Rex M. Tyrrell

UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen.

This study describes the damage that occurs to lipids and proteins that have been irradiated in vitro or in human skin fibroblasts with physiological doses of UVA radiation. Thiobarbituric acid-reactive species were formed from phosphatidylcholine after UVA radiation in vitro. By using iron chelators, this process was shown to involve iron. Ferric iron associated with potential physiological chelators was reduced by UVA radiation, but iron within ferritin was not. By enhancing the half life-time with deuterium oxide or by using scavengers, singlet oxygen was also shown to be involved in the UVA radiation-dependent peroxidation of phosphatidylcholine. UVA radiation-generated singlet oxygen reacted with phosphatidylcholine to form lipid hydroperoxides, and the breakdown of these hydroperoxides to thiobarbituric acid-reactive species was dependent on iron. We have shown that iron and singlet oxygen are also involved in the UVA radiation-dependent formation of thiobarbituric acid-reactive species in human skin fibroblasts, and we propose that a similar concerted effect of iron and singlet oxygen is involved in UVA radiation-dependent damage to fibroblast lipids. Sulphydryl groups of bovine serum albumin and human gamma-globulin were oxidised upon UVA irradiation in vitro. The use of scavengers and deuterium oxide showed that UVA radiation-dependent sulphydryl oxidation was dependent on singlet oxygen. By adding or chelating iron, UVA radiation-dependent oxidation of sulphydryl groups of bovine serum albumin and human gamma-globulin was shown to be iron-dependent. The use of catalase and hydroxyl radical scavengers demonstrated that hydrogen peroxide, but not the hydroxyl radical, was involved. The oxidation of sulphydryl groups of proteins in human skin fibroblasts that occurs as a result of UVA irradiation was also shown to involve iron, singlet oxygen, and hydrogen peroxide. We conclude that iron, singlet oxygen, and hydrogen peroxide are important redox active species involved in the deleterious effects of UVA radiation on lipids and proteins of human skin cells.

New trends in photobiology the interaction of UVA radiation with cultured cells

Recent work concerning the interaction of UVA radiation (320-380 nm) with cultured cells is reviewed with particular emphasis on the involvement of cellular oxidative stress in the biological effects of this radiation on eucaryotic cells. Possible chromophores are considered and their role in generation of various oxidant species including hydrogen peroxide, superoxide anion, singlet oxygen and hydroxyl radical. DNA and membranes are discussed as possible targets for the lethal action of long wavelength radiation. Four mechanisms of cellular defence are proposed: (1) DNA repair; (2) antioxidant enzymes; (3) endogenous free radical quenchers; (4) inducible protection.

ACTIVATION OF NF‐KB IN HUMAN SKIN FIBROBLASTS BY THE OXIDATIVE STRESS GENERATED BY UVA RADIATION

We have examined the role of the nucleus and the membrane in the activation of nuclear factor (NF)‐KB by oxidant stress generated via the UVA (320–380nm) component of solar radiation. Nuclear extracts from human skin fibroblasts that had been irradiated with UVA at doses that caused little DNA damage contained activated NF‐KB that bound to its recognition sequence in DNA. The UVA radiation‐dependent activation of NF‐KB in enucleated cells confirmed that the nucleus was not involved. On the other hand, UVA radiation‐dependent activation of NF‐KB appeared to be correlated with membrane damage, and activation could be prevented by a‐tocopherol and butylated hydroxytol‐uene, agents that inhibited UVA radiation‐dependent peroxidation of cell membrane lipids. The activation of NF‐KB by the DNA damaging agents UVC (200–290nm) and UVB (290–320nm) radiation also only occurred at doses where significant membrane damage was induced, and, overall, activation was not correlated with the relative levels of DNA damage induced by UVC/UVB and UVA radiations. We conclude that the oxidative modification of membrane components may be an important factor to consider in the UV radiation‐dependent activation of NF‐KB over all wavelength ranges examined.

Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes: implications for protection against oxidant stress.

Abstract— Activation of expression of the heme oxygenase (HO) gene appears to be involved in a cellular defense system in mammalian cells. We now demonstrate that while HO‐1 mRNA levels are strongly inducible in dermal fibroblasts they are barely inducible in human epidermal keratinocytes following oxidative stress (UVA radiation and hydrogen peroxide). Paralleling this result was the observation that HO‐2 mRNA levels were low in dermal fibroblasts but were high in epidermal keratinocytes. In neither case was the HO‐2 gene inducible. The expression of the two HO genes led to enzymatic activity in both types of skin cells with an approximately 2.5‐fold higher level of enzymatic activity present in keratinocytes compared with fibroblasts derived from the same biopsy. In addition, ferritin levels, which have been found to be augmented via the HO‐dependent release of iron from endogenous heme sources, were two‐ to three‐fold higher in keratinocytes compared with matching fibroblasts. This higher ferritin pool would result in an enhancement of cellular iron sequestering capacity that may confer increased resistance to oxidative stress. Indeed, keratinocytes showed less UVA radiation‐dependent cell membrane damage than fibroblasts. These results are consistent with the hypothesis that HO expression in human epidermis and dermis is related to cellular defense mechanisms that operate in human skin.

A common pathway for protection of bacteria against damage by solar UVA (334 nm, 365 nm) and an oxidising agent (H2O2).

Pre-exposure of growing bacterial populations to low concentrations of hydrogen peroxide (H2O2) protects a repair-proficient strain of Escherichia coli (AB1157) very strongly and a rec A strain (AB2463) to a lesser extent from the lethal action of subsequent exposure to 5 mM H2O2 in buffer. The conditioning procedure also protects AB1157 and AB2463 from the toxic effects of UVA (334 nm, 365 nm) radiation but not UVB (313 nm) or UVC (254 nm) radiations. Pretreatment of growing AB1157 with low fluences of UVA (365 nm) radiation leads to the induction of resistance to H2O2, an effect which apparently requires protein synthesis. As in a previous report, the treatment of growing populations with low concentrations of H2O2 enhanced the resistance of such populations to H2O2 challenge in the growth medium. However, when H2O2 (+ Cu2+)-treated bacteriophage were subsequently infected into AB1157 under optimal inducing conditions, their resistance was not enhanced relative to infection into untreated bacteria. We conclude that the primary mechanism for the inducible effects observed could be the induction of H2O2 scavenging activity by low concentrations of H2O2 either introduced into the growth medium directly or produced by low fluences of UVA irradiation.

Dark induction of haem oxygenase messenger RNA by haematoporphyrin derivative and zinc phthalocyanine; agents for photodynamic therapy

Haematoporphyrin derivative is one of the main drugs currently used in clinical trials involving photodynamic therapy of cancer, and zinc phthalocyanine is being considered as one of several possible alternatives. We show that incubation of cultured human fibroblasts populations with either of the two drugs will lead to a sharp increase in the accumulation of the messenger RNA corresponding to haem oxygenase. Only cells incubated with haematoporphyrin derivative show additional enhancement of expression of this specific gene on exposure to red light. Since haem oxygenase induction appears to be a specific stress response that may be involved in cellular defence, such observations should be confirmed under conditions which would allow the clinical implications to be fully evaluated.

Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: Evidence that α- and β-polymerases act sequentially at the repolymerisation step

Abstract We have characterised far-ultraviolet-radiation-induced DNA-repair synthesis in permeabilised arrested (non-dividing) primary human skin fibroblasts. Approximately half the maximum repair synthesis is seen after a UV fluence of 4.0 Jm −2 and little additional incorporation was observed at fluences above 20.0 Jm −2 . UV-damaged permeable cells were treated with specific inhibitors of DNA polymerase α and β, both alone and in combination. The degree of inhibition of repair incorporation by aphidicolin indicates that polymerase α is involved in the majority (85–90%) of repair synthesis after both high and low ( −2 ) UV fluences. Dideoxythymidine triphosphate seems able to inhibit DNA-repair synthesis only when polymerase α is fully or almost fully functional, indicating that polymerase β is unable to substitute in repair for an α polymerase blocked by aphidicolin. These data suggest that the two enzymes may act sequentially to complete repair patches rather than acting independently.