Tobias W. Fischer

Melatonin increases survival of HaCaT keratinocytes by suppressing UV-induced apoptosis

Abstract:u2002 Melatonin is a potent antioxidant and direct radical scavenger. As keratinocytes represent the major population in the skin and UV light causes damage to these cells, the possible protective effects of melatonin against UV‐induced cell damage in HaCaT keratinocytes were investigated in vitro. Cells were preincubated with melatonin at graded concentrations from 10−9 to 10−3u2003m for 30u2003min prior to UV irradiation at doses of 25 and 50u2003mJ/cm2. Biological markers of cellular viability such as DNA synthesis and colony‐forming efficiency as well as molecular markers of apoptosis were measured. DNA synthesis was determined by [3H]‐thymidine incorporation into insoluble cellular fraction, clonogenicity through plating efficiency experiments and apoptosis by the terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) assay. DNA synthesis experiments showed a strong protective effect by preincubation with melatonin at concentrations of 10−4u2003m (Pu2003<u20030.01) and 10−3u2003m (Pu2003<u20030.001). Additional postirradiation treatment with melatonin showed no increase in the pre‐UV incubation protective effect. These results indicate that preincubation is a requirement for melatonin to exert its protective effects. The mechanism of melatonins protective effect (10−6 to 10−3u2003m) includes inhibition of apoptosis as measured by TUNEL assay. Moreover, the biological significance of these effects is supported by clonogenic studies showing a significantly higher number of colonies in cultures treated with melatonin compared to controls. Thus, pretreatment with melatonin led to strong protection against UVB‐induced damage in keratinocytes.

Direct and Non-Direct Measurement Techniques for Analysis of Skin Surface Topography

Estimation of skin smoothness is of ever-increasing interest especially in the field of cosmetic research. There are some established methods for assessing skin smoothness, e.g. optical and mechanical profilometry, but the presentation of recently developed new methods reflects the demand for alternatives which are more precise and practical. For fundamental research on ultrastructure of the stratum corneum surface scanning electron microscopy is a suitable method. A direct method is the surface evaluation of living skin, which is based on an optical system in a CCD camera measuring four parameters of roughness, scaling, smoothing and wrinkling. A similar but non-direct method is optical profilometry using skin replicas. Laser profilometry produces a variety of data which can be analysed using complex mathematical functions. A promising new method is transparency profilometry (skin visiometer) using a very thin skin print which allows parallel light to pass through and is analysed immediately after production. The different methods can be used for characterization of the skin microrelief in dermatoses or for dynamic measurements of time-dependent changes in skin surface topography after application of cosmetic or medical products.

Melatonin suppresses reactive oxygen species induced by UV irradiation in leukocytes

Abstractu2002 An investigation of the antioxidative UV protective effect of melatonin was performed in an in vitro irradiation model with leukocytes. Leukocytes were isolated from EDTA‐treated whole blood and taken up in phosphate‐buffered saline (PBS). Five of 10 aliquots were incubated with 2u2003mmol/L melatonin and 5 with PBS as a control. The samples were irradiated by UV light (280–360u2003nm, max: 310u2003nm) at doses between 75 and 300u2003mJ/cm2 or left unirradiated. Radical formation was measured using the chemiluminescence technique. Staining with trypan blue was performed to assess cell viability. Melatonin significantly suppressed radical formation in cell solutions irradiated from 75 to 300u2003mJ/cm2 (Pu2003≤u20030.001). Controls showed an increase of reactive oxygen species (ROS) formation as a sign of oxidative stress when irradiated with increasing UV doses and a maximum ROS formation under 300u2003mJ/cm2 UV light. The cytotoxicity of UV light was reduced by melatonin up to a UV dose of 1.5u2003J/cm2. Leukocytes were suitable cells for the evaluation of the efficacy of melatonin as a radical scavenger under UV light. The results confirm that the clinically observed UV protective effects of melatonin may be at least partially based on its radical scavenging properties.

Melatonin reduces UV-induced reactive oxygen species in a dose-dependent manner in IL-3-stimulated leukocytes

Reactive oxygen species (ROS) are presumed to be involved in inflammatory UV reactions of the skin. This in vitro study was performed to investigate the suppressive effect of melatonin in interleukin‐3 (IL‐3) stimulated leukocytes. Neutrophilic granulocytes were isolated from EDTA‐treated whole blood and placed in a phosphate‐buffered saline (PBS) containing IL‐3. Cell suspensions were either treated with PBS (control) or with increasing doses of melatonin (0.1, 0.5, 1, 2, 3, 5, 7.5, 10 mmol). One PBS solution was left unirradiated and the other nine solutions (PBS and melatonin) were irradiated with 750 mJ/cm2 UVB light (280–360 nm, max: 310 nm). Radical formation was measured by the chemiluminescence technique. UV‐irradiated leukocytes showed a 5‐fold higher radical formation than unirradiated leukocytes. Melatonin, in increasing doses in powers of ten, led to a maximum suppression of free radicals at 10 nmol (P=0.01) and 1 mmol melatonin (P=0.001), showing a biphasic, non‐linear, dose–response relationship. Melatonin, given in amounts of 0.1–10 mmol, led to a direct dose‐dependent suppression of ROS. Radical formation was suppressed significantly in a range from 0.5 to 10 mmol (P=0.001). Melatonin is known to function as a radical scavenger and antioxidant; some of these melatonin effects may be receptor independent, while others may be receptor dependent.

Effect of caffeine and testosterone on the proliferation of human hair follicles in vitro

Backgroundu2002 Androgenetic alopecia (AGA) is a common problem in men of all ages, affecting approximately 50% at 50 years of age. The underlying cause is an androgen‐dependent miniaturization of genetically predetermined hair follicles. Here, the hair organ culture model was used to investigate the effects of testosterone and caffeine; the latter being a promising candidate for hair growth stimulation.

Percutaneous penetration of topically applied melatonin in a cream and an alcoholic solution.

In a clinical study, the skin penetration properties of melatonin 0.01% in a cream and 0.01 and 0.03% in a solution were investigated by evaluation of the serum melatonin levels over a 24-hour time course in 15 healthy volunteers. Blood samples for melatonin measurements were taken at 9.00 a.m. before applying the test preparations and 1, 4, 8 and 24 h after application. The measurements were carried out by radioimmunoassay for melatonin. In 15 volunteers, the serum levels of melatonin before application of the topical preparations were between 0.6 and 15.9 pg/ml. After application of the 0.01% melatonin cream, there was a steady increase starting from 9.00 a.m. up to a mean serum value of 9.0 pg/ml at 9.00 a.m. the next day. The solution of 0.01% melatonin also showed an increase, starting from 5.00 p.m., up to a mean melatonin level of 12.7 pg/ml 24 h after application. The solution containing 0.03% melatonin resulted in elevated melatonin levels 1 and 8 h after application. The values were 18.1 and 19.0 pg/ml. The cumulative melatonin values for each preparation were 7.1, 8.6 and 15.7 pg/ml, respectively. This study shows that the strongly lipophilic substance melatonin is able to penetrate through the skin and leads to dose- and galenic-dependent melatonin levels in the blood. No increase of melatonin above the physiological range was observed.

Melatonin Suppresses Reactive Oxygen Species in UV-Irradiated Leukocytes More than Vitamin C and Trolox

To prove the relative potency of melatonin as a radical scavenger in UV-irradiated leukocytes, it was compared to other antioxidative substances such as trolox and vitamin C. Human leukocytes were isolated from EDTA whole blood and incubated with melatonin, trolox and vitamin C. The experiments were performed in a wide concentration range from 0.1 nM to 1 mM and in a small concentration range from 0.5 to 2 mM (mel), 5 mM (trolox) and 10 mM (vit. C). Irradiation was performed with UV-light (280-360 nm) at a dose of 750 mJ/cm2. Radical formation was measured by the chemiluminescence technique. The maximum effect of radical suppression was seen at a concentration of 10 nM (p = 0.003) and 1 mM melatonin (p < 0.001) and vitamin C (p = 0.002; p < 0.001), respectively. ROS suppression by trolox was only significant at 1 mM (p < 0.001). In the small concentration range, a linear dose-response relationship was found and melatonin showed the strongest radical suppression (IC50 = 0.21 mM) followed by vitamin C (IC50 = 0.26 mM) and trolox (IC50 = 1.03 mM).

Assessment of 'dry skin': current bioengineering methods and test designs.

Dry skin is a frequent problem in dermatology and a sign of dysfunction of the epidermis, especially of the stratum corneum as the morphological equivalent of the skin barrier. It may occur as an individual disposition or as the leading symptom of atopic dermatitis or ichthyosis. Besides the visual examination of the skin, various bioengineering methods have been developed to assess the different pathological and adaptive changes in the skin. In addition to the assessment of skin humidity, barrier function and desquamation, the quantification of skin surface topography and the mechanical properties of skin are suitable methods to characterize a dry skin condition. For clinical assessment of moisturizing products and emollients the parameters of investigation have to be defined and integrated in an adapted study design depending on the composition and content of the active agent in the test product. Newly developed cosmetic products have to be investigated for safety and efficacy. Modern bioengineering methods are suitable to fulfill these challenges.

Erythema multiforme-like drug eruption with oral involvement after intake of leflunomide.

Leflunomide is an antirheumatic agent of the type of a ‘disease-modifying antirheumatic drug’. In rare cases, severe skin reactions up to the extreme expression of toxic epidermal necrolysis have been observed. A female patient with rheumatoid arthritis had been treated with systemic steroids and methotrexate for 2 years. Five weeks prior to admission to our hospital methotrexate was replaced by leflunomide. Three weeks after initiation of leflunomide therapy a progressive generalized erythema with blistering formation occurred accompanied by increase of body temperature, chills and erosive lesions on the lips and oral mucosa. The palmar and plantar surfaces revealed edema, erythema and pulpitis with epidermolysis. On histologic examination necrotic keratinocytes and epidermal spongiosis were observed. After administration of high-dose prednisolone and topical treatment the patient recovered within 14 days. This is one of the few cases of severe drug reaction after intake of leflunomide. Therefore, the indication of this relatively new drug should be considered carefully.

HaCaT Cell Proliferation Influenced by Melatonin

The hormone melatonin is characterized by numerous pharmacological effects. The influence of melatonin on the growth of the human hair follicle was shown in previous investigations. In the present study, the effects of melatonin were investigated by means of proliferation tests of HaCaT keratinocytes using the [3H]thymidine incorporation, a fluorescence assay with Hoechst dye 33342 and the ATP bioluminescence assay. The aim of the study was to find melatonin concentrations suitable for treatments of the skin and whether there is a cytotoxic effect on HaCaT cells. The different proliferative activity of melatonin depending on its concentration and the time of incubation could be shown in all investigations.