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

Abstract

DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2′-deoxycytidine, 2′-deoxyguanosine, thymidine, and 2′-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2′-deoxycytidine were isolated and identified as N⁴-hydroxy-2′-deoxycytidine and N⁴,5-cyclic amide-2′-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A ¹⁵N-labeled uric acid, uric acid-1,3-¹⁵N₂, having two ¹⁴N and two ¹⁵N atoms per molecule, produced N⁴,5-cyclic amide-2′-deoxycytidine containing both ¹⁴N and ¹⁵N atoms from uric acid-1,3-¹⁵N₂. Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N⁴-hydroxy-2′-deoxycytidine nor N⁴,5-cyclic amide-2′-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.

Introduction

Uric acid is the final metabolic product of purine catabolism in humans; the enzyme uricase that catalyzes the conversion of uric acid to a further oxidation product, allantoin, became inactivated during human evolution.^1,2) Thus, humans have a relatively high concentration of uric acid in serum: 2.6–7.2 mg/dL (155–428 µM) is considered normal in many countries.³⁾ Uric acid is an important antioxidant in humans, since it safely eliminates various reactive oxygen species.^4,5) The effect of uric acid to scavenge reactive oxygen species may contribute to extending the human lifespan. However, uric acid can also act as a pro-oxidant. Exposure of cells to uric acid induces oxidative stress.^6,7) Epidemiological cancer studies have reported both positive and negative associations between the serum uric acid concentration and cancer incidence.⁸⁾ A study on this association in a large cohort (approx. 500000 persons) and with a long follow-up period (up to 25 years) was recently reported.⁹⁾ Positive, negative, and neutral associations between the concentration of serum uric acid and cancer incidence were reported by the type of cancer. For skin cancer, positive associations were obtained. The incidence of non-melanoma skin cancer in the highest quartile for the serum uric acid concentration compared with the lowest quartile showed hazard ratios of 1.42 for men and 1.67 for women, whereas that of melanoma skin cancer showed hazard ratios of 1.09 for men and 0.83 for women. Skin cancer in humans is closely associated with exposure to UV light from the sun.^10,11) DNA and its components directly absorb UV light with a wavelength shorter than 300 nm to generate photoproducts, including thymine dimers.^12,13) However, solar radiation shorter than 300 nm is almost completely absorbed by the atmosphere, primarily by molecular oxygen and ozone.¹⁰⁾ In addition to direct UV reactions, DNA indirectly reacts with UV light at a wavelength longer than 300 nm and even to visible (Vis) light in the presence of photosensitizers, i.e., substances that cause photosensitization.¹³⁾ Photosensitization comprises diverse processes including energy transfer, electron transfer, hydrogen atom abstraction, and formation of singlet oxygen (¹O₂) and reactive free radicals including hydroxyl radical (·OH).¹⁴⁾ Endogenous photosensitizers include riboflavin and bilirubin.^13,15) We recently reported that salicylic acid is a photosensitizer of the reaction of thymidine (dThd) with UV light between 300 and 350 nm, resulting in cyclobutane dThd dimers via energy transfer.¹⁶⁾ We conducted experiments to identify other compounds that enhance nucleoside consumption by UV irradiation. Among the biological substances we examined, uric acid decreased the concentration of nucleosides when a nucleoside mixture was irradiated with UV light at a wavelength longer than 300 nm. In the present study, we report the effects of uric acid on UV irradiation of nucleosides and identification of the products of 2′-deoxycytidine (dCyd). We discuss the reactive species in this reaction system based on the results using radical scavengers and ¹⁵N-labeled uric acid.

Results and Discussion

A mixed solution of nucleosides (dCyd, 2′-deoxyguanosine (dGuo), dThd, and 2′-deoxyadenosine (dAdo); 100 µM each) with uric acid in 100 mM potassium phosphate buffer at pH 7.4 was irradiated with UV light from a high-pressure mercury lamp through a 300-nm longpass filter at a temperature of 37 °C. The reaction mixture was analyzed by reversed phase (RP) HPLC. The nucleoside concentrations were determined from the absorbance area of HPLC detected at 260 nm. Figure 1A shows the uric acid dose-dependent changes in concentrations of nucleosides when a solution of nucleosides with 0–400 µM uric acid was irradiated by UV light for 10 min. At 0 µM uric acid, no nucleosides reacted. The concentrations of all nucleosides decreased with an increasing uric acid dose up to 100 µM. At 200–400 µM uric acid, the concentrations of dCyd, dThd, and dAdo were almost constant, whereas the consumption of dGuo diminished with an increasing uric acid dose. The most reacted nucleoside was dCyd in the presence of 200 to 400 µM uric acid. Figure 1B shows the irradiation time-dependence of the concentrations of nucleosides when a solution of nucleosides with 400 µM uric acid was irradiated by UV light for 0–30 min. The concentrations of all the nucleosides decreased with an increasing irradiation time. The most reacted nucleoside was dCyd for reaction times up to 20 min. The consumption of dGuo accelerated markedly from 20 to 30 min. In this range of irradiation times, the concentration of uric acid in the solution was low due to the decomposition of uric acid (data not shown). This acceleration of the dGuo reaction under prolonged irradiation is consistent with the profile of uric acid dose-dependence shown in Fig. 1A, in which high consumption of dGuo occurs at low concentrations of uric acid, and indicates that at least two mechanisms, one of which involves all four nucleosides and the other involves only dGuo, are associated with the present reaction system. Uric acid can act as a scavenger of various reactive species,¹⁷⁾ and at high concentrations it should eliminate the reactive species specific to dGuo efficiently and safely.

Fig. 1. (A) Uric Acid Dose-Dependence of the Concentration Changes in Nucleosides When a Solution of 100 µM Each of dCyd, dGuo, dThd, and dAdo 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; (B) Time–Course of the Concentration Changes in Nucleosides When a Solution of 100 µM Each of dCyd, dGuo, dThd, and dAdo with 400 µM Uric Acid in 100 mM Potassium Phosphate Buffer at pH 7.4 Was Irradiated with UV Light through a 300-nm Longpass Filter for 0–30 min at a Temperature of 37 °C

dCyd (closed circles), dGuo (closed triangles), dThd (closed squares), dAdo (closed rhombi). All the reaction mixtures were analyzed by RP-HPLC. Means ± standard deviation (S.D.) (n = 3) are presented.

To obtain information about the reaction mechanisms, the UV irradiation reactions of nucleosides were conducted in the presence of additives: ethanol (EtOH, a scavenger of radicals),^18,19) sodium azide (NaN₃, a scavenger of radicals and ¹O₂),^20–22) or deuterium oxide (D₂O, an enhancer of ¹O₂ reaction by prolongation of the lifetime of ¹O₂).²³⁾ Table 1 shows the effects of 1% EtOH, 10 mM NaN₃, and 99.9% D₂O on the UV irradiation reactions of nucleosides. The mixed nucleoside solution with 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer at pH 7.4 was irradiated with UV light at a temperature of 37 °C for 10 min. With 100 and 400 µM uric acid, EtOH and NaN₃ suppressed all the nucleoside reactions, whereas D₂O induced slight decreases of nucleoside concentrations, particularly dGuo. A slight decrease by D₂O was also observed with 0 µM uric acid. It has been reported that irradiation by UV light with a wavelength longer than 270 nm to 2′-deoxyGMP (dGMP) generates ¹O₂ and that the generated ¹O₂ reacts with dGMP itself, causing the subsequent loss of dGMP.²⁴⁾ It has also been reported that ¹O₂ readily reacts with dGuo, whereas the reactivities of ¹O₂ with dCyd, dThd, and dAdo are low.¹³⁾ The present UV irradiation with a wavelength longer than 300 nm may have generated small amounts of ¹O₂ by dGuo. Prolongation of the ¹O₂ lifetime by D₂O may cause a detectable decrease in the dGuo concentration. Table 2 shows the effects of EtOH, NaN₃, and D₂O on the reaction by methylene blue with Vis light, a well-established ¹O₂-producing system, as a comparison.²⁵⁾ A mixed solution of nucleosides with 0, 100, or 400 µM uric acid was irradiated with Vis light from a tungsten lamp through a 520-nm longpass filter in the presence of 25 µM methylene blue at pH 7.4 and 37 °C for 10 min. With 0 µM uric acid, only dGuo was consumed. EtOH showed no effect, whereas NaN₃ suppressed and D₂O strongly enhanced the reaction of dGuo. With 100 µM uric acid, the effects of the additives were similar to those with 0 µM uric acid. With 400 µM uric acid, the reaction of dGuo was suppressed. Table 3 shows the effects of EtOH, NaN₃, and D₂O on the reaction by nitrite with UV light, a ·OH-producing system, as another comparison. It has been reported that nitrite with UV light generates ·OH and nitric oxide (·NO), and the formed ·OH reacts with dCyd, dGuo, dThd, and dAdo with comparable reactivities.^26,27) A mixed solution of nucleosides with 0, 100, or 400 µM uric acid was irradiated by UV light from a high-pressure mercury lamp through a 350-nm longpass filter in the presence of 1 mM NaNO₂ at pH 7.4 and 37 °C for 20 min. With 0 µM uric acid, all the nucleosides were consumed. EtOH and NaN₃ suppressed the reactions, whereas D₂O enhanced the reaction of dGuo. It has been reported that a small amount of ¹O₂ is formed by UV (360 nm) irradiation of nitrite solution.²⁶⁾ The enhancement of the reaction of dGuo in D₂O may be due to this ¹O₂. In addition, in D₂O, deuteroxyl radical (·OD) but not ·OH is generated. The reactivity of ·OD is different from that of ·OH.²⁸⁾ Another conceivable explanation of enhancement of the reaction of dGuo is that the reactivity of ·OD may be higher than that of ·OH. With 100 µM uric acid, the effects of the additives were similar to those with 0 µM uric acid. With 400 µM uric acid, the reactions of all the nucleosides were slightly suppressed. The reaction profiles with 100 and 400 µM uric acid by the ·OH system (Table 3) were similar to those for the uric acid/UV system (Table 1). The results shown in Tables 1–3 suggest that the contribution of ¹O₂ is small and that ·OH and other radicals are the major reactive species in the uric acid/UV system.

Table 1. Effects of Additives on the Reactions of Nucleosides and Uric Acid with UV Light (>300 nm)^a)

Additives	dCyd (µM)	dGuo (µM)	dThd (µM)	dAdo (µM)
—0 µM Uric acid—
None	100.9 ± 0.8	99.0 ± 0.3	100.7 ± 0.2	100.4 ± 0.2
1% EtOH	100.3 ± 0.6	99.1 ± 0.4	100.3 ± 0.3	100.4 ± 0.3
10 mM NaN3	99.6 ± 0.9	100.2 ± 0.4	100.4 ± 0.5	100.5 ± 0.4
99.9% D2O	95.5 ± 0.3	91.7 ± 0.5	99.3 ± 0.0	99.7 ± 0.3
—100 µM Uric acid—
None	61.1 ± 2.0	58.1 ± 2.6	72.7 ± 0.8	88.7 ± 0.9
1% EtOH	98.5 ± 0.4	100.3 ± 0.7	99.1 ± 0.3	100.4 ± 0.5
10 mM NaN3	98.4 ± 1.0	99.4 ± 1.2	99.2 ± 1.0	100.0 ± 1.2
99.9% D2O	57.8 ± 0.2	49.4 ± 1.7	65.1 ± 0.3	83.0 ± 0.2
—400 µM Uric acid—
None	47.8 ± 1.2	81.2 ± 1.0	62.2 ± 0.7	85.0 ± 1.0
1% EtOH	99.9 ± 0.6	100.8 ± 0.8	99.1 ± 0.8	100.4 ± 1.1
10 mM NaN3	99.9 ± 0.3	100.3 ± 0.2	100.3 ± 0.2	100.6 ± 0.2
99.9% D2O	47.0 ± 1.6	78.2 ± 0.6	62.5 ± 1.3	82.2 ± 1.1

a)The nucleoside concentrations were determined by RP-HPLC analysis. Means ± S.D. (n = 3) are presented. A solution containing a mixture of nucleosides (dCyd, dGuo, dThd, and dAdo, 100 µM each) and 0, 100, or 400 µM uric acid containing the additives in 100 mM potassium phosphate buffer (pH 7.4) was irradiated with UV light (300-nm longpass filter) at 37 °C for 10 min.

Table 2. Effects of Additives on the Reactions of Nucleosides and Methylene Blue with Vis Light (>520 nm)^a)

Additives	dCyd (µM)	dGuo (µM)	dThd (µM)	dAdo (µM)
—0 µM Uric acid—
None	99.9 ± 0.2	60.9 ± 0.7	99.8 ± 0.3	99.8 ± 0.3
1% EtOH	100.8 ± 0.2	57.4 ± 0.9	100.4 ± 0.4	100.2 ± 0.2
10 mM NaN3	100.5 ± 0.4	98.7 ± 0.8	100.8 ± 0.3	100.5 ± 0.6
99.9% D2O	99.0 ± 0.4	0.3 ± 0.1	93.7 ± 0.3	98.3 ± 0.5
—100 µM Uric acid—
None	99.9 ± 0.9	61.9 ± 0.6	99.6 ± 0.7	99.8 ± 1.0
1% EtOH	100.5 ± 0.4	62.4 ± 0.5	100.2 ± 0.3	100.0 ± 0.4
10 mM NaN3	99.8 ± 0.4	98.3 ± 0.3	99.9 ± 0.2	100.0 ± 0.2
99.9% D2O	99.7 ± 0.2	0.6 ± 0.2	94.9 ± 0.2	99.0 ± 0.1
—400 µM Uric acid—
None	99.8 ± 0.5	90.2 ± 0.2	100.1 ± 0.4	99.7 ± 0.4
1% EtOH	100.0 ± 0.2	90.0 ± 0.8	100.4 ± 0.3	99.9 ± 0.1
10 mM NaN3	99.9 ± 0.1	99.4 ± 0.2	99.8 ± 0.1	99.5 ± 0.1
99.9% D2O	99.8 ± 0.8	58.8 ± 1.6	99.0 ± 0.6	99.6 ± 0.7

a)The nucleoside concentrations were determined by RP-HPLC analysis. Means ± S.D. (n = 3) are presented. A solution containing a mixture of nucleosides (dCyd, dGuo, dThd, and dAdo, 100 µM each), 25 µM methylene blue, and 0, 100, or 400 µM uric acid containing the additives in 100 mM potassium phosphate buffer (pH 7.4) was irradiated with Vis light (520-nm longpass filter) at 37 °C for 10 min.

Table 3. Effects of Additives on the Reactions of Nucleosides and Nitrite with UV Light (>350 nm)^a)

Additives	dCyd (µM)	dGuo (µM)	dThd (µM)	dAdo (µM)
—0 µM Uric acid—
None	64.1 ± 0.1	79.1 ± 0.7	64.1 ± 0.5	81.4 ± 0.2
1% EtOH	99.2 ± 0.2	98.4 ± 0.2	99.0 ± 0.5	99.8 ± 0.2
10 mM NaN3	96.4 ± 0.2	98.4 ± 0.1	96.1 ± 0.2	97.9 ± 0.2
99.9% D2O	62.1 ± 0.6	60.2 ± 0.9	62.2 ± 1.2	77.7 ± 0.6
—100 µM Uric acid—
None	65.4 ± 0.4	80.3 ± 0.3	64.7 ± 0.2	81.8 ± 0.2
1% EtOH	99.5 ± 0.1	99.7 ± 0.2	99.3 ± 0.1	100.2 ± 0.1
10 mM NaN3	97.5 ± 0.2	99.1 ± 0.3	96.5 ± 0.1	99.1 ± 0.2
99.9% D2O	61.4 ± 0.7	66.2 ± 0.3	62.0 ± 0.9	77.6 ± 0.7
—400 µM Uric acid—
None	70.5 ± 1.1	85.9 ± 0.8	76.2 ± 1.2	84.4 ± 0.8
1% EtOH	99.9 ± 0.6	101.1 ± 0.2	99.6 ± 0.5	100.6 ± 0.5
10 mM NaN3	97.7 ± 0.2	99.7 ± 0.2	97.4 ± 0.3	99.3 ± 0.2
99.9% D2O	64.7 ± 0.7	75.7 ± 0.5	67.9 ± 0.8	80.0 ± 0.5

a)The nucleoside concentrations were determined by RP-HPLC analysis. Means ± S.D. (n = 3) are presented. A solution containing a mixture of nucleosides (dCyd, dGuo, dThd, and dAdo, 100 µM each), 1 mM nitrite, and 0, 100, or 400 µM uric acid containing the additives in 100 mM potassium phosphate buffer (pH 7.4) was irradiated with UV light (350-nm longpass filter) at 37 °C for 20 min.

We then investigated the reaction of dCyd in the presence of uric acid to obtain information on the products of this UV irradiation system. A solution of 100 µM dCyd with 400 µM uric acid in 100 mM potassium phosphate buffer at pH 7.4 was irradiated with UV light through a 300-nm longpass filter at a temperature of 37 °C for 10 min. Figure 2 shows the RP-HPLC chromatogram of the reaction mixture. Two product peaks with retention times of 14.2 and 14.9 min, referred to as Product 1 and Product 2, respectively, were detected in addition to the peaks of dCyd, uric acid, and decomposition products of uric acid denoted by asterisks. UV spectra of the products are shown in the Fig. 2 insets. The products were isolated by RP-HPLC and subjected to MS and NMR. For Product 1, electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) showed a signal at m/z 242 in negative mode. The high-resolution (HR) ESI-TOF/MS value of the molecular ion agreed with the theoretical molecular mass for C₉H₁₂N₃O₅⁻ attributable to [dCyd + O−H⁺]⁻ within 3 ppm. Product 1 showed a ¹H-NMR spectrum similar to that reported for N⁴-hydroxy-2′-deoxycytidine (N⁴-OH-dCyd).²⁹⁾ The λ_max values and their relative intensities of Product 1 were similar to those reported for N⁴-OH-dCyd.²⁹⁾ Thus, Product 1 was identified as N⁴-OH-dCyd. It has been reported that N⁴-OH-dCyd is synthesized from dCyd by the reaction with hydroxylamine (NH₂OH).²⁹⁾ That reaction mechanism was proposed via addition of hydroxylamine to the 5,6-double bond of the cytosine moiety and replacement by hydroxylamine in the exocyclic amino group of cytosine.³⁰⁾ We could not find any report on the formation of N⁴-OH-dCyd by oxidation of dCyd. Regarding biological activities, it has been reported that 5′-monophosphate of N⁴-OH-dCyd is a strong inhibitor of thymidylate synthase.^29,31) For Product 2, ESI-TOF-MS showed a signal at m/z 267 in negative mode. HR-ESI-TOF/MS values of the molecular ion agreed with the theoretical molecular mass for C₁₀H₁₁N₄O₅⁻ attributable to [dCyd + C + N + O−H−H⁺]⁻ within 3 ppm. The ¹H-NMR spectrum of Product 2 showed the disappearance of an aromatic proton attributable to the 5-position of the cytosine base. The ¹³C spectrum of Product 2 showed the appearance of an aromatic carbon signal at 155.2 ppm. In conjunction with the results of two-dimensional NMR measurements (¹H–¹H correlation spectroscopy, ¹H–¹³C heteronuclear multiple quantum coherence, ¹H–¹³C heteronuclear multiple bond connectivity), Product 2 was identified as N⁴,5-cyclic amide-2′-deoxycytidine (N⁴,5-cAmd-dCyd). The cycloaddition of the amide group between N4 and C5 of the pyrimidine ring of dCyd generated a bicyclo structure, a purine. We found only two reports of nucleoside synthesis of the same base moiety as N⁴,5-cAmd-dCyd.^32,33) A compound with the base moiety of N⁴,5-cAmd-dCyd was synthesized from a 5-aminocytosine derivative treated by chloroethyl formate to insert a carbonyl group between the 4-amino and 5-amino groups of the 5-aminocytosine moiety. The structures of N⁴-OH-dCyd and N⁴,5-cAmd-dCyd are shown in Fig. 3. Uric acid dose-dependent changes in the reaction of dCyd with UV light were examined. A solution of 100 µM dCyd with 0–400 µM uric acid in 100 mM potassium phosphate buffer at pH 7.4 was irradiated with UV light through a 300-nm longpass filter at a temperature of 37 °C for 10 min. The concentrations were determined by RP-HPLC. Figure 4A shows the changes in concentrations of N⁴-OH-dCyd and N⁴,5-cAmd-dCyd. With 25–100 µM uric acid, N⁴-OH-dCyd was greater than N⁴,5-cAmd-dCyd, whereas with 200–400 µM uric acid, N⁴,5-cAmd-dCyd was greater than N⁴-OH-dCyd. Figure 4B shows the dCyd concentration and total concentration of N⁴-OH-dCyd plus N⁴,5-cAmd-dCyd. The consumption of dCyd was maximal with 100 µM uric acid. Irradiation time-dependent changes of the dCyd reaction with UV light were examined. A solution of 100 µM dCyd with 400 µM uric acid was irradiated with UV light at pH 7.4 and 37 °C for 0–30 min. Figure 5A shows the changes in concentrations of N⁴-OH-dCyd and N⁴,5-cAmd-dCyd. The concentrations of both products increased up to 10 min then decreased, indicating that both decompose quickly under UV irradiation. Figure 5B shows the dCyd concentration and total concentration of N⁴-OH-dCyd plus N⁴,5-cAmd-dCyd. As shown in Figs. 4B and 5B, the total concentration of N⁴-OH-dCyd plus N⁴,5-cAmd-dCyd was one-tenth or smaller than that of the consumed dCyd over the range of uric acid concentrations and irradiation times examined. This indicates that undetected products remained in the reaction solution.

Fig. 2. RP-HPLC Chromatogram of a Reaction Mixture of dCyd with Uric Acid Detected at 230 nm

A solution of 100 µM dCyd 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 the RP-HPLC, an Inertsil ODS-3 octadecylsilane column of 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 methanol. The methanol concentration was increased from 0 to 50% over 30 min in linear gradient mode. The column temperature was 40 °C and flow rate was 1 mL/min. The insets are the UV spectra of Products 1 and 2.

Fig. 3. Structures of the Products N⁴-OH-dCyd and N⁴,5-cAmd-dCyd

Fig. 4. (A) Uric Acid Dose-Dependence of Concentration Changes in N⁴-OH-dCyd (Open Triangles) and N⁴,5-cAmd-dCyd (Open Squares); (B) Uric Acid Dose-Dependence of Concentration Changes in dCyd (Closed Circles) and the Total of N⁴-OH-dCyd and N⁴,5-cAmd-dCyd (Open Circles)

A solution of 100 μM dCyd and 0–400 μM uric acid in 100 mM potassium phosphate buffer at pH 7.4 was irradiated with UV light from a high-pressure mercury lamp through a 300 nm longpass filter for 10 min at a temperature of 37°C. The whole reaction mixture was analyzed by RP-HPLC. Means ± S.D. (n = 3) are presented.

Fig. 5. (A) Time–Course of Concentration Changes in N⁴-OH-dCyd (Open Triangles) and N⁴,5-cAmd-dCyd (Open Squares). (B) Time-Course of Concentration Changes in dCyd (Closed Circles) and the Total of N⁴-OH-dCyd and N⁴,5-cAmd-dCyd (Open Circles)

A solution of 100 µM dCyd and 400 µM uric acid in 100 mM potassium phosphate buffer at pH 7.4 was irradiated with UV light from a high-pressure mercury lamp through a 300-nm longpass filter for 0–30 min at a temperature of 37 °C. The whole reaction mixture was analyzed by RP-HPLC. Means ± S.D. (n = 3) are presented.

In order to obtain information about the origin of the amide group added to dCyd generating N⁴,5-cAmd-dCyd, a similar UV irradiation experiment was conducted using ¹⁵N-labeled uric acid (uric acid-1,3-¹⁵N₂; 98 atom % ¹⁵N) with two ¹⁴N atoms and two ¹⁵N atoms per molecule. A solution of 100 µM dCyd with 400 µM uric acid-1,3-¹⁵N₂ in 100 mM potassium phosphate buffer at pH 7.4 was irradiated with UV light through a 300-nm longpass filter at a temperature of 37 °C for 10 min. The products of N⁴-OH-dCyd and N⁴,5-cAmd-dCyd were isolated by RP-HPLC and subjected to MS. For N⁴-OH-dCyd, the spectrum of ESI-TOF-MS obtained using uric acid-1,3-¹⁵N₂ was similar to that using unlabeled uric acid (data not shown). For N⁴,5-cAmd-dCyd, the spectrum of ESI-TOF-MS obtained using uric acid-1,3-¹⁵N₂ was different from that using unlabeled uric acid. Figure 6A shows the MS of N⁴,5-cAmd-dCyd formed using unlabeled uric acid. The molecular ion peak [M − H⁺]⁻ (m/z 267) and another peak [M − H⁺ + 1]⁻ (m/z 268) were observed with a naturally-occurring isotope ratio. Figure 6B shows the MS of N⁴,5-cAmd-dCyd formed using uric acid-1,3-¹⁵N₂. The relative peak area of m/z 268 increased, but the peak of m/z 267 was still present with a larger peak area than that of m/z 268. These results indicate that both the ¹⁴N atom and ¹⁵N atom of uric acid-1,3-¹⁵N₂ were incorporated into N⁴,5-cAmd-dCyd. In order to estimate the ratio of ¹⁴N:¹⁵N introduced into N⁴,5-cAmd-dCyd from uric acid-1,3-¹⁵N₂, the peak area of m/z 267 in Fig. 6B was compared with the peak area of m/z 268 originating from ¹⁵N of uric acid-1,3-¹⁵N₂, which was obtained by subtraction of the contribution of the naturally-occurring isotopes shown in Fig. 6A. The ratio of ¹⁴N:¹⁵N introduced into N⁴,5-cAmd-dCyd from uric acid-1,3-¹⁵N₂ was calculated as 8 : 5. This result indicates that the amide group added to dCyd originates from both the five-membered imidazole ring and six-membered pyrimidine ring of uric acid. The reaction scheme of dCyd with uric acid-1,3-¹⁵N₂ is shown in Fig. 7.

Fig. 6. Negative Ion ESI-LC-TOF-MS Spectra of (A) N⁴,5-cAmd-dCyd Obtained from the Reaction with Unlabeled Uric Acid and (B) N⁴,5-cAmd-dCyd Obtained from the Reaction with ¹⁵N-Labeled Uric Acid (Uric Acid-1,3-¹⁵N₂)

The samples isolated by RP-HPLC were directly infused into the MS systems using a syringe pump.

Fig. 7. The Reaction Scheme of dCyd with Uric Acid-1,3-¹⁵N₂ Induced by UV Irradiation

Uric acid can reportedly react with various reactive species including ¹O₂, ·OH, peroxynitrous acid (ONOOH), hypochlorous acid (HOCl), and hypobromous acid (HOBr), since the redox potential of uric acid is low. In reactions with these reactive species, uric acid generates various intermediates including radicals and products. We examined the possibility that these intermediates or products of uric acid react with dCyd to generate N⁴-OH-dCyd and N⁴,5-cAmd-dCyd. The reaction solution was analyzed by RP-HPLC. Table 4 shows the concentrations of N⁴-OH-dCyd, N⁴,5-cAmd-dCyd, dCyd, and uric acid in the reaction solutions. The detection limits were 0.05 µM for both N⁴-OH-dCyd and N⁴,5-cAmd-dCyd. Neither N⁴-OH-dCyd nor N⁴,5-cAmd-dCyd was detected in any of the reaction solutions except for the present uric acid/UV system. It has been reported that the reactivity of ¹O₂ to dCyd is low.^{13) 1}O₂ readily reacts with uric acid to form parabanic acid and its hydrolysate, and oxaluric acid via several intermediates.^34,35) The present methylene blue/Vis ¹O₂-generating system did not decrease dCyd in the presence or absence of uric acid, whereas uric acid readily reacted and decreased. For ·OH, although a detailed study of the Fenton reaction of dCyd reported the formation of 26 products, neither N⁴-OH-dCyd nor N⁴,5-cAmd-dCyd was formed.³⁶⁾ The reactivity of ·OH to uric acid is low, and the Fenton system generates small amounts of allantoin, oxonic/oxaluric acid, and parabanic acid.^5,37) A urate radical, in which the unpaired electron is highly delocalized, resulting in resonance stabilization, is generated by pulse radiolysis of uric acid solution with one-electron oxidation.^38,39) The present two ·OH-producing systems, NaNO₂/UV and Fe²⁺/H₂O₂, did not generate N⁴-OH-dCyd or N⁴,5-cAmd-dCyd. Uric acid reacted well with the NaNO₂/UV system, while it reacted only slightly with the Fe²⁺/H₂O₂ system. The low reactivity of ·OH to uric acid is consistent with the reported results using Fe²⁺/H₂O₂ systems.^5,37) The NaNO₂/UV system reportedly generates ·NO in addition to ·OH.²⁶⁾ ·NO can react with uric acid under aerobic and anaerobic conditions.^40,41) The marked consumption of uric acid by the NaNO₂/UV system may be due to the reaction with the formed ·NO. For ONOOH, the reported reactivity with dCyd is very low, while coexisting ammonium bromide facilitates the reaction to generate 5-hydroxy-dCyd and 5-bromo-dCyd.⁴²⁾ Uric acid reacts with ONOOH to generate several radicals including urate radical, triuretcarbonyl radical, and aminocarbonyl radical (H₂N−C^•=O), plus several products including allantoin and triuret.^43–45) In the present experiment, ONOOH did not react with dCyd in the presence or absence of uric acid, although uric acid reacted slightly. HOCl has been reported to react with dCyd, generating N⁴-chloro-dCyd and 5-chloro-dCyd.⁴⁶⁾ HOCl rapidly reacts with uric acid, forming allantoin, oxonic/oxaluric acid, and parabanic acid.⁵⁾ For HOBr, 5-bromo-dCyd is generated from dCyd as the major product.⁴⁷⁾ Uric acid reacts with HOBr with a reactivity similar to that of HOCl.⁴⁸⁾ In the present experiments, although HOCl and HOBr reactions were eliminated effectively by uric acid, neither N⁴-OH-dCyd nor N⁴,5-cAmd-dCyd was generated. Table 4 shows that in all the reactions examined, except for UV irradiation in the presence of uric acid, N⁴-OH-dCyd and N⁴,5-cAmd-dCyd were not generated. These results indicate that reactive species other than those in the reaction systems examined are generated in the uric acid/UV system.

Table 4. Concentrations of N⁴-OH-dCyd, N⁴,5-cAmd-dCyd, dCyd, and Uric Acid in the Reaction Solution of dCyd in the Presence or Absence of Uric Acid for Various Reaction Systems^a)

	N4-OH-dCyd (µM)	N4,5-cAmd-dCyd (µM)	dCyd (µM)	Uric acid (µM)
—0 µM Uric acid—
UV>300 nmb)	<0.05	<0.05	98.6 ± 1.0	—
Methylene blue/Vis>520 nmc)	<0.05	<0.05	100.8 ± 0.7	—
NaNO2/UV>350 nmd)	<0.05	<0.05	57.6 ± 0.6	—
Fe2+/H2O2e)	<0.05	<0.05	85.3 ± 0.5	—
ONOOHf)	<0.05	<0.05	100.1 ± 0.5	—
HOClg)	<0.05	<0.05	18.5 ± 2.0	—
HOBrh)	<0.05	<0.05	44.0 ± 1.9	—
—100 µM Uric acid—
UV>300 nm	2.2 ± 0.1	0.7 ± 0.0	15.1 ± 0.7	<1
Methylene blue/Vis>520 nm	<0.05	<0.05	100.9 ± 0.3	<1
NaNO2/UV>350 nm	<0.05	<0.05	63.7 ± 1.0	<1
Fe2+/H2O2	<0.05	<0.05	91.9 ± 0.2	97 ± 0
ONOOH	<0.05	<0.05	100.5 ± 0.4	93 ± 3
HOCl	<0.05	<0.05	99.8 ± 0.2	33 ± 3
HOBr	<0.05	<0.05	97.1 ± 0.5	15 ± 3
—400 µM Uric acid—
UV>300 nm	0.5 ± 0.0	5.9 ± 0.2	32.1 ± 2.8	79 ± 8
Methylene blue/Vis>520 nm	<0.05	<0.05	100.5 ± 0.5	10 ± 3
NaNO2/UV>350 nm	<0.05	<0.05	63.6 ± 0.7	40 ± 5
Fe2+/H2O2	<0.05	<0.05	98.2 ± 0.3	396 ± 0
ONOOH	<0.05	<0.05	100.3 ± 0.3	388 ± 7
HOCl	<0.05	<0.05	99.9 ± 0.5	341 ± 4
HOBr	<0.05	<0.05	97.7 ± 0.3	313 ± 7

a)The concentrations were determined by RP-HPLC analysis. Means ± S.D. (n = 3) are presented. b)A solution containing 100 µM dCyd and 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer (pH 7.4) was irradiated with UV light (300-nm longpass filter) at 37 °C for 10 min. c)A solution containing 100 µM dCyd, 25 µM methylene blue and 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer (pH 7.4) was irradiated with Vis light (520-nm longpass filter) at 37 °C for 10 min. d)A solution containing 100 µM dCyd, 1 mM NaNO₂, and 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer (pH 7.4) was irradiated with UV light (350-nm longpass filter) at 37 °C for 20 min. e)A solution containing 100 µM dCyd, 100 µM FeSO₄, 200 µM H₂O₂, and 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer (pH 7.4) was incubated at 37 °C for 30 min. f)A solution containing 100 µM dCyd, 100 µM peroxynitrite, and 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer (pH 7.4) was incubated at 37 °C for 30 min. g)A solution containing 100 µM dCyd, 100 µM HOCl, and 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer (pH 7.4) was incubated at 37 °C for 30 min. h)A solution containing 100 µM dCyd, 100 µM HOBr, and 0, 100, or 400 µM uric acid in 100 mM potassium phosphate buffer (pH 7.4) was incubated at 37 °C for 30 min.

In conclusion, the present study showed that uric acid is a photosensitizer for nucleoside reactions by UV light with a wavelength longer than 300 nm. The reactive species of this reaction involve an unidentified radical from uric acid with a delocalized unpaired electron. Since uric acid exists ubiquitously at high concentrations in human cells and fluids, we should pay attention to the genotoxicity of uric acid in terms of DNA damage by sunlight. This is a fundamental study using nucleosides and artificial UV light. Further studies are needed to clarify the reliable structure of transient reactive species from uric acid using spin trap ESR and pulse photolysis, and to confirm the generation of specific products by uric acid in DNA of human skin cells exposed to sunlight.

Experimental

Materials

dCyd, dGuo, dThd, dAdo, uric acid, and uric acid-1,3-¹⁵N₂ (98 atom % ¹⁵N) were purchased from Sigma-Aldrich (MO, U.S.A.). Other chemicals were obtained from Sigma-Aldrich, Nacalai Tesque (Kyoto, Japan), Tokyo Chemical Industry (Tokyo, Japan), or Santoku Chemical Industries (Tokyo, Japan). Water was purified with a Millipore Milli-Q deionizer (MA, U.S.A.). Peroxynitrite (ONOO⁻) was synthesized by the reaction between acidified H₂O₂ and NaNO₂ followed by rapid mixing with excess NaOH in a quenched-flow reactor, as previously described.⁴⁹⁾ Unreacted H₂O₂ was removed by MnO₂. The concentration of ONOO⁻ was determined spectrophotometrically from its absorbance at 302 nm in 0.1 M NaOH using a molar extinction coefficient of 1670 M⁻¹ cm⁻¹.⁵⁰⁾ Chloride-free sodium hypochlorite (NaOCl) was prepared as previously reported.⁵¹⁾ The concentration of NaOCl was determined spectrophotometrically at 290 nm using a molar extinction coefficient of 350 M⁻¹ cm⁻¹.⁵²⁾ Bromide-free hypobromous acid (HOBr) was prepared from bromine water by the addition of silver nitrate and subsequent distillation, as previously reported.⁵³⁾ The concentration of HOBr was determined spectrophotometrically at 331 nm in 10 mM NaOH using a molar extinction coefficient of 315 M⁻¹ cm⁻¹.⁵³⁾

Irradiation Conditions

For UV light irradiations, UV light originating from a 250 W high-pressure mercury lamp (SP9-250UB, Ushio, Tokyo, Japan) with an optical filter through a light guide was used to directly irradiate the surface of a solution (1 mL) in a glass vial (12 mm i.d.) without a cap at 37 °C. Longpass filters LU0300 (cut-on 300 nm) or LU0350 (cut-on 350 nm) (Asahi Spectra, Tokyo, Japan) were used as the optical filters. The intensity of radiation on the surface of the sample solution was measured with a photometer (UIT-150, Ushio, Tokyo, Japan) equipped with the sensor UVD-S254 or UVD-S365. The intensities of the UV light were 0 mW/cm² for 254 nm and 263 mW/cm² for 365 nm with the 300-nm longpass filter, and 0 mW/cm² for 254 nm and 187 mW/cm² for 365 nm with the 350-nm longpass filter. For visible light irradiation, visible (Vis) light originating from a 100-W halogen lamp (MHAA-100W, Moritex, Tokyo, Japan) through a light guide with an orange cellophane sheet (cut-on 520 nm) was used to directly irradiate the surface of a solution (1 mL) in a glass vial (12 mm i.d.) without a cap at 37 °C.

HPLC and MS Conditions

The HPLC system consisted of LC-10ADvp pumps and an SPD-M10Avp UV/Vis photodiode-array detector (Shimadzu, Kyoto, Japan). For the RP-HPLC, an Inertsil ODS-3 octadecylsilane column of 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 methanol. The methanol concentration was increased from 0 to 50% over 15 min in linear gradient mode, and maintained at 50% from 15 to 30 min. The column temperature was 40 °C and flow rate was 1 mL/min. The RP-HPLC chromatogram was detected at 200–500 nm. ESI-TOF/MS measurements were performed on a MicrOTOF spectrometer (Bruker, Bremen, Germany) in negative mode. The sample isolated by RP-HPLC was directly infused into the MS system by a syringe pump without a column.

Spectrometric Data

Product 1. N⁴-Hydroxy-2′-deoxycytidine (N⁴-OH-dCyd), 1-(2-deoxy-β-D-ribofuranosyl)-4-hydroxylamino-2(1H)-pyrimidinone. ESI-TOF/MS (negative mode): m/z 242. HR-ESI-TOF/MS (negative mode): m/z 242.078791 obsd, (calcd for C₉H₁₂N₃O₅⁻ 242.078244). UV (pH 7.0): λ_max = 236, 266 nm, ε_230 nm = 9540 M⁻¹ cm⁻¹, ε_260 nm = 5250 M⁻¹ cm⁻¹. ¹H-NMR (500 MHz, in dimethyl sulfoxide (DMSO)-d₆): δ (ppm/tetramethylsilane (TMS)) 7.03 (d, J = 8.6 Hz, 1H, H-5), 6.15 (dd, J = 6.0, 8.3 Hz, 1H, H-1′), 5.56 (d, J = 8.1 Hz, 1H, H-6), 4.19 (m, 1H, H-3′), 3.71 (m, 1H, H-4′), 3.51 (m, 2H, H-5′,5″), 2.01 (m, 1H, H-2′ or 2″), 1.95 (m, 1H, H-2′ or 2″).

Product 2. N⁴,5-Cyclic amide-2′-deoxycytidine (N⁴,5-cAmd-dCyd), 1-(2-deoxy-β-D-ribofuranosyl)-1H-purine-2,8(7H,9H)-dione. ESI-TOF/MS (negative mode): m/z 267. HR-ESI-TOF/MS (negative mode): m/z 267.073611 obsd, (calcd for C₁₀H₁₁N₄O₅⁻ 267.073493). UV (pH 7.0): λ_max = 219, 314 nm, ε_230 nm = 13280 M⁻¹ cm⁻¹, ε_260 nm = 2660 M⁻¹ cm⁻¹. ¹H-NMR (500 MHz, in DMSO-d₆): δ (ppm/TMS) 7.79 (s, 1H, H-6), 6.22 (dd, J = 6.3, 6.3 Hz, 1H, H-1′), 4.23 (m, 1H, H-3′), 3.83 (m, 1H, H-4′), 3.60 (m, 2H, H-5′,5″), 2.21 (m, 1H, H-2′ or 2″), 1.98 (m, 1H, H-2′ or 2″). ¹³C-NMR (125 MHz, in DMSO-d₆): δ (ppm/TMS) 158.0 (C-4), 155.2 (C-8), 153.7 (C-2), 118.4 (C-6), 112.3 (C-5), 87.5 (C-4′), 85.9 (C-1′), 70.1 (C-3′), 60.9 (C-5′), 40.8 (C-2′). The numbering of atoms in the base moiety of N⁴,5-cAmd-dCyd was based on that of a purine ring.

Quantitative Procedures

For the reactions of nucleoside mixtures, the concentrations of the nucleosides were evaluated according to the integrated peak areas on RP-HPLC chromatograms detected at 260 nm compared with the peak areas of the starting nucleoside mixture. For the reactions of dCyd, the concentrations of the products were evaluated according to the integrated peak areas on RP-HPLC chromatograms detected at 230 nm and by the molecular extinction coefficients at 230 nm (ε_230 nm). The ε_230 nm value of 9540 M⁻¹ cm⁻¹ for N⁴-OH-dCyd was calculated using the UV spectrum from this study and the reported ε_270 nm value of 5200 M⁻¹ cm⁻¹ at pH 7.³⁰⁾ The ε_230 nm value of N⁴,5-cAmd-dCyd was determined as 13280 M⁻¹ cm⁻¹ from the integration of proton signals of NMR and the HPLC peak area detected at 230 nm relative to that of dCyd (ε_230 nm = 8160 M⁻¹ cm⁻¹) in the mixed solution.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Wu X. W., Lee C. C., Muzny D. M., Caskey C. T., Proc. Natl. Acad. Sci. U.S.A., 86, 9412–9416 (1989).
• 2) Oda M., Satta Y., Takenaka O., Takahata N., Mol. Biol. Evol., 19, 640–653 (2002).
• 3) Desideri G., Castaldo G., Lombardi A., Mussap M., Testa A., Pontremoli R., Punzi L., Borghi C., Eur. Rev. Med. Pharmacol. Sci., 18, 1295–1306 (2014).
• 4) Ames B. N., Cathcart R., Schwiers E., Hochstein P., Proc. Natl. Acad. Sci. U.S.A., 78, 6858–6862 (1981).
• 5) Kaur H., Halliwell B., Chem. Biol. Interact., 73, 235–247 (1990).
• 6) Sautin Y. Y., Nakagawa T., Zharikov S., Johnson R. J., Am. J. Physiol. Cell Physiol., 293, C584–C596 (2007).
• 7) Lanaspa M. A., Sanchez-Lozada L. G., Choi Y. J., Cicerchi C., Kanbay M., Roncal-Jimenez C. A., Ishimoto T., Li N., Marek G., Duranay M., Schreiner G., Rodriguez-Iturbe B., Nakagawa T., Kang D. H., Sautin Y. Y., Johnson R. J., J. Biol. Chem., 287, 40732–40744 (2012).
• 8) Yan S., Zhang P., Xu W., Liu Y., Wang B., Jiang T., Hua C., Wang X., Xu D., Sun B., Mediators Inflamm., 2015, 764250 (2015).
• 9) Yiu A., Van Hemelrijck M., Garmo H., Holmberg L., Malmström H., Lambe M., Hammar N., Walldius G., Jungner I., Wulaningsih W., Oncotarget, 8, 42332–42342 (2017).
• 10) IARC, IARC monographs on the evaluation of carcinogenic risks to humans, “Solar and ultraviolet radiation,” Vol. 55. IARC Press, Lyon, 1992, pp. 50–58.
• 11) IARC, IARC monographs on the evaluation of carcinogenic risks to humans, “Solar and ultraviolet radiation,” Vol. 100D, IARC Press, Lyon, 2012, pp. 35–101.
• 12) Hicks M. R., Kowałski J., Rodger A., Chem. Soc. Rev., 39, 3380–3393 (2010).
• 13) Cadet J., Vigny P., “Bioorganic Photochemistry: Photochemistry and the Nucleic Acids,” ed. by Morrison H., Vol. 1, Chap. 1, Wiley, New York, 1990, pp. 1–272.
• 14) von Sonntag C., “Free-Radical-Induced DNA Damage and Its Repair: A Chemical Perspective,” Springer-Verlag, Berlin, 2006.
• 15) Baier J., Maisch T., Maier M., Engel E., Landthaler M., Bäumler W., Biophys. J., 91, 1452–1459 (2006).
• 16) Suzuki T., Ota H., Namba Y., Fujino T., Chem. Pharm. Bull., 67, 130–134 (2019).
• 17) Halliwell B., Gutteridge J. M. C., “Free Radicals in Biology & Medicine,” Oxford University Press, Oxford, 2015.
• 18) Alam M. S., Rao B. S. M., Janata E., Phys. Chem. Chem. Phys., 3, 2622–2624 (2001).
• 19) Alam M. S., Rao B. S. M., Janata E., Radiat. Phys. Chem., 67, 723–728 (2003).
• 20) Ye M., Madden K. P., Fessenden R. W., Schuler R. H., J. Phys. Chem., 90, 5397–5399 (1986).
• 21) Buettner G. R., Oberley L. W., FEBS Lett., 121, 161–164 (1980).
• 22) Bancirova M., Luminescence, 26, 685–688 (2011).
• 23) Rengaraj S., Li X. Z., Chemosphere, 66, 930–938 (2007).
• 24) Mohammad T., Morrison H., J. Am. Chem. Soc., 118, 1221–1222 (1996).
• 25) DeRosa M. C., Crutchley R. J., Coord. Chem. Rev., 233–234, 351–371 (2002).
• 26) Bilski P., Chignell C. F., Szychlinski J., Borkowski A., Oleksy E., Reszka K., J. Am. Chem. Soc., 114, 549–556 (1992).
• 27) Suzuki T., Inukai M., Chem. Res. Toxicol., 19, 457–462 (2006).
• 28) Paraskevopoulos G., Nip W. S., Can. J. Chem., 58, 2146–2149 (1980).
• 29) Felczak K., Miazga A., Poznański J., Bretner M., Kulikowski T., Dzik J. M., Gołos B., Zieliński Z., Cieśla J., Rode W., J. Med. Chem., 43, 4647–4656 (2000).
• 30) Brown D. M., Schell P., J. Chem. Soc., 47, 208–215 (1965).
• 31) Goldstein S., Pogolotti A. L. Jr., Garvey E. P., Santi D. V., J. Med. Chem., 27, 1259–1262 (1984).
• 32) Sayed Ahmed A. F., El-Kafrawi E., Egypt. J. Pharm. Sci., 37, 501–507 (1996).
• 33) Sayed Ahmed A. F., Bul. Pol. Acad. Sci. Chem., 44, 209–213 (1996).
• 34) Bregnhøj M., Dichmann L., McLoughlin C. K., Westberg M., Ogilby P. R., Photochem. Photobiol., 95, 202–210 (2019).
• 35) Iida S., Yamamoto Y., Fujisawa A., J. Org. Chem., 84, 3552–3558 (2019).
• 36) Luo Y., Henle E. S., Linn S., J. Biol. Chem., 271, 21167–21176 (1996).
• 37) Regoli F., Winston G. W., Toxicol. Appl. Pharmacol., 156, 96–105 (1999).
• 38) Simic M. G., Jovanovic S. V., J. Am. Chem. Soc., 111, 5778–5782 (1989).
• 39) Simic M. G., Jovanovic S. V., al-Sheikhly M., Free Radic. Res. Commun., 6, 113–115 (1989).
• 40) Suzuki T., Nitric Oxide, 16, 266–273 (2007).
• 41) Gersch C., Palii S. P., Kim K. M., Angerhofer A., Johnson R. J., Henderson G. N., Nucleosides Nucleotides Nucleic Acids, 27, 967–978 (2008).
• 42) Suzuki T., Ida K., Uchibe S., Inukai M., Bioorg. Med. Chem., 16, 5164–5170 (2008).
• 43) Santos C. X., Anjos E. I., Augusto O., Arch. Biochem. Biophys., 372, 285–294 (1999).
• 44) Robinson K. M., Morré J. T., Beckman J. S., Arch. Biochem. Biophys., 423, 213–217 (2004).
• 45) Imaram W., Gersch C., Kim K. M., Johnson R. J., Henderson G. N., Angerhofer A., Free Radic. Biol. Med., 49, 275–281 (2010).
• 46) Masuda M., Suzuki T., Friesen M. D., Ravanat J. L., Cadet J., Pignatelli B., Nishino H., Ohshima H., J. Biol. Chem., 276, 40486–40496 (2001).
• 47) Henderson J. P., Byun J., Williams M. V., McCormick M. L., Parks W. C., Ridnour L. A., Heinecke J. W., Proc. Natl. Acad. Sci. U.S.A., 98, 1631–1636 (2001).
• 48) Skaff O., Pattison D. I., Davies M. J., Chem. Res. Toxicol., 20, 1980–1988 (2007).
• 49) Noble R. W., Gibson Q. H., J. Biol. Chem., 245, 2409–2413 (1970).
• 50) Beckman J. S., Chen J., Ischiropoulos H., Crow J. P., Methods Enzymol., 233, 229–240 (1994).
• 51) Hazen S. L., Hsu F. F., Gaut J. P., Crowley J. R., Heinecke J. W., Methods Enzymol., 300, 88–105 (1999).
• 52) Morris J. C., J. Phys. Chem., 70, 3798–3805 (1966).
• 53) Wajon J. E., Morris J. C., Inorg. Chem., 21, 4258–4263 (1982).