Riboflavin activated by A1 irradiation induces oxidative DNA damage-mediated mutations inhibited by vitamin C

Ahmad Besaratinia*, Sang-in Kim, Steven E. Bates, and Gerd P. Pfeifer*

Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved February 13, 2007 (received for review November 29, 2006) An increasingly popular theory ascribes UVA (>320–400 nm) carci- duced by UVA has inconsistently matched the mutagenic potential nogenicity to the ability of this wavelength to trigger intracellular of the various induced lesions (4). For the most part, however, the photosensitization reactions, thereby giving rise to promutagenic apparent discrepancies are attributable to a failure to investigate oxidative DNA damage. We have tested this theory both at the UVA-induced DNA damage and mutagenesis simultaneously (4). genomic and nucleotide resolution level in mouse embryonic fibro- Ideally, the correlative nature of events pertaining to each aspect of blasts carrying the lambda phage cII transgene. We have also tested UVA mutagenicity should be established by using comparative the hypothesis that inclusion of a cellular photosensitizer (riboflavin) model systems under uniform experimental conditions. can intensify UVA-induced DNA damage and mutagenesis, whereas Recently, we have shown distinctive UVA-induced oxidative addition of an antioxidant (vitamin C) can counteract the induced DNA damage and mutagenesis in the cII transgene of Big Blue effects. Cleavage assays with formamidopyrimidine DNA glycosylase mouse embryonic fibroblasts (11, 12). In the present study, we have (Fpg) coupled to alkaline and ligation-mediated expanded our investigation to determine the underlying mechanism PCR (LM-PCR) showed that riboflavin treatment (1 ␮M) combined of induced mutagenesis in the same model system. To gain insights with UVA1 (340–400 nm) irradiation (7.68 J/cm2) or higher dose UVA1 into the mechanism of DNA damage-targeted mutagenesis, we irradiation alone induced Fpg-sensitive sites (indicative of oxidized have studied the effects of UVA irradiation alone and in combi- and/or ring-opened purines) in the overall and in the cII nation with a cellular photosensitizer, riboflavin (21), introduced transgene, respectively. Also, the combined treatment with riboflavin into our model system. To verify the role of oxidative DNA damage and UVA1 irradiation gave rise to single-strand DNA breaks in the in mutagenesis, we have also incorporated an antioxidant, vitamin genome and in the cII transgene determined by terminal transferase- C (22), into our experimental system. Subsequently, we have dependent PCR (TD-PCR). A cotreatment with vitamin C (1 mM) investigated the modulation of DNA damage and mutagenesis efficiently inhibited the formation of the induced lesions. Mutage- consequent to irradiation with UVA or treatment with UVA- nicity analysis showed that riboflavin treatment combined with UVA1 activated riboflavin in the presence and absence of vitamin C. irradiation or high-dose UVA1 irradiation alone significantly in- The relevance of vitamin C for the present study lies on the fact creased the relative frequency of cII mutants, both mutation spectra that unlike other antioxidants, e.g., ␣-tocopherol or ␤-carotene, exhibiting significant increases in the relative frequency of G:C 3 T:A vitamin C does not absorb radiation in the UVA range (23). Thus, transversions, the signature mutations of oxidative DNA damage. The the inability of vitamin C to absorb UVA rules out the possibility induction of cII mutant frequency was effectively reduced consequent of interference with our experimental design, which was tailored to to a cotreatment with vitamin C. Our findings support the notion that investigate the antioxidative but not filtrating effects of a biologi- UVA-induced photosensitization reactions are responsible for oxida- cally relevant compound against oxidative stress imposed by ribo- tive DNA damage leading to mutagenesis. flavin treatment combined with UVA irradiation or intense UVA irradiation alone. In addition, vitamin C is a major hydrophilic ultraviolet A radiation ͉ photosensitizer ͉ antioxidant ͉ skin cancer low-molecular weight nonenzymatic compound that belongs to a network of antioxidant defense mechanisms (22). It is known that large body of evidence exists regarding the association vitamin C is widespread throughout the body, especially in the skin Abetween solar UV irradiation and human skin carcinogenesis (24), the target organ for UV-associated cancers (22). On the other (1, 2). Sunlight UV wavelengths that reach the surface of the earth hand, riboflavin is relevant for the herein study because as an are UVA (Ͼ320–400 nm) and UVB (280–320 nm), with shorter endogenous photosensitizer, this compound is ubiquitously present wavelengths (UVC) being completely absorbed by stratospheric throughout the body, including in various highly UV-exposed organs, e.g., eye lens (21, 25). oxygen (O2) (1, 3). The biologically relevant UVA and UVB have been extensively studied as the etiologic factors for skin cancer (4). Results The mechanistic involvement of UVB in carcinogenesis rests upon the ability of this wavelength to induce promutagenic cis-syn Cytotoxicity Examination. In the absence of UVA1 irradiation, cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimi- riboflavin treatment at none of the tested concentrations (0.01, 0.1, done photoproducts ((6-4)PPs), and Dewar valence photoisomers (4). However, the underlying mechanism of action for UVA Author contributions: A.B. and G.P.P. designed research; A.B., S.-i.K., and S.E.B. performed carcinogenicity is not fully delineated (4). Despite the weak absor- research; A.B. analyzed data; and A.B. wrote the paper. bance of UVA by DNA (3), a genotoxic mode of action for UVA The authors declare no conflict of interest. has been demonstrated (1). Yet, the exact process through which This article is a PNAS Direct Submission. UVA exerts genotoxicity remains elusive (4). Abbreviations: (6-4)PP, pyrimidine (6-4) pyrimidone photoproducts; 8-oxo-dG, 8-oxo-7,8- A widely recognized theory ascribes UVA genotoxicity to its dihydro-2Ј-deoxyguanosine; CPD, cis-syn cyclobutane pyrimidine-dimer; Fpg, formami- ability to trigger intracellular photosensitization reactions, thereby dopyrimidine DNA glycosylase; LM-PCR, ligation-mediated PCR; TD-PCR, terminal trans- giving rise to promutagenic DNA lesions (5). In fact, UVA has been ferase-dependent PCR; T4 Endo V, T4 endonuclease V. shown to induce CPDs (6–10) and oxidative DNA damage (5, 6, *To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

11–14), as well as mutagenesis (12, 15–20). However, the correla- This article contains supporting information online at www.pnas.org/cgi/content/full/ GENETICS tion between UVA-induced DNA damage and mutations has not 0610534104/DC1. been straightforward inasmuch as the spectrum of mutations pro- © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610534104 PNAS ͉ April 3, 2007 ͉ vol. 104 ͉ no. 14 ͉ 5953–5958 Downloaded by guest on September 23, 2021 001

08

06

04 Viability (%) Fig. 1. Cytotoxicity of riboflavin and/or UVA1 irradiation in

02 the presence and absence of vitamin C to Big Blue mouse embryonic fibroblasts. Cell cultures were treated with increas- ing concentrations of riboflavin or solvent in the presence or 0 absence of vitamin C at different concentrations for 20 min at 1 1 .1 0 0 0 6 2 4 8 6 2 6 2 4 8 6 2 6 2 4 8 6 2 6 2 4 8 6 2 6 2 4 8 6 2 6 2 4 8 6 2 .0 1 0 9 9 8 6 3 7 9 8 6 3 7 9 9 8 6 3 7 9 9 8 6 3 7 9 9 8 6 3 7 9 9 8 6 3 7 0 0 1 ...... 9 ...... 0. 7 5 0 3 7 5 0 0 3 7 5 0 0 1 3 7 0 0 7 5 0 0 1 7 5 0 1 3 1 30 1 1 3 1 1 3 15 3 1 3 1 3 3 1 3 37°C in the dark. Subsequently, the treated cultures were irradiated with various doses of UVA1, and afterward cell Mµ mc/J 2 J/ mc 2 mc/J 2 J/ mc 2 mc/J 2 J/ mc 2 nivalfobiR AVU AVU AVU AVU AVU AVU viability was determined by the trypan blue dye exclusion + + + + + nivalfobiR R fobi lav ni R fobi lav ni R fobi lav ni R fobi l niva assay. Viability is expressed as a percentage of total cell num- )Mµ 10.0( )Mµ 1.0( )Mµ 1( )Mµ 01( )Mµ 001( )Mµ ber. Error bars ϭ SD.

1, 10, and 100 ␮M) was cytotoxic to mouse embryonic fibroblasts. embryonic fibroblasts. However, vitamin C at a minimum concen- In combination with UVA1 irradiation, however, riboflavin treat- tration of 1 mM could completely protect against the cytotoxic ment caused concentration dependent cytotoxicity at radiation effects of combined treatment with riboflavin and UVA1 irradia- doses that were nonlethal per se. Irradiation with UVA1 alone tion or of UVA1 irradiation alone at high doses. Supporting resulted in appreciable cytotoxicity only at doses exceeding 15.36 information (SI) Fig. 4 illustrates the protective effects of vitamin J/cm2 (Fig. 1). As a prerequisite for our mutagenicity experiments, C against cytotoxicity of UVA1-activated riboflavin treatment and we performed a series of preliminary tests to determine a riboflavin of UV irradiation at a high dose of 30.72 J/cm2. Next, we repeated concentration together with UVA1 irradiation dose, which was all cytotoxicity examinations 24 h posttreatment to determine minimally cytotoxic, yet, significantly mutagenic. This choice of possible delayed effects on cell viability consequent to various dosing is crucial for establishing viable and replicating cells in which treatments. In all cases, there was a slight decrease (3–8%) in cell the induced DNA damage can be efficiently translated into muta- viability in cultures harvested 24 h posttreatment relative to coun- tions. As shown in Fig. 1, a treatment with 1 ␮M concentration of terpart cultures harvested immediately. Nonetheless, the overall riboflavin together with 7.68 J/cm2 UVA1 irradiation was margin- pattern of cytotoxicity established immediately after all treatments ally cytotoxic but significantly mutagenic in the cII transgene (Table or 24 h afterward was very comparable. 1). Treatment with riboflavin at concentrations Ն10 ␮M combined 2 with UVA1 irradiation (7.68 J/cm ) was extremely lethal, thus, DNA Damage in the Genome. Cleavage assay with formamidopyri- precluding inclusion of this dose range in mutagenicity testing. In midine DNA glycosylase (Fpg) digestion and alkaline gel electro- keeping with the objective of our study, we have therefore used the phoresis showed that neither riboflavin treatment nor UVA1 2 combination treatment of 1 ␮M riboflavin and 7.68 J/cm UVA1 irradiation (up to 7.68 J/cm2) alone produced Fpg-sensitive sites in irradiation in all DNA damage and mutagenicity experiments. the genomic DNA of mouse embryonic fibroblasts. However, the Vitamin C at concentrations between 0.001 and 10 mM alone or combination treatment of riboflavin and UVA1 irradiation gave in combination with UVA1 irradiation was not cytotoxic in mouse rise to Fpg-sensitive sites. Irradiation with UVA1 alone at doses Ն15.36 J/cm2 also yielded considerable Fpg-sensitive sites. Treat- Table 1. Mutant frequency of the cII transgene in Big Blue ment with vitamin C (0.001–10 mM) alone or in combination with mouse embryonic fibroblasts treated with riboflavin and/or UVA UVA1 irradiation did not generate Fpg-sensitive sites. However, irradiation in the presence and absence of vitamin C vitamin C treatment (1 mM) efficiently prevented the formation of Fpg-sensitive sites consequent to riboflavin treatment combined Verified Mutant with UVA1 irradiation or UVA1 irradiation alone at high dose Total no. of mutant frequency, (Fig. 2a). Conversely, cleavage assay with T4 endonuclease V (T4 Ϫ Treatment plaques, pfu plaques ϫ 10 5* Endo V) digestion and alkaline gel electrophoresis showed no Nontreatment 6,235,000 161 2.8 Ϯ 0.7 formation of CPDs in the genomic DNA in cells treated with Riboflavin (1 ␮M) 2,865,000 81 3.0 Ϯ 0.7 riboflavin or irradiated with UVA1, individually or combined. UVA (7.68 J/cm2) 1,882,500 75 3.9 Ϯ 1.5 UVA (15.36 J/cm2) 3,595,000 304 8.4 Ϯ 0.9† DNA Damage in the cII Transgene. Ligation-mediated PCR (LM- Riboflavin (1 ␮M) ϩ UVA (7.68 J/cm2) 4,462,500 569 12.1 Ϯ 2.2‡ PCR) of the Fpg-digested DNA showed that there was no lesion Ϯ Vitamin C (1 mM) 2,387,500 68 2.8 0.1 formation in the cII transgene in mouse embryonic fibroblasts Riboflavin (1 ␮M) ϩ vitamin C 1,900,000 69 3.3 Ϯ 2.1 (1 mM) treated with either riboflavin or UVA1 irradiation (up to 7.68 2 UVA (7.68 J/cm2) ϩ vitamin C (1 mM) 1,272,500 46 3.7 Ϯ 1.4 J/cm ) alone. However, the combination treatment of riboflavin UVA (15.36 J/cm2) ϩ vitamin C 1,665,500 63 4.0 Ϯ 1.0 and UVA1 irradiation caused a significant formation of lesions (1 mM) throughout the cII transgene. Irradiation with UVA1 alone at doses Riboflavin (1 ␮M) ϩ UVA (7.68 J/cm2) 3,937,500 167 4.1 Ϯ 1.8 equal to or higher than 15.36 J/cm2 also produced detectable level ϩ vitamin C (1 mM) of lesions in the cII transgene (Fig. 2b). The specificity of Fpg- *Results are expressed as median Ϯ SD. sensitive sites induced by riboflavin treatment combined with †Statistically significant as compared with control, P Ͻ 0.01. UVA1 irradiation or high-dose UVA1 irradiation alone was veri- ‡Statistically significant as compared with control, P Ͻ 0.01. fied by showing the absence of these lesions in the respective

5954 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610534104 Besaratinia et al. Downloaded by guest on September 23, 2021 a Riboflavin (1 µM) - + - --- - + + - - Vitamin C (1 mM) - - + - + - + - + + - UVA (7.68 J/cm2) - - - ++- - + + + - UVA (30.72 J/cm2) - - - - - + + - - -- )

Methylene blue + Light ------+ 2

Fpg c 8 mc/J M -++ -+-+-+-+-+-+-+-+-+- 6 . 7 ( AV ( 10.0 kb

8.0 kb U + )Mµ 1( )Mµ + 6.0 kb 5.0 kb 1( )Mµ )

4.0 kb 2 ) 2 mc/J 86.7( AVU 86.7( mc/J detaer 3.0 kb m c/J n nivalfobiR ivalfo 1

2.0 kb .0( T4 Endo V t - +

1.5 kb no B b iR V CPD photolyase N 1.0 kb U M -+-+-+ -+ - + 0.5 kb 495-500 bp

b 460 bp Riboflavin (1 µM) - + - -- - + + - Vitamin C (1 mM) - - + - + - - + + UVA (7.68 J/cm2) - - - ++ - + + - End 2 - - - - - + - - + UVA (30.72 J/cm ) Fpg M -++ -+-+-+-+-+-+-+- 495-500 bp 400 bp

460 bp End 364 bp cII Transgene 350 bp

400 bp

243-253 nt: AAA 300 bp e ACGCCCGG n 364 bp 228-230 nt: GAT e g

350 bp s

206-207 nt: GA n

255 bp a rT 179-184 nt: GG

GGGG IIc 300 bp 156-162 nt: AAT GCTG 131-132 nt: AG 200-204 bp 255 bp 117-122 nt: GAT CAG 99-100 nt: GG 81-84 nt: GAAG 200-204 bp 73-74 nt: GG 145 bp 55-57 nt: AAC 47-48 nt: CG 40-42 nt: GAG Start 145 bp 24-26 nt: CGA 8-10 nt: GTG Start cII Transgene

Fig. 2. Quantification of induced DNA lesions. (a) DNA damage in the genome. Cleavage assay with Fpg digestion and alkaline gel electrophoresis of the genomic DNA of mouse embryonic fibroblasts treated with riboflavin or irradiated with UVA1, individually or combined, in the presence and absence of vitamin C. (b and c) LM-PCR mapping of DNA damage in the cII transgene. DNA footprinting in mouse embryonic fibroblasts treated with riboflavin or irradiated with UVA1, individually or combined: in the presence and absence of vitamin C with (ϩ) and without (Ϫ) predigestion with Fpg enzyme (b) and with (ϩ) and without (Ϫ) pretreatment with T4 Endo V plus CPD photolyase reactivation (c). bp, base pair; M, molecular size marker; nt, nucleotide position.

samples not predigested with Fpg (Fig. 2b). Treatment with vitamin dimer, yielding a single-stranded DNA with a normal base on the C (0.001–10 mM) alone or in combination with UVA1 irradiation 5Ј sugar-phosphate terminus (26). did not result in lesion formation in the cII transgene. However, Likewise, terminal transferase-dependent PCR (TD-PCR) anal- vitamin C treatment (1 mM) effectively inhibited the formation of ysis revealed no substantial formation of lesions in the cII transgene lesions induced by riboflavin treatment combined with UVA1 in mouse embryonic fibroblasts treated with either riboflavin or irradiation or high-dose UVA1 irradiation alone (Fig. 2b). LM- UVA1 irradiation alone (SI Fig. 5a). The combined treatment with PCR of the pretreated DNA with T4 Endo V plus CPD photolyase riboflavin and UVA1 irradiation, however, produced significant reactivation showed no detectable level of lesions in the cII trans- levels of lesions throughout the cII transgene. Treatment with gene in cells treated with riboflavin or UVA1 irradiation, individ- vitamin C (0.001–10 mM) alone or in combination with UVA1 ually or combined (Fig. 2c). For LM-PCR analysis, CPDs can be irradiation did not yield any detectable level of lesions in the cII converted to ligatable 5Ј ends only by successive enzymatic diges- transgene. However, vitamin C treatment (1 mM) efficiently pre- tion with T4 endo V and Escherichia coli CPD photolyase reacti- vented the formation of lesions consequent to combined treatment vation. The T4 endo V cleaves the glycosidic bond of the 5Ј with riboflavin and UVA1 irradiation. We presume the TD-PCR pyrimidine in a CPD and breaks the sugar-phosphate backbone quantified lesions to be single-strand DNA breaks because photo-

between the two dimerized pyrimidines. The resulting dissociated dimeric (6-4)PPs are not produced by UVA1 irradiation (6), and GENETICS 3Ј pyrimidine retains an overhang dimer, which makes it unligatable because the T4 Endo V cleavage assay and LM-PCR could rule out until the E. coli CPD photolyase reactivation step detaches the the possibility of formation of CPDs at the genomic or nucleotide

Besaratinia et al. PNAS ͉ April 3, 2007 ͉ vol. 104 ͉ no. 14 ͉ 5955 Downloaded by guest on September 23, 2021 resolution level, respectively. We additionally excluded the forma- 06 1AVU +nivalfobiR 1AVU tion of (6-4)PPs by confirming that digestion with UV damage 05 * endonuclease, which is known to cleave both CPDs and (6-4)PPs 1AVU (27), did not produce any cleavage in the genomic DNA in cells 40 lortnoC

03 treated with riboflavin or UVA1 irradiation, individually or com- †

bined. Thus, by way of elimination, the lesions detected by TD-PCR Mutations 02

can be considered as single-strand DNA breaks. We verified this % assumption by subjecting the nondigested genomic DNA to alkaline 01

gel electrophoresis, which showed clear smears indicative of strand 0 → → → → → → breaks in the genomic DNA in cells treated with riboflavin and G:C C:G G:C T:A G:C A:T A:T T:A A:T G:C A:T C:G Del/Ins UVA1 irradiation combined (SI Fig. 5b). Fig. 3. Comparative mutation spectra of the cII transgene in Big Blue mouse embryonic fibroblasts treated with riboflavin (1 ␮M) combined with UVA1 cII Mutation Analysis. In the absence of UVA1 irradiation, riboflavin irradiation (7.68 J/cm2), UVA1 irradiation alone (18.0 J/cm2), or control. Ins, treatment at none of the tested concentrations was mutagenic in the insertion; Del, deletion. *, As compared with ‘‘nontreated control’’; P Ͻ 0.001. cII transgene in mouse embryonic fibroblasts. In combination with †, as compared with ‘‘nontreated control’’; P Ͻ 0.05. UVA1 irradiation (7.68 J/cm2), however, riboflavin treatment at a concentration of 1 ␮M was significantly mutagenic, raising the cII (31, 32), was concurrent with a lower frequency of C 3 T mutant frequency 4.3-fold over the background (12.1 Ϯ 2.2 vs. 2.8 Ϯ Ϫ transitions targeted to dipyrimidine sites, which are typically 0.7 ϫ 10 5; P Ͻ 0.001). Although the UVA1 dose (7.68 J/cm2) induced by photo-dimeric CPDs or (6-4)PPs (4), in the induced administered in the combination treatment was nonmutagenic per mutation spectra relative to control (Fig. 3). se, higher doses of UVA1 irradiation alone significantly increased the cII mutant frequency relative to background. Treatment with Discussion vitamin C (0.001–10 mM) alone or in combination with UVA1 irradiation was not mutagenic in the cII transgene. However, Our cytotoxicity examination revealed that riboflavin treatment vitamin C treatment (1 mM) substantially reduced the cII mutant alone was not cytotoxic to mouse embryonic fibroblasts. However, frequency induced by the combination treatment with riboflavin in combination with UVA1 irradiation, riboflavin treatment could and UVA1 irradiation or UVA1 irradiation alone at high doses significantly impair cellular viability. It has been shown that UVA (Ն15.36 J/cm2) (Table 1). irradiation of riboflavin generates reactive intermediate species, For mutation spectrometry analysis, we randomly selected one such as singlet oxygen, superoxide anions, triplet state riboflavin hundred cII mutant plaques induced by riboflavin treatment radicals, and hydrogen peroxide (33–35). High-dose UVA irradi- combined with UVA1 irradiation as compared with ninety- ation alone has also been proven to produce reactive oxygen species seven spontaneously arisen control plaques. Of these plaques, 94 with known cytotoxic properties (36). In our experiments, vitamin and 92 plaques, respectively, contained a minimum of one C at a physiologically relevant concentration of 1 mM was com- mutation along the cII transgene verified by DNA sequencing. pletely protective against the cytotoxic effects of combined treat- The vast majority of the induced and spontaneous mutations ment with riboflavin and UVA1 irradiation or of UVA1 irradiation were single base substitutions and less frequently single dele- alone at high doses. To rule out the possibility of filtration of UVA1 tions/insertions, with one multiple mutations only in the control by vitamin C, we spectrophotometrically determined the wave- (SI Table 2). Detailed mutation spectra induced by riboflavin length absorption of vitamin C and riboflavin in our experimental treatment combined with UVA1 irradiation or derived sponta- system. The UV absorption spectrum for vitamin C was only in the Ϸ neously are presented in SI Fig. 6. Overall, the spectrum of UVC range, reaching a peak at 265 nm. However, the wavelength induced mutations was significantly different from the sponta- scan of riboflavin confirmed an absorption in the UVA1 range with neously arisen mutation spectrum (P Ͻ 0.04, 95% confidence a peak at 375 nm, which was well within the emission spectrum of interval ϭ 0.03–0.05). To underscore the difference(s) between our UVA source. Thus, the observed protective effects of vitamin induced and spontaneous mutation spectra, we compared the C can essentially be ascribed to its antioxidant and free radical frequency of each type of mutation, e.g., transitions, transver- scavenging capacities (22). sions, etc. in the respective mutation spectra. Because the cII Our quantification of DNA damage both at the genomic and transgene in the Big Blue system is a nontranscribed gene (28), nucleotide resolution level verified that riboflavin treatment in the strand bias of mutagenesis, a phenomenon caused by tran- combination with UVA1 irradiation or high-dose UVA1 irradia- scription-coupled DNA repair (29), is unlikely to affect the tion alone induced oxidized (ring opened) purines in the overall spectrum of mutations in this transgene (28). Hence, it is genome and in the cII transgene in mouse embryonic fibroblasts. justified to combine the strand mirror counterparts of all tran- DNA footprinting mapped the formation of most induced lesions sitions (e.g., G 3 A ϩ C 3 T) and transversions (e.g., G 3 T to guanine residues along the cII transgene. It is known that the ϩ C 3 A and G 3 C ϩ C 3 G) when comparing the specific lower ionization potential of guanine relative to other DNA bases types of mutation between different treatment groups. As shown makes it a favorable target for type-I photosensitized one-electron in Fig. 3, the spectrum of mutations induced by riboflavin oxidation and/or type-II photooxidation, mediated through singlet treatment combined with UVA1 irradiation was different from oxygen generation (37, 38). The resulting radical cations of guanine the spontaneous mutation spectrum in that there was a signif- can undergo hydration to produce 8-oxo-dG and 2,6-diamino-4- icant increase in the relative frequency of G:C 3 T:A transver- hydroxy-5-formamidopyrimidine (39), both lesions being substrates sions (52.1% vs. 14.0%; P Ͻ 0.001). In confirmation, we have of the DNA repair , Fpg (40). In our hands, cleavage assays previously shown that UVA1 irradiation alone induced predom- coupled to alkaline gel electrophoresis and LM-PCR showed that inantly G:C 3 T:A transversions in the cII (11, 12) and lacI (30) Fpg-sensitive sites were formed conspicuously in the overall ge- transgenes of the same model system. Obviously, the hallmark of nome and in the cII transgene in cells treated with riboflavin G:C 3 T:A transversion mutations induced by UVA1 irradia- combined with UVA1 irradiation or irradiated with high-dose tion alone was more pronounced in the mutation spectrum UVA1 alone. produced by riboflavin treatment combined with UVA1 irradi- We and others have previously shown that intense UVA irradi- ation (see Fig. 3). The frequent occurrence of G 3 T transver- ation can cause single-strand DNA breaks in various experimental sions, which are the signature mutation of oxidative DNA systems (6, 41–43). The formation of these lesions has been ascribed damage, e.g., 8-oxo-7,8-dihydro-2Ј-deoxyguanosines (8-oxo-dG) to hydroxyl radical generation, e.g., by means of conversion of

5956 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610534104 Besaratinia et al. Downloaded by guest on September 23, 2021 hydrogen peroxide in the presence of transition metals through is of significance because per joule basis, UVB radiation is up to Fenton-like reaction (44). Formation of hydrogen peroxide has also 50,000 times more genotoxic than UVA radiation (49). Ikehata et been reported in various test systems consequent to treatment with al. (50) have used an irradiation source emitting a minute but UVA-activated riboflavin or irradiation with high-dose UVA alone appreciable fraction of UVB radiation, and reported an induced (34, 45). In our experiments, single-strand DNA breaks were mutation spectrum predominated by single and tandem C 3 T formed in cells treated with riboflavin combined with UVA1 transitions at dipyrimidine sites in the lacZ transgene in mouse skin irradiation. However, irradiation with UVA1 alone at a moderately epidermis. Also, Woollons et al. (51) have demonstrated that the mutagenic dose did not produce detectable levels of single-strand 0.8% UVB component of a UVA sunlamp accounted for 75% of DNA breaks. These findings imply that in our experimental system, the CPDs induced in human keratinocytes irradiated in vitro. the presence of endogenous photosensitizers enables UVA1 to The fairly high detection limit of most available techniques for exert its DNA damaging effects primarily through type-II photo- quantification of photo-induced DNA damages (particularly at the sensitization reactions involving singlet oxygen generation. How- nucleotide resolution level) has necessitated intense UVA irradi- ever, the excess riboflavin introduced into our system may have ation, enabling sufficient production of DNA lesions (6, 9, 10, 52). prompted UVA1 to cause additional DNA damage by producing Because high-dose UVA irradiation causes severe cytotoxicity, the other reactive oxygen species such as hydrogen peroxide. In both induced DNA damage is unlikely to be translated into mutation in cases, the formation of DNA damage was effectively inhibited by a cells with no or low proliferation capacity (4). We have established cotreatment with vitamin C. The protective effect of vitamin C that the type of induced DNA lesions depends on UVA irradiation against oxidative stress-induced DNA damage has been demon- dose, i.e., at low irradiation dose, of relevance for mutagenicity strated by others (43) and ascribed to its antioxidant and/or free experiments, oxidative DNA damage is formed predominantly (11, radical scavenging properties (22), as well as its ability to regenerate 12). However, at high-dose UVA irradiation, both CPDs and other antioxidants, e.g., ␣-tocopherol and urate from their respec- oxidative DNA damage are induced (6). It is imperative that tive radicals (46). Vitamin C can also directly react with singlet UVA-induced DNA damage and mutagenesis be investigated oxygen, and the resulting peroxide may be less toxic owing to simultaneously and at a biologically relevant dose. Our studies are efficient peroxide removing systems (47). unique in that they compare DNA damage-targeted mutagenicity Our mutagenicity analysis showed that riboflavin treatment in of UVA at a relevant dose in a single test system, therefore, combination with UVA1 irradiation or high-dose UVA1 irra- accounting for the above-mentioned potential confounding diation alone significantly increased the cII mutant frequency variables. relative to background in mouse embryonic fibroblasts. A co- Under special circumstances, vitamin C has been shown to treatment with vitamin C, however, substantially reduced the cII exhibit pro-oxidant activities (53). For example, Fan et al. (25) have mutant frequency induced by the combination treatment with reported that overexpression of the human vitamin C transporter riboflavin and UVA1 irradiation or high-dose UVA1 irradiation SVCT2 in a mouse model accelerated the modification of lens alone. DNA sequencing of the cII mutants revealed a significant crystalline as a consequence of vitamin C glycation by the Maillard increase in the relative frequency of G:C 3 T:A transversions reaction. In humans, high concentrations of vitamin C have been induced by riboflavin treatment combined with UVA1 irradia- found in the stratum corneum, and viable epidermis and dermis, tion, which is consistent with our previously established muta- with the greatest levels being in the deeper layers (24) wherein tion spectra induced by UVA1 irradiation alone in the cII (11, penetrating UVA causes oxidative stress (22). Millimolar concen- 12) and lacI (30) transgenes of the same experimental model trations of vitamin C (up to 10 mM) have been reported in various system. As mentioned earlier, G 3 T transversions are the tissues, including hippocampus and the adrenal glands. The pres- hallmark mutations induced by oxidative DNA damage, includ- ence of vitamin C and glutathione in the millimolar range in the ing 8-oxo-dG and 2,6-diamino-4-hydroxy-5-formamidopyrimi- human lens takes on additional significance with the knowledge dine (11, 12, 31, 48). The established signature mutation of G:C that the riboflavin concentration in this tissue reaches 4.5 ␮M (25). 3 T:A transversions induced by UVA1 irradiation alone was In our experiments, vitamin C at a physiologic concentration more pronounced in the mutation spectrum produced by ribo- exerted antioxidant effects in response to oxidative stress imposed flavin treatment combined with UVA1 irradiation (25.2% vs. by UVA1-activated riboflavin treatment or high-dose UVA1 irra- 52.1% of all mutations in Fig. 3). These observations confirm diation alone. The implication of these findings for public health is that the addition of an endogenous photosensitizer, such as the formulation of sunscreens with optimal concentrations of riboflavin, to our experimental system magnifies the oxidative- antioxidants. DNA damage-mediated mutagenicity of UVA1 alone. The In conclusion, we have shown a characteristic oxidative DNA induced mutation spectra did not display the signature mutations damage-targeted mutagenicity of UVA1 in the cII transgene of Big of photodimeric CPDs or (6-4)PPs, i.e., single or tandem C 3 T Blue mouse embryonic fibroblasts, augmented by the addition of transitions targeted to dipyrimidine sites (4). The latter finding the photosensitizer riboflavin, and diminished by the inclusion of is in good agreement with the absence of photodimeric lesions the antioxidant vitamin C. Our findings reaffirm the notion that in the genomic DNA consequent to various UVA1 treatments. intracellular photosensitization reactions, which generate promuta- The spectrum of mutations produced by UVA irradiation in genic oxidative DNA damage, are responsible for UVA-induced different studies has not always matched the mutagenic potential of genotoxicity. the various induced lesions (4). The discrepancies have mostly manifested in studies where the two endpoints, i.e., DNA damage Materials and Methods and mutation, have not been determined simultaneously and/or Cell Culture and Treatment. Early passage Big Blue mouse embry- within a single test system (4). We have shown that cell cultures of onic fibroblasts were grown to monolayer Ϸ30% confluence in varying species and types are differently resistant toward cytotoxic DMEM supplemented with 10% FBS. Before treatment, the and genotoxic effects of UVA radiation (6, 11, 12). Such variability culture media were removed, and the cells were washed thoroughly might arise from the unique DNA repair capacity and diverse with PBS. Riboflavin and vitamin C (Sigma–Aldrich, St. Louis, content of intracellular photosensitizers, specific for each species MO) were dissolved in PBS. The cells were treated with increasing and cell type (4). In addition, experimental variables such as UVA concentrations of riboflavin (0.01–100 ␮M) or solvent in the source and dose may also lead to some discrepancies (4). For presence or absence of vitamin C at different concentrations

instance, irradiation sources, which emit contaminating UV wave- (0.001–100 mM) for 20 min at 37°C in the dark. After multiple GENETICS lengths, e.g., in the UVB range, could cause distorted mutation washes with PBS, the culture dishes were placed on ice and spectra by inducing UVB-specific DNA lesions. This latter concern irradiated (in PBS) with various doses of UVA1 (0.96, 1.92, 3.84,

Besaratinia et al. PNAS ͉ April 3, 2007 ͉ vol. 104 ͉ no. 14 ͉ 5957 Downloaded by guest on September 23, 2021 7.68, 15.36, and 30.72 J/cm2). The UVA source was a Sellas Sunlight tially similar to LM-PCR with the only differences being the System (Medizinische Gera¨teGmbH; Gevelsberg, Germany) with primer extension and ligation steps (54). Unlike LM-PCR, an average fluence rate of 60 mW/cm2. The source exclusively emits TD-PCR does not require chemical or enzymatic digestion of long wave UVA (UVA1: 340–400 nm) and not UVA at the DNA to produce single-strand DNA breaks with 5Ј phosphate borderline with UVB (see SI Fig. 7). Immediately after irradiation, groups at the lesion formation sites. Thus, TD-PCR can be the cells were harvested by trypsinization for determination of cell readily applied to all 5Ј phosphorylated or unphosphorylated survival, and for detection of DNA damage. Alternatively, the cells DNA templates that contain polymerase blocking lesions, i.e., were cultured in complete growth medium for an additional 4 days, single-strand breaks and/or bulky lesions, e.g., CPDs or (6-4)PPs. and afterward were analyzed for mutant frequency and mutational spectrum of the cII transgene. The 4-day growing period is essential cII Mutant Frequency and Mutation Spectrometry Determination. The for the fixation of all mutations into the genome (26). At the time ␭LIZ shuttle vectors were rescued from the genomic DNA, and of harvesting, all cultured cells had undergone 3–4 population packaged into viable phage particles by using the ‘‘lambda LIZ doublings and reached full confluence. All experiments were transpack packaging extract’’ according to the manufacturer’s in- conducted in triplicates. structions (Stratagene, La Jolla, CA). The phages were pread- sorbed to G1250 E. coli, and the bacterial culture was plated on TB1 Lesion-Specific Cleavage Assays with Fpg and T4 Endo V. The enzy- agar plates. The plates were incubated for 48 h at 24°C or overnight matic digestion assays use Fpg for cleavage of oxidized (ring- at 37°C. Putative cII mutant plaques were all verified after being opened) purines, and T4 Endo V for nicking at CPDs (26). The replated under the selective condition on a second TB1 agar plate. digested DNA is then subjected to alkaline gel electrophoresis for The cII mutant frequency was expressed as the ratio of the number assessment of damage in the overall genome. Alternatively, the of verified mutant plaques to the total number of screened plaques. DNA digest can be analyzed by footprinting methodologies, e.g., For quality assurance, control phage solutions containing mutant LM-PCR, enabling the detection of lesions at the level of nucleotide and wild-type lambda cII having known mutant frequencies were resolution (26). Briefly, DNA was digested with an excess amount assayed in all runs. As recommended by the manufacturer, a of Fpg (Trevigen, Gaithersburg, MD) or T4 Endo V (Epicentre, minimum of 3 ϫ 105 rescued phages were screened for each Madison, WI) in special buffers supplied by the respective manu- experimental condition. For mutation spectrometry analysis, the facturers. After ethanol precipitation, the digests were loaded onto verified plaques were cored, and subjected to PCR amplification by a 1.5% alkaline agarose gel, and run for4hat40V.Asstandard using the ‘‘lambda select-cII sequencing primers’’ according to the controls, genomic treated with methylene blue plus white manufacturer’s recommended protocol. The PCR products were light or irradiated with UVB, which contained known amounts of purified with a QIA quick PCR-purification kit (Qiagen GmbH, 8-oxo-dG or CPDs (6), respectively, were used in all runs. Hilden, Germany), and sequenced by using a Big Dye terminator cycle sequencing kit and an ABI-377 DNA Sequencer (ABI Prism; LM–PCR and TD–PCR. LM–PCR was used for DNA footprinting of PE Applied BioSystems, Foster City, CA). For more details, see SI specific UVA-induced lesions, including oxidized (ring-opened) Materials and Methods. purines and CPDs along the entire length of the cII transgene. Methodologically, LM–PCR is based on the concept that DNA We thank Dr. Stella Tommasi for help with video microscopy for polymerase cannot synthesize DNA past certain type of lesions, cytotoxicity examination. This work was supported by National Institute i.e., single-strand breaks. The principles of TD-PCR are essen- of Environmental Health Sciences Grant ES06070 (to G.P.P.).

1. de Gruijl FR (2002) Skin Pharmacol Appl Skin Physiol 15:316–320. 30. Kim S, Pfeifer GP, Besaratinia A, Mutat Res, in press. 2. Woodhead AD, Setlow RB, Tanaka M (1999) J Epidemiol 9:S102–114. 31. Wood ML, Dizdaroglu M, Gajewski E, Essigmann JM (1990) Biochemistry 29:7024–7032. 3. Setlow RB (1974) Proc Natl Acad Sci USA 71:3363–3366. 32. Shibutani S, Takeshita M, Grollman AP (1991) Nature 349:431–434. 4. Pfeifer GP, You YH, Besaratinia A (2005) Mutat Res 571:19–31. 33. Baier J, Maisch T, Maier M, Engel E, Landthaler M, Baumler W (2006) Biophys J 5. Kvam E, Tyrrell RM (1997) Carcinogenesis 18:2379–2384. 91:1452–1459. 6. Besaratinia A, Synold TW, Chen HH, Chang C, Xi B, Riggs AD, Pfeifer GP (2005) Proc Natl 34. Hockberger PE, Skimina TA, Centonze VE, Lavin C, Chu S, Dadras S, Reddy JK, White Acad Sci USA 102:10058–10063. JG (1999) Proc Natl Acad Sci USA 96:6255–6260. 7. Douki T, Reynaud-Angelin A, Cadet J, Sage E (2003) Biochemistry 42:9221–9226. 35. Sato K, Taguchi H, Maeda T, Minami H, Asada Y, Watanabe Y, Yoshikawa K (1995) J Invest 8. Perdiz D, Grof P, Mezzina M, Nikaido O, Moustacchi E, Sage E (2000) J Biol Chem Dermatol 105:608–612. 275:26732–26742. 36. Krutmann J (2000) J Dermatol Sci 23 (Suppl 1):S22–26. 9. Rochette PJ, Therrien JP, Drouin R, Perdiz D, Bastien N, Drobetsky EA, Sage E (2003) 37. Douki T, Cadet J (1999) Int J Radiat Biol 75:571–581. Nucleic Acids Res 31:2786–2794. 38. Wondrak GT, Roberts MJ, Jacobson MK, Jacobson EL (2004) J Biol Chem 279:30009– 10. Mouret S, Baudouin C, Charveron M, Favier A, Cadet J, Douki T (2006) Proc Natl Acad 30020. Sci USA 103:13765–13770. 39. Cadet J, Ravanat J-L, Martinez GR, Medeiros MHG, Di Mascio P (2006) Photochem 11. Besaratinia A, Bates SE, Synold TW, Pfeifer GP (2004) Biochemistry 43:15557–15566. Photobiol 82:1219–1225. 12. Besaratinia A, Synold TW, Xi B, Pfeifer GP (2004) Biochemistry 43:8169–8177. 40. Boiteux S, O’Connor TR, Laval J (1987) EMBO J 6:3177–3183. 13. Kielbassa C, Roza L, Epe B (1997) Carcinogenesis 18:811–816. 41. Alapetite C, Wachter T, Sage E, Moustacchi E (1996) Int J Radiat Biol 69:359–369. 14. Zhang X, Rosenstein BS, Wang Y, Lebwohl M, Mitchell DM, Wei H (1997) Photochem 42. Lan L, Nakajima S, Oohata Y, Takao M, Okano S, Masutani M, Wilson SH, Yasui A (2004) Photobiol 65:119–124. Proc Natl Acad Sci USA 101:13738–13743. 15. Drobetsky EA, Turcotte J, Chateauneuf A (1995) Proc Natl Acad Sci USA 92:2350–2354. 43. Lehmann J, Pollet D, Peker S, Steinkraus V, Hoppe U (1998) Mutat Res 407:97–108. 16. Agar NS, Halliday GM, Barnetson RS, Ananthaswamy HN, Wheeler M, Jones AM (2004) 44. Mello Filho AC, Hoffmann ME, Meneghini R (1984) Biochem J 218:273–275. Proc Natl Acad Sci USA 101:4954–4959. 45. Minami H, Sato K, Maeda T, Taguchi H, Yoshikawa K, Kosaka H, Shiga T, Tsuji T (1999) 17. Ikehata H, Kudo H, Masuda T, Ono T (2003) Mutagenesis 18:511–519. 18. Kappes UP, Luo D, Potter M, Schulmeister K, Runger TM (2006) J Invest Dermatol J Invest Dermatol 113:77–81. 126:667–675. 46. Buettner GR (1993) Arch Biochem Biophys 300:535–543. 19. Kappes UP, Runger TM (2005) Radiat Res 164:440–445. 47. Kramarenko GG, Hummel SG, Martin SM, Buettner GR (2006) Photochem Photobiol 20. Kozmin S, Slezak G, Reynaud-Angelin A, Elie C, de Rycke Y, Boiteux S, Sage E (2005) Proc 82:1634–1637. Natl Acad Sci USA 102:13538–13543. 48. Kalam MA, Haraguchi K, Chandani S, Loechler EL, Moriya M, Greenberg MM, Basu AK 21. Foraker AB, Khantwal CM, Swaan PW (2003) Adv Drug Deliv Rev 55:1467–1483. (2006) Nucleic Acids Res 34:2305–2315. 22. Catani MV, Savini I, Rossi A, Melino G, Avigliano L (2005) Nutr Rev 63:81–90. 49. de Gruijl FR, Sterenborg HJ, Forbes PD, Davies RE, Cole C, Kelfkens G, van Weelden H, 23. Ou-Yang H, Stamatas G, Saliou C, Kollias N (2004) J Invest Dermatol 122:1020–1029. Slaper H, van der Leun JC (1993) Cancer Res 53:53–60. 24. Shindo Y, Witt E, Han D, Epstein W, Packer L (1994) J Invest Dermatol 102:122–124. 50. Ikehata H, Nakamura S, Asamura T, Ono T (2004) Mutat Res 556:11–24. 25. Fan X, Reneker LW, Obrenovich ME, Strauch C, Cheng R, Jarvis SM, Ortwerth BJ, 51. Woollons A, Kipp C, Young AR, Petit-Frere C, Arlett CF, Green MH, Clingen PH (1999) Monnier VM (2006) Proc Natl Acad Sci USA 103:16912–16917. Br J Dermatol 140:1023–1030. 26. Besaratinia A, Pfeifer GP (2005) Carcinogenesis 27:1526–1537. 52. Douki T, Perdiz D, Grof P, Kuluncsics Z, Moustacchi E, Cadet J, Sage E (1999) Photochem 27. Yajima H, Takao M, Yasuhira S, Zhao JH, Ishii C, Inoue H, Yasui A (1995) EMBO J Photobiol 70:184–190. 14:2393–2399. 53. Sakagami H, Satoh K (1997) Anticancer Res 17:3513–3520. 28. Lambert IB, Singer TM, Boucher SE, Douglas GR (2005) Mutat Res 590:1–280. 54. Chen H-H, Kontaraki J, Bonifer C, Riggs AD (April 10, 2001) Sci STKE, 10.1126/ 29. Mellon I, Spivak G, Hanawalt PC (1987) Cell 51:241–249. stke.2001.77.pl1.

5958 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610534104 Besaratinia et al. Downloaded by guest on September 23, 2021