Open access peer-reviewed chapter

Biomolecules Oxidation by Hydrogen Peroxide and Singlet Oxygen

Written By

Kazutaka Hirakawa

Submitted: 23 June 2017 Reviewed: 05 October 2017 Published: 20 December 2017

DOI: 10.5772/intechopen.71465

From the Edited Volume

Reactive Oxygen Species (ROS) in Living Cells

Edited by Cristiana Filip and Elena Albu

Chapter metrics overview

2,160 Chapter Downloads

View Full Metrics


Hydrogen peroxide (H2O2) and singlet oxygen (1O2) are important reactive oxygen species (ROS) for biological and medicinal fields. Oxidation processes of chemical materials by molecular oxygen are important H2O2 source, whereas photochemical reaction is important for 1O2 production. Reactivity and biomolecule damage by these ROS depend on the surrounding conditions and targeting molecules. In this chapter, production mechanisms of H2O2 and 1O2, biomolecule oxidation by these ROS, their detection methods, and production control of 1O2 are briefly reviewed.


  • hydrogen peroxide
  • singlet oxygen
  • DNA damage
  • protein damage
  • photooxidation

1. Introduction

Biomolecule damage, for example, oxidation of DNA and/or protein, by reactive oxygen species (ROS) is closely related to carcinogenicity [1, 2, 3] and/or toxicity [4, 5, 6]. Furthermore, oxidative damage to unwanted tissue can be applied to the treatment of disease including cancer treatment [7, 8, 9], and similar reaction is applied to sterilization [10, 11, 12, 13, 14]. Hydrogen peroxide (H2O2) is a relatively long-lived ROS compared with a short-lived ROS such as superoxide anion radicals (O2•−) [15]. One of the most important producing mechanisms of H2O2 is a dismutation of O2•−, which is easily formed though oxidation of various materials by dioxygen molecule (O2). Various carcinogenic chemical compounds produce H2O2 through their oxidation processes. Relationship among molecular oxygen and ROS is shown in Figure 1. Oxygen molecules are easily reduced by surrounding materials, and various ROS and the intermediates are formed (Figure 1A). In the case of photosensitized reaction, excited states of oxygen molecules are produced (Figure 1B). Singlet oxygen (1O2), which is also an important ROS, can be easily generated via photosensitized reaction [16, 17, 18]. The 1g+ state (1O2(1g+)) is mainly produced through the excitation energy transfer from the excited state, in general triplet excited (T1) state, of photosensitizer [16, 17, 18]. The 1O2(1g+) has higher energy, 1.6 eV, corresponding to the ground state of oxygen molecule (3O2). The lifetime of 1O2(1g+) is several picoseconds, and 1O2(1g+) is rapidly converted to the 1Δg state (1O2(1Δg)) [16, 17, 18]. Because the lifetime of 1O2(1Δg) (several microseconds) is markedly longer than that of 1O2(1g+), 1O2(1Δg) is a more important ROS. After that, 1O2 indicates 1O2(1Δg) without explanation in this chapter. Visible light, other than ultraviolet radiation, has sufficient energy to produce 1O2 from the ground state of oxygen molecule. Therefore, 1O2 production is an important mechanism of phototoxicity and/or photo-carcinogenicity under strong light illumination with phototoxic materials. The purpose of this chapter is a review of the ROS-mediated biomolecule damage and the related topics.

Figure 1.

Relationship among ground-state oxygen molecule (3O2) and ROS (A) and the energy levels of oxygen molecule (B). HOMO and SOMO are the abbreviations of highest occupied molecular orbital and semi-occupied molecular orbital, respectively. The “arrows” in (B) indicate the electron spin.


2. Hydrogen peroxide

Hydrogen peroxide itself is not strongly ROS. However, other ROS including hydroxyl radicals (OH) are produced from H2O2. In general, H2O2 is produced from the dismutation of O2•−, and, in vivo, production of H2O2 and O2•− occurs in mitochondria [19]. In this section, H2O2 formation from compounds, specifically artificial materials, is introduced.

2.1. Hydrogen peroxide formation through oxidation of chemical compounds

One of the most important processes of H2O2 production is a dismutation of O2•−. Various chemical compounds or metals can be oxidized by oxygen molecules. In the case of a simple electron transfer-mediated oxidation, O2•− is produced by the electron extraction from chemical compounds or metals. The lifetime of O2•− in aqueous solution is about several milliseconds [15]. The produced O2•− in aqueous media is converted to H2O2 through the dismutation by proton (H+) as follows:

2O 2 + 2H + H 2 O 2 + O 2 E1

For example, hydroquinone, which is one of the metabolites of benzene, can produce H2O2 through the autoxidation process (Figure 2) [20]. This process is markedly enhanced by the presence of metal ions, specifically Cu2+ ions [20]. In the presence of sacrificial reductants, for example, nicotinamide adenine dinucleotide (NADH), the oxidized form of hydroquinone, p-benzoquinone, is reduced to the parent hydroquinone. Consequently, the redox cycle is formed, leading to the production of H2O2 abundantly. It has been also reported that hydrazine analogues produce H2O2 through their autoxidation processes (Figure 3) [21, 22, 23].

Figure 2.

Autoxidation process of hydroquinone and ROS production.

Figure 3.

Autoxidation process of hydrazine analogues and ROS production.

2.2. Hydrogen peroxide production through photochemical processes

Photochemical processes also contribute to the formation of H2O2. Because the reorganization energy of the reduction of small molecule, such as O2 molecules, through electron transfer becomes large due to the Marcus theory [24, 25], the O2•− production through photoinduced electron transfer is energetically difficult [26, 27]. However, ultraviolet radiation to reductive photosensitizer, such as NADH (Figure 4), can produce O2•− as follows [28]:

NADH + O 2 NAD + H + + O 2 E3

Figure 4.

Structure of NADH.

where NADH* is the photoexcited state of NADH and NAD is the radical form. NAD undergoes further oxidation by oxygen molecules to NAD+, the final oxidized product. The formed O2•− is also converted to H2O2 through the dismutation process of Eq. (1).

Photocatalytic reaction can also produce H2O2 [29, 30, 31, 32, 33, 34]. For example, the surface of titanium dioxide (TiO2) can reduce relatively oxidative molecules under ultraviolet A (UVA; wavelength, 315–400 nm) irradiation [29, 30, 31, 32]. Two crystalline forms of TiO2, anatase and rutile with band gap energies of 3.26 and 3.06 eV, respectively, are well-known semiconducting photocatalyst [29, 30, 31, 32]. The adsorbed oxygen molecules on the TiO2 surface is reduced to O2•− by the electron of conduction band, which is excited from the valence band by UVA energy (Figure 5). Similarly to the abovementioned reaction, O2•− is also converted to H2O2 through the dismutation process of Eq. (1). In addition, oxidation reaction of TiO2 photocatalyst also produces H2O2. The formed hole (h+) in the valence band by UVA irradiation oxidizes water molecules on the surface of TiO2 to OH. The reaction of two OH species can produce H2O2 as follows:

2 OH H 2 O 2 E4

Figure 5.

Photocatalytic production of ROS by TiO2.

Although TiO2 particles are barely incorporated into cell nucleus [35], cellular DNA damage was reported [36, 37, 38, 39]. Because H2O2 has a transparency for nuclear membrane, the cellular DNA damage can be explained by H2O2-mediated mechanism [32]. The activation of H2O2 and DNA damage by H2O2 are described later.

2.3. Secondary formation of hydrogen peroxide through photocatalytic reaction

Photocatalytic reaction can produce oxidized intermediates other than final oxidized products of chemical compounds. For example, photooxidized amino acids [40] and sugars [41] by TiO2 photocatalyst produce H2O2 through secondary oxidation reaction in the presence of metal ions (Figure 6). Titanium dioxide can photocatalyze the production of OH, a strong oxidant, through the decomposition of H2O. The formed h+ in the valence band by UVA irradiation can also oxidize various materials adsorbed on TiO2 surface. Hydroxyl radicals and h+ can oxidize these biomolecules, resulting in the production of oxidized intermediates. The formation of partly oxidized molecules leads to the secondary H2O2 production in the presence of metal ions. This H2O2 production process may cause a remote H2O2 generation in cells.

Figure 6.

The secondary production of ROS by TiO2 photocatalysis.

It has been reported that the photooxidized phenylalanine and tyrosine by TiO2 produce H2O2 in the presence of copper(II) ion [40]. Since TiO2 photocatalysis induces a hydroxylation of aromatic compounds [42], the formation of benzenediol derivatives from these aromatic amino acids is possible (Figure 7). As mentioned above, hydroquinone can produce H2O2 through the autoxidation (Figure 2) [20, 43]. The amount of H2O2 production from the photooxidized phenylalanine is significantly larger than that from tyrosine [40]. The difference of their autoxidation rates should affect the H2O2 production. It has been reported that the autoxidation rate of 1,4-form of benzenediol is markedly faster than that of 1,2-form [20]. Phenylalanine can be oxidized into various types of benzenediol, including the 1,4-form; however, tyrosine cannot be converted to the 1,4-form. Consequently, phenylalanine can produce relatively large amount of secondary H2O2 through the photocatalysis of TiO2. Furthermore, other amino acids can be also oxidized by TiO2 photocatalyst and induce the secondary ROS production. Specifically, photocatalyzed cysteine by anatase form produces significantly large amount of secondary H2O2. In the case of sugar oxidation by TiO2 photocatalyst, the activity of secondary H2O2 production by anatase form TiO2 is larger than that of rutile form [41].

Figure 7.

Tyrosine oxidation by TiO2 photocatalysis and the copper ion mediated ROS production. Phenylalanine is also oxidized by the similar processes, leading to the secondary ROS production.


3. DNA damage by hydrogen peroxide

Hydrogen peroxide itself barely induces DNA damage; however, it can oxidize nucleobases and cleave sugar-phosphate backbone in the presence of metal ions. In this section, the sequence-specific DNA damage by the H2O2-derived ROS and its biological effect are briefly introduced.

3.1. Sequence-specific DNA damage by hydrogen peroxide

Hydrogen peroxide causes alkali-labile products at guanine, thymine, and cytosine in the presence of cupper ion (Cu2+) [44]. Since copper ions are associated with chromatin [45] to form stable complexes with DNA [46, 47, 48, 49], Cu2+ can play an important role in the activation of H2O2 in cell nucleus. Polyacrylamide gel electrophoresis studies demonstrated that H2O2 itself cannot cleave and oxidize DNA [44]. However, the incubation of DNA with H2O2 and Cu2+ induce base modifications at guanine, thymine, and cytosine residues. These base modification sites can be cleaved by hot piperidine treatment [20, 21, 22, 44]. The derived reactive species from H2O2, for example, copper-peroxyl species (Cu(I)-OOH), are responsible for this DNA damage:

H 2 O 2 + Cu + Cu I OOH + H + E5

Cu(I)-OOH is not strongly reactive compared with OH; however, its lifetime is relatively long to induce DNA base modification. Single-stranded DNA is easier oxidized by these ROS. Therefore, DNA damage by H2O2 is enhanced by denaturation of DNA [44]. Abovementioned chemical compounds, benzenediol [20] and hydrazine [21], induce these base modification in the presence of Cu2+. In the case of relatively low concentration of TiO2 particles, similar sequence-specific DNA damage was observed after UVA irradiation with Cu2+ [32]. DNA damage mediated by H2O2 is effectively inhibited by catalase [50], which is an enzyme to decompose H2O2 to H2O and O2. Chelating molecules for copper ions also effectively suppress this DNA damage. In addition, 3-methylthiopropanal (methional) is an effective inhibitor of Cu(I)-OOH [20, 32, 44]. Cu(I)-OOH cannot be scavenged by free OH scavengers, such as sugars and alcohols [20, 22, 32, 44]. In the presence of Cu2+, UVA-irradiated NADH also induces DNA damage by the similar process through H2O2 production [28]. In general, photosensitized DNA damage could be explained by 1O2 formation mechanism or electron transfer-mediated oxidation [51]. The H2O2-mediated DNA is a rare case in the photochemical DNA damage.

Hydrogen peroxide and Cu2+ can induce tandem lesion at guanine and thymine residues [32]. Clustered DNA lesions including tandem damage have important mutagenic potential [52, 53, 54]. Furthermore, the repair of such DNA damage is more difficult than single-base damage [55, 56, 57, 58, 59, 60]. Therefore, oxidative DNA damage through H2O2 production may play an important role in carcinogenesis.

In the presence of iron ions (Fe2+), OH is formed as follows:

H 2 O 2 + Fe 2 + OH + OH + Fe 3 + E6

Formed OH induces base oxidation with non-sequence specificity, because OH can oxidize all nucleobases [44, 61]. In addition, direct cleavage of sugar-phosphate backbone is caused by OH. Hydroxyl radical-mediated DNA damage was reported by the case of ascorbate with Cu2+ [62]. As mentioned above, in the case of TiO2 photocatalysis, OH is directly produced from water decomposition [29, 30, 31, 32], and DNA damage without sequence specificity can be induced in the absence of metal ions [32]. Relatively high concentration of anatase form of TiO2 induce non-sequence-specific DNA damage under UVA irradiation without metal ions through OH production [32]. DNA damage by OH is effectively inhibited by sugars and alcohols [32, 44]. However, in the presence of metal ions, the addition of OH scavengers rather enhances DNA damage through the secondary generation of H2O2 from the oxidized products of scavengers themselves by OH [32, 41]. Base modifications can cause carcinogenesis. Because H2O2 can penetrate into nuclear membrane, DNA modification can be induced by H2O2 originally formed in the sphere of outer cell nucleus through the assistance of metal ions.

3.2. Mutagenicity and cytotoxicity caused by hydrogen peroxide production

As oxidized products of nucleobases by the H2O2-mediated mechanism, 8-oxo-7,8-dihydroguanine (8-oxo-G; oxidized guanine, Figure 8) [63, 64, 65]; 5,6-dihydroxy-5,6-dihydrothymine (OH-thy; oxidized thymine, Figure 9) [58, 66, 67]; 5-hydroxyuracil (OH-Ura; oxidized cytosine, Figure 9) [67, 68]; 5-hydroxyhydantoin (OH-Hyd; oxidized cytosine, Figure 9) [68], and 5-hydroxycytosine (OH-Cys; oxidized cytosine, Figure 9) [67] are well-known compounds. In a certain case, oxidative DNA damage induces cell death [69, 70]. As a minor oxidized product of guanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G, Figure 10) can be formed by H2O2 and metal ions [63, 71]. Mutagenicity of Fapy-G is low [72]; however, a related product, methyl-Fapy-G formation, is a lethal lesion [73]. Furthermore, a theoretical study suggested that the formation of Fapy-G contributes to mutation [74]. Cytotoxicity of TiO2 photocatalyst can be explained by oxidative damage of membrane protein [75, 76, 77]. In addition, cellular DNA damage was also reported [78, 79]. Because H2O2 has a transparency for nuclear membrane, the cellular DNA damage by TiO2 photocatalysis can be explained by H2O2 production. The formed H2O2 through TiO2 photocatalysis is incorporated into cell nucleus and activated by endogenous metal ions, leading to oxidative DNA damage [32]. Examples of the mutations caused by the oxidized guanines are described in Section 4.

Figure 8.

Guanine oxidation by ROS. This scheme is an example of the guanine oxidation by 1O2 to 8-oxo-G. Other H2O2-derived ROS, OH and Cu(I))-OOH, also produce 8-oxo-G through the oxidation of guanine.

Figure 9.

Oxidized products of thymine and cytosine by 1O2.

Figure 10.

Structure of Fapy-G.


4. Singlet oxygen

In general, the production mechanism of 1O2 involves photochemical processes. Various photooxidation processes can be explained by 1O2 production. In this section, the production mechanism of 1O2, its application, and biomolecule oxidation by 1O2 are briefly introduced.

4.1. General property of singlet oxygen

Singlet oxygen is an excited state of 3O2, ground triplet state of molecular oxygen [16, 17, 18]. In general, singlet excited (S1) states of O2 are 1Δg and 1g+; they have excitation energy of 0.98 eV and 1.63 eV above 3O2, respectively [16, 17, 18]. Because of the short lifetime of 1g+ (a few picoseconds), 1Δg, the lower S1 state of O2, plays an important role in various oxidation reactions. In this chapter, 1Δg is denoted throughout as 1O2. The highest occupied molecular orbital (HOMO) of 3O2 is a semi-occupied molecular orbital (SOMO), whereas this molecular orbital of 1O2 becomes the lowest unoccupied molecular orbital (LUMO) (Figure 1B). The oxidative activity of 1O2 is stronger than that of 3O2 due to the vacant molecular orbital. Commonly, 1O2 is produced through photosensitized reaction. Since the excitation energy of 1O2 is relatively small, which corresponds to the energy of photon with the wavelength of 1270 nm (smaller than that of visible light photon), photoexcited states of various dyes can sensitize the generation of 1O2 under visible light or ultraviolet irradiation. Various molecules become photosensitizer (PST) to generate 1O2. In general, the photosensitized reaction of 1O2 generation is an electron exchange energy transfer (the Dexter mechanism) [80]. These processes are presented as follows:

PST + PST S 1 E7
PST S 1 PST + fluorescence E8
PST S 1 PST T 1 E9
PST T 1 + 3 O 2 PST + 1 O 2 E10

where PST*(S1) and PST*(T1) are the S1 and T1 states of PST, respectively. In general, since the lifetime of PST*(T1) is markedly longer (several microseconds) than that of PST*(S1) (several nanoseconds), 1O2 is produced by PST*(T1). However, the formation of 1O2 by PST*(S1) is not impossible. The lifetime of 1O2 (τΔ) is relatively long (Table 1). Generated 1O2 can oxidize various materials, including biomolecules, within its long lifetime. The τΔ strongly depends on the surroundings, and a solvent deuterium effect on the reactivity of 1O2 is significant (Table 1). For example, the τΔ in deuterium oxide (D2O) is markedly longer than that in H2O, and the biomolecule oxidation by 1O2 is significantly enhanced in D2O compared with that in H2O.

Solvent Photosensitizer τΔ/μs Reference
Water (H2O) Cationic porphyrin 3.5 [81]
Rose bengal 3.77 [82]
Phosphate buffer (pH 7.6) P(V) porphyrin 3.5 [83]
Ethanol (C2H5OH) Rose bengal 15.4 [82]
Ethanol/H2O (1/1) Rose bengal 6.37 [82]
Water (D2O) Berberine with DNA 72 [84]
Methylene blue 32 [85]
Phenalenone 64.4 [86]
Tris(bipyridine)Ru(II) 59.47 [82]
Chloroform (CHCl3) Phenalenone 232 [86]
Tetrachloromethane (CCl4) Phenalenone 34,000 [86]

Table 1.

Solvent dependence of the lifetime of singlet oxygen.

4.2. Photodynamic therapy

One of the most important medicinal applications of 1O2 is photodynamic therapy (PDT) (Figure 11) [7, 8, 9]. Photodynamic therapy is a promising and less invasive treatment for cancer [7, 8, 9] and photosterilization [10, 11, 12, 13, 14]. For cancer PDT, in general, porphyrins are used for photosensitizers, for example, porfimer sodium [87] and talaporfin sodium [88]. Photosterilization, antimicrobial PDT, is also carried out using dyes, for example, methylene blue (MB) [11, 14, 89]. The important mechanism of PDT processes including photosterilization is oxidation of biomolecules of cancer cell or bacteria through 1O2 production under visible light irradiation. Visible light, especially longer wavelength visible light (wavelength > 650 nm), is less harmful for the human body and can penetrate into the tissue deeply. As mentioned above, 1O2 can be generated by longer wavelength visible light. Administered photosensitizers, porphyrins, or other dyes produce 1O2 through energy transfer to oxygen molecules with relatively large quantum yield (ΦΔ).

Figure 11.

Scheme of the general procedure of PDT.

4.3. Photocatalytic singlet oxygen generation

As mentioned above, TiO2 photocatalyzes the generation of various ROS. Singlet oxygen can be also produced through the photocatalysis of TiO2 [31, 90, 91, 92, 93, 94, 95, 96]. In general, photogenerated electron in the conduction band reduces the surface-adsorbed oxygen molecules to O2•−. Through the reoxidation of O2•−, 1O2 is formed. The possible reactions of photocatalytic 1O2 productions are as follows:

O 2 + h + 1 O 2 E11


O 2 + OH 1 O 2 + OH E12

The photogenerated h+ in the valence band and OH can act as the oxidants to produce 1O2. In addition, hydroperoxyl radical (OOH) generated from O2•− and H+ also produces 1O2 as follows:

O 2 + OOH + H + 1 O 2 + H 2 O 2 E13

The reported values of ΦΔ are depending on the experimental condition, for example, around 0.2 (0.2, Degussa P25 in water [92], and 0.22, rutile particle in chloroform [95]). Other cases reported relatively small values, for example, 0.003 [96] and 0.02 [94]. In the cases of airborne 1O2, quite small value (10−8–10−9) was reported [93]. It has been reported that the τΔ value of 1O2 produced by Degussa P25 aqueous suspension is 5 μs [92]. Other photocatalytic materials, for example, zinc oxide (ZnO) can photocatalyze 1O2 production through the similar reaction of TiO2 photocatalysis [97]. Recently, carbon quantum dots, which have been paid attention as interesting nano-materials, also photocatalyze 1O2 production [33].

Singlet oxygen is an important ROS for PDT. Other than 1O2, H2O2 production can be also applied for PDT mechanism. Photocatalytic materials can produce these ROS under photoirradiation. Therefore, application of photocatalysts, specifically TiO2 nanoparticles, for PDT has been also studied [29, 98, 99, 100, 101]. To realize the TiO2-utilized PDT, direct administration of small TiO2 powders into tumor assisted with an optical fiber was proposed [29]. In addition, it was reported that oral-administrated TiO2 nanoparticles are transported into the tumor of nude mouse skin transplanted from a human prostate cancer cell line [98]. As mentioned above, in general, TiO2 nanoparticles can be excited by UVA irradiation. To utilize visible light for TiO2 excitation, upconversion technique was also studied [100].

4.4. DNA oxidation by 1O2 and mutation

Singlet oxygen can oxidize only guanines without sequence specificity; however, it does not have the ability to induce the oxidation of other nucleobases or to cleave the sugar-phosphate backbone [44]. The main oxidized product of guanine by 1O2 is 8-oxo-G (Figure 8) [63, 64, 65]. Guanines undergo the Diels-Alder reaction by photoproduced 1O2, leading to the formation of [4 + 2] cycloaddition product with the imidazole ring to produce an endoperoxide. Through the subsequent proton transfer, this peroxide is converted to 8-hydroperoxyguanine [102, 103], which becomes 8-hydroxyguanine [63]. The keto-enol tautomerism produces 8-oxo-G from 8-hydroxyguanine. Because single-stranded DNA is easily oxidized by ROS, 8-oxo-G formation by 1O2 is increased by DNA denaturation [44]. The 8-oxo-G formation causes DNA misreplication (Figure 12), which can lead to mutations such as G-C:T-A transversion caused by the stable base-pair formation between 8-oxo-G and adenine [104, 105]. Since 8-oxo-G is more easily oxidized than guanine, 8-oxo-G undergoes further reaction, leading to the formation of imidazolone and oxazolone (Figure 13) [63, 106, 107]. Imidazolone forms more stable base pair with guanine than cytosine [106, 107]. Therefore, guanine oxidation by 1O2 may cause G-C:C-G transversion [108, 109] through imidazolone formation, a further oxidized product of 8-oxo-G. Indeed, it has been reported that UVA can induce these mutations [110].

Figure 12.

Hydrogen bonding between 8-oxo-G and adenine.

Figure 13.

Structures of imidazolone and oxazolone and the hydrogen bonding between guanine and imidazolone.

4.5. Protein oxidation by 1O2

Protein oxidation is also induced by 1O2. The following amino acids, tryptophan, tyrosine, cysteine, histidine, and methionine, can be oxidized by 1O2 [111]. In the case of tryptophan oxidation by 1O2, N-formylkynurenine (Figure 14) is a major oxidized product [112, 113]. The reported reaction rate coefficient between tryptophan and 1O2 is 3.0 × 107 s−1 M−1 [114]. Oxidation of tryptophan residue in a certain protein can be examined with a fluorometer [115]. For example, human serum albumin (HSA) has one tryptophan residue, and the intrinsic fluorescence of tryptophan at around 350 nm can be diminished by the oxidative damage. Porphyrin phosphorus(V) complexes (Figure 15), of which the ΦΔ is larger than 0.5, can induce oxidative damage to the tryptophan residue of HSA [116]. Photosensitized HSA damage is enhanced in D2O, in which the lifetime of 1O2 is markedly elongated compared in H2O (Table 1). Furthermore, sodium azide (NaN3), a strong physical quencher of 1O2 [117], effectively suppresses this HSA damage. From the analysis of the effect of NaN3 on the HSA damage, the contribution of 1O2-mediated oxidation to the total quantum yield of protein damage can be determined [115]. Photosensitized 1O2 production by porphyrin phosphorus(V) complexes induces the damage of tyrosinase, which is an enzyme to catalyze the hydroxylation of tyrosine, resulting in the deactivation of tyrosinase [118]. Oxidation of the amino acid residue by 1O2 can cause the deactivation of protein function. The protein oxidation photosensitized by porphyrins through ROS production is an important mechanism of PDT.

Figure 14.

Structures of tryptophan and N-formylkynurenine, an oxidized product of tryptophan by 1O2.

Figure 15.

Example of P(V)porphyrin photosensitizer.

Photocatalyzed 1O2 production by TiO2 may not play an important role in the oxidation reaction [31, 94]. Formed 1O2 on the TiO2 surface is quenched by TiO2 itself with relatively large quenching rate coefficient (e.g., 2.4 × 109 M−1 s−1 [95]). In the presence of bovine serum albumin, 1O2 produced by TiO2 photocatalysis is effectively quenched, suggesting the protein oxidation [94]. However, in the case of TiO2 photocatalyst, other ROS are more important for protein oxidation than 1O2-mediated reaction [29, 30, 31, 32].


5. Detection of ROS

ROS detection is an important theme to investigate a biological effect of ROS or evaluation of the activity of PDT photosensitizers [119, 120, 121, 122]. Fluorometry is one of the most important and effective methods of ROS detection. For example, 5-carboxyfluorescein-based probe has been developed (Figure 16) [123]. This probe can detect H2O2 in the living cell. As an inexpensive method, the fluorometry using folic acid (Figure 17) was reported [23, 119, 124]. Folic acid can be decomposed by H2O2 in the presence of Cu2+, resulting in the fluorescence enhancement. The limit of detection (LOD, at signal/noise = 3) for this method was 0.5 μM H2O2. This method is based on the oxidative decomposition of folic acid by Cu(I)-OOH. In the presence of Fe2+, OH slightly induces the folic acid decomposition; however, the effect of OH on this folic acid decomposition is negligibly small because of the very short lifetime [125, 126]. In addition, O2•− does not have the activity of folic acid decomposition. Using folic acid or its analogue, 1O2 can be also detected [124]. Specifically, in D2O, folic acid or methotrexate (Figure 17), an analogue of folic acid, is effectively decomposed by 1O2, resulting in the fluorescence enhancement [124]. Using this method, the values of ΦΔ of various water-soluble photosensitizers can be determined.

Figure 16.

Structure of 5-carboxyfluorescein-based fluorescence probe for H2O2 [123].

Figure 17.

Structures of folic acid and methotrexate and the fluorometry of ROS [119, 124].


6. Control of singlet oxygen production

Control of photosensitized 1O2 is an important theme for biology or medicine, for example, to realize target-selective PDT [127] or “theranostics” (therapy and diagnosis) [128]. The pH-dependent control [129] and target-selective control [127, 128, 130, 131, 132] methods have been reported. It has been reported that free base porphyrins were synthesized to control their photosensitized 1O2 generating activity by pH (Figure 18) [129]. The S1 state of this porphyrin is quenched by the electron-donating moiety in neutral or alkali solution. However, protonation of this electron-donating moiety under acidic condition suppresses the electron transfer, leading to the recovery of the 1O2 production activity of porphyrin ring. Because cancer cell is slightly a more acidic condition compared with normal cells [133, 134, 135], this pH-based control of photosensitized 1O2 production can be applied to cancer-selective PDT. DNA-targeting control of photosensitized 1O2 generation has been also reported [127, 128]. For example, electron donor-connecting porphyrins have been studied (Figure 19) [81, 130, 131, 132]. These compounds can be photoexcited by visible light irradiation, and their S1 states are effectively quenched through intramolecular electron transfer. The charge-transfer state energy can be raised through the binding interaction with DNA, an anionic polymer, resulting in the inhibition of the intramolecular electron transfer and enhancement of 1O2 generation.

Figure 18.

Example of the reported pH-responsive porphyrin [129].

Figure 19.

The examples of DNA-targeting porphyrins: Ant-P [81], Pyr-P [130], Phen-P [131], and Nap-Ps [132].


7. Conclusions

Hydrogen peroxide is easily produced from the oxidation processes of chemical compounds by oxygen molecules. In addition, UVA-irradiated NADH and semiconductor photocatalytic materials can also produce H2O2. Formed H2O2 in cells can be incorporated into cell nucleus and activated by endogenous metal ions. Copper ion induces Cu(I)-OOH formation from H2O2, whereas OH is produced from H2O2 and iron ion. These ROS cause base oxidation, and OH can induce strand break of DNA. Base modifications lead to carcinogenesis or lethal effect. Photoirradiation to various sensitizing materials induces 1O2 production. Visible light has sufficient energy to produce 1O2. Therefore, 1O2 is easily produced by various dyes under photoirradiation. Photocatalytic 1O2 formation through reoxidation of O2•− is also possible. Formed 1O2 can oxidize guanine residues of DNA without sequence specificity and several amino acid residues of protein within its lifetime, which depends on the surroundings. Various detection methods of these ROS have been developed. In addition, the target-selective or condition-selective productions of ROS become important strategies for PDT and cancer “theranostics.”



These works were partially supported by the Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS).


  1. 1. Ziecha D, Francob R, Pappac A, Panayiotidisd MI. Reactive oxygen species (ROS)-induced genetic and epigenetic alterations in human carcinogenesis. Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis. 2011;711:167-173. DOI: 10.1016/j.mrfmmm.2011.02.015
  2. 2. Wu Q, Ni X. ROS-mediated DNA methylation pattern alterations in carcinogenesis. Current Drug Targets. 2015;16:13-19. DOI: 10.2174/1389450116666150113121054
  3. 3. Kawanishi S, Ohnishi S, Ma N, Hiraku Y, Murata M. Crosstalk between DNA damage and inflammation in the multiple steps of carcinogenesis. International Journal of Molecular Sciences. 2017;18:1808. DOI: 10.3390/ijms18081808
  4. 4. Auten RL, Davis JM. Oxygen toxicity and reactive oxygen species: The devil is in the details. Pediatric Research. 2009;66:121-127. DOI: 10.1203/PDR.0b013e3181a9eafb
  5. 5. Liochev SI. Reactive oxygen species and the free radical theory of aging. Free Radical Biology and Medicine. 2013;60:1-4. DOI: 10.1016/j.freeradbiomed.2013.02.011
  6. 6. Dixon SJ, Stockwell BR. The role of iron and reactive oxygen species in cell death. Nature Chemical Biology. 2014;10:9-17. DOI: 10.1038/nchembio.1416
  7. 7. Dolmans DEJGJ, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nature Reviews Cancer. 2003;3:380-387. DOI: 10.1038/nrc1071
  8. 8. Castano AP, Mroz P, Hamblin MR. Photodynamic therapy and anti-tumour immunity. Nature Reviews Cancer. 2006;6:535-545. DOI: 10.1038/nrc1894
  9. 9. Chilakamarthi U, Giribabu L. Photodynamic therapy: Past, present and future. The Chemical Records. 2017;17:1-29. DOI: 10.1002/tcr.201600121
  10. 10. Arenas Y, Monro S, Shi G, Mandel A, McFarland S, Lilge L. Photodynamic inactivation of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus with Ru(II)-based type I/type II photosensitizers. Photodiagnosis and Photodynamic Therapy. 2013;10:615-625. DOI: 10.1016/j.pdpdt.2013.07.001
  11. 11. Diogo P, Gonçalves T, Palma P, Santos JM. Photodynamic antimicrobial chemotherapy for root canal system asepsis: A narrative literature review. International Journal of Dentistry. 2015;2015:269205. DOI: 10.1155/2015/269205
  12. 12. Oruba Z, Łabuz P, Macyk W, Chomyszyn-Gajewska M. Antimicrobial photodynamic therapy-A discovery originating from the pre-antibiotic era in a novel periodontal therapy. Photodiagnosis and Photodynamic Therapy. 2015;12:612-618. DOI: 10.1016/j.pdpdt.2015.10.007
  13. 13. Tim M. Strategies to optimize photosensitizers for photodynamic inactivation of bacteria. Journal of Photochemistry and Photobiology B: Biology. 2015;150:2-10. DOI: 10.1016/j.jphotobiol.2015.05.010
  14. 14. Wainwright M, McLean A. Rational design of phenothiazinium derivatives and photoantimicrobial drug discovery. Dyes and Pigments. 2017;136:590-600. DOI: 10.1016/j.dyepig.2016.09.015
  15. 15. Jajic I, Sarna T, Strzalka K. Senescence, stress, and reactive oxygen species. Plants. 2015;4:393-411. DOI: 10.3390/plants4030393
  16. 16. DeRosa MC, Crutchley RJ. Photosensitized singlet oxygen and its applications. Coordination Chemistry Reviews. 2002;233-234:351-371. DOI: 10.1016/S0010-8545(02)00034-6
  17. 17. Schweitzer C, Schmidt R. Physical mechanisms of generation and deactivation of singlet oxygen. Chemical Reviews. 2003;103:1685-1758. DOI: 10.1021/cr010371d
  18. 18. Ogilby PR. Singlet oxygen: There is indeed something new under the sun. Chemical Society Reviews. 2010;39:3181-3209. DOI: 10.1039/B926014P
  19. 19. Wong HS, Dighe PA, Mezera V, Monternier PA, Brand MD. Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions. Journal of Biological Chemistry. 2017;292:16804-16809. DOI: 10.1074/jbc.R117.789271
  20. 20. Hirakawa K, Oikawa S, Hiraku Y, Hirosawa I, Kawanishi S. Catechol and hydroquinone have different redox properties responsible for their differential DNA-damaging ability. Chemical Research in Toxicology. 2002;15:76-82. DOI: 10.1021/tx010121s
  21. 21. Ito K, Yamamoto K, Kawanishi S. Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related hydrazines: Non-Fenton-type hydroxyl radical formation. Biochemistry. 1992;31:11606-11613
  22. 22. Hirakawa K, Midorikawa K, Oikawa S, Semi KS. Carcinogenic semicarbazide induces sequence-specific DNA damage through the generation of reactive oxygen species and the derived organic radicals. Mutatation Research Genetic Toxicology and Environmental Mutagenesis. 2003;536:91-101. DOI: 10.1016/S1383-5718(03)00030-5
  23. 23. Hirakawa K. Fluorometry of hydrogen peroxide using oxidative decomposition of folic acid. Analytical and Bioanalytical Chemistry. 2006;386:244-248. DOI: 10.1007/s00216-006-0649-1
  24. 24. Marcus RA. On the theory of oxidation-reduction reactions involving electron transfer. I. The Journal of Chemical Physics. 1956;24:966-978. DOI: 10.1063/1.1742723
  25. 25. Marcus RA, Sutin N. Electron transfers in chemistry and biology. Biochimica et Biophysica Acta. 1985;811:265-322. DOI: 10.1016/0304-4173(85)90014-X
  26. 26. Kikuchi K, Sato C, Watabe M, Ikeda H, Takahashi Y, Miyashi Y. New aspects on fluorescence quenching by molecular oxygen. Journal of the American Chemical Society. 1993;115:5180-5184. DOI: 10.1021/ja00065a033
  27. 27. Sato C, Kikuchi K, Okamura K, Takahashi Y, Miyashi T. New aspects on fluorescence quenching by molecular oxygen. 2. Inhibition of long-distance electron transfer in acetonitrile. The Journal of Physical Chemistry. 1995;99:16925-16931. DOI: 10.1021/j100046a018
  28. 28. Ito K, Hiraku Y, Kawanishi S. Photosensitized DNA damage induced by NADH: Site specificity and mechanism. Free Radical Research. 2007;41:461-468. DOI: 10.1080/10715760601145240
  29. 29. Fujishima A, Rao TN, Tryk DA. Titanium dioxide photocatalysis. Journal of Photochemistry and Photobiology C: Photochemistry Reviews. 2000;1:1-21. DOI: 10.1016/S1389-5567(00)00002-2
  30. 30. Liu K, Cao M, Fujishima A, Jiang L. Bio-inspired titanium dioxide materials with special wettability and their applications. Chemical Reviews. 2014;114:10044-10094. DOI: 10.1021/cr4006796
  31. 31. Nosaka Y, Nosaka AY. Generation and detection of reactive oxygen species in photocatalysis. Chemical Reviews. 2017;117:11302-11336. DOI: 10.1021/acs.chemrev.7b00161
  32. 32. Hirakawa K, Mori M, Yoshida M, Oikawa S, Kawanishi S. Photo-irradiated titanium dioxide catalyzes site specific DNA damage via generation of hydrogen peroxide. Free Radical Research. 2004;38:439-447. DOI: 10.1080/1071576042000206487
  33. 33. Yen YC, Lin CC, Chen PY, Ko WY, Tien TR, Lin KJ. Green synthesis of carbon quantum dots embedded onto titanium dioxide nanowires for enhancing photocurrent. Royal Society Open Science. 2017;4:161051. DOI: 10.1098/rsos.161051
  34. 34. Donat F, Corbel S, Alem H, Pontvianne S, Balan L, Medjahdi G, Schneider R. ZnO nanoparticles sensitized by CuInZnxS2+x quantum dots as highly efficient solar light driven photocatalysts. The Beilstein Journal of Nanotechnology. 2017;8:1080-1093. DOI: 10.3762/bjnano.8.110
  35. 35. Cai R, Hashimoto K, Itoh K, Kubota Y, Fujishima A. Photokilling of malignant cells with ultra-fine TiO2 powder. Bulletin of the Chemical Society of Japan. 1991;64:1268-1273. DOI: org/10.1246/bcsj.64.1268
  36. 36. Wamer WG, Yin JJ, Wei RR. Oxidative damage to nucleic acids photosensitized by titanium dioxide. Free Radical Biology and Medicine. 1997;23:851-858. DOI: 10.1016/S0891-5849(97)00068-3
  37. 37. Meena R, Rani M, Pal P, Rajamani P. Nano-TiO2-induced apoptosis by oxidative stress-mediated DNA damage and activation of p53 in human embryonic kidney cells. Applied Biochemistry and Biotechnology. 2012;167:791-808. DOI: 10.1007/s12010-012-9699-3
  38. 38. Nakagawa Y, Wakuri S, Sakamoto K, Tanaka N. The photogenotoxicity of titanium dioxide particles. Mutation Research Genetic Toxicology and Environmental Mutagenesis. 1997;394:125-132. DOI: 10.1016/S1383-5718(97)00126-5
  39. 39. Kashige N, Kakita Y, Nakashima Y, Miake F, Watanabe K. Mechanism of the photocatalytic inactivation of Lactobacillus casei phage PL-1 by titania thin film. Current Microbiology. 2001;42:184-189. DOI: 10.1007/s002840010201TiO2
  40. 40. Hirakawa K, Suzuki T. Amino acids photocatalyzed by titanium dioxide can produce secondary hydrogen peroxide. Trends in Photochemistry and Photobiology. 2014;16:63-69
  41. 41. Hirakawa K. Titanium dioxide photocatalyzes DNA damage via the secondary generation of hydrogen peroxide in the presence of sugars. Trends in Photochemistry and Photobiology. 2012;14:69-73
  42. 42. Emeline AV, Zhang X, Murakami T, Fujishima A. Activity and selectivity of photocatalysts in photodegradation of phenols. Journal of Hazardous Materials. 2012;211-212:154-160. DOI: 10.1016/j.jhazmat.2011.11.078
  43. 43. Hirakawa K, Sano S. Platinum nanoparticle catalyst scavenges hydrogen peroxide generated from hydroquinone. Bulletin of the Chemical Society of Japan. 2009;82:1299-1303. DOI: 10.1246/bcsj.82.1299
  44. 44. Kawanishi S, Hiraku Y, Oikawa S. Mechanism of guanine-specific DNA damage by oxidative stress and its role in carcinogenesis and aging. Mutation Research Reviews in Mutation Research. 2001;488:65-76. DOI: 10.1016/S1383-5742(00)00059-4
  45. 45. Agarwal K, Sharma A, Talukder G. Effects of copper on mammalian cell components. Chemico-Biological Interactions. 1989;69:1-16. DOI: 10.1016/0009-2797(89)90094-X
  46. 46. Bach D, Miller IR. Polarographic investigation of binding of Cu++ and Cd++ by DNA. Biopolymers. 1967;5:161-172. DOI: 10.1002/bip.1967.360050204
  47. 47. Bryan SE, Frieden E. Interaction of copper(II) with deoxyribonucleic acid below 30 degrees. Biochemistry. 1967;6:2728-2734. DOI: 10.1021/bi00861a012
  48. 48. Stoewe R, Prutz WA. Copper-catalyzed DNA damage by ascorbate and hydrogen peroxide: Kinetics and yield. Free Radical Biology and Medicine. 1987;3:97-105. DOI: 10.1016/S0891-5849(87)80003-5
  49. 49. Prutz WA, Butler J, Land EJ. Interaction of copper(I) with nucleic acids. International Journal of Radiation Biology. 1990;58:215-234. DOI: 10.1080/09553009014551581
  50. 50. Glorieux C, Calderon PB. Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach. Biological Chemistry. 2017;398:1095-1108. DOI: 10.1515/hsz-2017-0131
  51. 51. Hirakawa K. DNA damage through photo-induced electron transfer and photosensitized generation of reactive oxygen species. In: Kimura H, Suzuki A, editors. New Research on DNA Damage. Nova Science Publishers Inc., New York; 2008. pp. 197-219. ISBN: 978-1-60456-581-2
  52. 52. Gentil A, Le Page F, Cadet J, Sarasin A. Mutation spectra induced by replication of two vicinal oxidative DNA lesions in mammalian cells. Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis. 2000;452:51-56. DOI: 10.1016/S0027-5107(00)00034-8
  53. 53. Kalam MA, Basu AK. Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells: Tandem mutations and enhancement of GfT transversions. Chemical Research in Toxicology. 2005;18:1187-1192. DOI: 10.1021/tx050119r
  54. 54. Yuan B, Jiang Y, Wang Y, Wang Y. Efficient formation of the tandem thymine glycol/8-oxo-7,8-dihydroguanine lesion in isolated DNA and the mutagenic and cytotoxic properties of the tandem lesions in Escherichia coli cells. Chemical Research in Toxicology. 2010;23:11-19. DOI: 10.1021/tx9004264
  55. 55. Chaudhry MA, Weinfeld M. The action of Escherichia coli endonuclease III on multiply damaged sites in DNA. Journal of Molecular Biology. 1995;249:914-922. DOI: 10.1006/jmbi.1995.0348
  56. 56. Venkhataraman R, Donald CD, Roy R, You HJ, Doetsch PW, Kow YW. Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII. Nucleic Acids Research. 2001;29:407-414. DOI: 10.1093/nar/29.2.407
  57. 57. Budworth H, Dianova II, Podust VN, Dianov GL. Repair of clustered DNA lesions. Sequence-specific inhibition of long patch base excision repair by 8-oxoguanine. Journal of Biological Chemistry. 2002;277:21300-21305. DOI: 10.1074/jbc.M201918200
  58. 58. Budworth H, Dianov GL. Mode of inhibition of short-patch base excision repair by thymine glycol within clustered DNA lesions. Journal of Biological Chemistry. 2003;278:9378-9381. DOI: 10.1074/jbc.M212068200
  59. 59. Lomax ME, Cunniffe S, O’Neill P. 8-OxoG retards the activity of the ligase III/XRCC1 complex during the repair of a single-strand break, when present within a clustered DNA damage site. DNA Repair. 2004;3:289-299. DOI: 10.1016/j.dnarep.2003.11.006
  60. 60. Eot-Houllier G, Eon-Marchais S, Gasparutto D, Sage E. Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins. Nucleic Acids Research. 2005;33:260-271. DOI: 10.1093/nar/gki165
  61. 61. Oikawa S, Kawanishi S. Distinct mechanisms of site-specific DNA damage induced by endogenous reductants in the presence of iron(III) and copper(II). Biochimica et Biophysica Acta. 1998;1399:19-30. DOI: 10.1016/S0167-4781(98)00092-X
  62. 62. Kobayashi S, Ueda K, Morita J, Sakai H, Komano T. DNA damage induced by ascorbate in the presence of Cu2+. Biochimica et Biophysica Acta. 1998;949:143-147. DOI: 10.1016/0167-4781(88)90065-6
  63. 63. Burrows CJ, Muller JG. Oxidative nucleobase modifications leading to strand scission. Chemical Reviews. 1998;98:1109-1151. DOI: 10.1021/cr960421s
  64. 64. Kasai H, Yamaizumi Z, Berger M, Cadet J. Photosensitized formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-hydroxy-2′-deoxyguanosine) in DNA by riboflavin: A non singlet oxygen-mediated reaction. Journal of the American Chemical Society. 1992;114:9692-9694. DOI: 10.1021/ja00050a078
  65. 65. Cullis PM, Malone ME, Merson-Davies LA. Guanine radical cations are precursors of 7,8-dihydro-8-oxo-2′-deoxyguanosine but are not precursors of immediate strand breaks in DNA. Journal of the American Chemical Society. 1996;118:2775-2788. DOI: 10.1021/ja9536025
  66. 66. Hayes RC, Petrullo LA, Huang HM, Wallace SS, LeClerc JE. Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions. Journal of Molecular Biology. 1988;201:239-246. DOI: 10.1016/0022-2836(88)90135-0
  67. 67. D'Ham C, Romieu A, Jaquinod M, Gasparutto D, Cadet J. Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: Kinetic and mechanistic aspects. Biochemistry. 1999;38:3335-3344. DOI: 10.1021/bi981982b
  68. 68. Samson-Thibault F, Madugundu GS, Gao S, Cadet J, Wagner JR. Profiling cytosine oxidation in DNA by LC-MS/MS. Chemical Research in Toxicology. 2012;25:1902-1911. DOI: 10.1021/tx300195f
  69. 69. Dizdaroglu M. Oxidatively induced DNA damage and its repair in cancer. Mutation Research Reviews in Mutation Research. 2015;763:212-245. DOI: 10.1016/j.mrrev.2014.11.002
  70. 70. Kasai H. What causes human cancer? Approaches from the chemistry of DNA damage. Genes Environment. 2016;38:19. DOI: 10.1186/s41021-016-0046-8
  71. 71. Delaney S, Jarem DA, Volle CB, Yennie CJ. Chemical and biological consequences of oxidatively damaged guanine in DNA. Free Radical Research. 2012;46:420-441. DOI: 10.3109/10715762.2011.653968
  72. 72. Tudek B. Imidazole ring-opened DNA purines and their biological significance. Journal of Biochemistry and Molecular Biology. 2003;36:12-19. DOI: 10.5483/BMBRep.2003.36.1.012
  73. 73. Asagoshi K, Terato H, Ohyama Y, Ide H. Effects of a guanine-derived formamidopyrimidine lesion on DNA replication: Translesion DNA synthesis, nucleotide insertion, and extension kinetics. Journal of Biological Chemistry. 2002;277:14589-14597. DOI: 10.1074/jbc.M200316200
  74. 74. Hirakawa K, Yoshida M. Theoretical study of the effects of amino acids on one-electron oxidation of a nucleobase: Adenine residue can be a hole-trapping site. Pure and Applied Chemical Sciences. 2014;2:41-48. DOI: 10.12988/pacs.2014.424
  75. 75. Cai R, Hashimoto K, Kubota Y, Fujishima A. Increment of photocatalytic killing of cancer cells using TiO2 with the aid of superoxide dismutase. Chemistry Letters. 1992:427-430. DOI: 10.1246/cl.1992.427
  76. 76. Cai R, Kubota Y, Shuin T, Sakai H, Hashimoto K, Fujishima A. Induction of cytotoxicity by photoexcited TiO2 particles. Cancer Research. 1992;52:2346-2348
  77. 77. Kubota Y, Shuin T, Kawasaki C, Hosaka M, Kitamura H, Cai R, Sakai H, Hashimoto K, Fujishima A. Photokilling of T-24 human bladder cancer cells with titanium dioxide. British Journal of Cancer. 1994;70:1107-1111
  78. 78. Dunford R, Salinaro A, Cai L, Serpone N, Horikoshi S, Hidaka H, Knowland J. Chemical oxidation and DNA damage catalysed by inorganic sunscreen ingredients. FEBS Letters. 1997;418:87-90. DOI: 10.1016/S0014-5793(97)01356-2
  79. 79. Petković J, Küzma T, Rade K, Novak S, Filipič M. Pre-irradiation of anatase TiO2 particles with UV enhances their cytotoxic and genotoxic potential in human hepatoma HepG2 cells. Journal of Hazardous Materials. 2011;196:145-152. DOI: 10.1016/j.jhazmat.2011.09.004
  80. 80. Dexter DL. A theory of sensitized luminescence in solids. The Journal of Chemical Physics. 1953;21:836-850. DOI: 10.1063/1.1699044
  81. 81. Hirakawa K, Nishimura Y, Arai T, Okazaki S. Singlet oxygen generating activity of an electron donor-connecting porphyrin photosensitizer can be controlled by DNA. The Journal of the Physical Chemistry B. 2013;117:13490-13496. DOI: org/10.1021/jp4072444
  82. 82. Ohara K, Kikuchi K, Origuchi T, Nagaoka S. Singlet oxygen quenching by trolox C in aqueous micelle solutions. Journal of Photochemistry and Photobiology B: Biology. 2009;97:132-137. DOI: 10.1016/j.jphotobiol.2009.08.010
  83. 83. Hirakawa K, Azumi K, Nishimura Y, Arai T, Nosaka Y, Okazaki S. Photosensitized damage of protein by fluorinated diethoxyphosphorus(V)porphyrin. Journal of Porphyrins and Phthalocyanines. 2013;17:56-62. DOI: 10.1142/S1088424612501258
  84. 84. Hirakawa K, Hirano T, Nishimura Y, Arai T, Nosaka Y. Dynamics of singlet oxygen generation by DNA-binding photosensitizers. The Journal of the Physical Chemistry B. 2012;116:3037-3044. DOI: 10.1021/jp300142e
  85. 85. Matheson IBC, Lee J, King AD. The lifetime of singlet oxygen (1Δg) in heavy water, a revised value. Chemical Physics Letters. 1978;55:49-51. DOI: 10.1016/0009-2614(78)85129-X
  86. 86. Shimizu O, Watanabe J, Imakubo K, Naito S. Absolute quantum yields and lifetimes of photosensitized phosphorescence of singlet oxygen O2 (1Δg) in air-saturated aqueous and organic solutions of phenalenone. Chemistry Letters. 1999;28:67-68. DOI: 10.1246/cl.1999.67
  87. 87. Moghissi K, Dixon K, Stringer M, Thorpe JA. Photofrin PDT for early stage oesophageal cancer: Long term results in 40 patients and literature review. Photodiagnosis and Photodynamic Therapy. 2009;6:159-166. DOI: 10.1016/j.pdpdt.2009.07.026
  88. 88. Wang S, Bromley E, Xu L, Chen JC, Keltner L. Talaporfin sodium. Expert Opinion on Pharmacotherapy. 2010;11:133-140. DOI: 10.1517/14656560903463893
  89. 89. Hirakawa K, Ishikawa T. Phenothiazine dyes photosensitize protein damage through electron transfer and singlet oxygen generation. Dyes and Pigments. 2017;142:183-188. DOI: 10.1016/j.dyepig.2017.03.035
  90. 90. Konaka R, Kasahara E, Dunlap WC, Yamamoto Y, Chien KC, Inoue M. Irradiation of titanium dioxide generates both singlet oxygen and superoxide anion. Free Radical Biology and Medicine. 1999;27:294-300. DOI: 10.1016/S0891-5849(99)00050-7
  91. 91. Konaka R, Kasahara E, Dunlap WC, Yamamoto Y, Chien KC, Inoue M. Ultraviolet irradiation of titanium dioxide in aqueous dispersion generates singlet oxygen. Redox Report. 2001;6:319-325. DOI: 10.1179/135100001101536463
  92. 92. Nosaka Y, Daimon T, Nosakaa AY, Murakamia Y. Singlet oxygen formation in photocatalytic TiO2 aqueous suspension. Physical Chemistry Chemical Physics. 2004;6:2917-2918. DOI: 10.1039/B405084C
  93. 93. Naito K, Tachikawa T, Cui S, Sugimoto A, Fujitsuka M, Majima T. Single-molecule detection of airborne singlet oxygen. Journal of the American Chemical Society. 2006;128:16430-16431. DOI: 10.1021/ja066739b
  94. 94. Hirakawa K, Hirano T. Singlet oxygen generation photocatalyzed by TiO2 particles and its contribution to biomolecule damage. Chemistry Letters. 2006;35:832-833. DOI: 10.1246/cl.2006.832
  95. 95. Li W, Gandra N, Courtney SN, Gao R. Singlet oxygen production upon two-photon excitation of TiO2 in chloroform. Chemphyschem. 2009;10:1789-1793. DOI: 10.1002/cphc.200900155
  96. 96. Buchalska M, Łabuz P, Bujak Ł, Szewczyk G, Sarna T, MaćKowski S, Macyk W. New insight into singlet oxygen generation at surface modified nanocrystalline TiO2—The effect of near-infrared irradiation. Dalton Transactions. 2013;42:9468-9475. DOI: 10.1039/C3DT50399B
  97. 97. Dunlap WC, Yamamoto Y, Inoue M, Kashiba-Iwatsuki M, Yamaguchi M, Tomita K. Uric acid photo-oxidation assay: In vitro comparison of sunscreening agents. International Journal of Cosmetic Science. 1998;20:1-18. DOI: 10.1046/j.1467−2494.1998.171731.x
  98. 98. Miyoshi N, Kume K, Tsutumi K, Fukunaga Y, Ito S, Imamura Y, Bibin AB. Application of titanium dioxide (TiO2) nanoparticles in photodynamic therapy (PDT) of an experimental tumor. AIP Conference Proceedings. 2011;1415:21. DOI: 10.1063/1.3667210
  99. 99. Zhang H, Shan Y, Dong L. A comparison of TiO2 and ZnO nanoparticles as photosensitizers in photodynamic therapy for cancer. Journal of Biomedical Nanotechnology. 2014;10:1450-1457. DOI: 10.1166/jbn.2014.1961
  100. 100. Lucky SS, Muhammad Idris N, Li Z, Huang K, Soo KC, Zhang Y. Titania coated upconversion nanoparticles for near-infrared light triggered photodynamic therapy. ACS Nano. 2015;9:191-205. DOI: 10.1021/nn503450t
  101. 101. Jukapli NM, Bagheri S. Recent developments on titania nanoparticle as photocatalytic cancer cells treatment. Journal of Photochemistry and Photobiology B: Biology. 2016;163:421-430. DOI: 10.1016/j.jphotobiol.2016.08.046
  102. 102. Shu C, Kang P, Khan S, Foote CS. Low-temperature photosensitized oxidation of a guanosine derivative and formation of an imidazole ring-opened product. Journal of the American Chemical Society. 2002;124:3905-3913. DOI: 10.1021/ja011696e
  103. 103. Kang P, Foote CS. Formation of transient intermediates in low-temperature photosensitized oxidation of an 8-(13)C-guanosine derivatives. Journal of the American Chemical Society. 2002;124:4865-4873. DOI: 10.1021/ja012038x
  104. 104. Sugiyama H, Saito I. Theoretical studies of GG-specific photocleavage of DNA via electron transfer: Significant lowering of ionization potential and 5′-localization of HOMO of stacked GG bases in B-form DNA. Journal of the American Chemical Society. 1996;118:7063-7068. DOI: 10.1021/ja9609821
  105. 105. Yoshioka Y, Kitagawa Y, Takano Y, Yamaguchi K, Nakamura T, Saito I. Experimental and theoretical studies on the selectivity of GGG triplets toward one-electron oxidation in B-form DNA. Journal of the American Chemical Society. 1999;121:8712-8719. DOI: 10.1021/ja991032t
  106. 106. Kino K, Saito I, Sugiyama H. Product analysis of GG-specific photooxidation of DNA via electron transfer: 2-aminoimidazolone as a major guanine oxidation product. Journal of the American Chemical Society. 1998;120:7373-7374. DOI: 10.1021/ja980763a
  107. 107. Kino K, Sugiyama H. Possible cause of GC→CG transversion mutation by guanine oxidation product, imidazolone. Chemistry and Biology. 2001;8:369-378. DOI: 10.1016/S1074-5521(01)00019-9
  108. 108. McBride TJ, Schneider JE, Floyd RA, Loeb LA. Mutation induced by methylene blue plus light in single-stranded M13mp2. Proceedings of the National Academy of Sciences of the United States of America. 1992;89:6866-6870
  109. 109. Negishi K, Hao W. Spectrum of mutations in single-stranded DNA phage M13mp2 exposed to sunlight: Predominance of G-to-C transversion. Carcinogenesis. 1992;13:1615-1618. DOI: 10.1093/carcin/13.9.1615
  110. 110. Drobetsky EA, Turcotte J, Chateauneuf A. A role for ultraviolet A in solar mutagenesis. Proceedings of the National Academy of Sciences of the United States of America. 1995;92:2350-2354
  111. 111. Michaeli A, Feitelson J. Reactivity of singlet oxygen toward amino acids and peptides. Photochemistry and Photobiology. 1994;59:284-289. DOI: 10.1111/j.1751-1097.1994.tb05035.x
  112. 112. Ehrenshaft M, Silva SO, Perdivara I, Bilski P, Sik RH, Chignell CF, Tomer KB, Mason RP. Immunological detection of N-formylkynurenine in oxidized proteins. Free Radical Biology and Medidcine. 2009;4:1260-1266. DOI: 10.1016/j.freeradbiomed.2009.01.020
  113. 113. Thomas AH, Serrano MP, Rahal V, Vicendo P, Claparols C, Oliveros E, Lorente C. Tryptophan oxidation photosensitized by pterin. Free Radical Biology and Medidcine. 2013;63:467-475. DOI: 10.1016/j.freeradbiomed.2013.05.044
  114. 114. Jensen RL, Arnbjerg J, Ogilby PR. Reaction of singlet oxygen with tryptophan in proteins: A pronounced effect of the local environment on the reaction rate. Journal of the American Chemical Society. 2012;134:9820-9826. DOI: 10.1021/ja303710m
  115. 115. Hirakawa K, Umemoto H, Kikuchi R, Yamaguchi H, Nishimura Y, Arai T, Okazaki S, Segawa H. Determination of singlet oxygen and electron transfer mediated mechanisms of photosensitized protein damage by phosphorus(V)porphyrins. Chemical Research in Toxicology. 2015;28:262-267. DOI: 10.1021/tx500492w
  116. 116. He XM, Carter DC. Atomic structure and chemistry of human serum albumin. Nature. 1992;358:209-215. DOI: 10.1038/358209a0
  117. 117. Li MY, Cline CS, Koker EB, Carmichael HH, Chignell CF, Bilski P. Quenching of singlet molecular oxygen (1O2) by azide anion in solvent mixtures. Photochemistyr and Photobiology. 2001;74:760-764. DOI: 10.1562/0031-8655(2001)0740760QOSMOO2.0.CO2
  118. 118. Ouyang D, Hirakawa K. Photosensitized enzyme deactivation and protein oxidation by axial-substituted phosphorus(V) tetraphenylporphyrins. Journal of Photochemistry and Photobiology B: Biology. 2017;175:125-131. DOI: 10.1016/j.jphotobiol.2017.08.036
  119. 119. Hirakawa K. Using folic acids to detect reactive oxygen species. In: Taylor JC. editors. Advances in Chemistry Research. Volume 26. Nova Science Publishers Inc., New York; 2015. pp. 111-126. ISBN: 978-1-63463-630-8
  120. 120. Garcia-Diaz M, Huang YY, Hamblin MR. Use of fluorescent probes for ROS to tease apart Type I and Type II photochemical pathways in photodynamic therapy. Methods. 2016;109:158-166. DOI: 10.1016/j.ymeth.2016.06.025
  121. 121. Kalyanaraman B, Hardy M, Podsiadly R, Cheng G, Zielonka J. Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling. Archives Biochemistry and Biophysics. 2017;617:38-47. DOI: 10.1016/
  122. 122. Guo H, Aleyasin H, Dickinson BC, Haskew-Layton RE, Ratan RR. Recent advances in hydrogen peroxide imaging for biological applications. Cell & Bioscience. 2014;4:64. DOI: 10.1186/2045-3701-4-64
  123. 123. Abo M, Urano Y, Hanaoka K, Terai T, Komatsu T, Nagano T. Development of a highly sensitive fluorescence probe for hydrogen peroxide. Journal of the American Chemical Society. 2011;133:10629-10637. DOI: 10.1021/ja203521e
  124. 124. Hirakawa K. Fluorometry of singlet oxygen generated via a photosensitized reaction using folic acid and methotrexate. Analytical and Bioanalytical Chemistry. 2009;393:999-1005. DOI: 10.1007/s00216-008-2522-x
  125. 125. Land EJ, Ebert M. Pulse radiolysis studies of aqueous phenol. Water elimination from dihydroxycyclohexadienyl radicals to form phenoxyl. Transactions of the Faraday Society. 1967;63:1181-1190. DOI: 10.1039/TF9676301181
  126. 126. Liao JC, Roider J, Jay DG. Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals. Proceedings of the National Academy of Sciences of the United States of America. 1994;91:2659-2663
  127. 127. Tørring T, Helmig S, Ogilby PR, Gothelf KV. Singlet oxygen in DNA nanotechnology. Accounts of Chemical Research. 2014;47:1799-1806. DOI: 10.1021/ar500034y
  128. 128. Hirakawa K. Control of fluorescence and photosensitized singlet oxygen-generating activities of porphyrins by DNA: Fundamentals for “theranostics”. In: Yilmaz Y, editor. Phthalocyanines and Some Current Applications. InTechOpen, London; 2017. p. 169-188. ISBN: 978-953-51-3255-4. DOI: 10.5772/67882
  129. 129. Horiuchi H, Kuribara R, Hirabara A, Okutsu T. pH-Response optimization of amino-substituted tetraphenylporphyrin derivatives as pH-activatable photosensitizers. The Journal of Physical Chemistry A. 2016;120:5554-5561. DOI: 10.1021/acs.jpca.6b05019
  130. 130. Hirakawa K, Harada M, Okazaki S, Nosaka Y. Controlled generation of singlet oxygen by a water-soluble meso-pyrenylporphyrin photosensitizer through interaction with DNA. Chemical Communications. 2012;48:4770-4772. DOI: 10.1039/c2cc30880k
  131. 131. Hirakawa K, Ito Y, Yamada T, Okazaki S. Relaxation process of the photoexcited state and singlet oxygen generating activity of water-soluble meso-phenanthrylporphyrin in a DNA microenvironment. Rapid Communication in Photoscience. 2014;3:81-84. DOI: 10.5857/RCP.2014.3.4.81
  132. 132. Hirakawa K, Taguchi M, Okazaki S. Relaxation process of photoexcited meso-naphthylporphyrins while interacting with DNA and singlet oxygen generation. The Journal of Physical Chemistry B. 2015;119:13071-13078. DOI: 10.1021/acs.jpcb.5b08025
  133. 133. Kuin A, Aalders M, Lamfers M, van Zuidam DJ, Essers M, Beijnen JH, Smets LA. Potentiation of anti-cancer drug activity at low intratumoral pH induced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) and its analogue benzylguanidine (BG). British Journal of Cancer. 1999;79:793-801. DOI: 10.1038/sj.bjc.6690127
  134. 134. Gupta SC, Singh R, Asters M, Liu J, Zhang X, Pabbidi MR, Watabe K, Mo YY. Regulation of breast tumorigenesis through acid sensors. Oncogene. 2016;35:4102-4111. DOI: 10.1038/onc.2015.477
  135. 135. Shi R, Huang L, Duan X, Sun G, Yin G, Wang R, Zhu JJ. Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe. Analytica Chimica Acta. 2017;988:66-73. DOI: 10.1016/j.aca.2017.07.055

Written By

Kazutaka Hirakawa

Submitted: 23 June 2017 Reviewed: 05 October 2017 Published: 20 December 2017