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Copyright © 2006 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 91, Issue 4, 1452-1459, 15 August 2006

doi:10.1529/biophysj.106.082388

Nucleic Acids

Singlet Oxygen Generation by UVA Light Exposure of Endogenous Photosensitizers

Jürgen Baier^*, Tim Maisch^*, Max Maier^†, Eva Engel^‡, Michael Landthaler^* and Wolfgang Bäumler^*, ,  

^* Department of Dermatology, University of Regensburg, Regensburg, Germany
^† Department of Physics, University of Regensburg, Regensburg, Germany
^‡ Department of Organic Chemistry, University of Regensburg, Regensburg, Germany

Address reprint requests to Dr. Wolfgang Bäumler, Dept. of Dermatology, University of Regensburg, 93042 Regensburg, Germany. Tel.: 49-941-944-9607; Fax: 49-941-944-8943.

The UVA component of solar radiation (320–400nm) has been shown to produce deleterious biological effects in which singlet oxygen (¹Δ_g of O₂) plays a major role 1. This must have an effect on all tissue that gets into contact with UVA light, particularly the skin and the eye.

Skin is the largest body organ and is frequently exposed to sunlight, and UVA exposure is thought to cause skin aging and skin cancer mainly by singlet oxygen 2,3. Singlet oxygen mediates gene regulation via the transcription factor AP-2 4. It activates stress-activated protein kinases 5, or it induces in skin fibroblasts a pattern of mitogen-activated protein kinase as well as an induction of p38 and c-Jun-N-terminal kinase 6. Additionally, exposure to UVA light has been recognized as a source of aging of eye lens proteins and as a risk factor for cataract formation 7.

However, the mechanisms by which UVA light-induced photodamage occur have not been fully understood 1. UVA light is weakly absorbed by a limited number of molecules in the tissue, which may act as photosensitizers. After UVA light absorption, the photosensitizer molecules cross over to a triplet state and transfer energy to generate singlet oxygen. Some of these endogenous photosensitizers have been identified, such as flavins 8, NADH/NADPH 9, urocanic acid 1, and some sterols 10.

To provide undoubted evidence for a correlation of UVA damage in tissue and singlet oxygen, the efficacy of singlet oxygen generation (the quantum yield) must be determined for these substances. Usually, the involvement of singlet oxygen is shown indirectly by adding singlet oxygen quenchers (e.g., sodium azide, beta-carotene) 3,11,12. However, in biological systems (e.g., lipids cells) singlet oxygen is short-lived (few μs), showing a very short diffusion length 13. Thus, the quencher molecules must be present directly at the site of singlet oxygen generation with a sufficiently high concentration, which is difficult and a source of ambiguous results.

In contrast to that, singlet oxygen can be directly detected by measuring its luminescence and there is no need for any additional substances. The luminescence signal is extremely weak, but we were able to detect singlet oxygen in lipids and even in living cells 14,15,16. When measuring the luminescence signal, the quantum yield of singlet oxygen generation can be calculated using an exogenous photosensitizer such as perinaphthenone as reference 17,18.

Moreover, after exciting a photosensitizer, there is always a competition between the generation of oxygen radicals (type I, e.g., superoxide anion) and singlet oxygen (type II reaction). That may depend on the respective microenvironment, which can be the solvent 19, aggregation of molecules 20, or the oxygen concentration 14. It has been recently shown for exogenous photosensitizers that the quantum yield of singlet oxygen depends critically on the oxygen concentration (oxygen partial pressure, i.e., pO₂) in the respective experimental setup 14,16. This is important when comparing experiments of in vitro (pO₂∼150mmHg) and in vivo (e.g., skin: pO₂<20mmHg) conditions 21. Therefore, the quantum yield of the endogenous photosensitizers should be determined not only in fully aerated solutions (∼150mmHg) but also at a very low pO₂.

The high sensitivity of our detection systems allows the measurement of the entire time course of the luminescence signal. This yields a more precise evaluation of the generation and decay of singlet oxygen as compared to the germanium diode detectors used several years ago, particularly in the spectral range of UVA and at very low luminescence intensities. The latter is important since we reduce the oxygen concentration and we usually apply small excitation pulse energies (μJ) to avoid nonlinear behavior in the luminescence signal.

To mimic UVA light excitation of endogenous photosensitizers, the third harmonic of an Nd:YAG laser was available (λ_em=355nm). That wavelength is in the middle of the UVA light spectrum ranging from 320 to 400nm.

Riboflavin (purity≥99%), flavin mononucleotide sodium (FMN, purity ∼ 95%), flavin adenine dinucleotide disodium salt hydrate (FAD, purity≥95%), β-nicotinamide adenine dinucleotide sodium salt (NAD, purity ∼ 95%), and β-nicotinamide adenine dinucleotide phosphate hydrate (NADP, purity ∼ 95%) were dissolved in H₂O (bi-distilled) at a concentration of 50μM. Urocanic acid (purity≥99%) and cholesterol (5-cholesten-3β-ol, purity≥99%) were dissolved in EtOH at a concentration of 3mM and 50μM, respectively. All substances were purchased from Sigma-Aldrich (Steinheim, Germany). Sodium azide was purchased from Merck KGaA (Darmstadt, Germany) and the nonpolar Perinaphthenone (PN) from Acros Organics (Geel, Belgium) showing a purity of ≥97%. The polar Perinaphthenone (PNS) was synthesized in the Institute of Organic Chemistry, Regensburg. The synthesis of PNS was performed according to the description given by Nonell et al. 18 and high-performance liquid chromatography revealed purity of >97% (for molecular structure, see Fig. 1).

The absorption spectra of each probe were recorded at room temperature with a Beckman DU640 spectrophotometer (Beckman Instruments, Munich, Germany).

The potential sensitizers in solutions were transferred into a cuvette (QS-1000, Hellma Optik, Jena, Germany). They were excited using a frequency-tripled Nd:YAG laser (PhotonEnergy, Ottensoos, Germany) with a repetition rate of 2.0kHz (wavelength 355nm, pulse duration 70ns). The laser pulse energy for luminescence experiments was 50μJ. The singlet oxygen luminescence at 1270nm was detected in near-backward direction with respect to the excitation beam using an infrared sensitive photomultiplier (R5509-42, Hamamatsu Photonics Deutschland, Herrsching, Germany) with a rise-time of ∼3ns. The details of the setup are described elsewhere 15. The number of laser pulses for excitation was 40,000.

As shown in Baumer et al. 16, the luminescence intensity is given by

(1)

(2)

(3)

Using the Wilkinson definition 22 and the assumption of a negligible energy transfer from sensitizer S₁ state to oxygen, the singlet oxygen quantum yield Φ_Δ is given by

(4)

(5)

The singlet oxygen quantum yield Φ_Δ was determined by measuring the luminescence intensity of singlet oxygen at 1270nm as a function of absorbed laser energy using PNS as a reference. The ratio of the singlet oxygen quantum yield Φ_Δ of two sensitizers is obtained from Eqs. (4). It is given by

(6)

(7)

After light absorption of the photosensitizer, a fraction of the excited molecules in the S₁ state will populate within nanoseconds the triplet T₁-state yielding a concentration . Then, the ratio of the singlet oxygen luminescence energy of two sensitizers is given by

(8)

If both sensitizers are solved in H₂O and the quenching of singlet oxygen by the sensitizer can be neglected, than (see Eq. (2)). The concentration [S₁]₀ of the S₁ state depends linearly on the absorbed laser energy. The absorbed laser energy is calculated from the absorption cross-section of each sensitizer at 355nm, which has been determined from transmission measurements of the solutions. The ratio of the slopes s of the luminescence energy of singlet oxygen versus the absorbed laser energy is as follows (see Eqs. (6)):

(9)

The absorption cross-section spectra of the different photosensitizers are shown in Fig. 1. The present experimental setup allows the time-resolved detection of singlet oxygen luminescence at an excitation wavelength of 355nm, which is the triplication of the frequency of an Nd:YAG laser at 1064nm. PN and PNS (Figure 1A) exhibit high absorption values at 355nm and are used as reference photosensitizers to calculate the singlet oxygen quantum yield of the endogenous photosensitizer. PN is a well-known, nonpolar molecule 17, which can be solved in the EtOH, whereas the polar PNS has been synthesized for the use in aqueous solvents 18. Both molecules have a high singlet oxygen quantum yield close to unity independent of the solvent.

Display large version of this figure

Figure 1

Absorption cross-section spectra of cell components: (A) PN in H₂O and PNS in EtOH. (B) Riboflavin, FMN, and FAD in H₂O. The vertical line is at 355nm.

To compare with our excitation wavelength, the absorption cross-section spectra of riboflavin, FMN, and FAD dissolved in H₂O are shown in Figure 1B. The molecules have high absorption values for wavelengths shorter than 300nm, but also from 350 to 550nm. The light of our excitation laser (355nm) is well absorbed in these molecules.

The absorption cross-section of NAD, NADP (dissolved in H₂O), urocanic acid, and cholesterol (dissolved in EtOH) at 355nm are shown in Table 1. Although the absorption cross sections are very low at 355nm, we included these compounds in our luminescence measurements due to the high sensitivity of our experimental setup. However, no luminescence at 1270nm could be detected for NAD, NADP, and cholesterol, which is very likely due to the very low absorption cross sections at the excitation wavelength, in particular for sterols 10.

Table 1 Spectroscopic data of photosensitizers

	Photosensitizer	σabs (355nm) (10−17cm2)	(kΔ)−1 (μs)	ΦT		ΦΔ
	PN	4.06	14±2*	1.00 (17)	0.98±0.08	17
					0.93±0.08	18
	PNS	2.62	3.4±0.5†	1.00 (17)	0.98±0.08	17
					0.97±0.06	18
	Riboflavin	3.64	3.2±0.5†	0.61 (28)	Using ΦT, PT, :
				0.38 (29)	0.59±0.07‡	This work (pO2∼150mmHg)
					0.09±0.03§
					Using PNS 0.54±0.07
	FMN	3.01	3.7±0.5†	—	Using PNS 0.51±0.07	This work (pO2∼150mmHg)
	FAD	2.66	3.5±0.5†	—	Using PNS 0.07±0.02	This work (pO2∼150mmHg)
	Urocanic acid	0.17	13±3*	—	¶
	NAD	0.04	**	—	—
	NADP	0.02	**	—	—
	Cholesterol	0.05	**	—	—

* Dissolved in EtOH. † Dissolved in H2O. ‡ ΦΔ,max calculated using Eq. (4), and ΦT given Chacon et al. 28. § ΦΔ,min calculated using Eq. (4), and ΦT given by Islam et al. 29. ¶ Signal/noise ratio was too low to determine ΦΔ. ** No signal was detected.

There was singlet oxygen luminescence upon exciting 3mM urocanic acid. The luminescence decay time in air-saturated solution (EtOH) was 13±3μs, which is the typical decay time of singlet oxygen in ethanol 22. By adding 500μM sodium azide to urocanic acid solution, the luminescence signal completely disappeared. Thus, the luminescence photons at 1270nm are a direct proof of singlet oxygen, which has been generated by irradiation of urocanic acid with UVA light at 355nm. Our result confirms the published results when using the photoacoustic detection of singlet oxygen 1. Due to the weak luminescence signal, the quantum yield of singlet oxygen could be not determined for urocanic acid.

When exciting flavin molecules, a clear luminescence signal was detected in air-saturated water. Figure 2B shows exemplarily the luminescence at 1270nm of riboflavin (50μM). By adding the singlet oxygen quencher sodium azide (500μM), the decay time was significantly shortened (Figure 2C). To compare with flavins, the singlet oxygen luminescence of 50μM PNS (Figure 2A) was detected. The solid lines in Fig. 2 are the respective fits. The singlet oxygen luminescence of excited PNS rises with 2.3±0.5μs and decays with 3.4±0.5μs. For riboflavin in air-saturated solution, the signal rises with a time constant of 3.3±0.5μs and decays with a time constant of 3.2±0.5μs. The respective rise times represent the values of singlet oxygen in pure water 14,15. By adding 500μM sodium azide to the 50μM riboflavin solution, both the luminescence intensity and the decay time of singlet oxygen decreased, yielding a decay time of 1.8±0.5μs, which confirms the singlet oxygen luminescence.

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Figure 2

Luminescence of singlet oxygen at 1270nm generated by aqueous solution of (A) 50μM PNS, (B) 50μM riboflavin, and (C) 50μM riboflavin with 50mM NaN₃ versus time. The solid curves have been fitted to the experimental data points using Eq. (1).

Comparable to exogenous photosensitizers, endogenous molecules absorb UVA light in the skin and can generate singlet oxygen. The efficacy of a molecule to generate singlet oxygen is expressed by the quantum yield of singlet oxygen (Φ_Δ). The molecules such as the flavins or urocanic acid are assumed to play a major role regarding the photooxidative damage of the skin 1,3,4,6,7,24,25 and the eye lens 8,26. Thus, the quantum yield must be determined as precisely as possible. When looking at the pathways within the photosensitizer after UVA-light absorption, the quantum yield Φ_Δ depends on the triplet yield Φ_T, the triplet decay rate , the rate constant , and the fraction (see Eqs. (4)). That approach is frequently used to calculate the quantum yield Φ_Δ27. Thus, these rates and rate constants must be determined, which was performed for the flavin molecules riboflavin, FMN, and FAD.

Additionally, we were interested in the quantum yield at different oxygen concentrations. Therefore, the rates were determined in a range of [O₂]=10–280μM corresponding to a range of oxygen partial pressure of ∼5–150Torr (mmHg). That covers the conditions of singlet oxygen generation in vitro (∼150mmHg) and in vivo (10–20mmHg).

Starting with riboflavin, the rates and were determined. Firstly, the singlet oxygen luminescence was measured at different photosensitizer concentrations (0.01mM to 0.1mM) at [O₂]=170±10μM. In Figure 3A, the Stern-Volmer shows a constant singlet oxygen relaxation rate K_Δ within the experimental accuracy. According to Eq. (2), this yields the quenching rate constant of singlet oxygen by riboflavin (k_ΔS=0). Extrapolation to zero riboflavin concentration yields the lifetime of singlet oxygen in pure water as τ_Δ=1/k_Δ=3.2±0.5μs, which is in excellent correlation with other experiments 15. According to Figure 3A, the relaxation rate of the triplet T₁ state of riboflavin is also constant (=0) within experimental accuracy, exhibiting a value of .

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Figure 3

Dependence of the relaxation rates K_Δ of singlet oxygen and of triplet state of riboflavin on the concentration of (A) riboflavin in H₂O (at 170μM oxygen concentration) and (B) of oxygen (at 50μM riboflavin). The solid lines have been fitted to the experimental data points using Eqs. (2). (C) Dependence of the riboflavin T₁ state deactivation efficacy P_T on oxygen concentration. The solid curve has been fitted to the experimental data points using Eq. (5). The range of the singlet oxygen quantum yield Φ_Δ is shown versus oxygen concentration by using or 1 and Φ_T=0.38 or 0.61. The solid curve has been fitted by using Eq. (4).

After that, the singlet oxygen luminescence was measured at different oxygen concentrations using a constant riboflavin concentration of 50μM. In Figure 3B, the Stern-Vollmer plot shows the dependence of the relaxation rates of the riboflavin triplet T₁ state and of singlet oxygen K_Δ on the oxygen concentration in solution for riboflavin. The relaxation rate shows a linear dependence on the oxygen concentration ([O₂]=10μM–280μM). According to Eq. (3), the slope of the linear fit of the data yields the rate constant for the deactivation of riboflavin triplet T₁ state by oxygen with . Extrapolation of the linear fit to [O₂]=0 (assuming ) yields the relaxation rate of riboflavin triplet T₁ state in pure water . Thus, the lifetime of the triplet T₁ state of riboflavin in pure water is . This value is larger than reported previously (τ=42μs) 8. According to Figure 3B, the relaxation rate of singlet oxygen K_Δ=k_Δ=0.31±0.06μs⁻¹ (k_ΔS=0, Figure 3A) is independent of the oxygen concentration within the experimental accuracy.

To begin, the efficacy P_T of the T₁ state deactivation by oxygen was determined by applying Eq. (5) and the measured relaxation rates and rate constant. An aerated solution ([O₂]≈280μM) yields P_T=0.97±0.10. With decreasing oxygen concentrations, P_T is decreasing in particular, for [O₂]<50μM. Since for [O₂]=0 the value for P_T should theoretically be zero, the values in Figure 3C are fitted (solid line) accordingly using Eq. (5), including the rates which were appointed before.

Using the values of P_T, the quantum yield of singlet oxygen can be calculated by using (Eq. (4)). However, for Φ_T, only two values are available, being quite different with Φ_T=0.61 28 and Φ_T=0.38±0.05 29. Additionally, no values are available for , which can range between 0.25 and 1, depending on the triplet state energy and the polarity of the solvent 23.

Thus, Φ_Δ of riboflavin can be calculated only within a range that is shown as a hatched area in Figure 3C, whereas the line Φ_Δ,max() and Φ_Δ,min () is calculated. Thus, in aerated solution ([O₂]=280μM), the highest value is Φ_Δ,max=0.59±0.07 and the minimal value is Φ_Δ,min=0.09±0.03.

This experimental approach shows the clear dependence of the quantum yield on the oxygen concentration. The value Φ_Δ decreases with decreasing oxygen concentrations, which is very impressive for the line Φ_Δ,max. When looking at the line Φ_Δ,min, the values are not so different in the entire range of oxygen concentration. It is therefore important to know the true value of Φ_Δ, which might be in the range of 0.09 to 0.59 under the in vitro conditions ([O₂]=280μM). This is disappointing but comparable to problems with exogenous photosensitizers. For example, when investigating the photosensitizer Photofrin I in water, Φ_Δ values were determined with 0.06 (H₂O), high oxygen concentration 30; 0.12 (D₂O) aerated 31; 0.35 (D₂O), high oxygen concentration 32; or even 0.77 (D₂O) aerated 33.

To assess the role of endogenous sensitizers regarding UVA light, especially when looking at the biological effects attributed to singlet oxygen, precise values are necessary. Since there are hardly any values available for Φ_T or , the determination of Φ_Δ of riboflavin and the other flavins was performed by using another approach.

Since the range of possible values of Φ_Δ is maximal at high oxygen concentrations, the following experiments were carried out at [O₂]=280μM. The values Φ_Δ of flavins (riboflavin, FMN, and FAD) were determined by comparing quantitatively the luminescence signal at 1270nm to luminescence signal of PNS. Both Perinaphthenones are well-characterized molecules exhibiting a Φ_Δ of close to unity (see Table 1). Fig. 4 shows the dependence of time-integrated signal of luminescence of singlet oxygen at 1270nm on absorbed laser energy for PNS and for the endogenous photosensitizers riboflavin, FMN, and FAD at equal concentrations of 50μM. The time-integrated signal increased linearly with increasing absorbed excitation energy, whereas the respective fits are shown as solid lines.

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Figure 4

Time-integrated signal of luminescence of singlet oxygen at 1270nm versus absorbed energy for air-saturated solutions of PNS, riboflavin, FMN, and FAD in H₂O. Each slope is corrected by the absorption of the sensitizers at 355nm. The solid lines have been fitted to the experimental data points using a simple linear fit (y(x)=ax+b).

The ratio of the slopes is the same as the ratio of the singlet oxygen quantum yields with PNS as reference, respectively (Eq. (8)). The absorbed energy has been calculated from the incident laser energy by using the different absorption cross sections of each sensitizer at 355nm. In Table 1 the singlet oxygen quantum yields are shown for PNS and PN as reference and the calculated values of riboflavin (Φ_Δ=0.54±0.07), FMN (Φ_Δ=0.51±0.07), and FAD (Φ_Δ=0.07±0.02). The Φ_Δ value of riboflavin in aerated solution is added to Figure 3C, which is in good correlation to Φ_Δ,max of 0.59 within the experimental accuracy and the value determined by Chacon et al. 28. This may lead to the suggestion that the value of Φ_T is ∼0.6 and . Consequently, for molecules such as riboflavin, the line Φ_Δ,max is valid (Figure 3C) regarding the dependence of quantum yield on the oxygen concentration.

Riboflavin and FMN exhibit quantum yields higher than for exogenous photosensitizers such as hematoporphyrin derivative (Photofrin, Φ_Δ=0.35) 20, which are used in photodynamic therapy to kill cancer cells. Our results confirm that riboflavin and FMN are potential type II sensitizers under fully aerated conditions. Even the complex molecule FAD retains the ability of the flavin group to generate singlet oxygen. Interestingly, the quantum yield decreases with complexity of molecules going from riboflavin, to FMN and to FAD.

The detection of singlet oxygen by its luminescence is a powerful tool even in living cells in vitro 15,34. As already stated above, the efficacy of singlet oxygen generation decreases with decreasing oxygen concentration, i.e., decreasing oxygen partial pressure. That is shown in Figure 3C (Φ_Δ,max) for riboflavin, which is similar to other sensitizers 14,16 and the other flavins. To elucidate the role of flavins, experiments are carried out frequently in vitro under aerated conditions, which is equivalent to an oxygen partial pressure of ∼150Torr (150mmHg or [O₂]=280μM). Under in vivo conditions, e.g., in living skin, the oxygen partial pressure is only 20Torr (20mmHg or [O₂]=37μM) at the dermal-epidermal junction or even less inside the cells 21. In view of this difference in oxygen partial pressure, the singlet oxygen generation by riboflavin decreases approximately twofold at most. These results are important when comparing experiments that are performed at different oxygen partial pressure.

Recently, it was shown that irradiated riboflavin can damage nicotine by antibody-catalyzed oxidative degradation 35. However, that experiment was performed in aerated solution and therefore at a high efficacy of singlet oxygen generation, which might not reflect the degradation under low oxygen conditions in vivo. Riboflavin-sensitized photodynamic modifications of high-molecular-weight Kininogen were also investigated only in vitro and singlet oxygen was found to be an important mediator 36. According to experiments under aerobic conditions it was stated that photoexcitation of riboflavin may also potentially occur in vivo in the organs and tissues that are permeable to light, such as the eye or skin, and damage hyaluronic acid and other cell-matrix components, to cause inflammation and accelerate aging 37. In view of our results, one must be careful when judging the relevance of singlet oxygen in vivo based on experiments in vitro.

Additionally, after excitation of sensitizers such as riboflavin, there is always a competition between the generation of oxygen radicals (type I) and singlet oxygen (type II reaction). That competition depends on the oxygen concentration in the respective experimental setup. At fully aerated conditions ([O₂]≈280μM), the UVA light is effectively converted to singlet oxygen (Φ_Δ=0.54). At low oxygen concentrations ([O₂]<2μM), the singlet generation decreases to Φ_Δ<0.20. This is important since most of the endogenous photosensitizers are located inside cells and the oxygen partial pressure inside a cell can be 4Torr ([O₂]=7.5μM) and even less 38. At the same time, the generation of other reactive oxygen species (e.g., oxygen radicals) may increase. This correlates well to findings that riboflavin solution showed stronger cytotoxicity during irradiation under hypoxia than under air due to the heightened generation of H₂O₂39. Our results also support the very recent findings that the inactivation of 6-phosphate dehydrogenase (G6PD) results from its direct oxidation by the excited triplet state of riboflavin in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration 40.

In the last decade, numerous articles have stated that UVA light exposure cause skin aging or even skin cancer mainly by singlet oxygen 1,3,4,5,6,7,24,41,42. However, precise measurements of singlet oxygen generation by endogenous photosensitizers were missing, in particular at different oxygen concentrations.

Applying UVA light to urocanic acid, singlet oxygen luminescence was clearly detected, but the signal was too weak to quantify the respective quantum yield. Exciting riboflavin, FMN, and FAD, strong luminescence signal of singlet oxygen was detected. For these substances the quantum yield were successfully determined in air-saturated solvents using PNS as reference (riboflavin Φ_{Δ =} 0.54±0.07, FMN Φ_Δ=0.51±0.07, and FAD Φ_Δ=0.07±0.02). Depending on their concentration in the skin, the flavins are potential generators of singlet oxygen, even more effective than exogenous porphyrins used for cell killing in photodynamic therapy. In view of these high values, it seems to be reasonable that these substances including urocanic acid can provide sufficient singlet oxygen during UVA exposure leading to gene regulation, photoaging, and even carcinogenesis.

When measuring the efficacy of singlet oxygen generation at different oxygen concentrations, the efficacy of singlet oxygen generation (P_T) decreased significantly for low oxygen concentrations. When irradiating, e.g., riboflavin with UVA light, at least a factor-2-less singlet oxygen is generated in the skin as compared to the condition in an aerated environment (e.g., in vitro).