Free Radicals in the Atmosphere

Like the oxidation in a flame, the oxidation in the atmosphere is mediated by free radicals. Unlike a flame, however, atmospheric oxidation needs an external source of energy: the sun light. In fact the most important radical acting in the lower atmosphere, the hydroxyl radical, OH, is produced following the UV-photolysis of ozone, O,which yields an excited oxygen atom, O'D:

OH reacts with most atmospheric trace gases, in many cases as the first and rate determining step in the reaction chain leading to oxidation. In this way a host of various other radicals (e.g. peroxy radicals), most of them very short lived, are generated. Usually these oxidation reactions form chains which regenerate OH, thus maintaining OH at a relatively high concentration level on the order of 10⁶cm∼³ during the day. The reactions which control the OH concentration will be discussed in detail. During the night radical formation is greatly diminished. It proceeds, for example, through the reaction of defines with O, and. in dry air, through reaction of defines and aldehydes with the nitrate radical, NO,.     

Atmospheric Oxidation of Hydrocarbons

Hydrocarbon oxidation in the atmosphere proceeds generally by the following sequence of reactions: hydrocarbon + OH → alkyl radical + H₂O, alkyl radical + O,(³I) → alkylperoxy radical, alkylperoxy radical + NO → alkoxy radical + NO₂, alkoxy radical + O₂(³X) -→ aldehyde + HO,. The atmospheric lifetimes of hydrocarbons are determined by their reactivity towards OH as well as by the average OH concentration level. They are compound specific and vary from several hours to several years. Hydrocarbon oxidation chains couple with other trace gases (O_v, HO_x, and NO_v). For the conditions of the average continental atmosphere an increase of the oxidative potential (HO_v, O_x) is predicted through hydrocarbon oxidation.     

Drugs with susceptible sites for free radical induced oxidative transformations: the case of a penicillin

Penicillins, as bactericidal antibiotics, have been widely used to treat infections for several decades. Their structure contains both aromatic and thioether moieties susceptible to free radical oxidation. The ^?OH induced oxidation mechanism of amoxicillin was investigated by pulse radiolysis techniques and by final product analysis performed after steady-state γ-irradiation. The predominant sites of the ^?OH attack are suggested to be the thioether group, initially yielding an ^?OH adduct to the sulfur, and the aromatic ring. This adduct to the sulfur converts to sulfur radical cation, which has three competitive reaction paths: (1) by deprotonation at the adjacent carbon α-(alkylthio)alkyl radicals form, which undergo disproportionation leading presumably to sulfoxide as main product; (2) via the pseudo-Kolbe mechanism it may transform to α-aminoalkyl radicals; (3) the radical cation can be stabilized through intramolecular S.˙.O bond formation. The reaction mechanism suggests the presence of a short-living and a stabilized (via hydrogen bonding) long-living ^?OH adduct to the sulfur. The three-electron bonded dimers of amoxicillin were not formed owing to steric hindrance. Thiyl radicals were also present in equilibrium with α-aminoalkyl radicals. In the presence of dissolved oxygen, aromatic ring hydroxylation occurred along with complex reactions resulting in e.g. oxidation of the methyl groups. The formation of the sulfoxide is especially effective in the presence of dissolved oxygen, under anaerobic condition, however, it is also generated owing to H₂O₂ and α-(alkylthio)alkyl radicals. The thioether moiety appears to be more sensitive to oxidation compared to the aromatic ring in case of amoxicillin.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

Spin Trapping by Use of N-Tert-Butylhydroxylamine. Involvement of Fenton Reactions

Spin trapping of short-lived R. radicals is done by use of N-tert-butylhydroxylamine (1) and H²O². The hydroxylamine is oxidized to the radical t-BuN(O)H (2) which is converted into the spin trap 2-methyl-2-nitrosopropane (3). Simultaneously, hydroxyl radicals. OH are formed from H²O². The latter radical species abstracts hydrogen atoms from suitable molecules HR to give R. radicals, which are trapped with the formation of aminooxyl radicals, i. e., t-BuN(O)R (4) detectable by EPR spectroscopy. The reaction is enhanced by the presence of iron ions. The cleavage of H²O² into. OH radicals is considered to involve both a radical-driven (t-BuN(O)H 2) and an iron-driven Fenton reaction.     

Oxidation of hydroxylamine to nitrite as an assay for the combined presence of superoxide anions and hydroxyl radicals.

The pulse-radiolytic oxidation of hydroxylamine by either hydroxyl radicals (OH), superoxide anions (O₂^?), or a combination of both radicals was investigated. It was found that only OH radicals efficiently attack the substrate, while O₂^? is necessary for the subsequent formation of nitrite. Determination of the latter reaction thus allows the detection of the combined presence of both oxygen radical species.     

The Natural History of Oxygen

The nuclear reactions occurring in the cores of stars which are believed to produce the element oxygen are first described. Evidence for the absence of free oxygen in the early atmosphere of the earth is reviewed. Mechanisms of creation of atmospheric oxygen by photochemical processes are then discussed in detail. Uncertainty regarding the rate of diffusion of water vapor through the cold trap at 70 km altitude in calculating the rate of the photochemical production of oxygen is avoided by using data for the concentration of hydrogen atoms at 90 km obtained from the Meinel OH absorption bands. It is estimated that the present atmospheric oxygen content could have been produced five to ten times during the earth's history. It is shown that the isotopic composition of atmospheric oxygen is not that of photosynthetic oxygen. The fractionation of oxygen isotopes by organic respiration and oxidation occurs in a direction to enhance the O¹⁸ content of the atmosphere and compensates for the O¹⁸ dilution resulting from photosynthetic oxygen. Thus, an oxygen isotope cycle exists in nature.     

Hydrogen atom abstraction from HOOOCl by chlorine atom and OH radical

Gas-phase reactions of HOOOCl with both Cl atom and OH radical are investigated using ab initio methods. The structures of all reactants, products, intermediates, and transition states have been optimized and characterized with the quadratic configuration interaction (QCISD) method. The overall mechanism for the Cl + HOOOCl and OH + HOOOCl reaction is the formation of HCl + O₂ + ClO and H₂O + O₂ + ClO, respectively. The rate-limiting step in each reaction is the abstraction of hydrogen from HOOOCl by either Cl or OH radicals and the barrier height is predicted to be 1.9 kcal mol⁻¹ and 8.1 kcal mol⁻¹ for abstraction by Cl atom and OH radical, respectively. Since both barriers for hydrogen abstraction are high, the reaction is suggested to be slow. These results also suggest that an atmospheric removal mechanism for HOOOCl may result from reaction with Cl atoms rather than with OH radicals, and that photolysis of HOOOCl may be the major removal mechanism for the intermediate.     

Intra- and intermolecular oxidation of oxymyoglobin and oxyhemoglobin induced by hydroxyl and carbonate radicals

The mechanism of the reactions of myoglobin and hemoglobin with *OH and CO3*- in the presence of oxygen was studied using pulse and gamma-radiolysis. Unlike *NO2, which adds to the porphyrin iron, *OH and CO3*- form globin radicals. These secondary radicals oxidize the Fe(II) center through both intra- and intermolecular processes. The intermolecular pathway was further demonstrated when BSA radicals derived from *OH or CO3*- oxidized oxyhemoglobin and oxymyoglobin to their respective ferric states. The oxidation yields obtained by pulse radiolysis were lower compared to gamma-radiolysis, where the contribution of radical-radical reactions is negligible. Full oxidation yields by *OH-derived globin radicals could be achieved only at relatively high concentrations of the heme protein mainly via an intermolecular pathway. It is suggested that CO3*- reaction with the protein yields Tyr and/or Trp-derived phenoxyl radicals, which solely oxidize the porphyrin iron under gamma-radiolysis conditions. The *OH particularly adds to aromatic residues, which can undergo elimination of H2O forming the phenoxyl radical, and/or react rapidly with O2 yielding peroxyl radicals. The peroxyl radical can oxidize a neighboring porphyrin iron and/or give rise to superoxide, which neither oxidize nor reduce the porphyrin iron. The potential physiological implications of this chemistry are that hemoglobin and myoglobin, being present at relatively high concentrations, can detoxify highly oxidizing radicals yielding the respective ferric states, which are not toxic.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical (^?OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with ^?OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary ^?OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and ^?OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary ^?OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the ^?OH sources in pathological processes such as Parkinson's disease.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.     

Gas-phase reaction of benzo[a]anthracene with hydroxyl radical in the atmosphere: products,oxidation mechanism,and kinetics

Polycyclic aromatic hydrocarbons (PAHs) have induced large-scale and long-term environmental contamination due to heavy emissions, toxicity, and persistence. The investigation of the ultimate sink of PAHs in the atmosphere is very important. In this work, using quantum chemistry methods, the reaction mechanism of hydroxyl radical-initiated oxidation of benzo[a]anthracene (BaA) in the atmosphere was studied. The products resulted from the gas-phase reaction of BaA with hydroxyl radical include benzo[a]anthracenols, dialdehydes, ketones, epoxides, etc. Applying Rice-Ramsperger-Kassel-Marcus (RRKM) theory, the overall rate constant for reactions of ?OH addition to BaA was estimated to be 4.82?×?10^?11 cm³ molecule^?1 s^?1 at 298 K and 1 atm. The lifetime of BaA in the atmosphere with respect to hydroxyl radical was calculated to be 5.92 h.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

Theoretical study of the kinetics and mechanism of the decomposition of trifluoromethanol, trichloromethanol, and tribromomethanol in the gas phase

Ab initio calculations at the G2 level were used in a theoretical analysis of the kinetics of unimolecular and water-accelerated decomposition of the halogenated alcohols CX₃OH (X = F, Cl, and Br) into CX₂O and HX. The calculations show that reactions of the unimolecular decomposition of CX₃OH are of no importance under atmospheric conditions. A considerably lower energy pathway for the decomposition of CX₃OH is accessible by homogenous reactions between CX₃OH and water. It is shown that CX₃OH + H₂O reactions proceed via the formation of intermediate complexes. The mechanism of the reactions appears to be complex and consists of three consecutive elementary processes. The calculated values of the second-order rate constants are of 2.5 × 10⁻²¹, 2.1 × 10⁻¹⁹, and 1.2 × 10⁻¹⁷ cm³molecule⁻¹s⁻¹ at 300 K for CF₃OH + H₂O, CCl₃OH + H₂O, and CBr₃OH + H₂O, respectively. The theoretically derived atmospheric lifetimes of the CX₃OH molecules indicate that the water-mediated decomposition reactions CX₃OH + H₂O may be the most efficient process of CF₃OH, CCl₃OH, and CBr₃OH loss in the atmosphere.     

New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

Reaction rate coefficients of hydroxyl radical-induced DNA single- and α-type double-strand breaks

With a model system of pBR322 plasmid DNA solution in vitro, the dose effects of radiation- induced single- and double-strand breaks (SSB and DSB) were measured and DSB was distinguished into α- and β-types. Under the condition of low scavenging capacity existing in the irradiated DNA solution, SSB and αDSB were mainly induced by hydroxyl radicals (^·OH). Moreover, a certain relationship was obtained between the SSB and αDSB yields and the DNA concentration. It was found that when the DNA solution was irradiated in the presence of 2.5 mmol dm^–3 mannitol, the reciprocals of G(SSB) and G(αDSB), respectively, were linearly related to the reciprocal of the DNA concentration, i.e. the competition reactions of DNA and mannitol for ^·OH radicals can be described by second-order kinetics. The rate coefficients and the efficiencies of the ^·OH radical inducing SSB were deduced. Also, the reaction rate coefficients and the efficiencies for the induction of αDSB from SSB by the ^·OH radical transfer mechanism, were first derived from the competition kinetics. Received: 27 October 1999 / Accepted: 15 March 2000     

Sulfite-mediated oxidation of myeloperoxidase to a free radical: Immuno-spin trapping detection in human neutrophils

Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of the reactive sulfur trioxide (^•SO₃⁻), peroxymonosulfate (⁻O₃SOO^•), and sulfate (SO₄^•−) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions, and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of this study was to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage, possibly leading to (bi)sulfite-exacerbated allergic reactions.     

Oxidative decarboxylation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes by peroxidases and related hemeproteins: Intermediate formation and characterization of the corresponding cations

Tris(p-carboxyltetrathiaaryl)methyl radicals (TAM) are good EPR probes for measurement of dioxygen concentration in biological systems and for EPR imaging. It has been previously reported that these radicals are efficiently oxidized by superoxide, O₂⁻, or alkylperoxyl radicals, ROO, and by liver microsomes via an oxidative decarboxylation mechanism leading to the corresponding quinone-methides (QM). This article shows that peroxidases, such as horseradish peroxidase (HRP), lactoperoxidase (LPO) and prostaglandin synthase (PGHS), and other hemeproteins, such as methemoglobin (metHb), metmyoglobin (metMb) and catalase, also efficiently catalyze the oxidation of TAM radicals to QM by H₂O₂ or alkylhydroperoxides. These reactions involve the intermediate formation of the corresponding cations TAM⁺ that have also been cleanly generated by K₂Ir(IV)Cl₆ and characterized by UV-Visible spectroscopy and mass spectrometry, and through their reactions with ascorbate or H₂O₂. Labelling experiments on HRP-catalyzed oxidation of TAM to QM using H₂¹⁸O or ¹⁸O₂ in the presence of glucose and glucose oxidase (GOX) showed that the oxygen atom incorporated into QM came both from O₂ and from H₂O. Mechanisms for these reactions in agreement with those data were proposed. Oxidative decarboxylation of TAM to QM is a new reaction catalyzed by peroxidases. Such reactions should be considered when using TAM as EPR oximetry probes invivo or in vitro in complex biological media.