Chem. Res. Toxicol. 1990, 3, 59-64
59
Benzo[ a Ipyrene Bay-Region Sulfonates, a Novel Class of Reactive Intermediates Justin L. Green and Gregory A. Reed* Department of Pharmacology, Toxicology, and Therapeutics and Center for Environmental and Occupational Health, University of Kansas Medical Center, Kansas City, Kansas 66103 Received October 2, 1989
The mutagenicity of 7r,8t-dihydroxy-9t,lOt-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (antiBPDE) toward Salmonella typhimurium strain TA98 is enhanced by over 1.5-fold by the addition of 1-10 mM sulfite to the incubations. Sulfite itself is neither mutagenic nor toxic to the bacteria under these conditions. Analysis of anti-BPDE-derived products from these bacterial incubations demonstrates that, in addition to the expected hydrolysis products of the epoxide, novel more polar metabolites are produced. These same more polar compounds are produced by the addition of anti-BPDE to buffered aqueous solutions of sulfite. The major product of this reaction has been characterized by UV/visible and fluorescence spectroscopy, NI-FAB mass spectrometry, and proton NMR spectroscopy and is identified as 7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[alpyrene-10-sulfonate(BPT-10-sulfonate). This derivative is formed by the nucleophilic addition of sulfite to the 9,lO-oxirane ring of anti-BPDE. This product is easily differentiated both spectrally and chromatographically from the isomeric 7,8,10-trihydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrene-9-sulfonatereported from the attack of the sulfite anion radical on the activated aliphatic double bond of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) [Curtis e t al. (1988) Carcinogenesis 9, 20151, The nucleophilic trapping of diol epoxides by water or thiols is assumed to represent a detoxication of this class of mutagen. In contrast, the extensive conversion of anti-BPDE to BPT-lO-sulfonate in the bacterial incubations correlates with a marked enhancement of resultant mutagenicity. Further support for a key role of BPT-10sulfonate in the enhancement of anti-BPDE mutagenicity is provided by our findings on the reactivity of this compound. While extremely resistant to simple hydrolysis a t neutral pH, BPT-10-sulfonate nevertheless binds to calf thymus DNA, resulting in a level of binding onefourth of that obtained with anti-BPDE under identical conditions. These findings demonstrate that trapping of diol epoxides by sulfite, unlike the corresponding reactions of the epoxides with water or thiols, yields a new class of reactive intermediate which retains the ability to bind covalently to nucleic acids. Such reactive intermediates may play an important role in the sulfite-dependent enhancement of the genotoxicity of B P and B P derivatives.
with BP alone. A significant correlation to respiratory tract malignancy also exists in cases of human exposure to high The environment contains a broad spectrum of comambient levels of SO, (2, 3). pounds that have the potential to affect human health. The mechanism for this apparent cocarcinogenic activity Interactions of these compounds may produce modified of SO,is not known but might involve the enhanced forbiological activities with respect to those of the individual mation of carcinogenic metabolites of BP, the inactivation components. Benzo[a]pyrene (BP)' is a widespread enof cellular scavengers of those metabolites, or alteration vironmental pollutant with well-established potential as of cellular systems involved in the repair or replication of a mutagen and carcinogen (1). The ability of sulfur dioxide DNA. Each of these mechanisms has been proposed to (SO,), another ubiquitous pollutant, to increase the tuexplain cocarcinogenesis (6),and the first two have been morigenicity of BP provides an example of a potentially studied specifically relative to the interaction of SO, and important interaction of environmental contaminants. BP. In regard to enhanced formation of active carcinogens, While SO, is not itself a carcinogen or a mutagen (2,3), we have shown previously that the proximate carcinogen chronic, concurrent exposure to SO, and BP has produced 7,8-dihydroxy-7,8-dihydrobenzo [a ]pyrene (BP-7,8-diol) is an increased incidence of upper respiratory tract carciconverted to the ultimate carcinogen 7r&-dihydroxynomas in rats ( 4 ) and hamsters (5) relative to that seen 9t,10t-epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrene (antiBPDE) during the autoxidation of sulfite (SO?-), the physiological form of SO2 (7,8). Such an additional source Abbreviations: B P , benzo[a]pyrene; BP-7,8-diol,trans-7,8-diof the ultimate carcinogenic species could enhance the hydroxy-7,8-dihydrobenzo[a]pyrene;anti-BPDE, 7r&-dihydroxy9t,10t-epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrene; syn-BPDE, 7r,8t-dicarcinogenicity of BP. Sulfite-dependent inhibition of BP tetraols, hydroxy-9c,10c-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; scavenging systems for diol epoxides also has been reBPT-97,8,9,10-tetrahydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrenes; sulfonate, 7,8,10-trihydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrene-9- ported. Specifically, reaction of sulfite with glutathione sulfonate;BPT-10-sulfonate,7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzodisulfide leads to inhibition of glutathione S-transferases [alpyrene-10-sulfonate;THF, tetrahydrofuran; T E A , triethylamine; (9), the only enzymatic scavenging system active against DMSO, dimethyl sulfoxide;NI-FAB,negative ion fast atom bombard7,8,9-trihydroxy-10-(S-glutathionyl)-7,8,9,lO-tetrahydro- diol epoxides. This inhibition of glutathione S-transferases ment; GS-BPT, has been suggested to explain the ability of sulfite to enbenzo[a]pyrene.
Introduction
0893-228~/90/2703-0059$02.50/0 0 1990 American Chemical Society
60 Chem. Res. Toxicol., Vol. 3, No. I, 1990
Green a n d Reed
hance the covalent binding of BP metabolites to DNA in mL of acetone. A solution of 300 pg of anti-BPDE in 300 pL of dry tetrahydrofuran-triethylamine (991) was added immediately subcellular systems (10) and i n cultured lung fibroblasts to the buffered sulfite solution. The mixture was stirred vigorously (11). a t room temperature for 72 h. The volume was reduced to apResults of our earlier studies appear consistent with proximately 1 mL by evaporation under reduced pressure, and effects on scavenging systems, i n that t r e a t m e n t of Salthe entire product mixture was applied to a C-18 solid-phase monella t y p h i m u r i u m strains TA98 and TAlOO with extraction column (Analytichem International, Harbor City, CA) sulfite greatly enhances the mutagenicity of b o t h antiand washed with 3 mL of deionized water. Less than 2% of BPDE a n d 7r,8t-dihydroxy-9c,lOc-epoxy-7,8,9,lO-tetra- BPDE-derived material was lost in the water wash. The hydrobenzo[a]pyrene ( s y n - B P D E ) ( 4 1 2 ) . These effects BPDE-derived products were eluted from the column with 3 mL a r e n o t seen if sulfite is added after the bacteria are exof ice-cold methanol. The methanol eluate was evaporated to dryness under reduced pressure. The crude product mixture was posed to BPDE (12). We report here, however, that a dissolved in 1 mL of 50% aqueous 2-propanol and purified by unique profile of anti-BPDE-derived products is obtained reverse-phase HPLC utilizing a 10 X 250 mm Partisil ODS-2 from these systems, which includes novel polar metabolites semipreparative column (Whatman)eluted with a methanol-water in addition to the expected hydrolysis products of the diol gradient. Total yield of BPT-10-sulfonate was 33%, with hyepoxide. The significant yield of these novel products drolysis products making up the remainder of the BPDE-derived suggests that t h e y too m a y play a role in the e n h a n c e d material. mutagenicity of BPDE. The present work serves to (*)-7,8,9-Trihydroxy-lo-(S-glutathionyl)-7,8,9,10-tetracharacterize these interactions between sulfite a n d a n t i hydrobenzo[a]pyrene (GS-BPT). A solution of 200 pg of BPDE in chemical a n d bacterial systems, and to examine anti-BPDE in 4 mL of THF-TEA (19:l) was added dropwise to 10 mL of 0.1 M NaHC03 containing 25 mg of reduced glutathione the role of these interactions in the enhanced genotoxicity (GSH). The solution was stirred a t room temperature for 4 h. seen with concurrent exposure to these compounds. We The volume was reduced to 5 mL by evaporation with a nitrogen have identified a novel class of BP derivatives which arise stream, and the remaining solution was applied to a C-18 solidby the addition of sulfite to anti-BPDE. Further, we phase extraction column. The column was washed with 2 mL suggest a possible role for these products i n the enof water, and GS-BPT was eluted from the column with 2 mL h a n c e m e n t of diol epoxide mutagenicity by sulfite in of methanol. The organic eluate was pooled and evaporated to bacterial systems. about 1 mL under nitrogen. Analysis by HPLC showed less than 3% production of B P tetraols. Retention time of the major GSH Experimental Section adduct is 14.7 min. Mutagenicity Assays. All mutagenicity assays were accomRacemic BP-7,8-diol, anti-BPDE, [3H]-anti-BPDE (302 plished as reported previously (8, 12,15). Cells from overnight mCi/mmol), and [14C]-anti-BPDE (57 mCi/mmol) were supplied cultures of Salmonella typhimurium strain TA98 in Oxoid nuby the NCI Repository. The synthesis of 7,8,10-trihydroxytrient broth were pelleted by centrifugation and resuspended in 7,8,9,10-tetrahydrobenzo[a] pyrene-9-sulfonate (BPT-9-sulfonate) sterile phosphate-buffered saline. A 100-wL aliquot of the bacterial was conducted as described previously with a yield of 37% (13). suspension [(1-2) x 108 viable cells] was added to 400 pL of sterile Hydrolysis of anti-BPDE to obtain the isomeric 7,8,9,10-tetra0.1 M potassium phosphate buffer, pH 7.4, and mixed thoroughly. hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetraols) was Sulfite was added and the tubes were placed in a shaking water carried out as described previously (14). All synthetic and anabath at 37 "C. After 3 min, anti-BPDE dissolved in THF-TEA lytical procedures were accomplished under subdued light. (99:l) was added to the tubes and the incubation was continued Products were stored as dry solids under nitrogen a t -20 "C. for another 30 min. THF-TEA content for all incubations was Silylation-grade tetrahydrofuran (THF) and dimethyl sulfoxide constant at 2% (v/v). For mutagenicity studies, incubation (DMSO) were purchased from Pierce Chemical Co., and 99+% mixtures were plated and incubated for 48 h at 37 "C, and retriethylamine (TEA) was from Aldrich. Deuterated DMSO vertant colonies were scored. For the determination of bacterial (99.96%)was from Cambridge Isotope Laboratories, Wobum, MA. viability, these incubation mixtures were diluted 106-fold in sterile All other chemicals and solvents were purchased from Fisher potassium phosphate buffer, and 500 pL of the diluted mixture Scientific unless otherwise specified. was plated on nutrient agar. Viable colonies were counted after Analytical Procedures. Analytical HPLC was conducted by 24 h a t 37 "C. All assays were performed in triplicate, and the using a 4.6 X 250 mm Ultrasphere ODS column (Beckman Inresults expressed as mean standard deviation. struments Co., Fullerton, CA) eluted with a methanol-water Analysis of Mutagenicity Assay Metabolite Profiles. gradient. The solvent profile consisted of elution with 10% methanol for 5 min, a linear increase to 52% methanol between Bacterial mutagenicity assays were accomplished as described by using [3H]-anti-BPDE (302 mCi/mmol) and with a total volume 5 and 10 min, elution with 52% from 10 to 30 min, a linear of 1.0 mL. A 500-pL aliquot of each incubation was removed gradient from 52% to 80% methanol (30-35 min), and elution following the incubation period and set aside for product profile with 80% methanol for 10 min. Flow rate was 1.4 mL/min. analysis. These incubation aliquots were centrifuged for 10 min Detection consisted of monitoring absorbance (344 nm) and to pellet particulate material, and the resultant supernatant was fluorescence (344-nm excitation, 380-nm emission). Detection analyzed by HPLC as described. Elution fractions were collected of 14C-labeled compounds was performed with a Radiomatic every 30 s, and radioactivity was determined by liquid scintillation FLO-ONE p detector with a solid scintillant cell. Radiochrocounting. The remaining 500 pL of each incubation was plated matographic profiles of tritiated compounds were determined by and scored for mutagenicity. collecting 30-s fractions of eluant throughout the chromatographic Synthetic Product Profile Determination. Formation of run for liquid scintillation counting. Preparative chromatography BPT-10-sulfonate as a function of sulfite concentration was employed a Whatman Magnum-9 ODS-2 column. The same gradient and detection parameters were used as in the analytical quantitated with incubations using [14C]-anti-BPDEas a precursor. Simultaneous addition of 1 pM [14C]-anti-BPDE (57 system; however, the flow rate was 3.0 mL/min. UV spectra were obtained by using a Shimadzu UV-260 mCi/mmol) and 0-10 mM sulfite to 0.1 M potassium phosphate spectrophotometer and fluorescence spectra with a Shimadzu buffer (pH 7.4) was followed by incubation at 37 "C, for 30 min. RF-540 fluorometer. All spectra were measured in 95% ethanol. HPLC analysis of 100 pL of each reaction mixture was performed Negative ion fast atom bombardment maas spectra (NI-FAB) were as described previously. Metabolites were quantitated by comdetermined on a VG analytical ZAB double-focusing mass parison to standard curves. It was assumed that the major and spectrometer using a xenon atom gun. NMR spectra were obminor forms of BPT-10-sulfonate produced the same detector tained with a Bruker AM-500 spectrometer. Deuterated DMSO response. was the solvent. DNA Binding Studies. Silanized glassware used throughout (f)-7,8,9-Trihydroxy-7,8,9,lO-tetrahydrobenzo[a Ipyrenethe incubation and purification procedures for these studies. Calf 10-sulfonate (BPT-IO-sulfonate). A 300-pL aliquot of 0.5 M thymus DNA was dissolved in 0.1 M potassium phosphate buffer, sodium sulfite in 0.1 M glycine buffer, pH 10, was added to 2.4 pH 7.4, a t a concentration of 1 mg/mL. Tritium-labeled anti-
Chem. Res. Toxicol., Vol. 3, No. 1, 1990 61
Benzo[a]pyrene Bay-Region Sulfonates
+
Table I. Effect8 of Sulfite and anti-BPDE on S . typhimurium Strain TA98' conditions viability revertants/ plate control (1.0 f 0.1) x 108 23 f 7 23 f 6 (1.0 & 0.1)x 108 10 mM SO3*51 f 8 (0.9 f 0.1) X 10' anti-BPDE (0.1 pM) 130 6 anti-BPDE + SO?(0.9 f 0.1) x 108
The effects of sulfite and anti-BPDE on the viability and the phenotype of S. typhimurium strain TA98 were assayed, and the results are shown in Table I. None of the experimental treatments resulted in significant loss of bacterial viability. Unlike BP-7,8-diol,anti-BPDE is itself an active mutagen. The addition of 0.1 pM anti-BPDE, a concentration on the lower half of the linear dose-response range (8,12),resulted in a modest but significant increase in revertants over control values. Sulfite, at a concentration of 10 mM, showed no mutagenic activity. The treatment of the bacteria with 10 mM sulfite prior to anti-BPDE addition, however, increased the number of resultant revertants by more than 1.5-fold relative to the response to the diol epoxide alone. Additional studies have demonstrated this potentiation of mutagenicity using anti-BPDE concentrations from 0.05 to 0.5 pM and sulfite concentrations from 1 to 20 mM (8, 12). Mutagenicity Assay Product Profiles. HPLC analyses of the supernatants from incubation mixtures of S. typhimurium strain TA98 with [3H]-anti-BPDEin the presence and the absence of sulfite were obtained (Figure 1). In those incubations without sulfite, the predominant metabolites were the expected trans and cis diastereomers of BP tetraols. The ratio of trans- to cis-tetraols was approximately 13:l and reflected the known stereoselectivity of hydrolysis of anti-BPDE (14). In incubations that contained various concentrations of sulfite, not only were the BP tetraols detected, but two previously unreported polar metabolites were also present. The ratio of the product eluting at 11.7 min to that eluting at 12.4 min was about 5:l. The retention times for these products were clearly distinct from those of the BP tetraols, and of the anti-BPDE-glutathione conjugate as well. Indeed, no detectable glutathione conjugates were found by HPLC analysis in any of the bacterial incubations containing anti-BPDE.
I
80,000 40,000
*
Results Enhancement of BPDE Mutagenicity by Sulfite.
SO,=
R 20,000
All incubations contained (1-2) X lo8 S. typhimurium strain TA98 in 0.1 M potassium phosphate buffer, pH 7.4. Addition of sulfite occurred at t = 0 min and anti-BPDE at t = 3 min of a 33-min incubation at 37 "C. All results represent means f SD of triplicate determinations. BPDE (302 mCi/mmol), BPT-10-sulfonate (62 mCi/mmol), and B P tetraols (57 mCi/mmol) were added to the DNA solution as stocks in DMSO. Final concentration of all BP derivatives was 20 pM. Two separate control incubations contained DNA without BP derivatives and BPT-10-sulfonate without DNA. After 1 h at 37 "C, the solutions were extracted exhaustively with buffersaturated ethyl acetate. Pooled organic extracts from each incubation were evaporated under reduced pressure and reconstituted in 250 p L of 50% aqueous 2-propanol for HPLC analysis. DNA was denatured by boiling for 10 min to release intercalated and otherwise physically bound B P derivatives and immediately placed on ice. DNA was isolated by ethanol precipitation, and each ethanol-buffer supernatant was prepared as described above for HPLC analysis. The purified DNA was resuspended in 10 mM NaCl-1 mM EDTA and quantitated by absorbance a t 260 nm [20 AU/(mg of DNAsmL)], and then bound labeled material was quantitated by liquid scintillation counting. All incubations were run in triplicate, and results are reported as mean & standard deviation.
10 mH
CONTROL
40,000
20,000
o
io
20
30
40
50
ao
70
IO
oo
io0
Fraction Numbar
Figure 1. HPLC profiles of anti-BPDE-derived products from bacterial mutagenicity incubations. Supernatants from incubations containing 0.1 pM [3H]-anti-BPDE and S. typhinurium strain TA98 were prepared and analyzed as described under Experimental Section. Radioactivity was quantitated by liquid scintillation counting of 30-s fractions. The bottom panel is from a control incubation, whereas the top panel shows the effects of 10 mM sulfite on the resultant product profile. The mutagenicity and viability data from these same incubations are shown in Table
I. Table 11. Characterizationof BPDE-DerivedProducts' fluorescence, nm UV/vis, excitation emission product tR, min nm 348.1 401.3 BPT-10-sulfonate 11.7 345.5 331.7 383.8 329.8 317.7 315.5 BPT-9-sulfonate 11.2 343.1 344.9 398.5 327.0 328.9 380.1 315.2 313.0 trans-anti-BP tetraol 20.2 343.1 344.8 398.6 329.0 380.9 327.5 315.4 313.5
'Retention times for BP derivatives were obtained by using the same chromatographic procedure as for the profiles shown in Fig. 1. Details appear under Experimental Section. All spectra were recorded on samples dissolved in 95% ethanol. Identification of Sulfite-Dependent Products. The major novel product from incubations of sulfite with anti-BPDE was isolated by HPLC and its structure characterized. These data were compared with the corresponding values obtained for the major BP tetraol and for BPT-9-sulfonate. Analysis by NI-FAB mass spectrometry demonstrated a strong apparent molecular ion at m/z = 383, identical with that of BPT-9-sulfonate (13), and consistent with addition of sulfite to produce a tetrahydrotriol sulfonate derivative of B[a]P. Although similar in chromatographic behavior, this product is distinct from the previously reported BPT-9-sulfonate (Table 11). Spectral characterization of this product revealed a UV/visible absorbance spectrum and fluorescence spectra diagnostic for a 7,8,9,10-tetrahydrobenzo[a]pyrene system, but all maxima for the new compound exhibit a 2.5-4-nm bathochromic shift. The final basis for identification is the proton NMR spectrum of the product (Figure 2). In addition to eight aromatic protons (7.98-8.60 ppm) and three hydroxyl protons (4.79 ppm), four aliphatic protons are detected. The C-7 proton signal is seen clearly (d, 5.43 ppm, 1 H, J = 7.2 Hz), whereas the C-8 and C-9 proton signals are superimposed (m, 4.66 ppm, 2 H). The C-10
62 Chem. Res. Toxicol., Vol. 3, No. I , 1990
Green and Reed Table 111. DNA Binding bs BP Derivativesa BP derivative DNA binding, pmol/mg of DNA BPT-10-sulfonate 307 f 45 anti-BPDE 1293 f 40
I
BP tetraols
4
"Calf thymus DNA (1 mg/mL) in 0.1 M potassium phosphate buffer was incubated with 20 pM of each tritium-labeled BP derivative for l h at 37 "C. DNA isolation was accomplished by ethyl acetate extraction followed by ethanol precipitation. Data represent means f SD of triplicate determinations.
J Figure 2. Proton NMR spectrum of the major sulfite addition product. Sample was dissolved in DMSO-d6. o/ ,n
0 20I
i
0.15-
0-0
Major Sulf. Minor Sulf.
0-0
~
/o
/
0.10-
/
/ 0 05-
23 f 32
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0
I
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: 4
:
5
6
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7
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:
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8
9
10
[so3=]. mM
Figure 3. Formation of BPT-10-sulfonatesas a function of sulfite concentration. anti-BPDE (1pM) was incubated for 30 min with the indicated concentration of sulfite. Products were identified and quantitated by HPLC as described under Experimental Section. Points represent the mean of triplicate determinations. proton resonance is observed at 4.98 ppm (d, 1 H, J = 2.9 Hz). On the basis of the data presented, we conclude that the major novel metabolite of anti-BPDE is 7,8,9-trihydroxy-7,8,9,1O-tetrahydrobenzo[a]pyrene10-sulfonate (BPT-10-sulfonate). A second sulfite-dependent product is also observed, eluting about 0.7 min after the major product (Figure 1). The maxima in the UV/visible spectrum (344.4 nm) and fluorescence spectra (ex = 346.3 n, em = 383.0 nm) show a bathochromic shift similar to but slightly less pronounced than that observed with the major product (Table 11). Isolated quantities of this product were too small to allow for further characterization. Formation of BPT-10-sulfonateand BP Tetraols as a Function of Sulfite Concentration. Increasing formation of BPT-10-sulfonate was noted over the sulfite concentration range of 0.510 mM (Figure 3). Formation of the BPT-10-sulfonate is detectable a t 0.5 mM sulfite and increased linearly with sulfite concentration from 1 to 10 mM. A corresponding decrease in the formation of BP tetraols also was observed (data not shown). Under the given conditions, the ratio of trans- to cis-BP tetraols formed was approximately 7:l and the ratio of major to minor forms of BPT-10-sulfonate was approximately 5:l. When sulfite was replaced with 10 mM sulfate in incubations with anti-BPDE, BP tetraols were the only detectable products (data not shown). Incubations substituting the trans-anti-tetraol for anti-BPDE were performed in an identical fashion and failed to show either the formation of more polar products or a decrease in the amount of BP tetraols in the final incubation mixture (data not shown). DNA Binding Studies. Diol epoxides possess potent activities for the covalent modification of DNA. Not
surprisingly, the addition of 20 pM anti-BPDE to a solution of calf thymus DNA led to substantial modification of the DNA (Table 111). Further, hydrolysis of the epoxide to yield the relatively inert BP tetraols abolishes this covalent binding activity, as confirmed by the lack of binding in our study. The DNA-associated label from BP tetraols serves as a control for determining the level of noncovalent, intercalative binding in these systems, and it is less than 2% of the level of covalent binding observed with antiBPDE. We find, however, that the sulfite addition product BPT-10-sulfonate retains the ability to react with DNA (Table 111). The label from BPT-10-sulfonate does not simply copurify with DNA by these proceduresincubations of [3H]BPT-10-sulfonatewithout DNA which were subjected to the identical extraction and precipitation procedure contained no detectable label in the final NaC1-EDTA solution. Analysis of unbound materials from these incubations demonstrated that with anti-BPDE the BP tetraols derived from the epoxide are the only detectable products. This is in stark contrast to the results using BPT-10-sulfonate, where the unreacted starting compound is the only product recovered after a 1-h incubation (data not shown). For a BP derivative to bind to DNA with an affinity comparable to BPDE, and yet to exhibit such remarkable hydrolytic stability relative to BPDE, is indeed remarkable.
Discussion The mechanism of the cocarcinogenicity of SO2for BP is not known. It has been suggested that this enhancement of carcinogenicity may be due to alterations in the known pathways for activation and detoxication of BP derivatives. Although evidence favoring both increased production of anti-BPDE (7, 8) and decreased ability of biochemical systems which detoxicate this activated species (8-12) has been obtained in various model systems, the present work suggests that additional novel products and reactions may also be involved. We suggest that the formation of bayregion BP sulfonates, derivatives that are capable of direct interaction with nucleic acids, may play a role in the enhancement of genotoxicity produced by sulfite. Characterization of the major sulfite-dependent product from anti-BPDE and consideration of its synthetic route establish the identity of this product as 7,8,9-trihydroxy7,8,9,10-tetrahydrobenzo[a]pyrene10-sulfonate (BPT-10sulfonate). The observed molecular ion in the NI-FAB mass spectrum supports addition of a single sulfonate group to anti-BPDE, and the known reactions of diol epoxides with nucleophiles all occur by addition to the benzylic carbon of the epoxide (14, 16). That this is the case for the addition of sulfite as well is conclusively shown by the spectral characterization of the product. As detailed in Table 11, both the absorbance and fluorescence spectra for this product demonstrate a pyrene chromophore yet display a distinct bathochromic shift of all maxima. This is consistent with a change in the substitution on the C-10 benzylic carbon, a sensitive site for electronic alteration of the pyrene chromophore. This is in marked contrast
Benzo[a]pyrene Bay-Region Sulfonates to the spectra of the isomeric BPT-9-sulfonate. With a hydroxyl group on the benzylic position and the sulfonate isolated from the pyrene system, BPT-9-sulfonate exhibits spectral characteristics indistinguishable from those of BP tetraols. The NMR spectrum obtained on this compound (Figure 2) provides the final confirmation of the C-10 substitution by sulfite. The observed signals for the aromatic protons and the C-7, C-8, and C-9 protons appear as expected on the basis of previously reported spectra of BPDE-derived products (1620). The key observation is the chemical shift of the C-10 proton-instead of appearing at about 5.7 ppm, as would be predicted on the basis of the spectrum of the trans-anti-BP tetraol (20),this signal is seen at 4.98 ppm. This is consistent with the markedly lower deshielding activity of a sulfonate group relative to a hydroxyl group and has been reported previously for the NMR spectra of sulfite addition products to simple aliphatic epoxides (21). Both the known chemistry of anti-BPDE and sulfite and the results of our chromatographic and spectral analysis of the products of this reaction establish that the products are indeed BPT-10-sulfonates. On the basis of the stereoselectivity of all known nucleophilic additions to anti-BPDE (14,16-19), we propose that the major product (tR = 11.7 min) is the product of the trans addition of sulfite relative to the epoxide-derived C-9 hydroxyl, whereas the minor product ( t R = 12.4 min) results from the corresponding cis addition. Not surprisingly, the formation of bay-region sulfonates of BP is determined by the chemistry of both reactants. The highly reactive diol epoxide is efficiently trapped by many different nucleophiles. As the sulfite anion is an extremely potent nucleophile (22), it readily adds to the epoxide. This occurs with regiospecificity determined by the electronic contributions of the pyrene system and stereoselectivity enforced by the conformation of the benzo ring. BPT-10-sulfonates result, with a tentative assignment of trans and cis addition as the major and minor routes, respectively. In the reaction with BP-7,8-diol(13), sulfite itself will not attack the bay-region double bond, but the sulfite anion radical will. Here too the electronic character of the pyrene system dominates the regiospecificity of the attack, and a proposed secondary addition of molecular oxygen and subsequent reduction yields BPT-9-sulfonate (13). BP tetraols, however, lack both the epoxide group of BPDE and the alkene moiety of BP7,8-diol. No reaction has been detected between sulfite or sulfite-derived species with BP tetraols. This is consistent with the previous description of BP tetraols as biologically and chemically inert final metabolites of BP. Bay-region diol epoxides, as extremely reactive electrophiles, react readily with a wide range of nucleophiles. Reaction with water to form BP tetraols and trapping by glutathione or other cellular thiols are detoxication reactions, whereas covalent binding to nucleic acids is thought to account for the toxic and the mutagenic activities of the diol epoxides (1). That BPT-10-sulfonate is not a "deadend" metabolite like the BP tetraols and the glutathione conjugates is demonstrated by the results of our preliminary DNA binding studies. The ability of BPT-10sulfonate to bind covalently to DNA and the fact that the extent of this binding approaches the level obtained with anti-BPDE suggest that this bay-region sulfonate derivative may have significant biological activity. This apparent covalent reaction with DNA is even more unique when compared with the resistance to hydrolysis of BPT-10sulfonate. Although the authentic BPDE exhibits a much higher general reactivity toward nucleophiles, BPT-10-
Chem. Res. Toxicol., Vol. 3, No. 1, 1990 63 sulfonate appears far more selective in its susceptibility to such attack. BPT-10-sulfonate may represent a longer lived, more selective agent for DNA modification than are the diol epoxides. The biological consequences of forming BPT-10-sulfonate-derived lesions in DNA will define the role of this reactive intermediate in the comutagenicity and cocarcinogenicity of sulfur oxides. Our initial determinations of the effects of BPT-10-sulfonate with 5'. typhimurium tester strains revealed that this derivative exhibits only weak mutagenicity but that it greatly enhances the mutagenicity of anti-BPDE when both compounds are added to the bacterial incubations.2 This enhancement of BPDE genotoxicity suggests that the formation of bay-region sulfonates derived from BP may comprise a significant contribution to the cocarcinogenicity of sulfur oxides.
Acknowledgment. The work reported here was supported by NIH Grant ES-04092 (awarded to G.A.R.) and by Biomedical Research Grant SO7 RR-05373. The excellent technical assistance of Ms. Marilyn Ryan is greatly appreciated. Registry No. anti-BPDE, 58917-67-2; BP tetraol, 59957-91-4; BPT-10-sulfonate, 124443-16-9;GS-BPT, 93245-73-9;sulfite, 14265-45-3.
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