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2.
Am J Dermatopathol ; 31(5): 427-31, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19542914

ABSTRACT

Recently, the antioxidant repair enzymes methionine-S-sulfoxide reductase A (MSRA) and methionine-R-sulfoxide reductase B (MSRB) were described in human epidermal keratinocytes and melanocytes. Methionine sulfoxide reductases (MSRs) are thought to protect against reactive oxygen species-induced oxidative damage in many organs, including the most environmentally exposed organ, human skin. We sought to examine the expression and distribution of this enzyme family (MSRA, MSRB1, MSRB2, and MSRB3) within the various compartments of healthy and diseased human skin. Expression was assessed using polyclonal MSR antibodies and immunohistochemical staining of human skin biopsies from various anatomical sites. Remarkably, MSRA expression was not only found in the epidermis as previously described but also in hair follicles and eccrine glands and was most pronounced in sebaceous glands. Furthermore, MSRB2 expression was found in melanocytes while MSRB1 and MSRB3 were both expressed within vascular endothelial cells. In conclusion, MSR enzymes are differentially expressed in human skin. Thus, modulation of MSR repair antioxidants may have implications for cutaneous aging and carcinogenesis.


Subject(s)
Gene Expression , Oxidoreductases/biosynthesis , Skin/enzymology , Eccrine Glands/enzymology , Endothelium, Vascular/enzymology , Hair Follicle/enzymology , Humans , Immunohistochemistry , Keratinocytes/enzymology , Melanocytes/enzymology , Methionine Sulfoxide Reductases , Sebaceous Glands/enzymology , Skin/blood supply , Skin Diseases/enzymology
3.
Cutis ; 83(5): 243-9, 2009 May.
Article in English | MEDLINE | ID: mdl-19537282

ABSTRACT

Xanthogranuloma (XG) is a rare, non-Langerhans cell histiocytosis (LCH) that most commonly presents in infancy or early childhood. The condition is typified by the formation of reddish to yellow papules and nodules that are usually solitary. Xanthogranuloma rarely occurs in adults with immunohistochemical features similar to those seen in juvenile XG. Lesions in the adult form seen in juvenile XG. Lesions in the adult form also are typically solitary. We describe a 70-year-old white man who presented with widespread flat-topped, reddish to yellow papules and nodules with histologic and immunohistochemical findings consistent with XG. We explore the pathogenesis, differential diagnosis, prognosis, and treatment of this rare eruption. Comparison of adult and juvenile XG will facilitate a better understanding of the disease. Although rare, XG is an important disease to consider in the differential diagnosis of xanthomatous disease in adults.


Subject(s)
Xanthogranuloma, Juvenile/pathology , Aged , Diagnosis, Differential , Humans , Male
4.
J Appl Toxicol ; 28(8): 1040-4, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18626891

ABSTRACT

Forty years have passed since the accidental poisoning with polychlorinated biphenyls (PCB) in Japan in 1968, named Yusho. High concentrations of PCB are still detected in the serum of the Yusho victims. PCB produces superoxide (O(2) (-)) in the metabolic process and we reported high concentrations of serum nitrite, a stable metabolite reflecting nitric oxide (NO), in the Yusho victims. NO reacts with O(2) (-) and immediately produces peroxynitrite (ONOO(-)). ONOO(-) causes nitration of tyrosine residues and produces nitrotyrosine (NT). Therefore, we measured urinary concentrations of nitrite and NT in the victims and age-matched controls. The mean urinary concentrations of nitrite and NT were significantly higher than in the controls. There was a positive correlation between urinary nitrite and NT in the Yusho victims. Furthermore, there was a positive correlation between the ratio of urinary NT to nitrite and serum PCB concentrations in the Yusho victims. It was considered that the emergence of some ailments could be presumed to have been caused by high levels of urinary nitrite and NT in the Yusho victims.


Subject(s)
Environmental Pollutants/poisoning , Food Contamination , Foodborne Diseases/epidemiology , Nitrites/urine , Oryza/poisoning , Plant Oils/poisoning , Polychlorinated Biphenyls/poisoning , Tyrosine/analogs & derivatives , Adult , Aged , Disease Outbreaks , Environmental Pollutants/blood , Female , Humans , Japan/epidemiology , Male , Middle Aged , Poisoning/diagnosis , Polychlorinated Biphenyls/blood , Tyrosine/urine
6.
Mol Aspects Med ; 28(5-6): 646-67, 2007.
Article in English | MEDLINE | ID: mdl-17719081

ABSTRACT

Vitamin E has been used for more than 50 years in experimental and clinical dermatology. While a large number of case reports were published in this time, there is still a lack of controlled clinical studies providing a rationale for well defined dosages and clinical indications. In contrast, advances in basic research on the physiology, mechanism of action, penetration, bioconversion and photoprotection of vitamin E in human skin has led to the development of numerous new formulations for use in cosmetics and skin care products. This article reviews basic mechanisms and possible cosmetic as well as clinical implications of the recent advances in cutaneous vitamin E research. Experimental evidence suggests that topical and oral vitamin E has antitumorigenic, photoprotective, and skin barrier stabilizing properties. While the current use of vitamin E is largely limited to cosmetics, controlled clinical studies for indications such as atopic dermatitis or preventions of photocarcinogenesis are needed to evaluate the clinical benefit of vitamin E.


Subject(s)
Dermatology , Skin/metabolism , Vitamin E/metabolism , Animals , Antioxidants/metabolism , Dermatology/methods , Humans , Organ Specificity , Skin Diseases/metabolism , Vitamin E/administration & dosage , Vitamin E/adverse effects , Vitamin E/pharmacology
7.
J Appl Toxicol ; 27(2): 195-7, 2007.
Article in English | MEDLINE | ID: mdl-17186569

ABSTRACT

Thirty-five years have passed since the accidental poisoning with polychlorinated biphenyls (PCB) in Japan and yet high concentrations of PCB are still detected in the serum of the victims. PCB produces superoxide and thus victims are considered to be in a persistent state of oxidative stress. Urinary concentrations of 8-isoprostane (8IP) in the victims and age-matched controls were measured to assess this hypothesis. The mean urinary concentration of 8IP was significantly higher than that in the controls. There was a positive correlation between urinary 8IP and serum concentrations of cholinesterase. It was considered that Yusho is an oxidative stress and 8IP is a useful tool for checking the oxidative condition in Yusho victims.


Subject(s)
Environmental Illness/metabolism , Environmental Pollutants/poisoning , Food Contamination , Foodborne Diseases/metabolism , Lipid Peroxidation/drug effects , Oxidative Stress/drug effects , Polychlorinated Biphenyls/poisoning , Aged , Biomarkers/urine , Dinoprost/analogs & derivatives , Dinoprost/urine , Environmental Illness/etiology , Foodborne Diseases/etiology , Humans , Japan , Polychlorinated Biphenyls/blood
8.
J Invest Dermatol ; 126(5): 1128-34, 2006 May.
Article in English | MEDLINE | ID: mdl-16514415

ABSTRACT

Recently, we reported that photoaging correlates well with the amount of oxidized protein accumulated in the upper dermis, while protein oxidation levels in the viable epidermis are very low. We hypothesized that this might be due to epidermal expression of the repair enzymes methionine sulfoxide reductases (MSRs). The expression of human methionine sulfoxide reductase A (MSRA) was investigated in HaCaT cells, primary human keratinocytes, and in human skin. High MSRA mRNA and protein levels as well as MSR activity were found in cultured human keratinocytes. MSRA was expressed in human epidermis, as shown by immunohistochemistry in healthy human skin. Repetitive in vivo exposure of human skin to solar-simulated light on 10 consecutive days (n=10 subjects) significantly increased epidermal MSRA expression. To further assess the functional relevance of the enzyme, its expression in response to UVB, UVA, and H(2)O(2) was investigated in HaCaT cells. While UVB lowered protein expression of MSRA, an upregulation was observed in response to low doses of UVA and H(2)O(2). In summary, MSRA represents the only enzyme so far identified in human skin that is capable of repairing oxidative protein damage. In addition to melanogenesis and DNA repair systems, a wavelength-specific activation of epidermal MSRA may be involved in epidermal photoprotection.


Subject(s)
Gene Expression Regulation, Enzymologic/radiation effects , Oxidoreductases/genetics , Skin/enzymology , Cells, Cultured , Humans , Hydrogen Peroxide/pharmacology , Keratinocytes/enzymology , Methionine Sulfoxide Reductases , Reactive Oxygen Species , Skin/radiation effects , Ultraviolet Rays
9.
Dermatol Surg ; 31(7 Pt 2): 805-13; discussion 813, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16029671

ABSTRACT

BACKGROUND AND OBJECTIVE: The lipophilic antioxidant vitamin E has been used for more than 50 years in clinical and experimental dermatology. However, although a large number of case reports were published, there is still a lack of controlled clinical studies providing a rationale for clinical indications and dosage. In contrast, advances in basic research on the physiology, mechanism of action, penetration, bioconversion, and photoprotection of vitamin E in human skin have led to the development of numerous new formulations for use in cosmetics and skin care products. RESULTS: This article reviews the basic mechanisms and possible cosmetical and clinical implications of the recent advances in cutaneous vitamin E research. Experimental evidence suggests that topical and oral vitamin E has anticarcinogenic, photoprotective, and skin barrier-stabilizing properties. CONCLUSION: Although its current use is largely limited to cosmetics, controlled clinical studies for indications such as atopic dermatitis or prevention of photocarcinogenesis are needed to evaluate the clinical benefit of vitamin E.


Subject(s)
Antioxidants/administration & dosage , Dermatologic Agents/administration & dosage , Skin Aging/drug effects , Skin Diseases/drug therapy , Vitamin E/administration & dosage , Administration, Oral , Administration, Topical , Humans
10.
Dermatol Surg ; 30(12 Pt 2): 1556-60, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15606840

ABSTRACT

BACKGROUND: Keratoacanthomas are fast-growing squamous tumors, which usually show spontaneous regression. The development of giant variants with an aggressive behavior has been described. Although surgical excision remains the treatment of choice for very large keratoacanthomas, other therapeutic options including laser surgery and topical chemotherapy may be superior in special situations. OBJECTIVE: The objective was to evaluate the efficacy of Er:YAG laser surgery combined with topical 5-fluorouracil treatment in a case of recurrent giant keratoacanthoma. METHODS: A 64-year-old woman presented for evaluation and treatment of recurrent tumors in her face and extremities. Despite repeated invasive surgical removal of these lesions, recurrence of fast-growing giant keratoacanthomas developed in the pretibial region of her left lower leg. Owing to recurrence after conventional surgery and the tumor size, a novel treatment method using ablative Er:YAG laser combined with topical 5-fluorouracil was performed. RESULTS: After four treatments with excellent patient compliance, histologic analysis of punch biopsies revealed tumor-free ulcerations. Complete epithelization was obtained after 9 weeks. Six months after the treatment, no recurrence was observed. CONCLUSION: The combined use of ablative Er:YAG laser and topical 5-fluorouracil chemotherapy may be considered as an effective treatment option in cases of giant keratoacanthoma when conventional surgery is not indicated.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Fluorouracil/therapeutic use , Keratoacanthoma/diagnosis , Skin Diseases/diagnosis , Administration, Cutaneous , Antimetabolites, Antineoplastic/administration & dosage , Combined Modality Therapy , Diagnosis, Differential , Drug Administration Schedule , Female , Fluorouracil/administration & dosage , Humans , Keratoacanthoma/drug therapy , Keratoacanthoma/pathology , Keratoacanthoma/surgery , Laser Therapy , Leg , Middle Aged , Recurrence , Skin Diseases/drug therapy , Skin Diseases/pathology , Skin Diseases/surgery
11.
Free Radic Biol Med ; 37(5): 620-31, 2004 Sep 01.
Article in English | MEDLINE | ID: mdl-15288120

ABSTRACT

Alpha-tocopherol has been shown to increase nitric oxide (NO)-dependent relaxation but the underlying mechanisms have not been fully characterized. The present study investigates the effect of alpha-tocopherol and its derivative trolox on the synthesis of NO in human umbilical vein endothelial cells. NO was assayed as citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) on ionomycin stimulation of cells. Ionomycin induced citrulline and cGMP formation partially through phosphorylation of endothelial NO synthase (eNOS) at its serine residue 1177, which was mediated mainly by calmodulin-dependent kinase II. Preincubation of cells with alpha-tocopherol or trolox increased eNOS activity in a concentration-dependent manner without changing eNOS expression. The effect of the water-soluble trolox was due to chemical stabilization of the eNOS cofactor tetrahydrobiopterin. On the contrary, alpha-tocopherol, located mainly in cellular membranes, did not affect tetrahydrobiopterin but increased ionomycin-induced eNOS phosphorylation at serine 1177. The effects of alpha-tocopherol on citrulline and cGMP formation and eNOS phosphorylation were amplified by co-incubation with ascorbate, which is suggested to regenerate oxidized alpha-tocopherol and to act synergistically with alpha-tocopherol. Our data describe a new vasoprotective function of alpha-tocopherol that may contribute to the prevention of endothelial dysfunction in vivo.


Subject(s)
Biopterins/analogs & derivatives , Biopterins/metabolism , Chromans/pharmacology , Endothelium, Vascular/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Nitric Oxide Synthase/genetics , Serine , alpha-Tocopherol/pharmacology , Antioxidants/pharmacology , Binding Sites , Cells, Cultured , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Humans , Ionomycin/pharmacology , Nitric Oxide Synthase/drug effects , Nitric Oxide Synthase Type III , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction , Umbilical Veins
13.
Int J Dermatol ; 43(5): 326-35, 2004 May.
Article in English | MEDLINE | ID: mdl-15117361

ABSTRACT

Melanoma and nonmelanoma skin cancers are among the most prevalent cancers in the human population. Solar ultraviolet radiation is considered a major etiological factor but the relationship between dose, timing, and nature of exposure to tumor development is still unclear. Free radicals are generated by normal physiologic processes, including aerobic metabolism and inflammatory response, but may inflict cellular damage when generation is increased and antioxidant defense mechanisms are overwhelmed. Important findings supporting the free radical hypothesis in skin carcinogenesis are: (1) Reactive oxygen species (ROS) are generated in UVA- and UVB-irradiated skin in excessive doses, (2) the natural cutaneous antioxidant defense is impaired upon UV-exposure, (3) free radicals are involved in all steps of carcinogenesis, (4) supplementation with antioxidants can inhibit skin carcinogenesis, and (5) conditions that increase ROS generation enhance photocarcinogenesis. These findings provide a promising rationale for the development of powerful new antioxidant strategies in the prevention and therapy of skin cancer.


Subject(s)
Antioxidants , Oxidative Stress , Skin Neoplasms/drug therapy , Skin Neoplasms/physiopathology , Antioxidants/administration & dosage , Humans , Ultraviolet Rays/adverse effects
14.
J Am Acad Dermatol ; 50(6): 946-8, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15153900

ABSTRACT

We report a case of generalized cutaneous chronic graft-versus-host disease in a 17-month-old infant. Topical treatment with the novel ascomycin substance pimecrolimus once daily for 4 weeks led to a near complete clearance of skin lesions. Importantly, this excellent clinical response was obtained without additional use of systemic immunosuppressive treatment.


Subject(s)
Graft vs Host Disease/drug therapy , Skin Diseases/drug therapy , Tacrolimus/analogs & derivatives , Tacrolimus/adverse effects , Administration, Topical , Humans , Infant , Leukemia, Myelomonocytic, Acute/therapy , Male , Peripheral Blood Stem Cell Transplantation/adverse effects , Treatment Outcome
15.
Ann N Y Acad Sci ; 1031: 184-94, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15753144

ABSTRACT

In human skin, highest alpha-tocopherol levels are found in facial sebum. We hypothesized that the bioavailability of vitamin E in human skin is, at least in part, dependent on sebaceous gland secretion. To test this, 24 volunteers were subjected to a randomized daily supplementation with either 400 mg RRR-alpha-tocopheryl acetate (RRR-alpha-toc) or 400 mg all-rac-alpha-tocopheryl acetate (all-rac-alpha-toc) for 14 days. Fasting blood samples, facial sebum samples, and lower-arm skin-surface lipids (SSL) were taken at time-points between 0-21 days. Samples were analyzed by HPLC for alpha-tocopherol and squalene concentrations. Increased serum alpha-tocopherol levels were detectable as early as 12 h after supplementation of RRR-alpha-toc or all-rac-alpha-toc and peaked on day 7. No significant changes were observed in lower-arm SSL. Remarkably, while unchanged until day 14, alpha-tocopherol sebum levels were increased on day 21 in both the RRR-alpha-toc and the all-rac-alpha-toc group by 87% and 92%, respectively. With respect to dietary supplementation of vitamin E and its bioavailability in human skin, these results suggest that (1) sebaceous gland secretion is a relevant delivery mechanism; (2) the bioavailabilities of RRR-alpha-toc and the all-rac-alpha-toc are similar; and (3) significant accumulation requires a daily supplementation period of at least 2-3 weeks.


Subject(s)
Sebum/chemistry , Vitamin E/analysis , alpha-Tocopherol/analogs & derivatives , alpha-Tocopherol/administration & dosage , alpha-Tocopherol/chemistry , Adult , Chromatography, High Pressure Liquid , Dietary Supplements , Face , Female , Humans , Lipids/analysis , Male , Skin/chemistry , Squalene/analysis , Squalene/blood , Time Factors , Tocopherols , alpha-Tocopherol/analysis , alpha-Tocopherol/blood , alpha-Tocopherol/pharmacokinetics
16.
Free Radic Res ; 37(6): 655-63, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12868492

ABSTRACT

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Previously, it has been reported that UVB-irradiation of keratinocytes leads to intracellular generation of hydrogen peroxide (H2O2) and that antioxidants can inhibit ROS-induced apoptosis. Although both UVB-irradiation and H2O2-incubation led to increased intracellular H2O2 levels, the antioxidants catalase and glutathione monoester (GME), inhibited apoptosis only when induced by H2O2, not by UVB. Furthermore, extracellular signal-regulated kinase (ERK), a prominent member of the mitogen-activated protein kinase (MAPK) family, was found to be activated by treatment with both UVB and H2O2. Inhibition of ERK phosphorylation by pre-treatment with PD98059 resulted in enhanced apoptosis after H2O2-exposure. However,no significant difference of apoptosis was observed between cells with and without inhibitor pre-treatment upon UVB-irradiation. DNA damage in the form of cyclobutane pyrimidine dimers was observed after exposure to UVB, but no photoproducts were found in H2O2-treated cells. These results suggest a ROS-independent pathway of UVB-induced apoptosis. Although UVB-irradiation causes moderate increase in H2O2, the generation of H2O2 does not contribute to the induction of apoptosis. Moreover, activation of ERK only blocks H2O2-dependent apoptosis but has no impact on UVB-induced apoptosis.


Subject(s)
Apoptosis , Hydrogen Peroxide/chemistry , Keratinocytes/pathology , Ultraviolet Rays , Antioxidants/pharmacology , Catalase/metabolism , Cells, Cultured , Dimerization , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Enzyme Activation , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Glutathione/metabolism , Humans , Hydrogen Peroxide/metabolism , MAP Kinase Signaling System , Microscopy, Fluorescence , Mitogen-Activated Protein Kinases/metabolism , Pyrimidine Dimers , Reactive Oxygen Species , Time Factors
17.
J Invest Dermatol ; 120(6): 915-22, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12787115

ABSTRACT

At the outermost surface of human skin, skin surface lipids are first-line targets of solar ultraviolet radiation. Therefore, we hypothesized that ultraviolet A and ultraviolet B irradiation induce photo-oxidation of skin surface lipids. To test this, sebum samples were collected from facial skin of 17 healthy volunteers, weighed, and immediately irradiated with either ultraviolet B or ultraviolet A. Squalene, the major sebum lipid, as well as photo-oxidation products were identified in sebum lipid extracts by high-performance liquid chromatography analysis. Upon ultraviolet A exposures squalene was depleted in a concentration-dependent manner, whereas an unidentified sebum lipid photo-oxidation product was detected. Using high-performance thin layer chromatography, high-performance liquid chromatography, atmospheric pressure chemical ionization mass spectrometry, and nuclear magnetic resonance, unidentified sebum lipid photo-oxidation product was identified as a mixture of squalene monohydroperoxide isomers. Squalene monohydroperoxide isomers purified from sebum were identical with squalene monohydroperoxide isomers synthesized by preparative photo-oxidation of squalene. Squalene monohydroperoxide isomers were formed even after small suberythematogenic doses of ultraviolet A (5 J per cm2). Whereas physiologic baseline levels of squalene monohydroperoxide isomers in human skin were only slightly above detection limits, squalene monohydroperoxide isomer levels were strongly increased by suberythematogenic doses of ultraviolet A both in vitro and in vivo. High-performance liquid chromatography results could be complemented by a straightforward thin layer chromatography method for rapid screening of lipid peroxide formation in human sebum/skin surface lipids. In conclusion, specific squalene monohydroperoxide isomers were identified as highly ultraviolet A sensitive skin surface lipid breakdown products that may serve as a marker for photo-oxidative stress in vitro and in vivo.


Subject(s)
Lipid Metabolism , Sebum/metabolism , Skin/metabolism , Squalene/metabolism , Ultraviolet Rays , Adult , Chromatography, Thin Layer , Dose-Response Relationship, Radiation , Female , Humans , In Vitro Techniques , Male , Oxidation-Reduction/radiation effects , Protein Isoforms/metabolism , Sebum/radiation effects , Skin/radiation effects , Squalene/analogs & derivatives
18.
Free Radic Biol Med ; 34(2): 170-6, 2003 Jan 15.
Article in English | MEDLINE | ID: mdl-12521598

ABSTRACT

Benzoyl peroxide (BPO) is a commonly used drug in the treatment of acne vulgaris, but it induces unwanted side effects related to stratum corneum (SC) function. Since it has been recently shown to oxidize SC antioxidants, it was hypothesized that antioxidant supplementation may mitigate the BPO-induced SC changes. To test this, 11 subjects were selected to be topically supplemented with alpha-tocotrienol (5% w/vol) for 7 d on defined regions of the upper back, while the contralateral region was used for vehicle-only controls. Starting on day 8, all test sites were also treated with BPO (10%) for 7 d; the alpha-tocotrienol supplementation was continued throughout the study. A single dose of BPO depleted 93.2% of the total vitamin E. While continuing the BPO exposure for 7 d further depleted vitamin E in both vehicle-only and alpha-tocotrienol-treated sites, significantly more vitamin E remained in the alpha-tocotrienol-treated areas. Seven BPO applications increased lipid peroxidation. Alpha-tocotrienol supplementation significantly mitigated the BPO-induced lipid peroxidation. The transepidermal water loss was increased 1.9-fold by seven BPO applications, while there was no difference between alpha-tocotrienol treatment and controls. The data suggest that alpha-tocotrienol supplementation counteracts the lipid peroxidation but not the barrier perturbation in the SC induced by 10% BPO.


Subject(s)
Benzoyl Peroxide/pharmacology , Lipid Peroxidation/drug effects , Skin/drug effects , Vitamin E/analogs & derivatives , Vitamin E/administration & dosage , Vitamin E/pharmacology , Water Loss, Insensible/drug effects , Administration, Topical , Adult , Chromatography, High Pressure Liquid , Double-Blind Method , Female , Humans , Male , Tocotrienols
19.
Biol Chem ; 383(3-4): 703-8, 2002.
Article in English | MEDLINE | ID: mdl-12033459

ABSTRACT

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Poly (ADP-ribose) (PAR) is formed upon activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), and therefore was suggested as a new marker of apoptosis. Since DNA of epidermal cells represents a well-known chromophore for UVB irradiation, and UVB is known to generate H2O2 in keratinocytes, we hypothesized that PAR is a very sensitive marker of UVB- and H2O2-induced apoptosis in keratinocytes. In order to test this hypothesis, human immortalized keratinocytes (HaCaT) were UVB-irradiated or treated with H2O2, and subsequently apoptosis was identified by comparing conventional parameters such as morphological analysis, DNA laddering, and TUNEL assay, with PAR formation. Both, UVB and H2O2 treatment induced PAR formation in HaCaT cells in a dose-dependent manner, and its formation was detected as early as 4 h after irradiation, and at lower UVB doses (10 mJ/cm2) than observed by DNA laddering and the TUNEL assay. In conclusion, the detection of PAR formation is a very sensitive and early method for the identification of apoptotic cells in UVB-induced apoptosis of human keratinocytes.


Subject(s)
Apoptosis/radiation effects , Keratinocytes/physiology , Poly Adenosine Diphosphate Ribose/analysis , Ultraviolet Rays , Apoptosis/drug effects , Cell Survival/drug effects , Cell Survival/radiation effects , Cells, Cultured , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Keratinocytes/drug effects , Keratinocytes/radiation effects , Sensitivity and Specificity
20.
J Invest Dermatol ; 118(4): 618-25, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11918707

ABSTRACT

There is increasing evidence for the generation of reactive oxygen species in skin upon ultraviolet exposure, but little is known about their pathophysiologic relevance in human skin in vivo. We hypothesized that chronic and acute photodamage is mediated by depleted antioxidant enzyme expression and increased oxidative protein modifications. Biopsies from patients with histologically confirmed solar elastosis, from non-ultraviolet-exposed sites of age-matched controls, and from young subjects were analyzed. To evaluate the influence of acute ultraviolet exposures, buttock skin of 12 healthy subjects was irradiated repetitively on 10 d with a solar simulator and compared intraindividually to non-ultraviolet-treated contralateral sites. The antioxidant enzymes catalase, copper-zinc superoxide dismutase, and manganese superoxide dismutase were investigated by immunohistochemistry. Protein carbonyls were analyzed by immunohistochemical and immunoblotting techniques in human skin and in cell models. Whereas overall expression of antioxidant enzymes was very high in the epidermis, low baseline levels were found in the dermis. In photoaged skin, a significant depletion of antioxidant enzyme expression was observed within the stratum corneum and in the epidermis. Importantly, an accumulation of oxidatively modified proteins was found specifically within the upper dermis of photoaged skin. Upon acute ultraviolet exposure of healthy subjects, depleted catalase expression and increased protein oxidation were detected. Exposures of keratinocytes and fibroblasts to ultraviolet B, ultraviolet A, and H2O2 led to dose-dependent protein oxidation and thus confirmed in vivo results. In conclusion, the correlation between photodamage and protein oxidation was demonstrated for the first time, which hence may be a relevant pathophysiologic factor in photoaging.


Subject(s)
Dermis/enzymology , Skin Aging/physiology , Aged , Catalase/metabolism , Dermis/cytology , Dermis/radiation effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Humans , Hydrogen Peroxide/pharmacology , Immunoblotting , Keratinocytes/metabolism , Keratinocytes/radiation effects , Middle Aged , Oxidants/pharmacology , Oxidation-Reduction , Oxidative Stress/drug effects , Oxidative Stress/physiology , Proteins/analysis , Proteins/metabolism , Superoxide Dismutase/metabolism , Ultraviolet Rays
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