Effects of oxygen tension on the growth and pigmentation of normal human melanocytes.

The effects of oxygen tension on human melanocyte growth, tyrosinase activity, and melanin production were assessed. Melanocytes, seeded at 10(4) cells/cm2, were grown in modified Eagle's medium (MEM) with 5% fetal bovine serum (FBS) and 10 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Flasks were equilibrated with gas mixtures containing 5% CO2 and various partial pressures of oxygen (PO2 7-620 mm Hg) and kept in incubators, which were electronically maintained at the desired oxygen tensions. Melanocytes grew best at PO2 from 6-34 mm Hg. Growth was reduced by 30% at PO2 142 mm Hg, and even more at O2 tensions greater than 230 mm Hg. A PO2 of 603 mm Hg was cytotoxic. Tyrosinase activity (assayed by the method of Pomerantz) was 300 microU/mg protein at PO2 7-34 mm Hg. At PO2 235 and 355 mm Hg tyrosinase activity decreased to about 100 microU/mg protein. The apparent Km for tyrosine was unchanged in melanocytes cultured at all experimental oxygen tensions. The Vmax, however, was decreased at the higher oxygen tensions (PO2 235 mm Hg). At PO2 6-135 mm Hg the melanin content was proportional to tyrosinase activity. At cytostatic oxygen tensions (PO2 235 and 355 mm Hg) the intracellular melanin content increased somewhat, although tyrosinase activity was decreased. Low oxygen tension is favorable for both melanocyte proliferation and tyrosinase activity.

Superoxide dismutase induces differentiation of Friend erythroleukemia cells.

Friend erythroleukemia cells (FELC) served as a model system for cell differentiation because these cells can be triggered to differentiate by a variety of chemical agents. Treatment with the classical inducer of differentiation, hexamethylene bisacetamide (HMBA), stimulated superoxide dismutase (SOD) activity, which increased in parallel with HMBA-induced differentiation. Furthermore, FELC were shown to differentiate in response to the addition of liposomes containing SOD. Oxidative treatment with liposomes containing D-amino acid oxidase or xanthine oxidase, cumene peroxide, or potassium superoxide also induced differentiation, whereas antioxidants such as alpha-tocopherol, butylated hydroxytoluene, or beta-carotene did not induce differentiation. Also, HMBA induction of differentiation was suppressed by treatment with antioxidants.

Physiological consequences of human skin aging.

The expression and treatment of cutaneous disease in the elderly differ from those applicable to younger adults. Anatomical changes in aging skin result in altered physiological behavior and susceptibility to disease. Decreased epidermal renewal and tissue repair accompany the aging process. The rate of hair and nail growth declines, as well as the quantity of eccrine, apocrine, and sebum secretion. There are alterations in immune surveillance and antigen presentation with aging. The cutaneous vascular supply is decreased, leading to decreases in inflammatory response, absorption, and cutaneous clearance. Impaired thermal regulation, tactile sensitivity, and pain perception occur as one ages. We summarize the major changes that occur during the intrinsic aging process of the skin to facilitate the recognition and treatment of skin disease in the older patient.

Molecular bases of biologic aging.

The authors outline the progression of thought on the mechanism of the aging process, giving emphasis to environmental factors that influence genetic events. Discussion is limited to those theories that explain fundamental causes of aging and have a firm thermodynamic basis. The authors propose that the cumulative result of continual oxidative stress and other thermodynamic processes (such as aminoacid racemization and nonenzymatic glycosylation), resulting in altered function and increasing the net entropy of living systems, governs the rate of the aging process.

Nonmelanoma skin cancers in the elderly.

This article discusses the relationship between aging and development of nonmelanoma skin cancer. Possible age-related risk factors for development of such cancers are cumulative exposure to ultraviolet irradiation from the sun, decreased DNA repair capacity, decreased immunosurveillance, reduced melanocyte density, and altered dermal matrix. Clinical features of basal-cell carcinoma, squamous-cell carcinoma, and keratoacanthoma are discussed.

Oxidative influence on development and differentiation: an overview of a free radical theory of development.

Metabolic gradients exist in developing organisms and are believed to influence development. It has been postulated that the effects of these gradients on development result from differential oxygen supplies to tissues. Oxygen has been found to influence the course of development. Cells and tissues in various stages of differentiation exhibit discrete changes in their antioxidant defenses and in parameters of oxidation. Metabolically generated oxidants have been implicated as one factor that directs the initiation of certain developmental events. Also implicated as factors that modulate developmental processes are the cellular distribution of ions and the cytoskeleton both of which can be influenced by oxidants. The interaction of oxidants with ion balance and cytoskeleton is discussed.

Developmental changes in the superoxide dismutase activity of human skin fibroblasts are maintained in vitro and are not caused by oxygen.

Confluent cultures of human skin fibroblast lines established from fetal and postnatal donors were exposed to a broad range of oxygen tensions (10-600 mmHg) for 1 wk; superoxide dismutase (SOD) activity was subsequently determined. Hyperoxia increased SOD activity slightly in postnatal lines but not in fetal lines. The magnitude of the increase in postnatal lines was not significant. Fetal lines exhibit only about one-fifth the SOD activity observed in postnatal lines. The results indicate that, while development-associated changes in SOD do occur in human cells, these alterations do not result from variations in ambient oxygen tension.

Normal replicative lifespan of Alzheimer skin fibroblasts.

Cultured skin fibroblasts from four patients with Alzheimer's disease had life spans comparable to those of six age-sex matched controls, whether measured by passages to phase-out, cumulative population doublings to phase out, or percentage of nuclei incorporating [3H]thymidine (Cristofalo index). These results provide direct experimental evidence that Alzheimer's disease is not simply a form of accelerated aging. They suggest that the abnormalities, described by several groups, in Alzheimer fibroblasts reflect the disease rather than the physiological age of the donor, making the cultured cell a valid tool for studying the cellular pathophysiology of this disorder. Together with other data, these observations raise the possibility that some forms of Alzheimer's disease may represent inborn error(s) of metabolism of late clinical onset.

Superoxide dismutase induces differentiation in microplasmodia of the slime mold Physarum polycephalum.

Evidence is presented that supports a role for the enzyme superoxide dismutase (SOD) in the differentiation of the slime mold, Physarum polycephalum. SOD activity increases 46-fold during differentiation. A strain of Physarum that does not differentiate exhibits no change in SOD activity. Addition of SOD, via liposomes, to the nondifferentiating strain induces differentiation; this effect is enhanced by an inhibitor of glutathione synthesis. Other antioxidants selected for study failed to induce differentiation. Conversely, oxidative treatments including introduction of D-amino acid oxidase, via liposomes, induced differentiation. Cellular oxidation is the probable cause of the SOD effect.

Superoxide dismutase activity and glutathione concentration during the calcium-induced differentiation of Physarum polycephalum microplasmodia.

Microplasmodia of Physarum polycephalum differentiate into spherules when the CaCl2 concentration of their nutrient medium is increased to 54mM (high-calcium). The salts starvation medium routinely used to induce differentiation contains 8mM CaCl2. This medium will not induce spherulation in the absence of a calcium salt; no other metal is essential. High-calcium also induces the spherulation of a strain of Physarum that had not been previously observed to spherulate. The striking increase in superoxide dismutase activity (SOD) and the decrease in glutathione concentration (GSH) that are characteristic of salts-induced spherulation do not occur in salts media containing high-calcium. In the absence of calcium, no significant change in SOD is observed and very little change in GSH occurs. The immediate effect of the oxidative stress associated with spherulation may be the release of calcium stores into the cytosol. The parameters modulating this stress are, in turn, sensitive to exogenous calcium concentrations.

Effect of mupirocin on the growth and lifespan of human fibroblasts.

We have examined the effect of the antibiotic mupirocin on the growth and proliferative lifespan of human fibroblasts. Human embryonic lung fibroblasts were grown in Dulbecco's minimal essential medium, containing 10% fetal bovine serum and various concentrations of mupirocin ranging from 0.5-756 micrograms/ml. There is little difference in the rate of exponential growth and the final saturation density reached between control cultures and those grown in 0.5 microgram, 72 micrograms, or 76 micrograms/ml mupirocin. Growth was retarded or inhibited in cultures containing 650-750 micrograms/ml mupirocin. The proliferative lifespan of adult skin fibroblasts was examined in cultures serially subcultivated in medium containing either 7 micrograms/ml or 100 micrograms/ml mupirocin. Cultures incubated in these concentrations of mupirocin were capable of reaching their full proliferative potential. This study shows that the growth and proliferative lifespan of human fibroblasts is unaltered in the presence of at least 100 micrograms/ml mupirocin although growth of human fibroblasts is inhibited by 700 micrograms/ml mupirocin. These observations suggest that long-term use of low but bactericidal concentrations of mupirocin will not impair growth of fibroblasts in healing wounds.

Mechanisms of biologic aging.

The authors outline the progression of thought on the mechanism of the aging process, giving emphasis to environmental factors that influence genetic events. Discussion is limited to those theories that explain fundamental causes of aging and have a firm thermodynamic basis. The authors propose that the cumulative result of continual oxidative stress and other thermodynamic processes (such as amino-acid racemization and nonenzymatic glycosylation), resulting in altered function and increasing the net entropy of living systems, governs the rate of the aging process.

Effect of oxygen on the growth of human epidermal keratinocytes.

We studied the growth of secondary cultures of neonatal human keratinocytes at oxygen concentrations between 1 and 89%. Keratinocytes were grown in MCDB medium with 5% fetal bovine serum, 10 ng/ml epidermal growth factor, 5 micrograms/ml insulin, 0.5 microgram/ml hydrocortisone, and 0.1 mM ethanolamine and phosphorylethanolamine. Medium in the flasks was equilibrated with gas mixtures containing 5% CO2, various percentages of oxygen from 0-95% and nitrogen to balance. Cells were seeded at 10(4) cells/cm2 in sealed flasks (25 cm2). These were incubated at 37 degrees C in incubators maintained at the experimental oxygen tensions. Cells grew best at PO2 (partial pressure of oxygen) 133 mm Hg (18% O2), with a mean population doubling time of 2.8 days. Growth was retarded by 60% at PO2 38 mm Hg (5% O2) and by 98% at PO2 7 mm Hg (1% O2). However, the oxygen tension that resulted in the best plating efficiency was at PO2 12 mm Hg (2% O2). When oxygen tensions were shifted to 78-133 mm Hg, cells seeded under low oxygen tensions began to proliferate. These data suggest that a better harvest of keratinocytes is obtained when cells are seeded under low oxygen tension and then shifted to ambient oxygen tensions. At high oxygen tensions, above 20%, growth was inhibited by 75% at PO2 241 mm Hg (34% O2) and 98% at PO2 374 mm Hg (52% O2). At PO2 637 mm Hg (89% O2) no cell growth occurred. These findings showed that high oxygen tensions, above 20%, have no beneficial effect on the growth of keratinocytes.

Grafting of skin ulcers with cultured autologous epidermal cells.

We treated five adult individuals with six full-thickness chronic ulcerations in the skin caused by venous insufficiency, sickle cell anemia, or surgical wounds. Each patient received applications to the ulcerations of sheets of autologous epidermal cells grown in culture. All patients experienced relief of pain after grafting. Four of the six ulcers healed completely in 21 to 35 days, and three of the four remained healed for up to 2 years. One ulceration recurred within 2 months. Our experience suggests that cultured autologous epidermal grafts can provide continuous covering, relief from pain, and rapid healing of chronic debilitating ulcerations of the skin.

Local environment of chronic wounds under synthetic dressings.

Local wound environment under oxygen-permeable and oxygen-nonpermeable dressings in patients with chronic ulcers was investigated. The oxygen tensions under both these dressings were very low or zero. Wound fluid was more acidic under the nonpermeable hydrocolloid dressing than under the oxygen-permeable polyurethane dressing. Bacterial growth studied in vitro was retarded at the more acidic pH similar to that found under the hydrocolloid dressing. Viable and functioning neutrophils were found under both the polyurethane and hydrocolloid dressings, with a greater percentage of viable cells under the polyurethane film. Our data suggest that these synthetic dressings create hypoxic conditions in which wound healing occurs whether or not the dressing is permeable to oxygen. Furthermore the local wound environment can be modified by use of synthetic dressings.