Effects of additives on reaction of nucleosides with UV light in presence of uric acid and salicylic acid

Abstract

Recently, we reported that uric acid and salicylic acid are photosensitizers of the reaction of nucleosides with UV light via radical formation and energy transfer, respectively. In the present study, effects of 45 biologically relevant compounds on nucleoside reactions photosensitized by uric acid and salicylic acid were examined. When a mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine with uric acid was irradiated with UV light of a wavelength longer than 300 nm, all the nucleosides decreased. The addition of antioxidants suppressed the consumption of nucleosides. When the UV reaction of nucleosides was conducted with salicylic acid, thymidine decreased almost exclusively. Several antioxidants such as ascorbates, thiols, catecholamines, trans-2-hexen-1-ol, penicillin G, and NaHSO₃ enhanced the consumption of thymidine, although the other antioxidants suppressed it. The results suggest that antioxidants may be beneficial to protect against DNA damage by photosensitization via radical formation, but that several of them may be detrimental as they facilitate DNA damage by photo­sensitization via energy transfer.

Introduction

UV radiation from the sun is the most harmful factor for human skin, causing skin cancer.‍^(1,2) UV light of a wavelength shorter than 300 nm is absorbed by DNA directly to generate photoproducts.‍^(3,4) However, since the atmosphere absorbs this almost completely, the strength of UV light at wavelengths shorter than 300 nm in sunlight is very low on the ground surface.‍⁽¹⁾ This suggests that the mechanism of skin cancer in humans caused by sunlight involves substances promoting photosensitization, photosensitizers. Photosensitization includes many different prosesses such as radical formation including electron transfer and hydrogen atom abstraction, and energy transfer including direct excitation of substrates and singet oxygen formation.‍^(4,5) We recently reported that uric acid is a photosensitizer of the reaction of nucleosides, 2'-deoxycytidine (dC), 2'-deoxyguanosine (dG), thymidine (dT), and 2'-deoxyadenosine (dA), with UV light at wavelengths longer than 300 nm.‍⁽⁶⁾ These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. For the reaction of dC, N‍⁴,5-cyclic amide-2'-deoxycytidine was formed by cycloaddition of an amide group from uric acid. When a ‍¹⁵N-labeled uric acid, having two ‍¹⁴N and two ‍¹⁵N atoms in the molecule, was used, N‍⁴,5-cyclic amide-2'-deoxycytidine containing both ‍¹⁴N and ‍¹⁵N atoms was generated. For both the UV reactions of dG and dA acetylated on their ribose moiety, several products are generated.‍⁽⁷⁾ All the identified products of the acetylated dG and dA had already been reported in the reaction with reactive free radicals and oxidants.‍^(5,8,9) These results suggest that unidentified radicals derived from uric acid with a delocalized unpaired electron are generated and they react with nucleosides, generating the products. We also reported that salicylic acid is a photosensitizer of the reaction of nucleosides with UV light at wavelengths longer than 300 nm.‍⁽¹⁰⁾ The photosensitization reaction by salicylic acid occurred for dT almost exclusively. The major products of dT are isomers of cyclobutane thymidine dimers. Energy transfer may be involved in the reaction mechanism for the thymidine dimer formation reaction by salicylic acid, since similar results have been reported for other energy transfer photosensitizers such as acetophenone and p-aminobenzoic acid.‍^(4,11) When the photosensitization reaction of dT by salicylic acid was conducted in the presence of ascorbic acid, two pairs of diastereomers of ascorbic acid adducts of dT and 5,6-dihydrothymidine were produced.‍⁽¹²⁾ dT consumption was markedly increased in an ascorbic acid dose-dependent manner. These results suggest that photosensitization reactions are strongly affected by co-existing compounds. However, there is limited information on this. In the present study, effects of 45 biologically relevant compounds on reactions photosensitized by uric acid and salicylic acid were examined.

Materials and Methods

Materials

The chemicals dC, dG, dT, dA, uric acid, (±)-α-tocopherol phosphate disodium (α-tocopherol phosphate), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), l-glutathione reduced (glutathione), l-glutathione oxidized (glutathione disulfide), N-acetyl-l-cysteine (N-acetylcysteine), l-cysteine (cysteine), l-methionine (methionine), glycine, l-lysine monohydrochloride (lysine), l-tyrosine (tyrosine), (−)-epinephrine (adrenaline), sodium fluoride (NaF), sodium chloride (NaCl), sodium bromide (NaBr), sodium iodide (NaI), sodium nitrite (NaNO₂), and sodium nitrate (NaNO₃) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium salicylate (salicylic acid), l-ascorbic acid sodium salt (ascorbic acid), l-ascorbic acid 2-phosphate trisodium salt (ascorbic acid 2-phosphate), taurine, l-tryptophan (tryptophan), l-DOPA, dopamine hydrochloride (dopamine), l-noradrenaline (noradrenaline), d-(+)-glucose (glucose), d-(−)-fructose (fructose), sucrose, pyruvic acid sodium salt (pyruvic acid), lactic acid, fumaric acid disodium salt (fumaric acid), maleic acid disodium salt (maleic acid), trans-p-coumaric acid, trans-2-hexenal, hexanoic acid, donepezil hydrochloride (donepezil), penicillin G potassium salt (penicillin G), urea, sodium hydrogensulfite (NaHSO₃), sodium sulfate (Na₂SO₄), and sodium hydrogen carbonate (NaHCO₃) were obtained from Nacalai Tesque (Kyoto, Japan). The chemicals 2-glyceryl ascorbate, trans-2-hexen-1-ol, and hexanal were from Tokyo Chemical Industry (Tokyo, Japan). Allantoin was obtained from Kanto Chemical (Tokyo, Japan). Metformin hydrochloride (metformin) was obtained from LKT Labs (St. Paul, MN). Other chemicals were obtained from Sigma-Aldrich, Nacalai Tesque, and Kanto Chemical. Water was purified with a Millipore Milli-Q deionizer (Billerica, MA).

Irradiation conditions

UV light from a high-pressure mercury lamp (250 W, 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.) at 37°C. A longpass optical filter with a cut-on of 300 nm (LU0300, Asahi Spectra, Tokyo, Japan) was used. The intensity of radiation on the surface of the sample solution was measured by a photometer (UIT-150, Ushio) equipped with the sensor UVD-S254 or UVD-S365.

Spectrophotometer and HPLC conditions

Transmittance and absorption spectra were obtained by a MultiSpec-1500 UV/Vis spectrometer (Shimadzu, Kyoto, Japan). The UV/Vis spectra were measured at room temperature in the wavelength range of 200–800 nm. An HPLC system including an SPD-M10Avp UV/Vis photodiode-array detector (Shimadzu) with 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, and maintained at 50% from 15 to 30 min. The column temperature was 40°C and flow rate was 1 ml/min. The reversed phase (RP)-HPLC chromatogram was detected at 200–500 nm.

Results and Discussion

Irradiated UV light

UV light from an Hg lamp through a 300-nm longpass filter was used for the present UV irradiation reaction. Figure 1 shows the transmittance spectrum of the longpass filter for wavelengths ranging from 200 to 400 nm. The value of percentage transmittance was smaller than 1% at wavelengths from 200 to 290 nm. It increased from 290 to 310 nm. At wavelengths longer than 310 nm, it was around 90%. The intensities of the irradiated UV light on the surface of the sample solution through the filter were 0 mW/cm‍² for 254 nm and 244 mW/cm‍² for 365 nm.

Fig. 1.

A UV transmittance spectrum of the longpass filter for wavelengths ranging from 200 to 400 nm. The percentage transmittance (%) was measured by a spectrophotometer at room temperature.

UV/Vis absorption spectra of additives

Forty-five biologically relevant compounds were used as additives. UV/Vis absorption spectra of 1 mM additives in 100 mM potassium phosphate buffer of pH 7.4 at room temperature were measured by a UV/Vis spectrometer at wavelengths ranging from 200 to 800 nm. Spectra of 16 out of the 45 compounds showed absorbance larger than 0.01 absorbance unit (a.u.) at wavelengths above 300 nm. Figure 2 shows UV spectra of the 16 compounds at wavelengths ranging from 200 to 400 nm. Among these compounds, trans-p-coumaric acid and donepezil showed strong absorption up to 350 nm.

Fig. 2.

UV absorption spectra from 200 to 400 nm of the 16 compounds used as additives, which showed an absorbance value larger than 0.01 (a.u.) at wavelengths ranging from 300–800 nm. UV spectra were measured by a spectrophotometer for 1 mM additives in 100 mM potassium phosphate buffer of pH 7.4 at room temperature.

UV irradiation without photosensitizers

At first, to clarify the effects of the 45 additives themselves on nucleosides irradiated by UV light, UV reactions were investigated in the absence of the photosensitizers, uric acid and salicylic acid. A mixed solution of dC, dG, dT, and dA (100 μM each) with 5 mM additives in 100 mM potassium phosphate buffer at pH 7.4 and 37°C was irradiated with UV light from an Hg lamp through a 300-nm longpass filter for 20 min. The reaction mixture was analyzed by RP-HPLC. The nucleoside concentration was determined from the HPLC peak area at 260 nm. Table 1 shows the concentrations of nucleosides in the UV-irradiated solutions. Without additives, no nucleoside reacted. Among the 45 additives, those that clearly induced a decrease of nucleoside concentrations were α-tocopherol phosphate, Trolox, tryptophan, l-DOPA, dopamine, noradrenaline, adrenaline, pyruvic acid, donepezil, NaNO₂, and NaNO₃. α-Tocopherol phosphate decreased the nucleosides, especially dT. α-Tocopherol phosphate is a naturally occurring water-soluble α-tocopherol analog detected in biological tissues and fluids.‍⁽¹³⁾ α-Tocopherol phosphate is used as an ingredient of cosmetics due to its more favorable hydrophilicity and stability than α-tocopherol.‍⁽¹⁴⁾ Recently, we reported that α-tocopherol phosphate induces the UV reaction of nucleosides, especially dT, generating 5,6-dihydrothymidine as the major product.‍⁽¹⁵⁾ Trolox slightly decreased dC, dG, and dT. Trolox is another water-soluble α-tocopherol analog, substituting a hydrocarbon chain for a carboxy group.‍⁽¹⁶⁾ Reportedly, Trolox induces the reaction of nucleosides by UV irradiation, although 5,6-dihydrothymidine is not generated.‍⁽¹⁵⁾ Tryptophan reduced the dT concentration. It has been reported that tryptophan reacts with thymine to generate photoadducts by UV irradiation.‍⁽¹⁷⁾ l-DOPA, dopamine, noradrenaline, and adrenaline mainly induced the consumption of dT. The color of all solutions of catecholamines changed from clear to brown-black or dark-red by UV irradiation. These catecholamines can be polymerized by light in the presence of oxygen, resulting in a certain type of melanin.‍^(18–20) Although melanin absorbs UV light and protects the skin against DNA damage induced by UV irradiation, it also acts as a photosensitizer, generating reactive oxygen species including superoxide anion radicals and singlet oxygen, and also hydroxyl radicals in the presence of metal-ions.‍⁽²¹⁾ Pyruvic acid markedly decreased the dG concentration. It has been reported that singlet oxygen is generated by pyruvic acid with UV irradiation.‍⁽²²⁾ Singlet oxygen can react with dG preferentially, generating several products including 8-oxo-7,8-dihydro-2'-deoxyguanosine and imidazolone and oxazolone derivatives.‍^(23–25) Donepezil mainly decreased dG, and also dT and dC with lower efficiencies. Donepezil is an acetylcholinesterase inhibitor used for Alzheimer’s disease.‍⁽²⁶⁾ We could not find any report on the photosensitization activity of donepezil. NaNO₂ reduced the concentrations of all nucleosides. Reportedly, UV irradiation at around 350 nm of the nitrite ion (NO₂‍⁻) generates both the hydroxyl radical (‍^•OH) and nitric oxide (‍^•NO), inducing base release from nucleosides by ‍^•OH attack of their deoxyribose moiety and its subsequent decomposition.‍^(27,28) NaNO₃ reduced all the nucleosides with efficiency lower than that of NaNO₂. Reportedly, UV irradiation of the nitrate ion (NO₃‍⁻) generates both ‍^•OH and nitric dioxide (‍^•NO₂).‍⁽²⁸⁾

Table 1.

Effects of additives on the reaction of nucleosides with UV‍^a)

Additives	Absorption‍b) (300–800 nm)	dC (μM)	dG (μM)	dT (μM)	dA (μM)
None	−	99.7 ± 0.2	99.7 ± 0.4	98.8 ± 0.1	99.8 ± 0.3
Ascorbic acid	+	98.2 ± 0.2	98.3 ± 0.7	94.0 ± 1.5	98.4 ± 0.5
Ascorbic acid 2-phosphate	−	100.0 ± 1.4	98.1 ± 0.4	96.9 ± 1.2	98.6 ± 1.5
2-Glyceryl ascorbate	+	98.1 ± 0.2	97.9 ± 0.1	96.0 ± 0.1	97.9 ± 0.2
α-Tocopherol phosphate	+	86.0 ± 0.7	92.9 ± 1.0	37.6 ± 2.4	90.5 ± 0.6
Trolox	+	92.7 ± 0.5	95.4 ± 0.2	91.2 ± 2.2	98.6 ± 1.0
Glutathione	−	100.0 ± 0.6	100.7 ± 0.6	99.0 ± 0.4	100.6 ± 0.5
Glutathione disulfide	+	100.0 ± 0.2	99.4 ± 0.2	98.3 ± 0.4	100.2 ± 0.2
N-Acetylcysteine	−	100.3 ± 0.1	100.6 ± 0.1	99.2 ± 0.2	100.8 ± 0.1
Cysteine	−	100.9 ± 0.4	101.3 ± 0.3	99.3 ± 0.3	101.8 ± 0.6
Methionine	−	100.6 ± 0.4	101.0 ± 0.5	98.6 ± 1.1	100.6 ± 0.5
Taurine	−	100.4 ± 0.1	100.0 ± 0.2	98.7 ± 0.2	100.4 ± 0.2
Glycine	−	100.0 ± 0.1	100.0 ± 0.2	98.9 ± 0.4	100.3 ± 0.1
Lysine	−	101.7 ± 1.0	99.0 ± 1.0	98.2 ± 1.0	99.7 ± 1.1
Tryptophan	+	95.0 ± 0.5	98.9 ± 0.8	74.4 ± 2.6	94.9 ± 0.5
Tyrosine	+	97.3 ± 0.4	97.5 ± 0.4	94.8 ± 0.7	97.3 ± 0.4
l-DOPA	+	92.8 ± 2.4	95.4 ± 1.8	56.2 ± 1.3	91.7 ± 1.5
Dopamine	+	93.2 ± 0.8	97.7 ± 0.7	81.5 ± 2.5	97.9 ± 0.7
Noradrenaline	+	96.6 ± 2.4	95.9 ± 0.9	79.8 ± 2.6	95.6 ± 0.6
Adrenaline	+	93.7 ± 0.6	95.5 ± 1.3	81.0 ± 0.9	95.0 ± 1.6
Glucose	−	100.6 ± 1.1	100.7 ± 1.2	99.5 ± 1.2	100.8 ± 1.3
Fructose	−	99.6 ± 0.4	98.6 ± 1.4	98.6 ± 0.4	100.4 ± 0.0
Sucrose	−	100.2 ± 0.9	99.8 ± 0.8	98.5 ± 0.8	100.1 ± 0.7
Pyruvic acid	+	96.1 ± 1.6	14.3 ± 1.6	91.3 ± 1.9	97.2 ± 1.4
Lactic acid	−	99.6 ± 0.7	99.6 ± 0.6	99.9 ± 0.9	99.7 ± 0.9
Fumaric acid	−	99.1 ± 0.6	99.5 ± 0.2	98.1 ± 0.5	99.5 ± 0.4
Maleic acid	−	99.5 ± 1.2	99.0 ± 1.1	97.8 ± 1.2	99.4 ± 1.2
trans-p-Coumaric acid	+	99.9 ± 0.7	98.9 ± 0.7	98.6 ± 0.8	99.5 ± 0.5
trans-2-Hexenal	+	99.7 ± 0.2	97.9 ± 0.1	99.3 ± 0.3	100.5 ± 0.2
trans-2-Hexen-1-ol	−	100.1 ± 0.7	100.0 ± 0.6	98.5 ± 0.8	100.1 ± 0.8
Hexanoic acid	−	100.0 ± 0.4	99.8 ± 0.8	98.9 ± 0.5	100.4 ± 0.2
Hexanal	−	100.4 ± 1.0	99.2 ± 1.4	99.6 ± 1.0	100.2 ± 1.2
Donepezil	+	94.8 ± 1.0	72.0 ± 1.5	90.0 ± 0.9	101.8 ± 0.4
Penicillin G	−	98.7 ± 1.1	98.5 ± 1.3	98.2 ± 1.4	98.7 ± 1.1
Metformin	−	100.7 ± 1.0	101.1 ± 1.0	99.5 ± 0.9	101.3 ± 0.9
Allantoin	−	98.5 ± 0.2	98.6 ± 0.3	97.0 ± 0.4	98.7 ± 0.2
Urea	−	100.0 ± 0.5	99.1 ± 0.4	97.8 ± 0.8	99.4 ± 0.7
NaF	−	98.5 ± 0.5	98.3 ± 0.9	97.9 ± 0.6	98.6 ± 0.5
NaCl	−	99.1 ± 1.6	100.2 ± 0.1	98.3 ± 0.8	100.4 ± 0.2
NaBr	−	98.9 ± 0.3	98.3 ± 0.8	98.2 ± 0.2	99.0 ± 0.4
NaI	−	99.4 ± 0.3	99.8 ± 0.4	98.4 ± 0.4	99.5 ± 0.5
NaNO2	+	64.0 ± 1.4	75.6 ± 0.2	63.1 ± 1.4	80.9 ± 0.3
NaNO3	−	92.2 ± 0.6	93.4 ± 0.7	91.7 ± 0.6	97.3 ± 0.7
NaHSO3	−	96.2 ± 0.9	97.2 ± 1.1	93.2 ± 1.0	100.3 ± 0.8
Na2SO4	−	99.3 ± 0.7	99.8 ± 0.6	98.5 ± 0.6	99.9 ± 0.8
NaHCO3	−	100.7 ± 0.3	100.9 ± 0.3	99.6 ± 0.4	101.3 ± 0.3

‍^a)A mixed solution of 100 μM dC, dG, dT, and dA in 100 mM potassium phosphate buffer at pH 7.4 in the presence of 5 mM additives was irradiated with UV light through a 300-nm longpass filter at a temperature of 37°C for 20 min. ^b)“+” means that 1 mM additive in 100 mM potassium phosphate buffer at pH 7.4 shows an absorbance larger than 0.01 a.u. at wavelengths from 300 to 800 nm.

UV irradiation with uric acid

The experiments to examine the effects of the 45 additives on nucleosides irradiated by UV light were conducted in the presence of uric acid. A solution of dC, dG, dT, dA (100 μM each), and 400 μM uric acid with 5 mM additives in 100 mM potassium phosphate buffer at pH 7.4 and 37°C was irradiated with UV light for 20 min. The nucleoside and uric acid concentrations were determined from the RP-HPLC peak area at 260 or 300 nm. Table 2 shows the concentrations of nucleosides and uric acid in the UV-irradiated solutions. Without additives, all nucleosides were consumed in the order of dC>dT>dG>dA with the disappearance of almost all uric acid. Among the 45 compounds, additives that strongly suppressed the consumption of nucleosides were ascorbic acid and its derivatives, Trolox, thiols and its derivatives, tryptophan, tyrosine, catecholamines, trans-p-coumaric acid, trans-2-hexenal, trans-2-hexen-1-ol, donepezil, penicillin G, metformin, NaBr, NaI, and NaHSO₃. Ascorbic acid is an effective antioxidant and a radical scavenger.‍^(29,30) Various ascorbic acid derivatives are synthesized as ingredients of cosmetics for antioxidant protection against UV-induced photodamage.‍^(31–33) Trolox acts as a multiple free radical scavenger.‍⁽³⁴⁾ Thiols are important antioxidants in cells and body fluids.‍⁽³⁵⁾ In addition to thiols, glutathione disulfide, a disulfide, and methionine, a thioether, also reduce oxidative DNA damage.‍^(36,37) Although tryptophan is not a good antioxidant, its oxidative metabolites scavenge peroxyl radicals with high efficiency.‍⁽³⁸⁾ Tyrosine, a nomophenolic compound, shows antioxidant and antiradical activities.‍⁽³⁹⁾ Catecholamines, diphenolic compounds, are scavenger of radicals.‍^(40,41) trans-p-Coumaric acid, a monophenolic acid, is ubiquitously distributed in fruits and vegetables and shows antioxidant and radical scavenging activities.‍⁽⁴²⁾ trans-2-Hexenal, an α,β-unsaturated aldehyde, and trans-2-hexen-1-ol, an α,β-unsaturated alcohol, are major volatile compounds in green leaves and olive oil.‍⁽⁴³⁾ Although trans-2-hexenal is mutagenic due to generating a cyclic adduct with dG, it acts as a hydroxyl radical scavenger.‍^(44,45) It has been reported that cis-2-hexen-1-ol, a cis isomer of trans-2-hexen-1-ol, shows radical scavenging activity greater than that of trans-2-hexenal.‍⁽⁴⁶⁾ Donepezil can act as an antioxidant and a radical scavenger.‍⁽⁴⁷⁾ Penicillin G, an antibiotic, can act as a scavenger of radicals, since it has a cyclic thioether moiety, which is acceptable for free radicals.‍^(48,49) Metformin, an anti­hyperglycemic drug for type 2 diabetes, shows radical scavenging effects.‍⁽⁵⁰⁾ The bromide ion (Br‍⁻) and iodide ion (I‍⁻) show scavenging effects for ‍^•OH.‍⁽⁵¹⁾ The bisulfite ion (HSO₃‍⁻), a reducing reagent, is widely added to tap water and food because of its antioxidant, antibacterial, and bleaching effects.‍⁽⁵²⁾ All of the above compounds, which strongly suppress the reaction, can act as good antioxidants or radical scavengers, and they would scavenge uric acid radicals effectively. Compounds that moderately suppressed the UV reaction were taurine, lysine, glucose, fructose, sucrose, pyruvic acid, lactic acid, fumaric acid, maleic acid, hexanoic acid, hexanal, NaNO₂, and Na₂SO₄. These compounds may react with radicals formed from uric acid with relatively lower efficiencies. Although pyruvic acid induced decrease of dG concentration almost exclusively in the absence of uric acid probably due to generation of ‍¹O₂ (Table 1),‍⁽²²⁾ the consumption of dG was reduced in the presence of uric acid (Table 2). Uric acid may scavenge ‍¹O₂ generated by pyruvic acid, since uric acid is an excellent scavenger of ‍¹O₂.‍⁽⁵³⁾ Compounds showing no or little effects were α-tocopherol phosphate, glycine, allantoin, urea, NaF, NaCl, NaNO₃, and NaHCO₃. Although α-tocopherol phosphate induced decrease of dT concentration almost exclusively in the absence of uric acid probably due to hydrogenation of dT (Table 1),‍⁽¹⁵⁾ the consumption of dT was reduced in the presence of uric acid (Table 2). Uric acid may suppress the hydrogenation of dT by α-tocopherol phosphate. Among the 45 compounds tested, no additive accelerated the reaction of nucleosides with UV light in the presence of uric acid.

Table 2.

Effects of additives on the reaction of nucleosides with UV in the presence of uric acid‍^a)

Additives	dC (μM)	dG (μM)	dT (μM)	dA (μM)	Uric acid (μM)
None	36.5 ± 1.4	70.8 ± 1.5	49.8 ± 1.2	81.1 ± 0.5	13 ± 7
Ascorbic acid	97.9 ± 0.3	100.1 ± 0.2	94.5 ± 0.7	99.3 ± 0.2	391 ± 8
Ascorbic acid 2-phosphate	83.4 ± 2.7	97.8 ± 1.0	88.6 ± 2.2	97.9 ± 1.2	114 ± 31
2-Glyceryl ascorbate	88.2 ± 1.4	98.7 ± 1.8	93.5 ± 1.4	99.5 ± 2.0	ND
α-Tocopherol phosphate	50.4 ± 1.8	85.0 ± 1.8	57.1 ± 1.6	86.0 ± 1.8	33 ± 4
Trolox	96.0 ± 1.8	101.6 ± 1.3	95.1 ± 0.8	103.9 ± 1.2	268 ± 15
Glutathione	97.1 ± 0.6	99.6 ± 0.6	98.4 ± 0.3	99.3 ± 0.6	375 ± 6
Glutathione disulfide	97.0 ± 0.7	100.8 ± 0.9	96.4 ± 0.9	101.3 ± 1.3	144 ± 6
N-Acetylcysteine	100.3 ± 0.4	101.0 ± 0.0	100.1 ± 0.7	101.5 ± 0.5	387 ± 5
Cysteine	98.9 ± 0.2	100.8 ± 0.1	99.7 ± 0.7	100.9 ± 0.3	385 ± 4
Methionine	95.8 ± 1.2	98.2 ± 0.6	97.9 ± 0.5	98.2 ± 0.5	359 ± 22
Taurine	75.0 ± 3.2	88.2 ± 3.2	81.0 ± 3.6	91.6 ± 1.2	234 ± 17
Glycine	37.1 ± 0.3	74.0 ± 0.2	51.2 ± 0.0	83.5 ± 0.0	15 ± 0
Lysine	72.3 ± 3.3	90.9 ± 1.1	78.3 ± 2.9	93.0 ± 0.4	46 ± 24
Tryptophan	97.8 ± 1.5	97.8 ± 1.3	89.3 ± 2.7	88.3 ± 2.2	45 ± 2
Tyrosine	84.4 ± 1.9	98.3 ± 2.4	88.9 ± 1.5	98.7 ± 3.2	13 ± 4
l-DOPA	100.5 ± 0.4	100.8 ± 0.2	87.9 ± 7.4	101.1 ± 0.1	380 ± 4
Dopamine	89.2 ± 0.6	101.3 ± 0.5	96.2 ± 0.8	101.7 ± 1.7	425 ± 69
Noradrenaline	98.6 ± 2.0	100.8 ± 2.6	98.0 ± 2.5	101.4 ± 2.2	ND
Adrenaline	97.0 ± 0.4	97.2 ± 0.4	89.9 ± 0.4	97.2 ± 0.1	ND
Glucose	75.4 ± 0.8	90.5 ± 2.1	82.5 ± 0.8	97.0 ± 0.5	163 ± 18
Fructose	79.5 ± 2.4	91.0 ± 2.1	85.1 ± 2.0	97.3 ± 1.7	217 ± 7
Sucrose	71.7 ± 2.2	90.6 ± 2.2	81.7 ± 3.3	98.3 ± 2.2	54 ± 10
Pyruvic acid	70.7 ± 1.4	79.0 ± 0.6	78.1 ± 1.4	89.7 ± 0.8	117 ± 16
Lactic acid	65.4 ± 1.9	90.2 ± 0.8	77.5 ± 1.7	91.7 ± 1.7	54 ± 13
Fumaric acid	78.7 ± 5.3	94.0 ± 5.7	90.3 ± 5.9	99.3 ± 6.3	62 ± 24
Maleic acid	75.7 ± 2.5	87.2 ± 1.6	83.4 ± 3.7	93.2 ± 2.4	178 ± 21
trans-p-Coumaric acid	95.4 ± 0.6	96.5 ± 0.3	98.4 ± 0.6	96.7 ± 0.6	349 ± 19
trans-2-Hexenal	90.0 ± 0.4	92.0 ± 1.7	95.5 ± 0.7	95.4 ± 3.8	114 ± 49
trans-2-Hexen-1-ol	98.7 ± 0.7	101.0 ± 0.4	100.5 ± 0.8	101.3 ± 0.8	371 ± 15
Hexanoic acid	76.7 ± 1.0	94.7 ± 2.1	84.5 ± 2.3	97.0 ± 1.6	74 ± 11
Hexanal	79.3 ± 1.5	87.9 ± 2.2	83.5 ± 1.4	96.3 ± 1.1	56 ± 5
Domepezil	96.8 ± 4.3	87.1 ± 4.2	98.2 ± 4.4	98.6 ± 4.3	26 ± 3
Penicillin G	93.9 ± 1.6	95.6 ± 1.5	96.0 ± 1.3	95.2 ± 2.2	196 ± 11
Metformin	84.2 ± 0.5	87.5 ± 0.1	86.9 ± 0.5	92.9 ± 0.4	255 ± 10
Allantoin	45.9 ± 1.7	78.2 ± 3.2	58.9 ± 1.6	82.8 ± 4.4	14 ± 7
Urea	43.3 ± 1.5	73.9 ± 4.1	55.0 ± 2.6	82.8 ± 2.0	16 ± 4
NaF	37.9 ± 2.5	73.3 ± 3.0	52.5 ± 3.6	82.2 ± 2.8	22 ± 14
NaCl	57.9 ± 3.0	85.1 ± 2.0	67.9 ± 3.0	87.6 ± 1.2	36 ± 8
NaBr	84.2 ± 1.1	94.2 ± 2.4	90.0 ± 1.2	96.2 ± 1.6	126 ± 18
NaI	96.2 ± 0.2	98.9 ± 0.3	98.5 ± 0.2	98.7 ± 0.2	374 ± 20
NaNO2	61.8 ± 5.0	78.7 ± 3.3	63.8 ± 6.6	81.6 ± 3.1	113 ± 3
NaNO3	48.6 ± 2.8	75.4 ± 4.3	62.1 ± 2.0	83.6 ± 2.4	85 ± 45
NaHSO3	88.1 ± 7.3	97.2 ± 5.9	86.5 ± 6.4	97.7 ± 5.7	237 ± 29
Na2SO4	72.7 ± 5.3	88.5 ± 2.0	77.8 ± 4.1	91.7 ± 1.8	189 ± 47
NaHCO3	48.4 ± 2.4	73.2 ± 3.4	55.3 ± 2.7	83.8 ± 0.9	16 ± 5

‍^a)A mixed solution of 100 μM dC, dG, dT, and dA in 100 mM potassium phosphate buffer at pH 7.4 in the presence of 400 μM uric acid and 5 mM additives was irradiated with UV light through a 300-nm longpass filter at a temperature of 37°C for 20 min. ND denotes not determined due to overlapping peaks on the PR-HPLC chromatogram.

UV irradiation with salicylic acid

The experiments to examine the effects of 45 additives on the nucleosides irradiated by UV light were conducted in the presence of salicylic acid. A solution of dC, dG, dT, dA (100 μM each), and 1 mM salicylic acid with 5 mM additives in 100 mM potassium phosphate buffer at pH 7.4 and 37°C was irradiated with UV light for 20 min. The nucleoside and salicylic acid concentrations were determined from the RP-HPLC peak area at 260 nm. Table 3 shows the concentrations of nucleosides and salicylic acid in the UV-irradiated solutions. Without additives, almost exclusive consumption of dT was observed. The consumption of dT was markedly increased by additions of ascorbic acid and its derivatives, thiols and its derivatives, catecholamines except for adrenaline, trans-2-hexen-1-ol, penicillin G, and NaHSO₃. All the additives strongly enhancing the reaction were antioxidants. Among these compounds, the effects of additions of ascorbic acid and glutathione on the UV reaction of dT or thymine have been reported. Ascorbic acid accelerates the reaction of dT with UV in a reaction photosensitized by salicylic acid, generating products of two adducts of ascorbic acid, 5-(2-deoxy-2-l-ascorbyl)-5,6-dihydrothymidine and 5-(2-l-ascorbyl)-5,6-dihydrothymidine, and an adduct of two hydrogen atoms, 5,6-dihydrothymidine.‍⁽¹²⁾ Glutathione reacts with thymine to generate several adducts including 5-S-glutathione-5,6-dihydrothymine and 5-S,S-glutathione-5,6-dihydrothymine by both direct UV excitation and photosensitization.‍⁽⁵⁴⁾ On the other hand, Trolox, tryptophan, pyruvic acid, trans-p-coumaric acid, trans-2-hexenal, donepezil, and NaI strongly suppressed the dT reaction. Among them, NaI showed no absorption at wavelengths from 270 to 800 nm (<0.01 a.u. at 1 mM, data not shown). I‍⁻ did not suppress the consumption of salicylic acid (Table 3). It has been reported that I‍⁻ efficiently deactivates excited states of pterin and lumazine caused by UV light with rate constants close to the diffusion-controlled limit.‍⁽⁵⁵⁾ In the present system, I‍⁻ would quench excited states of dT but not those of salicylic acid. Pyruvic acid and trans-2-hexenal showed only weak absorption at above 300 nm (Fig. 2C), and they did not suppress the consumption of salicylic acid. Trolox and tryptophan showed only weak absorption at above 300 nm (Fig. 2A), but they partially suppressed consumption of salicylic acid. trans-p-Coumaric acid and donepezil showed strong absorption at wavelengths shorter than 350 nm (Fig. 2C). Since donepezil did not suppress the consumption of salicylic acid, it may quench the excited states of dT. trans-p-Coumaric acid showed the strongest suppression of nucleoside consumption by UV irradiation among the 45 compounds examined in the present study (Table 3). In addition, no consumption of salicylic acid was observed. trans-p-Coumaric acid would quench the excited state of salicylic acid or block excitation of salicylic acid by absorbing UV light. The other compounds not mentioned above showed little or no effect.

Table 3.

Effects of additives on the reaction of nucleosides with UV in the presence of salicylic acid‍^a)

Additives	dC (μM)	dG (μM)	dT (μM)	dA (μM)	Salicylic acid (μM)
None	97.0 ± 1.2	95.4 ± 1.1	59.1 ± 3.9	101.4 ± 1.2	719 ± 30
Ascorbic acid	101.7 ± 0.1	93.4 ± 1.0	2.4 ± 0.5	89.0 ± 1.6	828 ± 3
Ascorbic acid 2-phosphate	94.9 ± 0.4	95.3 ± 0.3	5.4 ± 4.3	98.2 ± 0.2	834 ± 16
2-Glyceryl ascorbate	93.5 ± 0.7	97.6 ±3.1	22.7 ± 7.8	99.1 ± 2.1	857 ± 39
α-Tocopherol phosphate	96.9 ± 0.3	97.2 ± 0.8	31.8 ± 3.0	99.8 ± 0.4	820 ± 21
Trolox	92.8 ± 0.9	93.2 ± 0.9	93.0 ± 1.2	102.0 ± 1.5	870 ± 21
Glutathione	95.2 ± 3.5	100.8 ± 3.5	0.2 ± 0.0	97.6 ± 1.5	712 ± 20
Glutathione disulfide	101.2 ± 2.8	99.4 ± 3.0	22.6 ± 2.1	100.3 ± 2.7	846 ± 6
N-Acetylcysteine	97.0 ± 0.6	101.9 ± 0.1	13.5 ± 1.8	98.3 ± 0.4	761 ± 4
Cysteine	99.0 ± 0.8	100.7 ± 0.5	2.7 ± 0.9	96.9 ± 0.8	831 ± 3
Methionine	97.9 ± 5.5	98.3 ± 5.5	10.3 ± 5.3	103.4 ± 1.0	767 ± 24
Taurine	98.3 ± 3.2	95.0 ± 2.9	66.5 ± 5.6	100.5 ± 3.1	713 ± 18
Glycine	99.1 ± 1.3	95.9 ± 1.8	59.4 ± 2.9	100.9 ± 2.7	734 ± 12
Lysine	98.7 ± 0.5	94.3 ± 0.6	64.9 ± 5.7	100.3 ± 0.8	744 ± 22
Tryptophan	88.9 ± 1.9	100.7 ± 0.5	84.5 ± 2.8	94.4 ± 0.1	913 ± 13
Tyrosine	96.7 ± 0.4	94.4 ± 0.7	36.1 ± 4.5	97.3 ± 0.5	712 ± 32
l-DOPA	97.7 ± 0.6	100.1 ± 0.4	3.4 ± 0.8	100.0 ± 0.6	864 ± 11
Dopamine	98.2 ± 2.8	99.9 ± 3.0	5.0 ± 0.4	98.8 ± 2.9	858 ± 24
Noradrenaline	100.3 ± 2.4	99.7 ± 2.7	11.7 ± 2.0	100.8 ± 2.4	860 ± 8
Adrenaline	100.4 ± 0.6	99.4 ± 0.2	43.9 ± 4.7	99.6 ± 0.2	935 ± 8
Glucose	98.5 ± 0.6	95.7 ± 0.8	66.9 ± 2.2	100.8 ± 0.7	727 ± 2
Fructose	98.5 ± 1.0	95.6 ± 1.0	65.3 ± 5.0	101.5 ± 0.5	720 ± 17
Sucrose	90.9 ± 0.2	87.2 ± 4.0	62.5 ± 3.3	94.4 ± 1.0	736 ± 76
Pyruvic acid	96.7 ± 0.2	97.1 ± 1.3	94.1 ± 0.3	98.4 ± 0.2	648 ± 27
Lactic acid	91.9 ± 1.8	92.3 ± 0.3	58.3 ± 1.5	97.4 ± 1.7	745 ± 21
Fumaric acid	96.0 ± 2.4	95.0 ± 1.7	54.2 ± 1.0	98.5 ± 2.7	805 ± 25
Maleic acid	93.4 ± 2.0	92.0 ± 1.9	40.7 ± 2.0	97.2 ± 2.0	707 ± 69
trans-p-Coumaric acid	98.6 ± 0.3	97.7 ± 0.2	98.8 ± 0.2	98.2 ± 0.2	989 ± 5
trans-2-Hexenal	97.2 ± 0.3	94.5 ± 0.7	91.1 ± 0.5	99.7 ± 0.5	637 ± 19
trans-2-Hexen-1-ol	100.2 ± 4.5	94.1 ± 3.1	6.4 ± 2.4	100.2 ± 3.6	715 ± 31
Hexanoic acid	93.5 ± 2.3	91.5 ± 2.0	56.3 ± 4.6	97.4 ± 2.4	737 ± 51
Hexanal	99.3 ± 0.5	90.8 ± 0.4	30.7 ± 3.8	96.8 ± 0.4	675 ± 18
Donepezil	97.2 ± 2.3	86.0 ± 2.6	95.7 ± 1.8	98.8 ± 1.7	521 ± 20
Penicillin G	99.3 ± 2.0	99.7 ± 2.4	11.6 ± 0.9	101.0 ± 2.3	892 ± 9
Metformin	102.6 ± 0.2	93.1 ± 0.6	65.2 ± 4.4	99.2 ± 0.4	717 ± 7
Allantoin	98.2 ± 3.9	92.6 ± 1.5	70.5 ± 2.8	100.4 ± 4.0	808 ± 19
Urea	99.0 ± 1.7	92.8 ± 1.4	65.0 ± 7.2	98.0 ± 0.7	744 ± 11
NaF	94.7 ± 1.1	94.3 ± 1.8	67.2 ± 2.4	100.7 ± 0.8	723 ± 20
NaCl	92.4 ± 1.9	90.8 ± 2.0	60.1 ± 0.1	96.4 ± 1.9	726 ± 84
NaBr	97.4 ± 2.3	91.6 ± 3.8	54.6 ± 3.8	98.4 ± 3.9	722 ± 55
NaI	98.6 ± 0.4	95.1 ± 0.5	91.9 ± 2.4	100.4 ± 0.1	725 ± 19
NaNO2	72.9 ± 0.7	82.5 ± 0.6	79.5 ± 0.5	85.2 ± 0.6	467 ± 32
NaNO3	88.5 ± 1.7	87.1 ± 1.8	61.9 ± 0.5	92.6 ± 1.8	646 ± 25
NaHSO3	80.0 ± 3.0	93.6 ± 0.4	4.0 ± 0.5	98.3 ± 0.5	747 ± 7
Na2SO4	92.7 ± 2.9	91.3 ± 2.8	57.3 ± 5.3	97.1 ± 2.9	679 ± 42
NaHCO3	93.7 ± 2.0	89.8 ± 1.9	56.4 ± 3.5	97.6 ± 2.4	712 ± 20

‍^a)A mixed solution of 100 μM dC, dG, dT, and dA in 100 mM potassium phosphate buffer at pH 7.4 in the presence of 1 mM salicylic acid and 5 mM additives was irradiated with UV light through a 300-nm longpass filter at a temperature of 37°C for 20 min.

Possible scheme for effects of antioxidants

In the present results, the nucleoside reaction photosensitized by uric acid was suppressed by antioxidants with radical scavenging ability. A possible scheme for inhibition of the reaction is shown in Fig. 3, upper part. Uric acid is activated by UV light at wavelengths longer than 300 nm to form radicals (Uric acid‍^•) through excited states (Uric acid*). Uric acid‍^• reacts with nucleosides to generate products.‍^(6,7) Some antioxidants quench Uric acid* and return it to a ground state. Other antioxidants react with Uric acid‍^•, returning it to uric acid or generating unknown products. On the other hand, regarding the dT reaction photosensitized by salicylic acid, some antioxidants facilitated the reaction but other antioxidants suppressed it. A possible scheme for the reaction is shown in Fig. 3, lower part. Salicylic acid is excited by UV light at wavelengths longer than 300 nm. Energy of excited salicylic acid (Salicylic acid*) is transferred to dT, resulting in excited dT (dT*). Although the lifetime of dT* in H₂O is short and it returns to a ground state spontaneously,‍⁽⁵⁶⁾ a small amount of dT* exists continuously during UV irradiation. When dT* encounters ground-state dT, thymidine dimers (dT=dT) are formed. Several antioxidants such as ascorbates, thiols, catecholamines, trans-2-hexen-1-ol, penicillin G, and NaHSO₃ react with dT*. When dT* reacts with the antioxidants more efficiently than with dT at the reaction concentrations, the consumption of dT is increased by the addition of antioxidants.

Fig. 3.

Schemes for possible roles of the antioxidants in the reaction of nucleosides with UV irradiation in the presence of uric acid and salicylic acid.

Conclusion

The present study showed that although the reaction of nucleosides with UV light in the presence of uric acid was suppressed by antioxidants, the reaction of dT in the presence of salicylic acid was enhanced by several antioxidants. This suggests that antioxidants would suppress UV DNA damage via radical formation, but several of them may enhance UV DNA damage caused via energy transfer on thymine sites. Various antioxidants are used as ingredients of cosmetics to suppress cell damage by UV light. We should pay attention to the genotoxicity of antioxidants in terms of DNA damage caused by sunlight.

Conflict of Interest

No potential conflicts of interest were disclosed.