6 Antioxidant Properties of Singlet Oxygen Suppressors
6.3 Singlet Oxygen: General Properties, Detection Methods
and Biological Roles
Molecular oxygen is unusual amongst com-mon molecules because it has an electronic configuration that has two highest energy electrons unpaired in the degenerated high-est occupied molecular orbitals (HOMOs) (Fig. 6.2). The ground-state oxygen is there-fore a triplet state. We may expect this spe-cies to have radical character and to react readily with any radical species and there-fore to facilitate the progression of radical
chain reactions (McCord and Fridovic, 1969; Halliwell, 2009).
The two electronically excited states immediately above the ground state are both singlet states, which are generically called singlet oxygen (1O2). The first, designated sigma state (1Sg+), has a very short lifetime (less than a picosecond) and rapidly decays to the lower singlet state (Schmidt and Bodesheim, 1994); the other designated delta state (1Dg) has a lifetime varying from microseconds to milliseconds in the con-densed phase (Schmidt, 2006). Singlet oxy-gen 1Sg+ lifetime in solution is so short that, for practical purposes, it is deactivated immediately to the 1Dg state. 1O2 (1Sg+) and (1Dg) are, respectively, 96 kJ mol−1 (22.4 kcal mol−1) and 159.6 kJ mol−1 (38.2 kcal mol−1), above the ground-state oxygen and, although they are not directly accessible in a spin-allowed transition from ground state, it can be trivially formed by photosensitization by triplet states. The triplet state of photosensi-tizers, with energy higher than 96 kJ/mol, can efficiently transfer energy to molecular ground-state oxygen forming ground-state photosensitizers and excited-state oxygen (Abdel-Shafi and Wilkinson, 2002), as detailed below. Living organisms use sev-eral molecules that absorb UV–VIS photons;
therefore, photosensitization is a frequent way in which singlet oxygen is generated in living organisms. Because 1O2 has an empty orbital, it can directly react with double bonds, engaging in chemical reactions that were not allowed with ground-state oxygen.
Consequently, many biological molecules and macromolecules have evolved to pro-tect living organisms from forming singlet oxygen and from its effect after it has been
Singlet 1Sg+
Ground 3Sg– Singlet 1Dg
Fig. 6.2. Simplified electronic configuration of oxygen in the triplet ground 3Sg+ and singlet states,
1Dg / 1Sg+.
formed. Singlet oxygen can also be formed by chemical/biochemical reactions, espe-cially by the Russell Mechanism present in peroxidation processes (Miyamoto et al., 2007), which may indicate a possible role for singlet oxygen in signalling events mainly related to the cellular stress response (Klotz et al., 2003).
A small fraction of the population of singlet oxygen molecules that is formed decays to the ground state emitting light in the near infrared region (NIR) lMAX = 1268 nm (Krasnovskii, 1976; Khan and Kasha, 1979;
Wilkinson et al., 1993) and this is the spec-tral fingerprint of singlet oxygen molecules (Fig. 6.3). This detection method has been used to observe singlet oxygen in vivo in tissues and in vitro in different types of solutions and/or suspensions (Niedre et al., 2002; Kuimova et al., 2009). Usual detection
equipment includes a laser system to provide light excitation and generation of singlet oxygen and a NIR fluorometer to detect its characteristic emission (Fig. 6.3).
Singlet oxygen can also be detected and quantified using chemical trap methods and extremely selective probes have been designed. Natural molecules, such as beta-nidines found in beetroot, have also been shown to work well as a probe to detect and quantify 1O2 (Bonacin et al., 2009).
Singlet oxygen can be generated in a controlled and reproducible way either by chemical or by physical methods. The most used chemical methods are: the reaction between hydrogen peroxide and sodium hypochlorite, N-chlorosuccinimide and alkaline hydrogen peroxide; and the ther-molysis of several endoperoxides (Baptista, 1998). By physical methods, it is possible
SAMPLE
MONO NIR-PMT
CONTROL
COMPUTER F
LASER (a)
(b) (c)
Emission at 1270 nm
Emission (a.u.)
1.0
12.0
6.0
0.0 (ii) (i)
5.0 10.0
Time (µs) 0.8
0.6 0.4
Wavelength (nm)
1200 1240 1280 1320
Fig. 6.3. (a) Experimental set-up to prove and study the generation and reactivity of singlet oxygen.
Equipment is built with laser sources that are used to excite photosensitizer molecules that form triplet states and react with oxygen forming 1O2. The light emission is filtered with silicon and/or interference filters (F), passes through a monochromator and is detected either by a NIR-PMT (faster and more sensitive) or by a Germaniun detector. (b) Characteristic NIR emission spectra of 1O2 generated in aqueous solution of Methylene Blue (10 mM) after excitation at 532 nm (10 mJ/pulse, 10 Hz). (c) Transient decay of 1O2 in aqueous solution, the lifetime of which is ~3 ms (i) and in aqueous solution in the presence of 1 mM sodium azide (ii) which is a commonly used agent that suppresses 1O2 and consequently reduces its lifetime.
Antioxidant Properties of Singlet Oxygen Suppressors 69
to obtain singlet oxygen by direct excita-tion of molecular oxygen using irradiaexcita-tion with an intense light source in the 0–1 tran-sition (1070 nm), but this is a spin-forbidden process and is therefore inefficient. It requires a pressure cell in which oxygen is dissolved in a good solvent (such as hex-afluorobenzene) under high pressure (140 atmospheres). It is also possible by micro-wave discharge in a steam of oxygen at 1–10 nm, which generates a mixture of sin-glet oxygen and atomic oxygen, the latter being scrubbed out by passing the gas stream over mercuric oxide (Baptista, 1998). Finally, it is possible to generate it chemically by thermal decomposition (Foote, 1968); however, the most common method for producing singlet oxygen in the laboratory is by photosensitization with a strongly absorbing dye such as methylene blue (Severino et al., 2003) or chlorophyll (Krasnovskii, 1976).
Photosensitization is a process in which a molecule absorbs light and gets excited from the ground-state (PS) into a singlet, a short-lived (~10−9 s) excited state (1PS*) that can be deactivated by chemical reactions, or
by radiative and non-radiative processes.
A good photosensitizer (PS) will undergo a spin-forbidden intersystem crossing that requires a spin inversion, converting the PS to a triplet state (3PS*). The triplet states relax back to ground states via a spin-forbidden radiative pathway (phosphorescence), which imposes relatively long lifetimes. The triplet state can also be disabled by electron or proton transfer, originating radicals, as in mechanism type I (Fig. 6.4). In oxygenated environments, PS can undergo a type II photochemical process that involves energy transfer between the excited triplet state of photosensitizer (3PS*) and the triplet state of molecular oxygen (3O2), producing short-lived and highly reactive excited singlet oxygen (1O2) (Wilkinson et al., 1993; Abdel-Shafi and Wilkinson, 2002; Junqueira et al., 2002; Schmidt, 2006). The competition between type I and type II reactions is diffi-cult to predict in the biological environment because the presence of biomolecules or interfaces can shift the relative rates of these processes that are observed in anisotropic solutions (Macpherson et al., 1993; Baptista and Indig, 1998).
SUBSTRATE
PRODUCT
PRODUCT
Reactions Type I
Reactions Type II MECHANISMS OF PHOTOSENSITIZED
OXIDATIONS
PRODUCT RADICALS
BM BM 1PS∗
PS
e–+2H+
3PS∗
O2
O2.–
O2 O2
H2O2
OH– OH Fe2+
Fe3+
1O2∗
Fig. 6.4. Photosensitization mechanisms, where PS is a photosensitizer that absorbs light going to the first singlet state (1PS*), converting into a triplet state (3PS*) by intersystem crossing. The excited species, especially 3PS*, can react by electron transfer forming radical species (Type I mechanism) and start radical chain reactions or react with molecular oxygen by energy transfer forming singlet oxygen (Type II mechanism). BM, biomolecules.
Plants are living organisms that survive the interaction with light. Therefore, in plant tissues, photosensitization reactions and singlet oxygen generation always com-pete with normal electron transfer reactions of the energy conversion process in photo-synthesis. Formation of 1O2 in photosystem II (PSII) of plants was invariably confirmed by the detection of its characteristic NIR emission at 1270 nm (Vass et al., 1992;
Telfer et al., 1994). The generation mecha-nism involves the reduction of quinone acceptors and back-electron transfer between reduced pheophytin and oxidized P680, leading to the formation of triplet spe-cies (Durrant et al., 1990). However, the details of 1O2 generation are still a matter of debate and triplets derived from other pho-tosynthetic reaction centre (RC) pigments have also been detected (Rinalducci et al., 2004). The formation of 1O2 in antenna- complex trimer proteins has been suggested to be the result of direct generation of 1O2 by oxygen quenching of triplet chlorophyll species formed in antenna complexes after light absorption and intersystem crossing (Krieger-Liszkay, 2005; Uchoa et al., 2008;
Triantaphylides and Havaux, 2009). In the case of Rhodobacter sphaeroides RCs, Uchoa and coworkers have shown that bac-teriopheophytin triplets are another possi-ble source of 1O2 (Uchoa et al., 2008).
It is becoming clear that understanding and controlling singlet oxygen generation in plants may be a key factor for improving crop yield, because overproduction of 1O2
can lead to photo-inhibition of photosyn-thesis and destruction of the photo-synthetic RC. Plants have developed macromolecular supra-structures and a myriad of antioxidant molecules to decrease the rate of formation of singlet oxygen by suppressing triplets and also to directly suppress singlet oxygen molecules that may be formed (Uchoa et al., 2008; Triantaphylides and Havaux, 2009). The quantum yield of singlet oxygen (fD, number of times that sin-glet oxygen molecules are generated per pho-ton absorbed), from the RCs of R. sphaeroides is 0.03 (Uchoa et al., 2008), which is consid-erably smaller than fD calculated for PSII of plants, which was calculated to be 0.2
(Telfer et al., 1994). This fact is in agreement with the smaller tendency of photo-inhibition in wild-type R. sphaeroides compared with plants and also with carotenoidless strains of purple bacteria (Uchoa et al., 2008).
Carotenoids are especially efficient sup-pressors of PS triplets and of 1O2. The main role of carotenoids in photosynthesis is to quench triplet states that are eventually formed in the RCs before they photosensitize
1O2 formation. Carotenoids may, however, also suppress 1O2 molecules that are formed in the RCs. Proof of this role for carotenoids may be obtained by comparing the efficiency of 1O2 generation in different strains of pur-ple bacteria. In RCs of R. sphaeroides, carote-noids are located within van der Waals distance of bacteriochlorophylls (~3.7 Å) and at 10 Å of a dimer pair of bacteriochloro-phylls suppressing triplets and singlet oxy-gen that are formed. Rhodopseudomonas viridis is a strain of purple bacteria that lacks carotenoids. Consequently, one could expect a higher efficiency of singlet oxygen genera-tion. In fact, Uchoa and coworkers have most exposed areas and therefore are the tis-sues most prone to have photodamage (Fattorusso, 1974; Krishna et al., 1991;
Halliwell et al., 1992; Chiarelli-Neto et al., 2011). Riboflavin derivatives are widely spread in living organisms, absorb light in the UVA spectral region (Speck et al., 1975;
Lu et al., 2000) and are known to efficiently produce 1O2 (FD = 0.5) (Wilkinson et al., 1993; Morita et al., 1997; Baier et al., 2006).
In fact, flavin co-enzymes FAD, FADH and FMN, which are of vital importance in cel-lular metabolism, are considered responsi-ble for a series of endogenous photodamage in the skin, which is started by UVA absorp-tion and generaabsorp-tion of 1O2 (Berneburg et al., 1999; Kessel, 2000). It has been shown recently that melanin itself can generate 1O2
under visible light exposure, showing the importance of understanding in more detail
Antioxidant Properties of Singlet Oxygen Suppressors 71
the photosensitization processes occurring in biological surfaces in contact with sun-light (Chiarelli-Neto et al., 2011).
Singlet oxygen has been shown to mediate the induction of expression of several redox defence genes (Klotz et al., 2003; Luo et al., 2006) as well as to cause the mitochondrial common deletion, which is associated with skin photo-ageing (Berneburg et al., 1999; Wertz et al., 2005).
Gene expression induced by UVA in HaCaT keratinocytes is highly altered by the presence of b-carotene, an effect that was related to the suppression of 1O2, as well as with direct effects of b-carotene in HaCaT cells (Wertz et al., 2005). Several other workers have reported evidence of the role of singlet oxygen in UVA photo-induced damage (Krishna et al., 1991;
Halliwell et al., 1992; Sander et al., 2004).
Photodamage in DNA molecules induced by endogenous and exogenous riboflavin (vitamin B2) and derivatives were reported, as well as the role of vitamin C acting as an 1O2 suppressor (Cross et al., 1998;
Besaratinia et al., 2007).
Humans have several defence systems to protect from photodamage, including a small-molecule antioxidant present in the intercellular compartments of korneocytes and melanin, which is a biopolymer that effi-ciently absorbs UV–VIS radiation as well as being an efficient antioxidant agent (Krol and Liebler, 1998; Callado, 2007) and also generates singlet oxygen (Chiarelli-Neto et al., 2011). Even so, over-exposure to sun can lead to several skin manifestations including wrinkles, advance senescence of dermal fibroblasts and epidermal keratino-cytes, which eventually can lead to the development of a cancer (Callado, 2007).
Clearly these effects are more pronounced in less protected skins that have, among other differences, lower amounts of melanin (Slominski et al., 2004). Overproduction of
1O2 can lead to a human degenerative disease called porphyria, caused by the accumula-tion of porphyrins or porphyrin precursors (Straka et al., 1990; Baptista, 1998). The symptoms arise mostly from effects on the nervous system and on the skin. Skin mani-festations can include burning, blistering
and scarring of sun-exposed areas (Bickers et al., 2006).
The study of porphyria and its action mechanisms have contributed significantly to the development of therapies based on photodynamic therapy (PDT). PDT is a promising modality for the management of various tumours and non-malignant dis-eases, based on the combination of a photo-sensitizer that is selectively localized in the target tissue and illumination of the lesion with visible light, resulting in photodamage and subsequent cell death, which is mainly due to singlet oxygen (Fattorusso, 1974;
Wilson et al., 1992; Kalka et al., 2000; Kessel, 2000; Tardivo et al., 2006). The photosensi-tizer can also get involved in electron trans-fer reactions, initiating radical-induced damage in biomolecules (Baptista and Indig, 1998; Junqueira et al., 2002; Severino et al., 2003). Although the initial PDT protocols were very expensive and only performed in a few hospitals with expensive laser sys-tems, recently much attention has been paid to developing inexpensive PDT protocols to treat several diseases (Tardivo et al., 2006;
Tardivo and Baptista, 2009).
6.4 Chemical Reactivity