Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

Mutation Research 688 (2010) 3–11

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Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation A. Viviana Pinto a,b,∗, Elder L. Deodato a,b, Janine S. Cardoso b, Eliza F. Oliveira a, Sérgio L. Machado a, Helena K. Toma a , Alvaro C. Leitão b , Marcelo de Pádula a a Laboratório de Diagnóstico Molecular e Hematologia, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde - Ilha do Fundão, CEP 21941-540, Rio de Janeiro, Brazil b Laboratório de Radiobiologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde - Ilha do Fundão, CEP 21949-900, Rio de Janeiro, Brazil

a r t i c l e

i n f o

Article history: Received 20 May 2009 Received in revised form 27 January 2010 Accepted 9 February 2010 Available online 16 February 2010 Keywords: Titanium dioxide Oxidatively generated DNA damage Saccharomyces cerevisiae 8-Oxo-7,8-dihydroguanine

a b s t r a c t Although titanium dioxide (TiO2 ) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO2 is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO2 –UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO2 associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO2 plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO2 protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO2 plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO2 plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Normal-sized (>100 nm) titanium dioxide is a white pigment widely used in industrial products like paints, papers, plastic, etc. [1–3]. As a result of TiO2 reflection capacity, it scatters mainly above 415 nm (UV radiation) [4]. Thus, TiO2 is utilized as a photo-blocking component in sunscreens and cosmetic products [5,6]. However, ultraviolet radiation is also absorbed by the electronic band structure of this semiconductor (band gap energies between 3.23 and 3.06 eV, corresponding to light between 385 and 400 nm) [7]. This results in the formation of electron–hole pairs, which, subsequent to their separation, migrate towards

∗ Corresponding author at: Instituto Nacional de Controle de Qualidade em Saúde, Fundac¸ão Oswaldo Cruz, Avenida Brasil, 4365–Manguinhos, CEP 21040-900, Rio de Janeiro, Brazil. Tel.: +55 21 3865 5139; fax: +55 21 3865 5139. E-mail address: alicia.pinto@incqs.fiocruz.br (A.V. Pinto). 0027-5107/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2010.02.003

the crystallite surface of TiO2 and initiate photo-reductions and photo-oxidations. The electrons react with pre-adsorbed molecular oxygen to yield superoxide radical anion (O2 •− ), whereas the valence band holes oxidize the surface hydroxyl groups (OH− ) to generate hydroxyl radicals (• OH) [8,9]. When • OH are produced within the cell cytoplasm they give rise to carboxyl radicals anions (• CO2 − ), which, subsequently, oxidize other cell components and form other damaging radical species [10]. Therefore, the damaging effects of reactive oxygen species generated by TiO2 associated with UV irradiation (essentially UVA  = 320–400 nm) were studied in both prokaryotic and eukaryotic cells [11–15]. It was extensively documented that TiO2 associated with UVA (photo-activated TiO2 ) inactivates, via cell membrane damage, a wide spectrum of microorganisms [11,13,16]. In eukaryotic cells, oxidatively generated damage induced by photo-activated TiO2 compromises the cellular membrane of T24 cells [17] and also DNA and RNA, leading to guanine hydroxylation [12]. Photo-activated TiO2 is also able to damage human fibroblasts [5,12,18], human

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bronchial epithelial cells [14], goldfish skin [15] and rainbow trout gonad cell lines [19]. In vitro, photo-activated TiO2 is able to damage DNA, especially at guanine residues [5,20,21], which may be due to DNA attack by • OH generated via iron-catalyzed Fenton reaction [22] and/or by the action of copper metallic ions [21]. Despite the large amount of information about the consequences of TiO2 associated with UVA radiation, the biological effects from the association of TiO2 photosensitized with UVB (band gap energies between 4.2 and 3.8 eV corresponding to light of about 290 and 320 nm) are almost unknown [23]. Since UVB radiation is the major environmental carcinogen that induces nonmelanoma skin cancer [24–26], it is extremely relevant to be acquainted with the putative biological consequences of UVB–TiO2 association. As photosensitized DNA oxidations were previously detected with TiO2 plus UVA, specially in guanines residues, the aim of this study was to characterize the chemical nature of DNA lesions induced by TiO2 plus UVB light, the biological effects and the mechanisms involved in the generation/repair of these DNA lesions. The identification of DNA damage was based on the substrate specificity of the base excision repair (BER) enzymes from Escherichia coli [27–30], which belong to the most effective DNA repair pathway (BER) that bacterial cells possess to counteract oxidatively generated damage [31,32]. The enzymes used were: (i) Fpg and Nth endonucleases for photo-oxidized purines and pyrimidines, respectively [33]; (ii) XthA exonuclease for regular and modified abasic sites [34], and (iii) Nfo endonuclease for regular/modified abasic sites [35] and incision of oxidized pyrimidines, including 5-hydroxycytosine [36]. In addition, T4 endonuclease V enzyme was used to detect the contribution of directly UVB-induced cyclobutane pyrimidine dimers (CDPs) [37–42] in comparison with oxidatively generated DNA damage. The biological consequences and the mechanisms involved in DNA damage induced by TiO2 plus UVB were assessed using Saccharomyces cerevisiae model. Cell survival and mutagenesis were determined in S. cerevisiae wild-type, ogg1 and rad1 mutants (deficient) strains after TiO2 plus UVB treatment. Additionally, ion chelators neocuproine (for copper) and dipyridyl (for iron) were used to test if oxidatively generated DNA damage would occur via metal-catalyzed reactions [43]. Although ogg1 strain is not particularly sensitive to most oxidants, it is clearly sensitive to UVA mutagenesis, as well as to a series of different oxidants [44]. The Ogg1 protein is the functional homolog of the bacterial Fpg protein and both are responsible, in vivo, for the repair of 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) [27,45]. The ogg1 mutant accumulates GC to TA transversions, which is a hallmark of 8-oxoG. In its turn, rad1 mutant is highly sensitive to UV [46], specially due to the formation of CPDs in DNA, which are capital UV induced lesions and biologically relevant in terms of both toxicity and mutagenesis [46,47]. In sum, these features render S. cerevisiae a useful model to study oxidatively generated DNA damage induced by physical and chemical agents [29,43]. The results reported herein demonstrate that, although TiO2 protected cells against lethal damage induced by UVB light, there was a clear increase in the number of Fpg-sensitive sites in DNA exposed to TiO2 plus UVB when compared to isolated treatments. In addition, S. cerevisiae wild-type strain, and specially the ogg1 mutant, displayed higher mutation frequencies when exposed to TiO2 plus UVB than UVB or TiO2 alone. Overall, the association of TiO2 plus UVB potentates UVB oxidatively generated DNA damage, possibly via Fenton reactions involving the production of guanine oxidation products, such as 8-oxo-7,8-dihydroguanine.

2. Materials and methods 2.1. Chemicals Titanium(IV) oxide anatase (<44 ␮m, 1317-70-0), ion chelators 2,2 dipyridyl (366-18-7) and neocuproine (484-11-7), as well as methylene blue (7220-79-3), were purchased from Sigma. Phosphate Buffered Saline (PBS) [8.0 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na2 HPO4 and 0.24 g/l of KH2 PO4 , at pH 7.2] was purchased from Laborclin, Brazil. Extemporaneous solutions of TiO2 were prepared at 100 mg/ml in sterile PBS as recommended [14,15]. Extemporaneous solutions of dipyridyl and neocuproine were prepared at 100 mM in H2 O:ethanol (50%, v/v). Methylene blue (MB) stock solution was prepared at 200 ␮g/ml in sterile PBS [29,43]. 2.2. Plasmid and enzymes Plasmid pUC18 (AmpR ) was prepared from transformed E. coli DH5␣ using the PureLinkTM Quick Plasmid Miniprep Kit (InvitrogenTM ) and stored at −20 ◦ C. Fpg, Nth, XthA and Nfo were a generous gift from Dr. S. Boiteux, CEA, Fontenay-aux-Roses, France. T4 endonuclease V was purchased from New England Biolabs. 2.3. Phototreatment of plasmid DNA Supercoiled plasmid pUC18 (25 ␮g/ml) was diluted in TE buffer (50 mM Tris–HCl, pH 7.8, 2 mM EDTA) and incubated in the presence of 100 ␮g/ml of TiO2 or 1 ␮g/ml of MB [29,48]. Immediately, each mixture (200 ␮l final volume) was placed in a 96-well microliter plate and then irradiated at 0 ◦ C. For TiO2 treatment, irradiation was performed using a 15-W Vilber Lourmart Ultraviolet lamp (broad spectrum 290–320 nm with emission peak at 312 nm) with a Nunc Petri lid that filters out light below 300 nm, as previously described [49]. Its UVB fluence was approximately 15 J/m2 /s and the UVA emission was negligible (0.06 J/m2 /s). UVB and UVA radiation doses were determined with a VL–215 LM radiometer (Vilbert Lourmart, Marne la Vallée, France) and Blak-Ray model J-221 longwave UV Measuremeter (UVP, San Gabriel, CA, USA), respectively. For MB treatment, the solution was irradiated with visible light (VL) using a GE PAR 38 “Cool beam” 220 V/150 W lamp as previously described [50]. Visible light fluence was measured with an YSI-Kettering model 65A radiometer (Yellow Spring Instruments, Yellow Spring, OH, USA). After treatment, DNA was ethanol precipitated, washed with ethanol 70% and resuspended in 50 ␮l of TE. 2.4. Quantification of strand breaks and enzyme-sensitive sites 200 ng modified pUC18 DNA dissolved in 20 ␮l BE16 buffer (25 mM Tris–HCl, pH 7.5, 2 mM EDTA, 50 mM KCl) was incubated at 37 ◦ C with 10 ng of Fpg, Nth, XthA and Nfo. In the case of exonuclease III, EDTA in the buffer was substituted by 15 mM CaCl2 [27]. For T4 endonuclease V enzyme, reactions were performed according to manufacturer instructions. After 30 min, the reactions were stopped by the addition of 3 ␮l 10% SDS. For determination of directly produced strand breaks, the incubation was performed without enzymes. Reaction products were separated using 1% agarose gel electrophoresis. The fraction of supercoiled DNA and open circular DNA was determined after ethidium bromide staining, analyzed using the Kodak 1D Scientific Imaging System and quantified with the Scion Image program. The average number of strand breaks per 10,000 base pairs was calculated assuming a Poisson distribution of the lesions [27]. 2.5. Yeast strains, media and growth conditions Yeasts were S. cerevisiae parental strain FF18733 (Mat a, his7, leu2, lys1, ura3, trp1) and their derivatives CD138 (ogg1::TRP1) and FF181481 (rad1::LEU2). Wild-type and FF181481 strains were a kind gift from Dr. F. Fabre, CEA, Fontenayaux-Roses, France and CD138 strain was from our laboratory stock [51]. Yeast strains were grown at 30 ◦ C in YPD medium (1% yeast extract, 1% bactopeptone, 2% glucose, with 2% agar for plates) or YNBD medium (2% glucose, 0.7% yeast nitrogen base without amino acids with 2% agar for plates) supplemented with appropriate amino acids and bases. Supplemented YNBD medium lacking arginine but containing canavanine (Sigma) at 60 mg/l was used for the selective growth of canavanine-resistant (CanR ) mutants [43]. 2.6. UVB treatment Yeast cultures (10 ml) were grown to a cell density of ∼1 × 108 cells/ml at 30 ◦ C with agitation (stationary phase). Cells were harvested, washed twice with PBS and resuspended in the same buffer. Glass Petri plates (5.0 cm diameter) accompanied by Nunc Petri lids with a final volume of 15 ml of PBS containing 1 × 107 cells/ml and 100 ␮g/ml of TiO2 were exposed to increasing UVB doses under agitation. The lids, UVB source and dosimeter utilized in these treatments were described above (phototreatment of plasmid DNA). After each dose, aliquots were taken, properly diluted in PBS buffer, plated on YPD medium, and colonies were counted after 2 days at 30 ◦ C. All survival experiments were independently performed at least three times.

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Fig. 2. Survival of wild-type (FF18733), ogg1 (CD138) and rad1 (FF181481) strains to UVB radiation in the presence and absence of TiO2 (100 ␮g/ml). Data represent means ± standard error (S.E.). Fig. 1. Comparison among different treatments, with exo and endonucleases, of pUC18 plasmid DNA exposed to: UVB (25 kJ/m2 ; 0 ◦ C), TiO2 (100 ␮g/ml; 30 min; 0 ◦ C), UVB plus TiO2 (25 kJ/m2 ; 100 ␮g/ml; 0 ◦ C), or methylene blue (1 ␮g/ml) in the presence of visible light (150 W, 40 cm; 2 min; 0 ◦ C). The different columns indicate the number of sites sensitive to (2) Fpg, (3) Nth, (4) exonuclease III (5) endonuclease IV, and (6) T4 endonuclease V. Column (1) gives the number of direct single strand breaks generated by the different agents. In the case of MB plus visible light, (1) indicates direct DNA breaks induced by MB (visible light had no effect on DNA break generation—data not shown). The number of spontaneous strand breaks was already discounted from each treatment. (*) Indicates significant versus control.

2.7. Induced mutation frequencies After cell treatment, quantification of UVB plus TiO2 -induced canavanineresistant mutants (UVB/TiO2 -CanR ) was performed by plating appropriate dilutions of treated cells on selective medium, and colonies were counted after 4 days at 30 ◦ C. Frequency of UVB/TiO2 -induced CanR mutants was calculated in terms of cell survivors and normalized to 107 cells. All these experiments were carried out independently at least five times, and all mutation frequencies were determined and expressed as averages with standard errors. 2.8. Pretreatment with ion chelators 15 ml of PBS containing 1 × 107 cells/ml was incubated at 30 ◦ C with 1.0 mM ion chelators dipyridyl or neocuproine for 20 min in PBS buffer. After, 100 ␮g/ml of TiO2 was added and UVB treatments were performed as described above. The use of ion chelators was based on previous reports examining the role of transition metals on oxidatively generated DNA damage and mutagenesis induced by H2 O2 [43,52,53] and by UVB [49]. 2.9. UV absorption spectra of neocuproine and dipyridyl The UV intensity absorption spectra, in arbitrary units, of ion chelators neocuproine and dipyridyl were obtained with a Hitachi U-3300 spectrophotometer coupled to its UV-solutions Application Program. 1 mM solutions of each ion chelator were extemporaneously prepared in sterile PBS and scanned from 200 up to 400 nm. Baseline was obtained with sterile PBS buffer.

overestimated, as they may result from the conversion of labile DNA modifications during DNA analysis or upon UVB irradiation itself, as the number of spontaneous strand breaks was already discounted from each treatment (Fig. 1, UVB). In higher extents, both oxidized purines and pyrimidines could also be detected (Fpgand Nth-sensitive sites, respectively, p < 0.05). Sites sensitive to XthA were not increased (p > 0.05), suggesting that AP sites are not being produced by UVB, or poorly detected, in our conditions. In fact, Nfo-sensitive sites were significant (p < 0.05), indicating that other species, different from regular AP sites, are generated by UVB radiation. As expected, T4 endonuclease V-sensitive sites were extensively increased (p < 0.001) confirming the marked contribution of CPD generation upon UVB irradiation [54]. TiO2 was able to produce few direct DNA strand breaks. After TiO2 treatment, sensitive sites to all five enzymes were not increased (p > 0.05, Fig. 1, TiO2 ), indicating that TiO2 alone was unable to generate oxidatively DNA damage or CPDs. In UVB plus TiO2 treatment, the production of DNA breakage, oxidized pyrimidines and abasic sites was comparable to those produced by respective controls UVB and TiO2 . In these conditions, the most relevant features are the decrease of T4 endonuclease V-sensitive sites (p < 0.05 when compared with those produced by UVB treatment alone) and the drastic increase of Fpg-sensitive sites (p < 0.001). Essentially, the association of UVB plus TiO2 protected DNA from CPD formation whilst specifically potentates the production of modified purines, such as 8-oxoG and FapyG, in detriment of other DNA modifications. Although different in terms of direct DNA strand break generation, MB plus VL and UVB plus TiO2 treatments share a particular profile similarity concerning DNA base modifications. Indeed, MB plus VL treatment was performed as a positive control for generation of oxidized purines via oxidatively generated damage [29,31].

2.10. Statistical analysis All data were subjected to statistical analysis using ANOVA followed by the Kruskal–Wallis test. A significance level of 5% was adopted to evaluate the data.

3. Results 3.1. Identification of UVB plus TiO2 -induced DNA lesions in vitro Supercoiled plasmid pUC18 was treated as follows: (i) 25 kJ/m2 of UVB, (ii) 100 ␮g/ml of TiO2 in darkness, (iii) 25 kJ/m2 of UVB plus 100 ␮g/ml TiO2 and (iv) 1 ␮g/ml of MB plus VL. To characterize the chemical nature of the putative DNA lesions generated by these treatments, 200 ng of treated plasmid DNA was incubated with the enzymes Fpg, Nth, XthA, Nfo and T4 endonuclease V, respectively. Direct DNA breaks and enzymic-sensitive sites are shown in Fig. 1. Direct DNA breakage could be detected after UVB treatment. However, it should be considered that these breaks may be

3.2. UVB/TiO2 association and biological consequences The survival and mutagenesis of S. cerevisiae to UVB plus TiO2 were determined to evaluate the biological effects of this combination in living organisms. The concentration of TiO2 and treatment condition, in all experiments, was 100 ␮g/ml in PBS at 30 ◦ C, under agitation. This was established as the maximal nontoxic concentration for S. cerevisiae in this experimental condition. In addition, TiO2 suspensions containing more than 100 ␮g/ml tend to produce sedimentation, even with vigorous agitation. As well, spontaneous mutation frequencies of all strains were not altered (3 h of direct contact, data not shown). Fig. 2 points up the response of S. cerevisiae wild-type (WT), ogg1 and rad1 strains to increasing UVB doses in presence or absence of TiO2 . In the absence of the blocking agent, rad1 strain displayed a marked sensitivity to UVB, followed by WT. Interestingly, both WT and ogg1 displayed comparable sensitivities

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Fig. 3. Effect of TiO2 on UVB-induced mutagenesis. Yeast strains were treated with UVB alone or associated with TiO2 as described in Section 2. (A) WT and ogg1 strains. (B) rad1 strain. Spontaneous mutation frequencies (CanR /107 cells) are: FF18733 (WT) 18 ± 5, CD138 (ogg1) 123 ± 17 and FF181481 (rad1) 35 ± 11. Data represent means ± S.E.

up to 10 kJ/m2 doses (p > 0.05). However, from 15 up to 30 kJ/m2 , the ogg1 was more resistant to UVB than the WT strain (p < 0.05). This suggests that active Ogg1 repair may contribute to cell toxicity at high UVB doses, as 8-oxoG in DNA may be less toxic than DNA

strand breaks introduced by Ogg1 protein in the initial repair step of 8-oxoG removal. Similar survival profiles have been previously documented when bacteria and yeast were exposed to oxidative stress induced by hydrogen peroxide [43,55]. The addition of TiO2

Fig. 4. Sensitivity and mutability of WT strain to UVB and UVB plus TiO2 in the presence of metallic chelators. (A) Cell survival in the presence of 1 mM dipyridyl. (B) Induced mutagenesis in the presence of 1 mM dipyridyl. (C) Cell survival in the presence of 1 mM neocuproine and (D) induced mutagenesis in the presence of 1 mM neocuproine. Data represent means ± S.E. For comparison, the survival and mutagenesis without chelators were added to each figure, respectively. Data represent means ± S.E.

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protected all WT, ogg1 and rad1 strains against lethal UVB damage (Fig. 2). Conversely, UVB-induced mutagenesis displayed different results among strains (Fig. 3). In the presence or absence of TiO2 , at 2.5 and 5.0 kJ/m2 UVB doses (Fig. 3A), WT strain displayed similar levels of UVB-induced mutagenesis. However, at 10 kJ/m2 , the association of UVB and TiO2 was more mutagenic than UVB alone. The ogg1 response to these treatments (UVB and UVB plus TiO2 ) was analogous to that of the WT. However, for higher doses (from 15 up to 30 kJ/m2 ), UVB and specially UVB plus TiO2 were far more mutagenic to the ogg1 than to the WT strain (p < 0.01). For the rad1 mutant, TiO2 was unable to enhance UVB-induced mutagenesis (Fig. 3B). In fact, for doses up to 0.25 kJ/m2 , TiO2 tends to protect rad1 cells against UVB mutagenesis (p < 0.05 for 0.25 kJ/m2 ). From 0.5 up to 1.0 kJ/m2 UVB doses, the rad1 mutant was refractory to the UVB plus TiO2 effect (p > 0.05) that enhanced mutagenesis in WT and ogg1 strains. These data suggest that, at low UVB doses, TiO2 is able to protect cells against CPD mutagenic and toxic effects. However at higher doses; TiO2 could also be sensitized by UVB and simultaneously damage cells through oxidatively generated DNA damage.

3.3. Mechanisms involved in the generation/repair of DNA lesions by UVB plus TiO2 According to recent reports, UVA plus TiO2 generate a series of reactive oxygen species (ROS) such as superoxide anion radical (O2 •− ), hydrogen peroxide (H2 O2 ), singlet oxygen (1 O2 ) and • OH via Fenton and/or Fenton-like reactions [22,56]. In the case of UVB plus TiO2 , if • OH is generated via Fenton or Fenton-like reactions, the use of transition metal chelators would attenuate the effects of TiO2 -mediated photosensitization by UVB. Fig. 4 illustrates survival and mutagenesis of WT strain to UVB and UVB plus TiO2 in iron (dipyridyl) and copper (neocuproine) depletion conditions. Dipyridyl and neocuproine were able to protect cells against lethal lesions induced by UVB and UVB plus TiO2 (Fig. 4A and C, p < 0.05). In fact, the chelator (dipyridyl or neocuproine) association with TiO2 reflects a sum of the protective effects of the isolated treatments. This can be due to two simultaneous effects: (i) dipyridyl and neocuproine were also able to absorb UVB light (Fig. 5A and B), which may contribute to UV protection (CPD damage), (ii) in parallel to their metallic chelating properties. While

Fig. 5. Ultraviolet absorption of metallic chelators. (A) 1 mM dipyridyl in sterile PBS. (B) 1 mM neocuproine in sterile PBS. Absorbance is in arbitrary units, as described in Section 2.

dipyridyl and neocuproine had no effect on the spontaneous mutagenesis (p > 0.05), the cell pretreatment with these chelators was able to drastically reduce both (p < 0.01) UVB and UVB plus TiO2 induced mutagenesis (Fig. 4B and D). For ogg1 strain, in order to examine chelator protective effects, doses were increased due to ogg1 relative resistance to UVB when compared to the WT strain. At these higher doses, both chelators protected (p < 0.01) ogg1 cells from UVB and UVB plus TiO2 lethality and mutagenesis (Fig. 6). As well as in WT strain, the additive effect in survival recovery produced by chelator association with TiO2 was also observed in ogg1 mutant. For rad1 strain, although both chelators were able to reduced (p < 0.01) UVB and UVB plus TiO2 lethality (Fig. 7A and C),

Table 1 Comparison of cellular protection and induced mutagenesis at LD37 . CanR mutants × 10−7 cells

WT strain

UVB (kJ/m2 ) at LD37

UVB UVB + TiO2 UVB + dipyridyl UVB + TiO2 + dipyridyl UVB + neocuproine UVB + TiO2 + neocuproine

3.15 6.15 15.00 30.00 22.50 30.00

1.00 1.95 4.76 9.52 7.14 9.52

2900 4650 1201 1084 1103 1230

± ± ± ± ± ±

235 479 172 237 217 378

1.00 1.60 0.41 0.37 0.38 0.42

ogg1 strain UVB UVB + TiO2 UVB + dipyridyl UVB + TiO2 + dipyridyl UVB + neocuproine UVB + TiO2 + neocuproine

5.15 13.60 33.50 78.00 43.00 73.00

1.00 2.64 6.50 15.15 8.35 14.17

4485 7000 1703 3170 1665 1828

± ± ± ± ± ±

389 826 211 445 562 150

1.00 1.56 0.38 0.71 0.37 0.41

rad1 strain UVB UVB + TiO2 UVB + dipyridyl UVB + TiO2 + dipyridyl UVB + neocuproine UVB + TiO2 + neocuproine

0.20 0.50 0.68 1.00 0.70 1.00

1.00 2.50 3.40 5.00 3.50 5.00

566 627 595 674 565 696

± ± ± ± ± ±

95 79 60 51 73 72

1.00 1.11 1.05 1.19 1.00 1.23

a b

UVB fold-protectiona

(LD37 of a certain treatment)/(LD37 of UVB treatment). (CanR mutants/107 of a certain treatment at LD37 )/(UVB-induced CanR mutants/107 at LD37 ).

Mutation fold-increaseb

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Fig. 6. Sensitivity and mutability of ogg1 strain to UVB and UVB plus TiO2 in the presence of metallic chelators. (A) Cell survival in the presence of 1 mM dipyridyl. (B) Induced mutagenesis in the presence of 1 mM dipyridyl. (C) Cell survival in the presence of 1 mM neocuproine and (D) induced mutagenesis in the presence of 1 mM neocuproine. Data represent means ± S.E. For comparison, the survival and mutagenesis without chelators were added to each figure, respectively. Data represent means ± S.E.

mutagenesis was not altered (p > 0.05) [Fig. 7B and D]. In order to properly compare and depict the effects of these chelators on mutagenesis and cell survival in different repair backgrounds, results were summarized in Table 1 at equivalent LD37 (lethal dose producing 37% survival). For WT strain, TiO2 was mutagenic, despite its ability in protecting cell against lethality, increasing in 60% UVB mutagenesis (Table 1). Concerning the association of TiO2 and dipyridyl, there was higher UVB protection against cell lethality than the isolated treatments. This indicates that other effect, besides UVB blockage, is involved in cell protection. Additionally, the reduction of UVB and UVB plus TiO2 mutagenesis relies specifically on dipyridyl. These data suggest that, apart CPD avoidance (due to UVB absorption), chelation can weaken the oxidative counterpart of UVB DNA damage at these dose levels in the WT strain. This phenomenon is more pronounced in the ogg1 mutant. In this strain, the absolute number of CanR mutants induced by UVB and UVB plus TiO2 is higher than that of the WT strain. Cell protection conferred by TiO2 and dipyridyl against UVB lethality was also higher in the ogg1 strain. Particularly, UVB plus TiO2 -induced mutagenesis was mildly reduced by dipyridyl, while UVB-induced mutagenesis in the presence of dipyridyl was reduced to those of the WT levels. Interestingly, this is not observed in the rad1 mutant, in which only UVB blockage by dipyridyl and/or TiO2 is important to reduce cell lethality, as mutagenesis remains unaltered.

Regarding neocuproine, for WT and rad1 strains, its association with TiO2 had a protective effect against UVB cell lethality equivalent to the sum of the isolated treatments. Concerning mutagenesis, neocuproine had no effect in rad1 strain while it was able to reduce UVB and UVB plus TiO2 -induced mutagenesis in WT strain, corroborating the notion that the oxidative counterpart of UVB DNA damage can be also neutralized by copper chelation. For ogg1 strain, neocuproine conferred higher UVB protection against cell lethality than the isolated treatments. It is worthy to note that neocuproine was able to reduce UVB-induced mutagenesis (with or without TiO2 ) to WT levels. This denotes the importance of copper ions in the mutagenesis induced by UVB sensitized TiO2 in an ogg1 deficient background. 4. Discussion Following UV radiation to the skin, sunburn cells can be perceived. Besides these typical effects, the initiation of photocarcinogenesis might begin [57,58]. Both UVB and UVA radiations have been described as mutagenic [59]. Thus, to wear sunscreens during solar exposure that could reduce cellular alterations, especially DNA damage, is recommended. Previous reports have demonstrated that TiO2 photo-blocking agent produces DNA breakage in vitro [5], but no specific lesions and/or mechanisms of action were described. On the other hand,

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Fig. 7. Sensitivity and mutability of WT strain to UVB and UVB plus TiO2 in the presence of metallic chelators. (A) Cell survival in the presence of 1 mM dipyridyl. (B) Induced mutagenesis in the presence of 1 mM dipyridyl. (C) Cell survival in the presence of 1 mM neocuproine and (D) induced mutagenesis in the presence of 1 mM neocuproine. Data represent means ± S.E. For comparison, the survival and mutagenesis without chelators were added to each figure, respectively. Data represent means ± S.E.

it was verified that, besides DNA damage, UVB light might generate oxidative species [60], which probably had a minor role in mutagenesis and cell toxicity [61,62]. In order to evaluate the DNA damage, repair and mutagenesis of TiO2 after UVB radiation, we have assessed the impact of this association in genomic DNA (genotoxicity). The in vitro treatment of supercoiled DNA plasmid coupled with enzymic digestion allowed the identification of the chemical nature of DNA lesions [27,29,48,50]. Through this in vitro approach, we demonstrated that oxidatively generated DNA damage induced by UVB is in agreement with previous reports [60], and UVB DNA damage profile points to the oxidation of purines and pyrimidines as important base modifications, besides the major generation of CPDs (Fig. 1). UVB plus TiO2 treatment generated a large excess of Fpg-sensitive sites in plasmid DNA. This fact supports that the oxidized purines, mainly guanines, are as important as CPDs upon UVB plus TiO2 treatment (Fig. 1). The amounts of Fpg-sensitive sites generated by UVB plus TiO2 was comparable to those produced by MB plus visible light treatment, which is known to produce, almost exclusively, singlet oxygen damage to guanine residues in DNA [63]. The excess of Fpgsensitive sites draws attention to another interesting aspect of DNA damage originated by UVB plus TiO2 : the type of DNA lesions. Most available evidence indicates that 8-oxoG is a pre-mutagenic rather

than lethal lesion and FapyG is a strong block to DNA polymerases and can be lethal if not repaired [64]. Thus, considering the survival and mutagenesis of WT and ogg1 strains upon UVB plus TiO2 treatment, 8-oxoG, rather than FapyG, may constitute the most plentiful lesion produced in DNA by UVB-irradiated TiO2 . The lower frequencies of direct strand breaks and Nth-sensitive sites with respect to guanine degradation products may be accounted for by a significant contribution of type I (one-electron oxidation) and/or type II photosensitization (singlet oxygen) [56,65] mechanisms which both give rise to 8-oxoG without DNA cleavage [66]. In addition, the contribution of type I photosensitization mechanism would require a close proximity between TiO2 and DNA, which is still controversial in terms of living organisms [67]. It is important to note that chemical damage observed in isolated plasmid DNA could be partially different from those occurred in cellular DNA [68]. The analysis of the yeast strain survival, as part of the biological effects of UVB plus TiO2 association, denotes an interesting property of TiO2 . While TiO2 photosensitization by UVA is known to efficiently inactivate bacteria and human colon carcinoma cells [13,69], the presence of TiO2 during UVB irradiation not only protected yeast cells from UVB lethal damage, but also strongly rescued yeast survival in the course of UVB irradiation (Fig. 2). The cellular targets of UVA and UVB radiations could explain the

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discrepancy between TiO2 plus UVA and TiO2 plus UVB effects, i.e. TiO2 -mediated photosensitization by UVA means a second source of ROS that reinforces the damages produced by UVA. Meanwhile, for UVB irradiation, the physical interference of TiO2 prevents yeast from direct lethal damages (like CPDs and pyrimidine (6-4) pyrimidone photoproducts). The analysis of wild-type strain mutagenesis proved the mutagenic potential of UVB plus TiO2 (Fig. 3A). In ogg1 strain, the absolute UVB plus TiO2 -induced increment in mutagenesis over UVB alone is equivalent to the total number of wild-type CanR mutants produced by UVB (Table 1). It is important to stress that, in the rad1 strain, UVB plus TiO2 -induced mutagenesis remained unaltered. Altogether, these features highlight the biological significance of TiO2 as a ROS generator. In this way, even if UVB represents a minor portion of the solar spectrum, a potential risk is involved upon solar UVB exposure in the presence of TiO2 . According to Rampelotto et al. [70], in a 10-year survey at the south of Brazil (29◦ S, 53◦ W), solar UVB radiation presented a typical fluence season profile, with lower values in Junes (6.8 J/m2 /s) and the highest in Januaries (34.7 J/m2 /s). It means that, in summer at this region, a 10 min exposure to sun is equivalent to 20.82 kJ/m2 of UVB. These data confirm that UVB incidence is relevant and may be enough to sensitize TiO2 as well as other molecules of biological interest. Therefore, the actual risk would reside in TiO2 penetration in the human skin upon solar exposure. However, there is still a great deal of controversy regarding TiO2 safety and skin penetration, specially when the Food and Drug Administration (FDA) allowed the use of nanoparticles for sunscreens in 1999, altering their permeability [67]. In fact, studies are still necessary to establish a series of biological parameters of TiO2 nanoparticles such as penetration, permeability/absorption, bioavailability in intact, traumatized or diseased skin. But the most important issue would be the reproduction of a human model for the real application of sunscreens, accounting for UV exposure [67]. We also appraised the involvement of transition metals in the generation of DNA damage [21,22]. The ensemble of the results with iron and copper chelators conferred a better picture of the TiO2 plus UVB combination in terms of DNA damage, repair and mutagenesis mechanisms. Both chelators rescued the mutagenesis of the WT and the ogg1 (in large extent), but not that of the rad1 strain (Figs. 4, 6 and 7). Thus, Fenton reactions may be at the origin of a significant part of the DNA damage and mutagenesis induced by UVB plus TiO2 association, despite the formation of CPD lesions. Comparisons among mutagenesis results at LD37 (Table 1) allowed the discrimination of the blocking effect of dipyridyl and neocuproine and their effects as chelators. As neocuproine was highly effective in the reduction of mutagenesis in the ogg1 strain, copper ions appear to be particularly important in the generation of mutagenic lesions (probably 8-oxoG) induced by UVB radiation (in the presence or absence of TiO2 ), which are recognized by Ogg1 protein. It is also important to note that, although chelators are able to drastically reduce UVB mutagenesis in combination or not with TiO2 , the residual absolute number of CanR mutants are still extremely higher than the spontaneous mutator levels of all three strains [51,72], which may be due to CPDs that are predominant upon UVB irradiation (Fig. 1). The results presented here provide important clues concerning the chemical nature of DNA damage induced by UVB plus TiO2 and a dual effect of TiO2 during UVB irradiation: cell lethality prevention and mutagenesis induction. The results of the depletion of the transition metals provided support to the mechanism of UVB plus TiO2 involving ROS production: via copper and iron mediated reactions. Finally, our results also implicate Ogg1 protein in the repair of mutagenic damage induced by UVB plus TiO2 in S. cerevisiae. At last, our contribution may constitute interesting clues for the cosmetic industry, which may improve TiO2 sunscreens, either avoiding the

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