Near-ultraviolet photolysis of β-phenylpyruvic acid generates free radicals and results in DNA damage

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Journal of Photochemistry and Photobiology B: Biology 89 (2007) 110–116 www.elsevier.com/locate/jphotobiol

Near-ultraviolet photolysis of b-phenylpyruvic acid generates free radicals and results in DNA damage A. Hargreaves a, F.A. Taiwo b, O. Duggan a, S.H. Kirk a, S.I. Ahmad a

a,*

School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK b School of Pharmacy, De Montfort University, Leicester LE1 9BH, UK Received 23 January 2007; received in revised form 6 August 2007; accepted 10 September 2007 Available online 20 September 2007

Abstract Ultraviolet A (UVA) light (315–400 nm) is ubiquitously found in our environment and constitutes about 95% of the total solar UV; all UVC and most UVB being absorbed by the stratospheric ozone layer. Compared with UVB and C, UVA does not show any direct effect on biological systems. Indirect effects of UVA, however, have been recognised overwhelmingly and this includes photosensitization of biological and non-biological compounds and production of free radicals many of which include oxygen and are hence known as reactive oxygen species or ROS. Several types of free radicals have been identified although their impacts on various macro- and micro-biomolecules are yet to be fully elucidated. b-Phenylpyruvic acid is ubiquitously found in eukaryotic cells as a metabolite of phenylalanine, which is subsequently converted to phenyllactate and/or to 2-hydroxyphenylacetate and mandelate. In patients suffering from phenylketonuria the hydroxylation of phenylalanine to tyrosine is defective due to lack of phenylalanine hydroxylase. These result in accumulation and excretion of this compound in the urine. Here we present evidence that photolysis of b-phenylpyruvic acid by a skin tanning lamp, emitting 99% UVA (315–400 nm) and 1% UVB (290–315 nm) generates carboxyl radicals ðCO2 Þ and also possibly causes direct electron transfer (or type 1) reactions. Electron paramagnetic resonance was used to detect the free radicals. To determine the biological effects of this photolytic reaction, T7 was exposed to these photolytic reactive agents and found to lead to high levels of phage inactivation. Damage to DNA and/or components such as tail fibre proteins may be involved in T7 inactivation. In addition, our unpublished data suggest that certain phenylketonuria cell lines are more sensitive to PPA + NUV, lending importance to photolytic studies of this agent.  2007 Elsevier B.V. All rights reserved. Keywords: Phenylpyruvate; Near-ultraviolet light; Reactive oxygen species; DNA damage; T7 inactivation

1. Introduction Of the three components of solar ultraviolet (UVC: 180– 280 nm, UVB: 280–315 nm and UVA: 315–400 nm) almost all UVC and most UVB are absorbed by the stratospheric ozone layer [1]. Hence exposure of living cells to sunlight involves UVA (95%) and UVB (5%) radiation (here

*

Corresponding author. Address: School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, England. Tel.: +44 115 98483325; fax: +44 115 9486636. E-mail addresses: [email protected], Shamim.ahmad@fbs. osaka-u-ac.jp (S.I. Ahmad). 1011-1344/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2007.09.007

referred to as Near UV or NUV). The finding of ozone depletion in the stratosphere, resulting in increased NUV penetration, is proposed to further increase the risk of biological damage [1,2]. Although significant damaging effects of UVC are well documented under laboratory conditions, its scarcity in nature reduces its likelihood to play any significant role in damaging biological systems. Unlike UVC and UVB however, UVA has been recognized to exert only a small direct effect on cellular systems, such as production of a small amount of pyrimidine dimers [3], but a significant indirect effect is reported via a variety of biological and man-made sensitizers. Effects include premature skin aging, carcinogenesis in mice and mutagenesis in mammalian cells, damage to eyes (cataract, photokeratitis, ocular

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melanoma and a variety of corneal/conjuctival effects), damage to and suppression of the immune system leading to general carcinogenesis and susceptibility to infection. Skin cancer (epithelioma, basal and squamous cell carcinoma and melanoma), and changes in the appearance of skin associated with aging, and also cataracts have been largely associated with NUV [4–8]. A number of UVA sensitizers have been identified including flavins, furocoumarins, porphyrins, chlorophylls, quinones, bilirubin and retinal, which upon photolysis, can transfer their excitation energy onto an adjacent dioxygen molecule, converting it to singlet oxygen (1O2) while the photosensitizer molecule returns to its ground state [9,10]. Other UVA induced photosensitizers are thiouridine, 5-methylamino-2-thiouridine, thiouracil, NADH, NADPH, cyclosporine and acridine dyes which upon photolysis generate a variety of reactive oxygen species (ROS); e.g. superoxide anions ðO 2 Þ, hydroxyl radicals (OH), hydrogen peroxide (H2O2) and singlet oxygen (1O2) [10–13]. A wealth of information is available that ROS interact with cellular systems including DNA, proteins and lipids and cause damage to them [14,15]. Damage to DNA includes strand breaks, formation of 8-oxo-7, 8-dihydroguanine (8-oxo-dG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine of 2 0 -deoxyguanosine (Fapy Gua) and pyrimidine dimers, and oxidation of pyrimidine bases [16–21]. Other damage includes lipid hydroperoxidation [22], and conversion of tryptophan, tyrosine, histidine, lysine, methionine and cysteine residues to a variety of derivatives. Singlet oxygen primarily has been implicated in the damage [11,17,18,23–26 and our unpublished data]. ROS are also associated with increased transcription and translation of certain proteins [23] and apoptosis [19]. 8Oxo-dG, if not removed from the DNA, has been shown to be a pre-mutational site mis-incorporating adenine opposite this modified base and cause an A:T ! C:G transversion [27]. In previous studies we have shown that phenylalanine, tyrosine, tryptophan, mandalate, histidine and H2O2 can generate a variety of ROS upon NUV photolysis. Furthermore the production of ROS is pH dependent and at higher pH the activation is more pronounced. ROS production was shown to inactivate T7 implying damage to cellular systems; again being pH dependent [24–26,28]. In this study we show that when b-phenylpyruvic acid (PPA) is photolysed by a NUV lamp (Philips UV HP3148/A) carboxyradicals ðCO2 Þ are generated, and direct electron transfer (type 1) reactions may occur [29]. Exposure of T7 to this sensitizer plus NUV results in significant phage inactivation, which was reduced by relevant ROS scavenger. Also exposure of human fibroblasts cultured in the presence of PPA + NUV, resulted in enhanced DNA single-strand breaks and alkali labile lesions. PPA is an important metabolite found commonly in eukaryotic cells as a metabolite of phenylalanine, which

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is subsequently converted to phenyllactate and/or to 2hydroxyphenylacetate and mandelate. In phenylketonuria patients the hydroxylation of phenylalanine to tyrosine cannot occur due to lack of phenylalanine hydroxylase. This results in its accumulation and subsequent excretion in the urine at concentrations up to 600 lM compared to 1–15 lM in normal controls [30]. Furthermore we have found that certain cell lines from patients suffering from phenylketonuria are more sensitive to PPA + NUV (our unpublished results), lending importance to photolytic studies of this agent. 2. Materials and methods 2.1. Microorganisms human cell lines and growth media The microorganisms employed in these experiments were phage T7 and Escherichia coli B. Difco nutrient broth and nutrient agar plates were used for growth and maintenance of E. coli cultures. Tryptone soya agar (TSA) and tryptone soya broth (TSB) were purchased from Oxoid Ltd., Basingstoke, UK. TS soft agar was prepared by adding 0.5% Oxoid agar to TSB. TSA plates were used to grow and titrate phage T7. Human lymphocyte B cells, RJK 853 were employed in the comet assays. This is an EBV-transformed B-lymphoblastoid cell line with a complete deletion in the gene encoding hypoxanthine guanine phosphoribosyltransferase (HPRT). It was a gift from Dr. Jane Cole, MRC Cell Mutation Unit, University of Sussex, England. 2.2. Reagents Nitroblue tetrazolium (NBT), superoxide dismutase (SOD, cat. no., S2515), and PPA were purchased from Sigma Chemical Co., Poole, UK. K2HPO4, KH2PO4 were purchased from BDH Ltd., Poole, UK, and used at 50 mM to generate buffer of appropriate pH as required. 2.3. Near UV lamp The NUV source employed was a Philips UVA lamp (HP3148/A, half body containing eight TL09 lamps of 40 W each, from Philips Electronics, Croydon, UK) with an average output of 60.3 J m2 s1 UVA and 0.7 J m2 s1 UVB. Light intensity and absorption spectra were measured using a Glen Spectral Radiometer, model 1680B. The emission profile is shown in [26]. It is likely that the 1% UVB radiation noted from the lamp may be due to leakage from the two ends. We did not seal the leakage because this lamp is used by the public for skin tanning purpose. 2.4. Nitroblue tetrazolium assay This was essentially carried out as described [26].

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2.5. Preparation, titration and NUV exposure of phage T7 These were carried out as previously described [25]. Briefly this involved adding 1 mM PPA to a suspension of phage T7 in phosphate buffer (pH 7.0) in a glass petri dish and exposure to various doses of NUV. Samples were removed at intervals, diluted, added to a freshly grown culture of E. coli B in nutrient broth and plated in soft agar on tryptone agar plates. 2.6. Electron paramagnetic resonance study Aqueous solutions of substrates in buffer were made up with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (50 mM) in a quartz flat cell and exposed to near UV radiation (7.9 · 104 J m2) at room temperature. The flat cell was immediately transferred to the EPR spectrometer cavity and spectra recorded as in our previous studies [26]. First derivative EPR spectra were recorded on a Bruker EMX 6/1 EPR spectrometer operating at X-Band frequency range (9.7 GHz) and a modulation of 100 kHz. Instrument settings were as follows: field set 3488 G, scan width 100 G, microwave power 10 mW, modulation amplitude 1 G, time constant 10.24 ms, scan time 83.89 s. To confirm radical species obtained, the OH was independently generated by the FeII/H2O2 reaction and the O 2 by the xanthine oxidase catalysed oxidation of hypoxanthine to uric acid during which dissolved dioxygen captured electrons to produce the O 2 . In the presence of the spin trap DMPO EPR features of DMPO, radical adducts were recorded. Comparative spectral simulations were generated using the SimFonia program (Bruker Biospin).

NUV absorption spectrum of ß-phenylpyruvic acid (PPA) in aqueous solution is illustrated in Fig. 1. From the result it is clear that this compound absorbs UV of broad wavelength including UVA. Therefore it is feasible that UV photolysis of this compound must also be occurring in the UVB range, the UV that we receive from the sun. In our earlier studies, and those of others, it has been shown that one mechanism by which nitroblue tetrazolium (NBT) is reduced to formazan blue is via the action of O 2 [34]. Therefore in this study NBT test was employed to determine if the formation of formazan blue occurs by the photolysis of PPA. Results (Fig. 2) show that indeed blue colour (i.e. formazan) is produced at pH 7, indicative of the production of O 2 . Irradiation of the NBT mixture without PPA, or incubation of NBT with PPA but without NUV generates no blue colour [26], indicative that O 2 only evolves when PPA is photolysed in the presence of oxygen.

2.7. Comet assay The levels of DNA damage (single-strand breaks and alkali-labile lesions) in RJK8853 cells, exposed to NUV and/or 2 mM PPA were determined using the alkaline comet assay according to the method described [31], and also the modified alkaline comet assay as described [32]. The modified comet assay slides were treated with endonuclease III which recognizes oxidatively modified pyrimidines [33]. This enzyme creates single-strand breaks by nicking the DNA at sites of oxidatively damaged nucleotides, which can be detected with the alkaline comet assay. The slides were then stained with 50 ll ml1 ethidium bromide and digitally analysed using UV microscopy and Komet 5.0 (Andor Technology plc, UK) analysis software. Fifty cells per slide were counted and the DNA damage results are expressed as percentage DNA in the comet tail.

Fig. 1. UV absorption spectrum of b-phenylpyruvic acid, 10 mM bphenylpyruvic acid was made up in phosphate buffer pH 7.0 and absorbance between 260 and 400 nm was measured on a Beckman DU7 spectrophotometer.

3. Results 3.1. ROS production from NUV photolysis of PPA Photochemical reactions implicitly require that the compound must absorb light of the reactive wavelength(s). The

Fig. 2. The effects of b-phenylpyruvic acid and NUV on the NBT reaction: NBT reaction mixture at pH 7.0 was supplemented with 1 mM b-phenylpyruvic acid and subjected to increasing dose of NUV. Absorbance values were subsequently read at 560 nm. Each point represents the mean ± the standard error of the mean of 8 observations.

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To confirm the production of O 2 , photolysis was carried out in the presence of 0.5 mg ml1 (2550 U ml1) superoxide dismutase (SOD). Results show that a 31% reduction occurs in the formation of blue colour when SOD is added in the NUV + PPA system (slope [DA (J m2)1] (·103 being 14.5 ± 0.9 without SOD and 10.0 ± 0.5 with SOD). Based on the specificity of SOD  for removal of O 2 the result points to O2 being generated. 3.2. Electron paramagnetic resonance (EPR) studies to detect ROS

1000

EPR Intensity /Arb. Unit

3.3. Phage T7 inactivation To determine the effect of PPA photolysis on phage inactivation, T7 (approximately 105 pfu ml1) was exposed to NUV in the presence of PPA (1 mM) at pH 7.0. Whilst in the absence of PPA there is a low level of NUV inactivation, and none when PPA is present without NUV, phage is synergistically inactivated when both agents are present (Fig. 4). If O 2 is directly or indirectly responsible for T7 inactivation it may be anticipated that SOD should prevent this. Results (Fig. 4) indicate that SOD does have a positive effect and that phage inactivation is decreased. 3.4. DNA damage To determine if the photolysis of PPA by NUV generates a damaging species leading to DNA damage, comet assays were carried out. Results (Fig. 5) show that indeed DNA can be damaged when human lymphocyte B cells are exposed to PPA and NUV. DNA damage levels were tested for significance using paired 2-sample, 1-tailed Student’s t-tests assuming equal variance. Fig. 5 also shows that cells exposed to PPA alone do not display an increase in the level of basal DNA damage but the level of oxidatively damaged pyrimidines does increase significantly (p < 0.05). When exposed to PPA combined with NUV light both the levels of basal and oxidatively damaged pyrimidines show a significant increase (p < 0.05), by doubling the level of DNA damage compared to the levels in the control. 100

% Survival

To investigate if the production of O 2 indeed occurs as a result of NUV photolysis of PPA, EPR analysis was carried out in the presence of the spin trap DMPO. Although a result was obtained for the control containing xanthine and xanthine oxidase under identical condition [26], indicating that the detection system was working, no signal for this DMPO-O2 was observed. Nevertheless, its generation could not altogether be ruled out due to its rapid decay to DMPO-OH. When the water-based solution of the spin trap DMPO was photolysed and analysed it showed formation of hydroxyl radicals, trapped as DMPO-OH, identified by its characteristic 4-line spectrum (aN = 14.8264, H a = 14.8264) (Fig. 3a). Solutions of histidine, mandelate, and phenylalanine gave similar results, though to varying intensities, suggesting different concentrations of the OH produced [25,26 and our unpublished data]. Photolysis of phenylpyruvate with DMPO, however, showed an additional signal predominating over that of DMPO-OH, shown in Fig. 3b. The more prominent 6-line spectrum is ascribed to carbon dioxide radical ðCO2 Þ trapped by DMPO. The simulated spectrum gave parameters (aN = 15.698 G, aH = 18.780 G) characteristic of carbon dioxide radicals [35].

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1 -1500 3450

0 3470

3490

3510

3530

4

8

12

Dose (x104Jm-2)

Field /Gauss

Fig. 3. First derivate EPR spectra showing (a) spectrum of unirradiated sample; (b) 4-line spectrum of DMPO-OH obtained from solvent water and (c) a predominant 6-line spectrum ascribed to DMPO-CO2 adduct from decarboxylation of phenyl pyruvate.

Fig. 4. b-phenylpyruvic acid plus NUV-induced inactivation of phage T7 at pH 7.0. Phage suspension incubated with 1 mM b-phenylpyruvic acid alone (n = 8) or with b-phenylpyruvic acid plus SOD (n = 2) and were irradiated with varying doses of NUV. Standard errors on all points were within 2% or number as appropriate.

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Base 90

Endo III

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% DNA in Comet Tail

70

60

50

40

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0 Control

ControlUV

Control H2O2

Phenylpyruvate

Phenylpyruvate UV

Treatment

Fig. 5. Basal level of DNA damage and oxidatively damaged pyrimidines in control untreated cells (RJK853 cell line) and cells treated with to H2O2, 2 mM phenylpyruvate, and 2 mM phenylpyruvate combined with NUV light, for 5 min. It also shows that cells exposed to PPA alone do not display an increase in the level of basal DNA damage but the level of oxidatively damaged pyrimidines does increase significantly (p < 0.05). When exposed to PPA combined with NUV light both the levels of basal and oxidatively damaged pyrimidines show a significant increase (p < 0.05), by doubling the level of DNA damage compared to the levels in the control.

4. Discussion Radiolysis of water by gamma and X-rays is known to cause formation of several water derived radical of which hydroxyl is one and also the O 2 from dissolved molecular oxygen (although in minute quantities). The trapped O 2 complex, DMPO-O2, has been show by one of us [36] to decay very rapidly to DMPO-OH, the species more often observed. The trapping of OH may therefore arise from its direct production or from the rapid decay of a primary  O 2 . In the present study, the characteristic O2 12 line EPR spectrum [37] was not observed even when the dead time of spectral recording post-photolysis was reduced to 90 s. Though occurrence of O 2 is not ruled out, the amount generated was probably too little to be observed under our experimental condition. Thus, although NBT reduction via NUV photolysis of PPA (Fig. 2) is thought to be mediated by O 2 generation, the EPR study shows that this may not exclusively be the case, as CO2 is also generated (Fig. 3). Moore and Wang [38] have suggested that reduction of NBT can also occur by a direct electron transfer (type 1 reaction) mechanism [29]. In their studies they used benzydiamine, which was shown to reduce NBT without mediation by O 2 . In addition we have shown that NBT reduction by electrons, released from the xanthine + xanthine oxidase reaction,

can occur in the absence of oxygen. This observation led to the conclusion that reduction of PPA other than by an O 2 route is possible [26]. Thus this study points to the possibility that more than one reaction occurs when PPA is photolysed by NUV; these are (i) direct electron transfer reaction as observed by Moore and Wang [38] and (ii) gen eration of ðCO 2 Þ and OH. Furthermore, any of these may contribute to reduction of NBT to formazan blue. If it is the case that NBT can be reduced to formazan by PPA photolysis without involving O 2 , the question arises as to how SOD partially prevents the reduction reaction. A possible explanation is that in the experimental systems we have employed the SOD may also act as an electron trapper. This possibility needs to be explored, because it is not only when studying PPA with that we have observed this phenomenon; we have shown in our earlier studies that in the presence of SOD the reduction of NBT by photolysis of phenylalanine, tyrosine, tryptophan, histidine and mandelate is also reduced [24,25,28]. Based on the EPR studies we propose that radiolysis of water produces OH which induces secondary damage to the substrate PPA leading to decarboxylation. Any small amount of O 2 formed in ambient solution would be undetectable but would enhance the hydroxyl radical effect by the well known protonation to hydrogen peroxide and homolytic cleavage to hydroxyl ions.

A. Hargreaves et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 110–116 þ O 2 þ 2H ! HOOH which is broken symmetrically between the oxygen to 2[OH]. A proposed mechanism for the generation of hydroxyl radicals via Fenton type reaction between Fe(II) and hydrogen peroxide and via UV radiolysis of water is given below:

H2 O * H2 Oþ þ e

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Acknowledgments We wish to thank Nottingham Trent and De Montfort Universities for research facilities, the HEFCE, Thomas Sivewright Catto Charitable Trust and N. Smith Charitable Settlement for funding this project, and the Boots Company, plc for their help in measuring NUV intensities.

hv

H2 Oþ þ H2 O !  OH þ H3 Oþ

References

þ  e solv þ H3 O ! H2 O þ H

H þ H ! H2 and from this OH induced decarboxylation of phenyl PPA may occur as shown in Fig. 6. The employment of phage T7 in our studies reveals the importance of these reactions to biological systems. Phage T7 is inactivated at a rapid rate implying that damage to biological systems indeed takes place. The result is not surprising as both the protein coat and DNA are targets of free radicals. Also, our previous studies have shown that T7 inactivation by the photolytic product of mandelate may occur through OH [26]. For T7 inactivation by the photolysis of PPA + NUV we propose that one or both of the following routes for damage exist (i) the primary solvent damage causing release of OH, and/or (ii) subsequent formation of CO2 . Both or one of these radicals may attack DNA or phage tail proteins leading to T7 inactivation [39]. Results from the comet assays confirm the damage to DNA (Fig. 5). We have yet to determine if it is CO2 , direct electron transfer reactions [29] or other free radicals such as Æ OH which produce the damage. The reduction of NBT to formazan blue and the importance of SOD in decreasing the reduction reaction are intriguing. In our previous studies we observed similar phenomena with mandelate and proposed that this reduction reaction may occur by direct electron transfer from an excited state of the sensitizer to the substrate without involving O 2 . Furthermore, the decrease in reduction reaction in the presence of SOD may be explained by the scavenger trapping electrons before they can interact with NBT. If this is the case a new role for SOD exists and this needs further investigation. In our studies we have identified and reported a number of biological compounds, tryptophan, histidine, tyrosine, mandelate and phenylalanine, whose UVA photolysis can damage biological systems. These findings are of particular importance in studies of skin ageing and skin cancer as a result of solar UV exposure, rich in UVA [39,40].

O

O

O

O

O

O

O HO

OH O

+

.C O

.OH

Fig. 6. Hydroxyl radical induced decarboxylation of phenylpyruvate.

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