Tetramethylammonium Hydroxide (TMAH ... - ACS Publications

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Chem. Res. Toxicol. 2003, 16, 1484-1492

A New Approach to Evaluating the Extent of Michael Adduct Formation to PAH Quinones: Tetramethylammonium Hydroxide (TMAH) Thermochemolysis with GC/MS Mary K. Briggs,† Emmanuel Desavis,‡ Paula A. Mazzer,† R. B. Sunoj,† Susan A. Hatcher,† Christopher M. Hadad,† and Patrick G. Hatcher*,† Department of Chemistry, The Ohio State University, Columbus, Ohio, 43210, and ESCOM (Ecole Superieure de Chimie Organique et Minerale), Paris, France Received July 15, 2003

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are converted to cytotoxic and carcinogenic metabolites, quinones, by detoxifying enzyme systems in animals. PAH metabolites such as the quinones can form Michael adducts with biological macromolecules containing reactive nucleophiles, making detection of exposure to PAHs difficult using conventional techniques. A technique has been developed for detecting exposure to PAHs. Tetramethylammonium hydroxide (TMAH) thermochemolysis coupled with GC/MS is proposed as an assay method for PAH quinones that have formed Michael adducts with biological molecules. Three PAH quinones (1,4-naphthoquinone, 1,2-naphthoquinone, and 1,4-anthraquinone) and 1,4-benzoquinone were reacted with cysteine, and the TMAH thermochemolysis method was used to assay for both thiol and amine adduction between the quinones and the cysteine. Additional studies with 1,4-naphthoquinone adducts to glutathione and bovine serum albumin showed the same thiol and amine TMAH thermochemolysis products with larger peptides as was observed with cysteine adducts. The TMAH GC/MS method clearly shows great promise for detecting PAH quinones, produced by enzymatic conversion of PAHs in biological systems, that have been converted to respective Michael adducts.

Introduction PAHs1

are ubiquitous environmental pollutants. Because these compounds require metabolic activation to exhibit their carcinogenic and cytotoxic effects (1), common assays for exposure to PAHs seek to identify toxic metabolites rather than parent compounds. While multiple biotransformation pathways exist for PAH conversions in mammalian systems, probably the most prevalent is through the P450 system (Scheme 1), which leads to the formation of quinone or diol epoxide intermediates (2-5). Both of these metabolites are reactive electrophiles, which readily undergo Michael addition to a multitude of intracellular nucleophiles, such as amino acids, glutathione (GSH), proteins, and nucleic acids (1). In vitro assays for the formation of quinone metabolites have routinely used excess sacrificial thiols, such as 2-mercaptoethanol, to aid detection (6-10). Such an approach is untenable in living systems, so in vivo exposure studies, both in the lab and in the field, have relied on assays of bile- or urine-excreted metabolites, * To whom correspondence should be addressed. Tel: (614)688-8799. E-mail: [email protected]. † The Ohio State University. ‡ Ecole Superieure de Chimie Organique et Minerale. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbons; TMAH, tetramethylammonium hydroxide; 1,4-NQ, 1,4-naphthoquinone; 1,2NQ, 1,2-naphthoquinone; BQ, 1,4-benzoquinone; AQ, 1,4-anthraquinone; BSA, bovine serum albumin; SIC, selective ion chromatogram; Me-S-Np, thioether-1,4-dimethoxynaphthalene; Me-S-No, thioether1,2-dimethoxynaphthalene; Me2-N-Np, dimethylamine-1,4-dimethoxynaphthalene; Me-N-Np, methylamine-1,4-dimethoxynaphthalene; MeS-An, thioether-1,4-dimethoxyanthracene.

which represent only a small proportion of the total dose (11-14) or on adduction to known targets such as hemoglobin (15, 16). The former methods are, at best, an indirect means of evaluating the extent of metabolite formation, and the latter suffer from low product yields. Therefore, to create a sensitive bioassay for exposure of living systems to PAHs, we have developed a procedure to detect the major products of PAH biotransformationss regardless of the extent of adduction to the enormous variety of biological nucleophiles. We here report our discovery of a technique having great potential in this regard. TMAH thermochemolysissa technique developed initially as pyrolysis with in situ methylationshas been used in environmental analysis to aid in identifying the structural components of large biopolymers (17-21). In this method, TMAH is used to depolymerize complex macromolecular environmental compounds and simultaneously methylate the polar monomeric products to form less polar derivatives, making them more amenable to detection via GC/MS. We and others have demonstrated that TMAH thermochemolysis can cleave peptide bonds (22-25) and have established a library of TMAH thermochemolysis products from the 20 naturally occurring amino acids, as well as from several of the more common fatty acids (26). TMAH thermochemolysis GC/ MS also possesses an inherent ability to cleave adducted molecules from conjugated macromolecules such as humic substances and plant biopolymers (27), which makes this process suitable for the detection of PAH metabolites.

10.1021/tx0341512 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/25/2003

PAH Michael Adduction Formation via TMAH GC/MS Scheme 1. Metabolic Activation of PAH (4)

This transformation should permit the detection of reactive PAH metabolites, quinones, that have formed Michael adducts with a variety of biological nucleophiles. Furthermore, the induced cleavage should retain the nucleophilic atom from the Michael addition reaction on the metabolite. Thus, the PAH metabolites detected via TMAH thermochemolysis should carry with them the signature of having undergone adduction reactions. To demonstrate the usefulness of this method as a qualitative assay tool, we have undertaken the detection of Michael addition products of the amino acid L-cysteine (Cys) to BQ and the model PAH quinones 1,4-NQ, 1,2NQ, and AQ. It is important to point out that while the term “PAH” is frequently used to describe larger, four or five ring compounds, the use of two and three ring aromatic hydrocarbons can serve as model members of the PAH class of compounds. Additional reactions were carried out with 1,4-NQ to demonstrate the utility of this assay for identification of adducts with larger molecules, such as proteinssin this case, using the tripeptide GSH and the 66 kDa protein BSA. Together, these experiments provide a qualitative basis for the development of the TMAH thermochemolysis method as a viable assay for PAH quinone formation.

Materials and Methods Caution: This work involved reactions with PAH quinones; therefore, the NIH guidelines for the Laboratory Use of Chemical Carcinogens were followed. Sodium hydroxide, L-Cys, BSA, and TMAH 25% (w/w) solution in methanol were purchased from Sigma. N-Tetracosane and AQ were purchased from Lancaster Synthesis, Inc. Diethyl ether, acetone, and ethyl acetate were obtained from Fisher

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1485 Chemicals. Ethanol was purchased from Pharmco. The reduced free acid form of GSH, BQ, 1,2-NQ, and 1,4-NQ were obtained from Aldrich Chemicals. Reaction of Cys with Quinones. The syntheses of the Cysquinone adducts used in this study were modified from the method of Thornton et al. (28). Cys (60 mM) was placed in a 40% (v/v) ethanol/water solution. The appropriate quinonesBQ, 1,4-NQ, 1,2-NQ, or AQswas dissolved to a final concentration of 120 mM in a 95% (v/v) ethanol/water solution, and the Cys mixture was added slowly while stirring. The resulting mixture was allowed to react overnight on an orbital shaker at 250 rpm at room temperature. The solution slowly changed from yellow to greenish brown. The reaction mixture was then cooled to 4 °C for 10 h before filtration. The precipitate was collected, washed with acetone, and allowed to air dry. Products were identified by electrospray ionization MS, using a 3 Tesla ion cyclotron resonance mass spectrometer housed in the Chemistry Department Mass Spectrometry Facility. This system is interfaced to an Analytica electrospray ionization source. Reaction of GSH with 1,4-NQ. A solution of GSH (43 mM) was made up in 60% (v/v) ethanol/water and then added to an 87 mM solution of 1,4-NQ in 95% (v/v) ethanol/water. The mixture was kept at room temperature overnight and then cooled to 4 °C for 10 h. Precipitate formed and was isolated by Bu¨chner filtration. The crystals were washed with acetone, redissolved in water, and then reprecipitated in acetone to produce fine, off-white crystals, representing a purified adduct. This adduct was also identified by electrospray ionization MS. Reaction of BSA with 1,4-NQ. BSA was dissolved in deionized water to a concentration of 16.7 mg/mL, added to a 0.0658 M solution of 1,4-NQ in 90% (v/v) ethanol/water, and mixed on the orbital shaker (200 rpm) for 24 h at room temperature. Acetone was added at three times the reaction volume, and the mixture was cooled to -20 °C overnight. The reaction product was centrifuged at 15000 rpm for 5 min, washed once with cold acetone, and then dried under a nitrogen stream. TMAH Thermochemolysis. Batch thermochemolysis in the presence of TMAH was carried out on all of the precipitated products as well as on each of the individual reactants using procedures similar to those described in McKinney et al. (29). Approximately 0.5 mg of product and 150 µL of TMAH (25% TMAH in methanol) were placed in Pyrex ampules and mixed by vortexing for 30 s. The samples were carefully dried under nitrogen, and vacuum-sealed prior to being baked at 250 °C for 30 min. These ampules were allowed to cool before cutting the glass seal and adding an internal standard, N-tetracosane (107 ng/µL). The sides of the ampule were washed with ethyl acetate (3 × 0.5 mL), and all washings were combined and concentrated under a stream of nitrogen. A 100 µL Hamilton syringe was used to measure the final volume of the extract. The samples were analyzed by capillary GC/MS on a HewlettPackard 6890 series GC system connected to a Pegasus II timeof-flight mass spectrometer (LECO corporation, MI). The column used for the GC separation was a 30 m × 0.25 mm (i.d.) fused silica capillary column with a 5% methylsilicone bonded phase, with a film thickness of 0.25 µm (SUPELCO DB-5). The samples (1 µL) were injected onto a split/splitless injector operating in the splitless mode. The column was run with constant flow (1.5 mL/min), and the temperature was ramped from an initial 50 to 200 °C, at 15 °C/min, and then to 300 °C at 25 °C/min, whereupon the temperature was held for 3 min before recycling. The ionization mode on the mass spectrometer was electron impact at 70 eV. Data acquisition and analysis were performed by use of the LECO Pegasus II version 1.33 software. Computational Methods. We employed molecular modeling to aid in the identification of isomeric products obtained from TMAH thermochemolysis. Full geometry optimization of a variety of initial starting geometries was performed at both the B3LYP and the MP2 methods using the 6-31G* basis set. All optimized geometries were characterized as true minima on the potential energy surface by vibrational frequency calculations.

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Figure 1. GC/MS total ion chromatogram of TMAH thermochemolysis products of the 1,4-NQ-Cys adduct. Peaks labeled with A are adduct-derived peaks; the remaining peaks are reactant peaks (IS, internal standard). Peak labels correspond to labels in Table 1. Energies were further refined at the B3LYP method using a more flexible basis set, 6-311+G**. All calculations were performed with Gaussian 98 (30) at the Ohio Supercomputer Center.

Results 1,4-NQ-Cys Adduct. The reaction of 1,4-NQ with Cys produced a dark green solid, which precipitated from the reaction mixture. Initial attempts to make a positive identification of the product were hampered by the low solubility of the 1,4-NQ-Cys adduct in most solvents amenable to electrospray ionization MS or NMR analysis. The product was finally solubilized in a solution of 1:1:1 THF:methanol:H2O with 3% acetic acid. In this solvent system, the product was stable for 30 min to an hour before slowly oxidizing and was able to be identified by electrospray ionization MS. The mass observed (m/z) was 322.01080 amu, corresponding to (M + 2Na - H)+. Additional confirmation of the identity of the adduct was attempted by 1H NMR, in deuterated Me2SO. However, the 1,4-NQ-Cys adduct also slowly oxidized in Me2SO, perhaps forming a multitude of additional adducts and limiting the applicability of this technique. After verification by MS, the 1,4-NQ-Cys adduct was subjected to TMAH thermochemolysis. A typical total ion chromatogram for the TMAH products of this adduct is shown in Figure 1, with adduct peak identifications listed in Table 1. Many of the peaks in the 1,4-NQ-Cys TMAH chromatogram were also observed in TMAH GC/MS control runs performed on the two reactants, 1,4-NQ and Cys. Peaks corresponding to TMAH reaction products of

Briggs et al.

the 1,4-NQ-Cys adduct are listed in Table 1. Several of these compounds are of special interest in the identification of 1,4-NQ adducts. The most important of these is peak A6, which is identified based on mass spectral fragmentation and which represents Me-S-Np. The mass spectrum of this peak (Figure 2A) shows characteristic ion intensities at m/z 234, 219, and 187. Using this ion signature, we were able to use the SIC to observe the presence of the Me-S-Np adduct fragment in more complicated systems. Three compounds originating from the TMAH thermochemolysis of the 1,4-NQ-Cys adduct appear to be nitrogen-substituted 1,4-NQ products: peaks A1, A2, and A5 in Figure 1. None of these three compounds are observed in the TMAH reaction with 1,4-NQ alone, leading to the conclusion that they derive from the 1,4NQ-Cys adduct, rather than an artifact due to the TMAH process. Peak A2 is Me2-N-Np, generated by the 1,4-NQ-Cys adduct bonded through the amine group of Cys. The mass spectrum of this compound (Figure 2B) has characteristic ion intensities at m/z of 231 and 216. Two other products (peaks A1 and A5) share virtually identical mass spectra, which show prominent intensities at m/z 217 and 202. These peaks could be positional isomers of the Me-N-Np. The area under peaks A1 and A5 has a relative ratio of one to five, which implies that peak A5 is the more stable isomer. While characteristic mass spectrometric peak intensities reveal the formation of the monomethylamine products, distinctions may not be clearly drawn between these two key isomeric products. Because Michael reactions typically occur under thermodynamic control, we have therefore sought to estimate the thermodynamic stabilities by computational methods. To differentiate between two possible positional isomers, we have estimated the relative energies using standard ab initio and density functional theory calculations (3134). Computed relative energies of fully optimized geometries of the two methylamine products at the B3LYP and MP2 methods with the 6-31G* basis set indicate that the 2-methylamine isomer is more stable than the 8-methylamine isomer (Table 2). The isomeric energy difference between the two isomers is predicted to be 1.4 kcal/mol at the B3LYP/6-311+G**//B3LYP/6-31G* level (Table 3). The thermodynamic stability of the 2-methylamine isomer is five times larger than that of the 8-methylamine isomer, which suggests that the observed peaks A1 and A5 correspond to 8-methylamine and 2-methylamine products, respectively (Figure 3). In addition to adducts with nitrogen and sulfur, 1,4NQ also forms adducts, albeit more slowly, to compounds with oxygen-containing functional groups (6). Thus, we were not surprised when we found evidence of adduction to the carboxyl group of Cys in our 1,4-NQ-Cys reaction. This finding is based on the peak representing trimethoxynaphthalene (peak A3 in Table 1). However, this peak was also evident in the TMAH reaction of 1,4-NQ alone, although in lower apparent amounts. Methanol is present in the TMAH thermochemolysis reaction and may lead to the formation of the trimethoxynaphthalene product observed in the thermochemolysis of 1,4-NQ alone. It is therefore difficult to differentiate between the trimethoxynaphthalene formed from 1,4-NQ alone and that formed by 1,4-NQ adduction to the carboxyl group of Cys. The rate of reaction of 1,4-NQ with oxygencontaining functional groups is up to 3 orders of magni-

PAH Michael Adduction Formation via TMAH GC/MS

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Table 1. Identified Peaks from 1,4-NQ-Cys Adduct TMAH Chromatogram in Figure 1

tude slower than with nitrogen-containing functional groupssand up to 9 orders of magnitude slower than with sulfur-containing groups (6)sthus, it is likely that 1,4NQ adduction to oxygen is of less importance in vivo. Nevertheless, further research is currently underway to determine the contribution of methanol to the formation of the trimethoxynaphthalene product. 1,4-NQ will often undergo autoxidation following an addition reaction, causing the reactive quinone form to reestablish, and a second Michael addition reaction can take place. Two of the compounds generated by the TMAH thermochemolysis of the 1,4-NQ-Cys adduct appear to represent the adduction of both the thiol and the amine groups of Cys to 1,4-NQ. The presence of the combined methylamine and thioether product from TMAH thermochemolysis, in the absence of the dithioether product, leads to the conclusion that the Michael reaction of 1,4-NQ with Cys involves formation of an adduct by bonding at both the sulfur and the nitrogen atoms of Cys (Scheme 2). Such binding has been observed previously for the addition of the sulfur and oxygen in 2-mercaptoethanol to 1,4-NQ (8), and the ability of Cys to form a six-membered, doubly conjugated ring with 1,4-NQ suggests that similar chemistry is taking place in this system. The two compounds (peaks A9 and A10 in Figure 1) are not major reaction products and differ only by degree of methylationspotentially due to steric hindrance on the doubly adducted quinone. The mass spectrum of peak A9 contains peaks at m/z 263, 248, and 233. Peak

A9 was identified as the monomethylamine and thioether-substituted 1,4-dimethoxynaphthalene. Peak A10 shows MS peaks at m/z 249 and 234 and is believed, based on mass spectral fragmentation, to represent the amine and thioether-substituted 1,4-dimethoxynaphthalene. The goal of the present study was to establish qualitative evidence that this assay technique could be used to detect quinone adducts. An accurate quantification of this assay requires a surrogate of the product of interest, which can be used to find the response factor of the desired compound. Through the use of internal standards, we can approximate the method’s sensitivity as 3.9 × 106 area units/µmol of quinone product, with a detection limit of less than 150 pmol of product based on current protocols. Initial studies with Sprague-Dawley rat liver microsomal conversion of naphthalene (data not shown) have also suggested that adduct concentrations in the 0.003 µmol of adduct/mg protein range are easily detected. It should be noted that all of these experiments used minimal amounts of starting material (approximately 0.5 mg of sample) and scaling up the initial sample ought to expand the ability of this assay to detect even very minute amounts of adduct expected from biological samples. Cys Adducts with Other Quinones. To demonstrate the versatility of the TMAH thermochemolysis assay for quinone adducts, further experiments were carried out with other commercially available quinones, including


Briggs et al.

Figure 3. Isomeric structures of Me-N-Np. (A) N-Methyl-1,4dimethoxy-8-naphthylamine (peak A1); (B) N-Methyl-1,4dimethoxy-2-naphthylamine (peak A5). Table 3. Key Geometrical Parameters of Methylamine Adduct Isomers Obtained at the B3LYP/6-31G* Level

Figure 2. SICs, mass spectra (inset), and structure of 1,4-NQCys adduct peaks after TMAH thermochemolysis. (A) Me-S-Np (peak A6), the peak at 801 s is due to the presence of a m/z fragment in peak A10; (B) Me2-N-Np (peak A2). Table 2. Relative Energies (kcal/mol) of Methylamine Adduct Isomers B3LYP 6-31G* isomera

∆E

2 8

0.00 0.98

6-311+G**

∆H298K ∆G298K 0.00 0.97

MP2

0.00 1.26

∆E 0.00 1.38

∆H298K ∆G298K 0.00 1.37

0.00 1.66

6-31G* ∆E 0.00 0.69

a Isomers 2 and 8 are N-methyl-1,4-dimethoxy-2-naphthylamine and N-methyl-1,4-dimethoxy-8-naphthylamine, respectively. The computed equilibrium constant is 0.12 based on the ∆G298K values obtained at the B3LYP/6-31G* level.

AQ, BQ, and 1,2-NQ. The dark product formed from the reaction of BQ with Cys was identified by electrospray ionization MS, again using the 1:1:1 THF:methanol:H2O, NaCl solvent system. The observed mass (m/z) 371.03423 amu ((1.0 ppm) corresponds to the (M + Na)+ peak. This BQ-Cys adduct was also subjected to TMAH thermochemolysis GC/MS, and the resulting adduct peak is shown in Figure 4. The primary adduct peak corresponded to the methylamine, thioether-substituted product, formed by the sequential addition of the thiol and then the amine groups of Cys to BQ. Interestingly, this product was derived from the quinone, and not the hydroquinone, form of the adduct. After Michael adduction, excess free quinone can reoxidize the hydroquinone adduct (4). Analogous product peaks were observed in the TMAH chromatogram of the 1,4-NQ-Cys adduct

(Figure 1, peaks A7 and A8). The quinone form is the major product from the BQ-Cys adduct, possibly due to the greater solubility of BQ in the reaction mixture. Greater solubility would lead to a higher actual excess of BQ at any given point during the 1,4-Michael addition reaction, thus facilitating more complete reoxidation. The reaction with AQ and Cys resulted in a dark, green-black product, which was also identified by electrospray ionization MS. Similar to the problems encountered with the 1,4-NQ-Cys adduct, this product also proved difficult to solubilize, with only a small amount of the product dissolving in the 1:1:1 THF:methanol:H2O, NaCl solution. The mass observed for this adduct was 469.10745 amu ((84 ppm), corresponding to the (M + Na)+ ion. The electrospray ionization MS analysis suggested that the AQ was adducted to Cys at only one location. After TMAH thermochemolysis, a peak corresponding to the Me-S-An was observed (Figure 5A). The dimethylamine-substituted product was observed but only in trace amounts. The reoxidized quinone form of this adduct was not observed, possibly due to the low solubility of AQ in the aqueous ethanol reaction mixture. The major product of the dihydrodiol dehydrogenase activation pathway for PAHs, as shown in Scheme 1, is expected to be the ortho-quinone derivative. For this reason, we also conducted experiments with 1,2-NQ and Cys, which resulted in a light tan product. The TMAH thermochemolysis of the 1,2-NQ-Cys adduct produced



Scheme 2. Proposed Steps in the Formation of the Doubly Adducted 1,4-NQ-Cys Product

Figure 4. SIC, mass spectrum (inset), and structure of BQCys adduct peaks.

peaks, similar in molecular weight to the 1,4-NQ-Cys adduct peaks. The major peak of interest was that of the Me-S-No product (Figure 5B). This peak shows similar spectra, and virtually identical retention time, as the MeS-Np product derived from the para-quinone, 1,4-NQ (Figure 2A). The difference in the spectra is expected to be due to the different fragmentation patterns seen in the NIST library for 1,2-NQ and 1,4-NQ. Note also that the signal-to-noise ratio is not as good as depicted in Figure 2A. This is because the analysis was performed at or near the detection limit of 150 pmol of the adduct. 1,4-NQ-GSH Adduct. Additional reactions were carried out between 1,4-NQ and larger peptides to demonstrate that the TMAH assay technique is applicable to PAH adduction to larger, more complicated molecules. The tripeptide GSH was selected because of its known ability to bind to PAH quinones (2). The solid adduct was readily soluble in water and was identified by electrospray ionization MS. The observed masses (m/z) were 464.11177, which corresponds to the (M + H)+ peak, and at m/z 486.09485, which corresponds to the (M + Na)+ peak. SICs for the TMAH thermochemolysis products of the GSH-1,4-NQ adduct are shown in Figure 6. Both the Me2-N-Np (Figure 6A) and the Me-S-Np (Figure 6B) products were observed. In both cases, two peaks (*) with different GC retention times but virtually identical mass spectra were observed, probably corresponding to the two possible positional isomers. 1,4-NQ-BSA Adducts. To investigate the value of this assay as an in vivo bioassay, the TMAH thermochemolysis procedure was applied to the 1,4-NQ adduct formed with the soluble protein BSA. There has been some speculation in the literature that nucleophilic

Figure 5. SIC, mass spectrum (inset), and structure of (A) MeS-An and (B) Me-S-No after TMAH thermochemolysis.

groups on BSA may react with 1,4-NQ (35). The Me2-NNp product was observed, and the SIC for this product (m/z ) 231) is shown in Figure 7. It should be noted that in the total ion chromatogram (data not shown), the peaks corresponding to the amino acid breakdown products after TMAH treatment primarily have retention times in the 200-600 s range, while the Me2-N-Np peak eluted at 820 s, so the analyte peak is separated in time from the background contribution of unreacted protein. Although BSA is reported to have one free thiol group (Cys 34), this Cys residue is often oxidized during protein purification (36), and no Me-S-Np was observed after the TMAH process. The detection of the Me2-N-Np peak,


Briggs et al. Scheme 3. Proposed Reaction Scheme for TMAH Thermochemolysis of PAH Quinone Adducts

Figure 6. SICs of TMAH thermochemolysis products of the 1,4-NQ-GSH adduct. (A) Me2-N-Np and (B) Me-S-Np.

Figure 7. SIC of TMAH thermochemolysis products from the 1,4-NQ-BSA adduct, showing the Me2-N-Np product.

however, demonstrates the ability of the TMAH process to detect adducts between PAH and whole proteins.

Discussion The reaction of the quinone adducts with TMAH cleaves a quinone-derived fragment from the conjugated biomolecule and methylates the resulting hydroxyl, thiol, or amino groups (Scheme 3). Thus, this assay creates fragments that are amenable to GC/MS analysis. By creating a reference library of PAH quinone adduct products, identification of the thioether and methylamine adducts was possible. GC parameters described in this study were developed to optimize separation of the quinone-adduct peaks from remnants of proteinaceous material, reducing the background signal. TMAH ther-

mochemolysis therefore allows simultaneous detection of thiol and amine adducts of PAH quinones without requiring separation from extraneous cellular material prior to analysis. One important finding of this work is the ability of this assay to identify quinone adducts through nitrogen functional groups. While the Michael addition of PAH quinones to the sulfhydryl group of GSH is well-known, these nitrogen adducts have received little attention in the literature (5, 37, 38). Early studies of the ability of PAH quinones to react with potential nucleophiles established initial rates of reaction with sulfhydryls that were 5 orders of magnitude greater than those for glycine (6). It is clear from these studies that addition by thiols is strongly kinetically favored. As PAH exposure is known to deplete intracellular GSH (1, 3), further exposure is likely to increase reaction with the much more abundant intracellular amine group associated with various biochemical molecules. The speed and sensitivity of the TMAH thermochemolysis assay are a significant advance over existing PAH exposure assays. In vivo studies on PAH exposure and bioaccumulation have tended to focus on select metabolites or specific adducts present in only trace amounts, reducing the inherent sensitivity of detection. Commonly used assay techniques include HPLC and LC/ MS detection of the monohydroxy forms of PAHs, such as 1-hydroxypyrene, in bile or urine (11, 12); identification of glutathion-S-yl derivatives (2, 39, 40); and the identification of whole protein adducts (15, 16, 41) or hydrolyzed protein fragments (42). The difficulty of using these techniques, however, is that in each case the analyte molecule represents only a trace portion of the total dose. In one study in which hydroquinone was administered to male Sprague-Dawley rats, the combined total of all mono-, di-, tri-, and tetra-glutathion-Syl derivatives detected represented only 4% of the total dose (2). In other studies, 17β-estradiolsan ortho-quinone form of estrogen, which shares much structural similarity to the PAH quinonesshas been shown to form adducts with GSH and Cys in human liver homogenates (43), but these conjugates have not been found in human urine, suggesting that the thioether conjugates may be metabolized to products that become bound to tissue macromolecules (13). Similarly, in studies with hamsters, only 11.7% of the total dose of 17β-estradiol is recovered as the GSH conjugate (44). In all of these cases, the reliance of the measurement technique on only defined total


adducts puts an inherent limitation on the sensitivity of the assay technique. One previous technique that has shown promise is the alkaline permethylation technique developed by Slaughter and Hanzlik, which is specific for the identification of quinone-thiol adducts (45). In their assay, purified proteins containing potential sulfhydryl adducts to benzoquinones are converted to thioether fragments for analysis. The alkaline permethylation technique has been used successfully for in vivo detection of sulfhydryl quinone conjugates with hydroquinone and substituted benzoquinones (40, 41, 46, 47). While the technique avoids the sensitivity limitations of the single adduct assays, the gain comes at a cost. It is a multistep process taking several days and requires a large amount of starting material (20-200 mg) and protein purification prior to analysis. Additionally, this technique does not show the amine adducts, which are easily detected by the TMAH thermochemolysis technique. In conclusion, TMAH thermochemolysis with GC/MS provides a rapid and potentially highly sensitive assay for PAH quinone Michael adduct formation. As an assay for PAH exposure, TMAH thermochemolysis is a significant improvement over existing techniques that sample only a subset of potential PAH metabolites. Instead of using multiple assays to detect quinone adducts with biological macromolecules, our technique permits simultaneous detection of all thiol and amine adducts. Work is currently underway to develop TMAH thermochemolysis as a quantitative bioassay for PAH exposure, for which this technique shows great promise.


(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

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Acknowledgment. This study was supported by the National Science Foundation (CHE-0089147) and at the Ohio Supercomputer Center with computational resources. We thank Beena Thomas and Sunghwan Kim for their valuable help in starting the project, Rakesh Sachdeva for continuous technical support, and Sarah Pilkenton and William Hockaday for their helpful comments on the text. We are also indebted to the two anonymous reviewers whose comments significantly added to this manuscript.

References (1) Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000) Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135-160. (2) Hill, B. A., Kleiner, H., Ryan, E. A., Dulik, D. M., Monks, T. J., and Lau, S. S. (1993) Identification of multi-S-substituted conjugates of hydroquinone by HPLC-coulometric electrode array analysis and mass spectroscopy. Chem. Res. Toxicol. 6, 459-469. (3) Monks, T. J., and Lau, S. S. (1997) Biological reactivity of polyphenolic-glutathione conjugates. Chem. Res. Toxicol. 10, 1296-1313. (4) Penning, T. M., Burczynski, M. E., Hung, C.-F., McCoull, K. D., Palackal, N. T., and Tsuruda, L. S. (1999) Dihydrodiol dehydrogenases and polycyclic aromatic hydrocarbon activation: Generation of reactive and redox active o-quinones. Chem. Res. Toxicol. 12, 1-18. (5) Schweigert, N., Zehnder, A. J. B., and Eggen, R. I. L. (2001) Chemical properties of catechols and their molecular modes of toxic action in cells, from microorganisms to mammals. Environ. Microbiol. 3, 81-91. (6) Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) Spectroscopic identification of ortho-quinones as the products of polycyclic aromatic trans-dihydrodiol oxidation catalyzed by dihydrodiol dehydrogenasesa potential route of proximate carcinogen metabolism. J. Biol. Chem. 263, 1814-1820. (7) Rogan, E. G., Devanesan, P. D., RamaKrishna, N. V. S., Higginbotham, S., Padmavathi, N. S., Chapman, K., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1993) Identification

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin. Chem. Res. Toxicol. 6, 356-363. Sugiyama, K., Wang, T.-C. L., Simpson, J. T., Rodriguez, L., Kador, P. F., and Sato, S. (1999) Aldose reductase catalyzes the oxidation of naphthalene-1,2-dihydrodiol for the formation of ortho-naphthoquinone. Drug Metab. Dispos. 27, 60-67. Sato, S., Sugiyama, K., Lee, Y. S., and Kador, P. F. (1999) Prevention of naphthalene-1,2-dihydrodiol-induced lens protein modifications by structurally diverse aldose reductase inhibitors. Exp. Eye Res. 68, 601-608. Palackal, N. T., Burczynski, M. E., Harvey, R. G., and Penning, T. M. (2001) The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: Potential role in polycyclic aromatic hydrocarbon activation. Biochem. J. 40, 10901-10910. van der Oost, R., van Schooten, F.-J., Ariese, F., Heida, H., Satumalay, K., and Vermeulen, N. P. E. (1994) Bioaccumulation, biotransformation, and DNA binding of PAHs in feral eel (Anguilla anguilla) exposed to polluted sediments: A field survey. Environ. Toxicol. Chem. 13, 859-870. Ruddock, P. J., Bird, D. J., and McCalley, D. V. (2002) Bile metabolites of polycyclic aromatic hydrocarbons in three species of fish from the Severn estuary. Ecotoxicol. Environ. Saf. 51, 97105. Elce, J. S., Bird, C. F., and Chandra, J. (1973) Water soluble products of the estrogens. Bile metabolites of 2-hydroxyestradiol17β in man. J. Clin. Endocrinol. Metab. 36, 1027-1030. C ˇ erna´, M., Pastorkova´, A., Myers, S. R., Ro¨ssner, P., and Binkova´, B. (1997) The use of a urine mutagenicity assay in the monitoring of environmental exposure to genotoxins. Mutat. Res. 391, 99110. Myers, S. R., Spinnato, J. A., and Pinorini, M. T. (1996) Chromatographic characterization of hemoglobin benzo[a]pyrene-7,8diol-9,10-epoxide adducts. Fundam. Appl. Toxicol. 29, 94-101. Myers, S. R., and Pinorini, M. T. (2000) Hemoglobin adducts of benzo[a]pyrene in tobacco smokers: Characterization of benzo[a]pyrene adducts in maternal and fetal blood samples. Polycyclic Aromat. Compd. 21, 167-186. Challinor, J. M. (1989) A pyrolysis-derivatization-gas chromatograph technique for the elucidation of some synthetic polymers. J. Anal. Appl. Pyrolysis 16, 323-333. de Leeuw, J. W., and Baas, M. (1993) The behaviour of esters in the presence of tetramethylammonium salts at elevated temperatures, flash pyrolysis or flash chemolysis. J. Anal. Appl. Pyrolysis 26, 175-184. Saiz-Jimenez, C. (1994) Analytical pyrolysis of humic substances: Pitfalls, limitations, and possible solutions. Environ. Sci. Technol. 28, 1773-1778. Filley, T. R., Hatcher, P. G., Shortle, W. C., and Praseuth, R. T. (2000) The application of C-13-labeled tetramethylammonium hydroxide (C-13-TMAH) thermochemolysis to the study of fungal degradation of wood. Org. Geochem. 31, 181-198. Knicker, H., del Rio, J. C., Hatcher, P. G., and Minard, R. D. (2001) Identification of protein remnants in insoluble geopolymers using TMAH thermochemolysis/GC-MS. Org. Geochem. 32, 397-409. Abbas-Hawks, C., Voorhees, K. J., and Hadfield, T. L. (1993) In situ methylation of nucleic acids using pyrolysis mass spectrometry. Rapid Commun. Mass Spectrom. 10, 1802-1806. Voorhees, K. J., Basile, F., Beverly, M. B., Abbas-Hawks, C., Hendricker, A., Cody, R. B., and Hadfield, T. L. (1997) The use of biomarker compounds for the identification of bacteria by pyrolysis mass spectrometry. J. Anal. Appl. Pyrolysis 40, 111134. Knicker, H., and Hatcher, P. G. (1997) Survival of protein in an organic-rich sediment: Possible protection by encapsulation in organic matter. Naturwissenschaften 84, 231-234. Zang, X., van Heemst, J. D. H., Dria, K. J., and Hatcher, P. G. (2000) Encapsulation of protein in humic acid from a histosol as an explanation for the occurrence of organic nitrogen in soil and sediment. Org. Geochem. 31, 679-695. Zang, X., Brown, J. C., van Heemst, J. D. H., Palumbo, A., and Hatcher, P. G. (2001) Characterization of amino acids and proteinaceous materials using online tetramethylammonium hydroxide (TMAH) thermochemolysis and gas chromatographymass spectrometry technique. J. Anal. Appl. Pyrolysis 61, 181193. del Rio, J. C., and Hatcher, P. G. (1998) Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography mass spectrometry. Org. Geochem. 29, 1441-1451.

1492 Chem. Res. Toxicol., Vol. 16, No. 11, 2003 (28) Thornton, D. E., Jones, K. H., Jiang, Z., Zhang, H., Liu, G., and Cornwell, D. G. (1995) Antioxidant and cytotoxic tocopheryl quinones in normal and cancer cells. Free Radical Biol. Med. 18, 963-976. (29) McKinney, D. E., Bortiatynski, J. M., Carson, D. M., Clifford, D. J., deLeeuw, J. W., and Hatcher, P. G. (1996) Tetramethylammonium hydroxide (TMAH) thermochemolysis of the aliphatic biopolymer cutan: Insights into the chemical structure. Org. Geochem. 24, 641-650. (30) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98; Gaussian, Inc.: Pittsburgh, PA, 1998. (31) Hehre, W. J., Radom, L., Schleyer, P. v. R., and Pople, J. A. Ab Initio Molecular Orbital Theory; Wiley: Pittsburgh, PA, 1986. (32) Becke, A. D. (1988) Density-functional exchange-energy approximation with correct asymptotic-behavior. Phys. Rev. A: At., Mol., Opt. Phys. 38, 3098-3100. (33) Becke, A. D. (1993) Density-functional thermochemistry 0.3. The role of exact exchange. J. Chem. Phys. 98, 5648-5652. (34) Lee, C. T., Yang, W. T., and Parr, R. G. (1988) Development of the Colle-Salvetti correlation-energy formula into a functional of the electron-density. Phys. Rev. B: Condens. Matter Mater. Phys. 37, 785-789. (35) Shen, L., Pisha, E., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J. L. (1997) Bioreductive activation of catechol estrogen-ortho-quinones: Aromatization of the B ring in 4-hydroxyequilenin markedly alters quinoid formation and reactivity. Carcinogenesis 18, 1093-1101. (36) Andersson, L.-O. (1966) The heterogeneity of bovine serum albumin. Biochim. Biophys. Acta 117, 115-133. (37) Murty, V. S., and Penning, T. M. (1992) Polycyclic aromatic hydrocarbon (PAH) ortho-quinone conjugate chemistry: Kinetics

Briggs et al.

(38) (39)

(40) (41) (42)

(43)

(44)

(45)

(46)

(47)

of thiol addition to PAH ortho-quinones and structuers of thioether adducts of nasphthalene-1,2-dione. Chem.-Biol. Interact. 84, 169-188. Gottlieb, A. J., and Wurzel, H. A. (1974) Protein-quinone interaction: in vitro induction of indirect antiblobulin reactions with methylDOPA. Blood 43, 85-97. Lunte, S. M., and Kissinger, P. T. (1983) Detection and identification of sulfhydryl conjugates of p-benxoquinone in microsomal incubations of benzene and phenol. Chem.-Biol. Interact. 47, 195212. Thompson, C. B. (1995) Apoptosis in the treatment and pathogenesis of disease. Science 267, 1456-1462. Mason, D. E., and Liebler, D. C. (2000) Characterization of benzoquinonespeptide adducts by electrospray mass spectrometry. Chem. Res. Toxicol. 13, 976-982. Izzotti, A., Balansky, R. M., D’Agostini, F., Bennicelli, C., Myers, S. R., Grubbs, C. J., Lubet, R. A., Kelloff, G. J., and De Flora, S. (2001) Modulation of biomarkers by chemopreventive agents in smoke-exposed rats. Cancer Res. 61, 2472-2479. Ball, P., Favthmann, E., and Knuppen, R. (1976) Comparative studies on the metabolism of estradiol-17β and 2-hydroxyextradiol-17β in man in vitro and in vivo. J. Steroid Biochem. 1, 139143. Butterworth, M., Lau, S. S., and Monks, T. J. (1997) Metabolism and mild nephrotoxicity of 17β-estradiol in the golden Syrian hamster. Formation of catechol estrogen glutathione conjugates and implications for 17β-estradiol-mediated nephrocarcinogenicity. Carcinogenesis 18, 561-567. Slaughter, D. E., Zheng, J., Harriman, S., and Hanzlik, R. P. (1993) Identification of Covalent Adducts to Protein Sulfur Nucleophiles by Alkaline Permethylation. Anal. Biochem. 208, 288-295. Boatman, R. J., English, J. C, Perry, L. G., and Fiorica, L. A. (2000) Covalent protein Adducts of Hydroquinone in Tissues from Rats: Identifiation and Quantitation of Sulfhydryl-Bound Forms. Chem. Res. Toxicol. 13, 853-860. Boatman, R. J., English, J. C., Perry, L. G., and Fiorica, L. A. (2000) Covalent protein adducts of hydroquinone in tissues from rats: Quantitation of sulfhydryl-bound forms following single gavage or intraperitoneal administration or repetitive gavage administration. Chem. Res. Toxicol. 13, 861-872.

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