FASEB J. Cell Migration Consortium
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.06-7513com.
 (The FASEB Journal. 2007;21:2101-2107.)
© 2007 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Erratum (v22,p1287a)
Right arrow All Versions of this Article:
fj.06-7513comv1
21/9/2101    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Offer, T.
Right arrow Articles by Ayling, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Offer, T.
Right arrow Articles by Ayling, J. E.

5-Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA

Tal Offer*,{dagger}, Bruce N. Ames*, Steven W. Bailey{dagger}, Elizabeth A. Sabens{dagger}, Mamoru Nozawa{dagger} and June E. Ayling{dagger},1

* Nutrition and Metabolism Center, CHORI, Oakland, California; and

{dagger} Department of Pharmacology, University of South Alabama, Mobile, Alabama, USA

1Correspondence: Department of Pharmacology Rm. 3370, MSB, 307 North University Blvd., University of South Alabama, Mobile, AL 36688. E-mail: jayling{at}jaguar1.usouthal.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The known functions of folate are to support one-carbon metabolism and to serve as photoreceptors for cryptochromes and photolyases. We demonstrate that 5-methyltetrahydrofolate (5-MTHF, the predominant folate in plasma) is also a potent, near diffusion limited, scavenger of singlet oxygen and quencher of excited photosensitizers. Both pathways result in decomposition of 5-MTHF, although ascorbate can protect against this loss. In the absence of photosensitizers, 5-MTHF is directly decomposed only very slowly by UVA or UVB. Although synthetic folic acid can promote DNA damage by UVA, submicromolar 5-MTHF inhibits photosensitization-induced strand breaks. These observations suggest a new role for reduced folate in protection from ultraviolet damage and have bearing on the hypothesis that folate photodegradation influenced the evolution of human skin color.—Offer, T., Ames, B. N., Bailey, S. W., Sabens, E. A., Nozawa, M., Ayling, J. E. 5-Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA.


Key Words: folate • folic acid • ultraviolet radiation • singlet oxygen • supercoiled DNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ADEQUATE TISSUE LEVELS OF TETRAHYDROFOLATES are vital for one-carbon metabolism. Tetrahydrofolate derivatives serve as cofactors in the de novo synthesis of purine nucleotides, for thymidylate synthase, and in the remethylation of homocysteine to methionine. Folate deficiencies have been associated with megaloblastic anemia, birth defects, low spermatogenesis, cognitive decline, and increased risk of cardiovascular disease and cancer (1 2 3 4 5) . The factors currently considered to determine folate status include dietary intake, bioavailability, excretion rate, and susceptibility to catabolic processes (6 , 7 , 8) .

The photoreceptive function of folate is linked to a variety of growth and adaptive processes in organisms ranging from bacteria to humans (9) . In many organisms 5,10-methylene-tetrahydrofolate serves as a light-harvesting cofactor in DNA photolyases (10) . The analogous folate-containing photoreceptors, cryptochromes, in humans use the energy from near-UV/blue light and are thought to play a role in the regulation of circadian rhythms (9) .

Photochemical degradation of folate by solar radiation has been suggested to play a role in the evolution of human skin color (5 , 11) . The worldwide pattern of light skin color has been proposed to be a product of natural selection acting to maximally capture solar ultraviolet radiation, which is necessary for catalyzing the production of vitamin D. It was hypothesized that dark skin evolved to protect the folate pool in the blood and dermal tissues from photodegradation (11) . The latter hypotheses was based, in part, on the photolability of folic acid, a synthetic oxidized form of the vitamin (12 13 14 15) . In plant and animal tissues, however, folate is predominantly present in a reduced form, mostly as one-carbon derivatives of tetrahydrofolate.

A major part of the ultraviolet radiation reaching the surface of the earth is UVA (320–400 nm). UVA, though less energetic, is more abundant than UVB (290–320 nm) and penetrates to the actively dividing basal layer of the skin (16) . Much of the oxidative effects of UVA radiation are mediated by excitation of cellular photosensitizers, such as porphyrins, via photochemical generation of singlet oxygen (17) .

Singlet oxygen is sufficiently long lived to allow its reaction with a variety of biomolecules. Oxidative damage in DNA and strand breaks have been implicated in UVA–induced photosensitization reactions via either type I (electron transfer) or type II (singlet oxygen) mechanisms (12 , 18) , both reported to involve mainly guanines as the site of damage in double-stranded DNA (18 , 19) . UVA-irradiated folic acid and its photodegradation product, pterin-6 carboxylic acid (PCA), have recently been reported to catalyze DNA cleavage, which was suggested to proceed via a type I mechanism (12) .

The photochemical properties of 5-methyltetrahydrofolate (5-MTHF), the predominant form of folate in the circulation, have not been well characterized. In this study, we monitored 5-MTHF exposed to UVA, UVB or visible light, in the presence and absence of photosensitizers, and tested its effect on the photocleavage of plasmid DNA. Quenching of the excited state of photosensitizers and scavenging singlet oxygen were examined, as well as the ability of ascorbate to regenerate 5-MTHF, which becomes oxidized in these reactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Sodium azide (>99%) was purchased from Fluka. Folic acid (98% + 8% H2O), sodium ascorbate, diethylenetriaminepentaacetic acid (D-6518), and superoxide dismutase (SOD) were purchased from Sigma. High-purity NaOH (B10252) and high-purity phosphoric acid (PX0996) were from EMD Chemicals. 5-Methyl-6S-tetrahydrofolic (5-MTHF) acid calcium salt (99.9% chiral purity, 99.4% chemical purity) was provided by Eprova (Schaffhausen, Switzerland). Supercoiled plasmid DNA, PBR 322, (4361 base pairs, molecular weight 2.83x106 Daltons, found to contain 5% each of the linear and relaxed forms) was obtained from Fermentas. Pterin-6-carboxylic acid (PCA) and 6-formyl-pterin (6-FP) were purchased from Schirck’s Laboratories (Jona, Switzerland), Rose Bengal (95% sodium salt) (RB) from Aldrich (St. Louis, MO), and BlueJuiceTM gel loading buffer and SYBR Safe DNA gel stain from Invitrogen (Carlsbad, CA, USA). Electrophoresis was performed on either a Bio-Rad (Hercules, CA, USA) mini GT or Fisher Biotech midi horizontal systems.

General UVA and UVB irradiation conditions
Samples were exposed to UV light at a distance of 30 cm from either a 15 W UVA lamp, Sylvania 350 BL, (lambda max=365 nm, 820 µW/cm2) or 15 W UVB lamp, UVP (lambda max=302 nm, 770 µW/cm2); both purchased from UVP, Inc. (Upland, CA, USA). The lamps were mounted in a UVP Model XX-15 lamp holder rested on a XX exposure stand. To give a solution depth of ~3 mm, 0.5-ml reaction volumes were used in clear 24-well plates. To prevent evaporation during the UVA exposure, DNA samples were covered with MicroAmpTM Optical Adhesive Film (ABI), allowing greater than 80% transmission above 330 nm. All reactions were performed in 10 mM potassium phosphate pH 7.4 and at ambient temperature equilibrated to air, unless otherwise noted. Diethylenetriaminepentaacetic acid (100 µM) was included in reactions with DNA to prevent the effect of adventitious redox-active metal ions, pre-empt the Fenton reaction and avoid hydroxyl radical-induced damage.

Direct photodegradation of 5-MTHF
Experiments examining the direct photodegradation of 5-MTHF in the absence of photosensitizers were carried out in 10 mM sodium phosphate, pH 7.4 (10 mM high-purity sodium hydroxide titrated to pH 7.4 with high purity phosphoric acid). Samples were placed 12.7 cm from the lamps, providing according to the manufacturer’s specifications 2,300 µW/cm2 of UVA, or 2,170 µW/cm2 of UVB. In addition to 24-well plastic plates, these reactions were also performed in similarly shaped Pyrex dishes, which were sonicated successively in 1 M NaOH, 1 M HCl and finally, 100 mM Na2EDTA before use. The direct decomposition reactions using UVA were covered with quartz windows. UVB reactions were covered either with a quartz window, or with a WG305 filter (3-mm thickness) from Schott to remove the UVC content and thus more closely simulate the UVB in sunlight. The temperature of these reactions with a sample-lamp distance of 12.7 cm rose from initially ambient, typically 25°C as measured by a thermistor probe in an adjacent well, to ~31°C over the course of 80 min. Reactions were analyzed by HPLC assay 1 (see below).

Detection of strand breaks in DNA
Supercoiled plasmid DNA is converted into a nicked circular-form (relaxed) due to single-strand breaks, and subsequently into a linear form as a result of double-strand breaks. The three forms can be separated by agarose gel electrophoresis. Supercoiled DNA migrates further than the linear form, which, in turn, migrates further than the relaxed form.

A mixture of 0.1 µg of plasmid PBR 322 DNA per 0.5 ml and either folic acid, 5-MTHF or PCA in 10 mM potassium phosphate, pH 7.4, was incubated for 80 min under the above UVA-irradiation conditions for a total exposure of 4 J/cm2. In some experiments both folic acid and 5-MTHF were present together each at an initial concentration of 50 µM. In other reactions containing 0.25 µg of plasmid DNA per 0.5 ml and 50 µM PCA, 5-MTHF was also present at an initial concentration of either 0.50 µM or 1.25 µM, and this was approximately maintained by continuous addition of a 0.25 mM solution using a Harvard syringe pump and magnetic stirring. Gradually increasing the rate of addition to the reaction with the lower initial concentration during 80 min from 1.1 µl/min to 2.1 µl/min kept the 5-MTHF between 0.48 and 0.62 µM, as determined by HPLC (assay 1, below) of samples taken every 20 min. The higher concentration reaction was maintained between 1.25 and 4.3 µM (average 2.0 µM) by gradually increasing the flow rate from 1.5 µl/min to 3.0 µl/min.

Duplicate 20-µl samples of the reaction mixtures for each time were then subjected to agarose gel electrophoresis (Fisher midi) after addition of 2 µl of 10x gel loading buffer. Agarose gel for electrophoresis was prepared by dissolving 0.9% agarose in 89 mM Tris borate buffer (pH 8.3), containing 2 mM EDTA (1xTBE). Electrophoresis was run at 4.7 V/cm for 4 h. In experiments using the Bio-Rad mini electrophoresis apparatus, volumes and run times were decreased accordingly. Gels were incubated with 1 µg/ml SYBR Safe stain for 30 min with shaking in the dark and the DNA bands were scanned using Fuji FLA-5000 phosphor-imaging system. The relative densities of the bands were analyzed using Fuji ImageGauge V4.0 software.

Reactions of 5-MTHF with pterin-6-carboxylic acid or folic acid as photosensitizers
Pterin-6-carboxylic acid was used as a model photosensitizer for the UVA region. Samples of 25-µM 5-MTHF in 0.5 ml of 10 mM potassium phosphate pH 7.4 were exposed in 24-well plates to UVA light (820 µW/cm2) containing either 50 µM folic acid or PCA (the latter in the absence or presence of varied sodium ascorbate). Reactions were carried out at ambient temperature, in atmospheric oxygen under the above general illumination conditions. Samples were taken by syringe and injected directly into the HPLC (with PCA, assay 1; with folic acid, assay 2, below).

Photochemical reactions with Rose Bengal
Reaction mixtures containing 5-MTHF (25 µM initial concentration in 10 mM potassium phosphate pH 7.4), 900 U SOD/ml, and 5 µM or 10 µM Rose Bengal (a well-characterized generator of singlet oxygen) were assembled in septum-stoppered glass cuvettes and equilibrated with air or sparged with 100% or 1.8% O2 in argon, or extensively with 100% argon. Reactions were run at ambient temperature and illuminated at a distance of 12 cm by light from a 40 W tungsten lamp passed through a Wratten #16 gelatin filter (50% transmittance at ~540 nm). Samples were taken through the septum by syringe and injected directly into the HPLC (assay 1, below).

HPLC assays
All chromatography was performed at 25°C, with detection by UV absorbance using a Waters 996 photodiode array spectrophotometer.

Assay 1: Samples taken from reactions containing Rose Bengal, PCA, or no photosensitizer were analyzed for 5-MTHF by HPLC on Luna phenyl-hexyl 3 µm (15x0.46 cm) (Phenomenex) eluted at 1.5 ml/min with ammonium phosphate (20 mM in ammonium) pH 5.9/acetonitrile (33:1). Assay 2: Photolysis reactions containing both folic acid and 5-MTHF were monitored by HPLC on Kromasil C-18 5 µm (25x0.40 cm) eluted at 1.0 ml/min with ammonium formate (20 mM in ammonium) pH 3.3/acetonitrile (10:1). Assay 3: The photolysis of folic acid alone was followed by HPLC on Luna phenyl-hexyl 3 µm (15x0.46 cm) eluted at 1.5 ml/min with ammonium phosphate (20 mM in ammonium), pH 2.8/acetonitrile (23:2).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Direct photodegradation of 5-MTHF and folic acid
To establish a baseline for comparison with the natural folate, the photochemical properties of synthetic folic acid were reexamined. Monitoring the photolysis reactions at pH 7.4 by HPLC, we confirmed that folic acid exposed to UVA initially yields p-aminobenzoylglutamate and 6-formyl-pterin (6-FP), which in turn is oxidized to pterin-6-carboxylic acid (PCA). In the chromatographic system used, 6-FP elutes as an unusually broad peak probably due to on-column equilibration of the aldehyde with its hydrate. Exposure to 820 µW/cm2 UVA consumed greater than 97% of the folic acid within 40 min (Fig. 1 A). Similar results were found with exposure to UVB.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		Figure 1. A) The photodegradation of folic acid by UVA (820 µW/cm2) at neutral pH monitored by HPLC shows the initial cleavage products p-aminobenzoylglutamate and 6-formyl-pterin (6-FP), which is then further oxidized to pterin-6-carboxylic acid (PCA). B) The photodegradation of 5-MTHF under UVA (2,300 µW/cm2) or UVB (2,170 µW/cm2) or in the dark. When exposed to UVB, the sample was covered either with a quartz window or a WG305 filter to remove the UVC output of the lamp.

The decay of 5-methyltetrahydrofolate, followed while being exposed to 2.8 times the intensity of UVA as with folic acid, revealed an entirely different outcome. In the absence of intentionally added photosensitizers, i.e., buffer only, 97% of the initial 5-MTHF was still present after 80 min when carried out in a glass dish (Fig. 1B ). Fitting data sampled every 20 min to a first-order decay gave a rate constant of 0.0004 min–1. Identical reactions performed in plastic 24-well plates showed about twice this decay rate. A control reaction performed in the dark at 37°C showed a loss of only ~1% of the initial 5-MTHF over 80 min due to autooxidation.

The rate of loss of 5-MTHF with UVB was faster than with UVA, but was dependent on the reaction cover window. Rates of 0.002 min–1 and 0.0009 min–1 were observed for quartz and WG305 covers, respectively (Fig. 1B ). No significant difference was seen with UVB between plastic and glass containers with either cover. In both UVA and UVB experiments, considerably higher rates were found when using buffers made from less pure reagents.

When added together with folic acid, 5-MTHF inhibited the photolysis of folic acid in a sacrificial way. Folic acid was maintained until 5-MTHF decayed below 1 µM concentration (Fig. 2 ). Thereafter, 6-FP, p-aminobenzoylglutamate, and PCA were produced as in the absence of 5-MTHF.


Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		Figure 2. Photodegradation of folic acid is inhibited by 5-MTHF. Folic acid, at 50 µM, was exposed in the presence of 25 µM 5-MTHF at pH 7.4 to UVA, (lambda max=365 nm, 820 µW/cm2) for 100 min. Samples analyzed by HPLC showed protection of folic acid (circles) by 5-MTHF (squares) until the latter was almost totally consumed.

Effect of photosensitizers on 5-MTHF
To study the activity of 5-MTHF in a well-characterized photosensitization reaction, Rose Bengal was used as a singlet oxygen generator. Since superoxide radicals are also generated by illuminated Rose Bengal (20) , superoxide dismutase was included in the reaction mixtures. 5-MTHF was found to be depleted in the presence of visible-light sensitized Rose Bengal (Fig. 3 A) giving rise to the same products that are generated by autooxidation. To differentiate the reaction of 5-MTHF with singlet oxygen from its reaction with the excited state of Rose Bengal, experiments were carried out at various oxygen levels. The rate of depletion of 5-MTHF slowed as the concentration of O2 increased (Fig. 3A ). However, vigorous sparging of the reaction with pure argon resulted in a very slow rate of consumption, consistent with the need for some oxygen to be present. PCA, an efficient generator of singlet oxygen (21) , also promoted 5-MTHF photodecomposition by UVA in a concentration-dependent manner. As with Rose Bengal, a similar inverse dependence of depletion rate on oxygen concentration was seen in reactions with PCA as photosensitizer.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Figure 3. Photodegradation of 5-MTHF by Rose Bengal (RB) depends on oxygen concentration and singlet oxygen and is slowed by ascorbate. 5-MTHF, at 25 µM, pH 7.4 in the presence of 900 U/ml. SOD was illuminated by light from a tungsten lamp passed through a Wratten #16 filter. A) The effect of oxygen concentration in the presence of 5 µM Rose Bengal under: 100% O2 (circles), air (squares), or 1.8% O2 in argon (triangles). B) The effect of the singlet oxygen scavenger azide on loss of 5-MTHF in the presence of 10 µM RB, and 100% O2 with no azide (triangles), 0.5 mM azide (squares), or 5 mM azide (circles). C) The maintenance of 5-MTHF in the presence of 10 µM RB and 100% O2 with initial ascorbate concentrations: none (diamonds), 0.2 mM (triangles), 1 mM (squares), or 2 mM (circles).

Competition for singlet oxygen with azide
To further confirm that the depletion is mediated at least in part by singlet oxygen, the rate of loss of 5-MTHF was again determined with Rose Bengal under 100% O2, but in the presence of increasing concentrations of azide, a potent 1O2 scavenger. The initial rate was decreased by ~80% with 5 mM azide, indicating the involvement of singlet oxygen (Fig. 3B ). A concentration of 0.5 mM azide decreased the initial depletion rate of 0.025 mM 5-MTHF by ~50% (Fig. 3B ).

Maintenance of 5-MTHF in photosensitization reactions by ascorbate
Photosensitized oxidation of 5-MTHF physiologically would occur in a milieu of reducing agents, such as ascorbate (vitamin C), which might promote its regeneration. The initial rate of loss of 5-MTHF in Rose-Bengal photosensitization reactions with 100% O2 was found to be decreased in the presence of millimolar levels of sodium ascorbate (Fig. 3C ). These results underestimate the ability of ascorbate to maintain 5-MTHF since a significant fraction is rapidly converted to dehydroascorbate even by the time of the first HPLC analysis. In the presence of 10 µM PCA the 5-MTHF was decomposed by 50% within 20 min under UVA exposure. Under the same conditions, 200 µM ascorbate maintained the majority of the 5-MTHF for at least 60 min, and 1 mM ascorbate for greater than 160 min.

Inhibition of strand-breaks in DNA by 5-MTHF
Although DNA is not a chromophore for UVA radiation, it can be damaged by oxidative reactions initiated by photosensitizers. Relaxation and subsequent linearization of supercoiled plasmid-DNA by UVA were used to compare the effects of folic acid with 5-MTHF. UVA exposure of supercoiled DNA for 80 min in the presence of 50 µM PCA or 50 µM folic acid yielded a high percentage of strand-breaks (Fig. 4 A and 4 B, respectively). At the total radiant energy used, UVA exposure by itself did not cause nicks (Fig. 4A , 4B , controls). These results are analogous to those previously reported for DNA damage and formation of 8-oxo-7,8-dihydro-2-deoxyguanosine by folic acid under UVA (12) . Neither folic acid nor PCA, at 50 µM, had any effect when incubated with supercoiled DNA for 80 min in the dark.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Figure 4. 5-MTHF inhibits UVA-mediated DNA damage catalyzed by PCA or folic acid. A) Supercoiled plasmid DNA, PBR 322, was exposed for 80 min to UVA with 50 µM PCA alone, or together with continuous addition of 5-MTHF to maintain a concentration near 0.5 µM. B) Supercoiled plasmid DNA with 50 µM folic acid alone, 50 µM folic acid added together with 50 µM initial 5-MTHF, or 50 µM 5-MTHF alone was exposed for 80 min to UVA. The positions of the DNA following agarose gel electrophoresis are indicated as the supercoiled form (S), a nicked circular form (relaxed) (R), and a linear form (L). In both (A) and (B) the control contained the DNA exposed to UVA by itself.

In view of the rapid quenching of the excited states of Rose Bengal or PCA and scavenging of 1O2 by 5-MTHF, we examined the ability of this natural folate to intervene in UVA-induced cleavage of DNA. To maintain a relatively constant level, 0.25 mM 5-MTHF was slowly pumped into reaction mixtures containing 50 µM PCA and its concentration was analyzed by HPLC at various times during exposure. The rate of UVA-mediated cleavage of supercoiled DNA by PCA was inhibited 10-fold by 5-MTHF at a concentration kept near 0.5 µM (Fig. 4A ). No damage was observed in reactions with 5-MTHF maintained at an average concentration of 2 µM. Sodium azide at 10 mM also afforded full protection under the same conditions, confirming that the damage is largely mediated by singlet oxygen. DNA damage induced by 50 µM folic acid was abrogated by inclusion of 50 µM 5-MTHF (added only initially) (Fig. 4B ), which decayed in a manner similar to that shown in Fig. 2 .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the absence of photosensitizers, photodegradation of the natural folate 5-MTHF directly by UVA was only marginally faster than that due to autooxidation in the dark. This is clearly related to the lack of significant absorbance of 5-MTHF above 330 nm. Conceivably, the small increase of decompostion with UVA above the dark control might have been due to residual photosensitizers even in the high purity buffer or 5-MTHF used. Interestingly, the stability of 5-MTHF was only slightly more affected by UVB irradiation (which somewhat overlaps its absorption maximum at 290 nm) than with UVA. With a quartz cover, which passes a small UVC component of the nominally UVB lamp, the rate of decay was only 0.002 min–1. Recently, a rate of decay of 0.0092 min–1 with 6(R,S)-5-MTHF has been reported with a similar lamp and intensity (22) . Our observations suggest that this 4.5-fold higher rate may have been due to photosensitizers or metal impurities in the buffer used in those experiments. When a filter is used to more closely simulate range of wavelengths present in UVB from sunlight, the rate of decay decreases to 0.0009 min–1. Thus, the excited state of 5-MTHF presumably undergoes nonradiative decay and/or releases energy by fluorescence more quickly than its interaction with the triplet molecular oxygen. The dose rate of UV reaching the earth depends on the geographic location, altitude, season, time of day, and ozone column. However, even the maximum level of UVB (23 , 24) is only about a quarter of that used (without the WG305 filter) to irradiate the 5-MTHF in the absence of photosensitizers. The intensity of UVA was ~3-fold less than the maximum level in solar radiation. Thus, the observed rates of photodegradation are so low that a meaningful physiological impact of UVA or UVB in sunlight on 5-MTHF in the absence of photosensitizers seems unlikely, especially taking into account the integrated exposure to solar UV over an entire day or season rather than the maximum level during the day.

The pteridine photoproduct of folic acid was originally proposed to be 6-formyl-pterin (6-FP) (13) . We confirmed more recent studies that also identified the additional p-aminobenzoylglutamate product and showed that 6-FP is further degraded to yield pterin-6-carboxylic acid (PCA) (12 , 14 , 15) . However, the rate of photodegradation of folic acid by UVA was many orders of magnitude faster than 5-MTHF. Thus, the fate of the excited state of the fully aromatic synthetic folate is very different from the natural reduced form.

The rate of 5-MTHF consumption with Rose Bengal as photosensitizer decreases with increasing oxygen concentration. This can be understood as competition for the activated state of Rose Bengal between quenching by 5-MTHF and formation of singlet oxygen, where the latter also reacts with 5-MTHF (Fig. 5 ). As the oxygen concentration increases, a greater percentage of the activated Rose Bengal is used to produce 1O2, which then consumes 5-MTHF, but at a slower rate. The same mechanism appears to be the case with UVA when PCA is photosensitizer.


Figure 5
View larger version (8K):
[in this window]
[in a new window]

  		Figure 5. Proposed mechanism of 5-MTHF photoantioxidative activity. 5-MTHF is depleted at a faster rate in low oxygen when singlet oxygen concentration is low due to a very rapid direct reaction with photoactivated RB. At high O2 levels the formation of singlet oxygen is increased at the expense of excited RB, and loss of 5-MTHF proceeds through the slower pathway.

The competition reaction between azide and 5-MTHF at high concentrations of O2 indicates that 5-MTHF is ~20-fold more effective in scavenging singlet oxygen. Since the kinetic constant for the reaction of azide with singlet oxygen in water is already quite high (4.5x108 M–1s–1) (25) , the reaction of 5-MTHF with singlet oxygen is nearly diffusion limited. Not only is this much faster than with folic acid (3.0x107 M–1s–1) (14 , 26) , but it also occurs without side-chain cleavage. Since the interaction of 5-MTHF with singlet oxygen was monitored by loss of 5-MTHF, physical quenching not leading to folate oxidation may also have occurred in concert with the scavenging reaction. The lack of full inhibition of 5-MTHF depletion by competition with 5 mM azide may be due to a residual reaction with photoactivated Rose Bengal, since saturation even with 100% oxygen (i.e., {approx}1.4 mM) may have not been sufficient to make the singlet oxygen pathway completely dominant (Fig. 5) .

Ascorbate may play a synergistic role by maintaining 5-MTHF in photosensitization reactions. Since the second-order rate constant for the reaction of singlet oxygen with ascorbate in water is 8.3 x 106 M–1s–1 (27) , the pseudo first-order rate with 2 mM ascorbate is 1.7 x 104 s–1. This is more than 10 times slower than the spontaneous decay rate of singlet oxygen (28) . Moreover, ascorbate has previously been reported to have no effect on the generation of protein-derived peroxides in viable Rose Bengal-loaded THP-1 cells (29) . Thus, ascorbate at this concentration does not significantly protect 5-MTHF by directly removing singlet oxygen or by directly quenching Rose Bengal. Ascorbate, at levels found in many cell types, may restore 5-MTHF by reducing the initial intermediate in its photodecomposition reactions (most likely its radical cation).

Generation of strand breaks in DNA has previously been reported to be associated with formation of singlet oxygen in photosensitization reactions (17 18 19 , 30 31 32) . Singlet oxygen is produced by PCA and 6-FP and has been suggested to participate in the photodecay of folic acid (21) . In the present study, we confirmed PCA serves as a photosensitizer and catalyzes formation of DNA strand breaks during exposure to UVA as reported by Hirakawa et al. (12) . In contrast to Hirakawa’s proposed mechanism of electron transfer (type I), the finding that azide inhibited DNA damage in photosensitization reactions mediated by PCA implies that singlet oxygen may also be involved in the damaging effect of folic acid and its photoproducts.

There are many endogenous photosensitizers that may promote photodamage (17 , 33 , 34) . Although unmetabolized folic acid has been detected in the plasma of individuals consuming greater than 200 µg of folic acid (35) , cellular levels of unmetabolized folic acid in skin are currently unknown, and its contribution to photosensitization is uncertain. However, the risk of squamous cell carcinoma in people with a past history of skin cancer has recently been reported to be decreased by over 2-fold in those with the highest intake of green leafy vegetables, but not of other vegetables. It was proposed that folate in the green vegetables helps to maintain genetic integrity (36) . Our results suggest that in addition to its possible role in facilitating DNA repair, adequate folate status might hinder incipient damage caused by photosensitization reactions.

Branda and Eaton reported a significant decrease of folate in plasma from psoriasis patients treated with methoxalen phototherapy. They also found loss of folate in plasma exposed ex vivo to UVA. They suggested that "Prevention of ultraviolet photolysis of folate and other light sensitive nutrients by dark skin may be sufficient explanation for maintenance of this characteristic in human groups indigenous to regions of intense solar radiation." (11) . Our results demonstrate that the photolysis of folate observed in their study was not due to the intrinsic photolability of 5-MTHF but instead may have been mediated by the methoxalen or other photosensitizers in plasma. An association between exposure to light and clinical folate deficiency has not yet been clearly established. Nonetheless, our results suggest that, depending on the presence of photosensitizers and possibly ascorbate status in the skin and/or plasma, sunlight or UVA may affect the folate pool. The hypothesis that skin color evolved to balance the need for adequate vitamin D production while minimizing not only burns as a major factor, but also destruction of folate should be expanded to consider the (5 , 11) effects of endogenous photosensitizers on 5-MTHF.

Preliminary studies on the nature of the decay products of 5-MTHF indicate that they still contain the p-aminobenzoylglutamate sidechain. Earlier studies of folate catabolites in human urine only looked for the presence of p-aminobenzoylglutamate and its N-acetylated form (6 , 7) . Therefore, the loss of 5-MTHF due to oxidative or photosensitized decay would have been undetected. Thus, the total loss of folate per day in humans may be underestimated. A good understanding of this loss is needed to optimally establish the appropriate daily intake for folate.

In conclusion, the present results show that, unlike folic acid, 5-MTHF is not significantly photolyzed by UV in the absence of photosensitizers and does not induce cleavage of plasmid DNA. By quenching the excited state of the photosensitizers and scavenging singlet oxygen 5-MTHF may, at concentrations lower than those of the total cytoplasmic or nuclear pools in many tissues, afford protection to DNA and other biomolecules. Moreover, ascorbate may have a synergistic effect by maintaining the folate pool against photolytic degradation.


   ACKNOWLEDGMENTS
 
This work was funded by the National Foundation for Cancer Research grant M2661, a Research Scientist Award K05 AT001323 (B.N.A.), NSF-EPSCoR 0091853–353, and NIH Grant HL068165 (J.E.A.).

Received for publication December 14, 2006. Accepted for publication February 1, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fenech, M. (2001) The role of folic acid and Vitamin B12 in genomic stability of human cells. Mutat. Res. 475,57-67[Medline]
  2. Morrison, H. I., Schaubel, D., Desmeules, M., Wigle, D. T. (1996) Serum folate and risk of fatal coronary heart disease. JAMA 275,1893-1896[Abstract]
  3. Ames, B. N., Wakimoto, P. (2002) Are vitamin and mineral deficiencies a major cancer risk?. Nat. Rev. Cancer. 2,694-704[CrossRef][Medline]
  4. Tucker, K. L., Qiao, N., Scott, T., Rosenberg, I., Spiro, A., 3rd (2005) High homocysteine and low B vitamins predict cognitive decline in aging men: the Veterans Affairs Normative Aging Study. Am. J. Clin. Nutr. 82,627-635[Abstract/Free Full Text]
  5. Jablonski, N. G., Chaplin, G. (2000) The evolution of human skin coloration. J. Hum. Evol. 39,57-107[CrossRef][Medline]
  6. Higgins, J. R., Quinlivan, E. P., McPartlin, J., Scott, J. M., Weir, D. G., Darling, M. R. (2000) The relationship between increased folate catabolism and the increased requirement for folate in pregnancy. BJOG. 107,1149-1154[CrossRef][Medline]
  7. Gregory, J. F., 3rd, Caudill, M. A., Opalko, F. J., Bailey, L. B. (2001) Kinetics of folate turnover in pregnant women (second trimester) and nonpregnant controls during folic acid supplementation: stable-isotopic labeling of plasma folate, urinary folate and folate catabolites shows subtle effects of pregnancy on turnover of folate pools. J. Nutr. 131,1928-1937[Abstract/Free Full Text]
  8. Stover, P. J. (2004) Physiology of folate and vitamin B12 in health and disease. Nutr. Rev. 62,S3-S12discussion S13[CrossRef][Medline]
  9. Partch, C. L., Sancar, A. (2005) Cryptochromes and circadian photoreception in animals. Methods Enzymol. 393,726-745[CrossRef][Medline]
  10. Lipman, R. S., Bailey, S. W., Jarrett, J. T., Matthews, R. G., Jorns, M. S. (1995) Stereospecificity of folate binding to DNA photolyase from Escherichia coli. Biochemistry 34,11217-11220[CrossRef][Medline]
  11. Branda, R. F., Eaton, J. W. (1978) Skin color and nutrient photolysis: an evolutionary hypothesis. Science 201,625-626[Abstract/Free Full Text]
  12. Hirakawa, K., Suzuki, H., Oikawa, S., Kawanishi, S. (2003) Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product. Arch. Biochem. Biophys. 410,261-268[CrossRef][Medline]
  13. Lowry, O. H., Bessey, O. A., Crawford, E. J. (1949) Photolytic and enzymatic transformations of pteroyglutamic acid. J. Biol. Chem. 180,389-398[Free Full Text]
  14. Thomas, A. H., Suarez, G., Cabrerizo, F. M., Martino, R., Capparelli, A. L. (2000) Study of the photolysis of folic acid and 6-formylpterin in acid aqueous solutions. J. Photochem. Photobiol. A. 135,147-154[CrossRef]
  15. Off, M. K., Steindal, A. E., Porojnicu, A. C., Juzeniene, A., Vorobey, A., Johnsson, A., Moan, J. (2005) Ultraviolet photodegradation of folic acid. J. Photochem. Photobiol. B. 80,47-55[CrossRef][Medline]
  16. Bruls, W. A., Slaper, H., van der Leun, J. C., Berrens, L. (1984) Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem. Photobiol. 40,485-494[Medline]
  17. Fiel, R. J., Datta-Gupta, N., Mark, E. H., Howard, J. C. (1981) Induction of DNA damage by porphyrin photosensitizers. Cancer Res. 41,3543-3545[Medline]
  18. Ravanat, J. L., Douki, T., Cadet, J. (2001) Direct and indirect effects of UV radiation on DNA and its components. J. Photochem. Photobiol. B. 63,88-102[CrossRef][Medline]
  19. Ito, K., Kawanishi, S. (1997) Site-specific DNA damage induced by UVA radiation in the presence of endogenous photosensitizer. Biol. Chem. 378,1307-1312[Medline]
  20. Lee, P. C., Rodgers, M. A. (1987) Laser flash photokinetic studies of Rose Bengal-sensitized photodynamic interactions of nucleotides and DNA. Photochem. Photobiol. 45,79-86[Medline]
  21. Thomas, A. H., Lorente, C., Capparelli, A. L., Martinez, C. G., Braun, A. M., Oliveros, E. (2003) Singlet oxygen (1{Delta}g): production by pterin derivatives in aqueous solutions. Photochem. Photobiol. Sci. 2,245-250[CrossRef][Medline]
  22. Steindal, A. E., Juzeniene, A., Johnsson, A., Moan, J. (2006) Photodegradation of 5-methyltetrahydrofolate. Photochem. Photobiol. 82,1319-1325[CrossRef][Medline]
  23. Huovinen, P., Gomez, I., Lovengreen, C. (2006) A five-year study of solar ultraviolet radiation in southern Chile (39° S): potential impact on physiology of coastal marine algae?. Photochem. Photobiol. 82,515-522[CrossRef][Medline]
  24. Kift, R., Webb, A. R., Page, J., Rimmer, J., Janjai, S. (2006) A Web-based tool for UV irradiance data: predictions for European and Southeast Asian sites. Photochem. Photobiol. 82,579-586(www.soda-is.com/eng/services/health_eng.html#)[CrossRef][Medline]
  25. Miskoski, S., Garcia, N. A. (1993) Influence of the peptide bond on the singlet molecular oxygen-mediated (O2(1{Delta}g)) photooxidation of histidine and methionine dipeptides. A kinetic study. Photochem. Photobiol. 57,447-452[Medline]
  26. Cabrerizo, F. M., Dantola, M. L., Petroselli, G., Capparelli, A. L., Thomas, A. H., Braun, A. M., Lorente, C., Oliveros, E. (2007) Reactivity of conjugated and unconjugated pterins with singlet oxygen (O2(1{Delta}g)): physical quenching and chemical reaction. Photochem. Photobiol. (in press)
  27. Chou, P. T., Khan, A. U. (1983) L-ascorbic acid quenching of singlet delta molecular oxygen in aqueous media: generalized antioxidant property of vitamin C. Biochem. Biophys. Res. Commun. 115,932-937[CrossRef][Medline]
  28. Studer, S. L., Brewer, W. E., Martinez, M. L., Chou, P. T. (1989) Time-resolved study of the photooxygenation of 3-hydroxyflavone. J. Am. Chem. Soc. 111,7643-7644[CrossRef]
  29. Wright, A., Hawkins, C. L., Davies, M. J. (2003) Photo-oxidation of cells generates long-lived intracellular protein peroxides. Free. Radic. Biol. Med. 34,637-647[CrossRef][Medline]
  30. Blazek, E. R., Peak, J. G., Peak, M. J. (1989) Singlet oxygen induces frank strand breaks as well as alkali- and piperidine-labile sites in supercoiled plasmid DNA. Photochem. Photobiol. 49,607-613[Medline]
  31. Ravanat, J. L., Di Mascio, P., Martinez, G. R., Medeiros, M. H. (2001) Singlet oxygen induces oxidation of cellular DNA. J. Biol. Chem. 276,40601-40604[Medline]
  32. Devasagayam, T. P., Steenken, S., Obendorf, M. S., Schulz, W. A., Sies, H. (1991) Formation of 8-hydroxy(deoxy)guanosine and generation of strand breaks at guanine residues in DNA by singlet oxygen. Biochemistry 30,6283-6289[CrossRef][Medline]
  33. Fiel, R. J., Beerman, T. A., Mark, E. H., Datta-Gupta, N. (1982) DNA strand scission activity of metalloporphyrins. Biochem. Biophys. Res. Commun. 107,1067-1074[CrossRef][Medline]
  34. Mahns, A., Melchheier, I., Suschek, C. V., Sies, H., Klotz, L. O. (2003) Irradiation of cells with ultraviolet-A (320–400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide. Free Radic. Res. 37,391-397[CrossRef][Medline]
  35. Kelly, P., McPartlin, J., Goggins, M., Weir, D. G., Scott, J. M. (1997) Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements. Am. J. Clin. Nutr. 65,1790-1795[Abstract/Free Full Text]
  36. Hughes, M. C., van der Pols, J. C., Marks, G. C., Green, A. C. (2006) Food intake and risk of squamous cell carcinoma of the skin in a community: the Nambour skin cancer cohort study. Int. J. Cancer. 119,1953-1960[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Erratum (v22,p1287a)
Right arrow All Versions of this Article:
fj.06-7513comv1
21/9/2101    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Offer, T.
Right arrow Articles by Ayling, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Offer, T.
Right arrow Articles by Ayling, J. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS