CHAPTER 1
UVB and UVA induced formation of photoproducts within cellular DNA
Jean Cadet, Thierry Douki, Jean-Pierre Pouget and Jean-Luc Ravanat
Table of contents
Abstract
1.1 Introduction 3
1.2 Distribution of UVB radiation-induced dimeric
pyrimidime photoproducts within cellular DNA 5
1.3 UVC and UVB-induced formation of monomeric base photoproducts 7
1.4 Damage induced by UVA radiation to cellular DNA 8
1.5 Conclusions 10
References 10
Abstract
Emphasis is placed in this short survey on recent aspects of the photochemistry of cellular DNA that involve both the effects of UVB and UVA radiations. Direct excitation of the purine and pyrimidine bases of DNA is known to mostly generate dimeric pyrimidine photoproducts in oxygen independent photoreactions. Interestingly, the twelve possible dimeric photoproducts at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates. This is achieved using a specific and sensitive assay that associates high performance liquid chromatography to tandem mass spectrometry (HPLC-MS/MS) operating in the electrospray ionization (ESI) mode. Thus, it was found that UVB irradiation of human monocyte cells gives rise predominantly to cis-syn cyclobutadithymine, thymine-cytosine pyrimidine(6-4) pyrimidone adduct and related cyclobutyl dimer. In contrast the dimeric photoproducts at (di)cytosine sites are generated in very low yields although characteristic tandem mutations of UV-B irradiation are observed at the latter CC sequences. Further, cytosine photohydrate and Dewar valence isomers of the (6-4) photoproducts are at the best minor UV-B photoproducts. Relevant information on UVA-sensitized oxidative damage to cellular DNA was gained from measurements using chromatographic methods and the modified comet assay. Thus, it was shown that 8-oxo-7,8-dihydro-2'-deoxyguanosine is the predominant DNA oxidation product, as mostly, the result of singlet oxygen oxidation. In addition, oxidized pyrimidine bases and DNA strand breaks whose formation involves •OH radical, are produced in much lower yields. Work is in progress to assess the UVA-induced formation of other markers of oxidative stress. These include on the one hand DNA-protein crosslinks and on the other hand DNA adducts with reactive aldehydes that arise from the breakdown of initially generated lipid peroxides.
1.1 Introduction
Solar UV radiation appears to be the main etiological factor responsible for the induction of skin cancers in human population. It is well established that UVB and UVA radiations act mostly on cellular DNA via direct and photosensitized reactions respectively [for earlier reviews, see 1,2]. Precise assessment of the final products of these photoreactions has been hampered for years by the lack of accurate and quantitative methods of measurement. This particularly concerns the individual determination of dimeric pyrimidine photoproducts including cis-syn cyclobutadi-pyrimidines (CPDs), pyrimidine (6-4) pyrimidone photoadducts (6-4PPs) and related valence Dewar isomers (DewarPPs) for which only limited information was available until recently. However, relevant data on the distribution and repair of the three latter classes of photoproducts within the DNA of plasmids, isolated cells and tissues were gained mostly from serological approaches. These include ELISA, RIA and immuno-dot-blot measurements together with immunostaining detection through the availability of monoclonal and polyclonal antibodies. We may also mention the recent development of an immunological method aimed at measuring CPDs and 6-4PPs in the DNA of isolated cells in association with the comet assay. Another suitable method that is receiving major attention is the ligation-mediated polymerase chain reaction (LM-PCR). This allows the mapping within DNA at the nucleotide resolution of dimeric pyrimidine photoproducts and particularly of CPDs, the latter lesions being revealed through the nicking activity of T4 endonuclease V. Thus, it was found that methylation of cytosine residues in 5'-CCG and 5'-TCG sequences leads to a 10-fold increase in the UVB formation of CPDs. Interestingly the latter lesions were found to constitute major p53 mutation hot spots in mouse skin tumors. It was also shown that accumulation of dimeric DNA photoproducts takes place at the same locations of the p53 gene in both human skin and epidermal cells of Hupki (human p53 knock-in) mice. Another striking information inferred from LC-PCR analysis is the predominant implication of CPDs in a vast majority of UVB-induced mutations in mammalian cells. LM-PCR measurement of CPDs in the DNA of the basal layer of engineered human skin led to the conclusion that upper layers of epidermis protected against the genotoxic effect of UVC but not from those of solar UVB radiation. Evidence was also provided, still using LM-PCR detection of CPDs, that human cells either genetically or functionally compromised for p53 function, are defective in both global and transcription-coupled nucleotide excision repair (TCNER). Interestingly, cells functionally compromised for retinoblastoma tumor suppressor protein function are only defective in TCNER. Preferential repair of CPDs was found to occur in the promoter and quiescent initiation domain of the CDC2 gene in both quiescent and proliferating human fibroblasts.
There is an increasing attention devoted to the assessment of the molecular effects of UVA radiation on DNA in relation with the increased formation of reactive oxygen species (ROS) that for the bulk is mediated by endogenous photosensitizers. This interest is explained, at least, partly by the likely association of UVA with skin cancer risk and particularly with skin melanoma incidence whose observation in heavily pigmented hybrids of Xiphophorus fish requires, however, further support before to be considered as a suitable model for human. In that respect, UVA does not appear to be a specific mutagen in contrast to UVB that induces a characteristic mutation fingerprint at bipyrimidine sites and more precisely at TC and CC sequences. Thus, the incidence of p53 mutations in UVA-induced skin tumors in hairless mice is very low without any specificity. The relatively high incidence of A:T -> T:A point mutations observed on the LacZ gene upon exposure of human cells to UVA radiation cannot be correlated with the formation of any known DNA lesions. In contrast, the UVA-mediated increase in the frequency of T -> G transversions in the aprt locus of Chinese hamster ovary cells that was not observed in the nucleotide excision repair-deficient cells may be accounted for by the damaging effects of ROS on DNA. It may be pointed out that indirect evidence for the exaltation of the formation of ROS upon UVA irradiation of human skin fibroblasts was provided by the observed induction of heme oxygenase and the release of free iron from ferritin. More direct proofs for the occurrence of oxidation reactions within human and CHO cells upon exposure to UVA radiation was the observed increase in the level of 8-oxo-7,8-dihydroguanine, an ubiquitous biomarker of oxidative processes, in both nuclear DNA and RNA. In addition, relevant information on several classes of oxidative damage induced by UVA and visible radiation to cellular DNA was gained from application of the alkaline elution technique that involves the use ot two DNA repair enzymes, namely ormamido-pyrimiidne DNA N-glycosylase (Fpg) and endonuclease III (endo III). Thus it was shown that the formation of Fpg-sensitive sites, likely to mostly involve 8-oxoGua, are predominant with respect to DNA strand breaks and endo III-sensitive sites (mostly oxidized pyrimidine bases).
Emphasis is placed in this short survey on recent aspects on the formation of UVB and UVA-mediated damage to cellular DNA that mostly involved dimeric pyrimidine photoproducts and oxidative lesions. The bulk of the measurement of DNA photoproducts was achieved using the recently available HPLC-tandem mass spectrometry technique and the modified comet assay.
1.2 Distribution of UVB radiation-induced dimeric pyrimidine photoproducts within cellular DNA
The advent of the HPLC in the mid 70's together with the availability of new stationary phases including octadecylsilyl silica gel (ODS) packing material has provided a strong impetus to the development of sensitive and highly resolutive analytical method aimed at monitoring the formation of tiny amounts of lesions within cellular DNA. Interestingly, it was shown as early applications that the cis-syn isomer of cyclobutadithymine (c,s Thy <> Thy) was efficiently separated from the DNA hydrolysis products. It should be reminded that the only one available sensitive detection approach at that time was the radioactive measurement of the content of HPLC fractions; however, this was not achieved on line due to the lack of suitable detector. One of the main advantages provided by the HPLC separation on the ODS columns is that thymine is eluted less rapidly than the targeted c,s Thy <>Thy, avoiding any contamination of the fractions containing the latter minor photoproduct due to the trailing of [H]-thymine. However, the measurement of cis-syn cyclobutane dimer involving cytosine and [H]-thymine that is released as UraoThy was difficult due to a co-eluting radioactive contaminant, the likely 5-hydroxy-5-methylhydantoin that arises from self-radiolysis process. Interestingly, the assay despite some limitations has received several relevant applications including the assessment of repair kinetic of c,s Thy < > Thy in UVC-irradiated of normal and xeroderma pigmentosum fibroblast cells. Subsequently, a suitable HPLC method that does not require radioactive pre-labeling of DNA has become available for monitoring the formation of fluorescent pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). This has required the use of HF-pyridine at room temperature as a mild hydrolytic reagent to obtain a quantitative release of relatively unstable 6-4PPs as nucleobase derivatives. The detection of the latter photoproducts that exhibit a fluorescence emission spectra with maxima around 380 nm upon excitation in the UVB range was achieved at the output of the HPLC column using a fluorescence detector. The distribution of the 6-4PPs including either two thymine or one thymine and a cytosine was assessed in UVC irradiated DNA. However, the relatively low sensitivity of the steady-state fluorescence detection technique has prevented the application of the assay for monitoring the formation of 6-4PPs within nuclear DNA of UVC or UVB irradiated cells.
Interestingly, most of the limitations that have been encountered for measuring the dimeric pyrimidine photoproducts (vide supra) were overcome using the accurate HPLC-MS/MS method operating in the electrospray ionization (ESI) mode. This method that recently became available has already found interesting applications for the measurement of DNA damage including several oxidized pyrimidine and purine nucleosides and nucleobases. The overall strategy involves enzymatic digestion of DNA from UVB irradiated cells by a cocktail of several enzymes including 3'-and 5'-exonucleases after the quantitative conversion of Cyt <> Cyt and 5'-end cytosine 6-4PPs into the corresponding uracil derivatives through deamination. Therefore, in one HPLC analytical run it is possible to accurately measure at the output of the chromatographic column the twelve possible bipyrimidine adducts at TT, TC, CT and CC sequences both in isolated and cellular DNA upon exposure to low doses of UVC and UVB radiations. Interestingly, the tandem mass spectrometric measurement which is achieved in the sensitive multiple reaction monitoring (MRM) mode provides also a specific way of distinguishing CPD from 6-4PP for a given bipyrimidine site due to the occurrence of a different fragmentation pattern. Similar trends in the photoproduct distribution are observed in isolated and cellular DNA upon either UVC or UVB irradiation. As a first remark it may be noted that the formation of the Dewar valence isomers of 6-4-PPs is barely detectable upon exposure of cellular DNA to biologically relevant doses of UVB radiation. Under the latter conditions, only the Dewar isomer at CC sites (Figure 1) was found to be produced, however, in a very low yield. The three main UVB-induced dimeric photoproducts appear to be generated in the following decreasing order of importance: c,s Thy <> Thy > 6-4PP at TC sequence > Thy <> Cyt (Table 1). In contrast, the CT sites and to a lower extent, the CC sequences exhibit a low photoreactivity as inferred from the low yield formation of both CPDs and 6-4-PPs. It should be reminded that the CC photoproducts although generated with a low efficiency exhibit a high mutagenic potential that leads to the observation of the characteristic UV-induced tandem mutation CC [right arrow] TT. However, further work is required to definitively establish the nature of the highly mutagenic UVB-induced photolesion(s). As a final remark, it may be stressed that the comparison of the HPLC-MS/MS measurements and LM-PCR analysis of dimeric pyrimidine photoproducts in the DNA of UVC irradiated cells shows that application of the latter method leads to a strong underestimation of the yield of 6-4PPs at TT and TC sites. This is likely due to the low efficiency of the piperidine-mediated conversion of the 6-4PPs into strand cleavage at the 3'-side since only the related valence Dewar isomers appear to be strongly alkali-labile.
1.3 UVC and UVB-induced formation of monomeric base photoproducts
Is was recently confirmed that UVB is able to generate 8-oxoGua in DNA of mouse keratinocytes, mouse epidermis and. Chines hamster ovary cells. However, as shown in a comparative study on the UVB-induced-formation of several classes of photodamage to cellular DNA, the contribution of 8-oxoGua is rather low. Thus, the yield of 8-oxoGua measured as the corresponding 2'-deoxyribonucleoside by HPLC-electrochemical detection in the DNA of UVB irradiated CHO cells was 2.1 lesions per 106 bases and kJ.m-2. This is about two orders of magnitude lower than the level of CPDs that was assessed by immunodetection. Further work is required to better delineate the mechanisms of 8-oxoGua formation that may involve a Fenton type chemistry, hole migration from initially photo-ionized pyrimidine and adenine bases or singlet oxygen oxidation. In that respect, we may anticipate a notable contribution of "OH radical or related reactive oxygen through the Fenton reaction since UVB irradiation of cellular DNA was found to lead to similar yields of strand breaks and Fpg-sensitive sites when detected using the alkaline elution technique.
Another putative UVB DNA photodamage that has received a lot of attention in early model studies is 6-hydroxy-5, 6-dihydrocytosine the so-called "cytosine photohydrate" that arises from hydration of singlet excited state cytosine [for a comprehensive review, see 1]. A relevant piece of information on the formation of cytosine photohydrate in both isolated and cellular DNA was recently gained from the application of a suitable HPLC-MS/MS assay. This allows the measurement of 2'-deoxycytidine photohydrates as the 6R and 6S diastereomers of 6-hydroxy-5,6-dihydro-2'-deoxyuridine upon DNA enzymatic hydrolysis and quantitative deamination. Thus, it was found that UVC-induced formation of cytosine hydrate in isolated DNA is a minor photochemical event with a yield of formation which is about 2 orders of magnitude lower than that of CPDs. The formation ratio CPDs/cytosine hydrate was found to be even lower by a factor of 10 in cellular DNA as the likely result of lower accessibility of water molecules for hydration of the cytosine moieties in compacted cellular DNA. These data that, at the best, suggest a minor contribution of cytosine hydrate to the overall biological effect of far-UV radiation are in agreement with a previous estimation of endonuclease Ill-sensitive sites that were to be 2 orders of magnitude lower than the level of CPDs.
