Chemico-Biological Interactions 141 (2002) 189– 210

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Measurement of hemoglobin and albumin adducts of naphthalene-1,2-oxide, 1,2-naphthoquinone and 1,4-naphthoquinone after administration of naphthalene to F344 rats Suramya Waidyanatha a, Melissa A. Troester a, Andrew B. Lindstrom b, Stephen M. Rappaport a,* a

Department of En6ironmental Sciences and Engineering, CB 7400, School of Public Health, Uni6ersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599 -7400, USA b National Exposure Research Laboratory, United States En6ironmental Protection Agency, Mail Drop D205-56 Research Triangle Park, NC 27711, USA Received 17 April 2002; received in revised form 2 June 2002; accepted 4 June 2002

Abstract Naphthalene-1,2-oxide (NPO), 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are the major metabolites of naphthalene that are thought to be responsible for

Abbre6iations: Alb, albumin; 1,2-NPQ-Alb and 1,4-NPQ-Alb, adducts resulting from reaction of 1,2and 1,4-NPQ with cysteinyl residues in Alb; NPO1-Alb and NPO2-Alb, adducts resulting from reaction of NPO with cysteinyl residues in Alb; 1,2-NPQ-Hb and 1,4-NPQ-Hb, adducts resulting from reaction of 1,2- and 1,4-NPQ with cysteinyl residues in Hb; NPO1-Hb and NPO2-Hb, adducts resulting from reaction of NPO with cysteinyl residues in Hb; 1,2-NPQ-NAC and 1,4-NPQ-NAC, reaction products of 1,2- and 1,4-NPQ with N-acetyl-L-cysteine; NPO1-NAC and NPO2-NAC, reaction products of NPO with N-acetyl-L-cysteine; 1,4-NPQ-S-TFA, trifluoroacetyl derivatives of 1,4-NPQ adduct after the MT assay; 1,2-NPQ-S-TFA, trifluoroacetyl derivative of 1,2-NPQ adduct after the MT assay; NPO1-S-TFA and NPO2-S-TFA, trifluoroacetyl derivatives of NPO1 and NPO2 adducts after the MT assay; Hb, hemoglobin; MT assay, methanesulfonic acid and trifluoroacetic anhydride assay; NAC, N-acetyl-L-cysteine; NPO, naphthalene-1,2-oxide; 1,2-NPQ, 1,2-naphthoquinone; 1,4-NPQ, 1,4-naphthoquinone; NICI, negative ion chemical ionization; SE, standard error; TFAA, trifluoroacetic anhydride. * Corresponding author. Tel.: + 1-919-966-5017; fax: +1-919-966-0521 E-mail address: [email protected] (S.M. Rappaport). 0009-2797/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 2 ) 0 0 0 4 8 - 0

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the cytotoxicity and genotoxicity of this chemical. We measured cysteinyl adducts of these metabolites in hemoglobin (Hb) and albumin (Alb) from F344 rats dosed with 100 –800 mg naphthalene per kg body weight. The method employs cleavage and derivatization of these adducts by trifluoroacetic anhydride and methanesulfonic acid followed by gas chromatography–mass spectrometry in negative ion chemical ionization mode. Cysteinyl adducts of both proteins with NPO, and 1,2- and 1,4-NPQ (designated NPO-Hb and -Alb, 1,2-NPQ-Hb and -Alb, and 1,4-NPQ-Hb and -Alb, respectively) were produced in a dose-dependent manner. Of the two structural isomers resulting from NPO, levels of NPO1 adducts were greater than those of NPO2 adducts in both Hb and Alb, indicating that aromatic substitution is favored in vivo at positions 1 over 2. Of the quinone adducts, 1,2-NPQ-Hb and -Alb were produced in greater quantities than 1,4-NPQ-Hb and -Alb, indicating either that the formation of 1,2-NPQ from NPO is favored or that more than one pathway leads to the formation of 1,2-NPQ. The shapes of the dose –response curves were generally nonlinear at doses above 200 mg naphthalene per kg body weight. However, the nature of nonlinearity differed, showing evidence of supralinearity for NPO-Hb, NPQ-Hb and NPQ-Alb and of sublinearity for NPO-Alb. Low background levels of 1,2-NPQ-Hb and -Alb and 1,4-NPQ-Hb and -Alb were detected in control animals without known exposure to naphthalene. However, the corresponding NPO-Hb and -Alb adducts were not detected in control animals. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hemoglobin adducts; Albumin adducts; Naphthalene

1. Introduction Naphthalene is an important industrial chemical used in the manufacture of synthetic leather tanning agents, resins, dyes, surfactants, phthalic anhydride and the insecticide, carbaryl [1]. Due to its crystalline nature and pleasant odor, small amounts of naphthalene have household uses, including mothballs and deodorants [1]. It is also a constituent of jet fuel [2,3], diesel fuel [4], cigarette smoke [5] and a by product of combustion, and hence is regarded as an ubiquitous environmental pollutant. In humans, acute exposure to naphthalene has been associated with cataracts [6,7] and hemolytic anemia [8]. However, little evidence is available regarding possible toxicity following chronic exposures in occupational or environmental settings. Naphthalene produces pulmonary alveolar/bronchiolar adenomas and necrosis of pulmonary bronchiolar epithelial cells in mice, which is the species most sensitive to the toxic effects of naphthalene [9–11]. In rats, naphthalene exposure produced nasal respiratory epithelial adenomas and olfactory epithelial neuroblastomas [12]. The species- and tissue-specific toxicity of naphthalene has been attributed to differences in metabolism [13,14]. As shown in Fig. 1, naphthalene is metabolized by cytochrome P450 isozymes (CYP 1A1, 1A2, 2A1, 2E1, 2F2) to naphthalene-1,2oxide (NPO) and subsequently to 1,4-naphthalenediol and 1,2-naphthalenediol [15,16]. These diols are oxidized, either enzymatically or nonenzymatically, to 1,4-naphthoquinone (1,4-NPQ) and 1,2-naphthoquinone (1,2-NPQ), respectively. In human lymphocytes and mononuclear leukocytes, cytotoxicity and genotoxicity of

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naphthalene has been associated with the NPQs but not with NPO in vitro [16,17]. However, all of these metabolites have demonstrated toxicity to mouse-lung Clara cells [17,18] and rat-lung L2 cells in vitro [19]. Since NPO and the NPQs are chemically reactive, indirect methods such as measurement of their adducts are needed to study their disposition in vivo. Covalent binding of total reactive metabolites to various tissues and proteins has been studied using radiochemical and immunochemical assays [20–23]. Alkaline permethylation of proteins followed by gas chromatography–mass spectrometry (GC – MS), first introduced by Slaughter and Hanzlik [24,25], has been used to measure specific cysteinyl adducts of naphthalene in vitro [17]. However, this assay has not been extended to in vivo studies, probably because of limited sensitivity. We have previously used cysteinyl adducts of hemoglobin (Hb) and albumin (Alb) to

Fig. 1. Proposed pathway for the metabolism of naphthalene.

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investigate the production of benzene oxide and the benzoquinones in humans and animals exposed to benzene [26– 28]. Here we report an extension of that method to quantitate cysteinyl adducts of the analogous naphthalene metabolites, NPO and 1,2- and 1,4-NPQ. The method was successfully applied to Hb and Alb from F344 rats dosed with 100– 800 mg naphthalene per kg body weight.

2. Methods

2.1. Chemicals Racemic NPO was kindly provided by Dr Alan Buckpitt from the University of California, Davis. N-Acetyl-S-1-naphthyl cysteine and N-acetyl-S-2-naphthyl cysteine were kindly provided by Dr Avram Gold from The University of North Carolina. N-Acetyl-L-cysteine (NAC), 1,2-NPQ (98%), 1,4-NPQ (97%) and desferoxamine were obtained from Sigma– Aldrich Inc. (St. Louis, MO). Methanesulfonic acid was from Fluka Chemical Company (Switzerland). [2H8]naphthalene was from Cambridge Isotope Laboratories (Woburn, MA). Ascorbic acid, hydrochloric acid (concentrated), acetone (nanograde), hexane (pesticide grade), methanol, and ethyl acetate were from Fisher Scientific (Pittsburgh, PA). Tri-sil® reagent, trifluoroacetic anhydride (TFAA) and trifluoroacetic acid were purchased from Pierce (Rockford, IL). TFAA was distilled once before use. Caution: TFAA reacts 6iolently with water and should only be used to deri6atize samples that are completely dry.

2.2. Animal experiments Fifteen male F344 rats (175– 200 g) were obtained from Charles River Breeding Laboratories (Raleigh, NC) and were housed in polycarbonate cages on a 12-h light/dark cycle for 2 weeks before use. Food and water were provided ad libitum. Rats were divided into five groups of three and each group received a single oral dose of naphthalene via corn oil gavage at 0 (corn oil), 100, 200, 400, or 800 mg per kg body weight. Twenty-four hours following dosing, rats were anesthetized with methoxyflurane and blood was removed by direct cardiac puncture into a heparinized syringe. (The metabolism appears to be complete within 24 h, because the maximum adduct levels were observed for the 24-h time point [29], in a concurrent time course study from 1 to 42 days). Approximately, 7–9 ml of blood were collected from each animal. Blood was stored immediately on ice. Plasma and red cell fractions were separated within 2 h and globin and Alb were isolated as previously reported [26].

2.3. Synthesis of isotopically-labeled protein bound internal standards A Fenton-type hydroxyl radical-generating system [30] was employed to synthesize deuterium-labeled internal standards from [2H8]naphthalene. One hundred

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milligrams of human Alb in 2 ml of deionized water was incubated with 100 mg of [2H8]naphthalene in 2 ml acetone in the presence of 1 mM iron(II) sulfate, 50 mM ascorbate and 100 mM H2O2. The reaction was allowed to proceed at 37 °C for 12–15 h with shaking and acetone was removed under a stream of nitrogen. After adding 5 ml of deionized water, unreacted [2H8]naphthalene was removed by extracting twice with 2 ml ethyl acetate. The aqueous phase was reduced to about 3 ml in vacuo and the product was partially purified by solid-phase microextraction using C18 Sep-Pak cartridges (Waters chromatography, Millipore Corporation, Milford, MA).

2.4. Synthesis and characterization of adducts of naphthalene oxide and the naphthoquinones with N-acetyl-L-cysteine Adducts were synthesized by reacting NPO and the NPQs with NAC. Standards of these prototype adducts were helpful in characterizing the analogous adducts resulting from reactions with protein sulfhydryl groups. Standards of NPO-bound NAC [N-acetyl-S-1-(1,2-dihydro-2-hydroxynaphthyl)cysteine (NPO1-NAC) and Nacetyl-S-2-(1,2-dihydro-1-hydroxynaphthyl)cysteine (NPO2-NAC)] were synthesized following procedures of Marco et al. [31] and Buonarati et al. [32]. A solution of 76 mg (0.54 mmol) of a mixture of racemic NPO in 3 ml of tetrahydrofuran was added dropwise to an ice-cold solution of 163 mg (1 mmol) of NAC in 1.5 ml of degassed 1 N NaOH. The reaction was allowed to proceed for 3 h at room temperature with continuous stirring under nitrogen. The mixture was neutralized with 1% acetic acid and evaporated in vacuo and the residue was reconstituted in 5 ml of deionized water for purification by reverse-phase HPLC as described below. Standards of 1,2-NPQ-bound NAC [(N-acetyl-S-4-(1,2-dihydroxynaphthyl)cysteine (1,2-NPQ-NAC)] and 1,4-NPQ-bound NAC [N-acetyl-S-2-(1,4-dihydroxynaphthyl)cysteine (1,4-NPQ-NAC)] were synthesized according to Zheng and Hammock [23], with minor modifications. Briefly, to a solution of 163 mg (1 mmol) of NAC, dissolved in 8 ml of water (purged with N2), 174 mg (1.1 mmol) of either 1,2- or 1,4-NPQ, in 4 ml of acetone (purged with N2) was added dropwise and the reaction mixture was stirred at room temperature for 30 min. After removing acetone under N2, ascorbic acid was added to a concentration of 100 mM and the final product was purified by reverse-phase HPLC as described below. Samples of modified NAC were purified by HPLC using a Hewlett-Packard 1100 series HPLC system, equipped with a quaternary pump (including a vacuum degasser), a manual injector, and variable wavelength and diode array detectors. A Beckman Ultrasphere, 5 m, 250× 10 mm2 ODS column (Beckman Instruments Inc., Fullerton, CA) was used, and the eluents were monitored by UV absorbance at 254 nm with a flow rate of 2.5 ml/min. The solvent system consisted of solvent A: 0.1% aqueous trifluoroacetic acid, and solvent B: 100% acetonitrile. For purification of NPO1- and NPO2-NAC, a linear gradient was run by varying solvent B from 5 to 30% over 20 min. For purification of 1,4-NPQ-NAC the linear gradient varied solvent B from 20 to 30% over 20 min and held solvent B at 30% for 10 min. For purification of 1,2-NPQ-NAC two linear gradients were run by varying solvent B

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from 0 to 18% in 18 min and 18 to 20% over 12 min. In all cases, late eluting compounds were removed by increasing solvent B to 50% over 1 min and holding solvent B at 50% for 10 min. The purified products were characterized by GC– MS in EI mode after conversion to the corresponding trimethyl silyl derivatives. To about 50 mg of each compound, 100 ml of Tri-Sil® reagent was added, samples were heated at 70 °C for 30 min and analyzed by GC– MS in scan mode as described below.

2.5. Characterization and measurement of adducts after reaction with TFAA and methanesulfonic acid (MT assay) Naphthalene-derived products of the MT assay were initially prepared by reacting 50 mg portions of 1,4-NPQ-NAC, 1,2-NPQ-NAC and the mixture of NPO-NACs with TFAA and methanesulfonic acid to yield O,O%,S-tristrifluoroacetylnaphthalenes (1,4- and 1,2-NPQ-S-TFA Fig. 2) and 1- and 2-naphthyltrifluorothioacetates (NPO1- and NPO2-S-TFA). After bringing samples to complete dryness in a vacuum oven (70– 80 °C, 15 mmHg), 750 ml of TFAA and 20 ml of methanesulfonic acid were added and the vials were capped with Teflonlined caps. Samples were heated at 100 °C for 40 min, cooled to room temperature, and unreacted anhydride was removed under a gentle stream of N2. One milliliter of hexane was added to the residue and the hexane layer was washed once with 1 ml of 0.1 M Tris buffer (pH 7.3) and twice with 1 ml of deionized water. After concentrating the samples to 200 ml, a 2-ml aliquot was analyzed by GC–MS with NICI in the selected ion-monitoring mode. After characterizing the products of the MT reaction with the prototype adducts of NAC, the identities of protein adducts derived from naphthalene in vivo were confirmed by performing the MT assay on 50 mg of Alb, combined from F344 rats dosed with 400 and 800 mg naphthalene per kg body weight, as described above. The reaction products from both experiments were analyzed by GC–MS in both NICI and EI scan modes as described below. Routine quantification of adducts in experimental samples was performed as follows: to 2.5– 5 mg of protein in a 4-ml vial, 5 mg of isotopically-labeled protein-bound internal standards ([2H5]1,4-NPQ-Alb, [2H5]1,2-NPQ-Alb, [2H7]NPO1-Alb and [2H7]NPO2-Alb) were added in 10 mM ascorbic acid/10 mM desferoxamine. (Ascorbic acid was added to maintain the adducted quinones in reduced form, thereby enabling the derivatization of hydroxyl groups with TFAA. Desferoxamine was included to scavenge trace metals from the medium, thereby preventing radical-mediated processes and stabilizing ascorbic acid.) Then samples were carried through the MT assay as described above. Standard curves were prepared by adding 0–500 pmol 1,2-NPQ-NAC, 1,4-NPQNAC, and a mixture of NPO1-NAC, NPO2-NAC, to 2.5 mg portions of human Alb or Hb (Sigma– Aldrich Inc., St. Louis, MO) and performing the assay as described above for the experimental samples.

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2.6. Gas chromatography – mass spectrometry All analysis were conducted using a HP 5890 series II gas chromatograph coupled to a HP 5989B MS engine. A DB-5 fused silica capillary column (60 m, 0.25-mm i.d., 0.25-mm film thickness) was used with He (99.999%) as the carrier gas at a flow rate of 1.5 ml/min. The MS transfer-line temperature was 280 °C and the chemical ionization reagent gas (methane) pressure were 2 torr. For the characterization of the trimethylsilyl derivatives of NPO1-, NPO2-, 1,2-NPQ- and 1,4-NPQ-NAC, samples were analyzed by GC– EI –MS at a source temperature of 200 °C and a injector port temperature of 270 °C. The GC oven was held at 75 °C for 2 min and was increased at 10 °C/min to 270 °C where it was held for 25 min. The mass spectra were recorded by scanning the mass range from m/z 45 to 650. For the characterization of adducts in the assay, the ion source temperature was set at 200 °C for both NPO- and NPQ-adducts in EI mode and at 100 and 150 °C for NPO- and NPQ adducts, respectively, in NICI mode. The mass spectra were recorded by scanning the mass range from m/z 45 to 650 in EI mode and from m/z 100 to 650 in NICI mode. The injection-port temperature was 250 °C in all cases. For both NPO-Hb and -Alb, the GC oven temperature was held at 75 °C for 2 min and increased at 4 °C/min to 160 °C, where it was held for 15 min. For 1,2-NPQ-Alb, 1,4-NPQ-Alb and 1,4-NPQ-Hb, the GC oven temperature was held at 75 °C for 2 min and increased at 6 °C/ min to 150 °C, where it was held for 28 min. For 1,2-NPQ-Hb, the best separation was achieved when the oven temperature was held at 75 °C for 2 min, then increased at 6 °C/min to 145 °C, where it was held for 35 min. Late-eluting compounds were removed by increasing the oven temperature at 50 °C/min to 260 °C, where it was held for 15 min. For the quantitation of adducts, all conditions were similar to those described above for GC– NICI – MS except that the mass spectrometer was operated in selected ion monitoring mode. The following ions were monitored: m/z 383 for 1,2- and 1,4-NPQ-S-TFA, 388 for [2H5]1,2- and 1,4-NPQ-S-TFA, 256 for NPO1and NPO2-S-TFA and 263 for [2H7]NPO1- and [2H7]NPO2-S-TFA. The quantitation was based on peak areas relative to the internal standards.

2.7. Precision, limit of detection and statistical analyses The limit of detection of the assay was estimated by spiking 2.5 mg portions of unmodified protein with 0.2 – 1 pmol NPQ- and NPO-NAC and performing the MT assay and GC– NICI – MS as described above. The precision of the assay was estimated by assaying 12 samples of human Alb modified in vitro with 10 mM each of NPO, 1,2- and 1,4-NPQ. The differences between adduct levels in control animals and exposed animals were tested by Student’s t-tests (two-sample test, assuming equal variance) using statistical software provided by Microsoft Excel (Redmond, WA).

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3. Results

3.1. Characterization and quantification of deuterated internal standards The radical-mediated activation of naphthalene via Fenton chemistry provided a simple indirect method of synthesizing deuterated analogs of NPO- and NPQ-adducts. The reaction of [2H8]naphthalene with NAC or Alb in a hydroxyl-radical generating system resulted in the formation of variety of products including the anticipated adducts: i.e. (2H5)1,2-NPQ-Alb, (2H5)1,4-NPQ-Alb, (2H7)NPO1-Alb, (2H7)NPO2-Alb. Although the exact mechanisms of formation of these adducts are unclear, we postulate the following based on the free radical chemistry of benzene ’ [33]. The OH attack at the 1- or 2-position of naphthalene should produce either naphthalene or naphthoxy radicals which can react with sulfhydryl radicals on Alb to form deuterated products, as shown in the upper portion of Fig. 3. Further hydroxylation of the naphthoxy radicals should form deuterated naphthalene-1,2and 1,4-diols that in turn react with sulfhydryl radicals to form the corresponding deuterated NPQ adducts, as shown in the lower portion of Fig. 3. Following reaction with TFAA and methanesulfonic acid, all deuterated adducts should produce derivatives analogous to those shown in Fig. 2. A stock solution of this modified Alb was prepared for use as an internal standard in 10 mM ascorbic/10 mM desferoxamine acid and stored at − 80 °C prior to use. Five microgram portions of this internal standard were repeatedly assayed to determine the apparent levels of modification of each adduct using 1,4-NPQ-, 1,2-NPQ-, NPO1- and NPO2-NAC as calibration standards.

3.2. Characterization of adducts of N-acetylcysteine with naphthalene oxide and the naphthoquinones NPO and 1,2- and 1,4-NPQ were reacted with NAC to form the cysteinyl adducts shown in Fig. 2. The standards of 1,4- and 1,2-NPQ were characterized by proton NMR on a Bruker AMX-500 spectrometer and spectra were similar to those described previously [23]. Since a limited amount of purified product was available for NPO-NAC, the NMR signal was too weak to allow characterization of adducts. The position of the S-substitution on the ring of NPO-NAC (NPO1- and NPO2NAC) was confirmed using the standards of N-acetyl-S-1-naphthyl cysteine and N-acetyl-S-2-naphthyl cysteine, which after the MT assay (see below) give rise to the same derivatives as those arising from NPO1- and NPO2-NAC fFig. 2
. Standards of NPO1-, NPO2-, 1,4-NPQ- and 1,2-NPQ-NAC were also characterized by GC –EI – MS in scan mode after trimethylsilyl derivatization. All spectra displayed high degrees of fragmentation with characteristic ions for NPO derivatives [m/z 451 (M+), 437 (M − CH3)+, 361 (M − SiCH3OH)+, 217 (C10H8OSi(CH3)3)]+ and NPQ derivatives [m/z 537 (M+), 478 (M− NH2COCH3)+ , 336 (C10H5(OSi(CH3)3)2SH)+]. Fig. 4A and B show EI mass spectra of trimethylsilyl derivatives of NPO1- and 1,4-NPQ-NAC, respectively.

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Fig. 2. Formation of cysteinyl adducts of naphthalene oxide, 1,2- and 1,4-NPQ and subsequent reaction of these adducts in the MT assay to yield the corresponding trifluoroacetylated derivatives NPO1-, NPO2-, 1,2-NPQ- and 1,4-NPQ-S-TFA.

3.3. Characterization of adducts of naphthalene oxide and the naphthoquinones following reaction with TFAA and methanesulfonic acid Reactions of cysteinyl adducts of NPO and 1,2- and 1,4-NPQ with TFAA and methanesulfonic acid produced the trifluoroacetyl derivatives shown in Fig. 2. Structures of these derivatives were confirmed by GC–EI – and –NICI –MS in scan mode after carrying NPO-NAC and 1,2- and 1,4-NPQ-NAC through the MT assay. The EI mass spectra showed high degrees of fragmentation with characteristic ions. Spectra of 1,2- and 1,4-NPQ-S-TFA gave molecular ions at m/z 480 as the base peak and analyte-specific major fragment ions at m/z 383 (M − COCF3). The EI spectrum of NPO1-S-TFA gave an analyte-specific fragment ion at m/z 159

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Fig. 3. Pathways for the formation of deuterated NPO and NPQ adducts during radical-mediated activation of deuterated naphthalene via Fenton chemistry.

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Fig. 4. GC– EI mass spectra of trimethyl silyl derivatives of: (A) NPO1-; and (B) 1,4-NPQ-NAC.

(M−COCF3) as the base peak and the molecular ion at m/z 256 representing 90% of the base peak. The spectrum of NPO2-S-TFA gave a molecular ion at m/z 256 as the base peak and the major fragment ion at m/z 159. Mass spectra obtained for 1,4-NPQ- and NPO1-S-TFA in EI mode are shown in Fig. 5. When the same standards were run in NICI mode, fewer fragment ions were observed (data not shown), with the base peaks being either the molecular ion at m/z 256 (NPO-adducts) or an analyte-specific fragment ion at m/z 333 (NPQ-ad-

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ducts). Fig. 6 displays mass spectra of 1,2-NPQ- and NPO2-S-TFA, respectively, obtained in NICI mode from 50 mg of Alb combined from animals dosed with 400 and 800 mg naphthalene per kg body weight. The EI and NICI mass spectra of all

Fig. 5. GC – EI mass spectra of: (A) 1,2-NPQ-; and (B) NPO1-S-TFA, products resulting from the reaction of cysteinyl adducts of 1,2-NPQ and NPO1 with TFAA and methanesulfonic acid.

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Fig. 6. GC– NICI mass spectra of: (A) 1,4-NPQ-; and (B) NPO2-S-TFA obtained by assaying 50 mg of Alb combined from F344 rats dosed with 400 and 800 mg naphthalene per kg body weight.

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TFA-derivatives of naphthalene-derived adducts measured in these animals were very similar to those of authentic standards.

3.4. Precision and limit of detection of the MT assay Based upon a signal-to-noise ratio of 3, the limit of detection of the assay corresponds to about 50 pmol adduct per g protein for all adducts, assuming 2.5 mg of protein, an injection volume of 2 ml, and a final sample volume of 200 ml. The precision, as indicated by estimated coefficients of variation were 14, 16, 13, and 18%, respectively, for NPO1-, NPO2-, 1,2-NPQ- and 1,4-NPQ-Alb (n= 12).

3.5. Production of Alb and Hb adducts of naphthalene oxide and the naphthoquinones following administration of naphthalene to rats The production of cysteinyl adducts of NPO and the NPQs with Alb and Hb was investigated in male F344 rats dosed with 100–800 mg naphthalene per kg body weight. Fig. 7 shows a chromatogram obtained by GC–NICI – MS in selected ion monitoring mode for 2.5 mg of Alb from an animal dosed with 400 mg naphthalene per kg body weight. NPO adducts were measured in all naphthalene-dosed animals but not in control animals. As shown in Table 1, levels of NPO1 adducts were higher than those of NPO2 in both proteins. When levels of the NPO adducts are plotted as a function of dose, as shown in Fig. 8, nonlinear production is seen above 200 mg naphthalene per kg body weight. Interestingly, the curves display sublinear (greater-than-proportional to dose at high doses) production of NPO-Hb and supralinear (less-than-proportional to dose at high doses) production of NPO-Alb. As shown in Table 2, levels of 1,2-NPQ-Alb were greater than those of 1,4-NPQAlb at all doses while those of 1,2- and 1,4-NPQ-Hb were generally comparable at all doses. Also, levels of NPQ-Alb were much greater than those of NPQ-Hb at all doses. Adducts of 1,2- and 1,4-NPQ, but not of NPO-adducts, were detected in Table 1 Levels of NPO adducts in Hb and Alb following administration of naphthalene to F344 rats and in controls Dose (mg/kg)

0 100 200 400 800

Alb adducts (nmol/g) (SE)

Hb adducts (nmol/g) (SE)

NPO1

NPO2

NPO1

NPO2

NDa 16.4 (1.08) 83.4 (6.45) 174 (17.8) 265 (23.8)

NDa 8.64 (1.31) 26.9 (1.55) 64.8 (8.66) 80.9 (10.1)

NDa 12.8 (2.52) 62.6 (5.11) 254 (74.1) 1546 (451)

NDa 4.30 (0.616) 15.9 (0.403) 42.1 (4.58) 188 (34.7)

Mean adduct concentration (SE), n= 3 for all groups. a ND, not detectable.

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Fig. 7. Typical GC –NICI–MS chromatogram obtained in selected ion monitoring mode following the reaction of 2.5 mg Alb (from a F344 rat dosed with 400 mg naphthalene per kg body weight) with trifluoroacetic and methanesulfonic acids. Ions m/z 256 and 383 corresponds to trifluorocetyl derivatives of NPO (NPO1- and NPO2-S-TFA) and NPQ adducts (1,2-NPQ- and 1,4-NPQ-S-TFA), respectively. Corresponding deuterated internal standard ions were m/z 263 and 388.

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Fig. 8. Dose-dependent formation of NPO adducts in Hb and Alb following administration of naphthalene to male F344 rats. Each point represents the mean ( 9 SE) of three animals per dose group.

both Hb and Alb from control animals. However, levels of 1,2- and 1,4-NPQ-Hb did not differ significantly from controls at doses below 400 mg/kg (P B 0.05) (Table 2). When plotted as a function of dose in Fig. 9, levels of 1,4-NPQ-Alb increased linearly with dose while those of 1,2-NPQ-Alb displayed sublinear production at doses above 200 mg naphthalene per kg body weight. The shapes of the corresponding curves for the NPQ-Hb adducts are difficult to gauge due to the presence of relatively large background levels of the same adducts (Fig. 9).

4. Discussion The evidence that naphthalene causes tumors of the respiratory tract in rodents [9 –12], and the widespread environmental exposure to this chemical from cigarette

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smoke and various fuels [2,3], notably diesel fuel [4,5] has increased interest in the metabolism and disposition of this environmental contaminant. The cytotoxicity of naphthalene has been attributed to its reactive metabolites, NPO, 1,2-NPQ, and 1,4-NPQ, perhaps due to binding to cellular macromolecules [16–19]. Zheng et al. have shown that 1,2-NPQ is the major metabolite covalently bound to cysteine residues in proteins from Clara cells exposed to naphthalene in vitro [17]. However, structural evidence for the formation of these reactive metabolites in vivo and subsequent binding to macromolecules is very sparse. Here, we present an assay with which we characterized and quantified cysteinyl adducts of NPO, 1,2- and 1,4-NPQ in F344 rats. All of the characterized adducts of NPO and the NPQs were easily detected in Hb and Alb of F344 rats, 24 h after receiving a single dose of 100–800 mg naphthalene per kg body weight. However, the relative amount of these adducts varied greatly with both the binding species and the protein. Regarding adducts of NPO, levels of NPO1 adducts were higher than those of NPO2 adducts in both Hb and Alb, indicating that aromatic substitution is favored in vivo at position 1 over 2. This is in contrast to what has been reported by other researchers, based on the mercapturic acids excreted in the urine of animals administered with racemic NPO, where the substitution at C1- and C2-positions were 15 and 85%, respectively [32]. Our experience, based on quantifying 1- and 2-napthyltrifluorothioacetates obtained after the MT assay of blood proteins modified in vitro with racemic NPO, resulted in substitution at C2- and C1-positions of 75 and 25% in Alb, and of 14 and 86% in Hb, respectively (data not shown). When a similar experiment was carried out on Alb adducts of NPO, derived via Fenton chemistry (as described for the synthesis of internal standards with naphthalene as the starting material), substitution at the C2- and C1-positions were 90 and 10%, respectively (data not shown). These results indicate that, perhaps, factors other than the chemical reactivity of NPO play roles in determining the extent of substitution of this epoxide with nucleophilic sites. Levels of NPO-Alb were greater than those of NPO-Hb at naphthalene doses up to 200 mg/kg body weight but less than those of Table 2 Levels of 1,2- and 1,4-NPQ adducts in Hb and Alb following administration of naphthalene to F344 rats and in controls Dose (mg/kg)

0 100 200 400 800

Alb adducts (nmol/g) (SE)

Hb adducts (nmol/g) (SE)

1,2-NPQ

1,4-NPQ

1,2-NPQ

1,4-NPQ

0.359 (0.092) 8.73* (2.77) 32.5* (8.83) 105.0* (30.3) 321* (81.4)

0.039 (0.021) 3.37* (0.596) 7.93* (1.67) 19.1* (4.45) 38.3* (6.67)

0.828 (0.060) 0.892 (0.302) 1.01 (0.162) 2.44* (0.504) 6.11* (0.772)

0.913 0.861 0.964 1.36* 2.09*

Mean adduct levels (SE) without subtracting background levels, n = 3 for all groups. * Indicates significant difference between control levels and dose-group (PB0.05).

(0.049) (0.164) (0.072) (0.055) (0.245)

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Fig. 9. Dose-dependent formation of NPQ adducts in Hb and Alb following administration of naphthalene to male F344 rats. Each point represents the mean ( 9 SE) of three animals per dose group.

NPO-Hb at higher doses, leading to supra- and sublinear dose–response curves for NPO-Alb and -Hb, respectively (Fig. 8). Regarding NPQ adducts, levels of NPQ-Alb were much greater than those of NPQ-Hb over the entire dose range. Indeed, levels of NPQ-Hb were only significantly greater than control values at the two highest naphthalene doses (400 and 800 mg/kg body weight). This was unexpected given the presence of an additional free cysteine at i 125 in rat Hb, which is readily accessible to electrophiles [34]. We speculate that this effect was due to higher concentrations of glutathione in red blood cells relative to plasma [35]; increased levels of glutathione could encourage additional substitution reactions with quinone adducts, leading to multi-S-substituted adducts [26,36]. Similar observations were noted with quinone metabolites of pentachlorophenol where mono-S-substituted Alb adducts were elevated over Hb

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adducts [36,37] and of benzene metabolites where levels of Alb-adducts increased with dose while those of Hb-adducts were not elevated above control values [26]. Of the two NPQ adducts, those of 1,2-NPQ were more abundant in both Hb and Alb than 1,4-NPQ. As shown in Fig. 1, this could reflect a single metabolic pathway for 1,4-NPQ (via aromatic hydroxylation of 1-naphthol by P450s) and multiple pathways for 1,2-NPQ (via hydrolysis of NPO by epoxide hydrolase or via a second oxidation of 1- or 2-naphthol). The dose – response curves shown for NPQ-Alb in Fig. 9 point to linear or sublinear production of adducts with naphthalene dose over the range of 100–800 mg/kg body weight. This finding contrasts with that noted above for the NPO-Alb adducts, where the curves showed evidence of supralinearity. Since NPO is generally thought to be a necessary precursor of the NPQs (Fig. 1), this difference in dose – response curves of NPO- and NPQ-Alb cannot be explained solely by saturation of CYP450 oxidation of naphthalene. Nor can the curves be explained solely by saturation of detoxification, due to depletion of glutathione, a factor that has been offered to explain increased covalent binding at high naphthalene doses in mice [20,22]. Perhaps a combination of these saturable pathways and other factors, such as differential rates of adduct instability [29], can explain the apparent contradiction in dose– response curves for NPO- and NPQ-Alb. In any case, further experimental confirmation of the observed effects is needed. Finally, we point to the presence of background levels of adducts of NPQ but not of NPO in control rats. Several previous studies have reported the presence of macromolecular adducts arising from nonspecific sources in rodents and humans [26,28,38–41]. The levels of NPQ adducts observed in this investigation ranged from about 0.04 to 1 nmol/g of protein, depending upon the particular adduct and protein (Table 3). These levels are much lower than those of 1,2- and 1,4-benzoquinone reported in control F344 rats at levels of 2.7–11.4 nmol/g [26]. The lower background levels of NPQ adducts compared to those of the benzoquinones suggest that naphthols and other naphthyl-containing compounds are much less abundant than phenols and phenyl-containing compounds in the diet and gut flora.

Table 3 Background levels of naphthalene derived quinone adducts in Hb and Alb in humans and rats Adduct

Human

Rat

1,2-NPQ-Hb 1,4-NPQ-Hb 1,2-NPQ-Alb 1,4-NPQ-Alb

0.327 (0.099) NDa 0.064 (0.013) 0.079 (0.015)

0.828 0.913 0.359 0.039

(0.060) (0.049) (0.092) (0.021)

Mean adduct levels (nmol/g) (SE), n= 3 for rats and n= 10 for humans. NPO adducts were not detected in both human and rats. Human Alb was obtained from Sigma–Aldrich Inc. (St. Louis, MO); this protein had been pooled from many presumably unexposed blood donors in the U.S. a ND, not detected.

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5. Conclusion An assay is presented to simultaneously measure cysteinyl adducts of major naphthalene metabolites, NPO, 1,2- and 1,4-NPQ in Hb and Alb. Formation of these adducts was dose-dependent in Alb and Hb from F344 rats receiving a single dose of 100–800 mg naphthalene per kg body weight. Low levels of 1,2- and 1,4-NPQ adduct, but not NPO adducts, were detected in Hb and Alb from both humans and rats without known exposure to naphthalene. Our data demonstrate the potential applicability of these adducts not only to study the metabolism and disposition of naphthalene metabolites, but also as biomarkers of exposure to naphthalene.

Acknowledgements The authors thank Dr Alan Buckpitt for providing racemic naphthalene oxide and Dr Avram Gold of our Chemistry Core for providing of N-acetyl-S-1-naphthyl cysteine and N-acetyl-S-2-naphthyl cysteine. This work was supported in part by the National Institute of Environmental Health Sciences through grants P42ES05948, P30ES10126, and T32ES07018. The research described in this document was also supported in part by the U.S. Environmental Protection Agency. It has been subjected to Agency review and approved for publication.

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