Detection of free radicals in aqueous extracts of cigarette tar by electron spin resonance

Free Radical Biology & Medicine, Vol. 19, No. 2, pp. 161-167, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00

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Original Contribution DETECTION OF FREE RADICALS IN AQUEOUS EXTRACTS OF CIGARETTE TAR BY ELECTRON SPIN RESONANCE L u N - Y I ZANG, KONI STONE, and WILL1AM A. PRYOR Biodynamics Institute, Louisiana State University, Baton Rouge, LA, USA

(Received 2 May 1994; Revised 17 November 1994; Accepted 2 December 1994) A b s t r a c t Aqueous extracts of cigarette tar (ACT) autooxidize to produce semiquinone, hydroxyl, and superoxide radicals in Mr-saturated buffered aqueous solutions. The semiquinone species were detected by direct electron spin resonance (ESR) measurements and identified as o- and p-benzosemiquinone radicals by comparison with the ESR signals of catechol and hydroquinone radicals under similar conditions. The rate of formation of these radicals was dependent on pH. Hydroxyl and superoxide radicals were detected as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adducts by ESR spin trapping. Superoxide dismutase (SOD) (20 units/ml) inhibited the formation of the superoxide spin adduct of DMPO completely. Addition of Fe 2+ to this system increased the ESR signal intensity of hydroxyl radical spin adduct of DMPO three to five times. These results indicate that superoxide and hydroxyl radicals are produced during the autooxidation of hydroquinone- and catechol-related species in ACT. Keywords--Electron paramagnetic resonance, Electron spin resonance, Hydroxyl, Semiquinone, Spin trapping, Superoxide, Free radicals

ide (H202) and hydroxyl radicals were detected in aqueous extracts of cigarette tar (ACT) by polarography and ESR spin trapping techniques, respectively. 17AsSince dioxygen cannot accept two electrons simultaneously (because of spin restrictions), it is reasonable to postulate that the hydrogen peroxide produced during the autooxidation of cigarette tar arises from the superoxide radical. In the present study, we report evidence of the formation of superoxide from o- and p-benzosemiquinones during the autooxidation of aqueous extracts of cigarette tar.

INTRODUC~ON

Tobacco use is believed to be a factor in the etiology of ischemic heart disease, pulmonary cancer, chronic obstructive lung disease, and peripheral vascular occlusive disease.~ Cigarette tar binds to DNA and causes single-strand breaks in the DNA of cultured human cells. 2-5 Reactive free radical species produced by the autooxidation or metabolism of smoke constituents are thought to play a role in smoke toxicity.6-g Cigarette smoke contains catechol and hydroquinone,9-1~ which oxidize to form semiquinone radical species. 2''3 Semiquinone radicals have been observed previously in many systems by photochemical, photosensitization, or metal stabilization techniques in combination with ESR detection, ~2.14A5and it might be expected that they would be generated in extracts of cigarette smoke. In fact, p-benzosemiquinone radicals can be observed in alkaline-treated cigarette tar extracts by direct ESR measurement.6 Recently, the generation of oxygen radicals during the oxidation of cigarette tar components was suggested, 16based on the fact that hydrogen perox-

MATERIALS AND METHODS

Research-grade cigarettes (1R2) were obtained from the Kentucky Tobacco Research Council. The spin trap 5,5-dimethyl- 1-pyrroline- N-oxide (DMPO) was purchased from Aldrich Chemical Company (Milwaukee, WI) and was purified as previously des c r i b e d , t9'2° Bovine erythrocyte superoxide dismutase (SOD), catalase, catechol, hydroquinone, 3-cyanoproxyl free radical (to be used as the nitroxide standard for determining relative concentrations of free radicals), and diethylenetriamine pentaacetic acid (DTPA)

Address correspondence to: William A. Pryor, Biodynamics Institute, 711 Choppin Hall, Louisiana State University, Baton Rouge, LA 70803-1800, USA. 161

162

L.-Y. ZANt3et

were obtained from Sigma Chemical Company (St. Louis, MO). All other chemicals were obtained at the highest obtainable purity. Aqueous cigarette tar (ACT) extracts were prepared as described previously. 21 The tar from five cigarettes on a Cambridge filter was extracted with 10 ml of distilled water, and the solution was dried under vacuum at 45°C overnight. The dried tar is a sticky material with dark brown color and was again taken up into distilled water. These ACT solutions contain about 10 mg tar per milliliter. Unless otherwise stated, the reaction mixture consisted of ACT ( 1 mg/ml) in 50 mM carbonate buffered aqueous solutions or in 50 mM carbonate buffered DMSO (7.0 M) solutions (pH 9.0), both in the presence of 0.25 mM DTPA in 2.0 ml final volume. Individual samples were placed into a Varian E-9 spectrometer cavity for ESR measurements. The ESR parameters were set at 100 KHz, X-band; microwave frequency, 9.363 GHz; microwave power, 5 roW; modulation amplitude, 0.63 G; time constant, 0.25 s; and receiver gain, 1.25 × 104. RESULTS The incubation of an air-saturated reaction mixture of aqueous extracts of cigarette tar (ACT) in 50 mM carbonate-buffered aqueous solution, pH 10.0, generates free radicals (Fig. l ). In the absence of ACT, no ESR signal is observed. The ESR spectrum in Fig. la contains two free radical species. The signals marked by p exhibit a quintet pattern with an intensity ratio of 1:4:6:4:1,20 a hyperfine splitting constant (hfsc) of a4H = 2.37 G, and a g value of 2.0040, values consistent with the p-benzosemiquinone anion. 22 The species marked with an open circle shows a triplet of triplets in a ratio of 1:2:1 and an hfsc of a2H = 3.68 G, ash = 0.76 G, and g value of 2.0039, values in accord with the those for the o-benzosemiquinone anion radical. ~4 The p-benzosemiquinone radical was found to decay rapidly. As shown in Fig. lb, the ESR spectrum of the o-benzosemiquinone radical dominates after a 10-min incubation at 25°C. To further confirm the generation of o- and p-benzoserniquinone radicals in ACT, we compared the ESR spectra obtained from freshly prepared, aerated catechol and hydroquinone solutions with those species generated in ACT under the same conditions. As shown in Figs. lc and d, the ESR spectra of these radicals are the same as those obtained in ACT. Catechol or hydroquinone solutions were aged for 6 months in 50 mM phosphate buffer, pH 7.4, under air in an open bottle. These solutions were investigated in an air-saturated 50-mM carbonate-buffered aqueous

al.

solution, pH 9.0; the ESR spectra are shown in Fig. 1, traces e and f, respectively. These spectra are similar to the ESR spectra for fresh autooxidized catechol and hydroquinone solutions under same conditions (Fig. 1, traces c and d). The similar hfsc and g values for catechol and hydroquinone solutions that were freshly prepared or were aged for 6 months are compared in Table 1. The pH plays an important role in the formation of free radicals during the autooxidation of cigarette tar in the aqueous system. As shown in Fig. 2, the o- and p-benzosemiquinone radicals increased in concentration with increasing pH when ACT (1 mg/ml) solutions at the indicated pH values were incubated for 1 min. Below pH 8.0, the rates of accumulation of these radicals are slow. The use of aqueous DMSO solutions is known to allow detection of the superoxide adduct of DMPO. The incubation of an air-saturated reaction mixture of ACT ( 1 mg/ml) and DMPO ( 100 mM) in a carbonatebuffered aqueous dimethyl sulfoxide (7 M) solution at pH 9.0 gives ESR signals that can be assigned to superoxide and carbon-centered radical spin adducts (Fig. 3a). The carbon-centered radical probably is a methyl radical adduct resulting from the reaction of hydroxyl radicals with the DMSO present in our solutions. (A p-benzo semiquinone ESR signal is observed initially, but it rapidly decays and is replaced with the superoxide spin adduct signal, which is about 30% of the intensity of the original semiquinone.) In the absence of ACT, no ESR signals were observed. The hfsc of the signal (aN = 13.4 G, ~aH = 10.3 G, and Van = 1.3 G) are consistent with previously reported values for the DMPO superoxide spin adduct in buffered dimethyl sulfoxide solutionsY -25 The reaction between xanthine and xanthine oxidase is known to produce superoxide. 26 As shown in Fig. 3b, the ESR spectrum obtained by this reaction system is very similar to the spectrum observed for ACT (Fig. 3a), supporting the conclusion that superoxide is generated during the autooxidation of ACT. Superoxide dismutase at 20 units/ml completely eliminates the ESR signal of DMPO superoxide spin adduct in ACT (Fig. 3c). It follows that autooxidation of ACT generates superoxide, and DMPO is capable of reacting with this primary species to form the spin adduct with the characteristic ESR signal shown in Fig. 3a. The superoxide spin adduct of DMPO is known to decompose to the D M P O - O H adduct; in addition, superoxide can dismutate to form hydrogen peroxide, which can give the hydroxyl radical. 27 Therefore, these ACT and DMPO aqueous solutions (50 mM carbonate buffer, pH 9.0) also give a spin adduct signal consisting of a four-line pattern with an intensity ratio of

Cigarette tar and free radicals

163 O °

p,o

a4H - 2 37 G

P

g - 2 0040

o

,/

o

o

V

ACT\

I/

.....

" %

B2H,,, 0.76 G a2H I 3 68 G

u

-

P

"

O

V

'

p,o ~

. . . . . V " -'.lNOa

II

O

~

~1" °

a[

.

0

a2H-, 0.76 G a2H- 3.68 G

Catechol

/~

1

g - 2.0039

~

I~ ~ ~

"" O -

.,. a4H=2.37aI ......

Hydroqulnone

A

U

,o.c._o!

o.,oo,, Jr•

a4H

A "

~.J

/I " I'''''"

2 37"G I V

"

i0.,oo,o

o.

"

Fig. I. Comparison of the ESR spectra of semiquinone radicals obtained from different sources. All of the hydroxyaromatics (traces c, d, e, and f) were 1.0 mM and were in air-saturated 50-mM carbonate-buffered solution, pH 9.0. In traces a and b, the ACT solutions contained 1.0 mg/ml tar and were at pH 10.0. Traces c and d were freshly prepared catechol and hydroquinone solutions. Traces e and f were catechol and hydroquinone solutions, pH 7.4, aged for 6 months (see the footnote for Table 1). All ESR spectra were recorded after 1-min incubation except the ACT trace shown in line b, which was recorded after 9-min incubation. The ESR parameters were set at 5-mW microwave power and a 0.25-s time constant for all ESR spectra; modulation amplitude, 0.25 G, and receiver gain, 8 x 103 for (a) and (b); and modulation amplitude, 0.1 G, and receiver gain, 2.5 x 103 for (c) through (f).

1:2:2:1 and hfsc o f aN -- an = 14.95 G (Fig. 3 d ) , consistent with the values for D M P O - O H . 28 Ethanol, a w e l l - k n o w n s c a v e n g e r o f h y d r o x y l radicals, 29-31 can react to p ro d u ce an a - h y d r o x y e t h y l radical, w h i c h adds to D M P O to f o r m a spin adduct. Thus, w h e n ethanol ( 1 . 5 % ) was added to the aqueous system, the E S R spectrum exhibited 3 x 2 lines with identical intensity

(Fig. 3 e ) , with hfsc (aN = 16.1 G and an = 23.1 G ) consistent with values for D M P O - C H ( O H ) CH3,32.33 indicating that the production o f D M P O - O H is the result o f trapping h y d r o x y l radicals. A d d i t i o n o f Fe 2÷ ( 5 0 # M ) to the mixture o f A C T and D M P O e n h a n c e d the E S R signal intensity o f D M P O - O H by three to five times the control (Fig.

164

L.-Y. ZANG et al. Table 1. Comparison of Semiquinone Radicals Detected from Different Sources Sources

Radicals Detected

Hyperfine Splitting

ACT

p-benzosemiquinone o-benzosemiquinone

Catechol

o-benzosemiquinone

Aged catechol*

o-benzosemiquinone

Hydroquinone Aged hydroquinone*

p-benzosemiquinone p-benzosemiquinone

a4H = a2. = a2H = a2a = a2. = am = a2H = a4n = aaH =

2.37 3.68 0.76 3.68 0.76 3.68 0.76 2.37 2.37

G G G G G G G G G

g Value 2.0040 2.0039 2.0039 2.0039 2.0040 2.0040

* Stock (50 mM) was aged for 6 months in 50 m M phosphate buffer, pH 7.4. The final concentrations for all experiments above were 1 raM.

3f). In this Fe 2÷-catalyzed system, SOD had little effect on the D M P O - O H signal intensity, indicating that the enhancement of the ESR signal of the hydroxyl radical spin adduct of DMPO by Fe 2÷ is due to a Fenton-type reactions involving hydrogen peroxide. 34 Catalase at the level of 15 #g/ml suppressed the ESR signal of the hydroxyl radical spin adduct completely (data not shown), further implicating hydrogen peroxide as the precursor of the hydroxyl spin adduct. DISCUSSION

The results reported here demonstrate that semiquinone radicals are produced during the autooxidation of aqueous cigarette tar extracts, and these primary radicals lead to superoxide radicals, hydrogen peroxide, and the hydroxyl radical. When cigarette tar was incubated with DMPO in buffered aqueous DMSO, ESR signals due to semiquinone radicals were observed, but these rapidly decayed and were replaced by the superoxide spin adduct (Fig. 3a). 23'35 This is

150

i ,00

_c=

0

'-'

7

'±

8

9

10

11

12

pH Fig. 2. Effect of pH on the formation of radicals at a fixed A C T concentration of 1.0 mg/ml. All points were measured after a lmin incubation at 25°C. ESR spectroscopy settings were microwave power, 10 mW; modulation amplitude, 0.63 G; time constant, 0.5 s; receiver gain, 2.0 × 104.

confirmed by the following observations: ( 1 ) Superoxi d e d i s m u t a s e 36'37 virtually eliminates the ESR signal (Fig. 3c), and (2) the ESR spectrum obtained by the reaction of the xanthine/xanthine oxidase system, a known superoxide radical source, 38gives ESR parameters in agreement with those from ACT. In an aqueous solution, an ESR signal with four lines in an intensity ratio of 1:2:2:1 (Fig. 3d) and an hfsc of aN = aH = 14.95 G is observed, consistent with the D M P O - O H adduct. 28 Addition of ethanol yields the a-hydroxyethyl radical adduct, further confirming ~7the formation of hydroxyl radicals in the aqueous cigarette tar system. Since superoxide can spontaneously dismutate to form hydrogen peroxide, 39 H202 accumulates in ACT solutions. ~8 The reaction in which superoxide radicals react with and destroy hydrogen peroxide is s l o w , 4°'41 but ferrous iron rapidly reduces hydrogen peroxide to the hydroxyl radical. As little as 50 /zM of Fe 2÷ enhanced the ESR signal for the hydroxyl radical spin adduct to three to five times of the intensity of the control (Fig. 3f), confirming that the enhancement of the ESR signal is due to the Fenton reaction. Catalase (at 15.0 #g/ml ), a scavenger of hydrogen peroxide, 42 virtually eliminates the hydroxyl radical spin adduct signal, confirming ~s that hydrogen peroxide is produced in the autooxidation of cigarette tar. The autooxidation of polyhydroxyaromatics is pH dependent. 43 As shown in Scheme I, after one proton is lost, the polyhydroxyaromatic QH2 forms an anion, Q H - , which has two possible pathways to form semiquinone radicals. In the first, the semiquinone radical could be formed after a hydrogen atom transfer from Q H - . In the second, Q H - loses a second proton to form a dianion, Q 2-, which forms a semiquinone radical after transferring an electron to dioxygen. Loss of a second electron from the semiquinone radical generates the quinone. As shown in Scheme I, dioxygen can accept an electron or hydrogen atom to form superox-

Cigarette tar and free radicals

ACT

,

j~

ouso fl

It

Xan/Xan Oxldase A in buffered DMSO Jl

(c)

A | IIII

A ! II11

165

h

A II,

t~.

0 O_

+ SOD

inbuffer~:~Tqueous ~~

~"~.~HH

~

OH

CH 3

Fig. 3. The ESR spectra of spin adducts of radicals observed during the incubation of aqueous cigarette tar extracts in airsaturated 50-mM carbonate-buffered aqueous DMSO (7 M) or aqueous solutions, pH 9.0, The reaction mixtures consisted of ACT ( 1 mg/ml), DMPO ( 100 raM) in (a) through (c) pH 9.0 carbonate-bufferedDMSO solutions and (d) through (f) buffered aqueous solutions. (a) ACT + DMPO; (b) xanthine (80 #M)/xanthine oxidase (0.15 mg protein/ml) + DMPO; (c) same as (a) but in the presence of SOD (20 units/ml); (d) same as (a) but in buffered aqueous solution; (e) same as (d) but in the presence of ethanol (1.5%); and (f) same as (d) but in the presence of Fe2÷ (50 ~M). The ESR settings were microwave power, 20 mW; modulation amplitude, 0.63 G; time constant, 0.5 s; receiver gain, 2.0 × 104 for (a) and (d), 4 × 103 for (e) and (f). The ESR setting for (b) was microwave power, 10 mW; modulation amplitude, 0.5 G; time constant, 0.25 s; receiver gain, 4 × 103.

ide in each electron or h y d r o g e n - a t o m transfer step. Superoxide radicals can then be converted to form hydroxyl radicals via h y d r o g e n peroxide (Eqs. 1-3)26,44: 202"- + 2H20 ~ H202 + 02 + 2 O H -

(1)

02"- + HO~ + H ÷ ~ H202 + 02

(2)

2HO2 ~ H202 + 02

(3)

H y d r o g e n peroxide is destroyed in this system b y Haber-Weiss reactions (Eqs. 4 - 5 ) : H202 + O2"- --~ "OH + 0 2 + O H -

(4)

H202 + HO2 ~ "OH + 02 + H20

(5)

In the p r e s e n c e of S O D a n d ferrous iron, Eqs. 1 - 3

166

L.-Y. ZANG et al.

(OH-) OH

o_

1111

OH

OH

-2H +

-m

...11-..

(OH,)

o-

0

--e"

•

O"

0

O.

0

o,- 1Q'3

(Q)

°. O-

(o2-) Scheme I. Proposed mechanism for the generation of superoxide radical during the autoxidation of aqueous extracts of cigarette tar. The mechanism is shown for 1,4 benzoquinone; a similar mechanism can be written for 1,2-benzoquinone (catechol).

are replaced by the SOD-catalyzed reaction 24'28'36 and Eqs. 4 - 5 are replaced by the faster Fenton reaction (Eq. 7 ) 40'41" SOD

2 0 2 " - + 2H + -* H 2 0 2 + 0 2 H 2 0 2 + F e 2+ ~ "OH + O H - + Fe 3+

(6) (7)

Thus, we conclude that polyhydroxyaromatic compounds, including catechol and hydroquinone, are responsible for the radicals observed in aqueous solutions of cigarette tar.

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ACT--aqueous extracts of cigarette tar DMPO--5,5-dimethyl-l-pyrroline-N-oxide DTPA--diethylenetriamine pentaacetic acid EPR--electron paramegnetic resonance ESR--electron s p i n r e s o n a n c e ; a l s o k n o w n as E P R , electron paramegnetic resonance hfsc--hyperfine splitting constant o-Q'---o-benzosemiquinone p-Q'---p-benzosemiquinone Q--quinone SOD--superoxide dismutase