Yasuko Shindo

TIME COURSE OF CHANGES IN ANTIOXIDANT ENZYMES IN HUMAN SKIN FIBROBLASTS AFTER UVA IRRADIATION

Examining damage (inactivation) of antioxidant enzymes in the cells and the pattern of recovery after a single UV exposure might be a useful method for analyzing the mechanisms of chronic UV irradiation, because chronic UV irradiation means repeated single exposures. We irradiated human skin fibroblasts with a single exposure to UVA (1, 6 or 12 J/cm2) and examined the time course of changes in antioxidant enzymes over several days. Only catalase activity was inactivated at the end of the irradiation (66% for 6 J/cm2, 53% for 12 J/cm2), recovering by day 5. Superoxide dismutase (SOD) activity decreased on day 3 (63% for 6 J/cm2, 72% for 12 J/cm2), and recovered on day 5, although it was not changed at the end of exposure. The activities of glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were nearly unchanged by irradiation. If repeated UV exposures occur before the inactivated enzyme activities recover, cellular damage will be significant due to the lowered antioxidant defense system. We examined the rates of synthesis and degradation of catalase in response to UVA irradiation. Both synthesis and degradation rates were changed by irradiation. These data indicate that catalase activity was still low on day 2 due to the existence of inactivated catalase produced at the end of UV irradiation, and catalase activities recovered by day 5 due to a significant increase in the synthesis rate. To elucidate the role of bound NADPH in catalase in response to UV irradiation, we measured the NADPH released from catalase after UVA irradiation using bovine liver catalase. UVA irradiation caused a release of NADPH from catalase (25% for 12 J/cm2), but this was not directly related to the inactivation of catalase.

Ultraviolet B-induced cell death in four cutaneous cell lines exhibiting different enzymatic antioxidant defences: involvement of apoptosis

Reactive oxygen species (ROS) play a role in the modulation of apoptosis. Antioxidant defence mechanisms against cell death involving apoptosis due to UVB irradiation were studied on three established cell lines (SCC derived from human skin squamous cell carcinoma, F-SV and F-ST derived from human skin fibroblasts) which were susceptible to cell death by UVB irradiation (12.5-250 mJ/cm2), and one cell line (N-F) derived from primary cultured human skin fibroblasts which was resistant to cell death. We compared antioxidant defences between the three established cell lines and N-F, measuring four antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) and a non-enzymatic antioxidant glutathione. The greatest difference was that Cu, Zn-SOD activity in N-F was 3-4-times the three other cell lines. Though SCC had much larger amounts of glutathione and higher antioxidant enzyme activities except for Cu, Zn-SOD than N-F, SCC was very susceptible to cell death. After UVB irradiation (at 16 h after 12.5 mJ/cm2), in all cell lines, SOD activity increased 1.1-1.3-times that of non-irradiated cells, while other enzyme activities remained constant. This presumably represents a protective response against ROS generated during UVB irradiation. N-F which was resistant to UVB-induced cell death had higher Cu, Zn-SOD activity before UVB irradiation, and a larger increase of SOD (mainly Mn-SOD) after UVB exposure than the other cell lines which were susceptible to cell death. Therefore, we conclude that the most important enzymatic antioxidant to protect cells from UVB damage is SOD.

Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence

A 23-year-old man with recurrent myoglobinuria had low muscle-free carnitine levels and deficient fasting ketogenesis. Urinary organic acid analysis showed large amounts of C6-C14 3-hydroxydicarboxylic acids. Mitochondrial trifunctional protein (TP), harboring long-chain enoyl-coenzyme A (CoA) hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase showed markedly decreased activity in fibroblasts. On immunoblot analysis, the TP content of his fibroblasts was less than 2% that of the control cells. TP deficiency can be a life-threatening disorder with early infantile onset, but it can also present in adolescence with recurrent myoglobinuria.

CHANGES IN ENZYME ACTIVITIES IN SKIN FIBROBLASTS DERIVED FROM PERSONS OF VARIOUS AGES

We examined antioxidant enzyme activities (catalase, glutathione peroxidase, and superoxide dismutase) in cultured skin fibroblasts (passage number 2-3) derived from 30 persons of various ages. With increasing ages, catalase activity decreased, glutathione peroxidase activity increased slightly, and superoxide dismutase activity was unchanged. After UVA irradiation (4.8 joule/cm2) of the fibroblasts, only catalase activity decreased by 70%. This suggests that catalase may play an important role in the aging of human skin fibroblasts.

Antioxidant defence mechanism of the skin against UV irradiation: Study of the role of catalase using acatalasaemia fibroblasts

To clarify the role of catalase, an antioxidant enzyme, in response to UV irradiation, we compared the effects of irradiation on cytotoxicity, activities of antioxidant enzymes, total glutathione concentrations, lipid peroxidation and the rate of collagen synthesis in skin fibroblasts from a patient with acatalasaemia and in those from a normal individual. The cells were irradiated with UVA (6 and 12 J/cm2 or UVB (0.5 and 1 J/cm2). Cell survival curves after UV irradiation were similar in cells from both subjects. Although superoxide dismutase activity in acatalasaemia cells was higher than in the control cells before irradiation, after irradiation the activity decreased in acatalasaemia cells (76% with 12 J/cm2 UVA, 47% with 1 J/cm2 UVB), but remained unchanged in control cells. Total glutathione concentrations also decreased in acatalasaemia cells (60% with 12 J/cm2) in response to UVA irradiation, but remained unchanged in control cells. Lipid peroxidation did not increase significantly in either cell type. The rate of collagen synthesis decreased to a similar extent in response to UV exposure in the two cell types (60–80% with 8.2 J/cm2 UVA, 40–50% with 10 mJ/cm2 UVB). We conclude from the results of cytotoxicity and lipid peroxidation that although acatalasaemia cells were killed by hydrogen peroxide at low concentrations with a single UV exposure, catalase functions only to a small degree as an antioxidant enzyme. There remains the possibility, however, that a deficiency of catalase may chronically damage the skin resulting in a reduced defence function of Superoxide dismutase and glutathione with repeated exposures to UV, which is becoming more common in our daily life.

The effect of tryptophan administration on fatty acid synthesis in the liver of the fasted normal rat

Abstract 1. 1. Tryptophan was administered to the fasted normal rats. The rates of the incorporation of radioactivities from tritiated water and [1- 14 C]acetate into hepatic fatty acids in vivo were enhanced about 2-fold by the treatment. 2. 2. Contents of intermediates in relation to the changes in carbohydrate and fatty acid metabolisms were determined. 3. 3. The rate of hepatic fatty acid synthesis was closely related to the content of citrate in the tissue. 4. 4. Hepatic pyruvate dehydrogenase was converted into an inactive form by tryptophan administration. The level of the activity of the enzyme was found to be related to the increase in fatty acid synthesis and accumulation of intermediates of the tricarboxylic acid cycle.

VERRUCIFORM XANTHOMA OF THE SCROTUM

We report two cases of verruciform xanthoma of the scrotal skin. In each case, the tumor was a single pink lesion with firm consistency and a granular surface. Each lesion was characterized histopathologically by papillomatous proliferation of the epidermis and dermis and dense foam‐cell infiltration of the region corresponding to the dermal papillary layer. These cells proved to be xanthoma cells that stained negatively or weakly positively with PAS, colloidal iron, and Alcian blue and strongly positively with Sudan III. No medical problems were found in either patient, and serum lipid levels were normal. This report also presents electron microscopic findings from scrotal verruciform xanthoma in one case and the results of lipid analysis of tumor tissue from both cases.

Squamous Cell Carcinoma Developing in Thermal Keratosis

Two cases of squamous cell carcinoma (SCC), both of them developed from thermal keratosis arising in areas of erythema ab igne (EAI) are reported. The lesions were located on the legs of two elderly Japanese women who had histories of chronic exposure to heat from a “kotatsu”, a type of leg‐warmer widely used in Japan. In one case, metastasis to regional lymph nodes occurred. It is emphasized that keratotic lesions arising in an area of EAI should be excised or biopsied because of the possibility of metastasizing SCC developing from them.

Cutaneous Infection Caused by Fusarium solani and Fusarium oxysporum

A case of cutaneous infection by Fusarium solani and Fusarium oxysporum on the middle finger of the right hand of a 58‐year‐old farmer is reported. On the extensor aspect of the distal phalanx of the middle finger, there was a pale reddish, edematous lesion with some vesicles, pustules, and an ulcer. Scrapings from the lesion showed fungal elements on direct microscopic examination, and Fusarium solani and Fusarium oxysporum were identified on slide culture. Hyperthermia treatment with a chemical pocket warmer led to conspicuous healing of the lesion.

An acantholytic variant of seborrheic keratosis.

A case of an acantholytic seborrheic keratosis, first reported by Tagami and Yamada, was seen in a 63‐year‐old woman. It appeared to belong to the irritated type of Levers classification.