Journal of Photochemistry
B: Biology, 4 (1990)
NEW TRENDS IN PHOTOBIOLOGY (Invited Review) THE INTERACTION OF UVA RADIATION WITH CULTURED CELLS R. M. TYRRELL
and S. M. KEYSET
ISREC, Ch. des Boveresses, CH-1066 Epalinges (Switzerland) (Received
Keywords. UVA, oxidative
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.
1. Introduction Solar ultraviolet radiation can be conveniently divided into two wavelength regions usually designated as UVB (290 - 320 nm) and UVA (320 380 nm). The UVB region overlaps with the tail of DNA absorption and it is believed to be this component of sunlight which is mainly responsible for the majority of human skin cancers. Nevertheless, the UVA region of sunlight is potentially carcinogenic and is certainly involved in photoaging so that there are good reasons to challenge the assumption that the long-term exposures to sunlight that are permitted by the use of UVB-absorbing sunscreens or the high level exposures of UVA delivered by modern tanning equipment are without risk [ 11. The scientific literature now includes an increasing number of studies concerning the cellular effects of these radiations. The purpose of ?resent address: Potters Bar, Herts. EN6 loll-1344/90/$3.50
ICRF Clare 3LD, U.K.
in The Netherlands
this review is to highlight certain recent findings which provide new insights into the interaction of solar radiation with cells and in particular the action of UVA on mammalian cells. Special emphasis will also be placed on the available information suggesting that solar UV radiation constitutes an important oxidative challenge to cells which must be met by the available antioxidant defence systems. Considerable information has been derived from studies with bacteria and bacteriophage and these will be mentioned where appropriate. However, no attempt has been made to cover all the literature, either past or present, in such a short overview. Prior to the last decade, most studies of the action of UVA and UVB radiation on cells employed bacteria, in particular Escherichiu coli, and this is reflected in several major reviews on the topic [2 - 41. More recent progress on studies with bacteria has been reviewed by Eisenstark . Several key observations were made very clearly on in the history of this research. Hollaender [ 61, in his pioneering studies, demonstrated that UVAirradiated bacteria became sensitive to salt solutions, thereby providing the first indication that membrane damage could be involved in UVA effects. It was also shown by Webb  that the lethal action of UVA on bacteria is strongly dependent upon the presence of oxygen. A similar oxygen dependence for UVA lethality was later found in mammalian cells . Related studies showed that hydrogen peroxide can be generated by the irradiation of tryptophan with UVA , thus providing at least one pathway for generation of active oxygen species by UVA. Despite these original observations, relatively little attention has been given to the obvious central role of oxygen in UVA effects until recently. A flurry of studies in the mid 1970s demonstrated that broad spectrum UVA was a mild mutagen to mammalian cells. In the following years an increasing number of laboratories employed cultured mammalian cell populations with particular emphasis on studies of DNA damage. Despite this work it is still not clear whether DNA is an important target for the lethal action of UVA in mammalian cells or which specific lesions are responsible. 2. UVA chromophores
and the generation
of active oxygen
Both DNA and proteins absorb UVA extremely weakly and, although direct photochemical events may occur, it appears most likely that the initial event in the biological effects of UVA radiation is absorption by a non-DNA chromophore which results in the generation of active oxygen species or energy transfer to the critical target molecules. Most of our knowledge of such potential chromophores has been obtained from in vitro experiments or from bacteria and is summarised in the recent review by Eisenstark [ 51. Indirect evidence has indicated a role for porphyrins in the UVA inactivation of Propionibacterium acnes . It has also been shown that E. coli mutants defective in the synthesis of &aminolevulinic acid are resistant to UVA inactivation [lo] thereby strongly suggesting that porphyrin components of the respiratory chain may act as
endogenous photosensitisers. This was supported by the finding that cytochrome overproducing strains were sensitive to broad-band UVA radiation [ll]. Porphyrins are also essential to human cellular metabolism and overproduction of iron-free porphyrins in erythropoietic or hepatic tissue is the underlying cause of the skin photodestruction seen in the group of diseases known as porphyrias. Although direct evidence is lacking, free porphyrins and heme-containing proteins (such as catalase, peroxidases and cytochromes) are also potentially‘ important chromophores in skin cells from normal individuals. Many other cellular compounds which contain unsaturated bonds such as flavins, steroids and quinones should also be considered as potential chromophores. Although normal levels of catalase (which contains heme) and alkyl hydroperoxide reductase (which contains flavin adenine dinucleotide) would be expected to exert a protective role in bacteria (see below), the overproduction of these enzymes is correlated with an increase in sensitivity to UVA irradiation [ 121. Porphyrins are an important class of photodynamic sensitisers which are believed to exert their biological action via the generation of singlet oxygen. Recent experiments have shown that deuterium oxide (which extends singlet oxygen lifetime) sensitises human fibroblast cell populations to the lethal action of UVA radiation while sodium azide (which quenches singlet oxygen), protects them . Although this is consistent with porphyrin involvement in UVA lethality, other cellular compounds may also generate singlet oxygen. It is also important to consider other active oxygen species which may be generated intracellularly. In addition to the generation of hydrogen peroxide by UVA irradiation of tryptophan , both superoxide anion and hydrogen peroxide can be generated by photo-oxidation of NADH and NADPH [ 14,151. The involvement of hydrogen peroxide in UVA effects has been given considerable attention. One reason is that naturally occurring iron complexes in viuo will react with this oxidant to generate the highly reactive hydroxyl radical [16, 171 in what is known as a Haber-Weiss (or superoxide driven Fenton) reaction. The reaction is driven by the continual reduction of ferric iron to the ferrous state by superoxide anion H,O, + Fe2+ O,- + Fe3+ --+
OH’ + OH- + Fe3+ Fe2+ + 0,
(2) If the above reaction is responsible for the lethal effects of UVA irradiation in cells then it would be expected that the common anti-oxidant enzymes, catalase and superoxide dismutase, would play a protective role against UVA induced cytotoxicity and that chemicals which are able to inhibit these enzymes or to either scavenge or prevent the formation of the hydroxyl radical or superoxide anion would modulate the effects of UVA irradiation on cells. Studies on bacteria have shown that xthA strains of E. coli (lacking exonuclease III) show cross sensitivity to hydrogen peroxide and broad-band UVA radiation indicating a common repair pathway for damage induced in DNA by both agents [18, 191. In addition, the presence
of added catalase in the plating medium protects UVA irradiated E. coli from inactivation [ 201. Peak et al.  have also found that glycerol, a scavenger of the hydroxyl radical, has a protective effect on UVA irradiated E. coli and conclude that this radical is involved in cell killing. However, the real relationship between hydrogen peroxide generated by UVA and lethality is not entirely clear as the catalase protection seen in the former study only occurred when catalase was present after irradiation and E. coli catalase mutants are either not sensitive to UVA (katE, katG, ref. 22) or are sensitive only because of an indirect effect on the activity of Exe III (katF, ref. 23). In addition Kramer and Ames  failed to find any evidence for the involvement of hydrogen peroxide in the inactivation of Salmonella typhimurium by broad-band UVA radiation at low fluence rates and found that hydrogen peroxide concentrations in irradiated cells did not exceed 1 PM even at fluences causing significant cell death. Despite these doubts as to the role of hydrogen peroxide generated in UVA irradiated bacteria, this oxidant appears to play a role in the induction of protective mechanisms against oxidative stress. E. coli pretreated with micromolar concentrations of hydrogen peroxide became resistant to UVA radiation [24, 251 and cells preirradiated with UVA are resistant to challenge with normally lethal concentrations of hydrogen peroxide [ 241. Considerably less is known about the involvement of hydrogen peroxide in the UVA inactivation of mammalian cells in culture. Parshad et al.  studied the induction of chromosome damage by fluorescent light in mouse and human cells and found that this was an oxygen dependent process that could occur when the cells were irradiated in saline. The damage could be prevented by the addition of catalase or by addition of mannitol, a scavenger of the hydroxyl radical, and they concluded that the clastogenic factor was hydrogen peroxide. In a recent study in this laboratory a range of agents known to modify the survival response of mammalian cells treated with hydrogen peroxide were used in an attempt to find evidence for the involvement of the hydroxyl radical in UVA induced lethality in human skin fibroblasts [ 131. Diethyldithiocai-bamate and aminotriazole, inhibitors of superoxide dismutase and catalase respectively, both sensitised cells to treatment with hydrogen peroxide but were without effect on UVA irradiated cells. Dimethyl sulfoxide a potent scavenger of the hydroxyl radical protected cells against killing by hydrogen peroxide as did the powerful iron chelating agents desferrioxamine and ortho-phenanthroline. None of these agents had a significant effect on cell killing when present during UVA irradiation. These results would suggest that the formation of hydrogen peroxide during UVA irradiation of human cells makes no contribution towards the lethal effects of this radiation treatment. However, in more recent work with a different human cell line we have observed some degree of protection against the lethal effects of irradiation in the presence of the same two iron chelators [ 271. Clearly further work with additional cell lines will be required to clear up this apparent contradiction and to clarify the role (if any) played by this photoproduct in UVA induced cell killing.
3. The target 3.1. DNA Despite the literature now available concerning damage to the DNA of mammalian cells as a result of UVA radiation, it is still not clear whether damage to the genetic material is an important factor in cellular inactivation. The types of DNA damage induced by the longer wavelengths have been reviewed previously (see ref. 8 for references) and will only briefly be considered here. The induction of pyrimidine dimers and single-strand breaks by UVA radiation was first observed in bacteria and bacteriophage. Both classes of lesions were later observed in mammalian cells but neither is believed to be critical for UVA inactivation of mammalian cells (see ref. 1 for references). As a result, other types of damage, in particular DNAprotein crosslinks, have become a focus for consideration as possible lethal lesions induced by the UVA and near-visible regions of the solar spectrum. Using the alkaline elution technique , Gantt et al.  first detected DNA to protein crosslinking in the DNA of both human and mouse cells following irradiation with a broad-band fluorescent light source (2 X Westinghouse F15T8/cw 15 W lamps). This was inferred from a decrease in the elution rate of DNA from exposed cells compared with controls which could be abolished by treatment with proteinase K. In addition, these workers showed that the formation of these crosslinks was a strictly oxygen dependent process and probably occurred via an indirect photosensitisation mechanism. The wavelengths responsible for photoproduct induction were between 380 and 490 nm. Since 1979, when this study appeared, more sophisticated monochromatic light sources have been employed to study the wavelength dependence for the induction of DNA-protein crosslinks. These studies have confirmed that near visible radiations are more efficient inducers of these lesions than UVA radiation at 365 nm  and that crosslink formation is an oxygen dependent process, probably involving singlet oxygen . However, no further insights into the biological significance of these lesions in terms of solar exposure have been revealed. The action spectrum for induction of DNA-protein crosslinks shows no obvious correlation with that for cell killing and although cell killing is also an oxygen dependent process this is also the case for the formation of single strand breaks in DNA and is likely to hold for many other endpoints measured over this region of the solar spectrum. Although the link between DNA damage induced by UVA radiation and lethality is not clear, the observation of mutational changes is clearly an expression of changes in DNA structure. Several studies have shown that broad-band sources that include UVA wavelengths are mutagenic to mammalian cells [33 - 361 but the effects could have been due entirely or in part to shorter wavelengths. Later work with monochromatic radiation in the UVA range demonstrated that radiation at 365 nm (but not 334 nm) was mutagenic to a human teratocarcinoma cell line . Radiation at both 334 and 365 nm was mutagenic to cultured human fibroblast cells  but not
human lymphoblastoid cells [ 391. However, these differences almost certainly reflect inherent variations in sensitivity according to the cell type and assay conditions, and there is little doubt that sufficient fluences of UVA radiation lead to pre-mutagenic damage. Broad-spectrum UVA radiation, rigorously filtered to exclude short wavelengths, leads to a small but significant increase in the in vitro transformation of 3T3 cells cultured from Balb/c mice [ 401. This is almost certainly a further biological manifestation of the genetic damage induced by radiation in this range. 3.2. Membranes The presence of free radicals leads to lipid peroxidation and membrane damage (for review see ref. 41) so that irreversible damage to membranes has been considered as a possible cause of cell death. Data accumulated in bacteria include studies on salt toxicity, membrane leakage and lipid peroxidation, and these provide substantial evidence that membrane damage which occurs as a result of UVA irradiation will be a factor in cell death, at least under certain conditions (for references see ref. 42). However, both in bacteria and in mammalian cells, it is extremely difficult to establish a clear correlation between membrane damage and cell death. Although peroxidation of membrane lipids is certainly a likely cellular consequence of generation of singlet oxygen (see above), this active species is also able to damage DNA. The two pathways that we propose for constitutive (glutathione) and inducible (heme oxygenase) defence against UVA damage in the following section are also consistent with, but far from substantiate, a membrane target. 4. Cellular defence 4.1. DNA repair Studies with excision deficient human fibroblast cell lines (derived from Xeroderma pigmentosum patients) have shown that these lines are more sensitive to UVA radiation than are cell lines from normal individuals [ 43, 441. However, the differences are much less marked than with UVB or UVC (200 - 290 nm) radiations so that it appears that excision repair plays at most a minor role in cellular defence against damage induced by longer wavelengths. In addition, it has been shown with both Hela cells  and human lymphoblastoid cells  that the UVA component of sunlight or radiation at monochromatic UVA wavelengths (334 and 365 nm) can actually inhibit repair synthesis stimulated by short wavelength (UVC and UVB) radiations. The persistence of a covalent link between a protein and the genetic material (see above) would be expected to pose a sizeable obstruction to normal replication and transcription. However, these cells appear to be able to tolerate significant numbers of these lesions following UVA radiation [ 311. Thus, the repair of UVA induced DNA-protein crosslinks must either
be a very efficient process or these lesions do not cause the expected disruption of normal cellular functions. DNA-protein crosslinks induced by certain chemical agents can be repaired via the nucleotide excision repair pathway  but it is not yet clear whether this is also the case for UVA induced crosslinks. Studies of the removal of DNA-protein crosslinks induced by UVA and near visible radiations have revealed that, following irradiation, the numbers of crosslinks detected may actually increase transiently [47, 481. This observation has been interpreted as evidence that crosslinks may actually arise as a consequence of a cellular repair process operating on UVA induced damage. These rather indirect ways of studying UVA and near visible radiation induced DNA-protein crosslinking have so far yielded little information as to their true nature and significance. One priority of future studies must be to attempt to identify the proteins which become associated with the DNA following irradiation. Recent studies using a combination of gel electrophoresis and sensitive immunological detection have led to the isolation and identification of certain of the proteins crosslinked to DNA by chromate and cis-Pt (II) diamminedichloride. These non-histone proteins include lamins and cytokeratins, both components of the nuclear matrix [49,50]. Interestingly the anti-tumor agent methylene dimethanesulfonate also crosslinks a subset of non-histone proteins but unlike chromate also crosslinks DNA to all five major histones . The application of such techniques to the identification of the proteins crosslinked to DNA following UVA and near visible irradiation of mammalian cells and a study of their repair could significantly advance our understanding of the nature and biological significance of these lesions. 4.2. Defence against oxidative damage As described earlier in the review, UVA radiation induces an oxidant stress which almost certainly involves the generation of active species including hydroxyl radicals and singlet oxygen. Both are known to damage DNA so that cellular defence will involve the mobilisation of DNA repair systems with specificity for oxidant damage. However, oxidative stress can be caused by many other factors including the metabolism of xenobiotics and normal cellular metabolism, and since most cellular components are susceptible to potentially deleterious oxidation, cells possess a variety of anti-oxidant mechanisms . The role of these in protection against UVA stress is only just beginning to be understood. 4.2.1. Anti-oxidant enzymes Mammalian cells possess several anti-oxidant enzymes which include catalase (specific for hydrogen peroxide), superoxide dismutase (dismutates superoxide to hydrogen peroxide) and glutathione peroxidase which uses glutathione as a hydrogen donor to reduce lipid peroxides. Phospholipase a2 appears to be required to break down peroxidised lipids to a suitable substrate for glutathione peroxidase . As mentioned previously, the evidence for the involvement of catalase in protecting bacterial cells from
UVA induced damage is equivocal and preliminary studies in mammalian cells using inhibitors of both catalase and superoxide dismutase have so far indicated that neither of these enzymes plays an important protective role. Despite the fact that they possess glutathione reductase, enteric bacteria do not have glutathione peroxidase. Instead these cells possess a novel alkyl hydroperoxide reductase activity which may serve an equivalent role in protection against alkyl hydroperoxides [ 531. Interestingly deletions of the gene encoding this activity resulted in increased sensitivity to broadspectrum UVA radiation in S. typhimurium indicating that lipid hydroperoxides might be important photoproducts . No direct evidence is yet available for a role for glutathione peroxidase in protection of mammalian cells against UVA radiation. However, inhibition of phospholipase a, by P-mercaptosuccinate does not lead to the large sensitisation to UVA radiation that would be expected if glutathione peroxidase were important [541. 4.2.2. Free radical quenchers Many cellular components may act as free radical scavengers including ascorbate, uric acid, carotenoids and sulfhydryl compounds (see ref. 41, for review). Amongst these, reduced glutathione appears to play a major role in protecting cultured mammalian cells against the cytotoxic action of both UVA and UVB radiations [55, 561. There is a direct correlation between the levels of endogenous glutathione in human cell populations and sensitivity to UVA radiation. At short wavelengths (including 302 nm), the protection appears to be related to the free radical scavenging properties of this ubiquitous tripeptide whereas at longer wavelengths (313 and 365 nm) the role of glutathione appears to be more specific and may involve the requirement of glutathione as the unique substrate for glutathione peroxidase. The protective role of glutathione, which constitutes the most abundant nonprotein thiol in cells, is also strongly indicated in studies with mouse skin that have shown rapid depletion of cutaneous glutathione by both UVA and UVB radiations [ 57,581. Actinic reticuloid is a common idiopathic photodermatosis which affects principally exposed skin areas of middle-aged and elderly men. Cells cultured from patients with this disease show a persistent inhibition of RNA synthesis and cytopathic changes when irradiated with broad-band UVA radiation . The authors suggest that there is a cellular defect which predisposes to actinic reticuloid and that this could involve inefficient quenching of free radicals. 4.2.3. Inducible responses and the possible role of heme oxygenase Recent studies on bacteria have revealed the existence of a positive regulon for control of a variety of genes involved in anti-oxidant defence in S. typhimurium and E. coli which requires the product of the oxyR1 gene . Among the proteins coordinately induced by oxyR1 are catalase and alkyl hydroperoxidase. This inducible response was initially characterised
in cells treated with hydrogen peroxide but was later observed in cells irradiated with broad-spectrum UVA radiation [ 121. Strains carrying a deletion of either oxyR1 or the gene coding for one of the inducible proteins, alkyl hydroperoxidase, are both more sensitive than wild type bacteria to the lethal effects of UVA radiations suggesting a role for these inducible proteins in defence against UVA induced damage. These findings are consistent with earlier studies on E. coli by Peters and Jagger  who found that irradiation of cells with low fluences of UVA (365 nm) radiation induced both a specific stress protein and resistance to subsequent radiation exposure at the same wavelength, and with studies which demonstrated the crossover of induced resistance to both hydrogen peroxide and UVA radiation [24,25]. The induction of protective responses by oxidative stress, including UVA radiation, may not be restricted to bacteria. Recently we detected high levels of a unique 32-kDa stress protein after treatment of human skin fibroblasts with both hydrogen peroxide and UVA radiation [ 621. The gene encoding this protein was later isolated and the protein identified as the catabolic enzyme heme oxygenase . In view of the high levels of this enzyme induced in cells from a tissue not involved in hemoglobin breakdown, we have suggested that breakdown of heme and heme containing proteins is involved in cellular protection against UVA and other oxidant stress. Various mechanisms exist in cells for maintaining the lowest possible level of free iron thereby preventing its catalytic role in the Fenton reaction. Since the iron contained in heme and heme-containing proteins can readily become available to participate in this reaction, the level of cellular and circulating heme pools may influence dramatically the generation of active oxygen species under conditions of oxidant stress. For example it has been proposed that haptoglobin and hemopexin (respectively hemoglobin and heme-transporting proteins) may play an important role in protecting extracellular fluids against radical reactions [64,65]. The generation of high levels of heme oxygenase by UVA and other oxidants, including hydrogen peroxide, provides an additional cellular pathway by which heme levels may be rapidly reduced and thereby reduce oxidation to a minimum. The rapid breakdown of hemecontaining proteins will also remove chromophores capable of generating singlet oxygen upon irradiation with UVA (see above). Finally the catabolism of heme and heme-containing proteins will produce biliverdin and bilirubin (in the presence of biliverdin reductase) both of which are known to be powerful anti-oxidants (e.g. ref. 66). Although this pathway may also contribute to the overall anti-oxidant capacity of cells, the levels that may be produced in skin cells following heme oxygenase induction would appear small when compared with the 3 - 5-mM concentrations of glutathione present constitutively in cells. Nevertheless there does appear to be a relationship between cellular glutathione levels and the inducible response since lowering the levels of cellular glutathione also lowers the threshold fluence for UVA stimulation of protein levels .
Since we now know that UVA-enhanced stimulation of heme oxygenase gene expression is almost exclusively at the transcriptional level [6’7], we are encouraged to speculate that a positive regulator molecule may exist with similar properties to the oxyR protein of E. coli [ 681. Such a protein would normally be protected from oxidation by glutathione but would be activated to stimulate transcription under conditions of oxidant stress that include UVA irradiation of cells. Despite a considerable amount of work, it is clear from the literature reviewed that the target(s) for the lethal action of UVA on mammalian cells remains a mystery. Since genetic damage is clearly induced, there is little doubt that cellular mechanisms for monitoring and repairing DNA damage play an important role in cellular protection. However, it is equally clear that UVA generates an oxidant stress which demands the operation of a wide variety of cellular anti-oxidant mechanisms. Recent work has pointed to certain mechanisms which may be involved. The elucidation of the interrelationship and relative importance of these pathways presents a challenge for the future. Acknowledgments Research from this laboratory mentioned in this review has been supported by grants from the Swiss National Science Foundation (3.186.088) and the Swiss League Against Cancer. References 1
3 4 5 6 7
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