©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Scavenging of Nitrogen Dioxide, Thiyl, and Sulfonyl Free Radicals by the Nutritional Antioxidant -Carotene (*)

(Received for publication, August 14, 1995; and in revised form, November 10, 1995)

Steven A. Everett (§) Madeleine F. Dennis Kantilal B. Patel Susan Maddix Subhas C. Kundu Robin L. Willson

From the  (1)Gray Laboratory, P. O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom (2)Department of Biology and Biochemistry, Brunel University, Middlesex UB8 3PH, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mechanisms of free radical scavenging by the nutritional antioxidant beta-carotene have been investigated by pulse radiolysis. Free radicals, which can initiate the chain of lipid peroxidation, including nitrogen dioxide (NO(2)), thiyl (RS), and sulfonyl (RSO(2)) radicals, are rapidly scavenged by beta-carotene. Absolute rate constant k[NO(2) + beta-carotene] = (1.1 ± 0.1) times 10^8M s and for the glutathione thiyl radical k[GS + beta-carotene] = (2.2 ± 0.1) times 10^8M s have been determined. The mechanisms however are mutually exclusive, the former involving electron transfer to generate the radical-cation [beta-carotene] and the latter by radical-addition to generate an adduct-radical [RSbulletbulletbulletbeta-carotene]. Rate constants for thiyl radical-addition reactions vary from 10^6 to 10^9M s and correlate with the lipophilicity of the thiyl radical under study. Sulfonyl radicals undergo both electron abstraction, [beta-carotene] and radical-addition, [RSO(2)bulletbulletbulletbeta-carotene] in an approximate 3:1 ratio. The beta-carotene radical-cation and adduct-radicals are highly resonance stabilized and undergo slow bimolecular decay to non-radical products. These carotenoid-derived radicals react differently with oxygen, a factor which is expected to influence the antioxidant activity of beta-carotene within tissues of varying oxygen tension in vivo.


INTRODUCTION

beta-Carotene (provitamin A) is a major lipid soluble antioxidant nutrient present in a range of fresh fruit and vegetable produce(1) . Epidemiological evidence that correlates beta-carotene dietary supplementation with a reduced risk of contracting cancer and heart disease (2) has provided the impetus for ongoing human intervention trials(3, 4, 5, 6) , particularly on smokers(7, 8) . The antioxidant properties of beta-carotene have been implicated in the molecular basis for disease prevention(9, 10) , primarily because of the putative role of oxidative stress in disease initiation and progression(11, 12, 13, 14) . In vitro studies on cellular membrane damage (15, 16) and low density lipoprotein (LDL) (^1)oxidation (17) indicate that beta-carotene may modulate free radical processes in vivo by behaving as a chain-breaking antioxidant in lipid peroxidation(9, 18) Paradoxically, other studies have shown that beta-carotene offers little protection from metal-catalyzed LDL oxidation (19, 20) and can kill tumor cells via prooxidative pathways(21) . beta-Carotene in combination with alpha-tocopherol has recently been found to reduce radiation effects in normal tissues, suggesting a potential application as a radioprotector in clinical cancer radiotherapy(22) .

Although the role of beta-carotene as a singlet oxygen quencher has been thoroughly characterized(23, 24, 25, 26, 27) , surprisingly fewer mechanistic studies on radical-scavenging properties of the carotenoid have been pursued. Pulse radiolysis has shown that halogenated peroxyl radicals (e.g. CCl(3)OO) are rapidly scavenged by beta-carotene (28, 29, 30) and that the reactivity toward the superoxide radical-anion (O(2)) is much less significant than for the related carotenoid lycopene(31) .

Nitric oxide, a major component of cigarette smoke (500-1000 ppm), may contribute to the development of oral/lung cancers and heart disease in smokers by generating more damaging nitrogen oxides, including nitrogen dioxide (NO(2)) radicals (32) . In vitro studies have demonstrated that NO(2) radicals can initiate the autoxidation of polyunsaturated fatty acids (33) and that cigarette smoke can induce LDL oxidation(34) . Paradoxically, although beta-carotene does not appear to prevent the oxidation of LDL by cigarette smoke(34) , it can protect lymphocytes from NO(2) radical-induced membrane damage(16) .

A number of thiols, including the endogenous thiol antioxidant glutathione (GSH), have been shown to enhance nitrogen dioxide-induced lipid peroxidation in model systems by generating thiyl (RS) and thiyl-derived radical species(35) . Thiyl radicals are synonymous with the classical repair reactions of thiols in vivo(36, 37) and are also generated by peroxidase-catalyzed oxidation of thiols (38, 39) . Although the fate of RS radicals within cells remains a matter for debate(40) , there is increasing evidence to suggest that they are capable of initiating the chain of lipid peroxidation at least in model systems(41, 42, 43) . Thiyl radicals undergo conjugation with molecular oxygen to generate the thiyl peroxyl (RSOO) radical (44) which can rearrange to thiyl-sulfonyl (RSO(2)) radical(45) , another potent initiator of lipid peroxidation(46) . A thiol-specific antioxidant enzyme considered widely distributed in mammalian tissues which catalyzes the removal of thiyl radicals has provided evidence for thiyl radical-induced damaging reactions(47) . Moreover, human (THP-1) macrophages oxidize LDL by a thiol-dependent mechanism, which is believed to involve thiyl and thiyl-derived radical species.

Following a preliminary communication(48) , we now report a detailed pulse radiolysis study on the oxidation of beta-carotene by NO(2), RS, and RSO(2) radicals and characterization of the resultant beta-carotene-derived radical species.


EXPERIMENTAL PROCEDURES

Materials

All-trans-beta-carotene, beta-mercaptoethanol, ethanesulfonyl chloride, tert-butyl alcohol, and 2-propanol were obtained from Aldrich and used as received. The thiols, glutathione (GSH), cysteine (CysSH) as well as sodium nitrate, thiocyanate, and phosphate salts were from Sigma (Analar grade). The radioprotective thiol drug 2-(3-aminopropylamino)ethanethiol (WR-1065) was kindly donated by the National Cancer Institute.

Methods

The pulse radiolysis technique is a useful way of generating specific radical species and studying their reactions with beta-carotene by kinetic spectrophotometry(49) . The pulse radiolysis facility at the Gray Laboratory Cancer Research Trust, including methodology, data acquisition, and analysis, have been described elsewhere(50) . In this study 30-ns pulses of 3.5 MeV electrons were used to deliver doses of typically 0.3-2 Gy as determined by thiocyanate dosimetry(51) . A tungsten lamp with a photodiode detector allowed the decay kinetics of beta-carotene radical species to be observed over 0.2 s. Free radical-induced bleaching of the beta-carotene ground-state absorption was also measured by a xenon lamp utilizing narrow (1.25-mm slits) to eliminate photobleaching.

The nitrogen dioxide radical (NO(2)) was generated by radiolysis of N(2)-saturated tert-butyl alcohol/water mixtures (50%, v/v) containing 0.1 mM NO(3) in 1 mM phosphate buffer at pH 5. Under these conditions the radiolysis of tert-butyl alcohol/water mixtures generates the primary radical species (52) in .

In and the hydrated electron (e) and solvated electron (e) reduce nitrate to nitrate radical anions (NO(3)^2) which rapidly protonate at pH 5, then dissociate (<1 µs) into the NO(2) radicals(53) .

Both hydroxy (OH) and hydrogen (H) radicals are scavenged by tert-butyl alcohol to generate a radical that is unreactive toward beta-carotene and does not absorb above 300 nm.

Thiyl radicals were generated by radiolysis of N(2)O-saturated tert-butyl alcohol/water mixtures (50%, v/v) containing 10 mM thiol and 1 mM phosphate buffer at pH 5. In this case hydrated electron is converted to OH radicals that becomes the principal product of water radiolysis in .

Thiyl radicals (RS) are then rapidly formed (<1 µs) after the pulse by the repair of the carbon-centered radicals () generated in and by the thiol(54, 55, 56) .

Thiyl-sulfonyl radicals were generated on radiolysis of N(2)-saturated tert-butyl alcohol/water mixtures (50%, v/v) containing 10 mM C(2)H(5)SO(2)Cl and 1 mM phosphate buffer at pH 5. In this case RSO(2) radicals are generated by dissociative electron capture (57) in , while both OH and H radicals are once again scavenged by the alcohol in .

The free radical yields are represented by their respective G-values, where G(NO(2)) = 0.9, G(RS) = 5.5 and G(RSO(2)) = 0.9 were determined by monitoring the oxidation of ABTS (2,2`-azinobis(3-ethylbenzothiazoline-6-sulfonate)) to the stable radical-cation ABTS (max = 417 nm, = 34,700 dm^3 mol s)(53, 58) .

Prior to experimentation a N(2)-saturated stock solution of beta-carotene in toluene was prepared and kept in the dark. Solutions were prepared immediately before experimentation using purified water from a Milli-Q system (Millipore). Solutions required for pulse radiolysis experiments contained no more than 20 µmol dm beta-carotene to allow adequate light transmission in the 300-500 nm region, where beta-carotene exhibits a strong ground-state absorption(59) . Before irradiation solutions were bubbled with nitrous oxide (N(2)O, oxygen content < 10 ppm) or with nitrogen (N(2), oxygen content < 10 ppm) from the British Oxygen Co., United Kingdom. All experiments were performed at ambient room temperature (20 ± 2 °C).

Bleaching of the beta-carotene ground-state absorption in the 300-500 nm has proven a useful method of probing the mechanism of free radical and enzymatic destruction of carotenoid(9, 10, 60) . To assess the possibility of beta-carotene-derived products interfering at these wavelengths. Steady-state radiolysis experiments were performed in a field of a 2000 Ci of Co -source at a dose rate of 5 Gy min (total dose 25 Gy equals < 30% conversion of beta-carotene to products) as determined by Fricke dosimetry(61) . EDTA 10 mmol dm was included to prevent metal-catalyzed degradation of either beta-carotene or beta-carotene-derived products (60) , otherwise the experimental conditions were as for pulse radiolysis. Products formed on irradiation of beta-carotene were separated by high performance liquid chromatography using a Hypersil 5ODS 125 times 4.6-mm column. Samples were separated isocratically with a methanol/acetonitrile/chloroform (25:60:15%, v/v) eluent (62) and components detected spectrophotometrically at 451 and 300 nm by a Waters 486 variable wavelength detector. Although NO(2), RS, RSO(2) radicals depleted beta-carotene in a dose-dependent manner no products of the reactions were detected above 420 nm which would interfere with the pulse radiolysis kinetic measurements.


RESULTS

Oxidation of beta-Carotene by Nitrogen Dioxide

The absorption spectra obtained by pulse radiolysis of N(2)-saturated tert-butyl alcohol/water mixture (50%, v/v) containing 0.1 mM NO(3), 1 mM phosphate buffer, and 10 µM beta-carotene at pH 5 are shown in Fig. 1. These are attributed to the beta-carotene radical species generated by the oxidation of beta-carotene by NO(2) radicals. Electron pulses of low dose typically 1 Gy were used to eliminate competition from the fast bimolecular decay of NO(2) radicals via (2k(8) = 4.5 times 10^8M s) and to ensure that all NO(2) radicals generated by reacted solely with beta-carotene.


Figure 1: Oxidation of beta-carotene by nitrogen dioxide. Absorption spectra showing the formation and decay of the beta-carotene radical-cation as measured by pulse radiolysis of N(2)O-saturated tert-butyl alcohol/water mixtures (50%, v/v) containing nitrate (0.1 M) and beta-carotene (10 µM) at pH 5. The spectra were built from measurements of absorbance at discrete wavelengths 10 ms (solid circles) and 0.1 s (open circles) after pulses of approximately 1 Gy. Insets: optical traces showing exponential bleaching of the beta-carotene ground-state absorption at 450 nm and the corresponding exponential growth of the beta-carotene radical-cation absorption at 910 nm.



The absorption of the solution measured 10 ms after the electron pulse showed a decrease in the wavelength range 300-600 nm (where beta-carotene absorbs) accompanied by an increase in the range 600-1000 nm. These spectral changes are ascribed to the reaction of NO(2) radicals with beta-carotene causing a bleaching of the parent chromophore and the formation of a new absorbing species with (max) approx 910 ± 5 nm. The extinction coefficient of the radical species was calculable from the radical yield of G = 8.5 ± 0.2 times 10^4M cm. Under the experimental conditions employed, the radiation chemical yield of nitrogen dioxide radicals was G(NO(2)) = 0.9 giving an extinction coefficient for the beta-carotene radical species of = 9.4 ± 0.2 times 10^4M cm. By comparison with an analogous transient generated by flash photolysis (25) and one-electron oxidation of beta-carotene by the halogenated peroxyl radical CCl(3)OO(28, 29) , the transient species was identified as the beta-carotene radical-cation [carotene] generated by electron abstraction from beta-carotene by NO(2) radicals via . The extinction coefficient of the [carotene] radical-cation compares well with that measured in aqueous Triton X-100 ((max) 1 times 10^5M cm)(63) .

By analogy to the reversible addition reactions of nitrogen dioxide with the double bonds of alkenes to form nitro alkyl radicals (64) , the nitrogen dioxide beta-carotene adduct-radical [NO(2)bulletbulletbulletbeta-carotene] was considered a possible precursor to radical-cation formation(16) . Indeed, a characteristic shoulder at 650-800 nm in the [beta-carotene] radical-cation absorption spectrum was observed and thought to be a separate radical species quite possibly a [NO(2)bulletbulletbulletbeta-carotene] radical. However, the reaction kinetics between 600 and 1000 nm were identical, and it was therefore concluded that the entire absorption in this region was due to the [carotene] radical-cation alone.

Both the decline in the beta-carotene ground-state absorption at 451 nm (left inset, Fig. 1) and the increase in the [beta-carotene] radical-cation absorption at 910 nm (right inset, Fig. 1) were exponential and first-order in beta-carotene concentration. The rate constant for the reaction obtained from the slope of the linear plot of the first-order rate constant of the build-up of absorption at 910 nm versus the concentration of beta-carotene was identical to the corresponding plot for the decline in beta-carotene absorption at 451 nm (not shown here), both giving k(9) = (1.1 ± 0.1) times 10^8M s. This provided further confirmation that no other radical species are generated under these experimental conditions and that NO(2) radicals react with beta-carotene exclusively by electron abstraction to form the carotenoid radical-cation. If intermediate [NO(2)bulletbulletbulletbeta-carotene] radicals are formed they must be highly unstable, rapidly eliminating NO(2) to generate the radical-cation in <10 µs.

After 0.1 s the [beta-carotene] radical-cation absorption has decayed almost to zero (see in Fig. 1). The absorption at 910 nm decays predominantly by second-order kinetics with a half-life which decreased with increased radiation dose (0.2-2 Gy) as shown in Fig. 2(i.e. with increasing initial concentration of radicals). This is attributed principally to the bimolecular decay of the radical-cation according to .


Figure 2: Decay kinetics of the beta-carotene radical-cation. Transient absorption at 910 nm observed on reaction of NO(2) radical with beta-carotene showing decay of the beta-carotene radical-cation (experimental conditions as per Fig. 1). Inset: dependence of the reciprocal of the first half-life of the beta-carotene radical-cation on the dose per pulse (approximately 0.2-2 Gy).



The reciprocal of the first half-life of the [beta-carotene] radical-cation varied linearly with the initial radical concentration and from the slope of the fitted straight line the rate constant 2k = 4.1 times 10^5M s was determined. An intercept of < 6 s indicated that a small fraction of the [beta-carotene] radical-cation decays by a first-order process, quite possibly solvation or base-catalyzed formation of the carotene hydroxy adduct-radical, and , respectively.

However, due to the constraints of pH on NO(2) radical (see and ) and beta-carotene concentration within this experimental system, this particular aspect of the mechanism could not be pursued. The decay of the radical-cation absorption after 0.1 s paralleled a partial restitution of the beta-carotene ground-state absorption in the 300-600 nm region. These spectral changes were consistent with the partial regeneration of beta-carotene as a consequence of .

beta-Carotene Scavenging of RS Radicals

The absorption spectra for the reaction of the beta-mercaptoethanol thiyl radical with beta-carotene in Fig. 3was obtained by pulse radiolysis of an N(2)O-saturated tert-butyl alcohol/water mixture (50%, v/v) containing beta-mercaptoethanol 10 mM, 10 µM beta-carotene, and 1 mM phosphate buffer at pH 5. In marked contrast to the absorption spectrum obtained from NO(2) radical attack on beta-carotene in Fig. 1, no absorption was observed above 600 nm, indicating a complete absence of the beta-carotene radical-cation. However, a decline in the beta-carotene ground-state absorption was observable, indicating that the RS radicals are scavenged by beta-carotene by an alternative mechanism to that observable with nitrogen dioxide.


Figure 3: Carotene scavenging of the beta-mercaptoethanol thiyl radical. Absorption spectra obtained on the pulse radiolysis of N(2)-saturated tert-butanol/water mixtures (50%, v/v) containing beta-mercaptoethanol (10 mM) and beta-carotene (10 µM) at pH 5. Bleaching of the beta-carotene ground-state absorption was observed 50 µs (solid circles) and 0.2 s (open circles) after approximately 1-Gy pulses. Insets: optical traces at 450 nm obtained in the absence and presence of beta-mercaptoethanol. In the presence of thiol a characteristic biphasic exponetial decline in the beta-carotene ground-state absorption is observed.



The decline in absorption at 451 nm is biphasic with a fast step complete in approx50 µs and a much slower step complete some 80 ms later. The fast step was exponential and first-order in beta-carotene concentration, while the slower phase was second-order, independent of beta-carotene concentration, but dependent on the radiation dose per pulse.

The observations have been attributed to a radical-addition process in which RS radicals are scavenged by beta-carotene to generate a resonance stabilized adduct-radical [RSbulletbulletbulletbeta-carotene] via . Two isobestic points are located near 380 and 520 nm, respectively, thus indicating that the adduct-radical absorbs in the same region of the spectrum as that of the beta-carotene ground-state absorption.

The rate constant, k = 2.5 ± 0.1 times 10^9M s for the beta-mercaptothiyl radical plus beta-carotene was obtained from the slope of the linear plot of the first-order rate constant for the fast step versus beta-carotene concentration. At such low doses per pulse (1 Gy), the slower decline in absorption at 451 nm could have been easily mistaken for first-order processes, including hydrogen abstraction from beta-carotene (), radical-addition at alternative sites along the polyconjugated backbone of beta-carotene or as a consequence of a reverse repair reaction, e.g.in which beta-carotene could perturb a possible equilibrium reaction () by irreversibly scavenging thiyl radicals via . In the latter case the observed biphasic bleaching of the beta-carotene chromophore could reflect the fast formation of the [RSbulletbulletbulletbeta-carotene] radical-adduct via and followed by a slower formation of the [RSbulletbulletbulletbeta-carotene] radical-adduct reflecting the contribution from the reverse repair reaction ().

However, the radical species decayed slowly by pure second-order kinetics with a half-life which decreased with increased radiation dose (0.2-2 Gy), suggesting the [RSbulletbulletbulletbeta-carotene] radicals undergo bimolecular decay to products via . In Fig. 4the plot of the reciprocal of the first half-life of the radical species varied linearly with the initial radical concentration and from the slope of the fitted straight line the rate constant 2k = (2.1 ± 0.1) times 10^6M s was determined.


Figure 4: Decay kinetics of the beta-carotene-thiyl adduct-radical. Transient absorption at 450 nm observed on the reaction of HO(CH(2))(2)S radical with beta-carotene at pH 5 initiated by a pulse of approximately 1.8 Gy. Inset: dependence of the reciprocal of the first half-life of the [RSbulletbulletbulletbeta-carotene] adduct-radical on the dose/pulse.



No intercept is observed in Fig. 4, ruling out any contribution from the aforementioned first-order processes. The O(2) radical-anion has been shown to undergo addition-elimination reactions with beta-carotene(31) . An analogous reversible reaction can be envisaged for the reaction of thiyl radicals with beta-carotene, .

However, in this system the elimination of the thiyl radical from the [RSbulletbulletbulletbeta-carotene] radical-adduct is unable to compete with , which effectively pulls the equilibrium position () toward [RSbulletbulletbulletbeta-carotene] radical-adduct formation.

No evidence was obtained for thiol repair of the [RSbulletbulletbulletbeta-carotene] adduct-radical, since hydrogen atom transfer via would have regenerated another RS radical-capable of initiating a chain reaction and further bleaching of the beta-carotene via .

Indeed no changes in the second-order decay at 450 nm were observed when [RSH] 5-30 mM, which would suggest that the resultant carbon-centered adduct-radical is relatively unreactive, presumably due to resonance stabilization through delocalization of the radical electron spin density along the polyconjugated backbone of beta-carotene.

Under the same experimental conditions the glutathione thiyl radical (GS) gave the same biphasic bleaching of the beta-carotene ground-state absorption and exhibited similar spectral changes previously observed for the beta-mercaptothiyl radical (see Fig. 5, left and right insets). Again no evidence was obtained for carotenoid radical-cation formation by the GS radical, which clearly favors the radical-addition pathway. In this case, however, the rate constant for GS radical-addition to beta-carotene, k = (2.2 ± 0.1) times 10^8M s is almost an order of magnitude slower than that previously measured for the beta-mercaptoethanol thiyl radical. Rate constants, k for a variety of thiyl radical reactions with beta-carotene (together with other rate constants measured during this study) are displayed in Table 1. Values for k vary by almost 2 orders of magnitude, depending on the degree of hydrophilicity of the thiyl radical under study. The lowest rate constant, k = (4.2 ± 0.3) times 10^6 mM s, was measured for the thiyl radical of WR-1065, which contains a doubly protonated polyamino side chain(65) . A similar trend has been observed for rates of thiyl radical abstraction of biallylic hydrogen atoms from PUFAs(42) .


Figure 5: beta-Carotene scavenging of the glutathione thiyl radical. Absorption spectra obtained on the pulse radiolysis of N(2)-saturated tert-butyl alcohol/water mixtures (50%, v/v) containing GSH (10 mM) and beta-carotene (10 µM) at pH 5. bleaching of the beta-carotene ground-state absorption was observed 50 µs (solid circles) and 0.1 s (open circles) after approximately 1-Gy pulses. Insets: optical traces at 450 nm showing exponential fast then slower bleaching of beta-carotene as observed for other thiols.





Since thiyl radicals are also capable of abstracting a hydrogen atom from the activated C-H bonds of alcohols and ethers(42) , it was necessary to rule out the possibility of hydrogen abstraction from beta-carotene (). Provided the experimental conditions remained constant the rate constants for the bimolecular decay of [beta-carotene] radicals following were expected to be independent of the nature of the attacking thiyl radical. However, further evidence supporting the assignment of the observed thiyl radical-mediated biphasic bleaching of the beta-carotene ground-state absorption to formation and decay of the [RSbulletbulletbulletbeta-carotene] adduct-radical via and was derived from comparing second-order rate constants, 2k for thiyl radicals of beta-mercaptoethanol and WR-1065. Addition of the WR-1065 thiyl radical to beta-carotene effectively lowered the rate of bimolecular decay of the adduct-radicals due to mutual electrostatic repulsion of the polyamino side chains(65) , i.e. where R = H(3)N(CH(2))(3)H(2)N(CH(2))(2)-. The measured rate constant 2k = (5.2 ± 0.1) times 10^5M s was slower than the glutathione thiyl radical-adduct (1.3 ± 0.1) times 10^6M s and almost an order of magnitude slower than that of the more hydrophobic beta-mercaptoethanol thiyl radical-adduct (2.1 ± 0.1) times 10^6M s. Thiyl radical-addition to beta-carotene is kinetically favored relative to hydrogen abstraction from the polyconjugated C-H bonds.

Scavenging of RSO(2)Radicals by beta-Carotene

The absorption spectra obtained by pulse radiolysis of an N(2)-saturated tert-butyl alcohol/water mixture (50%, v/v) containing 10 mM C(2)H(5)SO(2)Cl and 1 mM phosphate buffer at pH 5 are shown in Fig. 6. The observed spectra were attributed to the rapid scavenging of the thiyl-sulfonyl C(2)H(5)SO(2) radical by beta-carotene to generate carotenoid radical species. The characteristic absorption of the [beta-carotene] radical-cation peaking at 910 nm is accompanied by a biphasic decline in the beta-carotene ground-state absorption between 300 and 600 nm, characteristic of a carotenoid radical-adduct. Thiyl-sulfonyl radicals clearly react by both electron transfer and radical-addition pathways, and .


Figure 6: Carotene scavenging of sulfonyl radicals. Absorption spectra showing the bleaching of the beta-carotene ground-state absorption measured by pulse radiolysis (200 µs and 0.2 s after approximately 1.5-Gy pulse, respectively) of N(2)-saturated tert-butyl alcohol/water mixtures (50%, v/v) containing ethanesulfonyl chloride (10 mM) and beta-carotene (10 µM) at pH 5. Insets: optical traces showing a biphasic decline in the beta-carotene ground-state absorption at 450 nm and the exponential growth of the beta-carotene radical-cation absorption at 910 nm.



The radiation chemical yield of the [beta-carotene] radical-cation at 910 nm was determined as G = 6.1 ± 0.2 times 10^4M cm. Taking approx 9.4 ± 200 times 10^4M cm for the [beta-carotene] radical-cation gave a yield of G[beta-carotene] = 0.64, i.e. 71% of the original radical yield G(C(2)H(5)SO(2)) = 0.9. Formation of the [C(2)H(5)SO(2)bulletbulletbulletbeta-carotene] radical-adduct therefore accounts for the remaining 29% of the C(2)H(5)SO(2) radical yield.

The fast decline in absorption at 451 nm was exponential and first-order in beta-carotene concentration and reflects the contributions from both and . The overall rate constant for C(2)H(3)SO(2) radical attack on beta-carotene was obtained from the slope of the plot of observed first-order rate constant (k) versus beta-carotene concentration and was found to be k/k = 5.5 ± 0.1 times 10^9M s. From the relative yields of [beta-carotene] radical-cation to the [C(2)H(5)SO(2)bulletbulletbulletbeta-carotene] radical-adduct (i.e. 71 to 29%, respectively), the first-order rate constants k and k were determined. The observed rate constants for electron transfer (k) and radical-addition (k) are given by k = (0.71 times k) and k = [(1 - 0.71) times k], respectively. Fig. 7shows the linear plots of k and kversus [beta-carotene] the slopes of which correspond to the rate constants k = (3.9 ± 0.1) times 10^9M s and k = (1.6 ± 0.1) times 10^9M s. The rate constant for obtained from the slope of the plot of the first-order build up of the [beta-carotene] radical-cation absorption at 910 nm versus [beta-carotene] gave k = (3.4 ± 0.1) times 10^9M s, in excellent agreement with that obtained from the corresponding kinetics at 450 nm.


Figure 7: Kinetics associated with beta-carotene scavenging of the sulfonyl radical. Transient absorption at 450 nm observed on reaction of C(2)H(5)SO(2) radical with beta-carotene at pH 5 initiated by a pulse of 1.5 Gy. Inset: plots of the observed and calculated first-order rates: k at 450 nm (solid circles), k at 910 nm (open squares), k at 450 nm (solid triangles) and k at 450 nm (solid squares) versus beta-carotene concentration.




DISCUSSION

Free radical oxidants, which are recognized as potential initiators of the chain of lipid peroxidation, including NO(2), RS, and RSO(2) radicals, were rapidly scavenged by the lipid-soluble antioxidant beta-carotene. Although NO(2) and RS radicals are both moderately strong one-electron oxidants with similar redox potentials (e.g. E^o(NO(2)/NO(2)) = + 1.04 V) (66) and E^o(HO(CH(2))(2)S, H/HO(CH(2))(2)SH) = + 1.3 V)(67) , respectively), they interact with beta-carotene via distinct mechanisms. Nitrogen dioxide reacts exclusively by electron transfer to generate the [beta-carotene] radical-cation which decays via a bimolecular charge transfer process. In marked contrast all the RS radicals under study undergo rapid addition reactions to generate [RSbulletbulletbulletbeta-carotene] adduct-radicals. Thiyl radicals are particularly electrophilic, and it is therefore not surprising that they add to centers of relatively high electron density such as the polyconjugated -system of beta-carotene. This electrophilicity may be enhanced by the ability of sulfur to use d orbitals to accommodate negative charge. Thiyl-sulfonyl radicals can abstract an electron to generate the polyene radical-cation (71%) or add to the polyconjugated double bonds to generate [RSO(2)bulletbulletbulletbeta-carotene] adduct-radicals (29%).

Hydrogen abstraction by nitrogen dioxide, thiyl, and thiyl-sulfonyl radicals from PUFAs generate pentadienyl radicals(41, 42, 68) , which act as radical precursors in the chain of lipid peroxidation(43) . Although deviations from homogeneous reaction kinetics are expected within cells, rate constants for radical-scavenging by beta-carotene compare favorably with the corresponding rates of hydrogen abstraction from PUFAs. For example, the rate constant for the oxidation of beta-carotene by GS radical, k = (2.2 ± 0.1) times 10^8M s is almost 1 order of magnitude faster than with linoleic, linolenic, and arachidonic acids, k = 0.8-3.1 times 10^7M s(42) . However, more lipophilic thiyl radicals, for example the HO(CH(2))(2)S radical, the differential is greater, k = (2.5 ± 0.1) times 10^9M s as opposed to 3.1-6.8 times 10^7M s for the same range of PUFAs(42) .

The antioxidant properties of beta-carotene will of course not only reflect rates of free radical scavenging, but also the reactivity of the resultant beta-carotene-derived radicals. Carotenoid radical-cations and adduct-radicals are highly resonance stabilized and must therefore be relatively unreactive compared with the attacking free radical species(10) . Within lipid-rich environments, such as biomembranes or LDL carotene, the bimolecular decay of radical-adducts and radical-cations will generate non-radical products probably incapable of exerting prooxidative effects. Carotenoid radical-adducts are also likely to contribute to the antioxidant properties of beta-carotene by scavenging PUFA radicals, thereby terminating the chain of lipid peroxidation as exemplified by and .

The antioxidant efficiency of beta-carotene against peroxyl radical-initiated lipid peroxidation in homogeneous solutions is diminished with increasing partial pressure of oxygen (pO(2)) (18) Corroborating observations in liposomes (69, 70) and in LDL (19) suggest that carotenoid-peroxyl radicals may prove important in vivo particularly in tissues of high pO(2) such as the lung. Based on the classical mechanisms of peroxyl radical formation, the conjugation of molecular oxygen with beta-carotene adduct-radicals to generate a carotenoid-peroxyl radical () represents a most feasible decay pathway following RS radical oxidation of beta-carotene. Due to resonance stabilization within the carotenoid adduct-radical, the reaction with oxygen may be represented as a reversible equlibrium .

High pO(2) is likely to drive to the right, thereby promoting the autoxidation of beta-carotene or PUFAs in a similar manner to other carotenoid-peroxyl radicals(9, 18) . Theoretically, any radical species likely to generate carotenoid adduct-radicals when scavenged by beta-carotene, including thiyl-sulfonyl radicals, may initiate prooxidative pathways at high pO(2). No evidence was obtained for the reaction of carotenoid radical-cations with molecular oxygen. In the case of nitrogen dioxide, beta-carotene is likely to behave as a chain-breaking antioxidant, since the carotenoid radical-cations may undergo bimolecular decay by charge transfer to generate non-radical products, which are unlikely to exhibit prooxidative effects. However, GSH has been shown to enhance nitrogen dioxide-induced lipid peroxidation and DNA strand breaks in model systems(35) , possibly via the formation of thiyl and thiyl-derived radical species according to .

represents a stoichiometric conversion of nitrogen dioxide to the glutathione thiyl radical(53, 71) , which in the context of free radical scavenging by beta-carotene may produce a corresponding shift from carotenoid radical-cation to adduct-radical formation. represent the formation of glutathione thiylperoxyl (GSOO), sulfonyl (GSO(2)), sulfonylperoxyl (GSO(2)OO), and sulfinyl (GSO) radicals, moderately strong oxidants which may exert damaging effects in vivo(44, 45, 46, 72, 73) . The interplay of free radical reactions in oxidative stress has been largely overlooked in studies of the antioxidant effects of beta-carotene(9, 10) , a factor that may contribute to some of the contradictory results obtained from in vitro models.


FOOTNOTES

*
This work is supported by the Association for International Cancer Research and by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 44-1923-835-210; :everett{at}graylab.ac.uk.

(^1)
The abbreviations used are: LDL, low density lipoproetin; Gy, SI unit of radiation dose (1 Gy = 1 J Kg); G-value, radiation chemical yield of free radical species (G = 1 equals approx 0.1 µmol J in SI units); PUFAs, polyunsaturated fatty acids; ABTS, 2,2`-azino bis(3-ethylbenzothiazoline-6-sulfonate).


ACKNOWLEDGEMENTS

We thank Dr. Boris Voijnovic and the Advances in Technology Group at the Gray Laboratory for assistance with the pulse radiolysis.


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