Enzyme-Mediated Dialdehyde Formation - ACS Publications

Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and. Public Health, and Department of Biophysics and Biophysical Chemistr...
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Chem. Res. Toxicol. 2000, 13, 1174-1180

Enzyme-Mediated Dialdehyde Formation: An Alternative Pathway for Benzo[a]pyrene 7,8-Dihydrodiol Bioactivation Kevin H. Stansbury,† David M. Noll,‡ John D. Groopman,† and Michael A. Trush*,† Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, and Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 Received July 25, 2000

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are widespread environmental carcinogens of human concern. Several enzymatic systems have been shown to activate benzo[a]pyrene 7,8-dihydrodiol, the proximate carcinogenic metabolite of benzo[a]pyrene, to a reactive species which produces both a chemiluminescence response and genotoxic lesions. The chemiluminescence response has been proposed to be the result of the formation of a dioxetane which upon ring opening forms a reactive dialdehyde intermediate. In in vitro incubations involving phorbol ester-stimulated human polymorphonuclear leukocytes or an isolated enzyme system consisting of myeloperoxidase, taurine, and hydrogen peroxide, a prolonged (>60 min) chemiluminescence response was observed from benzo[a]pyrene 7,8-dihydrodiol. HPLC analysis of the reaction mixture revealed the existence of a product which is dependent upon both taurine and the hydrocarbon. Characterization of this product using UV, NMR, and MS indicated that the product is a pyrene with two side chains resulting from bond breakage of a ring, yielding a dialdehyde. These side chains contain a portion of taurine covalently attached through imine formation with the aldehydes resulting from dioxetane ring opening. Replacement of taurine with either protein or DNA also produced a prolonged chemiluminescence response. These results demonstrate for the first time the formation of a novel electrophilic species from benzo[a]pyrene 7,8-dihydrodiol which along with an increased production of photons from this activation mechanism may lead to DNA and/or protein damage that is different from that elicited by diol epoxides.

Introduction 1

It has long been recognized that benzo[a]pyrene (BP) is a ubiquitous environmental pollutant, possessing strong carcinogenic activity in several animal species (1). For BP to exert carcinogenic activity, it must be metabolically activated, ultimately producing electrophilic species. These electrophiles react covalently with various cellular macromolecules, producing addition products or adducts (2). One well-characterized mechanism of activation for BP involves sequential oxidations catalyzed by various isoforms of cytochrome P450. Through this pathway of oxidation, the procarcinogenic metabolite 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) and * To whom correspondence should be addressed: Division of Toxicological Sciences, Johns Hopkins School of Hygiene and Public Health, 615 N. Wolfe St., Room 7032, Baltimore, MD 21205. Phone: (410) 9554712. Fax: (410) 955-0116. E-mail: [email protected]. † Johns Hopkins School of Hygiene and Public Health. ‡ Johns Hopkins School of Medicine. 1 Abbreviations: BP, benzo[a]pyrene; BPDE, 7,8-dihydroxy-9,10epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BP-7,8-diol, (()-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene; BSA, bovine serum albumin; CL, chemiluminescence; cis-anti-BP tetrol, 7R,8R,9β,10β-tetrahydroxy7,8,9,10-tetrahydrobenzo[a]pyrene; cis-syn-BP tetrol, 7β,8R,9β,10βtetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; FaPy, formamidopyrimidine; HRP, horseradish peroxidase; MPO, myeloperoxidase; PMN, polymorphonuclear leukocyte; TPA, 12-O-tetradecanoyl phorbol 13acetate; trans-anti-BP tetrol, 7R,8β,9β,10R-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans-syn-BP tetrol, 7β,8R,9β,10R-tetrahydroxy7,8,9,10-tetrahydrobenzo[a]pyrene.

the proposed ultimate carcinogenic metabolite 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) are formed (3). Using 3-methylcholanthreneinduced rat liver microsomes, Seliger and co-workers showed that a chemiluminescence (CL) response could be elicited from both BP (4) and BP-7,8-diol (5). The CL response from BP-7,8-diol was 3 times greater than the response seen from BP. While at first this CL response was attributed to the formation of the diol epoxide metabolite (4), it was later ascribed to the breakdown of a dioxetane derivative (6). With the strain of a fourmembered ring and the weakness of the peroxide dioxygen bond, the decomposition of a dioxetane produces an electronically excited product which emits a photon as it returns to the ground state (7). A similar CL response can be seen when BP-7,8-diol is incubated with 12-O-tetradecanoyl phorbol 13-acetate (TPA)-stimulated human polymorphonuclear leukocytes (PMNs) (8, 9). In addition to this photon emission, the interaction of BP-7,8-diol with PMNs has been shown to produce intermediates which bind covalently to DNA (8, 9), induce mutagenesis in Salmonella typhimurium strain TA100 (8, 9), and increase the level of sister chromatid exchanges (SCEs) in cocultured V79 cells (10). Studies of BP-7,8-diol metabolism with either TPAstimulated PMNs or myeloperoxidase (MPO) produce a strikingly similar pattern of BP-7,8-diol-derived tetrols

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Enzyme-Mediated Dialdehyde Formation

resulting in an anti/syn ratio of 6/1 (11). Such ratios have been described as being indicative of a peroxyl radicaldependent epoxidation, since it is in contrast to a 1/1 ratio observed from a cytochrome P450-catalyzed epoxidation (12, 13). The CL response seen from BP-7,8-diol with both the cytochrome P450 and PMN systems (5, 8, 9), as well as a singlet oxygen-mediated oxidation of BP-7,8-diol (14), has been hypothesized to be derived from the formation of an excited state dialdehyde intermediate. In this metabolism scheme, BP-7,8-diol is oxidized to a dioxetane intermediate, which upon ring opening produces a photoemissive reactive dialdehyde intermediate. This study provides for the first time direct evidence that indeed a dialdehyde, and by inference a dioxetane, is a significant product of BP-7,8-diol metabolism. Moreover, it appears that this BP-7,8-diol dialdehyde is highly bioreactive toward amino acids, proteins, and DNA.

Experimental Procedures Caution: Benzo[a]pyrene and many of its metabolites are mutagens and/or carcinogens. All laboratory procedures involving these chemicals should be performed with extreme care, utilizing appropriate safety wear, and where possible in a wellventilated fume hood. [3H]BP-7,8-diol, BP-7,8-diol, BPDE, 7R,8β,9β,10R-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BP tetrol), 7β,8R,9β,10R-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-syn-BP tetrol), 7R,8R,9β,10β-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (cis-anti-BP tetrol), and 7β,8R,9β,10βtetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (cis-syn-BP tetrol) were obtained from the NCI Chemical Carcinogen Repository, Midwest Research Institute (Kansas City, MO). 1-Pyrenecarboxaldehyde, 1-pyrenecarboxylic acid, 1-pyrenemethylamine, and 1-pyrenemethanol were purchased from Aldrich Chemical Co. (Milwaukee, WI), and (R)-3-hydroxy-3-phenylpropanoic acid was obtained from ACROS Organics (Pittsburgh, PA). TPA was obtained from LC Laboratories (Woburn, MA). Horseradish peroxidase (HRP) type II, salmon sperm DNA, and bovine serum albumin (BSA) were obtained from Sigma Chemical Co. (St. Louis, MO). Human MPO was obtained from Calbiochem (San Diego, CA). The MPO and HRP enzyme activities are expressed in units based upon the rate of oxidation of o-dianisidine (15) and pyrogallol (16), respectively. All other chemicals were of the highest purity available. Isolation of PMNs. Blood was purchased from the Johns Hopkins Hemapheresis Center, and PMNs were isolated by dextran sedimentation and red blood cell lysis as previously described (8). The PMNs were washed, resuspended in phosphatebuffered saline (PBS) containing 0.5 mM MgCl2, 0.7 mM CaCl2, and 1% glucose (complete PBS), and counted on a hemocytometer. This procedure yielded a preparation of PMNs that was at least 95% pure. Measurement of Chemiluminescence (CL) Responses. CL was monitored in a Bioluminat LB9505 microcomputercontrolled luminometer (Berthold Analytical Instruments, Inc., Nashua, NH). BP-7,8-diol CL experiments were begun by incubating HRP (1.4 units) or human MPO (1.6 units), BP-7,8diol (5 µM), and taurine (5 mM) in 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 2 mL for 5 min at 37 °C in dark-adapted polystyrene tubes. The background CL of each sample tube was determined, and H2O2 (final concentration of 40 µM) was injected to initiate the reaction. The CL was monitored for 55 min at 0.6 min intervals, and the tubes were maintained at 37 °C throughout the counting process. All additions to the reactions, as well as the CL counting procedure, were performed in a darkened room. Results are expressed as counts per unit time minus background. Data are presented as temporal response curves and the area under the curve.

Chem. Res. Toxicol., Vol. 13, No. 11, 2000 1175 For incubations involving cellular macromolecules, either salmon sperm DNA (0.5 mg/mL) or BSA (0.5 mg/mL) was substituted for taurine in the incubation procedure described above. Samples were run in triplicate, and the results of the CL responses were analyzed using the paired t test. In addition, CL responses were monitored from unstimulated or stimulated (30 ng/mL TPA) human neutrophils (1 × 106 cells) incubated with BP-7,8-diol (5 µM) in complete PBS (final volume of 1 mL). Metabolite Preparation and Isolation. [3H]BP-7,8-diol (5 µM, specific activity of 100 mCi/mmol) was incubated with an enzyme system consisting of either MPO (1.6 units) or HRP (1.4 units) and 40 µM H2O2 in 50 mM potassium phosphate buffer (pH 7.4) for 6 h at 37 °C. The total reaction volume was 2 mL. Additions of either MPO (1.6 units) or HRP (1.4 units) were made every hour, and H2O2 (final concentration of 40 µM) was added after 3 h. The reactions were terminated by addition of ethyl acetate and the mixtures extracted with 4 × 4 mL of ethyl acetate. The organic phase from each extraction was combined and dried under nitrogen and the residue redissolved in 250 µL of methanol containing unlabeled standards of trans-anti-, trans-syn-, cis-anti-, and cis-syn-BP tetrols. For the purpose of metabolite isolation and identification, a total of 1720 incubation sample extracts using the procedure described above with unlabeled BP-7,8-diol, HRP, and H2O2 were combined and subjected to analysis by HPLC. The major metabolite was collected, and its solvent was evaporated and rechromatographed to ensure a single peak. HPLC Analysis. The analysis of the metabolites was performed using a Waters liquid chromatograph system and a Beckman/Altex ODS reverse phase column (5 µm, 4.6 mm × 250 mm) at ambient temperature with a flow rate of 1 mL/min using a methanol/water solvent system. Initial solvent conditions included 50% methanol in water with a linear gradient to 55% methanol over the course of 30 min, then a linear gradient from 55 to 75% methanol over the course of 20 min, and finally isocratic at 75% methanol over the course of 30 min. Unlabeled metabolites were detected by UV absorbance at 344 nm, while radiolabeled metabolites were detected by a Radiomatic FloOne Beta Model A-200 radioactive flow detector (Packard Instrument Co., Meriden, CN). BP tetrol products were identified by the cochromatography of radiolabeled peaks with the unlabeled BP tetrol standards. MS Analysis. MS analysis of the metabolite was performed at the Scripps Research Institute (La Jolla, CA) using electrospray ionization/mass spectrometry (ESI/MS) in both positive and negative ionization modes. NMR Analysis. 1H and 13C NMR spectra for all samples were recorded in CD3COCD3 or CDCl3 at 300 MHz on a Bruker AMX300 spectrometer. The chemical shifts are reported as parts per million referenced to TMS or residual solvent peaks.

Results The interaction of BP-7,8-diol with MPO and H2O2 has previously been shown to produce BP-7,8-diol-derived tetrols as well as covalent binding to DNA (11, 17). As shown in the inset of Figure 1A (solid line), a CL response with a short duration was seen from BP-7,8-diol in the presence of MPO and H2O2. This response was similar in duration to that observed by Seliger et al. (14) with a singlet oxygen-generating system. Taurine is an amino acid found at a millimolar concentration (∼15 mM) in PMNs (18), and exogenous taurine inhibits the SCE inductive effect of BP-7,8-diol with PMNs (10). Therefore, the effect of taurine on BP7,8-diol CL was examined. When taurine was included in the MPO/H2O2 system, the CL response of BP-7,8-diol was increased in both magnitude and duration (Figure 1A, dotted line). Interestingly, this CL profile of BP-7,8diol in the presence of MPO, H2O2, and taurine is similar

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Figure 1. CL responses from BP-7,8-diol (5 µM) when acted upon by either (A) MPO (0.8 unit/mL) in the absence (s) or presence (‚‚‚) of taurine (5 mM) or (B) isolated human neutrophils (1 × 106 cells) unstimulated (s) or stimulated (‚‚‚) by TPA (30 ng/mL).

Figure 2. HPLC separation and LS detection of the products formed from the 6 h incubation of [3H]BP-7,8-diol with either MPO in the presence (A) or absence (a) of taurine or HRP in the presence (B) or absence (b) of taurine. The chromatograms have been offset to allow for comparisons. The three primary radioactive peaks are the trans-anti-BP tetrol (peak 1), a taurine-dependent product (peak 2), and unmetabolized BP-7,8diol (peak 3). See Experimental Procedures for details.

to that of BP-7,8-diol with TPA-stimulated human PMNs (Figure 1B, dotted line). To better understand the basis for the prolonged CL from BP-7,8-diol in the presence of taurine, the reactions were analyzed by HPLC. Preliminary 1 h incubations indicated the presence of a new HPLC peak dependent upon the presence of taurine. To maximize production of this taurine-dependent product, incubations were conducted for 6 h. Shown in Figure 2 are the radioactive profiles from the HPLC analysis of these incubations conducted in the presence (profiles A and B) or absence (profiles a and b) of taurine with either MPO (profiles A and a) or HRP (profiles B and b). Two major products were detected: the trans-anti-BP tetrol (peak 1) and a taurine-dependent product (peak 2) which was slightly more polar than the starting material, BP-7,8-diol (peak 3). Both HRP and MPO yielded similar metabolite profiles. However, the MPO incubation (profile A) produced more polar metabolites which eluted near the solvent front (∼3 min) than did HRP. These polar metabolites were found only when taurine was present

Stansbury et al.

Figure 3. 1H NMR spectra of the product isolated from the metabolism of BP-7,8-diol with taurine, catalyzed by HRP. The inset contains the product’s 13C NMR spectra, the proposed structure with its ring carbons appropriately numbered, and the positive ion MS fragmentation losses indicated.

in the system, and since they were radioactive, a portion of the BP-7,8-diol must be a component of the metabolites’ structure. To gain further insight into the structure of the taurine-dependent product, a significant amount (∼500 µg) of the metabolite (peak 2) was isolated. Figure 3 shows the 1H NMR spectra of the taurine-dependent product. The doublet at 9.45 ppm corresponds to the H10 proton of the pyrene with the remaining pyrene proton resonances occurring between 8.1 and 8.5 ppm. These aromatic resonances integrate to eight protons, and the observed splitting pattern allows them to be unambiguously assigned to an intact pyrene ring system, thus ruling out the subsequent ring oxidation during the peroxidase-mediated activation of BP-7,8-diol. In addition to the aromatic resonances, three additional protons are observed in the 1H NMR spectrum of the taurine-dependent product (Figure 3) using the solvent CDCl3. The pair of doublets at 2.92 and 3.42 ppm are consistent with coupling of nonequivalent CH2 protons to the 5.8 ppm resonance. An additional proton resonance at 5.0 ppm was observed in the 1H NMR spectrum using the solvent CD3COCD3. This proton resonance is readily exchanged with D2O which suggests that it is an OH. Furthermore, the effect on the 5.8 ppm resonance upon exchange of the OH proton is consistent with these being coupled. This evidence in combination with a comparison of the 13C chemical shifts of the metabolite with a model compound, (R)-3-hydroxy-3phenylpropanoic acid (53.15 vs 49.07 ppm and 75.56 vs 75.02 ppm, respectively), allowed for the assignment of the diol-derived portion of the C2 side chain. This NMR data provide the first evidence of the opening of the diolcontaining ring on BP-7,8-diol, forming a pyrene having two aliphatic side chains. Further evidence for ring opening can be seen in Table 1, which lists the proton assignment, chemical shift, and splitting pattern for certain pyrene protons of the taurinedependent product, the starting material BP-7,8-diol, and 1-pyrenecarboxaldehyde. The chemical shifts for the pyrene ring protons of the isolated product differ slightly from those of 1-pyrenecarboxaldehyde, but significantly with those of BP-7,8-diol. The slight (>0.01 ppm) chemical shift change of the H3 proton indicates rather minor perturbations to the electronic character at the C2 position of the metabolite. In contrast, there is a large

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(m/z 14) along with cyclization of the two side chains gives rise to an ion peak at m/z 273. A structure of this isolated taurine-dependent product consistent with the MS and NMR data obtained is shown in Figure 3 (inset). In addition to the NMR and MS data, the proposed structure of the isolated taurine-dependent product is supported by experiments using either radiolabeled taurine or BP-7,8-diol. Radioactivity was associated with the UV peak when unlabeled taurine and tritiated BP-7,8diol were present in the incubation, unlike reactions conducted with taurine tritiated at both carbons and unlabeled BP-7,8-diol (20). The increased CL seen in the presence of taurine as well as the HPLC and MS analyses indicates that covalent bonding occurred between BP-7,8-diol and taurine. It was of interest then to obtain some evidence that this BP-7,8-diol-derived dialdehyde exhibited reactivity toward other biomolecules. Accordingly, DNA and protein were substituted for taurine in the incubation systems and the generation of CL was monitored. As shown in Table 2, increased CL responses were observed with these biomolecules, with BSA exhibiting a greater response (7fold greater than control) than the salmon sperm DNA (2-fold). It is interesting to note that both MPO and HRP produced the same magnitude of the response with either DNA or BSA. These results indicate that the dialdehyde derivative from BP-7,8-diol is reactive toward larger biomolecules.

Table 1. Comparison of the Proton Position, Chemical Shift, and Splitting Pattern for Pyrene Ring Protons at the Same Position of BP-7,8-Diol, 1-Pyrenecarboxaldehyde, and the Isolated Taurine-Dependent Product 1-pyrenecarboxaldehyde

taurine-dependent product

BP-7,8-diol

H2,a 8.60 ppm, d not determined H4, 8.40 ppm, d H5, 8.28 ppm, d H7, 8.22 ppm, m H10, 9.52 ppm, d

none H3,a 8.55 ppm, s H4, 8.37 ppm, d H5, 8.29 ppm, d H7, 8.21 ppm, m H10, 9.45 ppm, d

none H6,b 8.54 ppm, s H5, 8.18 ppm, d H4, 8.14 ppm, d H2, 8.03 ppm, m H11, 8.49 ppm, d

a Proton positions are shown in the inset of Figure 3. b Proton positions are shown in Scheme 1; the values are reported in ref 20.

chemical shift perturbation observed at H10 for the product, as compared to the corresponding proton H11 of BP-7,8-diol (Table 1). Since the H10 position of pyrene is extremely sensitive to the electronic nature of the substituent at C1, we analyzed a series of related pyrene compounds under solvent and acquisition conditions identical to those used to analyze the taurine-dependent product. Using 1-pyrenecarboxaldehyde (H10, 9.52 ppm), 1-pyrenecarboxylic acid (H10, 9.41 ppm), 1-pyrenemethanol (H10, 8.45 ppm), and 1-pyrenemethylamine hydrochloride (H10, 8.79 ppm), it was determined that the chemical shift of the H10 proton of the taurine-dependent product is indicative of an electron-withdrawing substituent at C1 and in conjunction with the MS analysis presented below is consistent with a carboxylic acid-like moiety at the C1 position of pyrene. As expected, no aldehyde proton signal (10-11 ppm) was observed, due to the highly reactive nature of dialdehydes. The isolated taurine-dependent product was subjected to MS analysis, and both positive and negative ion mass spectra were obtained. The molecular ion mass as determined from both ion spectra was 358. This is a mass difference of 72 from that of BP-7,8-diol, an increase which cannot be due solely to the addition of molecular oxygen to the parent compound. Since taurine is required to produce this metabolite, it is reasonable to assume that the mass increase is partially due to portions of taurine remaining from the further metabolism of the compound formed from the reaction of taurine with the highly reactive dialdehyde species. Major ion peaks were seen in the positive ion spectrum at m/z 381, 337, 311, 295, and 273 (data not shown), with the major peak (m/z 381) due to complexation of sodium with the molecular ion. The complete loss of the formamido group (m/z 44) gives rise to an ion peak at m/z 337. Fragment ions at m/z 311 and 295 are formed from the loss of the fragment (m/z 70) formed by the cleavage between the carbonyl and methylene groups on the C2 side chain and the loss of the amine (m/z 16), respectively, from a 381 ion peak. Further loss of the methylene group

Discussion Because of the concern for human exposure to polycyclic aromatic hydrocarbons (PAHs), there is much interest in understanding the mechanisms which contribute to their bioeffects. Both BP (21, 22) and BP-7,8diol (18, 22) are considered “complete” carcinogens, exhibiting both initiating and promoting activities, while BPDE has been found to possess only tumor-initiating activity (22, 23). It is generally believed that BPDE, the ultimate carcinogenic form of BP, is formed from the oxidation of the procarcinogenic BP-7,8-diol. A critical component in this activation pathway and a requirement for the carcinogenic activity of BP and BP-7,8-diol is the existence of a double bond at positions 9 and 10 of the molecule (22). This same double bond is also a prerequisite for a CL response and, therefore, formation of a proposed dioxetane product from BP-7,8-diol (8). Demonstration of the generation of a dioxetane and its dialdehyde breakdown product from BP-7,8-diol would indicate a previously unrecognized bioactivation pathway for this compound, one capable of producing potentially both genotoxic electrophiles and bioreactive photoemissive intermediates. As such, this pathway could contribute to the “complete” carcinogenicity of BP-7,8-diol. We have presented three lines of evidence which indeed point to the production of a dioxetane-dialdhyde intermediate during the metabolism of BP-7,8-diol by peroxi-

Table 2. CL Response of BP-7,8-Diol (5 µM) by either HRP (1.4 units) or MPO (1.6 units) in the Presence of Salmon Sperm DNA (0.5 mg/mL) or Bovine Serum Albumin (0.5 mg/mL)a integrated CL activity (counts × 105) myeloperoxidase calf thymus DNA bovine serum albumin a

horseradish peroxidase

control

with a biomolecule

control

with a biomolecule

6.38 ( 0.33 3.63 ( 0.36

12.71 ( 27.07 ( 1.26b

2.76 ( 0.04 2.76 ( 0.04

4.65 ( 0.22b 28.31 ( 0.83b

0.13b

Each value represents the mean ( SD of three measurements. b Denotes P < 0.001, using a paired Student’s t test.

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Scheme 1. Proposed Scheme of MPO Metabolism of BP-7,8-Diol Showing the Initial Formation of an Epoxide and a Dioxetanea

a The dioxetane derivative then cleaves to form an excited state dialdehyde intermediate (II) which can then react with taurine, producing an imine (III). Further oxidation of this imine intermediate gives rise to the product (IV).

dases: (a) observation of a prolonged CL response in the presence of taurine, (b) observation of new metabolic products being formed from BP-7,8-diol when incubated with either MPO or HRP, and (c) characterization by NMR and MS of an isolated BP-7,8-diol metabolite having an intact pyrene ring with two side chains arising from a ring-opening process. The data presented here support the following metabolic pathway for BP-7,8-diol (Scheme 1) in which both an epoxide and a dioxetane intermediate are formed initially. Ring opening of the dioxetane intermediate forms an excited state dialdehyde (II) which is photoemissive and capable of forming an imine (III) or Schiff base with taurine. Subsequent oxidation and bond breakage of the rearranged imine (IV) also produces photon emission and as such accounts for the observed prolonged CL response. The prolonged CL response may be similar to the photoemission reported by Nascimento and Cilento (24) resulting from the oxidation of a Schiff base consisting of phenylacetaldehyde and lysine. The characterization of the isolated taurine-dependent product gave several insights into its formation. The NMR data indicate that the number of protons in the pyrene ring system of BP-7,8-diol has not been reduced, demonstrating that ring oxidation on the pyrene moiety has not occurred. The NMR and MS data provide evidence for a ring-opened derivative of BP-7,8-diol, and the observed fragmentation pattern can be explained by losses occurring from those side chains. The mass difference of the product and the starting material indicates that taurine had interacted with the ring-opened deriva-

tive, and further metabolism had left portions of taurine bound to the ring-opened sections of the derivative. The formation of both a dioxetane and a dialdehyde from BP-7,8-diol and the increased CL in the presence of DNA have implications for the molecular actions of PAHs, not previously appreciated. As a class, dioxetanes have been shown to cause lesions in DNA. However, the type of DNA damage seen with dioxetanes is dependent upon the structural features of the dioxetane (25). In the superhelical DNA from bacteriophage PM2, dioxetanes primarily cause DNA modifications which are recognized by formamidopyrimidine (FaPy)-DNA glycosylase (i.e., 8-hydroxyguanine and FaPy residues) (26). This damage has been attributed to the formation of singlet oxygen and is similar to DNA lesions caused by other sources of singlet oxygen, either Rose Bengal and light (27) or the endoperoxide NDPO2 (28). In contrast to aliphatic dioxetanes, only the dioxetane of the cyclic compound benzofuran was shown to be highly mutagenic in S. typhimurium strain TA100. The mutagenic species appears to possess a lifetime of a few minutes, and is believed to act as an alkylating agent (25). Using the 32Ppostlabeling assay to analyze DNA modified in vitro with either the benzofuran epoxide or the benzofuran dioxetane, the observed adducts exhibited different chromatographic behavior, suggesting that they are structurally different (25). Aldehyde derivatives have also been shown to modify protein or nucleic acids. These include the lipid peroxidation product malondialdehyde (29) as well as proposed dialdehyde intermediates of aflatoxin (30) or 2-methylfuran (31).

Enzyme-Mediated Dialdehyde Formation

The results of this study have established that there are indeed two separate activation pathways for BP-7,8diol: the well-characterized conversion to an electrophilic diol epoxide and a previously uncharacterized dioxetanederived dialdehyde (Scheme 1). As indicated by the observations with taurine, this dialdehyde is highly reactive and is capable of forming adducts with aminecontaining molecules. Since both DNA and protein possess many amine groups, this metabolism pathway could produce cross-links between DNA and DNA, protein and protein, and DNA and protein. When the carcinogenic activity of certain cross-linking agents is considered (32), this DNA lesion may have profound genotoxic effects. Like the release of singlet oxygen, the subsequent release of photons from the breakdown of products formed from dialdehyde-amine interactions could also contribute to biological damage. The damage may be more pronounced in this case due to the particularly prolonged duration (>60 min) of the photoemissive response. The CL profile observed from BP-7,8-diol and PMNs (Figure 1B) may be reflective of such dialdehyde-target interactions within intact cells. Since PMNs can mediate this activation pathway, such a process may contribute to the wellrecognized relationship between chronic inflammation states and tumor development (33). In this regard, a G to A polymorphism in the promoter region of the MPO gene has been observed (34). When the occurrence of significant pulmonary inflammation following exposure to many environmental pollutants is considered (17, 33, 35), this polymorphism sets the stage for potential geneenvironment interactions. Interestingly, individuals who inherit two copies of the allele that reduces MPO gene transcription may be at decreased risk for lung cancer (34, 36). Although this study utilized peroxidases as an activating system, the significant CL previously observed from BP and BP-7,8-diol with rat liver microsomes (4, 5) suggests that cytochrome P450 can catalyze both activation pathways but that there are also molecules within the microsomes that serve as targets for the dialdehyde. The type and extent of BP-7,8-diol-derived dioxetanedialdehyde interactions with proteins and nucleic acids are currently under investigation. Clearly, such studies will contribute to further understanding the molecular mechanisms underlying both the toxicologic and carcinogenic actions of PAHs so widely prevalent in the human environment.

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Acknowledgment. This study was supported by NIH Grants ES03760 and ES06052, Training Grant ES007141, and Center Grant ES03819. K.H.S. was supported by NRSA Fellowship Grant ES05766. D.M.N. was supported by a grant from the Robert Leet and Clara Guthrie Patterson Trust. The NMR studies were performed in the Johns Hopkins University Biochemistry NMR Facility established by NIH Grant GM27512 and a Biomedical Shared Instrumentation Grant RR06262.

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References (1) International Agency for Research on Cancer (1973) Benzo[a]pyrene. In IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Certain Polycyclic Aromatic Hydrocarbons and Heterocyclic Compounds, Vol. 3, pp 91-136, International Agency for Research on Cancer, Lyon, France. (2) Jerina, D. M., Lehr, R., Schaeffer-Ridder, M., Yagi, H., Karle, J. M., Thakker, D. R., Wood, A. W., Lu, A. Y. H., Ryan, D., West, S., Levin, W., and Conney, A. H. (1977) Bay region epoxides of

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