Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Reactions of 3′,5′-di-O-acetyl-2′-deoxyguansoine and 3′,5′-di-O-acetyl-2′-deoxyadenosine to UV light in the presence of uric acid

Reactions of 3′,5′-di-O-acetyl-2′-deoxyguansoine and 3′,5′-di-O-acetyl-2′-deoxyadenosine to UV... Introduction: Recently, it was revealed that uric acid is a photosensitizer of reactions of nucleosides on irradiation with UV light at wavelengths longer than 300 nm, and two products generated from 2′-deoxycytidine were identified. In the present study, UV reactions of acetylated derivatives of 2′-deoxyguansoine and 2′-deoxyadenosine were conducted and their products were identified. Findings: Each reaction of 3′,5′-di-O-acetyl-2′-deoxyguansoine or 3′,5′-di-O-acetyl-2′-deoxyadenosine with UV light at wavelengths longer than 300 nm in the presence of uric acid generated several products. The products were separated by HPLC and identified by comparing UV and MS spectra of the products with previously reported values. The major products were spiroiminodihydantoin, imidazolone, and dehydro-iminoallantoin nucleosides for 3′,5′-di-O-acetyl-2′-deoxyguansoine, and an adenine base and a formamidopyrimidine nucleoside for 3′,5′-di-O- acetyl-2′-deoxyadenosine. Conclusions: If these damages caused by uric acid with sunlight occur in DNA of skin cells, mutations may arise. We should pay attention to the genotoxicity of uric acid in terms of DNA damage to dGuo and dAdo sites mediated by sunlight. Keywords: Uric acid, Deoxyguanosine, Deoxyadenosine, Photosensitizer, UV light Introduction study reported that the incidence of non-melanoma skin Since uric acid is the final metabolic product of purine ca- cancer showed a positive association with the serum uric tabolism in humans, it exists ubiquitously in various cells acid concentration [8]. Recently, we showed that uric acid and body fluids at relatively high concentrations [1, 2]. is a photosensitizer of reactions of nucleosides on irradi- Uric acid is an important antioxidant in humans [3]. How- ation with UV light at wavelengths longer than 300 nm ever, it can also act as a pro-oxidant inducing oxidative [9]. The reactions of nucleosides were suppressed by rad- stress of cells [4, 5]. It has been reported that uric acid in ical scavengers. Two products from 2′-deoxycytidine cultured mouse skin cells is increased by UV irradiation, (dCyd) were separated by reversed phase (RP) HPLC and and that uric acid on the human skin surface is increased identified by MS and NMR as N -hydroxy-2′-deoxycyti- by sunlight exposure [6, 7]. An epidemiological cancer dine and N ,5-cyclic amide-2′-deoxycytidine, formed by cycloaddition of an amide group from uric acid. The re- sults using N-labeled uric acid indicated that the amide * Correspondence: tsuzuki@shujitsu.ac.jp group added to dCyd originates from both the five- School of Pharmacy, Shujitsu University, 1-6-1 Nishigawara, Okayama 703-8516, Japan © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Suzuki et al. Genes and Environment (2022) 44:4 Page 2 of 9 membered imidazole ring and six-membered pyrimidine concentration was increased from 0 to 37.5% over 45 ring of uric acid, suggesting that an unidentified radical min in linear gradient mode. The column temperature derived from uric acid with a delocalized unpaired elec- was 40 °C and flow rate was 1 mL/min. The RP-HPLC tron is generated. To obtain information about reaction chromatogram was detected at 200–500 nm. ESI-TOF/ products of nucleosides other than dCyd, we analyzed the MS measurements were performed on a MicrOTOF reaction solutions of 2′-deoxyguanosine (dGuo) and 2′- spectrometer (Bruker, Bremen, Germany) in negative deoxyadenosine (dAdo) irradiated with UV light in the mode. The sample isolated by RP-HPLC was directly in- presence of uric acid. However, we failed to obtain prod- fused into the MS system by a syringe pump without a uct peaks with a good resolution on RP-HPLC. Thus, column. acetylated derivatives of dGuo (3′,5′-di-O-acetyl-2′-deox- yguanosine; AcdGuo) and dAdo (3′,5′-di-O-acetyl-2′- Quantitative procedures deoxyadenosine; AcdAdo) were prepared and used for The concentrations of the products were evaluated ac- analysis of the UV irradiation reaction to improve the re- cording to integrated peak areas on RP-HPLC chromato- tention and separation of the products by RP-HPLC. In grams detected at 245 nm and the ε value of each 245 nm the present study, we show identification and quantifica- product, compared with the peak area of the standard tion of the products from AcdGuo and AcdAdo by UV ir- solution of AcdGuo for the AcdGuo reactions or Ade radiation in the presence of uric acid. for the AcdAdo reactions. The ε values were used 245 nm − 1 − 1 − 1 − 1 as 12,400 M cm for AcdGuo and 8450 M cm for Materials and methods Ade. The used ε values of the products are indi- 245 nm Materials cated in Tables 1 and 2. dGuo, dAdo, and uric acid were purchased from Sigma- Aldrich (MO, USA). Other chemicals were obtained Results and discussion from Sigma-Aldrich, Nacalai Tesque (Kyoto, Japan), and Reaction of AcdGuo Tokyo Chemical Industry (Tokyo, Japan). Water was A solution of 100 μM AcdGuo with 400 μM uric acid in purified with a Millipore Milli-Q deionizer (MA, USA). 100 mM potassium phosphate buffer at pH 7.4 was irra- AcdGuo and AcdAdo were synthesized from dGuo and diated with UV light from a high-pressure mercury lamp dAdo, respectively, by acetylation using acetic anhydride through a 300-nm longpass filter at a temperature of as previously described [10]. AcdGuo and AcdAdo were 37 °C for 10 min. The reaction mixture was analyzed by purified by RP-HPLC. RP-HPLC equipped with a UV-Vis photodiode-array de- tector. As shown in Fig. 1, several product peaks ap- Irradiation conditions peared in addition to uric acid and its decomposition For UV light irradiation, UV light originating from a products, denoted by asterisks, and AcdGuo and its con- 250-W high-pressure mercury lamp (SP9-250UB, Ushio, taminants, denoted by crosses. Six products (Products Tokyo, Japan) with an optical filter through a light guide 1–6) were isolated by RP-HPLC and subjected to MS was used to directly irradiate the surface of a solution (1 analysis. The products were identified on the basis of the mL) in a glass vial (12 mm i.d.) without a cap at 37 °C. similarity of their UV and MS spectra with reported Longpass filter LU0300 (cut-on 300 nm) (Asahi Spectra, values using a reaction system of AcdGuo with hypobro- Tokyo, Japan) was used as the optical filter. The inten- mous acid [12]. Table 1 summarizes the characteristics sity of radiation on the surface of the sample solution of Products 1–6. Products 1 and 2 were identified as di- was measured with a photometer (UIT-150, Ushio, astereomers of a 3′,5′-di-O-acetyl derivative of spiroimi- Tokyo, Japan) equipped with a sensor, UVD-S254 or nodihydantoin deoxyribonucleoside (AcdSph). Product 3 UVD-S365. The intensities of the UV light were 0 mW/ was a 3′,5′-di-O-acetyl derivative of diamino-oxazolone 2 2 cm for 254 nm and 264 mW/cm for 365 nm. deoxyribonucleoside (AcdOz). Product 4 was a 3′,5′-di- O-acetyl derivative of amino-imidazolone deoxyribonu- HPLC and MS conditions cleoside (AcdIz). Product 5 was a 3′,5′-di-O-acetyl de- The HPLC system consisted of LC-10ADvp pumps and rivative of dehydro-iminoallantoin deoxyribonucleoside ox an SPD-M10Avp UV-Vis photodiode-array detector (AcdIa ). Product 6 was a 3′,5′-di-O-acetyl derivative of (Shimadzu, Kyoto, Japan). For the RP-HPLC, an Inertsil 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-AcdGuo). ODS-3 octadecylsilane column of size 4.6 × 250 mm and Authentic guanine (Gua) was eluted with the RP-HPLC particle size of 5 μm (GL Sciences, Tokyo, Japan) was retention time of 12.0 min as a broadened peak. The used. The eluent was 20 mM ammonium acetate (pH concentration of Gua in the present reaction solution 7.0) containing acetonitrile. For AcdGuo, the acetonitrile could not determined due to overlapping of a peak of a concentration was increased from 0 to 30% over 45 min decomposition product of uric acid. The structures of in linear gradient mode. For AcdAdo, the acetonitrile the reaction products from AcdGuo are shown in Fig. 2. Suzuki et al. Genes and Environment (2022) 44:4 Page 3 of 9 Table 1 Characteristics of Products Formed by UV Irradiation of AcdGuo with Uric Acid −1 − 1 a Products t (min) λ (nm) m/z (negative) ε (M cm ) R max 245 nm 1. AcdSph (fast) 25.2 230 (shoulder) 382 5480 2. AcdSph (slow) 25.6 230 (shoulder) 382 5480 3. AcdOz 28.7 232 329 6000 4. AcdIz 30.9 254, 320 311 20,500 ox 5. AcdIa 33.3 236 354 12,840 6. 8-oxo-AcdGuo 37.6 254, 295 366 15,560 The values of ε are those previously reported [10] 245 nm Uric acid dose-dependent changes in the reaction of When the solution was incubation at 37 °C for 10 min AcdGuo with UV light were examined. A solution of without UV irradiation, no product was detected. At 5 min 100 μMAcdGuowith0–400 μM uric acid in 100 mM po- UV irradiation, the major products were AcdIz and ox tassium phosphate buffer at pH 7.4 was irradiated with UV AcdIa .At15–30 min, the main product was AcdSph. light from a high-pressure mercury lamp through a 300- The concentration of 8-oxo-AcdGuo was maximal at 5 nm longpass filter at a temperature of 37 °C for 10 min. min, then decreased, with an intermediate kinetics profile. The product concentrations were determined from the ab- It has been reported that spirohydantoin nucleoside (dSph) sorbance area of HPLC detected at 245 nm using their re- is generated as a two-step oxidation product of dGuo via ported molar extinction coefficients [12]. Figure 3Ashows 8-oxo-dGuo [13]. In the present system, AcdSph should the changes in concentrations of the products. At 0 μM also lead to further oxidation of 8-oxo-AcdGuo. Reportedly ox uric acid, no product was detected. At around 100 μM uric AcdIa is generated as a three-step oxidation product of ox acid, concentrations of all products other than 8-oxo- dGuo [14]. Since AcdIa was one of the major products in AcdGuo were maximal, while the concentration of 8-oxo- the present reaction, the reaction rates of these three-steps AcdGuo increased with an increasing uric acid concentra- of oxidation should be relatively high. On the other hand, tion up to 400 μM. Over the uric acid concentration range imidazolone nucleoside (dIz) is generated by oxidation of examined, the major products were AcdSph, AcdIz, and dGuo and subsequent degradations without 7,8-dihydro-8- ox AcdIa with comparable yields. Figure 3Bshows the Acd- oxo-2′-deoxyguanosine (8-oxo-dGuo) [15]. dIzisnot Guo concentration and total concentration of all six prod- stable, converting to stable oxazolone nucleoside (dOz) ucts. At 0 μM uric acid, no consumption of AcdGuo was with a half-life of 2.5 h at 37 °C in a neutral solution at pH observed. At 100 μM uric acid, the consumption of Acd- 7[16]. The present results showing a gradual increase in Guo and total yield of the products were maximal. Over the AcdOz concentration with an increase in the irradi- the uric acid concentration range examined, the total yield ation time and decrease of AcdIz at 20–30 min would be of all products was approximately one-third of the con- explainable by the instability of AcdIz. Figure 3Dshows sumption of AcdGuo, suggesting that further reactions of the AcdGuo concentration and total concentration of all the products or other reactions without these products products. The consumption of AcdGuo increased in a occur. Irradiation time-dependent changes in the reaction time-dependent manner. Although the total concentration of AcdGuo with UV light were examined. A solution of of products increased with an increasing irradiation time, 100 μM AcdGuo with 400 μM uric acid in 100 mM potas- thechangeat15–30 min was slight, suggesting that further sium phosphate buffer at pH 7.4 was irradiated with UV reactions occur involving the products. lightatatemperatureof37°Cfor 0–30 min. Figure 3C Mutations caused by the sites of some of these shows the changes in concentrations of the products. products generated in DNA have been reported as Table 2 Characteristics of Products Formed by UV Irradiation of AcdAdo with Uric Acid −1 −1 a Products t (min) λ (nm) m/z (negative) ε (M cm ) R max 245 nm 7. Ade 16.5 260 134 8450 8. Fapy-AcdAdo (fast) 28.7 259 352 2860 9. Fapy-AcdAdo (slow) 29.3 259 352 2860 10.5′,8-cyclo-AcdAdo (fast) 33.9 274 332 8930 11.5′,8-cyclo-AcdAdo (slow) 34.6 274 332 6910 12.5′-deoxy-5′,8-cyclo -AcdAdo 35.3 264 274 9560 13. 8-oxo-AcdAdo 38.6 212, 269 350 10,220 The values of ε were calculated from the reported ε values for the products of dAdo at λ and their UV spectra obtained in the present study [11] 245 nm max Suzuki et al. Genes and Environment (2022) 44:4 Page 4 of 9 Fig. 1 RP-HPLC chromatogram of a reaction mixture of AcdGuo with uric acid detected at 245 nm. A solution of 100 μM AcdGuo and 400 μM uric acid was irradiated with UV through a 300-nm longpass filter in 100 mM potassium phosphate buffer at pH 7.4 and 37 °C for 10 min. The HPLC system consisted of LC-10ADvp pumps and an SPD-M10Avp UV-Vis photodiode-array detector (Shimadzu, Kyoto, Japan). For RP-HPLC, an Inertsil ODS-3 octadecylsilane column of size 4.6 × 250 mm and particle size of 5 μm (GL Sciences, Tokyo, Japan) was used. The eluent was 20 mM ammonium acetate (pH 7.0) containing acetonitrile. The acetonitrile concentration was increased from 0 to 30% over 45 min in linear gradient mode. The column temperature was 40 °C and flow rate was 1 mL/min follows: dSph in an oligonucleotide strongly blocks dAMP incorporation, suggesting that the formation of nucleotide incorporation by DNA polymerases, and dOz in DNA may cause G to T transversion [18]. For causes both G to T and G to C transversion muta- 8-oxo-dGuo, dCMP and dAMP are incorporated op- tions when duplication occurs over this lesion [17]. In posite 8-oxo-dGuo in an oligonucleotide by DNA vitro nucleotide insertion by Klenow fragment exo polymerases [11]. When dAMP is incorporated, G to opposite dOz in an oligonucleoside induces mainly T transversion mutation occurs. Fig. 2 The reaction products from AcdGuo by UV irradiation in the presence of uric acid Suzuki et al. Genes and Environment (2022) 44:4 Page 5 of 9 Fig. 3 Uric acid dose-dependence and time-course of the concentration changes of AcdGuo reaction products. Uric acid dose-dependence of the concentration changes of (a) each product and (b) AcdGuo and total products, when a solution of 100 μM AcdGuo with 0–400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 10 min at pH 7.4 and 37 °C. Time-course of the concentration changes of (c) each product and (d) AcdGuo and total products, when a solution of 100 μM AcdGuo with 400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 0–30 min at pH 7.4 and 37 °C. AcdSph (1 and 2) (closed circle), AcdOz (3) (closed square), AcdIz (4) (closed rhombus), ox AcdIa (5) (open circle), 8-oxo-AcdGuo (6) (open square), AcdGuo (closed triangle), and the total concentration of Products 1–6 (open triangle). All reaction mixtures were analyzed by RP-HPLC. Means ± standard deviation (S.D.) (n = 3) are presented Reaction of AcdAdo and 9 were diastereomers of a 3′,5′-di-O-acetyl deriva- A solution of 100 μM AcdAdo with 400 μM uric acid in tive of formamidopyrimidine deoxyribonucleoside (Fapy- 100 mM potassium phosphate buffer at pH 7.4 was irra- AcdAdo). Products 10 and 11 were diastereomers of a diated with UV light through a 300-nm longpass filter at 3′,5′-di-O-acetyl derivative of 5′,8-cyclo-2′-deoxyadeno- a temperature of 37 °C for 10 min. The reaction mixture sine (5′,8-cyclo-AcdAdo). Product 12 was a 3′-O-acetyl was analyzed by RP-HPLC. As shown in Fig. 4, several derivative of 5′-deoxy-5′,8-cyclo-2′-deoxyadenosine (5′- product peaks appeared in addition to uric acid and its deoxy-5′,8-cyclo-AcdAdo). Product 13 was a 3′,5′-di-O- decomposition products, denoted by asterisks, and acetyl derivative of 7,8-dihydro-8-oxo-2′-deoxyadenosine AcdAdo and its contaminants, denoted by crosses. Seven (8-oxo-AcdAdo). The structures of the reaction products products (Products 7–13) were isolated by RP-HPLC from AcdAdo are shown in Fig. 5. and subjected to MS analysis. The products were identi- Uric acid dose-dependent changes in the reaction of fied on the basis of coincidence of their UV and MS AcdAdo with UV light were examined. A solution of spectra with corresponding reported values using a reac- 100 μMAcdAdo with 0–400 μM uric acid in 100 mM po- tion system of dAdo with the Fenton system [19]. Table tassium phosphate buffer at pH 7.4 was irradiated with 2 summarizes the characteristics of Products 7–13. UV light at a temperature of 37 °C for 10 min. The prod- Product 7 was identified as adenine (Ade). Products 8 uct concentrations were determined from the absorbance Suzuki et al. Genes and Environment (2022) 44:4 Page 6 of 9 Fig. 4 RP-HPLC chromatogram of a reaction mixture of AcdAdo with uric acid detected at 245 nm. A solution of 100 μM AcdAdo and 400 μM uric acid was irradiated with UV through a 300-nm longpass filter in 100 mM potassium phosphate buffer at pH 7.4 and 37 °C for 10 min. The HPLC conditions were the same as shown in Fig. 1 excluding the acetonitrile concentration. The acetonitrile concentration was increased from 0 to 37.5% over 45 min in linear gradient mode Fig. 5 The reaction products from AcdAdo by UV irradiation in the presence of uric acid Suzuki et al. Genes and Environment (2022) 44:4 Page 7 of 9 area of HPLC detected at 245 nm using their molar ex- common radical intermediate formed from dAdo by oxi- tinction coefficients, which were calculated from the re- dative stress via subsequent reduction and oxidation, re- ported values at λ for corresponding products of dAdo spectively [20, 21]. In the present study, the reduction max and UV spectra of the products of AcdAdo obtained in reaction generating Fapy-AcdAdo may become dominant the present study [19]. Figure 6A shows the changes in in the presence of a higher concentration of uric acid. Fig- concentrations of the products. At 0 μMuricacid, no ure 6B shows the AcdAdo concentration and total con- product was detected. At 5–300 μM uric acid, the main centration of Products 7–13.At 0 μMuric acid, no product was Ade. At above 300 μM uric acid, the concen- consumption of AcdAdo was observed. The consumption tration of Fapy-AcdAdo markedly increased. At 400 μM of AcdAdo increased up to 100 μMuricacid, andthen de- uric acid, the main product was Fapy-AcdAdo. The con- creased moderately. The total generation of the products centration of 8-oxo-AcdAdo was almost constant at 50– was approximately one-seventh of the consumption of 400 μM uric acid. Reportedly, formamidopyrimidine deox- AcdAdo at 100 μM uric acid and one-half at 400 μMuric yribonucleoside (Fapy-dAdo) and 7,8-dihydro-8-oxo-2′- acid, suggesting that further reactions involving the prod- deoxyadenosine (8-oxo-dAdo) are generated from a ucts or other reactions without these products occur, Fig. 6 Uric acid dose-dependence and time-course of the concentration changes of AcdAdo reaction products. Uric acid dose-dependence of the concentration changes of (a) each product and (b) AcdAdo and total products, when a solution of 100 μM AcdAdo with 0–400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 10 min at pH 7.4 and 37 °C. Time-course of the concentration changes of (c) each product and (d) AcdAdo and total products, when a solution of 100 μM AcdAdo with 400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 0–30 min at pH 7.4 and 37 °C. Ade (7) (closed circle), Fapy-AcdAdo (8 and 9) (closed triangle), 5′,8-cyclo-AcdAdo (10 and 11) (closed square), 5′-deoxy-5′,8-cyclo-AcdAdo (12) (open circle), 8-oxo-AcdAdo (open triangle), AcdAdo (closed rhombus), total concentration of Products 7–13 (open rhombus). All the reaction mixtures were analyzed by RP-HPLC. Means ± standard deviation (S.D.) (n =3) are presented Suzuki et al. Genes and Environment (2022) 44:4 Page 8 of 9 especially at around 100 μM uric acid. Irradiation time- hydroxyl radical, peroxynitrous acid, hypochlorous acid, dependent changes in the reaction of AcdAdo with UV and hypobromous acid did not generate N ,5-cyclic light were examined. A solution of 100 μMAcdAdo with amide-2′-deoxycytidine in the presence of uric acid. 400 μM uric acid in 100 mM potassium phosphate buffer These results suggest that an unidentified radical derived at pH 7.4 was irradiated with UV light at a temperature of from uric acid with a delocalized unpaired electron is 37 °C for 0–30 min. Figure 6C shows the changes in con- generated. All the identified products formed from acet- centrations of the products. When the solution was incu- ylated dGuo and dAdo in the present UV irradiation bation at 37 °C for 10 min without UV irradiation, no study had already been reported in the reaction with re- product was detected. At up to 10 min UV irradiation, the active free radicals and oxidants [12, 19, 29]. It has also main product was Fapy-AcdAdo. At 15–30 min, the con- been reported that hydrogen atom abstraction on the centration of Fapy-AcdAdo markedly decreased, suggest- sugar moiety of nucleosides induces release of the base ing that further reactions occur involving Fapy-AcdAdo. and crosslinking between the sugar and the base, and At 15–30 min, the main product was Ade. Figure 6D that it on the base moiety of nucleosides induces various shows the AcdAdo concentration and total concentration products having modified bases [29]. A possible reaction of Products 7–13. The consumption of AcdAdo increased mechanism for the present UV reaction of AcdGuo and in a time-dependent manner, although the total gener- AcdAdo with uric acid is as follows: The radical derived ation of the products was maximal at 10 min and de- from uric acid by UV irradiation induces hydrogen atom creased gradually up to 30 min. abstraction from AcdGuo and AcdAdo. When hydrogen Mutations caused by the sites of some of these prod- atom abstraction from the deoxyribose moiety of ucts generated in DNA have been reported as follows: In AcdAdo occurs, Ade, 5′,8-cyclo-AcdAdo, and 5′-deoxy- vitro nucleotide insertion by the Klenow fragment exo 5′,8-cyclo-AcdAdo are generated. On the other hand, opposite Fapy-dAdo in an oligonucleoside induces when hydrogen atom is abstracted from the base moi- mainly dTMP incorporation, although the frequency is eties of AcdGuo and AcdAdo, the other products are one-fourth that of the native dAdo in the template [21]. generated. Further studies are needed to reveal the de- The frequency of misincorporation of dAMP and dGMP tailed reaction mechanism. opposite Fapy-dAdo was 50% greater than that opposite native dAdo, suggesting increasing rates of A to T and A Conclusions to C transversion mutation. For 8-oxo-dAdo, human The present study showed that in the presence of uric DNA polymerase η proficiently incorporated dGMP op- acid, a photosensitizer, AcdGuo and AcdAdo were posite 8-oxo-dAdo, suggesting an increase of A to C decomposed by UV light at wavelengths longer than transversion mutation [22]. The release of Ade base 300 nm. Several products generated in AcdGuo and from DNA is caused via abstraction of various hydrogen AcdAdo reactions were identified. All the identified atoms of deoxyribose by radicals with or without a single products were previously reported as products caused by strand break [23, 24]. In the absence of a single strand reactive oxygen species. Unlike the reaction of dCyd, break, an abasic site is generated in DNA. In vitro DNA products generated by the addition of a part of uric acid synthesis by human DNA polymerase ε is strongly were not detected. Reportedly, several of these products blocked at the abasic site analog [25]. In living cells, vari- generated in DNA induce mutation. If this DNA damage ous mutations are induced by abasic sites [26, 27]. caused by uric acid with sunlight occurs in skin cells, mutations may arise. We should pay attention to the Reaction mechanism genotoxicity of uric acid in terms of DNA damage to Photosensitization includes many different prosesses dGuo and dAdo sites mediated by sunlight. such as energy transfer, electron transfer, hydrogen atom abstraction, singet oxygen formation, and radical forma- tion [28, 29]. Recently we showed that uric acid is a Abbreviations photosensitzer on the reaction of nucleosides, dCyd, AcdGuo: 3′,5′-di-O-acetyl-2′-deoxyguanosine; AcdAdo: 3′,5′-di-O-acetyl-2′- deoxyadenosine; AcdSph: 3′,5′-di-O-acetyl derivative of dGuo, dAdo, and thymidine, by UV light with wave- spiroiminodihydantoin deoxyribonucleoside; AcdOz: 3′,5′-di-O-acetyl lengths longer than 300 nm [9]. These reactions were derivative of diamino-oxazolone deoxyribonucleoside; AcdIz : 3′,5′-di-O-acetyl ox inhibited by the addition of radical scavengers, ethanol derivative of amino-imidazolone deoxyribonucleoside; AcdIa :3′,5′-di-O- acetyl derivative of dehydro-iminoallantoin deoxyribonucleoside; 8-oxo-Acd- and sodium azide. For the reaction of dCyd, N ,5-cyclic Guo: 3′,5′-di-O-acetyl derivative of 7,8-dihydro-8-oxo-2′-deoxyguanosine; amide-2′-deoxycytidine was formed by cycloaddition of Ade: adenine; Fapy-AcdAdo: 3′,5′-di-O-acetyl derivative of an amide group from uric acid. When a N-labeled uric formamidopyrimidine deoxyribonucleoside; 5′,8-cyclo-AcdAdo: 3′,5′-di-O- 14 15 acetyl derivative of 5′,8-cyclo-2′-deoxyadenosine; 5′-deoxy-5′,8-cyclo- acid, having two N and two N atoms in the molecule, AcdAdo: 3′-O-acetyl derivative of 5′-deoxy-5′,8-cyclo-2′-deoxyadenosine; 8- was used, N ,5-cyclic amide-2′-deoxycytidine containing oxo-AcdAdo: 3′,5′-di-O-acetyl derivative of 7,8-dihydro-8-oxo-2′- 14 15 both N and N atoms was generated. Singlet oxygen, deoxyadenosine Suzuki et al. Genes and Environment (2022) 44:4 Page 9 of 9 Acknowledgements 13. Luo W, Muller JG, Rachlin EM, Burrows CJ. Characterization of Not applicable. spiroiminodihydantoin as a product of one-electron oxidation of 8-oxo-7,8- dihydroguanosine. Org Lett. 2000;2(5):613–7. https://doi.org/10.1021/ol9913643. 14. Luo W, Muller JG, Rachlin EM, Burrows CJ. Characterization of hydantoin Authors’ contributions products from one-electron oxidation of 8-oxo-7,8-dihydroguanosine in a T.S. designed the experiments. M.T. and A.O-T. performed the experiments. nucleoside model. Chem Res Toxicol. 2001;14(7):927–38. https://doi.org/10.1 T.S. wrote the manuscript. All authors read and approved the final 021/tx010072j. manuscript. 15. Cadet J, Douki T, Ravanat J-L. Oxidatively generated damage to isolated and cellular DNA. In: Geacintov NE, Broyde S, editors. The chemical biology of Funding DNA damage. Wiley-VCH, Weinheim: Germany; 2010. p. 53–80. https://doi. Not applicable. org/10.1002/9783527630110.ch3. 16. Henderson PT, Delaney JC, Muller JG, Neeley WL, Tannenbaum SR, Burrows Availability of data and materials CJ, et al. The hydantoin lesions formed from oxidation of 7,8-dihydro-8- Not applicable. oxoguanine are potent sources of replication errors in vivo. Biochemistry. 2003;42(31):9257–62. https://doi.org/10.1021/bi0347252. Declarations 1 13 17. Raoul S, Berger M, Buchko GW, Joshi PC, Morin B, Weinfeld M, et al. H, C and N nuclear magnetic resonance analysis and chemical features of the Ethics approval and consent to participate two main radical oxidation products of 2′-deoxyguanosine: oxazolone and This research does not require ethical approval, since it does not include imidazolone nucleosides. J Chem Soc Perkin Trans 2. 1996:371–81. in vivo experiments or clinical trials on humans and animals.. 18. Duarte V, Gasparutto D, Jaquinod M, Cadet J. In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides. Consent for publication Nucleic Acids Res. 2000;28(7):1555–63. https://doi.org/10.1093/nar/28.7.1555. Not applicable. 19. Chattopadhyaya R, Goswami B. Oxidative damage to DNA constituents by iron-mediated Fenton reactions: the deoxyadenosine family. J Biomol Struct Competing interests Dyn. 2012;30(4):394–406. https://doi.org/10.1080/07391102.2012.682206. The authors declare that they have no competing interests. 20. Vieira AJSC, Steenken S. Pattern of hydroxy radical reaction with adenine and its nucleosides and nucleotides. Characterization of two types of Received: 26 November 2021 Accepted: 11 January 2022 isomeric hydroxy adduct and their unimolecular transformation reactions. J Am Chem Soc. 1990;112(19):6986–94. https://doi.org/10.1021/ja00175a036. 21. Delaney MO, Wiederholt CJ, Greenberg MM. Fapy-dA induces nucleotide References misincorporation translesionally by a DNA polymerase. Angew Chem Int Ed 1. Wu XW, Lee CC, Muzny DM, Caskey CT. Urate oxidase: primary structure and Engl. 2002;41(5):771–3. https://doi.org/10.1002/1521-3773(20020301)41:5< evolutionary implications. Proc Natl Acad Sci U S A. 1989;86(23):9412–6. 771::AID-ANIE771>3.0.CO;2-V. https://doi.org/10.1073/pnas.86.23.9412. 22. Koag M-C, Jung H, Lee S. Mutagenesis mechanism of the major oxidative 2. Oda M, Satta Y, Takenaka O, Takahata N. Loss of urate oxidase activity in adenine lesion 7,8-dihydro-8-oxoadenine. Nucleic Acids Res. 2020;48(9): hominoids and its evolutionary implications. Mol Biol Evol. 2002;19(5):640– 5119–34. https://doi.org/10.1093/nar/gkaa193. 53. https://doi.org/10.1093/oxfordjournals.molbev.a004123. 23. Balasubramanian B, Pogozelski WK, Tullius TD. DNA strand breaking by the 3. Ames BN, Cathcart R, Schwiers E, Hochstein P. Uric acid provides an hydroxyl radical is governed by the accessible surface areas of the antioxidant defense in humans against oxidant- and radical-caused aging hydrogen atoms of the DNA backbone. Proc Natl Acad Sci U S A. 1998; and cancer: a hypothesis. Proc Natl Acad Sci U S A. 1981;78(11):6858–62. 95(17):9738–43. https://doi.org/10.1073/pnas.95.17.9738. https://doi.org/10.1073/pnas.78.11.6858. 24. Dizdaroglu M, Jaruga P. Mechanisms of free radical-induced damage to 4. Sautin YY, Nakagawa T, Zharikov S, Johnson RJ. Adverse effects of the classic DNA. Free Radic Res. 2012;46(4):382–419. https://doi.org/10.3109/10715762.2 antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/ 011.653969. nitrosative stress. Am J Physiol Cell Physiol. 2007;293(2):C584–96. https://doi. 25. Locatelli GA, Pospiech H, Tanguy Le Gac N, van Loon B, Hubscher U, org/10.1152/ajpcell.00600.2006. Parkkinen S, et al. Effect of 8-oxo-guanine and abasic site DNA lesions on 5. Lanaspa MA, Sanchez-Lozada LG,Choi YJ,CicerchiC,KanbayM,Roncal-Jimenez in vitro elongation by human DNA polymerase ϵ in the presence of CA, et al. Uric acid induces hepatic steatosis by generation of mitochondrial replication protein a and proliferating cell nuclear antigen. Biochem J. 2010; oxidative stress: potential role in fructose-dependent and -independent fatty liver. 429(3):573–82. https://doi.org/10.1042/BJ20100405. J Biol Chem. 2012;287(48):40732–44. https://doi.org/10.1074/jbc.M112.399899. 26. Kamiya H, Suzuki M, Ohtsuka E. Mutation-spectrum of a true abasic site in 6. Shi Y, Evans JE, Rock KL. Molecular identification of a danger signal that codon 12 of a c-ha-ras gene in mammalian cells. FEBS Lett. 1993;328(1-2): alerts the immune system to dying cells. Nature. 2003;425(6957):516–21. 125–9. https://doi.org/10.1016/0014-5793(93)80979-5. https://doi.org/10.1038/nature01991. 27. Cabral Neto JB, Cabral RE, Margot A, Le Page F, Sarasin A, Gentil A. Coding 7. Hayashi N, Togawa K, Yanagisawa M, Hosogi J, Mimura D, Yamamoto Y. Effect properties of a unique apurinic/apyrimidinic site replicated in mammalian cells. of sunlight exposure and aging on skin surface lipids and urate. Exp Dermatol. J Mol Biol. 1994;240(5):416–20. https://doi.org/10.1006/jmbi.1994.1457. 2003;12(Suppl. 2):13–7. https://doi.org/10.1034/j.1600-0625.12.s2.2.x. 28. Cadet J, Vigny P. The photochemistry of nucleic acids, chap. 1. In: Morrison 8. Yiu A, Van Hemelrijck M, Garmo H, Holmberg L, Malmström H, Lambe M, H, editor. Bioorganic Photochemistry: Photochemistry and the Nucleic Acids, et al. Circulating uric acid levels and subsequent development of cancer in vol. 1. New York: Wiley; 1990. p. 1–272. 493,281 individuals: findings from the AMORIS study. Oncotarget. 2017;8(26): 29. von Sonntag C. Free-radical-induced DNA damage and its repair: a chemical 42332–42. https://doi.org/10.18632/oncotarget.16198. perspective. Berlin: Springer-Verlag; 2006. https://doi.org/10.1007/3-540-30592-0. 9. Suzuki T, Ozawa-Tamura A, Takeuchi M, Sasabe Y. Uric acid as a photosensitizer in the reaction of deoxyribonucleosides with UV light of wavelength longer than 300 nm: identification of products from 2′-deoxycytidine. Chem Pharm Publisher’sNote Bull. 2021;69(11):1067–74. https://doi.org/10.1248/cpb.c21-00501. Springer Nature remains neutral with regard to jurisdictional claims in 10. Matsuda A, Shinozaki M, Suzuki M, Watanabe K, Miyasaka T. Convenient published maps and institutional affiliations. method for the selective acylation of guanine nucleosides. Synthesis. 1985; 1986(05):385–6. https://doi.org/10.1055/s-1986-31644. 11. Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature. 1991; 349(6308):431–4. https://doi.org/10.1038/349431a0. 12. Suzuki T, Nakamura A, Inukai M. Reaction of 3′,5′-di-O-acetyl-2′- deoxyguansoine with hypobromous acid. Bioorg Med Chem. 2013;21(13): 3674–9. https://doi.org/10.1016/j.bmc.2013.04.060. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes and Environment Springer Journals

Reactions of 3′,5′-di-O-acetyl-2′-deoxyguansoine and 3′,5′-di-O-acetyl-2′-deoxyadenosine to UV light in the presence of uric acid

Loading next page...
 
/lp/springer-journals/reactions-of-3-5-di-o-acetyl-2-deoxyguansoine-and-3-5-di-o-acetyl-2-7U9qIb615n
Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2022
eISSN
1880-7062
DOI
10.1186/s41021-022-00234-5
Publisher site
See Article on Publisher Site

Abstract

Introduction: Recently, it was revealed that uric acid is a photosensitizer of reactions of nucleosides on irradiation with UV light at wavelengths longer than 300 nm, and two products generated from 2′-deoxycytidine were identified. In the present study, UV reactions of acetylated derivatives of 2′-deoxyguansoine and 2′-deoxyadenosine were conducted and their products were identified. Findings: Each reaction of 3′,5′-di-O-acetyl-2′-deoxyguansoine or 3′,5′-di-O-acetyl-2′-deoxyadenosine with UV light at wavelengths longer than 300 nm in the presence of uric acid generated several products. The products were separated by HPLC and identified by comparing UV and MS spectra of the products with previously reported values. The major products were spiroiminodihydantoin, imidazolone, and dehydro-iminoallantoin nucleosides for 3′,5′-di-O-acetyl-2′-deoxyguansoine, and an adenine base and a formamidopyrimidine nucleoside for 3′,5′-di-O- acetyl-2′-deoxyadenosine. Conclusions: If these damages caused by uric acid with sunlight occur in DNA of skin cells, mutations may arise. We should pay attention to the genotoxicity of uric acid in terms of DNA damage to dGuo and dAdo sites mediated by sunlight. Keywords: Uric acid, Deoxyguanosine, Deoxyadenosine, Photosensitizer, UV light Introduction study reported that the incidence of non-melanoma skin Since uric acid is the final metabolic product of purine ca- cancer showed a positive association with the serum uric tabolism in humans, it exists ubiquitously in various cells acid concentration [8]. Recently, we showed that uric acid and body fluids at relatively high concentrations [1, 2]. is a photosensitizer of reactions of nucleosides on irradi- Uric acid is an important antioxidant in humans [3]. How- ation with UV light at wavelengths longer than 300 nm ever, it can also act as a pro-oxidant inducing oxidative [9]. The reactions of nucleosides were suppressed by rad- stress of cells [4, 5]. It has been reported that uric acid in ical scavengers. Two products from 2′-deoxycytidine cultured mouse skin cells is increased by UV irradiation, (dCyd) were separated by reversed phase (RP) HPLC and and that uric acid on the human skin surface is increased identified by MS and NMR as N -hydroxy-2′-deoxycyti- by sunlight exposure [6, 7]. An epidemiological cancer dine and N ,5-cyclic amide-2′-deoxycytidine, formed by cycloaddition of an amide group from uric acid. The re- sults using N-labeled uric acid indicated that the amide * Correspondence: tsuzuki@shujitsu.ac.jp group added to dCyd originates from both the five- School of Pharmacy, Shujitsu University, 1-6-1 Nishigawara, Okayama 703-8516, Japan © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Suzuki et al. Genes and Environment (2022) 44:4 Page 2 of 9 membered imidazole ring and six-membered pyrimidine concentration was increased from 0 to 37.5% over 45 ring of uric acid, suggesting that an unidentified radical min in linear gradient mode. The column temperature derived from uric acid with a delocalized unpaired elec- was 40 °C and flow rate was 1 mL/min. The RP-HPLC tron is generated. To obtain information about reaction chromatogram was detected at 200–500 nm. ESI-TOF/ products of nucleosides other than dCyd, we analyzed the MS measurements were performed on a MicrOTOF reaction solutions of 2′-deoxyguanosine (dGuo) and 2′- spectrometer (Bruker, Bremen, Germany) in negative deoxyadenosine (dAdo) irradiated with UV light in the mode. The sample isolated by RP-HPLC was directly in- presence of uric acid. However, we failed to obtain prod- fused into the MS system by a syringe pump without a uct peaks with a good resolution on RP-HPLC. Thus, column. acetylated derivatives of dGuo (3′,5′-di-O-acetyl-2′-deox- yguanosine; AcdGuo) and dAdo (3′,5′-di-O-acetyl-2′- Quantitative procedures deoxyadenosine; AcdAdo) were prepared and used for The concentrations of the products were evaluated ac- analysis of the UV irradiation reaction to improve the re- cording to integrated peak areas on RP-HPLC chromato- tention and separation of the products by RP-HPLC. In grams detected at 245 nm and the ε value of each 245 nm the present study, we show identification and quantifica- product, compared with the peak area of the standard tion of the products from AcdGuo and AcdAdo by UV ir- solution of AcdGuo for the AcdGuo reactions or Ade radiation in the presence of uric acid. for the AcdAdo reactions. The ε values were used 245 nm − 1 − 1 − 1 − 1 as 12,400 M cm for AcdGuo and 8450 M cm for Materials and methods Ade. The used ε values of the products are indi- 245 nm Materials cated in Tables 1 and 2. dGuo, dAdo, and uric acid were purchased from Sigma- Aldrich (MO, USA). Other chemicals were obtained Results and discussion from Sigma-Aldrich, Nacalai Tesque (Kyoto, Japan), and Reaction of AcdGuo Tokyo Chemical Industry (Tokyo, Japan). Water was A solution of 100 μM AcdGuo with 400 μM uric acid in purified with a Millipore Milli-Q deionizer (MA, USA). 100 mM potassium phosphate buffer at pH 7.4 was irra- AcdGuo and AcdAdo were synthesized from dGuo and diated with UV light from a high-pressure mercury lamp dAdo, respectively, by acetylation using acetic anhydride through a 300-nm longpass filter at a temperature of as previously described [10]. AcdGuo and AcdAdo were 37 °C for 10 min. The reaction mixture was analyzed by purified by RP-HPLC. RP-HPLC equipped with a UV-Vis photodiode-array de- tector. As shown in Fig. 1, several product peaks ap- Irradiation conditions peared in addition to uric acid and its decomposition For UV light irradiation, UV light originating from a products, denoted by asterisks, and AcdGuo and its con- 250-W high-pressure mercury lamp (SP9-250UB, Ushio, taminants, denoted by crosses. Six products (Products Tokyo, Japan) with an optical filter through a light guide 1–6) were isolated by RP-HPLC and subjected to MS was used to directly irradiate the surface of a solution (1 analysis. The products were identified on the basis of the mL) in a glass vial (12 mm i.d.) without a cap at 37 °C. similarity of their UV and MS spectra with reported Longpass filter LU0300 (cut-on 300 nm) (Asahi Spectra, values using a reaction system of AcdGuo with hypobro- Tokyo, Japan) was used as the optical filter. The inten- mous acid [12]. Table 1 summarizes the characteristics sity of radiation on the surface of the sample solution of Products 1–6. Products 1 and 2 were identified as di- was measured with a photometer (UIT-150, Ushio, astereomers of a 3′,5′-di-O-acetyl derivative of spiroimi- Tokyo, Japan) equipped with a sensor, UVD-S254 or nodihydantoin deoxyribonucleoside (AcdSph). Product 3 UVD-S365. The intensities of the UV light were 0 mW/ was a 3′,5′-di-O-acetyl derivative of diamino-oxazolone 2 2 cm for 254 nm and 264 mW/cm for 365 nm. deoxyribonucleoside (AcdOz). Product 4 was a 3′,5′-di- O-acetyl derivative of amino-imidazolone deoxyribonu- HPLC and MS conditions cleoside (AcdIz). Product 5 was a 3′,5′-di-O-acetyl de- The HPLC system consisted of LC-10ADvp pumps and rivative of dehydro-iminoallantoin deoxyribonucleoside ox an SPD-M10Avp UV-Vis photodiode-array detector (AcdIa ). Product 6 was a 3′,5′-di-O-acetyl derivative of (Shimadzu, Kyoto, Japan). For the RP-HPLC, an Inertsil 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-AcdGuo). ODS-3 octadecylsilane column of size 4.6 × 250 mm and Authentic guanine (Gua) was eluted with the RP-HPLC particle size of 5 μm (GL Sciences, Tokyo, Japan) was retention time of 12.0 min as a broadened peak. The used. The eluent was 20 mM ammonium acetate (pH concentration of Gua in the present reaction solution 7.0) containing acetonitrile. For AcdGuo, the acetonitrile could not determined due to overlapping of a peak of a concentration was increased from 0 to 30% over 45 min decomposition product of uric acid. The structures of in linear gradient mode. For AcdAdo, the acetonitrile the reaction products from AcdGuo are shown in Fig. 2. Suzuki et al. Genes and Environment (2022) 44:4 Page 3 of 9 Table 1 Characteristics of Products Formed by UV Irradiation of AcdGuo with Uric Acid −1 − 1 a Products t (min) λ (nm) m/z (negative) ε (M cm ) R max 245 nm 1. AcdSph (fast) 25.2 230 (shoulder) 382 5480 2. AcdSph (slow) 25.6 230 (shoulder) 382 5480 3. AcdOz 28.7 232 329 6000 4. AcdIz 30.9 254, 320 311 20,500 ox 5. AcdIa 33.3 236 354 12,840 6. 8-oxo-AcdGuo 37.6 254, 295 366 15,560 The values of ε are those previously reported [10] 245 nm Uric acid dose-dependent changes in the reaction of When the solution was incubation at 37 °C for 10 min AcdGuo with UV light were examined. A solution of without UV irradiation, no product was detected. At 5 min 100 μMAcdGuowith0–400 μM uric acid in 100 mM po- UV irradiation, the major products were AcdIz and ox tassium phosphate buffer at pH 7.4 was irradiated with UV AcdIa .At15–30 min, the main product was AcdSph. light from a high-pressure mercury lamp through a 300- The concentration of 8-oxo-AcdGuo was maximal at 5 nm longpass filter at a temperature of 37 °C for 10 min. min, then decreased, with an intermediate kinetics profile. The product concentrations were determined from the ab- It has been reported that spirohydantoin nucleoside (dSph) sorbance area of HPLC detected at 245 nm using their re- is generated as a two-step oxidation product of dGuo via ported molar extinction coefficients [12]. Figure 3Ashows 8-oxo-dGuo [13]. In the present system, AcdSph should the changes in concentrations of the products. At 0 μM also lead to further oxidation of 8-oxo-AcdGuo. Reportedly ox uric acid, no product was detected. At around 100 μM uric AcdIa is generated as a three-step oxidation product of ox acid, concentrations of all products other than 8-oxo- dGuo [14]. Since AcdIa was one of the major products in AcdGuo were maximal, while the concentration of 8-oxo- the present reaction, the reaction rates of these three-steps AcdGuo increased with an increasing uric acid concentra- of oxidation should be relatively high. On the other hand, tion up to 400 μM. Over the uric acid concentration range imidazolone nucleoside (dIz) is generated by oxidation of examined, the major products were AcdSph, AcdIz, and dGuo and subsequent degradations without 7,8-dihydro-8- ox AcdIa with comparable yields. Figure 3Bshows the Acd- oxo-2′-deoxyguanosine (8-oxo-dGuo) [15]. dIzisnot Guo concentration and total concentration of all six prod- stable, converting to stable oxazolone nucleoside (dOz) ucts. At 0 μM uric acid, no consumption of AcdGuo was with a half-life of 2.5 h at 37 °C in a neutral solution at pH observed. At 100 μM uric acid, the consumption of Acd- 7[16]. The present results showing a gradual increase in Guo and total yield of the products were maximal. Over the AcdOz concentration with an increase in the irradi- the uric acid concentration range examined, the total yield ation time and decrease of AcdIz at 20–30 min would be of all products was approximately one-third of the con- explainable by the instability of AcdIz. Figure 3Dshows sumption of AcdGuo, suggesting that further reactions of the AcdGuo concentration and total concentration of all the products or other reactions without these products products. The consumption of AcdGuo increased in a occur. Irradiation time-dependent changes in the reaction time-dependent manner. Although the total concentration of AcdGuo with UV light were examined. A solution of of products increased with an increasing irradiation time, 100 μM AcdGuo with 400 μM uric acid in 100 mM potas- thechangeat15–30 min was slight, suggesting that further sium phosphate buffer at pH 7.4 was irradiated with UV reactions occur involving the products. lightatatemperatureof37°Cfor 0–30 min. Figure 3C Mutations caused by the sites of some of these shows the changes in concentrations of the products. products generated in DNA have been reported as Table 2 Characteristics of Products Formed by UV Irradiation of AcdAdo with Uric Acid −1 −1 a Products t (min) λ (nm) m/z (negative) ε (M cm ) R max 245 nm 7. Ade 16.5 260 134 8450 8. Fapy-AcdAdo (fast) 28.7 259 352 2860 9. Fapy-AcdAdo (slow) 29.3 259 352 2860 10.5′,8-cyclo-AcdAdo (fast) 33.9 274 332 8930 11.5′,8-cyclo-AcdAdo (slow) 34.6 274 332 6910 12.5′-deoxy-5′,8-cyclo -AcdAdo 35.3 264 274 9560 13. 8-oxo-AcdAdo 38.6 212, 269 350 10,220 The values of ε were calculated from the reported ε values for the products of dAdo at λ and their UV spectra obtained in the present study [11] 245 nm max Suzuki et al. Genes and Environment (2022) 44:4 Page 4 of 9 Fig. 1 RP-HPLC chromatogram of a reaction mixture of AcdGuo with uric acid detected at 245 nm. A solution of 100 μM AcdGuo and 400 μM uric acid was irradiated with UV through a 300-nm longpass filter in 100 mM potassium phosphate buffer at pH 7.4 and 37 °C for 10 min. The HPLC system consisted of LC-10ADvp pumps and an SPD-M10Avp UV-Vis photodiode-array detector (Shimadzu, Kyoto, Japan). For RP-HPLC, an Inertsil ODS-3 octadecylsilane column of size 4.6 × 250 mm and particle size of 5 μm (GL Sciences, Tokyo, Japan) was used. The eluent was 20 mM ammonium acetate (pH 7.0) containing acetonitrile. The acetonitrile concentration was increased from 0 to 30% over 45 min in linear gradient mode. The column temperature was 40 °C and flow rate was 1 mL/min follows: dSph in an oligonucleotide strongly blocks dAMP incorporation, suggesting that the formation of nucleotide incorporation by DNA polymerases, and dOz in DNA may cause G to T transversion [18]. For causes both G to T and G to C transversion muta- 8-oxo-dGuo, dCMP and dAMP are incorporated op- tions when duplication occurs over this lesion [17]. In posite 8-oxo-dGuo in an oligonucleotide by DNA vitro nucleotide insertion by Klenow fragment exo polymerases [11]. When dAMP is incorporated, G to opposite dOz in an oligonucleoside induces mainly T transversion mutation occurs. Fig. 2 The reaction products from AcdGuo by UV irradiation in the presence of uric acid Suzuki et al. Genes and Environment (2022) 44:4 Page 5 of 9 Fig. 3 Uric acid dose-dependence and time-course of the concentration changes of AcdGuo reaction products. Uric acid dose-dependence of the concentration changes of (a) each product and (b) AcdGuo and total products, when a solution of 100 μM AcdGuo with 0–400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 10 min at pH 7.4 and 37 °C. Time-course of the concentration changes of (c) each product and (d) AcdGuo and total products, when a solution of 100 μM AcdGuo with 400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 0–30 min at pH 7.4 and 37 °C. AcdSph (1 and 2) (closed circle), AcdOz (3) (closed square), AcdIz (4) (closed rhombus), ox AcdIa (5) (open circle), 8-oxo-AcdGuo (6) (open square), AcdGuo (closed triangle), and the total concentration of Products 1–6 (open triangle). All reaction mixtures were analyzed by RP-HPLC. Means ± standard deviation (S.D.) (n = 3) are presented Reaction of AcdAdo and 9 were diastereomers of a 3′,5′-di-O-acetyl deriva- A solution of 100 μM AcdAdo with 400 μM uric acid in tive of formamidopyrimidine deoxyribonucleoside (Fapy- 100 mM potassium phosphate buffer at pH 7.4 was irra- AcdAdo). Products 10 and 11 were diastereomers of a diated with UV light through a 300-nm longpass filter at 3′,5′-di-O-acetyl derivative of 5′,8-cyclo-2′-deoxyadeno- a temperature of 37 °C for 10 min. The reaction mixture sine (5′,8-cyclo-AcdAdo). Product 12 was a 3′-O-acetyl was analyzed by RP-HPLC. As shown in Fig. 4, several derivative of 5′-deoxy-5′,8-cyclo-2′-deoxyadenosine (5′- product peaks appeared in addition to uric acid and its deoxy-5′,8-cyclo-AcdAdo). Product 13 was a 3′,5′-di-O- decomposition products, denoted by asterisks, and acetyl derivative of 7,8-dihydro-8-oxo-2′-deoxyadenosine AcdAdo and its contaminants, denoted by crosses. Seven (8-oxo-AcdAdo). The structures of the reaction products products (Products 7–13) were isolated by RP-HPLC from AcdAdo are shown in Fig. 5. and subjected to MS analysis. The products were identi- Uric acid dose-dependent changes in the reaction of fied on the basis of coincidence of their UV and MS AcdAdo with UV light were examined. A solution of spectra with corresponding reported values using a reac- 100 μMAcdAdo with 0–400 μM uric acid in 100 mM po- tion system of dAdo with the Fenton system [19]. Table tassium phosphate buffer at pH 7.4 was irradiated with 2 summarizes the characteristics of Products 7–13. UV light at a temperature of 37 °C for 10 min. The prod- Product 7 was identified as adenine (Ade). Products 8 uct concentrations were determined from the absorbance Suzuki et al. Genes and Environment (2022) 44:4 Page 6 of 9 Fig. 4 RP-HPLC chromatogram of a reaction mixture of AcdAdo with uric acid detected at 245 nm. A solution of 100 μM AcdAdo and 400 μM uric acid was irradiated with UV through a 300-nm longpass filter in 100 mM potassium phosphate buffer at pH 7.4 and 37 °C for 10 min. The HPLC conditions were the same as shown in Fig. 1 excluding the acetonitrile concentration. The acetonitrile concentration was increased from 0 to 37.5% over 45 min in linear gradient mode Fig. 5 The reaction products from AcdAdo by UV irradiation in the presence of uric acid Suzuki et al. Genes and Environment (2022) 44:4 Page 7 of 9 area of HPLC detected at 245 nm using their molar ex- common radical intermediate formed from dAdo by oxi- tinction coefficients, which were calculated from the re- dative stress via subsequent reduction and oxidation, re- ported values at λ for corresponding products of dAdo spectively [20, 21]. In the present study, the reduction max and UV spectra of the products of AcdAdo obtained in reaction generating Fapy-AcdAdo may become dominant the present study [19]. Figure 6A shows the changes in in the presence of a higher concentration of uric acid. Fig- concentrations of the products. At 0 μMuricacid, no ure 6B shows the AcdAdo concentration and total con- product was detected. At 5–300 μM uric acid, the main centration of Products 7–13.At 0 μMuric acid, no product was Ade. At above 300 μM uric acid, the concen- consumption of AcdAdo was observed. The consumption tration of Fapy-AcdAdo markedly increased. At 400 μM of AcdAdo increased up to 100 μMuricacid, andthen de- uric acid, the main product was Fapy-AcdAdo. The con- creased moderately. The total generation of the products centration of 8-oxo-AcdAdo was almost constant at 50– was approximately one-seventh of the consumption of 400 μM uric acid. Reportedly, formamidopyrimidine deox- AcdAdo at 100 μM uric acid and one-half at 400 μMuric yribonucleoside (Fapy-dAdo) and 7,8-dihydro-8-oxo-2′- acid, suggesting that further reactions involving the prod- deoxyadenosine (8-oxo-dAdo) are generated from a ucts or other reactions without these products occur, Fig. 6 Uric acid dose-dependence and time-course of the concentration changes of AcdAdo reaction products. Uric acid dose-dependence of the concentration changes of (a) each product and (b) AcdAdo and total products, when a solution of 100 μM AcdAdo with 0–400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 10 min at pH 7.4 and 37 °C. Time-course of the concentration changes of (c) each product and (d) AcdAdo and total products, when a solution of 100 μM AcdAdo with 400 μM uric acid was irradiated with UV light through a 300-nm longpass filter for 0–30 min at pH 7.4 and 37 °C. Ade (7) (closed circle), Fapy-AcdAdo (8 and 9) (closed triangle), 5′,8-cyclo-AcdAdo (10 and 11) (closed square), 5′-deoxy-5′,8-cyclo-AcdAdo (12) (open circle), 8-oxo-AcdAdo (open triangle), AcdAdo (closed rhombus), total concentration of Products 7–13 (open rhombus). All the reaction mixtures were analyzed by RP-HPLC. Means ± standard deviation (S.D.) (n =3) are presented Suzuki et al. Genes and Environment (2022) 44:4 Page 8 of 9 especially at around 100 μM uric acid. Irradiation time- hydroxyl radical, peroxynitrous acid, hypochlorous acid, dependent changes in the reaction of AcdAdo with UV and hypobromous acid did not generate N ,5-cyclic light were examined. A solution of 100 μMAcdAdo with amide-2′-deoxycytidine in the presence of uric acid. 400 μM uric acid in 100 mM potassium phosphate buffer These results suggest that an unidentified radical derived at pH 7.4 was irradiated with UV light at a temperature of from uric acid with a delocalized unpaired electron is 37 °C for 0–30 min. Figure 6C shows the changes in con- generated. All the identified products formed from acet- centrations of the products. When the solution was incu- ylated dGuo and dAdo in the present UV irradiation bation at 37 °C for 10 min without UV irradiation, no study had already been reported in the reaction with re- product was detected. At up to 10 min UV irradiation, the active free radicals and oxidants [12, 19, 29]. It has also main product was Fapy-AcdAdo. At 15–30 min, the con- been reported that hydrogen atom abstraction on the centration of Fapy-AcdAdo markedly decreased, suggest- sugar moiety of nucleosides induces release of the base ing that further reactions occur involving Fapy-AcdAdo. and crosslinking between the sugar and the base, and At 15–30 min, the main product was Ade. Figure 6D that it on the base moiety of nucleosides induces various shows the AcdAdo concentration and total concentration products having modified bases [29]. A possible reaction of Products 7–13. The consumption of AcdAdo increased mechanism for the present UV reaction of AcdGuo and in a time-dependent manner, although the total gener- AcdAdo with uric acid is as follows: The radical derived ation of the products was maximal at 10 min and de- from uric acid by UV irradiation induces hydrogen atom creased gradually up to 30 min. abstraction from AcdGuo and AcdAdo. When hydrogen Mutations caused by the sites of some of these prod- atom abstraction from the deoxyribose moiety of ucts generated in DNA have been reported as follows: In AcdAdo occurs, Ade, 5′,8-cyclo-AcdAdo, and 5′-deoxy- vitro nucleotide insertion by the Klenow fragment exo 5′,8-cyclo-AcdAdo are generated. On the other hand, opposite Fapy-dAdo in an oligonucleoside induces when hydrogen atom is abstracted from the base moi- mainly dTMP incorporation, although the frequency is eties of AcdGuo and AcdAdo, the other products are one-fourth that of the native dAdo in the template [21]. generated. Further studies are needed to reveal the de- The frequency of misincorporation of dAMP and dGMP tailed reaction mechanism. opposite Fapy-dAdo was 50% greater than that opposite native dAdo, suggesting increasing rates of A to T and A Conclusions to C transversion mutation. For 8-oxo-dAdo, human The present study showed that in the presence of uric DNA polymerase η proficiently incorporated dGMP op- acid, a photosensitizer, AcdGuo and AcdAdo were posite 8-oxo-dAdo, suggesting an increase of A to C decomposed by UV light at wavelengths longer than transversion mutation [22]. The release of Ade base 300 nm. Several products generated in AcdGuo and from DNA is caused via abstraction of various hydrogen AcdAdo reactions were identified. All the identified atoms of deoxyribose by radicals with or without a single products were previously reported as products caused by strand break [23, 24]. In the absence of a single strand reactive oxygen species. Unlike the reaction of dCyd, break, an abasic site is generated in DNA. In vitro DNA products generated by the addition of a part of uric acid synthesis by human DNA polymerase ε is strongly were not detected. Reportedly, several of these products blocked at the abasic site analog [25]. In living cells, vari- generated in DNA induce mutation. If this DNA damage ous mutations are induced by abasic sites [26, 27]. caused by uric acid with sunlight occurs in skin cells, mutations may arise. We should pay attention to the Reaction mechanism genotoxicity of uric acid in terms of DNA damage to Photosensitization includes many different prosesses dGuo and dAdo sites mediated by sunlight. such as energy transfer, electron transfer, hydrogen atom abstraction, singet oxygen formation, and radical forma- tion [28, 29]. Recently we showed that uric acid is a Abbreviations photosensitzer on the reaction of nucleosides, dCyd, AcdGuo: 3′,5′-di-O-acetyl-2′-deoxyguanosine; AcdAdo: 3′,5′-di-O-acetyl-2′- deoxyadenosine; AcdSph: 3′,5′-di-O-acetyl derivative of dGuo, dAdo, and thymidine, by UV light with wave- spiroiminodihydantoin deoxyribonucleoside; AcdOz: 3′,5′-di-O-acetyl lengths longer than 300 nm [9]. These reactions were derivative of diamino-oxazolone deoxyribonucleoside; AcdIz : 3′,5′-di-O-acetyl ox inhibited by the addition of radical scavengers, ethanol derivative of amino-imidazolone deoxyribonucleoside; AcdIa :3′,5′-di-O- acetyl derivative of dehydro-iminoallantoin deoxyribonucleoside; 8-oxo-Acd- and sodium azide. For the reaction of dCyd, N ,5-cyclic Guo: 3′,5′-di-O-acetyl derivative of 7,8-dihydro-8-oxo-2′-deoxyguanosine; amide-2′-deoxycytidine was formed by cycloaddition of Ade: adenine; Fapy-AcdAdo: 3′,5′-di-O-acetyl derivative of an amide group from uric acid. When a N-labeled uric formamidopyrimidine deoxyribonucleoside; 5′,8-cyclo-AcdAdo: 3′,5′-di-O- 14 15 acetyl derivative of 5′,8-cyclo-2′-deoxyadenosine; 5′-deoxy-5′,8-cyclo- acid, having two N and two N atoms in the molecule, AcdAdo: 3′-O-acetyl derivative of 5′-deoxy-5′,8-cyclo-2′-deoxyadenosine; 8- was used, N ,5-cyclic amide-2′-deoxycytidine containing oxo-AcdAdo: 3′,5′-di-O-acetyl derivative of 7,8-dihydro-8-oxo-2′- 14 15 both N and N atoms was generated. Singlet oxygen, deoxyadenosine Suzuki et al. Genes and Environment (2022) 44:4 Page 9 of 9 Acknowledgements 13. Luo W, Muller JG, Rachlin EM, Burrows CJ. Characterization of Not applicable. spiroiminodihydantoin as a product of one-electron oxidation of 8-oxo-7,8- dihydroguanosine. Org Lett. 2000;2(5):613–7. https://doi.org/10.1021/ol9913643. 14. Luo W, Muller JG, Rachlin EM, Burrows CJ. Characterization of hydantoin Authors’ contributions products from one-electron oxidation of 8-oxo-7,8-dihydroguanosine in a T.S. designed the experiments. M.T. and A.O-T. performed the experiments. nucleoside model. Chem Res Toxicol. 2001;14(7):927–38. https://doi.org/10.1 T.S. wrote the manuscript. All authors read and approved the final 021/tx010072j. manuscript. 15. Cadet J, Douki T, Ravanat J-L. Oxidatively generated damage to isolated and cellular DNA. In: Geacintov NE, Broyde S, editors. The chemical biology of Funding DNA damage. Wiley-VCH, Weinheim: Germany; 2010. p. 53–80. https://doi. Not applicable. org/10.1002/9783527630110.ch3. 16. Henderson PT, Delaney JC, Muller JG, Neeley WL, Tannenbaum SR, Burrows Availability of data and materials CJ, et al. The hydantoin lesions formed from oxidation of 7,8-dihydro-8- Not applicable. oxoguanine are potent sources of replication errors in vivo. Biochemistry. 2003;42(31):9257–62. https://doi.org/10.1021/bi0347252. Declarations 1 13 17. Raoul S, Berger M, Buchko GW, Joshi PC, Morin B, Weinfeld M, et al. H, C and N nuclear magnetic resonance analysis and chemical features of the Ethics approval and consent to participate two main radical oxidation products of 2′-deoxyguanosine: oxazolone and This research does not require ethical approval, since it does not include imidazolone nucleosides. J Chem Soc Perkin Trans 2. 1996:371–81. in vivo experiments or clinical trials on humans and animals.. 18. Duarte V, Gasparutto D, Jaquinod M, Cadet J. In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides. Consent for publication Nucleic Acids Res. 2000;28(7):1555–63. https://doi.org/10.1093/nar/28.7.1555. Not applicable. 19. Chattopadhyaya R, Goswami B. Oxidative damage to DNA constituents by iron-mediated Fenton reactions: the deoxyadenosine family. J Biomol Struct Competing interests Dyn. 2012;30(4):394–406. https://doi.org/10.1080/07391102.2012.682206. The authors declare that they have no competing interests. 20. Vieira AJSC, Steenken S. Pattern of hydroxy radical reaction with adenine and its nucleosides and nucleotides. Characterization of two types of Received: 26 November 2021 Accepted: 11 January 2022 isomeric hydroxy adduct and their unimolecular transformation reactions. J Am Chem Soc. 1990;112(19):6986–94. https://doi.org/10.1021/ja00175a036. 21. Delaney MO, Wiederholt CJ, Greenberg MM. Fapy-dA induces nucleotide References misincorporation translesionally by a DNA polymerase. Angew Chem Int Ed 1. Wu XW, Lee CC, Muzny DM, Caskey CT. Urate oxidase: primary structure and Engl. 2002;41(5):771–3. https://doi.org/10.1002/1521-3773(20020301)41:5< evolutionary implications. Proc Natl Acad Sci U S A. 1989;86(23):9412–6. 771::AID-ANIE771>3.0.CO;2-V. https://doi.org/10.1073/pnas.86.23.9412. 22. Koag M-C, Jung H, Lee S. Mutagenesis mechanism of the major oxidative 2. Oda M, Satta Y, Takenaka O, Takahata N. Loss of urate oxidase activity in adenine lesion 7,8-dihydro-8-oxoadenine. Nucleic Acids Res. 2020;48(9): hominoids and its evolutionary implications. Mol Biol Evol. 2002;19(5):640– 5119–34. https://doi.org/10.1093/nar/gkaa193. 53. https://doi.org/10.1093/oxfordjournals.molbev.a004123. 23. Balasubramanian B, Pogozelski WK, Tullius TD. DNA strand breaking by the 3. Ames BN, Cathcart R, Schwiers E, Hochstein P. Uric acid provides an hydroxyl radical is governed by the accessible surface areas of the antioxidant defense in humans against oxidant- and radical-caused aging hydrogen atoms of the DNA backbone. Proc Natl Acad Sci U S A. 1998; and cancer: a hypothesis. Proc Natl Acad Sci U S A. 1981;78(11):6858–62. 95(17):9738–43. https://doi.org/10.1073/pnas.95.17.9738. https://doi.org/10.1073/pnas.78.11.6858. 24. Dizdaroglu M, Jaruga P. Mechanisms of free radical-induced damage to 4. Sautin YY, Nakagawa T, Zharikov S, Johnson RJ. Adverse effects of the classic DNA. Free Radic Res. 2012;46(4):382–419. https://doi.org/10.3109/10715762.2 antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/ 011.653969. nitrosative stress. Am J Physiol Cell Physiol. 2007;293(2):C584–96. https://doi. 25. Locatelli GA, Pospiech H, Tanguy Le Gac N, van Loon B, Hubscher U, org/10.1152/ajpcell.00600.2006. Parkkinen S, et al. Effect of 8-oxo-guanine and abasic site DNA lesions on 5. Lanaspa MA, Sanchez-Lozada LG,Choi YJ,CicerchiC,KanbayM,Roncal-Jimenez in vitro elongation by human DNA polymerase ϵ in the presence of CA, et al. Uric acid induces hepatic steatosis by generation of mitochondrial replication protein a and proliferating cell nuclear antigen. Biochem J. 2010; oxidative stress: potential role in fructose-dependent and -independent fatty liver. 429(3):573–82. https://doi.org/10.1042/BJ20100405. J Biol Chem. 2012;287(48):40732–44. https://doi.org/10.1074/jbc.M112.399899. 26. Kamiya H, Suzuki M, Ohtsuka E. Mutation-spectrum of a true abasic site in 6. Shi Y, Evans JE, Rock KL. Molecular identification of a danger signal that codon 12 of a c-ha-ras gene in mammalian cells. FEBS Lett. 1993;328(1-2): alerts the immune system to dying cells. Nature. 2003;425(6957):516–21. 125–9. https://doi.org/10.1016/0014-5793(93)80979-5. https://doi.org/10.1038/nature01991. 27. Cabral Neto JB, Cabral RE, Margot A, Le Page F, Sarasin A, Gentil A. Coding 7. Hayashi N, Togawa K, Yanagisawa M, Hosogi J, Mimura D, Yamamoto Y. Effect properties of a unique apurinic/apyrimidinic site replicated in mammalian cells. of sunlight exposure and aging on skin surface lipids and urate. Exp Dermatol. J Mol Biol. 1994;240(5):416–20. https://doi.org/10.1006/jmbi.1994.1457. 2003;12(Suppl. 2):13–7. https://doi.org/10.1034/j.1600-0625.12.s2.2.x. 28. Cadet J, Vigny P. The photochemistry of nucleic acids, chap. 1. In: Morrison 8. Yiu A, Van Hemelrijck M, Garmo H, Holmberg L, Malmström H, Lambe M, H, editor. Bioorganic Photochemistry: Photochemistry and the Nucleic Acids, et al. Circulating uric acid levels and subsequent development of cancer in vol. 1. New York: Wiley; 1990. p. 1–272. 493,281 individuals: findings from the AMORIS study. Oncotarget. 2017;8(26): 29. von Sonntag C. Free-radical-induced DNA damage and its repair: a chemical 42332–42. https://doi.org/10.18632/oncotarget.16198. perspective. Berlin: Springer-Verlag; 2006. https://doi.org/10.1007/3-540-30592-0. 9. Suzuki T, Ozawa-Tamura A, Takeuchi M, Sasabe Y. Uric acid as a photosensitizer in the reaction of deoxyribonucleosides with UV light of wavelength longer than 300 nm: identification of products from 2′-deoxycytidine. Chem Pharm Publisher’sNote Bull. 2021;69(11):1067–74. https://doi.org/10.1248/cpb.c21-00501. Springer Nature remains neutral with regard to jurisdictional claims in 10. Matsuda A, Shinozaki M, Suzuki M, Watanabe K, Miyasaka T. Convenient published maps and institutional affiliations. method for the selective acylation of guanine nucleosides. Synthesis. 1985; 1986(05):385–6. https://doi.org/10.1055/s-1986-31644. 11. Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature. 1991; 349(6308):431–4. https://doi.org/10.1038/349431a0. 12. Suzuki T, Nakamura A, Inukai M. Reaction of 3′,5′-di-O-acetyl-2′- deoxyguansoine with hypobromous acid. Bioorg Med Chem. 2013;21(13): 3674–9. https://doi.org/10.1016/j.bmc.2013.04.060.

Journal

Genes and EnvironmentSpringer Journals

Published: Jan 21, 2022

Keywords: Uric acid; Deoxyguanosine; Deoxyadenosine; Photosensitizer; UV light

References