentiation. RESULTS: The 3 BPs affected the viability and proliferation of OKF6 and HFFF2 cells at concentrations in keeping with their known relative in vitro potencies. There was no effect on cell migration or tissue architecture in organotypic cultures at subtoxic concentrations. CONCLUSION: The lack of effect of these drugs on cell migration below concentrations known to affect cell viability suggests that BP-related osteonecrosis is not caused through suppression of keratinocyte or fibroblast motility.

Self-supported fibrin-polyvinyl alcohol interpenetrating polymer networks: an easily handled and rehydratable biomaterial.

A fibrin hydrogel at physiological concentration (5 mg/mL) was associated with polyvinyl alcohol (PVA) inside an interpenetrating polymer networks (IPN) architecture. Previously, PVA has been modified with methacrylate functions in order to cross-link it by free-radical polymerization. The fibrin network was synthesized by the enzymatic hydrolysis of fibrinogen by thrombin. The resulting self-supported materials simultaneously exhibit the properties of the fibrin hydrogel and those of the synthetic polymer network. Their storage modulus is 50-fold higher than that of the fibrin hydrogel and they are completely rehydratable. These materials are noncytotoxic toward human fibroblast and the fibrin present on the surface of PVAm-based IPNs favors cell development.

PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK).

Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism.

Testosterone exerts selective anti-inflammatory effects on human skin mast cells in a cell subset dependent manner.

Androgens are known to exert anti-inflammatory effects but their impact on mast cells (MCs) remains to be determined. Here, we show that MCs isolated from human foreskin samples (male) and those from breast skin (female) express the androgen receptor, albeit with a 10-fold difference between the subsets. While fundamental MC properties (FcεRI, c-Kit, tryptase; histamine release upon FcεRI cross-linking) were unaffected or slightly reduced (chymase) by testosterone, the hormone had a more profound impact on the production of cytokines, with IL-6 being a target (reduction by 53%). Interestingly, this effect was limited to breast skin MCs (15 of 16 donors displayed this phenomenon), but was not reproduced by foreskin MCs. Collectively, effector functions of human skin MCs are modulated by androgens in a gene-selective and MC subset-specific fashion. Possibly, MCs from women are more susceptible to testosterone. We also demonstrate that MC IL-6 production is highly variable among individuals.

Structural analysis of the phimotic prepuce in patients with failed topical treatment compared with untreated phimosis.

OBJECTIVES: To evaluate histological alterations in prepuce of patients with phimosis submitted to topic treatment with betamethasone in association with hyaluronidase. MATERIALS AND METHODS: We studied sixty patients (mean age 4.5), presenting true phimosis and treated with a topical treatment with betamethasone cream (0.2%) + hyaluronidase. The parents of seven of these patients opted for circumcision (control group). The other fifty-three patients were submitted to clinical treatment. The samples were stained with Weigert's resorcin-fuchsin (analysis of the elastic fibers) and Picro-Sirius Red, for analysis of the collagen. The volumetric density of the elastic fibers was determined by stereological methods. RESULTS: Only eight (15 %) of the fifty-three patients submitted to topical treatment presented failure, being indicated for circumcision (histological analysis). We observed an increase of the collagen type III of the patients submitted to topical treatment. The quantification showed a reduction of the volumetric density of the prepuce's elastic fibers of the patients submitted to the cream treatment, when compared to the control group (p = 0.056). The volumetric density of the elastic fibers of the prepuce at the group not submitted to topical treatment showed an average of 14.60% (11.06 to 21.64%); in the group submitted to the cream treatment, the volumetric density of the elastic fibers of the prepuce showed an average of 10.34% (3.45 to 17.9%). CONCLUSION: The topical treatment of phimosis with betamethasone 0.2 % + hyaluronidase had a success rate of 85 %. Patients with failure of the topical treatment with steroid had histological alterations in the prepuce.

Structural study of prepuce in hypospadias--does topical treatment with testosterone produce alterations in prepuce vascularization?

PURPOSE: Androgen stimulation before hypospadias surgery has resulted in increased penile size, fewer complications and improved cosmesis, and suggests increased neovascularization. To our knowledge the real effect on neovascularization remains to be proved. We studied the histological effects of testosterone on neovascularization. MATERIALS AND METHODS: A total of 26 boys with hypospadias were randomly allocated to 2 groups before surgical correction. Group 1 did not receive any treatment and group 2 received 1% testosterone propionate ointment twice daily for 30 days before surgery. During the surgical procedure a fragment of prepuce was excised and prepared for histological evaluation. The number and volume density of blood vessels were determined by labeling for von Willebrand's factor. Blood vessel quantification as volume density was done using a video microscopy system with a superimposed cycloid arch test system. RESULTS: The groups were similar in age and hypospadias classification. Testosterone treated prepuces (group 2) had an increased absolute number of blood vessels (mean ± SD 8.5 ± 1.3 vs 4.8 ± 1.8 vessels per field) and increased blood vessel volume density (mean 50.5% ± 7.8% vs 24.8% ± 8.6% vessels per point) (each p <0.001) compared to those in untreated patients (group 1). CONCLUSIONS: The use of 1% testosterone propionate ointment before hypospadias surgery produces neovascularization in absolute numbers and in volume density.

Phototoxic versus photoprotective effects of tattoo pigments in reconstructed human skin models: In vitro phototoxicity testing of tattoo pigments: 3D versus 2D.

The increasing number of tattooed persons urges the development of reliable test systems to assess tattoo associated risks. The alarming prevalence of 60 % phototoxic reactions in tattoos ask for a more comprehensive investigation of phototoxic reactions in tattooed skin. Here, we aimed to compare the cellular responses of human skin cells to ultraviolet (UV)A and UVB irradiation in doses of short to intermitted sun exposure (3-48 J/cm² and 0.05-5 J/cm², respectively) in the presence of tattoo pigments. Therefore, we used fibroblast monolayer culture (2D), our recently developed three dimensional full-thickness skin model with dermal-located tattoo pigments (TatSFT) and its dermal equivalents (TatSDE) that lack keratinocytes. We tested the most frequently used tattoo pigments carbon black, titanium dioxide (TiO2) anatase and rutile as well as Pigment Orange (P.O.)13 in ranges from 0.067 to 2.7 ng/cell in 2D. For TatSDE and TatSFT, concentrations were 1.3 ng/cell for TiO2, 0.67 ng/cell for P.O.13 and 0.067 ng/cell for carbon black. We assessed cell viability and cytokine release in all systems, and cyclobutane pyrimidine dimer (CPD) formation in TatSFT. Phototoxicity of tattoo pigments was exclusively observed in 2D, where especially TiO2 anatase induced phototoxic effects in all concentrations (0.067-2.7 ng/cell). In contrast, fibroblasts were protected from UV irradiation in TatSDE by TiO2 and carbon black. Neither toxic nor protective effects were recorded in TatSFT. P.O.13 showed altered cytokine secretion in 2D (0.067-1.3 ng/cell) and TatSDE, despite the absence of significant effects on viability in all systems. All pigments reduced the number of CPDs in TatSFT compared to the pigment-free controls. In conclusion, our study shows that within a 3D arrangement, intradermal tattoo pigments may act photoprotective despite intrinsic phototoxic properties in 2D. Thus, dermal 3D equivalents should be considered to evaluate acute tattoo pigment toxicology.