Materials and Methods

Experimental Animals.

Male, CFW (Swiss-Webster) mice (25–30-g body weight) and male, Sprague-Dawley rats (250–300-g body weight) were purchased from Charles River Breeding Laboratories (Portage, MI). Animals were housed over inert bedding in stainless steel cages within high-efficiency particulate air (HEPA)-filtered laminar air-flow cabinets. They were allowed free access to food and filtered, deionized water, and kept on a 12-h light/dark cycle in University of California Davis facilities, which are certified by the American Association for the Accreditation of Laboratory Animal Care. They were used no sooner than 7 days after receipt from the supplier.

Chemicals.

1-NN was purchased from Aldrich Chemical Company (Milwaukee, WI) and was recrystallized from ethanol before use (m.p. 61.5°C). GSH was purchased from Fluka Chemical Corp. (Milwaukee, WI). GST was purified from mouse liver cytosol by affinity chromatography. All other chemicals were reagent grade or better.

Radioactive Chemicals.

GSH, [glycine-2-³H], (specific activity 44,800 mCi/mmol) was purchased from DuPont-NEN Life Science Products (Boston, MA). The specified radiochemical purity was 97% and was verified by HPLC on a C₁₈ column. The material was used without additional purification. Impurities did not coelute with any of the metabolites and, accordingly, did not affect the analysis conducted during these studies. Each lot was opened and used immediately to lessen the possibility of oxidation to the disulfide. [³H]GSH was diluted with unlabeled compound to achieve specific activities of 5 to 7.4 mCi/mmol for the in vitro studies.

Microsome Preparation and Incubations.

Lungs were perfused with isotonic saline before removal from the animal, and microsomes were prepared by differential centrifugation as described earlier (Watt et al., 1999). Incubations were prepared on ice in a final volume of 300 μl of 0.1 M sodium phosphate buffer (pH 7.4) and consisted of 300 μg of microsomal protein, 1 mM 1-NN, 0.1 mM [³H]GSH, 10 1-chloro-2,4-dinitrobenzene U/ml GST and NADPH-generating system (consisting of 0.14 mM NADP, 3.8 mM glucose 6-phosphate, 0.1 U glucose 6-phosphate dehydrogenase, and 10 mM MgCl₂) (Watt et al., 1999). After a 2-min preincubation period with the NADPH-generating system, 1-NN was added with GSH and GST, and incubation was allowed to proceed for 20 min at 37°C. The reaction was terminated by adding one volume of methanol, and samples were stored at −20°C. All incubations were prepared in triplicate. Controls were prepared without NADPH-generating system.

Sample Preparation and HPLC Analysis of 1-NN GSH Conjugates.

Reaction mixtures were centrifuged to remove the protein. The remaining supernatant was evaporated under vacuum to approximately 50μl. Samples were stored at −80°C until analysis. Samples were chromatographed on a Phase Sep C₁₈reversed phase column (25 cm × 4.6 mm i.d.; 5-μm particle). The eluates were monitored by UV absorbance at 256 nm. A mobile phase of 0.06% triethylamine phosphate in water (pH 3.1) and acetonitrile was run at a flow rate of 1.0 ml/min with a linear increase from 5 to 16% acetonitrile in 60 min. The column eluate was collected directly into scintillation vials at 0.5-min intervals. Complete radiochromatographic profiles were obtained and radioactive peaks corresponding to 1-NN GSH conjugates were summed, with the appropriate background counts being subtracted.

Mass Spectrometry (MS).

1-NN GSH conjugates were analyzed on a Finnigan LCQ (Finnigan Corp., San Jose, CA) ion trap mass spectrometer with 2000 amu mass range and MS/MS capability using 50:50 v/v acetonitrile/water with 1% acetic acid as the mobile phase. Conditions were identical with those used in Watt et al. (1999).

Results and Discussion

Separation of 1-NN GSH Conjugates by HPLC and MS Analysis.

Metabolism of 1-NN by mouse liver microsomal proteins with [³H]GSH and GST resulted in seven radioactive peaks with elution times from 21 to 31 min at approximately 10 to 12% acetonitrile (Fig. 1). The second through the seventh peaks coeluted with the six conjugates, which have been identified previously by MS and proton NMR, from rat microsomal incubations. The earliest eluting peak (labeled peak 2) in extracts of mouse lung or liver microsomal incubations was absent in identical incubations prepared from rat lung and liver. MS in positive ion mode of peak 2 yielded a m/z 497 (M+H), and daughter ions of m/z 479, 306, and 177, which was consistent with the formation of 1-NN GSH conjugates with a molecular weight of 496. A daughter ion of m/z 479 corresponded to the loss of one water molecule from the parent compound, whereas that of m/z 306 corresponded to reduced GSH. The daughter ion of m/z 177 corresponded to 1-NN with the loss of the nitro group and the formation of hydroxy naphthyl thiolate ion. This was shown previously as a daughter ion in MS of naphthalene GSH conjugates (Buckpitt et al., 1987). There are 12 possible 1-NN GSH conjugates that could result from the three corresponding intermediate epoxides, C₇,C₈-, C₅,C₆-, and C₃,C₄-epoxides (Scheme 2 ofWatt et al., 1999). In the case of rat, conjugates 1, 3, and 4 are from the C₇,C₈-epoxide and are the first three to elute from the HPLC column. The first peak eluting from HPLC of mouse lung and liver microsomal incubations is labeled peak 2, and is either a diastereomer of conjugate 1 (1-nitro-7-hydroxy-8-glutathionyl-7,8-dihydronaphthalene) or a diastereomer of conjugate 7 (1-nitro-5-glutathionyl-6-hydroxy-5,6-dihydronaphthalene). The small quantities of this GSH adduct (peak 2, Fig. 1B) were not sufficient to obtain interpretable NMR signals and thus, definitive assignment of the regiochemistry of this conjugate was not possible. However, based on the elution pattern, it is more likely to be a diastereomer of conjugate 1. The absence of peak 2 (Fig. 1B) in rat indicated that there were differences in the regio- and stereoselectivity of GSH conjugation between the two species.

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Figure 1

A, HPLC radioactivity profile of extracts of incubations of 1-NN and GSH with mouse liver microsomal protein [1-NN (1 mM), [³H]GSH (0.1 mM, 5 mCi/mmol), GST (10 U/ml), and NADPH-generating system were incubated with 300 μg microsomal protein for 20 min; the profile demonstrated the production of seven radioactive peaks]; B, HPLC UV profile of the incubation with the corresponding seven peaks; C, control incubation without the NADPH-generating system.

Metabolism of 1-NN by Liver and Lung.

In mouse, the total rates of 1-NN GSH conjugate formation in the lung were approximately 33% that in the liver when calculated on a per microgram microsomal protein basis (Fig.2). The same was true in rat. If turnovers are calculated based on the level of CYP (assuming that lung microsomal CYP levels are approximately 10-fold less than liver; seeBuckpitt and Cruikshank, 1997), then overall metabolism of 1-NN to conjugate is approximately 4-fold higher in lung than in liver. As with other metabolically activated Clara cell toxicants, we assume that the high susceptibility of the lung (compared with liver) is in part due to the localization of CYP within the Clara cell, a cell population that only represents 5 to 10% of the total population of cells in the lung. However, this does not explain the apparent sensitivity of ciliated cells to 1-NN. A variety of techniques, including immunocytochemistry with a number of CYP antibodies, have failed to demonstrate the presence of CYP monooxygenase within ciliated cells (Plopper, 1993). Ciliated cell injury could result from diffusion of reactive metabolites from neighboring Clara cells. However, ciliated cell injury does not occur with naphthalene and there is considerable evidence showing that naphthalene epoxide diffuses across cells (Richieri and Buckpitt, 1987). Additional work comparing GSH depletion in ciliated and Clara cells of mice treated with 1-NN and naphthalene using fluorescence microscopy may provide additional data needed to begin to understand the underlying causes of ciliated cell toxicity.

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Figure 2

Comparison of the rates of 1-NN GSH conjugates generated from incubations of microsomal proteins obtained from liver and lung with 1-NN, GSH, and GST.

Values of mouse liver are mean ± S.D. of six separate incubations using pooled microsomes prepared on two different days. Values of mouse lung are mean ± S.D. of three separate incubations using pooled microsomes prepared on two different days. Values of rat liver and lung from Watt et al. (1999) are presented for comparison.

The composition of conjugates generated in mouse lung microsomal incubations differed substantially from those generated by liver microsomes. In lung microsomal incubations, conjugates 1, 2, 3, and 4 (derived from the C₇,C₈-epoxide) accounted for the majority (78%) of the total conjugates. In liver microsomal incubations, conjugates 5, 6, and 7 (derived from the C₅,C₆-epoxide) predominated and accounted for almost 65% of the total conjugates.

In both mouse and rat lungs, the rate of conjugate 4 generation was higher than that of any other conjugates. In mouse liver, the rate of conjugate 6 generation prevailed and accounted for nearly half of the total 1-NN GSH conjugates. This was different from rat liver, where the rate of conjugate 5 generation was the highest. Conjugates 5 and 6 were diastereomers that were derived from different enantiomeric C₅,C₆-epoxides. This indicated that different CYP in the liver could be involved in 1-NN metabolism between these two species, resulting in the generation of the 5R,6S-epoxide in one and the 5S,6R-epoxide in the other. Our preliminary studies in rat liver using suicide substrates for CYP indicated that CYP 2B, 2E, and 2F could all be involved in the metabolism of 1-NN (Watt et al., 1998). Additional studies using isoform-specific inhibitory antibodies could be useful in understanding the differences in regio- and/or stereoselectivity in 1-NN metabolism.