1,3-Dinitrobenzene Metabolism and Protein Binding - American

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1,3-Dinitrobenzene Metabolism and Protein Binding Ian T. Reeve* and Marion G. Miller† Department of Environmental Toxicology, University of California at Davis, Davis, California 95616 Received August 10, 2001

1,3-Dinitrobenzene is a testicular toxicant, which produces a lesion in the seminiferous tubules of the rat. In the present study, we investigated which subcellular fractions of the seminiferous tubules are capable of 1,3-dinitrobenzene metabolism and protein adduct formation. Subcellular fractions of the liver were used as positive controls and to further investigate potentially important binding proteins. Microsomes, cytosol, and mitochondria prepared from each tissue were incubated with 200 µM [14C]1,3-dinitrobenzene and 2 mM NADH or NADPH. Since nitroreduction is an oxygen sensitive metabolic pathway, incubations were carried out in the presence and absence of oxygen. Under anaerobic conditions, 1,3-dinitrobenzene was metabolized to nitroaniline and/or nitrophenylhydroxylamine. Metabolite formation was inhibited under aerobic conditions, suggesting the presence of an oxygendependent redox-cycle. For the seminiferous tubules, no metabolites were generated under aerobic conditions. In the absence of oxygen, only the mitochondria produced 1,3-dinitrobenzene metabolites. For the liver, under anaerobic conditions, all three subcellular fractions produced 1,3-dinitrobenzene metabolites with the microsomes containing the greatest activity. However, under aerobic conditions, only the microsomes generated metabolites. One-dimensional gel electrophoresis demonstrated that protein adduct formation within the liver and seminiferous tubule subcellular fractions correlated with metabolite formation. Addition of GSH to seminiferous tubule mitochondrial incubations decreased the amount of 14C-labeled protein. Moreover, when seminiferous tubule mitochondria were incubated with 1,3-dinitrobenzene at an increased protein concentration, radioactive labeling of a 54 kDa protein became more prominent. Two-dimensional gel electrophoresis of liver mitochondrial protein incubated with [14C]1,3-dinitrobenzene and NADPH yielded three predominantly radiolabeled proteins of the same approximate size (54 kDa). Amino acid sequencing identified each of these proteins as rat mitochondrial aldehyde dehydrogenase.

Introduction Nitroaromatic compounds are used widely as pesticides, explosives, pharmaceuticals, and chemical intermediates in industrial synthesis (1). Numerous nitroaromatics such as trinitrotoluene (2), 2,4- and 2,6-dinitrotoluene (3), nitrobenzene (4), and DNB (1,3-dinitrobenzene)1 (5) have been shown to cause testicular toxicity in laboratory animals. In this study, DNB was used as a model compound for investigating nitroaromatic induced testicular toxicity. This toxicant has been shown to produce histological alterations in the testis (6, 7), decreased sperm numbers, and altered sperm motility and morphology (8, 9). Work by Foster et al. demonstrated that DNB increased lactate secretion and germ cell release in Sertoli/germ cell cocultures (10). Other Sertoli/germ cell coculture experiments and studies utilizing isolated seminiferous tubules (i.e., digested away from the interstitial tissue) have demonstrated that DNB treatment decreased cellular ATP and mitochondrial GSH levels (11, 12). * To whom correspondence should be addressed. Phone: (530) 7523164. Fax: (530) 752-3516. E-mail: [email protected]. † Department of Environmental Toxicology, The University of California at Davis. Phone: (530) 754-8982. Fax: (530) 752-3394. E-mail: [email protected]. 1 Abbreviations: DNB, 1,3-dinitrobenzene; NNB, nitrosonitrobenzene; NPHA, nitrophenylhydroxylamine; NA, nitroaniline; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Reductive metabolism of DNB occurs both in vivo (13) and in vitro in isolated hepatocytes (14), Sertoli/germ cell cocultures (11), and isolated seminiferous tubules (12, 15). DNB, as with other nitroaromatics, is metabolized via a 6e- nitroreduction, ultimately forming an aniline metabolite (Scheme 1) (16). Nitroaromatic reduction proceeds via an one electron reduction to form a nitroxyl anion radical. Under aerobic conditions, this metabolite delivers an electron to molecular oxygen creating the superoxide anion radical. This process oxidizes the nitroxyl anion radical back to the parent compound. In the absence of oxygen, the nitroxyl anion radical is further reduced by an electron to form NNB (nitrosonitrobenzene), which accepts another two electrons to form NPHA (nitrophenylhydroxylamine). In the final reduction step, the NPHA is converted to NA (nitroaniline), by a two electrons. Numerous nitroaromatics have been shown to covalently adduct protein after metabolic activation (17). Enzymatic reduction of 5-nitrofuran derivatives such as N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide results in the generation of protein adducts (18). Metabolism and protein binding were also seen when each of the three isomers of dinitrobenzene were incubated with erythrocytes (19). In rat Sertoli and germ cell cocultures, Brown and Miller (11) demonstrated a correlation between DNB metabolism and protein adduct formation.

10.1021/tx015554+ CCC: $22.00 © 2002 American Chemical Society Published on Web 02/23/2002

1,3-Dinitrobenzene Metabolism and Protein Binding Scheme 1. Nitroreductive Metabolism of 1,3-Dinitrobenzene to Nitrophenylhydroxylamine and Nitroaniline

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 353 were >98% pure as determined by HPLC. NPHA (>91% pure as determined by HPLC) was synthesized via reduction of DNB with palladium and phosphinite as described in Entwistle and Gilkerson (23). NNB (>98% pure as determined by HPLC) was donated by Dr. Mark Kurth (Department of Chemistry, UC Davis). The synthesis was carried out by initially reducing DNB with a rhodium-charcoal catalyst, in the presence of hydrazine, to NPHA, which was subsequently oxidized to NNB using FeCl3 (23). Digestion medium was purchased from Gibco BRL (Gainsburg, MD). Enlightening fluorographic embedding solution was purchased from New England Nuclear Co. (Boston, MA). All other chemicals used were from Bio-Rad (Hercules, CA), Fisher Scientific (Pittsburgh, PA), and Sigma and were of the highest available purity. Structural Confirmation of NPHA. After synthesis, the dried NPHA product was solubilized in methanol. HPLC was then performed using an 8 mm i.d., 4 um pore size uBondapak C-18 radial compression column from Waters Associates (Milford, MA). The mobile phase consisted of an isocratic solvent system of methanol and potassium phosphate buffer (50 mM, pH 6.8) at a ratio of 35:65, respectively. The flow rate was 2 mL/min. The product was fraction-collected and extracted from the mobile phase using ethyl acetate. This extract was washed several times with water to remove any buffer salts and then evaporated under a stream of nitrogen. The dried product was solubilized in methanol, and negative ion MS was carried out using a triple quadrupole mass spectrometer. The m/z obtained for the product was 152.3, which, within the limit of instrument resolution, is the expected value for the molecular ion of NPHA. Animals. Male Sprague-Dawley rats (115-125 days old) were purchased from Charles-River (Hollinger, CA). Animals were housed in a constant temperature and humidity environment (22 ( 2 °C and 50 ( 10%, respectively). A light/dark cycle of 12 h was maintained. Food (Purina rat chow) and water were provided ad libitum.

Adduct formation after DNB treatment has been attributed to NNB, which, due to the electrophilic nitroso group, reacts covalently with protein. Eyer and Ascherl (20) established that nitrosobenzene can adduct hemoglobin through the formation of sulfinamide bonds. In addition, direct ligation to the heme iron has been suggested (20). Umemoto et al. (21) demonstrated that the cysteine sulfhydryl nonenzymatically complexes with 2-nitroso-6-methyldipyrido(1,2-a:3′,2′-d) imidazole to form a N-hydroxy-sulfonamide adduct. NNB has been shown to react with cysteine, N-acetylcysteine, and cysteamine (22) and adduct hematin, globin and hemoglobin.2 The goal of this project is to gain insight into the mechanism by which DNB causes testicular toxicity through investigating DNB metabolism, using NADH or NADPH as cofactors and protein adduct formation in subcellular fractions prepared from interstitia-free seminiferous tubules. Metabolic and binding profiles of DNB under aerobic and anaerobic conditions in microsomal, cytosolic and mitochondrial fractions were determined. Subcellular fractions of the liver were used as positive controls and to further investigate protein adduct formation.

Experimental Procedures Caution: DNB has been shown to produce methemoglobinemia, testicular toxicity, and neurotoxicity in laboratory animals (1, 5). Chemicals. [14C]DNB with a specific activity of 12.2 µCi/umol was purchased from Sigma Chemical Co. (St. Louis, MO). NA was purchased from Aldrich Chemical Co.(Milwaukee, WI). Both 2

Winder, B., and Miller, M., unpublished results.

Preparation of Subcellular Fractions from Seminiferous Tubules and Liver. Rats were euthanized by carbon dioxide inhalation and the testes were removed, detunicated, and quartered using a razor blade. To isolate the interstitiafree seminiferous tubules, the testes were digested using 1 mg/ mL Type IV collagenase (Sigma) for 30 min at 34 °C in a shaking water bath. The digestions were carried out in digestion medium (Dulbecco’s modified Eagle’s medium and nutrient mixture F12 [1:1] with 15 mM HEPES and 2.5 mM L-glutamine). After digestion, the tubules were washed several times with chilled isolation medium (0.25 M sucrose, 2 mM HEPES, pH 7.4). Washed, interstitia-free seminiferous tubules were homogenized using a glass/Teflon homogenizer. Mitochondria were isolated using the methods described previously by Meyers et al. (24). Microsomes and cytosol were isolated as described by Lake (25). The liver was removed, rinsed in ice-cold isolation medium, and minced. The pieces were homogenized (10% w/v in isolation medium), using a glass-Teflon homogenizer. Subcellular fractions of microsomes, mitochondria, or cytosol were prepared from homogenate using the above methods. Incubations. Subcellular fractions were incubated at protein concentrations of 1.5, 2.0, 2.5, or 20 mg/mL, as determined using a spectrophotometric bicinchoninic acid assay (26), with [14C]DNB (200 µM, 1.22 µCi/incubation) dissolved in tetrahydrofuran (incubation concentration of 0.5% v/v) and NADPH (2 mM) at 37 °C (liver fractions) or 32 °C (seminiferous tubule fractions) for 30 min. Incubations were carried out under either ambient aerobic or anaerobic conditions. To establish anaerobic conditions, the incubation buffer (Tris 0.12 M, pH 7.4) was degassed and sparged under nitrogen. Incubations were performed under a nitrogen atmosphere in a vial made airtight with a rubber septum. Duplicate sample aliquots were taken every 5 min. Reactions were stopped by the addition of 2 vol of chilled methanol. All incubations were preincubated for two minutes at the appropriate temperature and atmosphere before the addition of [14C]DNB.

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Separation of Parent Compound and Metabolites. The methanolic precipitate of each sample aliquot was centrifuged for 2 min at 2218g. DNB and its metabolites were separated by reverse-phase HPLC under the previously described conditions. Identification and Quantitation of Parent Compound and Metabolites. The HPLC eluent was monitored for levels of radioactivity using a Packard (Meridan, CT) Radiomatic inline radioactivity detector. The radioactive peaks were identified via co-chromatography with authentic standards (13). The identity of the radioactive peak correlating with the NPHA standard was confirmed by LC/MS using the conditions described for the synthesized standard. The m/z obtained for the product was 152.4, which, within the limit of instrument resolution, is the expected value for the molecular ion of NPHA. Quantitation of the parent compound and metabolites was by UV absorption (254 nm). Statistical Analysis of Metabolism Data. Differences in DNB metabolism were analyzed using the F-test followed by the Student’s t-test (27). In all analysis, the level of statistical significance was set at P < 0.05. Detection of Radiolabeled Proteins in Liver Fractions. Incubations were performed with 2.5 mg of protein/mL, 2 mM NADPH, and 200 µM [14C]DNB under anaerobic and aerobic conditions. After methanolic precipitation (T ) 0 and 30 min incubation timepoints), proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with 10% resolving and 3% stacking gels using the method of Laemmli (28). Proteins were stained overnight in a solution containing 0.1% Coomassie Blue R-250, 46.5% methanol, 46.5% water, and 7% acetic acid. The gel was destained in 46.5% methanol, 46.5% water, and 7% acetic acid and subsequently embedded with Enlightening fluorographic embedding solution according to the manufacturer’s instructions. The gel was then placed against filter paper and dried at 50 °C under a vacuum for 2.5 h. The dried gel was placed against Kodak (Rochester, NY) XAR-OMAT film for 3 weeks at -80 °C. The exposed film was developed and fixed according to the manufacturer’s instructions. Detection of Radiolabeled Proteins in Seminiferous Tubule Fractions. Incubations containing 2 mM NADPH and 200 µM [14C]DNB were performed under anaerobic conditions at 2.5 mg of protein/mL with and without the addition of 6 mM GSH or at 20 mg of protein/mL. Proteins were analyzed by SDS-PAGE as described previously. Preparation of Liver Mitochondrial Protein for TwoDimensional Gel Electrophoresis. Liver mitochondria (20 mg of protein/mL in a total incubation volume of 0.55 mL) were incubated at 37 °C for 90 min in Tris (0.12M) buffer, pH 7.4, with NADPH (2 mM) and [14C]DNB (200 µM, 1.34 µCi) solubilized in THF (0.95% v/v). Following the incubation, 0.5 mL of the incubation was stirred for 1 h at 4 °C in a proteinsolubilizing solution. The final mixture contained thiourea (2 M), urea (7 M), CHAPS (4%) and mitochondrial protein (10 mg/ mL). The solution was then centrifuged for 60 min at 100000g and 4 °C to pellet insoluble material (29). The supernatant was desalted using gel-filteration (exclusion limit of 5 × 103 globular protein) and the protein concentration of the eluent determined via the Bio-Rad protein assay. Detection of Radiolabeled Liver Mitochondrial Proteins by Two-Dimensional Gel Electrophoresis. A total of 2.4 mg of the desalted, solubilized liver mitochondrial protein was mixed with pH 3-10 nonlinear IPG Buffer (final concentration of 2% v/v) from Amersham Pharmacia Biotech, dithiothreitol (2.8 mg/mL), and trace amounts of bromophenol blue to a total volume of 300 µL. An Immobiline DryStrip (13 cm, pH 3-10 nonlinear) from Amersham Pharmacia Biotech was rehydrated with 250 µL of this mixture, containing 2 mg of mitochondrial protein, overnight in a rehydration tray. Isoelectric focusing of the proteins in the strip was carried out according to the manufacturer’s instructions. The proteins were electrophoresed with the following sequence of voltages: 15 min

Reeve and Miller at 300 V, 15 min at 588 V, 30 min at 1167 V, 30 min at 2333 V, and 20 h at 3500 V. The strip was equilibrated in SDS Equilibration Buffer and the proteins separated on a 10% polyacrylamide gel, using an Hoefer SE 600 unit (Amersham Pharmacia Biotech). Proteins were stained and the gel embedded and dried as described earlier. An aliquot (0.2 µL) of black ink containing [14C]DNB (50 000 dpm) was applied to each of two locations on the paper backing of the embedded and dried gel (lower left) in order to align the Coomassie blue stained proteins with the corresponding fluorogram. The gel was then laminated to Kodak XAR-OMAT film for 6 weeks at -80 °C. The exposed film was developed according to the manufacturer’s instructions. In-Gel Digestion and Peptide Sequencing. Protein digestion, peptide purification, and amino acid analysis were carried out at the Molecular Structure Facility at the University of California at Davis. Briefly, three Coomassie-stained protein spots on the 2D-gel containing highly 14C-labeled proteins were excised from the gel. The proteins within each spot were reduced with 10 mM dithiothreitol in 100 mM NH4HCO3, alkylated with 55 mM iodoacetamide in 100 mM NH4HCO3 and digested overnight at 37 °C in 50 mM NH4HCO3, pH 7.8, using modified porcine trypsin from Promega (Madison, WI) (0.5 ug/spot). The resulting peptides were extracted from the gel using 20 mM NH4HCO3 followed by 5% formic acid in 50% acetonitrile. The extracted peptides were resolved using an Applied Biosystems (Foster City, CA) 172 microbore HPLC system. The best resolved peptides were sequenced using an Applied Biosystems Model 477 Sequencing System.

Results Microsomal Metabolism of DNB. With liver microsomes (2.0 mg of protein/mL), after 20 min of incubation under anaerobic conditions, 94.1 ( 2.4% of the DNB was reduced. 4.6 ( 2.4% to NPHA and 89.5 ( 4.8% to NA (Figure 1A). Under aerobic conditions, the presence of oxygen inhibited metabolite formation for the first 5 min of the incubation (Figure 1B). At the end of this 5-min delay, oxygen stores could have been depleted, allowing reduction to proceed past the 1e- step, generating NPHA and NA. After 20 min of incubation, no further metabolism occurred. Aerobic conditions inhibited metabolite formation at all protein concentrations tested (Figure 1C). No DNB metabolism was detected in the seminiferous tubule microsomal fraction under either anaerobic or aerobic conditions. Mitochondrial Metabolism of DNB. With liver mitochondria (2.0 mg of protein/mL), after 30 min of incubation with NADPH, under anaerobic conditions, 64.1 ( 4.0% of the DNB was metabolized. 50.7 ( 7.7% to NPHA and 13.3 ( 4.0% to NA (Figure 2A). After 30 min of incubation with NADH as a cofactor and in the absence of oxygen, 18.2 ( 4.7% of added DNB was reduced to NPHA, the only metabolite detected (Figure 2B). Relatively greater levels of NADPH-supported DNB metabolism were also seen with higher protein concentrations (Figure 2C). Under aerobic conditions, no metabolism occurred within the liver mitochondrial incubations. With the seminiferous tubule mitochondria (2.0 mg of protein/mL) incubated anaerobically for 30 min with NADPH, 17.7 ( 1.4% of the DNB was metabolized. 14.3 ( 3.5 to NPHA and 1.31 ( 0.5% to NA (Figure 3A). Using the NADH cofactor, 12.6 ( 1.7% of the DNB was metabolized, 11.1 ( 1.5% to NPHA and 1.4 ( 0.2% to NA (Figure 3B). The higher level of NADPH-dependent DNB metabolism compared to that supported by NADH is small but significant (P < 0.05) at all three (1.5, 2.0,

1,3-Dinitrobenzene Metabolism and Protein Binding

Figure 1. Liver microsomal metabolism of DNB (anaerobic vs aerobic). (A) Anaerobic conditions at 2.0 mg of protein/mL in 2 mM NADPH and 200 µM [14C]DNB: (2) DNB, (b) NPHA, (0) NA. (B) As in panel A, but incubated under aerobic conditions. (C) Percentage of DNB metabolized in incubations carried out for 15 min at 1.5, 2.0, and 2.5 mg of protein/mL under anaerobic conditions (light shading) vs aerobic conditions (dark shading) with 2 mM NADPH and 200 µM [14C]DNB. Each data point represents the mean ( SD of three experiments. At each protein concentration, the percentage of DNB metabolized under anaerobic conditions was significantly greater than that which occurred aerobic conditions (P < 0.05).

and 2.5 mg of protein/mL) concentrations (Figure 3C). There was no seminiferous tubule mitochondrial metabolism of DNB under aerobic conditions. Cytosolic Metabolism of DNB. After 30 min of incubation with NADPH under anaerobic conditions, the liver cytosol (2.0 mg of protein/mL) metabolized 36.9 ( 2.7% of the DNB, 28.9 ( 2.5% to NPHA and 8.1 ( 0.4% to NA (Figure 4A). With NADH as a cofactor, 47.7 ( 2.1% of the DNB was metabolized, 38.1 ( 1.1% to NPHA and 9.6 ( 2.3% to NA (Figure 4B). Increasing the amount of cytosolic protein gave significantly (P < 0.05) higher levels of NADH-supported DNB metabolism (Figure 4C). Under anaerobic conditions, no DNB metabolism was detected with the seminiferous tubule cytosol. In both the liver and seminiferous tubule cytosolic incuba-

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Figure 2. Liver mitochondrial metabolism of DNB (NADPH vs NADH). (A) Anaerobic conditions at 2.0 mg of protein/mL in 2 mM NADPH and 200 µM [14C]DNB: (2) DNB, (b) NPHA, (0) NA. (B) As in panel A, but incubated with 2 mM NADH in place of NADPH. (C) Percentage of DNB metabolized in incubations carried out for 30 min at 1.5, 2.0, or 2.5 mg of protein/mL with 2 mM NADPH (light shading) vs 2 mM NADH (dark shading). Each data point represents the mean ( SD of three experiments. At each protein concentration, the percentage of DNB metabolized in incubations containing NADPH was significantly greater than the percentage of DNB metabolized in incubations containing NADH (P < 0.05).

tions, no metabolite formation occurred under aerobic conditions. SDS-PAGE/Fluorography of Liver Microsomal, Mitochondrial, and Cytosolic Proteins Incubated with [14C]DNB. Consistent with a metabolically derived [14C]-adduct, no radiolabeled proteins were found at the T0 timepoint in liver microsomes (Figure 5, lane B). However, after 30 min of incubation under anaerobic conditions, multiple radiolabeled bands were detected (Figures 5, lane C). Under aerobic conditions, [14C]-adduct formation in liver microsomal proteins at the T0 and 30 min incubation timepoints was similar to those seen under anaerobic conditions (data not shown). Like the liver microsomes, after 30 min of incubation under anaerobic conditions, many liver mitochondrial

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Figure 3. Seminiferous tubule mitochondrial metabolism of DNB (NADPH vs NADH). (A) Anaerobic conditions at 2.0 mg of protein/mL in 2 mM NADPH and 200 µM [14C]DNB: (2) DNB, (b) NPHA, (0) NA. (B) As in panel A, but incubated with 2 mM NADH in place of NADPH. (C) Percentage of DNB metabolized in incubations carried out for 30 min at 1.5, 2.0, or 2.5 mg of protein/mL with 2 mM NADPH (light shading) vs 2 mM NADH (dark shading). Each data point represents the mean ( SD of three experiments. At each protein concentration, the percentage of DNB metabolized in incubations containing NADPH was significantly greater than the percentage of DNB metabolized in incubations containing NADH (P < 0.05).

Figure 4. Liver cytosolic metabolism of DNB (NADPH vs NADH). (A) Anaerobic conditions at 2.0 mg of protein/mL in 2 mM NADPH and 200 µM [14C]DNB: (2) DNB, (b) NPHA, (0) NA. (B) As in panel A, but incubated with 2 mM NADH in place of NADPH. (C) Percentage of DNB metabolized in incubations carried out for 30 min at 1.5, 2.0, or 2.5 mg of protein/mL with 2 mM NADPH (light shading) vs 2 mM NADH (dark shading). Each data point represents the mean ( SD of three experiments. At each protein concentration, the percentage of DNB metabolized in incubations containing NADH was significantly greater than the percentage of DNB metabolized in incubations containing NADPH (P < 0.05).

proteins were radiolabeled (Figure 5, lane E). No [14C]adduct formation was seen at the T0 incubation timepoint (Figure 5, lane D). Under aerobic conditions, no radiolabeling was visible (data not shown). Multiple proteins were radioactively labeled after 30 min of incubation of cytosol with DNB (Figure 5, lane G). However, relative to the microsomal and mitochondrial proteins, the intensity of radiolabel was noticeably less. No label was detected at T ) 0 min., indicating that the adducting species is a metabolite of DNB (Figure 5, lane F). No protein radiolabeling was visible under aerobic conditions (data not shown). SDS-PAGE/Fluorography of Seminiferous Tubule Microsomal, Mitochondrial and Cytosolic Proteins Incubated with [14C]DNB. There was no protein

radiolabeling visible in seminiferous tubule microsomes or cytosol incubated under anaerobic or aerobic conditions. Under anaerobic conditions after 30 min of incubation, the seminiferous tubule mitochondria (2.5 mg of protein/mL) contained multiple radiolabeled bands (Figure 6, lane C). No label was detected at the T0 incubation timepoint (Figure 6, lane B). No adduct formation was visible under aerobic conditions (data not shown). Effect(s) of GSH and Increased Protein Concentration on [14C]Protein Adduct Formation in Seminiferous Tubule Mitochondria Treated with [14C]DNB. Prior to incubation under anaerobic conditions for 30 min, GSH (6 mM incubation concentration) was added to seminiferous tubule mitochondria (2.5 mg of protein/ mL). At the T0 incubation timepoint, there was no radiolabeling visible (Figure 6, lane D). However, after



Figure 5. Coomassie stained SDS-polyacrylamide gel (left) and corresponding fluorogram (right) of protein from anaerobic incubations containing 200 µM [14C]DNB, 2 mM NADPH, and 2.5 mg of protein/mL of liver microsomes at T0 (lane B) and after 30 min (lane C), mitochondria at T0 (lane D) and after 30 min (lane E) or cytosol at T0 (lane F) and after 30 min (lane G). Lane A contains the molecular weight markers in kilodaltons.

Figure 6. Coomassie stained SDS-polyacrylamide gel (left) and corresponding fluorogram (right) of protein from anaerobic incubations containing 200 µM [14C]DNB, 2 mM NADPH, and 2.5 mg of protein/mL of seminiferous tubule mitochondria at T0 (lane B) and after 30 min (lane C); 200 µM [14C]DNB, 2 mM NADPH, 6 mM GSH, and 2.5 mg of protein/mL of seminiferous tubule mitochondria at T0 (lane D) and after 30 min (lane E); 200 µM [14C]DNB, 2 mM NADPH, and 20 mg of protein/mL of seminiferous tubule mitochondria at T0 (lane F) and after 30 min (lane G). Lane A contains the molecular weight markers in kilodaltons.

30 min of incubation, this treatment, while having little or no effect on the number of radiolabeled proteins, did produce a visible decrease in the overall level of adduct formation (Figure 6, lane E). Changing the incubation protein concentration of seminiferous tubule mitochondria from 2.5 to 20 mg of protein/mL enhanced protein radiolabeling throughout the mitochondria. However, this increase in signal appeared to be especially prominent in a protein band with an estimated molecular weight of 54 kDa (Figure 6, lanes F and G). Two-Dimensional Gel Electrophoresis of Liver Mitochondrial Proteins Incubated with [14C]DNB. Fluorography of the second dimension (SDS-PAGE) indicated three predominantly radiolabeled proteins (1-3) which were chosen for identification. These three proteins were relatively high in abundance and approximately equal in size as indicated by Coomassie blue protein staining (Figure 7). Sequencing of Three Predominantly Radiolabeled Liver Mitochondrial Proteins. Proteins 1-3, visualized with Coomassie blue stain, were cut from the gel, tryptically digested in situ, and the peptides resolved using reverse-phase HPLC equipped with UV detection. In all cases the peptides sequenced were identical to rat mitochondrial aldehyde dehydrogenase (EC 1.2.1.3). For protein 1, two peptides, each consisting of 13 amino acids, were sequenced. The sequences were identical to those

of amino acids 162-174 and 397-409. For protein 2, one peptide (10 amino acids) was sequenced and found to be identical to amino acids 444-453. Protein 3 had two of its peptides, each containing 13 amino acids, analyzed. The sequences matched those of amino acids 162-174 and 397-409 (Table 1).

Discussion Nitroreduction is considered to play a role in the toxicity of numerous nitroaromatics. Investigations by O’Brien et al. have correlated the one-electron redox potentials of many nitroaromatics to their ability to cause cytotoxicity in isolated rat hepatocytes (30). Nitroaromatic compounds have also been shown to covalently adduct hemoglobin, after metabolic activation (17). DNB, which produces testicular toxicity, is metabolized in vivo and in vitro in testes and liver preparations (11-15). Numerous enzymes have nitroreductase activity and could be responsible for the observed metabolism of DNB. Known nitroreductases are xanthine oxidase (22, 31-33) and DT diaphorase of the cytosol (33), aldehyde dehydrogenase (34), found in the endoplasmic reticulum (35), mitochondria (36), and cytosol (22), and cytochrome p450 reductase in the microsomal fraction (16, 33). The mitochondrial localization of DNB metabolism in the seminiferous tubule suggests that aldehyde dehydrogenase may be a participant.

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Figure 7. Coomassie stained two-dimensional gel of liver mitochondrial proteins which were incubated with NADPH (2 mM) and [14C]DNB (200 µM, 1.34 µCi) for 90 min (top) and the corresponding fluorogram (bottom). The highly 14C-labeled proteins (1-3) are indicated with arrows. The 14C-ink spots used to align the gel with the corresponding fluorogram are located on the lower left.

In the present studies, for each subcellular fraction, the presence of oxygen inhibited metabolite formation, suggesting the presence of a redox-cycle. In this cycle, DNB accepts an electron to form the nitroxyl anion radical which donates an electron to molecular oxygen, forming the superoxide anion radical and regenerating DNB (Scheme 1). In most of the aerobic incubations, this putative redox-cycling completely eliminated the production of NPHA and NA. However, with liver microsomes, the presence of oxygen merely delayed the onset of metabolite formation. Liver microsomes have high nitroreductase activity and the active cycling could have depleted oxygen required to maintain the cycle. In the

absence of oxygen, the nitroxyl anion radical would be further reduced to form NPHA and NA. Electron spin resonance studies have demonstrated that the nitroxyl anion and superoxide anion radicals can be enzymatically generated from numerous nitroaromatic compounds. Studies using incubations containing liver microsomes in the presence of NADPH, have shown that nitroaromatics such as nitrobenzene (37), p-nitrobenzoic acid (38, 39) and nitrofurantoin (40, 41) produced the nitroxyl anion radical species. Other experiments have demonstrated NADPH-dependent enzymatic generation of the superoxide anion radical using metronidazole in aerobic liver microsomal incubations (42) and nitrofurantoin or nifurtimox in aerobic liver mitochondrial incubations containing either NADPH or NADH (43). These data provide strong evidence that nitroaromatics undergo redox-cycling under aerobic conditions. Hence, the observed inhibition of DNB metabolite formation in this study is likely due to the same mechanism. In the anaerobic incubations of liver microsomes, metabolite formation occurred until the DNB was depleted. However, under aerobic conditions, a plateau in metabolite formation occurs after twenty minutes of incubation (Figure 1B). This inhibition may be caused by depletion of the NADPH cofactor since futile cycling diverts electrons derived from reducing equivalents away from the nitroxyl anion radical to molecular oxygen. This diversion ultimately depletes the NADPH required for nitroreduction. Depletion of reducing equivalents may be contributing to the testicular lesion by decreasing the activity of GSSG reductase, which requires NADPH to maintain cellular glutathione in its reduced form (44). Hence, this inhibition may indirectly cause oxidative stress by diminishing the detoxification of active oxygen species via glutathione peroxidase. Another explanation for the observed cessation of metabolite formation is protein oxidation. The superoxide anion radical, generated via the redox-cycle, and its degradation products such as the hydroxyl radical, could oxidatively damage the DNB reductase(s), eliminating enzymatic activity (45). Oxidative stress generated via a nitroreductive redox-cycle is thought to be responsible for the pulmonary edema and fibrosis sometimes caused by nitrofurantoin therapy. (39). Another potential mechanism of DNB induced testicular toxicity is the formation of reactive metabolites, which


adduct critical biological macromolecules. Numerous nitroaromatics such as ronidazole, metronidazole, nitrofurazone, and nitrofurantoin have been shown to undergo metabolic activation with protein adduct formation (46, 47). In previous studies, DNB metabolism and protein binding occurred in liver hepatocytes and testicular cocultures (11, 14). The present study demonstrated that the protein adducting species is a metabolite of DNB. Treatment with [14C]DNB produced little or no binding at T ) 0 min. However, under anaerobic conditions, adduct levels increased with incubation time and, except for the liver microsomes, which contained relatively high DNB reductase activity, were substantially higher than those generated under aerobic conditions. These data suggest that a metabolite downstream of the nitroxyl anion radical was responsible for protein binding. As mentioned earlier, NNB has been shown to nonenzymatically react with numerous sulfhydryl-containing compounds such as hemoglobin, cysteine, and GSH (22). Hence, if the adducting species were this metabolite, elevating the concentration of available sulfhydryls via increasing GSH should decrease protein radiolabeling. This effect has been demonstrated with other nitroaromatics in incubations treated with GSH or other thiols (48). In the present investigation, this experiment was carried out using anaerobic incubations containing seminiferous tubule mitochondria, [14C]DNB, and NADPH. The results show that the addition of 6 mM GSH had little or no effect on the binding pattern (i.e., the number of proteins labeled) but did decrease overall binding levels (Figure 6, lanes C and E). This decrease in adduct formation and the absence of NNB in the metabolic profile suggest that this metabolite could be the binding species. Adduct formation occurred throughout the seminiferous tubule mitochondria with multiple bands exhibiting radiolabel. However, in experiments where whole seminiferous tubules were incubated with [14C]DNB, SDSPAGE/fluorography of the isolated mitochondrial protein yielded a single radiolabeled protein band with an estimated molecular weight of 54 kDa (15). In the present study, increasing the incubation protein concentration of the seminiferous tubule mitochondria to 20 mg/mL produced a similar binding profile with enhanced radiolabeling of a single protein band of comparable weight (∼54 kDa) (Figure 6, lanes C and G). Two-dimensional gel electrophoresis/fluorography of liver mitochondrial protein incubated with [14C]DNB and NADPH showed relatively enhanced radiolabeling of three proteins which were also ∼54 kDa in size (Figure 7). Each protein was sequenced via Edman degradation and identified as rat mitochondrial aldehyde dehydrogenase. The predominantly radiolabeled proteins (1-3) are relatively abundant, as viewed with Coomassie-blue protein staining, indicating that this enzyme exists at high levels in the mitochondria (Figure 7). However, aldehyde dehydrogenase is not the most abundant protein in the gel. Therefore, the enhanced radiolabeling must be due to other properties of the enzyme. Aldehyde dehydrogenase contains cysteine residues (49), which have been shown to react nonenzymatically with NNB (22). Aldehyde dehydrogenase also contains nitroreductase activity, making it a potential candidate for DNB metabolism and binding in situ. Previous studies have shown that this enzyme is capable of reducing numerous


nitroaromatics such as nitrofurazone, 2-bromo-5-nitrothiazole, 1,3-dimethyl-5-nitrouracil, 4-nitropyridine-N-oxide, and 2-amino-5-nitrothiazole using aldehydic cosubstrates, shown to occur endogenously via lipid peroxidation, as electron donors (50). The Cys-302 residue is critical to the activity of this enzyme (49). Therefore, it is possible that NNB, which has been shown to react with cysteine sulfhydryls (22), binds this residue, decreasing or eliminating the activity of the aldehyde dehydrogenase. Cys-302 adduct formation is thought to be involved in the inhibition of liver mitochondrial aldehyde dehydrogenase activity caused by acetaminophen treatment. Over 70% of acetaminophen’s reactive metabolites have been shown to bind cysteine residues on target proteins (49). Two important reactions carried out by aldehyde dehydrogenase are the reduction of NAD to NADH and oxidation of aldehydes to their corresponding carboxylic acids. Decreased levels of NADH in the mitochondria may reduce the rate of oxidative phosphorylation (49). In addition, decreasing NADH levels may adversely affect the integrity of the mitochondrial membrane and increase the release of calcium (51). Inhibition of aldehyde dehydrogenase could also generate elevated aldehyde levels within the mitochondria. Acetaldehyde is known to covalently bind proteins and may be involved in lipid peroxidation. It has also been associated with decreased oxidative phosphorylation (52). The data in this study suggest that DNB treatment could produce testicular toxicity through a mitochondrial mechanism in the seminiferous tubules. This mechanism could involve either oxidative stress, generated by redoxcycling, and/or the inhibition of aldehyde dehydrogenase. Future studies will focus on the ability of DNB treatment to produce these effects in seminiferous tubule mitochondria.

Acknowledgment. The authors’ research reported here was supported by the NIEHS Center for Environmental Health Sciences Grant ES05707 (P01) and the NIH Grant ES05701 (R01).

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