Biological hydroperoxides and singlet molecular oxygen generation

Critical Review

Biological Hydroperoxides and Singlet Molecular Oxygen Generation

Sayuri Miyamoto, Graziella E. Ronsein, Fernanda M. Prado, Miriam Uemi, Thais C. Correa,

Izaura N. Toma, Agda Bertolucci, Mauricio C. B. Oliveira, Flavia D. Motta, Marisa H. G. Medeiros and

Paolo Di MascioDepartamento de Bioquımica, Instituto de Quımica, Universidade de Sao Paulo, Sao Paulo, SP, Brazil

Summary

The decomposition of lipid hydroperoxides (LOOH) into

peroxyl radicals is a potential source of singlet molecular oxygen

(1O2) in biological systems. Recently, we have clearly demonstrated

the generation of1O2 in the reaction of lipid hydroperoxides with

biologically important oxidants such as metal ions, peroxynitrite

and hypochlorous acid. The approach used to unequivocally

demonstrate the generation of1O2 in these reactions was the use

of an isotopic labeled hydroperoxide, the18O-labeled linoleic acid

hydroperoxide, the detection of labeled compounds by HPLC

coupled to tandem mass spectrometry (HPLC-MS/MS) and the

direct spectroscopic detection and characterization of1O2 light

emission. Using this approach we have observed the formation of18O-labeled

1O2 by chemical trapping of

1O2 with anthracene

derivatives and detection of the corresponding labeled endoperoxide

by HPLC-MS/MS. The generation of1O2 was also demonstrated

by direct spectral characterization of1O2 monomol light emission in

the near-infrared region (l¼ 1270 nm). In summary, our studies

demonstrated that LOOH can originate1O2. The experimental

evidences indicate that1O2 is generated at a yield close to 10% by

the Russell mechanism, where a linear tetraoxide intermediate is

formed in the combination of two peroxyl radicals. In addition to

LOOH, other biological hydroperoxides, including hydroperoxides

formed in proteins and nucleic acids, may also participate in

reactions leading to the generation1O2. This hypothesis is currently

being investigated in our laboratory.

IUBMB Life, 59: 322–331, 2007

Keywords Lipid hydroperoxides; singlet molecular oxygen;18O-labeled oxygen; protein hydroperoxides; DNAhydroperoxides; mass spectrometry; near-infraredemission.

INTRODUCTION

Singlet molecular oxygen (1O2) is a strong oxidant

that displays considerable reactivity towards electron-

rich organic molecules including, nucleic acids, proteins

and lipids (1). Evidence has been accumulated indicat-

ing that 1O2 is implicated in the genotoxic effect of the

UVA (320 – 380 nm), component of solar radiation and is

likely to play an important role in the cell signaling

cascade associated with apoptosis (2, 3). Recently, elevated

level of 1O2 has been considered to be a key chemical

event associated with genetic controlled cell death in

plants (3).

Up to now, several photochemical and nonphotochemical

reactions have been shown to produce 1O2 in biological

system. Singlet molecular oxygen can be produced in

biological systems by photoexcitation (Type II reactions)

upon exposure of endogenous photosensitizers (porphyrins,

flavins, quinones, etc.) to UVA (4). The photochemical type

II generation of 1O2 induces tumor cell death, and plays very

important roles in photodynamic therapy. Singlet molecular

oxygen can be also generated by chemical reactions involving

peroxides. Among them, the reactions involved in the

generation of 1O2 during phagocytosis (5, 6), lipid peroxida-

tion (7), and the catalytic mechanisms of peroxidases (8, 9)

have been extensively studied due to their biological

relevance.

In this paper we describe the generation of 1O2 from

biological hydroperoxides, particularly the generation from

LOOH, which has been the focus of our studies in the past few

years. These peroxides are formed in biological systems and

they have multiple damaging effects on cellular macromole-

cules and are also important regulators of many cellular

processes. Evidence and strategies used to demonstrate 1O2

generation from LOOH will be presented and the possibility of1O2 generation from other biologically relevant hydroper-

oxides, such as protein hydroperoxides and DNA hydroper-

oxides, will be discussed.

Received 25 January 2007; accepted 25 January 2007Address correspondence to: Sayuri Miyamoto or Paolo Di Mascio,

Departamento de Bioquımica, Instituto de Quımica, Universidade deSao Paulo, CP 26077, CEP 05513-970, Sao Paulo, SP, Brazil.Tel: þ55 (11) 3091 3815 (ext. 224). Fax: þ55 (11) 38155579.E-mail: [email protected] or E-mail: [email protected]

IUBMBLife, 59(4 – 5): 322 – 331, April –May 2007

ISSN 1521-6543 print/ISSN 1521-6551 online � 2007 IUBMB

DOI: 10.1080/15216540701242508

HYDROGEN PEROXIDE

Hydrogen peroxide (H2O2) is generated endogenously

during electron transport in mitochondria. The ‘leakage’ of

electrons partially reduces oxygen to produce superoxide

anions (O2.7). Subsequent dismutation of O2

.7 generates

H2O2, which in the presence of transition metals produces

highly reactive hydroxyl radicals (HO.). H2O2 has been

demonstrated to act as an important messenger in signal

transduction pathways, particularly in the apoptotic machin-

ery (10).

It is well established that H2O2 reacts with hypochlorous

acid (HOCl) to yield stoichiometric amounts of 1O2 (11)

(Eq.1). Reactions of H2O2 with NO. (12) and peroxynitrite

(ONOO7) (13) have been also reported to generate 1O2.

Direct evidence of 1O2 in the reaction of H2O2 with HOCl was

first demonstrated by Khan and Kasha in 1963 (14). HOCl is a

potent oxidant generated in neutrophils by the reaction of

chloride ion with hydrogen peroxide catalyzed by myeloper-

oxidase (5, 6). This heme enzyme is stored at high concentra-

tions in the granules of phagocytic cells (neutrophils and

monocytes), and upon stimulation it is secreted into both the

extracellular milieu and the phagocytic vacuole (5, 6). It is

believed that the generation of 1O2 by the reaction of H2O2

with HOCl constitutes an important defense mechanism

against microorganisms (5).

HOClþH2O2 ! 1O2 þ Cl� þH2OþHþ ð1Þ

The involvement of 1O2 in the microbicidal action of

polymorphonuclear (PMN) leukocytes has been extensively

studied. In 1972, Allen et al. (15) observed an emission of light

in the visible region from human PMN leukocytes, which they

associated with the decay of 1O2 to the triplet ground state.

This observation prompted them to suggest the formation of1O2 as the microbicidal agent during phagocytosis of engulfed

microorganisms. Subsequently in 1974, Krinsky also obtained

evidence indicating 1O2 as one of the bactericidal agents in

human leukocytes using colorless mutant strains of Sarcina

lutea and carotenoid containing wild type strains (16). Later in

1992, Steinbeck et al. detected the production of 1O2 by

phagocytosing PMN leukocytes using particles coated with a

chemical trap (17).

The formation of 1O2 during phagocytosis is mostly

attributed to the reaction of H2O2 with HOCl (6), although

some studies also suggest the generation of 1O2 by the

interaction of O2.7 and H2O2, the so-called Haber-Weiss

reaction (Eq. 2) (18). However, the biological relevance of 1O2

by this reaction is still controversial. Recently, we have also

observed the formation of 1O2 in the reaction of LOOH with

HOCl (19) (more details in the next section). The physiological

relevance of this findings remains to be clarified. Nonetheless,

the generation of 1O2 in this reaction may be an additional

important mechanism that contributes to the microbicidal

activity of HOCl during phagocytosis, as well as, for the

propagation of lipid peroxidation in pathologic conditions

involving inflammatory processes, such as atherosclerosis and

cancer.

H2O2 þO2�� ! O2 þ �OHþOH� ð2Þ

LIPID HYDROPEROXIDES

Cell membranes are potential targets of attack by reactive

oxygen and nitrogen species. These species are capable of

abstracting a hydrogen atom from a bis-allylic methylene

group present in polyunsaturated fatty acids, thus initiating a

chain reaction known as lipid peroxidation. During this

process, membrane lipids are oxidized yielding LOOH as

primary products (20). LOOH are also generated in 1O2-

mediated oxidations and by the action of enzymes such as

lipoxygenases and cyclooxygenases (20) (Fig. 1).

Once formed, LOOH can participate in reactions that can

either decrease or increase its toxicity (Fig. 1). Normally cells

are endowed with enzymes capable of reducing LOOH to less

reactive lipid hydroxides (LOH) (21). The reductive detox-

ification is mediated by enzymes belonging to the glutathione

peroxidase (GPx) family, as well by glutathione transferases

(21). It has also been described that phospholipid hydroper-

oxides can be either directly reduced by phospholipid

hydroperoxide glutathione peroxidase (PHGPx) or cleaved

by phospholipase A2 (PLA2), releasing fatty acid hydroper-

oxides (abbreviated as LOOH).

Although at normal conditions most of LOOH are

reduced to the less reactive hydroxides, there are certain

conditions where LOOH are not efficiently detoxified and

thus can participate in reactions leading to increased toxicity.

It is well known that LOOH causes changes in the structural

organization and packing of membrane lipid components,

thus leading to alteration in membrane fluidity and proper-

ties. Moreover, LOOH can affect many cellular processes,

leading to increased cellular protection (22), or promoting

cell death by induction of apoptotic pathways (23). Several

classes of hydroperoxides including fatty acid hydroperox-

ides, phospholipid hydroperoxides, cholesterol hydroperox-

ides and cholesteryl ester hydroperoxides have been detected

and characterized both in vitro and in vivo (20) (Fig. 2).

These hydroperoxides may accumulate in several pathologi-

cal conditions and attention has been focused on elucidating

their pathophysiological role.

Most hydroperoxides are stable at room temperature, but

they can be decomposed by heating, exposure to UV light (in

some cases) or by the addition of transition metals. Hydro-

peroxides are readily broken in the presence of trace amounts

of catalytic transition metals (e.g., Fe2þ, Cu2þ), as well as by

heme-proteins. Metal-induced decomposition of LOOH yields

lipid alkoxyl (LO.) and/or lipid peroxyl radicals (LOO.) (20).

These oxyl radicals are responsible for the propagation of the

BIOLOGICAL HYDROPEROXIDES AND 1O2 GENERATION 323

oxidation process, as well as, the generation of other highly

reactive products capable to cause modifications in proteins

and mutagenic lesions. Among these products are electrophilic

aldehydes, epoxides, ketones, and excited species, such as 1O2

and electronically excited cabonyl species. Indeed, both excited

species have been identified as the chemiluminescence emitters

in the ultra-weak luminescence associated with lipid peroxida-

tion in biological systems (7).

The chemiluminescence (CL) produced by a chemical

reaction provides important information about the excited

species being generated. Two types of CL arise from 1O2:

dimol light emission, which corresponds to the simultaneous

deactivation of two molecules of 1O2 to the ground state

with photoemission in the visible red region at 634 and

703 nm; and monomol light emission, which corresponds to

the deactivation of one molecule of 1O2 to the ground state

with photoemission in the near-infrared region at 1270 nm.

On the other hand, excited carbonyl species emit between

380 and 460 nm. The acquisition of spectrally resolved CL

band can be used as an important tool to identify the

emitting species. This has proven to be a particularly useful

way for the unequivocal identification of 1O2 in the near-

infrared region.

It has been proposed that excited species generated in

the course of lipid peroxidation arise mainly from the

annihilation of peroxyl radicals by the Russell mechanism

(24 – 26). In 1957, Russell proposed that the combination of

two peroxyl radicals proceed through the generation of a

linear tetraoxide intermediate, which undergoes rapid

fragmentation generating a ketone, an alcohol and mole-

cular oxygen (24) (Fig. 3). It has been postulated that in the

case of primary and secondary peroxyl radicals this reaction

generates either an electronically excited oxygen molecule or

an electronically excited ketone (25). The tetraoxide decom-

position requires that the hydrogen-a of one peroxyl radical

is transferred to the second peroxyl radical in one of the

rate-controlling steps of the reaction (25). This step is

critical for the elimination of oxygen from the tetraoxide in

the singlet-excited state. Indeed, subsequent studies pointed

out that the termination reactions involving primary or

Figure 1. Scheme illustrating the generation and fate of lipid hydroperoxides in membranes. Phospholipid hydroperoxides can be

reduced to less reactive hydroxides by phospholipid glutathione peroxidase (PHGPx) or hydrolyzed by phospholipase A2

(PLA2) releasing fatty acid hydroperoxides (LOOH). These hydroperoxides can be reduced by glutathione peroxidases (GPx) or

decomposed by metal ions, ONOO7 or HOCl generating fatty acid peroxyl (LOO.) and/or alcoxyl radicals (LO.) that can

participate in reactions leading to the formation of lipid fragmentation products, propagation of lipid peroxidation and

generation of 1O2 by the Russell mechanism.

324 MIYAMOTO ET AL.

secondary peroxyl radical generates predominantly 1O2 (27).

The yield of excited ketones was 103 – 104 lower than of 1O2

(27). The 1O2 yield in the recombination of primary and

secondary peroxyl radicals has been estimated to be around

3.9 to 14% (27).

The generation of 1O2 during metal catalyzed decomposi-

tion of alkylhydroperoxides has been studied by several

authors (25, 26). Recently, we have clearly demonstrated the

generation of 1O2 by the Russell mechanism from LOOH,

specifically from linoleic acid hydroperoxides in the presence

of metal ions (28). The yield of 1O2 derived from this

hydroperoxide was 6%, close to the values determined for

other organic primary and secondary peroxyl radicals. To

study the mechanism of the reaction we synthesized 18-oxygen

labeled linoleic acid hydroperoxide (LA18O18OH) and de-

tected the products (1O2, ketone and alcohol) by HPLC-MS/

MS (28, 29). The incubation of labeled hydroperoxides

with Ce4þ or Fe2þ induced the formation of 18O-labeled

1O2 (18[1O2]), in accordance with the Russell’s mechanism,

where the bimolecular reaction of LA18O18O. generates 18[1O2]

(Fig. 4).

Besides metal ions, we have studied the ability of other

biologically relevant oxidants, such as ONOO7 and HOCl to

induce the generation of 1O2 from LOOH (19, 30).

Peroxynitrite is a strong oxidant of biological importance

produced by the reaction of the O2.7 and nitrogen monoxide.

Our study showed that 1O2 is generated in the reaction of

ONOO7 with LAOOH (30). The generation of 1O2 probably

occurs through the combination of peroxyl radicals by the

Russell mechanism as described above. Peroxyl radicals can be

formed upon reaction of LOOH with the radicals derived from

ONOO7 decomposition.

It is reported that at physiological condition ONOO7 is

protonated and rapidly decomposes to generate nitrogen

dioxide radicals (.NO2) and hydroxyl radicals (.OH) in

approximately 30% yields (31). On the other hand, in the

Figure 2. Structures of some lipid hydroperoxides that can be found in biological systems. The structure of the 13-

hydroperoxyoctadecadienoic acid is illustrated as an example of fatty acid hydroperoxide that can be found either free or

esterified to phospholipids or cholesteryl esters. For cholesterol, the structure of 7a-OOH is illustrated. LAOOH, linoleate

hydroperoxide; PLOOH, phospholipid hydroperoxide; ChOOH, cholesterol hydroperoxide; ChLAOOH, cholesteryl linoleate

hydroperoxide.


presence of bicarbonate, ONOO7 reacts rapidly with CO2

forming an unstable nitrosoperoxycarbonate anion adduct

(ONOOCO27), which decomposes giving carbonate radical

anion (CO3.7) and .NO2 in approximately 35% yields (32).

All these radical species formed from ONOO7 are highly

oxidizing agents, being able to promote the conversion of

LOOH into peroxyl radicals, which undergo a self-reaction

yielding 1O2 (Fig. 5).

Figure 3. Proposed Russell mechanism for the bimolecular reaction of primary and secondary peroxyl radicals (ROO.) involving

a cyclic mechanism from a linear tetraoxide intermediate (ROOOOR) and the corresponding products: (a) alcohol (ROH),

ketone (RO) and 1O2; or (b) alcohol (ROH), excited ketone (RO*) and O2.

Figure 4. Reaction of two labeled linoleate peroxyl radicals (LA18O18O.) leading to the formation of a labeled tetraoxide

intermediate (LA18O18O18O18OLA) that decomposes generating an 18[1O2] and the corresponding linoleate alcohol (LA18OH)

and ketone (LA18O) products.

326 MIYAMOTO ET AL.

HOCl is a highly reactive species capable of modifying a

variety of biomolecules (Fig. 6). Free amino and thiol groups

of amino acids and peptides constitute important targets for

HOCl, yielding unstable chloramines and sulfenyl chloride

intermediates, respectively (33). Another important target for

HOCl is lipids. It is known that HOCl adds across the

carbon-carbon double bonds in fatty acids and cholesterol

yielding chlorohydrins (34). Additionally, HOCl has been

also shown to induce the generation of reactive oxygen

species, such as .OH in the reaction with with O2.7 or

Fe2þ(35).

In a recent study we have demonstrated that HOCl also

reacts with LOOH, such as fatty acid hydroperoxides or

phosphatidylcholine hydroperoxides contained in liposomes,

to generate 1O2 at physiological pH (19). Interestingly the

generation of 1O2 did not occur with tertiary hydroperoxides,

which indicates the involvement of the Russell mechanism.

The generation of 1O2 by this mechanism is also supported by

the detection of peroxyl radical intermediates, as well as, the

formation of 18[1O2] in the reaction of LA18O18OH with

HOCl.

PROTEIN HYDROPEROXIDES

Proteins are also important targets of attack by reactive

oxygen and nitrogen species. A wide range of different

products can be formed on reaction of a protein with reactive

species. Among these products, protein and peptide hydro-

peroxides are of special interest, since they have a half-life of

several hours at room temperature and in the presence of

other oxidants, can further react amplifying the initial

damage (36).

It has been proposed that .OH can promote the

peroxidation of the polypeptide backbone as well as of the

amino acid side-chains (37). Hydroxyl radical-mediated

abstraction of the a-hydrogen atom of the polypeptide chain

gives rise to a carbon-centred radical which subsequently

reacts with oxygen to yield a peroxyl radical. Then, the

transient peroxyl radical is reduced in the presence of an

O2.7 being converted to a hydroperoxide upon protonation

(Fig. 7A). For amino acid side-chains, it was shown that six

amino acids (proline, glutamate, lysine, valine, leucine and

isoleucine) substantially reacted with .OH, acquiring hydro-

peroxide groups (38).

In this way, although many amino acid residues have been

shown to generate hydroperoxides, there is a paucity of

information about the chemical structures of these com-

pounds. However, the correct structural elucidation of these

hydroperoxides will allow us to investigate further biological

implications of these potentially damaging agents. For

example, some of the generated amino acids hydroperoxides

(Val-OOH, Lys-OOH, Leu-OOH) are believed to contain an

important structural characteristic (hydroperoxides having a

hydrogen-a) required for 1O2 generation (Fig. 7B). This

characteristic has been widely explored in the case of LOOH,

but still ignored in the case of proteins.

As already mentioned the self-reaction of peroxyl

radicals containing hydrogen-a generates 1O2. In analogy to

Figure 5. Possible reactions involved in the generation of 1O2 in the reaction of LOOH with ONOO7.


LOOH, amino acid hydroperoxides such as Val-OOH and

Leu-OOH, which are expected to contain a-hydrogen in their

structures, can generate 1O2 in the presence of oxidizing

agents. In addition, it is also probable that 1O2 can be

formed in a reaction containing primary and secondary

hydroperoxides (LOOH or amino acid hydroperoxides)

with tertiary hydroperoxides that lack the hydrogen-a(e.g., tryptophan or tyrosine hydroperoxides). The impor-

tance of this kind of reaction is reinforced by the fact that

amino acid hydroperoxides can be formed near lipid sites

(e.g., membranes), where they can react with pre-existing

LOOH.

DNA HYDROPEROXIDES

Along with proteins, nucleic acids are also important

targets of oxidative modifications mediated by reactive

species. Modification of cellular DNA upon exposure to

reactive oxygen species is involved in the induction of the

mutagenesis, carcinogenesis and aging. It is well known

that .OH generates a wide range of modification in DNA,

including base and sugar lesions, strand breaks, and DNA

protein crosslinks. It has been reported that production of

.OH close to DNA leads to the formation of 8-oxo-

7,8-dihydro-20-deoxyguanosine and other oxidation products,

such as DNA hydroperoxides (37).

The main reaction of .OH with DNA involves addition to

the carbon C5 and C6 of cytosine and thymine and to the C4,

C5, and C8 of guanine and adenine (37). It has been shown

that among nucleic acids, pyrimidine bases and in particular

thymine was the preferential target for the radiation-induced

formation of stable hydroperoxide at neutral and slightly

acidic pH values (37). Later on, it was confirmed that exposure

of naked nucleic acids to gamma rays, yielded a higher

consumption of molecular oxygen for pyrimidine bases than

for purine bases (37).

The reaction of .OH with thymidine has been shown to

generate thymidine hydroperoxides. The reaction proceeds

mainly by addition of .OH at carbon C5 (60%) or C6 (35%),

leading to the formation of 5-hydroxy-6-hydroperoxy-5,6-

dihidrothymidine (5-OH-6-OOHThd) and 6-hydroxy-5-

hydroperoxy-5,6-dihidrothymidine (6-OH-5-OOHThd), re-

spectively (Fig. 8A). Alternatively, .OH can also promote

the abstraction of H atom from the methyl group, yielding

5-(hydroperoxymethyl)- 20-deoxyuridine (5-OOHUrd) in a

lower yield (5%).

Figure 6. Reactions of HOCl with biomolecules leading to the formation of chlorinated products as well as reactive oxygen

species.

328 MIYAMOTO ET AL.

The attack of .OH to cytosine and cytidine generates

cytosine and 20-deoxycytidine hydroxyhydroperoxides (39).

Two major hydroperoxides are formed, the 5-hydroxy-6-

hydroperoxide-5,6-dihydro-20-deoxycytidine (5-OH-5-OOHd-

Cyd) and the 6-hydroxy-5-hydroperoxide-5,6-dihydro-20-

deoxycytidine (6-OH-5-OOHdCyd) (Fig. 8B). These hydro-

peroxides are relatively unstable and their decomposition

products have been detected from free cytosine and cultured

human cells (40).

Thymidine and cytidine hydroperoxides are the primary

products of radical induced DNA oxidation. These hydroper-

oxides are relatively stable and their decomposition products

have been shown to be very mutagenic. Similarly to lipids and

protein hydroperoxides, it is chemically probable that the

decomposition of thymidine or cytidine hydroperoxides

containing hydrogen-a, in the presence of metal ions can lead

to the generation of 1O2.

CONCLUSION

It is well known that hydrogen peroxide is an important

source of 1O2. Our studies strongly point that lipid derived

hydroperoxides can also potentially contribute to the genera-

tion of 1O2, particularly in situations where metal ions and

other oxidants, such as ONOO7 and HOCl are present. The

physiological relevance of 1O2 generation by these reactions

remains to be clarified. Nonetheless, our studies indicate that

fatty acid hydroperoxides, as well as phospholipid hydroper-

oxides formed in membranes can generate 1O2. Therefore it is

highly probable that 1O2 is involved in the cellular responses

elicited by LOOH.

Besides lipids, a great variety of other biological hydroper-

oxides, including protein and nucleic acid hydroperoxides, are

formed in biological systems. Analogously to lipids, these

hydroperoxides can undergo decomposition reactions leading

Figure 7. Generation of hydroperoxides in protein backbone mediated by .OH attack (A). Amino acid hydroperoxides

containing hydrogen-a. Valine hydroperoxide (Val-OOH), lysine hydroperoxide (Lys-OOH) and leucine hydroperoxide (Leu-

OOH) (B).


to the generation of peroxyl radical intermediates and singlet

molecular oxygen generation.

ACKNOWLEDGEMENTS

This work was supported by the Brazilian research funding

institutions FAPESP (Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo), CNPq (Conselho Nacional para o

Desenvolvimento Cientıfico e Tecnologico), Instituto do

Milenio: Redoxoma, Fundo Bunka de Pesquisa – Banco

Sumitomo Mitsui (S.M. Fellow), and L’OREAL-UNESCO

for Women in Science (S.M. Fellow) and The John Simon

Memorial Guggenheim Foundation (P.D.M. Fellow).

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Biological hydroperoxides and singlet molecular oxygen generation

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