218
Bioconjugate Chem. 1002, 3, 218-224
Characterization of Mercapturic Acid and Glutathionyl Conjugates of Benzo[a]pyrene-7,8-dione by Two-Dimensional NMRf Varanasi S. Murtyt and Trevor M. Penning* Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084. Received December 3, 1991
Non-K-region polycyclic aromatic hydrocarbon (PAH) o-quinones represent alternative metabolites of PAH trans-dihydro diol proximate carcinogens. These PAH o-quinones react readily with glutathione and N-acetyl-L-cysteine,and these adducts may be responsible for their detoxication. Reactions between benzo[a]pyrene-7,8-dioneand either N-acetyl-L-cysteineor glutathione gave three predominant products which were purified by semipreparative reverse-phase high-pressure liquid chromatography and characterized by homonuclear two-dimensional correlation spectroscopy (COSY). The first product corresponded to a Michael type, 1,4-addition product isolated at the level of quinone oxidation. The second product converted to the first and is a presumptive 1,4-addition product isolated at the level of hydroquinone oxidation. The third product was 7,8-dihydroxybenzo[alpyrene(a hydroquinone) and was formed as a result of the reductive potential of the thiol. Additional proof for the catechol structure was obtained by its conversion to its diacetate and its identity with authentic 7,8-diacetoxybenzo[alpyrene. The structures of these adducts and intermediates confirm that thiol addition involves formation of the ketol and rearrangement to give a catechol followed by oxidation to yield the quinone adduct. No evidence was obtained for the formation of either bisphenol or bisglutathionyl adducts. The COSY spectra provide the first complete structure of a benzo[alpyrenyl-peptide conjugate.
INTRODUCTION Polycyclic aromatic hydrocarbons (PAH)' are widespread environmental pollutants that cause cancer ( I , 2). PAH which contain a terminal benzo ring are activated by metabolism to form non-K-region trans-dihydro diols which act as immediate precursors of the anti-diol epoxides (3), which are ultimate carcinogens. For benzo[alpyrene this pathway of activation involves conversion of (f)-trans-7,8-dihydroxy-7,8-dihydrobenzo~ulpyrene (B[a] P-diol) to (f)-trans-7,8-dihydroxy-anti-9,10-epoxy7,8,9,10-tetrahydrobenzo[alpyrene(anti-BPDE), which then reacts with the 2-amino group of guanine within DNA
NMR and mass spectrometry have established the structure of the 2-mercaptoethanol adduct of benzo[alpyrene7,8-dione as a 1,4-Michael addition product, leading to the proposal that a ketol and catechol are also intermediates in adduct formation, but these were never isolated. To assess the contribution of DD to B[alP-diol metabolism, subcellular fractions of rat liver have been fortified with appropriate cofactors to optimize the activities of enzymesthat would compete for this proximate carcinogen. In these studies, rat liver Slw fortified with NAD(P)+ produced significant amounts of benzo[alpyrene-7,8-dioneS Indeed the amount of dione formed was only superseded by the formation of tetrols of benzo[al(4-7). pyrene (7,8,9,10-tetrahydroxy-7,8,9,lO-tetrahydrobenzoOne of several enzymes that can suppress the formation [alpyrenes) by microsomes fortified with an NADPH of the anti-diol epoxides is dihydrodiol dehydrogenase generating system, (Penning and Shou, in preparation). [DD; EC 1.3.1.20 (@I. Previous studies from this laboThese tetrols arise from the hydrolysis of the anti-BPDE ratory have shown that homogeneous dihydrodiol dehywhich is a product of the microsomaloxygenationof B [alpdrogenase catalyzes the oxidation of non-K-region transdiol. Together, these data imply that benzo[alpyrenedihydro diols to yield intermediate catechols which then 7,8-dione may be an important cellular metabolite of air oxidize to form the corresponding non-K-region o-quiB [a]P-diol. nones (9, IO). These PAH o-quinones react with buffer Once formed PAH o-quinones have the potential to be nucleophiles but can be trapped from the reaction mixture cytotoxic species, thus by entering redox cycles they could as thiol ether adducts with 2-mercaptoethanol(IO). Thus, generate semiquinone and superoxide anion radicals. An B[alP-diol is oxidized by DD to yield benzo[alpyreneexamination of the cytotoxicity of several PAH o-quino7,8-dione, which then reacts with the thiol scavenger. 'H nes (naphthalene-1,2-dione, 7,12-dimethylbenz [a]anthracene-3,4-dione, and benzo[alpyrene-7,8-dione)indi* Address all correspondence to Dr. Trevor M. Penning, Dept. cated that benm[a]pyrene-7,&dionewas the least cytotoxic of Pharmacology, University of Pennsylvania School of Medicine, to rat H-411e hepatoma cells (111, suggesting that elim37th & Hamilton Walk, Philadelphia, PA 19104-6084. ination pathways exist for this quinone. These findings + A preliminary account of this work was presented at the indicate that further examination of benzo[alpyrene-7,8American Association for Cancer Research Annual Meeting in Houston, Texas, and was published in abstract form: Proc. Am. dione conjugate chemistry is warranted if the elimination Assoc. Cancer Res. (1991) 32, Abst.# 735. toxicity of this PAH o-quinone is to be understood. * Present address: Uniroyal Chemical Co., Middlebury, CT. andlor In the present study we report the synthesis and * Abbreviations: PAH, polycyclic aromatic hydrocarbons; DD, by homonuclear two-dimensional corredihydrodiol dehydrogenase, trans-1,2-dihydrobenzene-1,2-diol: characterization lation (COSY)spectroscopy of the mercapturic acid and dehydrogenase (EC 1.3.1.20);B [a]P-diol, (f)-trans-7,gdihydroxyglutathionyl conjugates of benzo[al pyrene-'l,&dione. The 7,8-dihydrobenzo[a]pyrene;anti-BPDE,(f)-trans-7,&dihydroxystudies show that although 1,4-addition products can be anti-9,10-epoxy-7,8,9,l(ltetrahydrobenu,[a]pyrene COSY, correlaisolated the reactions are complicated by the formation of tion spectroscopy; TFA, trifluoroacetic acid; and RP-HPLC, hydroquinone conjugates. The elucidation of the strucreverse-phase high-pressure liquid chromatography. 1043-1802/92/2903-0218$03.00/0 0 1992 American Chemical Society
Conjugate Characterization by 2D-NMR
Bloconjugate Chem., Vol. 3, No. 3, 1992 218 E
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C tures of the N-acetyl-L-cysteine and glutathionyl conjugates of benzo[alpyrene-7,8-dionenow permit their use as synthetic standards for studies on the further metabolism of benzo[al pyrene-7,8-dione. The correlation spectra reported here provide unequivocal proton assignments of a benzoCaIpyreny1-glutathione conjugate which may be useful in assigning structure to other PAH-peptidyl conjugates. To date a complete structure of the S-glutathionyl adduct of anti-BPDE has not been described. EXPERIMENTAL PROCEDURES
General. The work described involves the synthesis and handling of hazardous or potentially hazardous agents. Work was therefore conducted in accordance with “NIH Guidelines for the Laboratory Use of Chemical Carcinogens”. Materials. Benzo[a]pyrene-7,8-dionewas synthesized according to published procedures (12). N-Acetyl-L-cysteine and reduced glutathione were purchased from Sigma (St.Louis, MO) and were used without further purification. HPLC-grade trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL). Deuterated solvents and trimethylsilane (TMS) were purchased from Aldrich (Milwaukee, WI). NMR Spectroscopy. Spectra were obtained on a Bruker AM-500 spectrometer equipped with an ASPECT 3000 computer operating at 500.13 MHz. Samples were dissolved in DMSO-& or dioxane-dlD20 mixtures. Chemical shifts are expressed relative to TMS. To acquire the COSY spectra the two-pulse experiment described by Aue et al. (13)was used. In this experiment the preparatory phase ends with a nonselective rf pulse at time t = 0 (preparatory pulse). A flip angle of 90” was employed to generate the off-diagonal elements, and at the end of the evolution period a second rf field (mixing pulse) was applied at a time t = tl (13,14). Synthesis of Mercapturic Acid and Glutathionyl Conjugates of Benzo[a]pyrene-7,8-dione.The synthesis of N-acetyl-S-(7,8-dihydro-7,&dioxobenzo[alpyren-
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Figure 2. RP-HPLC chromatography of reaction mixtures resulting from N-acetyl-L-cysteine addition to benzo[a] pyrene7,8-dione: Elution profile for benzo[a]pyrene-7,8-dione(A), elupyrene-7,Sdione adtion profile for N-acetyl-L-cysteine-benzo[al ducts obtained at 24 h (B),and UV spectra of N-acetyl-L-cysteinebenzo[alppene7,&dione adducts (C). S i m i i data were obtained for the addition of glutathione to benzo[a]pyrene-7,8-dione(not shown).
10-y1)cysteineand S-(7,&dihydro-7,8-dioxobenzo[alpyren10-y1)glutathione were conducted in a similar manner. Benzo[a]pyrene-7,8-dione(1equiv) was reacted with glutathione (2 equiv) in dioxane/water (2:1, v/v) for 24 h under Nz a t 25 “C. The dioxane was removed under vacuum, excess quinone was removed with ethyl acetate, and the aqueous layer was lyophilized. The residue was reconstituted in a small volume of aqueous dioxane and purified by semipreparative ion-pair RP-HPLC using an acetonitrile/water gradient containing 0.1 5% TFA, in which the acetonitrile concentration was increased from 40 to 60% over 50 min at a flow rate of 1.0 mL/min. Peak fractions were collected, concentrated, and analyzed by ion-pair RPHPLC using a Perkin-Elmer LC-480 with diode-array detection. RESULTS
NMR Chemical Shift Assignments and Coupling Constants for Benzo[a]pyrene-7,8-dione. A 2D-COSY spectrum of benzo[alpyrene-7,8-dionewas acquired (Fig ure 1)from which it was possible to assign all the protons unequivocally. The appropriate connectivities obtained from the cross-peaks are listed in Table I. All of the assignments obtained in this 2D experiment confirmed the chemical shifts reported for ‘H NMR (270 MHz) of the quinone (12)with one exception. In our experiments H-12 has a chemical shift of 8.21 ppm instead of 7.62 ppm;
Bioconlogete Chem., Vol. 3, No. 3, 1992
220
Murty and Penning
o-quinone. Further, proof of the structure of the hydroquinone came from trapping the unstable compound as its diacetate (acetic anhydridelpyridine). The product of this reaction gave an identical retention time and UV spectra by diode-array RP-HPLC to a synthetically prepared standard obtained via reductive acetylation of benzo[alpyrene-7,8-dione(16). 'H NMR of the trapped diacetate gave chemical shifts that corresponded to the methylgroups of the two acetates observed in the authentic standard at 2.55 and 2.35 ppm. 2D-COSY Spectra of Glutathionyl Adducts of Benzo[a]pyrene-7,8-dione. S-Glutathionyl conjugates of benzo[alpyrene-7,8-dionewere prepared as previously described in the Experimental Procedures. From the product profile it was anticipated that, of the three however, the coupling constant with H-11 is unchanged, products, one would correspond to a glutathionyl conjugate J11,lz = 9.5 Hz. It seemslikely that the originalassignment obtained at the level of quinone oxidation, one would was in error since others have reported the difficulty in correspond to a glutathionyl conjugate obtained at the assigning PAH aromatic protons based on 'H NMR data level of hydroquinone oxidation, and one would correspond (15). Formation of N-Acetyl-L-cysteinyland Glutathioto 7,8-dihydroxybenzo[alpyrene. Isolation of the putative nyl Conjugates of Benzo[a]pyrene-7,8-dione.Reacquinone-glutathionyl conjugate gave pure material as tions between benzo[alpyrene-7,8-dioneand either Njudged by RP-HPLC and this was subjected to 2D-COSY acetyl-L-cysteine or glutathione were monitored by RPspectroscopy. Examination of the spectrum indicates that HPLC using a diode-array detector. After 24 h three although a large solvent peak exists in the aliphatic region products had formed (I, 11, and 111). Peak I gave a UV it is possible by examination of the cross peaks to assign spectrum identical to benzo[alpyrene-7,8-dioneand is a all the aliphatic protons in glutathione (Table IV). It is presumptive thiol ether adduct obtained at the level of also possible to assign all the aromatic protons present in quinone oxidation (Figure 2a,b). The PD-COSY spectra benzo[alpyrene-7,8-dione,and together these support a of peak I obtained from both reactions are presented later. simple 1,CMichael addition product with thiol conjugation Attempts to isolate peak I1 showed that upon further occurring at C-10. Thus, there is an isolated vinyl proton purification and chromatography, peak I1 was converted that corresponds to H-9, while the benzylic vinyl proton to peak I. Inspection of the UV spectra for peak I1indicates at H-lo isabsent from the spectrum. Since all the aromatic that it contains a mixture of two chromophores, that protons can be accounted for, the product does not observed with the quinone (peak I) and that observed with correspond to a bisphenol adduct; similarly, since all the the hydroquinone (peak 111; presented later). Thus the aliphatic protons can be accounted for, the product does UV spectra support the view that, in each reaction, peak not correspond to a bisglutathionyl adduct. The comI1 is a hydroquinone conjugate which autooxidizes to yield pound thus corresponds to S-(7,8-dihydro-7,8-dioxobenzothe quinone conjugate. Peak I11 was isolated from each [a]pyren-IO-y1)glutathione. reaction and the OD-COSY spectrum indicates that this Recently, the confirmations of free and lanthanide-comis 7,8-dihydroxybenzo[alpyrene(also presented later). 2D-COSY Spectra of N-Acetyl-L-cysteineAdducts plexed glutathione have been determined in solution by of Benzo[a]pyrene-7,8-dione.The PD-COSY spectrum 'Hand 13CNMR (17). Comparison of the chemical shifts of product I obtained from the reaction of N-acetyl-Lfor the protons of the tripeptide with those obtained in cysteine and benzo[alpyrene-7,8-dioneis shown in Figure this study are in close agreement, with the exception of 3 and corresponds to N-acetyl-S-(7,8-dihydro-7,8-diox- Cyspl and Cysp2, which have been shifted downfield by obenzo[alpyren-10-y1)cysteine. The aliphatic region shows 0.50 and 0.65 ppm, respectively. This shift is consistent the presence of Cysa (4.83 ppm) and Cyspl (3.70 ppm) with the formation of a benzylic thiol. In the earlier study and CysB2 (3.50 ppm). A complete list of the connectivthe confirmation of glutathione was predicted by using ities obtained from the cross peaks is given in Table 11.In the coupling constants to predict torsion angles. Of the the aromatic region key chemical shifts show the presence pertinent coupling constants, the constants for Glua of a vinylic proton at 6.7 ppm which corresponds to H-9, coupling with Glup should be 2.8 and 13.5 Hz,if standard and confirms that the conjugate is a quinone rather than proton-proton vicinal trans and gauche couplings are a hydroquinone. The vinylic proton corresponding to Hobserved. In the structure of the benzo[alpyrenyl-glu10 (8.49 ppm) is also absent, indicating that this is the tathionyl conjugate these constants are 7.5 and 15.0 Hz, position of thiol substitution. respectively, indicating that within the adduct the conRapid anaerobic handling of product I11 isolated from firmation of glutathione has been distorted. It should be the reaction of N-acetyl-L-cysteine with benzo[alpyreneemphasized that it is not possible to predict the complete 7,8-dione led to the recovery of 7,8-dihydroxybenzo[a] confirmation of glutathione within the benzo[alpyrenyl pyrene. A 2D-COSY spectrum of this product permitted adduct since the spectra were taken in a dioxane-dlDzO the assignment of all the protons (Figure 4). The spectrum mixture which will promote the exchange of the amide indicates that all the protons are aromatic (see Table I11 and carboxyl group protons. Chemical shifts for these for a complete list of the connectivities). Absent from the protons along with the coupling constants observed with spectrum are vinylic protons that correspond to H-9 and adjacent methylene and methine protons are required for H-10. Careful inspection of the spectrum indicates that the complete confirmational analysis of the tripeptide. In a number of smaller cross-peaks exist that can be assigned to a structure that would coincide with benzo[alpyrenethe earlier work (I7) the spectrum of glutathione was taken 7,8-dione. It is estimated that the isolated 7,8-dihydroxin H20 containing 10% DzO, a solvent incompatible with ybenzo[alpyrene exists as a mixture of (6:l)hydroquinone/ the solubility of a benzo[alpyrene adduct. Table I. Proton Assignments for Benzo[a]pyrene-7,8-dione coupling chemical shift (ppm), constants, Droton multidicitv J (Hz) connectivities 8,1*2 H-1 or 8.25,-dd; H-3 8.24, dd H-1/H-3 (8.25/8.24)with 8 8.09, t H-2 H-2 (8.09) 9 H-4 (8.16) with H-5 (8.11) H-4 8.16, d H-5 (8.11) with H-4 (8.16) 9 H-5 8.11, d H-6 8.84, s 10.5 H-9 (6.59) with H-10 (8.48) H-9 6.59, d 10.5 H-10 (8.48) with H-9 (6.59) H-10 8.48,d H-11 (8.36) with H-12 (8.21) 9.5 H-11 8.36, d H-12 (8.21) with H-11 (8.36) H-12 9.5 8.21, d ~
Conjugate Characterlzatlon by PD-NMR
Bioconjugate Chem., Vol. 3, No. 3, 1992 221 Cyrlu
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Figure 3. The 500-MHz 1H-COSY spectrum of the N-acetyl-L-cysteineadduct of benzo[a]pyrene-7,8-dione(with solvent suppression). Panel A shows the aromatic region and panel B shows the aliphatic region. The sample is 1 mg/0.5 mL in dioxane-dlDz0. The pulse sequence applied is described in the Experimental Procedures and spectra were collected into 16K data points and 80 scans were taken. DISCUSSION
Benzo[a]pyrene-7,8-dionerepresenta a novel metabolite of B[a]P-diol which is a potent proximate carcinogen. It can form in vitro as a result of the reaction catalyzed by dihydrodiol dehydrogenase, in which B[al P-diol undergoes a formal dehydrogenation to yield a ketol which then rearranges to a catechol and undergoes air oxidation to form benzo[alpyrene-7,8-dione.Benzo[alpyrene-7,8-dione in turn is reactive and can be trapped as a thiol ether adduct (IO). Benzo[alpyrene-7,8-dionehas also been detected as a supplemajor metabolite of B[alP-diol in rat liver SIOO mented with NAD(P)+ (Penning and Shou, unpublished observations), suggesting that the conversion of B[alPdiol to benzo[a]pyrene-7,8-dionemay represent a significant route of B[alP-diol metabolism. This paper describes the synthesis and complete characterization of Nacetyl-L-cysteine (mercapturic acid) and glutathionyl conjugates of benzo[alpyrene-7,8-dionethat could represent the ultimate water-soluble forms of this o-quinone. The availability of these conjugates will permit their use as synthetic standards to identify their formation in vivo. ZD-COSY has permitted the assignment of each proton resonance in the thiol conjugates. In each case the predominant stable product is the simple 1,GMichael addition product obtained at the level of quinone oxidation.
This is supported by the presence of an isolated vinyl proton corresponding to H-9, and the lack of a benzylic vinyl proton at H-10. It has been proposed that a ketol and catechol are intermediates in thiol ether formation (IO). The studies described here support this mechanism since in the presence of excessthiol, adducts were detected as hydroquinones which autooxidize to quinone conjugates upon sample handling. In the pathway proposed (Scheme I), N-acetyl-L-cysteine or glutathione attacks benzo[a]pyrene-7,8-dioneto yield a ketol. Enolization then occurs to yield the corresponding hydroquinone conjugate, which could either autooxidize or cross-oxidize to yield the o-quinone conjugate. Although, these reactions were conducted under a nitrogen atmosphere, it is difficult to exclude all the oxygen,and therefore autooxidation with molecular oxygen acting as the oxidant is favored. For example, when the reductive addition of glutathione to 2-(hydroxymethy1)1,6naphthoquinone is followed, molecular oxygen is consumed and H202 is formed (18). Interestingly, the rates of oxygen consumption and H202 production were unaffected by superoxide dismutase. This suggests that superoxide anion is rapidly depleted by redox transitions with the hydroquinone and/or the anion of reduced glutathione to yield H202. In contrast, cross-oxidation, which requires one molecule of unconjugated o-quinone to oxidize
Murty and Penning
Bioconjugate Chem., Vol. 3, No. 3, 1992
222
12
12
1
1
Table 111. Proton Assignments for 7,8-Dihydroxybenzo[a]pyrenee chemical coupling shift (ppm), constants proton multiplicity (Hz),J connectivities H-1 8.00, dd; 7.5,7.9 or H-3 8.15, dd H-2 7.88, t 7.5 H-l/H-3 (8.00/8.15) with H-2 (7.88) H-4 (7.83) with H-5 (7.95) H-4 7.83, d 9.1 H-5 (7.95) with H-4 (7.83) H-5 7.95, d 9.1 H-6 8.72, s H-9 (7.45) with H-10 (8.45) H-9 7.45, d 9.2 H-10 (8.45) with H-9 (7.45) H-10 8.45, d 9.3 H-11 8.92, d 9.1 H-11 (8.92) with H-12 (8.22) H-12 8.22, d 9.1 H-12 (8.22) with H-11 (8.92)
OH
0 Ratio of catechol/quinone observed in this experiment is 6:l (based on peak ratios).
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Figure 4. The 500-MHz 'H-COSY spectrum of a 6:l mixture of 7,8-dihydroxybenzo[a] pyrene and benzo[a]pyrene-7,8-dione obtained from thiol addition reactions. The sample is 1 mg/0.5 mL in dioxane-d/DzO. The pulse sequence applied is described in the Experimental Procedures and spectra were collected into 16K data points and 112 scans were taken. Table 11. Proton Assignments for N-Acetyl-S(7,8-dihydro-7,8-dioxobenzo[a]pyren10-y1)cysteine chemical coupling shift (ppm), constant, proton multiplicity J (Hz) connectivities H-101 H-3 8.29, dd; 7.5 8.30. dd H-2 8.11, t 7.6 H-1/H-3 (8.29/8.30)with H-2 (8.11) H-4 8.24, d H-4 (8.24) with H-5 (8.19) H-5 (8.19)with H-4 (8.24) H-5 8.19, d H-6 8.79, s H-9 6.70, s H-10 absent H-11 9.00, d 9.5 H-11 (9.00)with H-12 (8.21) H-12 8.21, d 9.5 H-12 (8.21)with H-11 (9.00) CYSSl 3.70, dd 11.75 Cyspl (3.7) with Cysp2 (3.50) CYSP2 3.50, dd 13.20 Cysa2 (3.5) with Cyspl (3.7) Cysa 4.83, t Cysa (4.8) with Cysfll (3.70) Cysa (4.8)with CysS2 (3.50) CH3 (acetyl) 1.88, s
a molecule of hydroquinone conjugate is not favored. Thus, in the reductive addition of glutathione to 24hydroxymethyl)-1,4-naphthoquinone,cross-oxidation only occurred when the [GSHl:[quinonel ratio was 0.35. In the reactions described here the [RSHl:[benzo[alpyrene-7,8dionel ratio was 2.0. On this basis, it is predicted that the autooxidation of thiol conjugates of 7,8-dihydroxybenzo-
Table IV. Proton Assignments for S-(7,8-Dihydro-7,8dioxobenzo[alpyren-10-y1)glutathione chemical coupling shift (ppm), constants proton multiplicity (Hz), J connectivities H-1 or 8.21, dd; 10.0 H-3 8.19, dd H-2 8.04, t 9.0 H-1/H-3 (8.21/8.19) with H-2 (8.04) 8.08, dd; 10.0 H-4 with H-5 H-4 or H-5 8.10, dd H-6 8.53, s H-9 6.62, s H-10 absent 10.0 H-11 (8.82) with H-12 (7.96) H-11 8.82, d 8.10, d 10.0 H-12 (8.10) with H-11 (8.82) H-12 Cyspl 3.43, dd; Cysj3l (3.43) with Cysp2 (3.60) or @2 3.60, dd Cysa 4.78, t 15.0 Cysa (4.71) with Cysj31/@2 (3.4313.60) Gluy 2.40, t 7.5 Gluy (2.4) with GluP (2.0) Gluo 2.0, t 15.0 Gluj3 (2.0) with Gluy (2.5) Glua 3.7, t 5.0 Glua (3.7) with Glup (2.0) Glya 3.86,s
[alpyrene would be initiated by the reaction of the semiquinone radical with molecular oxygen to yield the o-quinone conjugate and superoxide anion. The superoxide anion would then propogate formation of the oquinone conjugate by oxidizing the hydroquinone and forming hydrogen peroxide (see eqs 1 and 2, where RQ'-
+ 0, = RQ + O,*RQH, + 02*= RQ'- + H,O, RQ'-
RQH,
+ 0, = RQ + H,O,
(1)
(2)
(net balance)
= semiquinone radical of a thiol ether conjugate of benzo[alpyrene-7,8-dione; RQ = thiol ether conjugate of benzo[alpyrene-7,8-dione; RQH, = thiol ether conjugate of 7,8-dihydroxybenzo[a]pyrene). Because the reactions described here were conducted in the presence of a molar ratio of [RSHl:[quinonel = 2.0, the thiol is consumed by at least two processes. First, reductive addition of the thiol to the quinone will deplete RSH (Scheme I). Second, RSH oxidation may be coupled to redox transitions that involve the thiyl radical. This radical may be produced by interaction of RSH with superoxide anion, hydroxyl radical, or semiquinone radical. Reaction of RSH with the semiquinone radical would produce the hydroquinone, while dimerization of resultant thiyl radicals would yield RSSR. These reactions have
Conjugate Characterlzatlon by PD-NMR
i
Bioconjugate Chem., Vol. 3, No. 3, 1992 223
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Figure 5. The 500-MHz 1H-COSY spectrum of the glutathionyl conjugate of benzo[alpyrene-7,8-dione(with solvent suppression). Panel A shows the aromatic region and panel B shows the aliphatic region. The sample is 1mg/0.5 mL in dioxane-d/DzO. The pulse sequence applied is described in the Experimental Procedures and spectra were collected into 16K data points and 80 scans were taken. Scheme I. Mechanism of Formation of Thiol Ether Conjugates of Benzo[a]pyrene-7,8-dione (R-SH = glutathione or N-acetyl-L-cysteine)
HO 0
6
been well-documented for the addition of GSH to 2-(hydroxymethyl)-l,4-naphthoquinone(18)and would provide an explanation for the chromatographic profiles shown in Figure 2. It is clear that the intermediates that form and their redox transitions are critically dependent on the [RSHI: [quinone] ratio. However, once these reactions are handled aerobically, fully oxidized thiol conjugates of benzo[alpyrene-7,8-dioneare obtained.
Although it is conceivable that N-acetyl-L-cysteine and glutathione might add directly to the hydroquinone (7,8dihydroxybenzo[alpyrene)this possibility is ruled out on mechanistic grounds (18-21). Since all the aliphatic and aromatic protons in the 2DNMR spectra can be assigned to mono adducts, the formation of bisphenol and bisglutathionyl adducts are ruled out. Our results are in contrast to other studies which have examined the formation of thiol ether adducts of p-quinones (22,23). Thus, bisglutathionyl conjugates have been described for 2-bromobenzoquinone (22). In addition, reactions betweenp-quinone and L-cysteine may not be simple 1,4-Michael additions. Studies on the Lcysteinyl adducts of 2-bromobenzoquinone indicate that intramolecular Schiff s base formation occurs, leading to cyclization reactions that result in the formation of 1,4benzothiazines (23). The studies described here may have avoided the formation of these complex products since N-acetyl-L-cysteine rather than cysteine was used as a reactant. We have recently described the cytotoxicity of a variety of PAH o-quinones in H-411e (rat hepatoma) cells. Benzo[a]pyrene-7,8-dione (20 pM) was the least cytotoxic of the PAH o-quinones examined. It reduced cell viability without having an adverse effect on cell survival. This effect was accompanied by a concomitant depletion of glutathione which was not accompanied by a change in GSH/GSSG ratios ( 1 1 ) . This implies that benzoCalpyrene-
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7,8-dione does not cause a change in redox state and generate superoxide anion as observed in vitro but rather depletes glutathione by forming glutathionyl conjugates. The glutathionyl conjugate characterized in this study may represent the metabolite that is responsible for the elimination of this quinone from hepatoma cells and its limited cytotoxicity. It is clear from our work that B[alP-diol can act as a branch point in benzo[alpyrene metabolism giving rise to either anti-BPDE or benzo[alpyrene-7,8-dione. Both metabolites can react with glutathione and can be eliminated as glutathione conjugates. This paper describes the first complete structure of a benzo[alpyrenylglutathionyl conjugate. The structure of the glutathionyl conjugate of anti-BPDE has not been fully characterized. ACKNOWLEDGMENT
This work was supported by U.S. Public Health Service Grant CA35904 (to T.M.P.) from the National Cancer Institute and NIH Research Career Development Award CA01335 (to T.M.P.). Portions of this work were supported by the High-Resolution NMR facility in the Department of Biochemistry and Biophysics at the University of Pennsylvania. Dr. Kurt Loening of Topterm, North American Division was responsible for the nomenclature of the compounds. LITERATURE CITED
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