Today, UVA is an accepted carcinogen (3
). Nevertheless, the types of DNA damage induced by UVA are still not completely understood (9
). The classical point of view was that UVA induces predominantly oxidative damages and among those the majority accounts for 8-oxo-guanine (42
). More recently, it was demonstrated that UVA is also able to induce thymidine dimers, especially TT-CPDs, although at a 1000-fold lower efficiency compared to UVC (43
). Even if CPDs are added to the UVA damage profile, there is still a gap to fully understand the mutagenic potential of UVA radiation, at least in hamster cells (45
). In addition, UVA was reported to induce dsbs (18
). Dsbs could be a third component in the damage profile of UVA and account a major damage class explaining UVA-induced mutagenicity.
The energy of a single UVA photon is too low to induce a covalent bound break or change. So all types of DNA damage, such as oxidative base damage, oxidative backbone damage, CPDs or dsbs, induced by UVA are strictly dependent on—so far—unknown cellular sensitizers (6
). Several chemical structures have been suggested as potential sensitizers, e.g. cytochromes, flavins or NAD(P)H (9
). Recent overviews of potential PS have been given in (49
). Since all damage induced by UVA is dependent on radicals formed by the cellular sensitizers, we need to consider a second fact: radicals are highly reactive, short lived and have very limited diffusion ranges [ranging from 2 nm (hydroxyl radical) to 100 nm (singlet oxygen) for different radical species], at least if we consider radical oxygen species to be the main source of radicals involved in UVA-dependent DNA damage (51
). Taken together, photo-induced damage by UVA has to be localized in a very restricted volume around the cellular sensitizers.
A proposed model of dsb-induction is, therefore, based on clustered ssbs in close proximity that are converted to a dsb when they occur simultaneously and within 1.5 helix turns (53
). It is known that clustered ssbs [also arising during DNA repair of OCDLs (26
)] are treated as dsbs by the cell (22
). Additionally, it was shown that clustered damage is especially mutagenic and cytotoxic and has a reduced repair kinetic (25
). This directly coincides with the results presented here and in previous studies, demonstrating that UVA-induced dsbs are generated with a temporal lag and have at least 6-h persistence (37
). In a recent article, Cadet and Douki (10
) argued that the frequency of these events is too low, since the frequency of 8-oxo-dGs induced by UVA and the ratio of 8-oxo-dGs to ssbs would not allow a clustered occurrence of ssbs to be converted to dsbs. This is true if one assumes a random distribution of ROS generated by randomly located sensitizers. Due to the short diffusion range of the ROS in the vicinity of cellular sensitizers, we have to assume that these are chromatin bound in one way or the other (55
). This would lead to the conclusion that the damage is more clustered and less random.
From our results, we can conclude several new facts and confirm several steps of the clustered damage model: (i) We confirm that ROS are intermediates of the DNA damaging process, especially for the dsb formation, since the presence of an anti-oxidant (Naringin) does prevent the formation of dsbs and ssbs. ROS also cause ssbs, but obviously this damage is repaired too fast to be detected in the split-dose experiments. This is reflected by the fact that the alkaline Comet-assay, which detects ssbs and alkali labile sites, does not show an increase in damage levels after split-dose irradiation. Thus, implying a repair mechanism faster than the split-dose recovery time of 2 h which is well within the accepted time frame of BER repair (41
). (ii) We could demonstrate that a split-dose irradiation scheme enhances the number of dsbs, suggesting that a sensitizer can also be depleted (most likely photooxidized) by photon absorption. If this happens, sensitizers can no longer function in the generation of ROS. However, given some time (as in split dose experiments) these sensitizers can be exchanged and produce ROS and dsbs again. The number of DNA dsbs not fully repaired after the first dose fraction plus those being produced with the second-dose fraction is then apparently higher than the one produced by a single (high) dose which might be able to exhaust the relevant PS pool. These findings are in agreement with an investigation by Hoffmann-Doerr et al.
) who explained their results of split-dose experiments of FPG-sensitive sites after UVA/visible light exposure by a photosensitizier exhaustion mechanism. It should be noted also that, in our investigation, the split-dose effect is stronger when detected by the γH2AX foci compared to the detection on the level of DNA fragmentation (neutral Comet-assay). This suggests a prolonged existence of the γH2AX foci exceeding the DNA re-ligation event. (iii) We were able to demonstrate that UVA induces a large quantity of clustered oxidative DNA lesions as detected with FPG as an enzymatic probe together with the neutral Comet-assay. This does not cover all possible oxidative damage, only FPG sensitive sites [7, 8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, 5-hydroxy-cytosine and 5-hydroxy-uracil] and ssbs, but these are the most common DNA lesions induced by UVA. Additionally, we could demonstrate that the dsb formation is dependent on the temperature. Reduced temperature during irradiation and post-incubation (4°C) leads to a significant decrease of detected DNA fragments in the neutral Comet-assay. (iv) When we determine DNA damage on stretched chromatin fibres, we see clusters of damage at distinct points. This fits the model of spatially fixed sensitizers at specific sites in the chromatin. Importantly it is seen for different UVA-induced DNA lesions, such as 8-oxo-dG and abasic sites. So it is a direct hint to a locally higher concentration of ROS, which would most likely be able to cause clustered damage. These findings are also supported by work of Ito et al.
), who showed that, e.g. 8-oxo-dG formation on double-stranded (naked) DNA proceeds through the direct interaction of (UVA-) photosensitized riboflavin with DNA. This reaction pathway of DNA-dsb induction might be similar or even identical to that described after the application of ionizing radiation, which produces a comparable pattern of ROS (59
Taken together, we conclude from our results that there is indeed a replication-independent induction of dsbs by UVA exposure, as reported earlier by several other studies (18
). Our results seem to be in disagreement with the recent report by Rizzo et al.
who did not find activation of the homologous recombination (HR) dsb repair pathway in UVA-irradiated human cells. In their study, only >15 γH2AX foci/cell were considered as dsb induction (17
). However, this might indicate problems in sensitivity of their γH2AX-assay and a biased focus on HR as the only dsb repair pathway. It is known that dsbs are predominantly repaired by the non-homologous end joining systems, especially if the cells are in G1 phase of the cell cycle and breaks are directly ligatable (60
), which is the predominant case for dsbs generated by clustered ssbs.
The relevance of these findings is highlighted by the fact that UVA doses used in this investigation (up to 600 kJ/m2
) can easily be accumulated under natural conditions from solar ambient UVA radiation. Based on a model by Green and colleagues (61
), which has been extensively validated by comparison with measured spectral irradiance at ground level (40
), a UVA dose of 600 kJ/m2
will be accumulated at latitude 50–55°N, at clear skies in the month of June between 11:30 am and 3:30 pm (40
The fact that we could demonstrate our findings in both primary human fibroblasts as well as in the keratinocyte cell line HaCaT indicates that we are describing a common mechanism for the induction of dsbs by UVA and not just a property of a given cell line. Dsbs are known to be precursor lesions of chromosome aberrations. We could recently show that UVA induces chromosomal aberration in human keratinocytes and that these cells give rise to squamous cell carcinoma after transplantation into nude mice (37
). As UVA represents the major component of solar UV radiation and artificial UV used in sunbeds, the results of our study might have important implications in skin cancer aetiology and risk assessment.