(free radicals/sulfur dioxide) ... peroxyl free radical, we soughtto obtain chemical support for .... organic extracts, however, demonstratedthat BP-7,8-diol.
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 7499-7502, October 1986
Medical Sciences
Epoxidation of (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene during (bi)sulfite autoxidation: Activation of a procarcinogen by a cocarcinogen (free radicals/sulfur dioxide)
GREGORY A. REED*, JOHN F. CURTIS, CAROLYN MOTTLEYt, THOMAS E. ELINGf, AND RONALD P. MASON: Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709
Communicated by Irwin Fridovich, June 16, 1986
ABSTRACT The (bi)sulfite ion undergoes extensive autoxidation in neutral aqueous media with the formation of sulfur trioxide radical anion that is detected by ESR. The radical anion subsequently reacts with molecular oxygen to form a peroxyl radical. We find that when (±)-trans-7,8-dihydroxy7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is included in this autoxidation system, BP-7,8-diol is converted to diolepoxides, ultimate carcinogenic derivatives of benzo[a]pyrene. This epoxidation occurs with a stereoselectivity consistent with either a peroxyl radical or a peracid as the epoxidizing agent. The epoxidation is dependent on the concentration of both (bi)sulfite and oxygen. In the presence of 10 IAM butylated hydroxyanisole, which abolishes (bi)sulflte autoxidation, no (bi)sulfite-dependent epoxidation occurs. These results are discussed in regard to the mechanism of (bi)sulfite autoxidation, and in relationship to the cocarcinogenicity of sulfur dioxide [anhydrous (bi)sulfite] for benzo[a]pyrene-induced pulmonary neoplasia.
Sulfur dioxide, an environmental pollutant, exists primarily as the (bi)sulfite anion in aqueous solution at near neutral pH. The metal-catalyzed autoxidation of the (bi)sulfite anion, studied for decades, is an oxygen-consuming chain reaction (1-3). The initiating one-electron oxidation yields the sulfur trioxide radical anion ('SO-), a predominantly sulfur-centered radical (4) that reacts with molecular oxygen. Superoxide initiates (bi)sulfite oxidation by forming 'SO (1, 5) but is not a chain propagator (2, 5), implying superoxide is not formed by reactions of the '0j radical. Presumably the reaction product of SO with oxygen is a peroxyl free radical.
S53
+ 02
-03SOO.
[1]
Huie and Neta (6) presented a kinetic argument favoring this oxygen addition reaction. Since this pathway produces a peroxyl free radical, we sought to obtain chemical support for the formation of -03SOO during (bi)sulfite autoxidation based on products of a specific trapping reaction. The trapping agent employed was (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). This compound is stereoselectively epoxidized to form (+)-7r,8t-di-
hydroxy-9t, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) by either peroxyl radicals (7-10) or peracids (11-13). The inclusion of BP-7,8-diol in an aqueous system for (bi)sulfite autoxidation did indeed lead to the production of anti-BPDE. In this paper we present a characterization of this epoxidation and consider the implications ofthis reaction in regard to the mechanism of (bi)sulfite autoxidation and the
cocarcinogenicity of sulfur dioxide in the presence of benzo[a]pyrene. EXPERIMENTAL PROCEDURES Materials. The following compounds were supplied by the National Cancer Institute Chemical Carcinogen Reference Standard Repository: BP-7,8-diol, [G-3H]BP-7,8-diol (416 mCi/mmol; 1 Ci = 37 GBq), (±)-7r,8t-dihydroxy-9t,10tepoxy-7,8,9,10-tetrahydro[7-14C]benzo[a]pyrene (antiBPDE) (17 mCi/mmol), and (+)-7r,8t-dihydroxy-9c,10cepoxy-7,8,9,10-tetrahydro[7-14C]benzo[a]pyrene (synBPDE) (17 mCi/mmol). Chromatographic standards were prepared by solvolysis of the [14C]-labeled diol epoxides (14). Butylated hydroxyanisole (BHA), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), superoxide dismutase, and diethylenetriaminepentaacetic acid (DETAPAC) were from Sigma. Potassium peroxomonosulfate (OXONE, DuPont) was obtained from Aldrich. Polyoxyethylene sorbitan monolaurate (Tween 20) and all chromatography solvents were from Fisher. Reaction Conditions. Tritiated and unlabeled BP-7,8-diol, in ethanol, were added to the reaction vessel, and the solvent was evaporated under a stream of N2. The residue was dissolved in 0.1 M potassium phosphate buffer, pH 7.0, containing 0.5% Tween 20. The specific activity and concentration of [3H]BP-7,8-diol for all experiments were 45 mCi/mmol and 10 ,uM, respectively. DETAPAC, when used, was dissolved directly in the buffer, while BHA was added as an acetone solution; the final acetone concentration was 0.1%. To study the oxygen-dependence of the system, buffer containing BP-7,8-diol was bubbled for 30 min with argon, air, or oxygen prior to initiation of the reaction, and reaction vessels were purged with the appropriate gas during the reaction. All incubations were performed at 37°C with continuous shaking. Incubations were initiated by the addition of (bi)sulfite or of OXONE. (Bi)sulfite and OXONE stock solutions (10-100 mM) were prepared in 0.1 M potassium phosphate buffer, pH 7.4, containing 1 mM DETAPAC to inhibit autoxidation. Aliquots of 1 ml were withdrawn from incubation mixtures at the indicated times, and the reaction was immediately quenched by the addition of BHA to a Abbreviations: BHA, butylated hydroxyanisole; BP-7,8-diol, (+trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; anti-BPDE, ()-
7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene;
DMPO, 5,5-dimethyl-1-pyrroline n-oxide; DETAPAC, diethylenetriaminepentaacetic acid; BP tetraols, isomeric 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene. *Present address: Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66103. tPermanent address: Department of Chemistry, Luther College, Decorah, IA 52101. tTo whom correspondence should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7499
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concentration of 100 AM. Organic-soluble components were extracted twice with 1.5 ml of ethyl acetate. For experiments using OXONE, samples were immediately extracted with ethyl acetate following the addition of 100 AM BHA. These extracts were evaporated under reduced pressure, and the residues were dissolved in 250 1.l of methanol for analysis. Analytical Procedures. UV/visible spectra were obtained using a Hewlett-Packard 8450A diode array spectrophotometer. HPLC was performed using a Waters C18 puBondapak column (10 Am, 3.9 mm x 30 cm) as described (9). Radioactivity eluting from the column was quantitated using a Radiomatic Flo-One 83 system and Hydrofluor scintillation cocktail. ESR Measurements. ESR spectra were done on an IBM Instruments ER 200D-SRC electron spin resonance spectrometer equipped with a TM cavity. Samples were prepared by diluting 2.5 M (bi)sulfite in pH 7.4 phosphate buffer (1 mM in DETAPAC) to a final concentration of 10 mM (bi)sulfite using water. The final signal intensity was very dependent on the purity of the water, and varied from day to day and incubation to incubation.
RESULTS Examination of (bi)sulfite autoxidation, as measured by oxygen consumption, verified that the autoxidation is rapid in the neutral buffer and detergent system. Upon addition of 1 mM (bi)sulfite, oxygen consumption began immediately, and more than 70% of the dissolved oxygen was consumed within 3 min. Oxygen consumption was totally abolished in the presence of 1% methanol or ethanol (2, 3) or in the presence of 1 mM DETAPAC (15, 16). The autoxidation of (bi)sulfite resulted in the formation of a *'SO radical. This labile free radical intermediate was detected by spin trapping with DMPO. Fig. 1 shows an ESR spectrum of a DMPO-'SO3 nitroxide adduct detected in these incubation systems. The hyperfine coupling constants are identical to those reported (15). This experiment confirms, for the first time with ESR, the existence of 'SO- radical in the autoxidation mechanism. The inclusion of [3H]BP-7,8-diol in the autoxidation system does not affect oxygen consumption. Analysis by HPLC of organic extracts, however, demonstrated that BP-7,8-diol was extensively consumed during (bi)sulfite autoxidation, yielding polar products. Some labeled material eluted in or near the solvent front. The yield of this material varied widely, ranging from 1% to 15% of the total organo-soluble labeled material. The major organo-soluble products coelute with isomeric 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene standards (BP tetraols) prepared by the hydrolysis of [14C]BP-7,8-diol-9,10-epoxides (11-13). The UV spectra of these isolated products confirmed their identity. All exhibited clear maxima at 236, 246, 255, 266, 277, 312,
Proc. Natl. Acad Sci. USA 83
(1986)
326, and 342 nm, a spectrum diagnostic for 7,8,9,10tetrahydrobenzo[a]pyrene derivatives. The ratio of tetraols derived from the diastereomeric anti- and syn-diolepoxides was approximately 6:1. Formation of water-soluble products was also observed. The addition of methanol to short-term incubations yielded organic extractable metabolites that coelutedwiththe 10-methoxy-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (17), indicating the intermediacy of the diolepoxide (data not shown). The dependence of product formation on (bi)sulfite concentration is shown in Fig. 2. Rapid epoxidation of BP-7,8diol occurs at (bi)sulfite concentrations of 0.5 and 1 mM, while with 0.1 mM (bi)sulfite, significant epoxidation is not observed until 60 or 120 min of reaction time. The formation of water-soluble material exhibited a similar time- and (bi)sulfite-dependence, yielding about 50%o as much product as the epoxidation reaction. These water-soluble products have not been reported (13) and are possibly sulfited products. To determine the role of (bi)sulfite autoxidation in the consumption of BP-7,8-diol, various modifiers of the autoxidation were employed. The metal ion chelator DETAPAC, an inhibitor of (bi)sulfite autoxidation (15, 16), caused some inhibition of epoxidation at 100 ,M and 60-70% inhibition at a concentration of 500 ILM (Fig. 3). At 1 mM, DETAPAC totally abolished both (bi)sulfite-dependent oxygen consumption and epoxidation (data not shown). The effect on epoxidation of fDETAPAC in concentrations far in excess of the trace transition metal concentrations may indicate that DETAPAC is breaking a free radical chain reaction-i.e., DETAPAC may be a weak antioxidant (1). Inhibition of the formation of water-soluble products by DETAPAC was identical to that shown for formation of the epoxidation products (data not shown). The phenolic antioxidant BHA proved to be a far more potent inhibitor of (bi)sulfite-dependernt epoxidation than was DETAPAC (Fig. 4). At a concentration of 10 ,uM, BHA completely blocked the epoxidation of BP-7,8-diol. Forma-
4.0 1 mm
/
3.0
a0 2.0
-
20
40
W 120 60
Time, min FIG. 1. Spectrum ofthe SO3 adduct of DMPO from the autoxidation of (bi)sulfite. Incubations contain 10 mM (bi)sulfite, 270 mM DMPO. The siknal intensity continued to increase with time. The spectrum here was recorded 10 min after mixing. Instrument conditions were as follows: 100-G scan (1 G = 1 x 10-4 T), 0.32-G modulation amplitude, 2 x 101 gain, 200-msec time constant, 200-sec sweep time, and 21-mW microwave power.
FIG. 2. (Bi)sulfite-dependent epoxidation of BP-7,8-diol. The indicated concentrations (0.1-1.0 mM) of sodium (bi)sulfite were incubated at 37°C with 10 AM [3H]BP-7,8-diol in 0.1 M potassium phosphate, pH 7.0, containing 0.5% Tween 20. Aliquots were removed at the noted times and quenched with BHA, and ethyl acetate extracts were analyzed by HPLC. Epoxide formation was inferred from the formation of BP tetraols, the spontaneous hydrolysis products of the diolepoxides.
Medical Sciences: Reed et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
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the oxygen concentration, as this process was inhibited under argon and stimulated under oxygen (data not shown). Our results demonstrate a requirement for (bi)sulfite autoxidation and for molecular oxygen for epoxidation to occur. Among the possible products of (bi)sulfite autoxidation is peroxomonosulfate. The potassium salt of this compound has been observed to epoxidize various alkenes (18). We find that peroxomonosulfate epoxidizes BP-7,8-diol, but is much slower than (bi)sulfite under identical conditions (Fig. 5). The formation of material(s) eluting in the solvent front and of nonextractable products was also observed with a time course similar to that shown for tetraol formation. In marked contrast to the (bi)sulfite-dependent reactions, 10 ,u M BHA had no effect in the peroxomonosulfate system (Fig. 5).
2.0
0
0.0
DISCUSSION (Bi)sulfite-dependent epoxidation of BP-7,8-diol clearly requires the autoxidation of (bi)sulfite. The ability of
.
I
20
40
I
60
Time, min FIG. 3. Inhibition of (bi)sulfite-dependent epoxidation of BP-7,8diol by DETAPAC. Reaction conditions and analytical procedures were as described in Fig. 2. (Bi)sulfite concentration was 1 mM, and DETAPAC concentrations were as noted.
tion of water-soluble products was inhibited by about 70%o under these conditions (data not shown). Superoxide dismutase (40 ,ug/ml) had no effect on either the formation of tetraols or water-soluble products. The oxygen sensitivity of (bi)sulfite-dependent BP-7,8-diol epoxidations was investigated by running the reactions under argon, air, or oxygen atmospheres. Under argon, no epoxidation products were observed during a 60-min incubation (data not shown). The requirement for oxygen was further established by the observed stimulation of the epoxidation reaction when done under an oxygen atmosphere. The formation of water-soluble products was not as sensitive to
DETAPAC or BHA to block both (bi)sulfite autoxidation and BP-7,8-diol epoxidation supports the requirement for the autoxidation. The effect of molecular oxygen on the epoxidation also points to a role for (bi)sulfite autoxidation. ESR experiments demonstrate the formation of 'SO during (bi)sulfite autoxidation. The observed epoxidation provides complementary evidence to that presented by Huie and Neta (6) in support ofthe formation of 03SOO' during (bi)sulfite autoxidation. Evidence for 03SOO- formation was based on the magnitude and pH dependence of the rates of oxidation of ascorbate and of a tocopherol analogue subsequent to the pulse radiolysis of (bi)sulfite in the oxygen-containing solutions. This report provides evidence for the production of 03SOO-. Epoxidation of BP-7,8-diol by peroxyl radicals (7-10) and by peracids (11-13) is well documented. Though other routes of epoxidation have been used (13), the oxygen insertion from peroxyl radicals or peracid proceeds with a pronounced stereoselectivity, producing anti-BPDE as the predominant product (7-13). This same stereoselectivity is observed for BP-7,8-diol epoxidation occurring either during (bi)sulfite autoxidation or when treated with peroxomonosulfate. For (bi)sulfite metal-catalyzed autoxidation to yield 6.0
0 AoM 3.0
4.02.0
c0
a.
0~ co
m
2.0-
1.0
10 AM 20
40
60
Time, min FIG. 4. Inhibition of (bi)sulfite-dependent epoxidation by BHA. Reaction conditions and analytical procedures were as described for Fig. 2. (Bi)sulfite concentration was 1 mM, and BHA was added in acetone at the indicated concentration. The acetone concentration in all cases, including the control, was 0.1%.
0
30
60
90
Time, min FIG. 5. Epoxidation of BP-7,8-diol by potassium peroxomonosulfate (OXONE). Reaction conditions and analytical procedures were as described in Fig. 2. Reactions were initiated with 1 mM (bi)sulfite (o), 1 mM KO3SOOH (o), 1 mM KO3SOOH and 10 AM BHA (A), or 1 mM (bi)sulfite and 10 ,uM BHA (n).
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Medical Sciences: Reed et al.
Proc. Natl. Acad. Sci. USA 83
such epoxidizing agents is supportive of the pathway shown in Scheme 1. The key step is oxygen addition to *SO. The
so4
S~ ~ I
I
-|e
OH
OH
RH
02
SO3 t
r- 03SOO
Re
'SO;
.tAJ
Scheme
1
resultant peroxyl radical, -O3SOO', may directly transfer oxygen to BP-7,8-diol as is the case with various organic peroxyl radicals (7-10). Alternatively, -03SOO may abstract a hydrogen to yield -03SOOH, the peracid anion. As we showed with BP-7,8-diol and as others have observed with a variety of alkenes (18), this peracid is also an efficient epoxidizing agent. Unlike the (bi)sulfite-dependent reaction, the peroxomonosulfate-initiated epoxidation does not consume molecular oxygen, nor is it inhibited by BHA. The absence of inhibition by BHA eliminates the involvement of a peroxomonosulfate-derived peroxyl radical and supports the direct action of the peracid as an epoxidizing agent. Humans are exposed to significant levels of (bi)sulfite in addition to those present naturally as a catabolite of sulfurcontaining amino acids (19). (Bi)sulfite itself is added to a variety of foods, beverages, and drugs as a preservative and is formed in the lung by the hydration of sulfur dioxide, a ubiquitous air pollutant (3, 20). (Bi)sulfite exposure can produce an induction of an acute asthmatic state (3, 20). (Bi)sulfite also appears to have genetic effects (21) and can act as a mutagen or comutagen (22) and a cocarcinogen (20, 23). Laskin and coworkers (20) reported that sulfur dioxide was cocarcinogenic for development of benzo[a]pyreneinduced pulmonary carcinoma, which has been confirmed (23). Our finding of (bi)sulfite-dependent generation of antiBPDE, the ultimate carcinogenic form of benzo[a]pyrene, must be considered in light of this observed cocarcinogenic relationship. Enhancement of anti-BPDE production in pulmonary tissues comprises a potential mechanism for (bi)sulfite cocarcinogenicity. As stated by Berenblum (24) in his review on cocarcinogenesis ".... it is surprising how little is known yet about possible cocarcinogenic influences favoring activation." The epoxidation of BP-7,8-diol during sulfite autoxidation may represent an example of such a mechanism for cocarcinogenesis, although other mechanisms are possible (25).
(1986)
In addition to autoxidation, (bi)sulfite is oxidized by sulfite oxidase, a mitochondrial enzyme (26), and by peroxidases, both plant (16) and mammalian (15). Oxidation by sulfite oxidase forms sulfate directly without forming a free radical intermediate (27), whereas peroxidases, like autoxidation, can form 'S3. In this regard, sulfite oxidase should inhibit, and peroxidases should stimulate, epoxidation of BP-7,8diol. 1. McCord, J. M. & Fridovich, I. (1969) J. Biol. Chem. 244, 6056-6063. 2. Hayon, E., Treinin, A. & Wilf, J. (1972) J. Am. Chem. Soc. 94, 47-57. 3. Neta, P. & Huie, R. E. (1985) Env. Health Perspect. 64, 209-217. 4. Chantry, G. W., Horsfield, A., Morton, J. R., Rowlands, J. R. & Whiffen, D. H. (1962) Mol. Phys. 5, 233-239. 5. McCord, J. M. & Fridovich, I. (1968) J. Biol. Chem. 243, 5753-5760. 6. Huie, R. E. & Neta, P. (1985) Chem.-Biol. Interact. 53, 233-238. 7. Dix, T. A. & Marnett, L. J. (1981) J. Am. Chem. Soc. 103, 6744-6746. 8. Dix, T. A. & Marnett, L. J. (1983) Science 221, 77-79. 9. Dix, T. A., Fontana, R., Panthani, A. & Marnett, L. J. (1985) J. Biol. Chem. 260, 5358-5365. 10. Reed, G. A., Brooks, E. A. & Eling, T. E. (1984) J. Biol. Chem. 259, 5591-5595. 11. Yagi, H., Hernandez, 0. & Jerina, D. M. (1975) J. Am. Chem. Soc. 97, 6881-6883. 12. McCaustland, D. J. & Engel, J. F. (1975) Tetrahedron Lett. 30, 2549-2552. 13. Yagi, H., Thakker, D. R., Hernandez, O., Koreeda, M. & Jerina, D. M. (1977) J. Am. Chem. Soc. 99, 1604-1611. 14. Whalen, D. L., Ross, A. M., Yagi, H., Karle, J. M. & Jerina, D. M. (1978) J. Am. Chem. Soc. 100, 5218-5221. 15. Mottley, C., Mason, R. P., Chignell, C. F., Sivarajah, K. & Eling, T. E. (1982) J. Biol. Chem. 257, 5050-5055. 16. Mottley, C., Trice, T. B. & Mason, R. P. (1982) Mol. Pharmacol. 22, 732-737. 17. Marnett, L. J. & Bienkowski, M. J. (1980) Biochem. Biophys. Res. Commun. 96, 639-647. 18. Bloch, R., Abecassis, J. & Hassan, D. (1985) J. Org. Chem. 50, 1544-1545. 19. Johnson, J. L., Waud, W. R., Rajagopalan, K. V., Duran, M., Beemer, F. A. & Wadman, S. K. (1980) Proc. Nati. Acad. Sci. USA 77, 3715-3719. 20. Laskin, S., Kuschner, M., Sellakuman, A. & Katz, G. V. (1976) in Air Pollution and the Lung, eds. Aharonson, E. F., Ben-David, A. & Klingberg, M. A. (Halsted, New York), pp. 190-213. 21. Shapiro, R. (1983) Basic Life Sci. 23, 35-60. 22. Shapiro, R. (1977) Mutat. Res. 39, 149-175. 23. Pauluhn, J., Thyssen, J., Althoff, J., Kimmerle, G. & Mohr, U. (1985) Exp. Pathol. 28, 31. 24. Berenblum, I. (1985) Cancer Res. 45, 1917-1921. 25. Leung, K.-H., Post, G. B., & Menzel, D. B. (1985) Toxicol. Appl. Pharmacol. 77, 388-394. 26. Rajagopalan, K. V. & Johnson, J. L. (1977) in Biochemical Effects of Environmental Pollutants, ed. Lee, S. D. (Ann Arbor Science Publishers, Ann Arbor, MI), p. 307-314. 27. Cohen, H. J. & Fridovich, I. (1971) J. Biol. Chem. 246, 359-366.
