Noritaka Ohkuma

Reduced superoxide dismutase activity in UVB-induced hyperproliferative pig epidermis

SummaryDecreased superoxide dismutase (SOD) activity has been reported in various hyperproliferative keratinocytes. In order to elucidate the relationship between epidermal SOD activity and keratinocyte proliferation, we employed in vivo UVB irradiation. Following UVB irradiation at twice the minimum erythema dose, pig epidermis revealed an initial decrease in thymidine incorporation and mitotic counts for at least 48 h, followed by a marked increase, the peak of which was observed at 96 h after irradiation, and a return to basal levels by 5–7 days. The SOD activity remained constant during the initial 48 h and then decreased to about 50% at 96 h, mainly due to a decresed Cu,Zn-SOD activity. Our results indicate that the increased keratinocyte proliferation induced by UVB irradiation is accompanied by a decrease in SOD activity, and that this decrease is mainly due to a decreased Cu,Zn-SOD activity. No alteration in SOD activity was noted during the initial hypoproliferative phase following irradiation.

Superoxide Dismutase in Epidermis: Its Relation to Keratinocyte Proliferation

Decreased superoxide dismutase (SOD) activity has been reported in various hyperproliferative tissues. In order to elucidate the significance of SOD activity in keratinocyte proliferation, we measured the SOD activity in several pathologic epidermal conditions. The SOD activity was significantly decreased in psoriatic hyperproliferative epidermis as well as in basal cell epithelioma, squamous cell carcinoma and seborrheic keratosis.

Superoxide dismutase in epidermis (1).

Superoxide dismutase (SOD), which catalytically scavenges superoxide anion (O–2), constitutes an essential defense against the toxicity of oxygen. We investigated the enzyme activity of pig skin epidermis. SOD activity was determined by monitoring the inhibitory effect of SOD on red formazan formation from neotetrazolium, which depends on (O–2) generation. (O–2) was generated by the hypoxanthine‐xanthine oxidase reaction. Pig epidermis contained significant amounts of heat‐labile SOD activity which was proportional to the added epidermal homogenate. The optimal pH of the reaction was between pH 8.2 and 8.5. Metallochelating agents such as cyanide, sodium azide, and diethyldithiocarbamate (DDC) inhibited the epidermal SOD activity in a dose‐dependent manner. It has been known that two types of SOD, a Cu, Zn‐type and a Mn‐type, are present in eukaryotes; that the latter is insensitive to cyanide inhibition. Using this property, the main SOD present in the epidermis was hypothesized to be the Cu, Zn‐type (8.6 ±1.1 unit/mg protein; around 75%); the Mn‐type was a minor component (2.8 ± 0.2 unit/mg protein; around 25%). SOD staining following acrylamide disc gel electrophoresis revealed two epidermal SOD bands, one of which was abolished by the addition of cyanide. These results are consistent with the view that pig epidermis contains two types of SOD, a Cu, Zn‐type and a Mn‐type; the former appears to be predominant.

Effects of UVB irradiation on epidermal adenylate cyclase responses in vitro: its relation to sunburn cell formation.

SummaryUVB irradiation augmented the betaadrenergic adenylate cyclase response of pig skin epidermis in vitro. The effect was observed 2–4 h following the irradiation and lasted at least for 48 h. There was no significant difference in cyclic AMP phosphodiesterase activity between control and UVB-irradiated epidermis at lower irradiation dose (150 mJ/cm2), which is the dose of the most marked beta-adrenergic augmentation effect. The augmentation effect was specific to the beta-adrenergic system; adenosine and histamine adenylate cyclase responses were unchanged or decreased depending on the irradiation dose. Histologically, marked sumburn-cell formation was observed following the UVB irradiation.It has been suggested that oxygen intermediates generated by ultraviolet radiation participate in sunburncell formation. The addition of superoxide dismutase (SOD) in the incubation medium significantly inhibited sunburn-cell formation. On the other hand, the beta-adrenergic augmentation effect was not affected by the addition of SOD. Other scavengers of oxygen intermediates (catalase, catalase+SOD, xanthine, or mannitol) did not inhibit the UVB-induced beta-adrenergic augmentation effect. Further, superoxide-anion generating systems (hypoxanthine-xanthine oxidase system and acetaldehyde-xanthine oxidase system) revealed no stimulatory effect on the beta-adrenergic response of epidermis.These results indicate that (a) the UVB-induced beta-adrenergic augmentation effect is inherent to skin and does not depend on systemic factors such as inflammatory infiltrates following UVB irradiation; (b) in contrast to sunburn-cell formation, induction of the beta-adrenergic adenylate cyclase response is not directly associated with oxygen intermediates generated by UVB irradiation.

Analysis of psoriatic patients registered in Asahikawa Medical College Hospital from 1983 to 2007

Psoriasis is a chronic inflammatory skin disease, which has been increasing during the last 50 years in Japan. The aim of the present study is to analyze psoriatic patients registered from 1983–2007 in Asahikawa Medical College Hospital, which is located in the northern part of Japan. A total of 607 cases were registered at the first inspection in the Department of Dermatology, Asahikawa Medical College. Men (403 cases, 66.4%) were predominant over women (204 cases, 33.6%). The clinical types of psoriasis were psoriasis vulgaris (91.5%), guttate psoriasis (4.2%), psoriasis arthropathica (2.8%), psoriatic erythroderma (0.6%), generalized pustular psoriasis (0.6%), localized pustular psoriasis (0.15%) and infantile psoriasis (0.15%). Topical corticosteroids (78.1%) and vitamin D3 (18.1%) products were the main previous topical agents. Previous systemic treatments included etretinate (7.7%), cyclosporine (1.5%) and methotrexate (0.3%). Use of topical vitamin D3 and cyclosporine therapies have been gradually increasing during the past 25 years. Regarding the previous phototherapy, topical psoralen and ultraviolet A therapy (PUVA) (4.9%) was predominant over ultraviolet B (0.9%), and systemic PUVA (0.7%). Use of ultraviolet B phototherapy has been increasing during the past 5 years. The results are essentially similar to those of a survey of psoriasis in Japan from 1982–2001. Although the incidence of psoriasis might be higher in Hokkaido Prefecture, there is essentially no variation in the disease profile of psoriatic patients.

Modulation of pig epidermal adenylate-cyclase responses by protein-synthesis inhibitors: Its relation to glucocorticoid and colchicine effects

SummaryThe effects of protein-synthesis inhibitors (actinomycin D, puromycin, and cycloheximide) on epidermal adenylate-cyclase responses were investigated. When pig skin (epidermis) was incubated in RPMI-1640 medium, the β-adrenergic adenylatecyclase response (epinephrine-induced cyclic-AMP accumulations) decreased, whereas the adenosine and histamine responses increased after long-term (up to 48 h) incubation. The addition of actinomycin D or puromycin to the incubation medium resulted in a marked increase in epinephrine-induced cyclic-AMP accumulations and a decrease in adenosine- and histamine-induced cyclic-AMP accumulations. Cycloheximide had a weak effect on the epinephrine response, and had apparently stronger effects on the adenosine and histamine responses than actinomycin D or puromycin. Histologically, various degenerative changes of keratinocytes (with or without acantholytic changes) were observed after long-term incubation with these protein-synthesis inhibitors. Both low- and high-Kmcyclic-AMP phosphodiesterase activities were moderately decreased by the protein-synthesis inhibitors. However, augmentation effects on the β-adrenergic response were also observed in the presence of the cyclic-AMP phosphodiesterase inhibitor, theophylline. We have described previously similar augmentation effects on the β-adrenergic response caused by glucocorticoids and colchicine. Comparison of the effects of these chemicals with those of protein-synthesis inhibitors revealed that the most marked effects on the β-adrenergic response were produced by actinomycin D, puromycin and colchicine; glucocorticoid had a moderate effect (hydrocortisone), while cycloheximide had only a weak effect. Furthermore, the simultaneous addition of actinomycin D (or puromycin) and colchicine (or hydrocortisone) at their optimal concentrations produced a more marked (additive or synergistic) increase in the β-adrenergic response than the addition of each chemical alone. The simutaneous addition of actinomycin D and puromycin at their optimal concentrations resulted in neither an additive nor a synergistic effect. Our results indicate that, as well as being affected by glucocorticoids and colchicine, epidermal adenylatecyclase responses are affected by various protein-synthesis inhibitors, the mechanism of which seems to be independent of that of glucocorticoids or colchicine.

Reversible Inhibition of Keratinocyte Thymidine Incorporation by the Calmodulin Antagonist, W-7

Although calmodulin has been suggested as an important regulator of keratinocyte proliferation, its precise role remains unknown. We employed a calmodulin antagonist, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), to examine the role of calmodulin on keratinocyte proliferation. N‐(6‐aminohexyl)‐1‐naphthalenesulfonamide (W‐5), a chlorine‐deficient analogue of W‐7 with little anti‐calmodulin activity, was used as the control. W‐7 markedly inhibited thymidine incorporation of pig epidermis at concentrations close to its anti‐calmodulin activity; W‐5 had no effect on the thymidine incorporation. The inhibitory effect of W‐7 was reversible; the removal of W‐7 from the incubation medium resulted in the reinitiation of the thymidine incorporation, suggesting that W‐7 is not a cytotoxic agent. These results are consistent with the view that calmodulin is an essential regulator of keratinocyte proliferation. The epidermal beta‐adrenergic response, which is decreased in various hyperproliferative epidermal abnormalities, was increased in W‐7‐treated hypoproliferative epidermis. The epidermal SOD activity, which is also decreased in the hyperproliferative epidermis, however, was not affected by the W‐7 treatment.

Effects of retinoids on DNA synthesis of pig epidermis: Its relation to epidermal beta-adrenergic adenylate cyclase response and to epidermal superoxide dismutase activity

It has been suggested that the epidermal beta-adrenergic adenylate cyclase response and the epidermal superoxide dismutase (SOD) activity is inversely associated with keratinocyte cell proliferation. Effects of various retinoids on the thymidine incorporation of pig epidermis were compared with their effects on the beta-adrenergic response and the SOD activity. Following 24 h incubation with synthetic retinoids (etretin and E-5166 (3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid)), thymidine incorporation of epidermis was significantly decreased. The effect of etretin was more potent than that of E-5166; the former revealed the inhibitory effect at lower concentrations than the latter. The effect of etretinate was not statistically significant. Following the 24 h incubation with the synthetic retinoids, the epidermal beta-adrenergic adenylate cyclase responses were increased. Etretin was again more potent than E-5166, while etretinate showed also little effect on the beta-adrenergic response of epidermis. Thus the inhibitory effect on the thymidine incorporation was inversely correlated with the beta-adrenergic augmentation effect among these synthetic retinoids. On the other hand, physiologic retinoids (retinol and retinoic acid) revealed no correlation between these two parameters; whereas both compounds decreased the thymidine incorporation to a similar extent, only retinoic acid revealed a marked beta-adrenergic augmentation effect. Decreased SOD activity has been observed in various hyperproliferative epidermis. The SOD activity, however, was totally unaffected by the retinoid-treatments.

Experimental hyperkeratosis. Effects of topical Vitamin A ointment in vivo.

The following conclusions will be drawn from the data obtained in this study. 1) Topical VA ointments which contain 5,000 IU and 10,000 IU VA respectively could not prevent n‐hexadecane‐induced epidermal proliferation and hyperkeratosis during the period studied. 2) After the epidermal hypertrophy and hyperkeratosis were established, continuous application of VA ointments of 5,000 IU and 10,000 IU resulted in even greater activity of the enzymes than those observed with the control (hydrophilic ointment alone). 3) Judging from the data obtained from the present study, VA ointment can be useful in the treatment of hyperkeratotic conditions characterized by the normal or decreased epidermal proliferation, because they promote the epidermal metabolic rate.