Isoform-Selective Inactivation of Human Arylamine N

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Chem. Res. Toxicol. 2009, 22, 1962–1974

Isoform-Selective Inactivation of Human Arylamine N-Acetyltransferases by Reactive Metabolites of Carcinogenic Arylamines Li Liu, Carston R. Wagner, and Patrick E. Hanna* Department of Medicinal Chemistry, UniVersity of Minnesota, 308 HarVard Street SE, Minneapolis, Minnesota 55455 ReceiVed August 3, 2009

Human arylamine N-acetyltransferases (NATs) are expressed as two polymorphic isoforms, NAT1 and NAT2, that have toxicologically significant functions in the detoxification of xenobiotic arylamines by N-acetylation and in the bioactivation of N-arylhydroxylamines by O-acetylation. NAT1 also catalyzes the N-acetylation of 4-aminobenzoylglutamic acid, a product of folic acid degradation, and is associated with endogenous functions in embryonic development. On the basis of earlier studies with hamster NAT1, hamster NAT2, and human NAT1, we proposed that human NAT2 would be more susceptible than NAT1 to inactivation by N-arylhydroxamic acid metabolites of arylamines. Kinetic analyses of the inactivation of recombinant NAT1 and NAT2 by the N-arylhydroxamic acid, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), as well as the inactivation of NAT2 by N-hydroxy-4-acetylaminobiphenyl (NOH-4-AABP), resulted in second-order inactivation rate constants (kinact/KI) that were several fold greater for NAT2 than for NAT1. Mass spectrometric analysis showed that inactivation of NAT2 in the presence of the N-arylhydroxamic acids was due to formation of a sulfinamide adduct with Cys68. Treatment of HeLa cells with N-OH-4-AABP and N-OH-AAF revealed that the compounds were less potent inactivators of intracellular NAT activity than the corresponding nitrosoarenes, but unexpectedly, the hydroxamic acids caused a significantly greater loss of NAT1 activity than of NAT2 activity. Nitrosoarenes are the electrophilic products responsible for NAT inactivation upon interaction of the enzymes with Narylhydroxamic acids, as well as being metabolic products of arylamine oxidation. Treatment of recombinant NAT2 with the nitrosoarenes, 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NOF), caused rapid and irreversible inactivation of the enzyme by sulfinamide adduct formation with Cys68, but the kinact/KI values for inactivation of recombinant NAT2 and NAT1 did not indicate significant selectivity for either isoform. Also, the IC50 values for inactivation of HeLa cell cytosolic NAT1 and NAT2 by 4-NO-BP were similar, as were the IC50 values obtained with 2-NO-F. Treatment of HeLa cells with low concentrations (1-10 µM) of either 4-NO-BP or 2-NO-F resulted in preferential and more rapid loss of NAT1 activity than NAT2 activity. Because of its wide distribution in human tissues and its early expression in developing tissues, the apparent high sensitivity of intracellular NAT1 to inactivation by reactive metabolites of environmental arylamines may have important toxicological consequences. Introduction 1

Arylamine N-acetyltransferases (NATs) (EC 2.3.1.5) are expressed as two polymorphic isoforms in human tissues (1, 2). NATs catalyze the acetyl coenzyme A (AcCoA)-dependent phase II biotransformation of arylamines (aromatic amines) (ArNH2) to N-arylacetamides (ArNHCOCH3), a process that is responsible for detoxification of numerous xenobiotics. In contrast, the NAT-catalyzed conversion of N-arylhydroxyl* To whom correspondence should be addressed. Tel: 612-625-4152. Fax: 612-624-0139. E-mail: [email protected]. 1 Abbreviations: AcCoA, acetyl coenzyme A; 2-AF, 2-aminofluorene; 4-ABP, 4-aminobiphenyl; DMEM, Dulbecco’s modified Eagle’s medium; ESI, electrospray ionization; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GR, glutathione reductase; GSH, glutathione; MALDI, matrix-assisted laser desorption/ionization; NAT, arylamine N-acetyltransferase; NO-B, nitrosobenzene; 4-NO-BP, 4-nitrosobiphenyl; 2-NO-F, 2-nitrosofluorene; N-OH-4-AABP, N-hydroxy-4-acetylaminobiphenyl; N-OH-AAF, N-hydroxy-2-acetylaminofluorene; 2-NO-T, 2-nitrosotoluene; PABA, p-aminobenzoic acid; PAS, p-aminosalicylic acid; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; PNPA, p-nitrophenyl acetate; Q-TOF, quadrupole time-of-flight; SMZ, sulfamethazine.

amines (ArNHOH) to reactive, electrophilic N-acetoxyarylamine conjugates (ArNHOCOCH3) is a bioactivation process that is considered to be central to the initiation of the mutagenic and carcinogenic effects of the N-hydroxylated metabolites of arylamines. Reactive N-acetoxyarylamines also are produced by an N,O-acetyl transfer reaction that occurs upon interaction of N-arylhydroxamic acids (ArNHOHCOCH3) with NATs (3-5). Human NAT2 and its isoform, NAT1, share 81% sequence identity and exhibit several distinct substrate specificities, with sulfamethazine (SMZ) being selectively N-acetylated by NAT2 and with p-aminobenzoic acid (PABA) being a specific substrate for NAT1 (6-8). The well-documented substrate specificities of NAT1 and NAT2 notwithstanding, a number of arylamines, including the carcinogens 4-aminobiphenyl (4-ABP) and 2-aminofluorene (2-AF), are readily acetylated by both NATs, and several N-arylhydroxylamines and N-arylhydroxamic acids are converted to N-acetoxyarylamines by both enzymes (9-13). Both NAT1 and NAT2 are expressed as proteins with varying degrees of stability and catalytic capability due to single

10.1021/tx9002676 CCC: $40.75  2009 American Chemical Society Published on Web 10/20/2009

InactiVation of NATs by Arylamine Metabolites

Figure 1. Structures of N-arylhydroxamic acids and nitrosoarenes used in this study.

nucleotide polymorphisms (SNPs) in the DNA coding exons. The NAT SNPs can dramatically influence the N-acetylation phenotype (1, 3, 14). The NAT2 slow acetylator genotype is associated with an elevated frequency of adverse reactions to hydrazine or arylamine drugs that are dependent upon NAT2 for metabolic disposition (15). An association between either NAT1 or NAT2 genotype and risk for a number of organspecific cancers is supported by the results of epidemiological investigations (14, 16). The most extensively studied and most thoroughly documented association is between the NAT2 slow acetylator genotype and bladder cancer risk, an association that is strengthened by aromatic amine exposure from cigarette smoke or other sources (17-21). The relationship between genetically attenuated NAT2 activity and the untoward effects of arylamines and other NAT2 substrates suggest the importance of identifying and characterizing environmental agents and their metabolic products that may impair NAT activity. Such metabolic products include N-arylacetohydroxamic acids, which are in vivo metabolites of both arylamines and N-arylacetamides in humans and animals (22-25). We reported that N-hydroxy-2-acetylaminofluorene (N-OH-AAF) (Figure 1), the N-arylacetohydroxamic acid metabolite of 2-AF, caused inactivation of hamster NAT1 in vivo and in vitro and that N-hydroxy-4-acetylaminobiphenyl (NOH-4-AABP) (Figure 1), a metabolite of the tobacco carcinogen, 4-ABP, inactivated both of the hamster recombinant NAT isoforms, as well as human recombinant NAT1 (26-28). Interaction of an N-arylacetohydroxamic acid with an NAT results in acetylation of the active site cysteine thiol and subsequent generation of a nitrosoarene (ArNdO), which reacts with the enzyme to form a sulfinamide adduct (27, 28). It was proposed that the susceptibilities of NATs to inactivation by N-arylacetohydroxamic acids are inversely related to the stabilities of the acetyl-enzyme intermediates and that human NAT2 might be expected to be more readily inactivated than human NAT1. In this paper, we report the results of our investigation of the in vitro kinetics and mechanism of inactivation of recombinant human NAT2 and NAT1 by N-OH-AAF, as well as the inactivation of NAT2 by N-OH-4-AABP. The unexpected finding that intracellular NAT1 is more susceptible than NAT2 to inactivation by N-OH-AAF and N-OH-4-AABP prompted a study of the inactivation of NAT2 by nitrosoarenes and a determination of the relative susceptibilities of NAT2 and NAT1 to inactivation by the nitroso metabolites of 4-ABP and 2-AF. The structures of N-OH-AAF, N-OH-4-AABP, and the nitrosoarenes used in this study are shown in Figure 1.

Experimental Procedures Caution: N-OH-4-AABP, N-OH-AAF, 4-nitrosobiphenyl (4-NOBP), 2-nitrosofluorene (2-NO-F), nitrosobenzene (NO-B), and

Chem. Res. Toxicol., Vol. 22, No. 12, 2009 1963 2-nitrosotoluene (2-NO-T) should be handled in accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens (29). Materials and Methods. Recombinant human NAT1 and NAT2 were prepared as described previously (10, 30). N-OH-4-AABP, N-OH-AAF, 4-NO-BP, and 2-NO-F were synthesized as reported (27, 28, 31, 32). AcCoA, p-anisidine, glutathione (GSH), 3-(Nmorpholino)propanesulfonic acid (MOPS), NO-B, p-nitrophenylacetate (PNPA), pepsin A, and SMZ were purchased from Sigma (St. Louis, MO). Bio-Spin 6 Tris columns were obtained from BioRad (Hercules, CA). 2-NO-T was purchased from Aldrich (Milwaukee, WI). HeLa cells were obtained from American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Mediatech (Herndon, VA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA). Protein concentrations were determined by the method of Bradford (33). All experiments were conducted in degassed buffers and under aerobic conditions. Spectrophotometric data were acquired on a Varian Cary 50 UV-vis spectrophotometer equipped with a circulation water bath. Kinetic data were analyzed with the JMP IN software suite (SAS Institute, Inc.). Statistical significance of differences between means was assessed with Student’s t test. IC50 values were calculated with Kaleidagraph 3.6 (Synergy Software). NAT2 Activity Assay. The assay was performed with PNPA as the acetyl donor and p-anisidine as the acetyl acceptor as described previously (10). Time-Dependent Inactivation of NAT2 by N-OH-4-AABP and N-OH-AAF at 37 °C. The incubation mixtures contained 24 µg/mL (0.71 µM) of NAT2 and either N-OH-4-AABP (100-1250 µM) or N-OH-AAF (5-150 µM) and tetrasodium pyrophosphate buffer (50 mM, pH 7.0; 0.1 mM DTT; 1% glycerol) in a total volume of 50 µL. The reaction was initiated by the addition of N-OH-4-AABP or N-OH-AAF dissolved in DMSO (2.5 µL). Aliquots (2 µL) were withdrawn at 30 s intervals (0-360 s) in experiments with N-OH-4-AABP or at 45 s intervals (0-270 s) in experiments with N-OH-AAF and were transferred to an assay cuvette. Control incubations contained DMSO but not N-OH-AAF or N-OH-4-AABP. Time-Dependent Inactivation of NAT1 by N-OH-AAF at 37 °C. The incubation mixtures contained NAT1 (50 µg/mL, 1.46 µM), N-OH-AAF (30-800 µM), and tetrasodium pyrophosphate buffer (50 mM, pH 7.0; 0.1 mM DTT; 1% glycerol) in a total volume of 50 µL. The reaction was initiated by the addition of N-OH-AAF dissolved in DMSO (2.5 µL). Aliquots (2 µL) were withdrawn at 45 s intervals (0-270 s) and transferred to an assay cuvette. The NAT1 activity was measured as described previously (28). Control incubations contained DMSO but not N-OH-AAF. Half-Life of Acetyl-NAT2 Intermediate. NAT2 (56.5 µg/mL, 1.67 µM) was incubated with PNPA (2 mM) in MOPS buffer (398 µL; 100 mM, pH 7.0; 150 mM NaCl; 0.1 mM DTT) at 37 °C. The reactions were initiated by addition of PNPA dissolved in DMSO (2 µL). The rate of the hydrolysis was determined by monitoring the increase in absorbance at 400 nm due to the formation of p-nitrophenol (ε400nm ) 9400 M-1 cm-1) for 5 min. Control incubations were conducted in the absence of enzyme. Formation of 2-NO-F and 4-NO-BP during the Incubation of NAT1 or NAT2 with N-OH-AAF or N-OH-4-AABP. In a total volume of 500 µL, NAT1 (4 µM) or NAT2 (4 µM) was incubated with either N-OH-AAF (200 µM) or N-OH-4-AABP (200 µM) in MOPS buffer (100 mM, pH 7.0; 150 mM NaCl; 0.025 mM DTT), which had been extensively degassed under vacuum, at 37 °C for 30 min. The reactions were initiated by addition of N-OH-AAF or N-OH-4-AABP dissolved in DMSO (4 µL). The formation of 2-NO-F was monitored continuously at A380nm (ε380nm ) 19800 M-1 cm-1), and the formation of 4-NO-BP was monitored at A350nm (ε350nm ) 11800 M-1 cm-1) for 30 min. Control experiments were conducted in which either enzyme or hydroxamic acid was omitted. Time-Dependent Inactivation of NAT2 by Nitrosoarenes at 37 °C. The incubation mixtures contained NAT2 (34.5 µg/mL, 1 µM), 4-NO-BP (0.1-5 µM), 2-NO-F (0.1-2 µM), NO-B (500-4000

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µM), or 2-NO-T (100-2000 µM) and potassium phosphate buffer (20 mM, pH 7.4; 1 mM EDTA) in a total volume of 75 µL. The reaction was initiated by addition of nitrosoarenes dissolved in 1.5 µL of DMSO. The final concentration of DMSO was 2%. Aliquots (2 µL) were withdrawn at 15 (4-NO-BP, 0-45 s), 10 (2-NO-F, 0-30 s), 30 (2-NO-T, 0-120 s), or 60 s (NO-B, 0-180 s) intervals and transferred to an assay cuvette. The controls contained DMSO but not nitrosoarene. Inactivation of NAT2 by NO-B and 2-NO-T in the Presence of AcCoA. In a total volume of 98 µL, NAT2 (34.5 µg/mL, 1 µM) was incubated with AcCoA (20 µM) for 1 min in potassium phosphate buffer (20 mM, pH 7.4; 1 mM EDTA) at 37 °C. 2-NO-T or NO-B dissolved in 2 µL of DMSO was added. The final concentration of 2-NO-T was 500 µM, and that of NO-B was 800 µM. The incubation was continued for 2 (2-NO-T) or 3 min (NO-B). Aliquots (2 µL) were withdrawn and assayed for NAT2 transacetylation activity. Inactivation of NAT2 by 4-NO-BP and 2-NO-F in the Presence of GSH. In a total volume of 147 µL, NAT2 was incubated with GSH for 2 min in potassium phosphate buffer (20 mM, pH 7.4; 1 mM EDTA) at 37 °C. 4-NO-BP or 2-NO-F dissolved in 3 µL of DMSO was added, and incubation was continued for 60 s at 37 °C. The final concentrations were as follows: NAT2, 25 µg/mL (0.73 µM); GSH, 0.5, 1, or 5 mM; and 4-NO-BP or 2-NO-F, 1 µM. Aliquots (2 µL) were withdrawn and assayed for NAT2 activity. Controls contained only potassium phosphate buffer and DMSO. There was no statistically significant difference between control activities determined in the presence and absence of GSH (p > 0.2). Sample Preparation for Nanoelectrospray Ionization (ESI)Quadrupole Time-of-Flight (Q-TOF) MS of N-OH-4-AABPTreated NAT2. To NAT2 (84 µg, 40 µM) in potassium phosphate buffer (60 µL, 20 mM, pH 7.4; 1 mM EDTA; 10% glycerol; 0.1 mM DTT) was added N-OH-4-AABP dissolved in 2 µL of DMSO. The final concentration of N-OH-4-AABP was 500 µM. DMSO only was added to the control incubation mixtures. After a 10 min incubation at 37 °C, the residual activity was less than 3% of the control activity. The reaction mixture was loaded onto a Bio Spin 6 Tris Column, which had been equilibrated with the potassium phosphate buffer. After centrifugation at 1000g for 4 min, 8 µL of the solution was used to measure the protein concentration and residual activity. The remaining solution was stored at -80 °C. Sample Preparation for Nano-ESI-Q-TOF MS of N-OHAAF-Treated NAT2. The experimental procedures were the same as described for N-OH-AAF-treated human NAT1 except that the concentration of human NAT2 was 25 µM and the incubation time was 9 min. Sample Preparation for Nano-ESI-Q-TOF MS of N-OHAAF-Treated NAT1. To NAT1 (150 µg, 44 µM) in potassium phosphate buffer (100 µL, 20 mM, pH 7.4; 1 mM EDTA; 10% glycerol; 0.1 mM DTT) was added N-OH-AAF dissolved in 2 µL of DMSO. The final concentration of N-OH-AAF was 500 µM. DMSO only was added to the control incubation mixtures. After a 12 min incubation at 37 °C, the residual activity was less than 3% of the control activity. The reaction mixture was loaded onto a Bio Spin 6 Tris Column. After centrifugation, the sample was concentrated to 30 µL by freeze drying. Sample Preparation for Nano-ESI-Q-TOF MS of 4-NO-BPTreated NAT2 and 2-NO-F-Treated NAT2. The experimental procedures were identical to those described for N-OH-4-AABPtreated NAT2 except that the incubation time with 4-NO-BP was 3 min and with 2-NO-F it was 5 min. The final concentrations of 4-NO-BP and 2-NO-F were 250 and 100 µM, respectively. Pepsin Digestion of N-OH-4-AABP-Treated NAT2. NAT2 was incubated with N-OH-4-AABP as described above for the sample preparation for nano-ESI-Q-TOF MS. The solution (50 µL) obtained with the Bio Spin 6 Tris column was concentrated to 30 µL by freeze drying. HCl (2.2 µL, 1 N) was added to adjust the pH to 1.3. Digestion was initiated by the addition of pepsin (1 mg/mL in H2O, pH 1.3). The ratio of pepsin to NAT2 protein was approximately 1:100. After incubation at 37 °C for 3 h, the reaction

Liu et al. was terminated by adjusting the pH to 8 with 1 N NaOH (1.7 µL). Acetonitrile (2.1 µL) was added, and the sample was stored at -80 °C. A control digest, to which DMSO had been added, rather than the N-OH-4-AABP-DMSO solution, was obtained by an identical procedure. Pepsin Digestion of 4-NO-BP, N-OH-AAF, or 2-NO-FTreated NAT2. The same procedures were carried out to inactivate NAT2 with 4-NO-BP, N-OH-AAF, or 2-NO-F as described above for the sample preparation for nano-ESI-Q-TOF MS. The digestion procedure was the same as described for pepsin digestion of N-OH4-AABP-treated NAT2. Pepsin Digestion of N-OH-AAF-Treated NAT1. The same procedures were carried out to inactivate NAT1 with N-OH-AA-F as described above for the sample preparation for nano-ESI-Q-TOF MS. The digestion procedure was the same as described for pepsin digestion of N-OH-4-AABP-treated NAT2. Nano-ESI-Q-TOF MS of Unmodified and Modified NAT1 and NAT2. NAT1 and NAT2 samples were desalted with Millipore C4 ZipTips and analyzed by nano-ESI-Q-TOF MS as previously described (28). Matrix-Assisted Laser Desorption/Ionization (MALDI)-TOF MS Screening and Sequencing of Peptides. The pepsin digests were desalted with Millipore C18 ZipTips and analyzed by MALDITOF MS and sequenced by MALDI-TOF MS/MS as previously described (28). MS Data Analysis. Theoretical masses of proteins, peptides, and fragment ions were generated with Protein Prosepector (http:// prospector.ucsf.edu) and with the ABI AS software package. Cell Culture Conditions. HeLa cells were maintained as monolayers in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin-fungizome in a humidified, 5% CO2 atmosphere at 37 °C. NAT2 Activity in HeLa Cell Cytosol. At approximately 90% confluence, cell monolayers (100 mm plate) were washed with 5 mL of phosphate-buffered saline (PBS). Cell monolayers were exposed to 2 mL of trypsin-EDTA and incubated for 3 min at 37 °C. After treatment, monolayers were washed with 10 mL of PBS buffer and centrifuged for 5 min at 1000g (4 °C). Cells were resuspended in 0.8 mL of lysis buffer [20 mM Tris-HCl, pH 7.5; 1 mM EDTA; 0.1% Triton X-100; 1 mM phenylmethanesulfonyl fluoride (PMSF); 1 mM benzamidine; 1.4 µM pepstatin A; 2.0 µΜ leupeptin] and disrupted at 4 °C by sonication (5 × 5 s bursts). The cell lysate was centrifuged for 15 min at 15700g (4 °C), and the supernatant was withdrawn for analysis. Cell cytosol (80 µL) and SMZ, dissolved in 10 µL of assay buffer (20 mM Tris-HCl, 1 mM EDTA, pH 7.5), were incubated at 37 °C for 5 min. AcCoA dissolved in 10 µL of assay buffer was added. The final concentrations were as follows: SMZ, 200 µM; and AcCoA, 800 µM. The incubation was continued at 37 °C for various lengths of time (30-90 min). Cold trichloroacetic acid (20% w/v) (100 µL) was added, followed by centrifugation for 5 min at 12000g. The supernatant was added to 4-dimethylaminobenzaldehyde (800 µL, 5% w/v in 9:1 acetonitrile/water). The Schiff base that forms with the unreacted SMZ was quantitated by measuring the absorbance at 450 nm (ε450nm ) 7300 M-1 cm-1). All assays were performed in triplicate under initial rate conditions. Either AcCoA or SMZ was omitted in control incubations. NAT1, Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), and Glutathione Reductase (GR) Activity in HeLa Cell Cytosol. NAT1, GAPDH, and GR activities were measured as reported previously (34). Effect of N-OH-AAF and N-OH-4-AABP on NAT1 and NAT2 Activities in HeLa Cell Cytosol. In a total volume of 50 µL, cell cytosol (48 µL) was incubated with either N-OH-AAF (10-500 µM for NAT1, and 2.5-20 µM for NAT2) or N-OH-4AABP (10-200 µM for NAT1 or 5-50 µM for NAT2) at 37 °C for 30 min. The reaction was initiated by the addition of the hydroxamic acids dissolved in DMSO (2 µL). PABA and AcCoA or SMZ and AcCoA dissolved in assay buffer (50 µL) and warmed to 37 °C for 5 min were added to the mixtures. The final concentrations were as follows: PABA or SMZ, 200 µM; AcCoA,


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Figure 2. Time- and concentration-dependent inactivation of NATs by N-OH-AAF. (a) Human NAT1 and (b) human NAT2. The results represent the means ( SDs of three experiments.

400 µM for NAT1; and 800 µM for NAT2. Residual NAT1 or NAT2 activity was measured as described above. The control incubations contained DMSO but not hydroxamic acids. Effect of AcCoA on N-OH-AAF and N-OH-4-AABP Inactivation of NAT1 and NAT2 in HeLa Cell Cytosol. To cell cytosol (46 µL) was added AcCoA dissolved in assay buffer (2 µL). The mixture was incubated for 5 min at 37 °C followed by the addition of either N-OH-AAF or N-OH-4-AABP in DMSO (2 µL). The final concentrations were as follows: AcCoA, 400 µM for NAT1 and 800 µM for NAT2; N-OH-AAF, 50 µM for NAT1 and 10 µM for NAT2; and N-OH-4-AABP, 50 µM for NAT1 and 20 µM for NAT2. After incubation for 30 min at 37 °C, PABA and AcCoA or SMZ and AcCoA dissolved in assay buffer (50 µL) were added. The final concentrations were as follows: PABA or SMZ, 200 µM; AcCoA, 400 µM for NAT1; and 800 µM for NAT2. The residual NAT1 and NAT2 activity was measured as described above. For experiments without AcCoA, cell cytosol (46 µL) was incubated with 2 µL of assay buffer for 5 min. Either N-OH-AAF or N-OH-4-AABP (2 µL) was added, and incubations were continued for 30 min. PABA and AcCoA or SMZ and AcCoA dissolved in 50 µL of assay buffer were added, and the NAT1 or NAT2 activity was measured. Effect of Nitrosoarenes on Endogenous NAT1 and NAT2 Activities in HeLa Cells. At approximately 90% confluence, cell monolayers were washed twice with PBS. The cells were exposed to 4-NO-BP (5-40 µM), 2-NO-F (2.5-20 µM), NO-B (100-250 µM), or 2-NO-T (100-250 µM) in 10 mL of serum-free DMEM media containing DMSO. The final concentration of DMSO was 0.1%. The incubation was continued at 37 °C for various periods of time. Cells were washed with PBS buffer, trypsinized, and harvested. The controls contained DMSO only. Cytosol was prepared as described above, and the NAT1 and NAT2 activities were determined. Cell viability was confirmed with the trypan blue assay as described previously (34). Effect of N-OH-AAF and N-OH-4-AABP on Endogenous NAT1, NAT2, GAPDH, and GR Activities in HeLa Cells. At approximately 90% confluence, cell monolayers were washed twice with PBS. The cells were exposed to either N-OH-AAF (25-250 µM) or N-OH-4-AABP (25-500 µM) in 10 mL of serum-free DMEM media containing DMSO. The final concentration of DMSO was 0.5%. The incubation was continued at 37 °C for either 2 or 6 h. Cells were washed with PBS buffer, trypsinized, and harvested.

The controls contained DMSO only. Cytosol was prepared as described above, and the NAT1 and NAT2 activities were measured, as well as the GAPDH and GR activities. Cell viability was confirmed with the trypan blue assay as reported previously (34). The cell viability in the presence of 0.5% DMSO was 104 ( 7% (6 h).

Results Inactivation of NAT2 and NAT1 by N-OH-AAF and N-OH-4-AABP. NAT2 was inactivated more rapidly than NAT1 when the recombinant enzymes were incubated with various concentrations of N-OH-AAF. A 3 min treatment of NAT2 with N-OH-AAF (10 µM) caused a 45% loss of activity, whereas a 3 min incubation of NAT1 with a 3-fold higher concentration of N-OH-AAF resulted in a loss of only 28% of the enzyme’s activity. Little reduction in NAT1 activity was detectable within the first 45 s of exposure to N-OH-AAF (Figure 2a), in contrast to the almost immediate onset of inactivation observed with NAT2 (Figure 2b). The delay in the onset of inactivation is similar to that which occurred when NAT1 was treated with N-OH-4-AABP (28). The loss of both NAT1 and NAT2 activities in the presence of N-OH-AAF was time-dependent and was concentrationdependent at lower concentrations but was saturable at higher concentrations. As shown in Figure 2, the inactivation of both NAT1 and NAT2 by N-OH-AAF occurred according to an apparent first-order kinetic process. First-order inactivation rate constants (kobs) for each concentration of N-OH-AAF were obtained from the slopes of the lines in Figure 2, and limiting inactivation rate constants (kinact) and dissociation constants (KI) were obtained by nonlinear regression analysis of the data according to eq 1, in which [I] is the inhibitor concentration (35). The second-order rate constant (kinact/KI) for inactivation of NAT2 by N-OH-AAF was approximately 8-fold greater than the corresponding value for inactivation of NAT1 (Table 1).

kobs ) kinact /(1 + KI /[I])

(1)

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Table 1. Inactivation of NAT1 and NAT2 by N-OH-AAF and N-OH-4-AABPa N-OH-AAF -1

enzyme

KI (µM)

kinact (s )

NAT1 NAT2

237 ( 25 48 ( 6

0.01 ( 0.001 0.02 ( 0.001

N-OH-4-AABP -1

kinact/KI (M

59 ( 12 459 ( 80

-1

s )

KI (µM) b

1260 193 ( 10

kinact (s-1) b

0.01 0.004 ( 0.001

kinact/KI (M-1 s-1) 7.9b 21 ( 6.3

a Results are expressed as the means ((SDs) of three experiments, except for the results for inactivation of NAT2 by N-OH-4-AABP, which represent the mean of two experiments. b From ref 28.

To further test the hypothesis that human NAT2 would be more susceptible to inactivation by N-arylhydroxamic acids than NAT1, NAT2 was treated with N-OH-4-AABP. The inactivation of NAT2 with N-OH-4-AABP also exhibited pseudo first-order kinetics and was saturable at higher concentrations (Figure S1 of the Supporting Information). Analysis of the data as described above for inactivation by N-OH-AAF provided the kinact and KI values listed in Table 1. The kinact/KI value for inactivation of NAT2 by N-OH-4-AABP is 2.7 times greater than the value previously observed for NAT1 (Table 1) (28). Thus, the results obtained with both N-OH-AAF and N-OH-4-AABP are consistent with the proposal that NAT2 should be more vulnerable to inactivation by N-arylhydroxamic acids than NAT1 (28). ESI Q-TOF MS Analysis, Proteolysis, and MALDI QTOF MS/MS Analysis of N-OH-AAF-Treated Human NAT1 and NAT2. Our previous investigations of NAT inactivation by N-arylhydroxamic acids revealed the formation of covalent sulfinamide adducts with Cys68 in the active sites of the enzymes (27, 28). Similar analyses were carried out in the present investigation to determine whether or not Cys68sulfinamide adducts accounted for the observed losses of NAT activity. The molecular mass of recombinant NAT1 is 34299.2 Da, which includes four additional N-terminal amino acids, Gly, Thr, Leu, and Glu, which are not present in endogenous human NAT1 (30). The ESI mass spectrum of NAT1 that had been treated with N-OH-AAF contains an intense peak (34495.1 Da), which corresponds to a mass increase of 196 Da over the unmodified NAT1 peak (34299.3 Da) and indicates the presence of a (2-fluorenyl)sulfinamide adduct (+195 Da) (Figure 3a). The third peak in the spectrum (34858.9 Da) represents a mass increase of 363.8 Da (169 + 195 Da) relative to the 34495.1 Da peak. MALDI Q-TOF MS analysis of the pepsin digest of NAT1 that had been incubated with N-OH-AAF revealed two peaks with monoisotopic masses of 1590.78 and 1727.87 Da that were not present in the pepsin digest of native NAT1 (Supporting Information, Figure S2a-d). MALDI Q-TOF MS/MS analysis of the peptide of mass 1590.78 revealed the modified (+32 Da) peptide sequence DQVVRRNRGGWCL, which corresponds to Asp57-Leu69 (unmodified theoretical mass 1558.78 Da), and includes the catalytically essential Cys68 (Supporting Information, Figure S3a). The m/z values for the b2-b10 ions and the b11-NH3 ion are consistent with the values expected for unmodified residues, whereas the b12 ion (1459.68 m/z) and the intense b12-H2SO2 ion (1393 m/z) verify that the thiol group of Cys68 had been converted to a sulfinic acid. It was established previously that under the conditions of pepsin digestion, sulfinamide adducts are hydrolyzed to sulfinic acids (27, 28). The peptide of mass 1727.87 Da corresponds to the modified (+179 Da) sequence LEDSKYRKIYSF (Leu181-Phe192), for which the unmodified theoretical mass is 1548.76 Da. The b5H2O (555.18 m/z) and the b6-H2O ion (897.38 m/z), as well as the peak at 315.14 m/z, which is 179 Da greater than the theoretical mass of a tyrosine immonium ion, support the conclusion that Tyr186 is covalently bound to a 2-AF group

Figure 3. Deconvoluted nano-ESI Q-TOF mass spectra: (a) N-OHAAF-inactivated NAT1, (b) N-OH-AAF-inactivated NAT2, and (c) N-OH-4-AABP-inactivated NAT2. The theoretical mass of recombinant human NAT1 is 34299.2 Da. The theoretical mass of recombinant human NAT2 is 33841.8 Da.

(Supporting Information, Figure S3b). Previous mass spectrometric analyses of NATs that had been treated with either N-OH-AAF or N-OH-4-AABP also revealed the presence of minor adducts formed between either 2-AF or 4-ABP and tyrosine residues (27, 28).


The ESI Q-TOF mass spectrum of N-OH-AAF-inactivated NAT2 is shown in Figure 3b. The theoretical mass of recombinant NAT2 is 33841.8 Da, including the three additional N-terminal amino acids, Gly, Leu, and Glu (10). The peak of second greatest intensity (34039.1 m/z) represents a mass increase of 197 Da as compared to the native protein, indicating a (2-fluorenyl)sulfinamide adduct. The peak of greatest intensity (34427.6 Da) is a result of a mass increase of 389 Da (195 + 194 Da) in comparison to the peak of 34039.1 Da. Pepsin digestion of N-OH-AAF-inactivated NAT2 and screening of the digest by MALDI Q-TOF MS resulted in the identification of only one modified (+32 Da) peptide of m/z 1613.81, which corresponds to the sequence DHIVRRNRGGWCL (Asp57Leu69; unmodified theoretical mass, 1581.82) (Supporting Information, Figure S4d). MALDI Q-TOF MS/MS was used to identify Cys68 as the modified residue, based on the presence in the spectrum of the b11 ion (1347.62 m/z), the b12 ion (1482.66 m/z), and the prominent b12-H2SO2 ion (1416.71 m/z) (Supporting Information, Figure S5c). Although the ESI Q-TOF mass spectra of N-OH-AAFinactivated NAT1 and NAT2 indicated the possible presence of 2-NO-F adducts other than the Cys68 sulfinamide, no additional sulfinamide conjugates were identified. Similar results were previously obtained upon treatment of NATs with N-OHAAF or N-OH-4-AABP, followed by analysis of peptide digests obtained with several proteases (27, 28). The additional apparent adducts may be unstable semimercaptals, which are the initial, reversible intermediates formed in reactions between thiols and nitrosoarenes (36). ESI Q-TOF MS Analysis, Proteolysis, and MALDI QTOF MS/MS Analysis of N-OH-4-AABP-Treated NAT2. We reported previously that inactivation of human NAT1 by N-OH4-AABP involved formation of a (4-biphenyl)sulfinamide conjugate (+185 Da) with Cys68 (28). Treatment of NAT2 with N-OH-4-AABP afforded a modified protein for which the ESI Q-TOF mass spectrum displayed a major peak (34024.2 Da) that represents the addition of 185 Da to the molecular mass of the native protein (33839.1 Da; theoretical mass, 33841.8 Da) (Figure 3c). The MALDI Q-TOF mass spectrum of the pepsin digest of the adducted NAT2 contained one modified peptide (+32 Da) with a monoisotopic mass 1613.80 Da, which corresponds to the 13 amino acid sequence DQIVRRNRGGWCL (Asp57-Leu69; unmodified theoretical mass, 1581.82 Da) (Supporting Information, Figure S4b). The presence of the b11 ion (1347.73 m/z), the b12 ion (1482.68 m/z), and the strong b12-H2SO2 ion (1416.72 m/z) in the MALDI Q-TOF MS/MS spectrum supports the conclusion that the thiol group of Cys68 has been converted to a sulfinic acid (Supporting Information, Figure S5a). Hydrolytic Stability of Acetylated NAT2. After confirmation by mass spectrometric analysis that sulfinamide adduct formation was responsible for the N-arylhydroxamic acidmediated inactivation of NAT2, experiments were conducted to test the hypothesis that the relative hydrolytic stabilities of acetylated NATs influence the susceptibility of the enzymes to inactivation (28). NATs catalyze the transfer of the acetyl group from N-arylacetohydroxamic acids to Cys68 in the active site of the enzyme (27, 28). The more rapid onset of inactivation of NAT2, as compared to NAT1 (Figure 2 and Table 1), should be facilitated by rapid hydrolysis of the Cys68 thioacetyl ester of NAT2 to make the Cys68 thiol group available for reaction with an electrophilic nitrosoarene. The first-order rate constant (kH2O) for hydrolysis of acetylated NAT2 was determined by incubation of the enzyme with PNPA

Chem. Res. Toxicol., Vol. 22, No. 12, 2009 1967 Table 2. Hydrolysis Rate Constants (kH2O) and Half-Lives (t1/2) of Acetylated NATs and Formation of 2-NO-F and 4-NO-BP from N-OH-AAF and N-OH-4-AABP in the Presence of NATsa

NAT1 NAT2

kH2O (s-1)

t1/2 (s)

N-OH-AAF 2-NO-F (µM)b

N-OH-4-AABP 4-NO-BP (µM)b

0.02 ( 0.001c 0.1 ( 0.002

33c 7 ( 1.4

10 ( 0.9 5 ( 0.7

20 ( 3c 4 ( 0.9

a Results are expressed as the means ((SDs) of three experiments. Concentration of nitrosoarene (2-NO-F and 4-NO-BP) produced during a 30 min incubation of N-arylhydroxamic acid (200 µM) with NAT1 (4 µM) or NAT2 (4 µM). c From ref 28. b

and monitoring the rate of formation of p-nitrophenol under steady state conditions as described previously for the hamster NAT isoforms and human NAT1 (28, 37). The half-life of acetyl-NAT2 was calculated from the relationship t1/2 ) 0.693/ kH2O. The hydrolytic rate constant was 0.10 s-1, which is 5-fold greater than the corresponding rate constant for hydrolysis of acetylated NAT1, and the t1/2 for acetylated NAT2 is 4.7-fold less than that of acetylated NAT1 (Table 2). The results are consistent with the proposal that the propensity of NAT2 to undergo more rapid inactivation than NAT1 in the presence of N-arylhydroxamic acids is related to the relatively facile hydrolysis of the acetyl-NAT2 intermediate. Additionally, the longer half-life of acetylated NAT1 and the lag period that occurs between exposure of NAT1 to N-OHAAF and the onset of activity loss suggest that more nitrosoarene should accumulate in the incubation medium when a hydroxamic acid is incubated with NAT1 than with NAT2. Thus, when identical concentrations of either NAT1 or NAT2 were incubated with identical concentrations of either N-OH-AAF or N-OH-4-AABP and the formation of 2-NO-F and 4-NO-BP was monitored continuously for 30 min, the final concentration of 2-NO-F that was produced by NAT1 was 2-fold greater than that produced by NAT2, and the amount of 4-NO-BP generated was four times greater with NAT1 than with NAT2 (Table 2). Effect of N-OH-AAF and N-OH-4-AABP on NAT1 and NAT2 Activities in HeLa Cell Cytosol. Cell cytosol was treated with a range of concentrations of N-OH-AAF and N-OH-4AABP, and the IC50 values for inhibition of NAT1 and NAT2 were calculated. Similar to the results obtained with the recombinant enzymes and consistent with the expectation that NAT2 would be more susceptible than NAT1 to inactivation by the hydroxamic acids, both compounds were more potent inactivators of NAT2 than NAT1. For N-OH-AAF, the IC50 values obtained with NAT1 and NAT2 were 27.1 ( 0.19 and 9.5 ( 0.6 µM, respectively, and for N-OH-4-AABP, the corresponding values were 34 ( 1.3 and 19.7 ( 0.57 µM. To obtain evidence that inactivation of NATs in HeLa cell cytosol was due to interaction of the hydroxamic acids with the active sites of the enzymes, AcCoA was added to the incubation mixtures prior to addition of N-OH-AAF or N-OH-4-AABP. In these experiments, the hydroxamic acids caused losses of both NAT1 and NAT2 activities of approximately 60%, but in the presence of AcCoA, the enzymes were inactivated to an extent of only 6-17% (Supporting Information, Table S1). Protection of the enzymes from inactivation by N-OH-AAF and N-OH-4-AABP in the presence of the endogenous acetyl donor, AcCoA, is consistent with the expected interaction of the hydroxamic acids with the active sites of the NATs. Effect of N-OH-AAF and N-OH-4-AABP on NAT1 and NAT2 Activities in HeLa Cells. Incubation of HeLa cells for 2 h with N-OH-AAF (25-250 µM) and with N-OH-4-AABP (25-500 µM) did not cause a significant change in either NAT1

1968


Liu et al. Table 3. Inactivation of NAT2 by Nitrosoarenesa KI (µM)

kinact (s-1)

4-NO-BP 0.71 ( 0.09 0.06 ( 0.002 NO-B 2260 ( 760 0.03 ( 0.005 2-NO-F 2-NO-T a

Figure 4. Inactivation of HeLa cell NAT1, NAT2, GAPDH, and GR by N-OH-4-AABP and N-OH-AAF. The incubation time was 6 h. (a) N-OH-4-AABP. Each bar represents the mean and standard deviation of the results of three experiments. Asterisks represent significant differences from control: *p < 0.01, and **p < 0.001. Pound symbols represent significant differences from percentage of NAT1 activity: #p < 0.05, and ##p < 0.001. (b) N-OH-AAF. Each bar represents the mean and standard deviation of the results of three experiments. Asterisks represent significant differences from control: *p < 0.05, and **p < 0.001. Pound symbols represent significant differences from percentage of NAT1 activity: #p < 0.001.

or NAT2 N-acetylation activity. After a 6 h incubation, however, N-OH-4-AABP (50 µM) had caused a 24% reduction in NAT1 activity without affecting NAT2 (Figure 4a). Treatment of the cells with N-OH-4-AABP (100 µM) inhibited NAT1 by 56% but lowered NAT2 activity by only 19%. Both NAT1 and NAT2 were inhibited by approximately 50% of control activities when the cells were exposed to a 250 µM concentration of N-OH4-AABP. Similarly, N-OH-AAF (50 µM) lowered intracellular NAT1 activity by 33%, but the reduction in NAT2 activity was only 11% (Figure 4b). The effects of higher concentrations of N-OH-AAF (100 and 250 µM) on NAT1 and NAT2 activities were not significantly different, with activity reductions being 45-55%. For purposes of comparison with other enzymes containing catalytically essential thiols, the effects of the N-arylhydroxamic acids on intracellular GAPDH and GR activities were determined, and even after 6 h, the highest concentrations of N-OH-4-AABP and N-OH-AAF had substantially greater inhibitory effects on NAT activities than on GAPDH and GR (Figure 4a,b). Thus, although both cytosolic NAT2 and recombinant NAT2 were more susceptible to inactivation by N-arylhydroxamic acids than the corresponding preparations of NAT1, intracellular NAT1 was more readily inhibited than NAT2 by the same compounds. Because nitrosoarenes are the ultimate molecular species responsible for the inactivation of NATs upon their interaction with N-arylhydroxamic acids and because of the unexpected finding that intracellular NAT1 is preferentially

kinact/KI (M-1 s-1) 80400 ( 12800 14 ( 6

k2 (M-1 s-1)

50500 ( 4300 16 ( 1.4

Results are expressed as the means ((SDs) of three experiments.

inactivated by N-OH-4-AABP and N-OH-AAF (Figure 4a,b), we conducted experiments to compare the susceptibility of NAT2 and NAT1 to inactivation by nitrosoarenes. We previously reported that 4-NO-BP and 2-NO-F are potent inactivators of NAT1 (34). Inactivation of Recombinant NAT2 by Nitrosoarenes. On the basis of our earlier hypothesis that NATs would be readily inactivated by nitrosoarene metabolites of arylamines that are efficiently N-acetylated by the enzymes, NAT2 was treated with 4-NO-BP, 2-NO-F, NO-B, and 2-NO-T (34). Each of the nitrosoarenes caused a concentration-dependent and timedependent loss of NAT2 activity, which exhibited apparent firstorder kinetics (Supporting Information, Figure S6). The inactivations of NAT2 by both 4-NO-BP and NO-B were saturable processes, with the rates becoming concentration-independent at higher concentrations of the nitrosoarenes. The KI and kinact values for 4-NO-BP and NO-B were calculated from eq 1 and are shown in Table 3. The second-order inactivation rate constant (kinact/KI) for 4-NO-BP was 5700-fold greater than that of NO-B, which is similar to the result obtained for the inactivation of NAT1 by the two compounds (34). Saturation was not observed at higher concentrations when NAT2 was inactivated with either 2-NO-F or 2-NO-T (Supporting Information, Figure S6). The second-order rate constants (k2) for inactivation of NAT2 by 2-NO-F and 2-NO-T were obtained by fitting the data to eq 2, in which [I] is the concentration of the inactivating agent and n is the order of the reaction with respect to the inactivator (38).

log kobs ) log k2 + n log [I]

(2)

For both 2-NO-F and 2-NO-T, the reaction order was 1.0, indicating that a single essential residue undergoes modification with each inactivation event. The k2 value for inactivation of NAT2 by 2-NO-F was 3160-fold greater than that of 2-NO-T and is comparable in magnitude to the kinact/KI value of 4-NOBP (Table 3). These results provide further support for the hypothesis that the relative effectiveness of nitrosoarenes as inactivators of NATs can be predicted from the efficiency of the NAT-catalyzed acetylation of the corresponding arylamines, as both 4-ABP and 2-AF are much more efficiently N-acetylated by NAT2 than either aniline or 2-methylaniline (o-toluidine) (10, 11, 34). MS Analysis of 4-NO-BP-Treated NAT2 and 2-NO-FTreated NAT2. After incubation of NAT2 with 4-NO-BP, the ESI-Q-TOF mass spectrum of the adducted protein indicated the presence of a (4-biphenyl)sulfinamide conjugate (+183 Da), as it exhibited a major peak (34022.0 Da), which corresponds to the addition of 184 Da to the native protein. A minor peak (34206.3 Da), which indicates the addition of 184 Da to the mass of 34022.0 Da also was present (Supporting Information, Figure S7a). Pepsin digestion of the modified protein and screening of the digest by MALDI Q-TOF MS (Supporting Information, Figure S4e) revealed a modified peptide with a mass of 1613.80 Da, corresponding to the Asp57-Leu69 sequence with a 32 Da increase in mass. Tandem MS analysis of the 1613.80 Da peptide allowed the identification of the


Chem. Res. Toxicol., Vol. 22, No. 12, 2009 1969

Figure 5. Inactivation of NAT2 (0.72 µM) by 4-NO-BP (1 µM) and 2-NO-F (1 µM) in the presence of GSH. The incubation time was 60 s. Each bar represents the mean ( SD of the results of three experiments. Asterisks represent significant differences from controls: *p < 0.05, and **p < 0.001.

Cys68 side chain as having been converted to a sulfinic acid, with the b11 ion (1347.73 m/z), b12 ion (1482.68 m/z), and intense b12-H2SO2 ion (1416.72 m/z) being diagnostic for the added mass of 32 Da (Supporting Information, Figure S5b). The ESI Q-TOF mass spectrum of 2-NO-F-treated NAT2 contained a major peak (34039.2 Da), which represents a 196 Da increment relative to the native protein peak of 33843.7 Da (theoretical mass, 33841.8 Da). A minor peak (34235.6 Da) represents an additional mass increment of 196 Da (Supporting Information, Figure S7b). Either sulfinamide formation or semimercaptal formation by 2-NO-F would produce a mass increment of 195 Da. The MALDI Q-TOF mass spectrum of the pepsin digest of 2-NO-F-treated NAT2 contained a single modified peptide (+32 Da), which matched the peptide sequence Asp57-Leu69 (unmodified theoretical mass 1581.82 Da) (Supporting Information, Figure S4e). Sequencing of the 1613.82 Da peptide by tandem mass spectrometry generated a b11 ion (1347.68 m/z), a b12 ion (1482.72 m/z), and a strong b12-H2SO2 ion (1417.72 m/z), all of which verify the presence of a sulfinic acid group on the Cys68 side chain (Supporting Information, Figure S5d). Effect of AcCoA on the Inactivation of NAT2 by NO-B and 2-NO-T. To obtain evidence that NO-B and 2-NO-T, which were relatively weak inactivators of NAT2 (Table 3), produce their effects on the enzyme by reaction with the active site cysteine thiol, NAT2 was incubated with AcCoA prior to treatment with either NO-B or 2-NO-T. Acetylation of the thiol by AcCoA should render it unavailable for reaction with nitrosoarenes and attenuate their inhibitory effects. Incubation of NAT2 with NO-B (800 µM) caused a 59 ( 5% loss of activity, but with AcCoA (20 µM) in the reaction mixture, NO-B inhibited NAT2 activity by only 13 ( 6%. Similarly, 2-NO-T (500 µM) caused 58 ( 1.7% inactivation of NAT2 in the absence of AcCoA and a 15 ( 6% loss of activity in the presence of the endogenous acetyl donor. Inactivation of NAT2 by 4-NO-BP and 2-NO-F in the Presence of GSH. We reported previously that physiological concentrations of the cellular nucleophile GSH provided only partial protection of human NAT1 from inactivation by 4-NOBP and 2-NO-F (34). In analogous experiments with NAT2, 4-NO-BP (1 µM) and 2-NO-F (1 µM) caused inactivations of 88 and 86%, respectively, during a 60 s incubation with NAT2 (0.73 µM) in the absence of GSH (Figure 5). In the presence of GSH (0.5 mM), 4-NO-BP caused a 32% reduction in activity

and 2-NO-F inhibited NAT2 by 31%. 2-NO-F caused 27 and 14% inactivation in the presence of 1 and 5 mM concentrations of GSH, whereas the corresponding losses in activity produced by 4-NO-BP were 14 and 7%. Although nitrosoarenes react with GSH and other thiols to form sulfinamides, these results support the conclusion that 4-NO-BP and 2-NO-F react much more readily with NAT2 than with GSH, as we observed previously for NAT1 (34, 36, 39, 40). Effects of 4-NO-BP and 2-NO-F on NAT2 and NAT1 Activities in HeLa Cells. Treatment of cells for 1 h with 4-NOBP (2.5 µM) caused a loss in NAT1 activity of 36% but had no effect on NAT2 activity (Figure 6a). At a concentration of 5 µM 4-NO-BP, the NAT1 activity was inhibited by 50%, with 26% of the NAT2 activity having been lost. At higher concentrations (10-40 µM) of 4-NO-BP, NAT1 and NAT2 activities were reduced by approximately 55-65%, and the differences in the reductions in NAT1 and NAT2 activities were not significant (Figure 6a). Similar to the results obtained with 4-NO-BP, 2-NO-F (1 µM) caused a 30% reduction in NAT1 activity but had no effect on intracellular NAT2 (Figure 6b). 2-NO-F (2.5 µM) inhibited HeLa cell NAT1 activity by 53% and caused a 25% reduction in NAT2 activity. Higher concentrations of 2-NO-F (5 µM - 20 µM) had similar inhibitory effects on NAT1 and NAT2 and had decreased the activities by approximately 50-70% after a 1 h of exposure to the nitrosoarene (Figure 6b). When HeLa cells were incubated with 4-NO-BP (10 µM) for 2 h, there was no significant difference in the extent of loss of NAT1 and NAT2 activities at any time (15-120 min) during exposure to the nitrosoarene (Figure 6c). NAT1 and NAT2 activities had decreased by approximately 40% after 15 min and by approximately 65% after 120 min (Figure 6c). In contrast, a 15 min exposure of the cells to 2-NO-F (10 µM) caused a 60% reduction in NAT1 activity, whereas NAT2 activity decreased by 30% (Figure 6d). The activities of the two NATs were approximately 30% of control values after 30 min, and a maximum loss of the two activities of approximately 80% was observed after 2 h. The data illustrated in Figure 6, therefore, establish that treatment of HeLa cells with low concentrations of 4-NO-BP and 2-NO-F causes a significantly greater reduction of NAT1 activity than of NAT2 activity, but a preferential inactivation of NAT1 is not observed when the cells are exposed for 1 h to higher concentrations of the nitrosoarenes. The greater suscep-

1970


Liu et al.

Figure 6. Inactivation of HeLa cell NAT1 and NAT2 by 4-NO-BP and 2-NO-F. (a) 4-NO-BP. Concentration dependence. The incubation time was 1 h. (b) 2-NO-F. Concentration dependence. The incubation time was 1 h. (c) 4-NO-BP. Time dependence. The concentration of 4-NO-BP was 10 µM. (d) 2-NO-F. Time dependence. The concentration of 2-NO-F was 10 µM. Each bar represents the mean and standard deviation of the results of three experiments. Asterisks represent significant differences from control: *p < 0.01, and **p < 0.001. Pound symbols represent significant differences between percentage of NAT1 and NAT2 activity: #p < 0.05, ##p < 0.02, ###p < 0.01, and ####p < 0.001. The NAT1 activities after treatment with 4-NO-BP (10-40 µM) and 2-NO-F (5-20 µM) are from ref 34.

tibility of NAT1 to inactivation is further emphasized by the substantial differences in NAT1 and NAT2 activities after exposure of the cells to 2-NO-F (10 µM) for 15 min (Figure 6d).

Discussion The irreversible inactivation of biotransformation enzymes by xenobiotics and their metabolites is of toxicological and clinical significance (41). Although numerous epidemiological and pharmacogenetic studies have identified toxicological and clinical consequences associated with NAT genetic polymorphisms, the effects of exogenous agents and their metabolites on NAT activity levels have received less attention (15-21, 42, 43). Bartsch et al. reported in 1972 that dietary administration of the 2-AF metabolite, N-OH-AAF, to rats for 4-8 days caused a 20-30% reduction in the acetyltransferase-catalyzed Oacetylation of N-arylhydroxylamines in liver cytosol (44). A decade later, N-OH-AAF was shown to inactivate the AcCoAdependent N-acetylation of SMZ in hamster liver cytosol but to have less effect on the AcCoA-dependent N-acetylation of PABA (45). The mechanism of inactivation of NATs by N-arylhydroxamic acids in vitro proceeds through a transfer of the acetyl group from the hydroxamic acid to NAT, resulting in the production

of an arylhydroxlamine (Scheme 1, B), which undergoes oxidation to a nitrosoarene and reacts with the catalytic nucleophile, Cys68, of NAT to form a covalent sulfinamide adduct (Scheme 1, C and E) (27). On the basis of the results of inactivation experiments with N-OH-4-AABP and hamster NAT1, hamster NAT2, and human NAT1, it was proposed that human NAT2 would be more readily inactivated by Narylhydroxamic acids than human NAT1 because hamster NAT1, the orthologue of human NAT2, is more rapidly inactivated than hamster NAT2. It was also proposed that the relative stabilities of the Cys68 thioacetyl esters formed in the first step of the inactivation process (Scheme 1, B) would be a determinant of susceptibility to inactivation by N-arylhydroxamic acids and that the human NAT2 thioacetyl ester would be more rapidly hydrolyzed (Scheme 1, D) than the corresponding ester of NAT1 (28). The results of the kinetic analyses of the inactivation of human NAT1 and NAT2 by N-OH-AAF and N-OH-4-AABP (Table 1) are consistent with the expectation of more rapid inactivation of NAT2 by the two hydroxamic acids, as the second-order rate constants for inactivation (kinact/KI) are approximately 8- and 3-fold larger for NAT2 than for NAT1. Additionally, the half-life of acetylated NAT1 is approximately 5-fold greater than that of NAT2, rendering the Cys68 thiol of


Chem. Res. Toxicol., Vol. 22, No. 12, 2009 1971 Scheme 1a

a

A ) P450, NAT; B ) NAT; C ) nonenzymatic oxidation; D ) nonenzymatic hydrolysis; E ) nonenzymatic sulfinamide formation; and F ) esterases.

NAT1 less available for reaction with a nitrosoarene and contributing to both the slower rate of inactivation of NAT1 and the accumulation of larger quantities of 2-NO-F and 4-NOBP in the incubation mixture (Table 2). The greater susceptibility of NAT2 to inactivation was also observed upon treatment of HeLa cell cytosol with N-OH-AAF and N-OH-4-AABP; the IC50 value for inhibition of NAT1 by N-OH-AAF was three times larger than the IC50 for inhibition of NAT2, and for N-OH4-AABP, the IC50 value for inhibition of NAT1 was approximately twice that obtained with NAT2. Treatment of HeLa cells with relatively high, but nontoxic, concentrations of N-OH-AAF and N-OH-4-AABP had inhibitory effects on intracellular NAT activities. A partial explanation for the much lower inhibitory potency of N-OH-4-AABP and N-OH-AAF, as compared to the corresponding nitrosoarenes, is that lower intracellular concentrations of the N-arylhydroxamic acids were achieved. The acetohydroxamic acid functional group is weakly acidic and more polar than the nitroso group and can markedly decrease the lipid solubility of a molecule. The calculated partition coefficient (Log P) for N-OH-AAF is reported to be 2.60-2.80, whereas the calculated Log P value for 2-NO-F is 3.96 (46). Similarly, the calculated Log P value for N-OH-4-AABP is 2.61-2.92, and the range of values reported for 4-NO-BP is 3.50-4.14 (46). Thus, it is probable that the ability of N-OH-AAF and N-OH-4-AABP to enter cells by passive diffusion is diminished in comparison with the corresponding nitrosoarenes, which appear to penetrate HeLa cells readily at low concentrations (34). Another factor contributing to the lower potencies of N-OH-AAF and N-OH-4AABP is the requirement for deacetylation, followed by oxidation of the resulting N-arylhydroxamines, prior to sulfinamide adduct formation (Scheme 1). It is possible that esterasecatalyzed hydrolysis of the hydroxamic acids may be required to produce sufficient quantities of the arylhydroxylamines to effect inhibition (Scheme 1, F), with NAT-catalyzed deacetylation (Scheme 1, B) being less important. It was found that treatment of hamsters with an esterase inhibitor prior to intraperitoneal administration of N-OH-AAF prevented the in vivo inactivation of NAT activity, whereas inactivation of partially purified hamster hepatic NAT was not prevented by the esterase inhibitor (26). Additional studies will be required to determine whether or not HeLa cells have the enzymatic capacity to hydrolyze either N-OH-AAF or N-OH-4-AABP and to what extent the compounds accumulate in the cells. Unexpectedly, and in contrast to the isoform selectivity observed for the inactivation of recombinant and cytosolic NAT1

and NAT2, intracellular NAT1 was more readily inactivated than NAT2 when HeLa cells were treated with N-OH-AAF and N-OH-4-AABP. Mass spectrometric analyses confirmed that the ultimate reactants responsible for inactivation of NAT1 and NAT2 in vitro by the two N-arylhydroxamic acids are 4-NOBP and 2-NO-F, which we previously found to be very potent inactivators of recombinant and intracellular NAT1 (34). The second-order rate constants for inactivation of recombinant human NAT1 by 4-NO-BP and 2-NO-F are 59200 and 34500 M-1 s-1, respectively (34). The very similar corresponding values for inactivation of recombinant NAT2 by 4-NO-BP and 2-NO-F (Table 3) do not indicate selective inactivation of either isoform in vitro. Treatment of HeLa cells with either 4-NO-BP or 2-NO-F caused a rapid loss of NAT2 activity. Although both nitrosoarenes were effective and potent inactivators of intracellular NAT2, a portion of the enzyme, representing approximately 20-40% of the total NAT2 activity, was not susceptible to inactivation. A similar fraction of HeLa cell NAT1 remained functional after incubation of the cells with concentrations of 4-NO-BP and 2-NO-F that caused maximal reductions in N-acetylation activity (34). It was suggested that the apparent resistance to inactivation may be attributed either to a portion of the enzyme existing in the acetylated state, which would render Cys68 unavailable for reaction with a nitrosoarene, or to a portion of the intracellular enzyme being inaccessible to the nitrosoarenes (34). Low concentrations of both 4-NO-BP and 2-NO-F caused a 30-40% greater loss of intracellular NAT1 activity than of NAT2 activity. Additionally, the onset of inactivation of NAT1 by 2-NO-F was more rapid than the onset of inactivation of NAT2. Coupled with the preferential loss of NAT1 activity that occurred upon treatment of HeLa cells with N-OH-4-AABP and N-OH-AAF, the results obtained with the nitrosoarenes strongly suggest that intracellular NAT1 is more vulnerable to inhibition by reactive metabolites of arylamines than is NAT2. On the basis of the comparative hydrolytic stabilities of the NATs, it seems unlikely that the more facile inactivation of NAT1 than NAT2 is due to a smaller fraction of NAT1 existing in the acetylated state. Although differences in either the intracellular compartmentalization of the NATs or the protein-protein interactions could contribute to differential reactivity with nitrosoarenes, data necessary to support a role for such factors have not been reported. NAT2 is the only mammalian enzyme known to catalyze the AcCoA-dependent N-acetylation of SMZ, and as the specific activity for N-acetylation of SMZ by recombinant NAT1 (0.51

1972


( 0.05 µmol/mg/min) is approximately 3% of that of recombinant NAT2, the contribution of NAT1 to the cytosolic N-acetylation of SMZ in HeLa cells can be considered to be negligible. In related experiments, specific activities very similar to that determined in HeLa cell cytosol for N-acetylation of SMZ were found in the soluble fractions of MCF7, MDA-MB-231, and MDA-MB-468 breast cancer cells, as well as in HEK293T cells (data not presented). There appear to be no previous reports of NAT2 activity in HeLa cells. Although it is generally considered that NAT2 activity is restricted almost exclusively to liver and intestinal tissues, Dairou et al. recently reported the quantification of AcCoA-dependent N-acetylation of the specific human NAT2 substrate, SMZ, in two murine skeletal muscle cell lines and in human lens epithelial cells (47, 48). Low levels of NAT2 activity and NAT2-specific mRNA have been detected in other human extrahepatic tissues (49, 50). Quantification of NAT1 in HeLa cell cytosol by use of a NAT1-specific antibody, as well as by comparison of cytosolic NAT1-specific activities with those exhibited by recombinant NAT1, revealed that NAT1 comprises approximately 0.002% of cytosolic protein (34). The magnitude of the specific activity for N-acetylation of SMZ by recombinant NAT2 (14.3 ( 0.6 µmol/mg protein/min) was 6% of the rate of recombinant NAT1-catalyzed N-acetylation of PABA (237 ( 2.4 µmol/mg protein/min) and was 4% of the rate of recombinant NAT1-catalyzed N-acetylation of p-aminosalicylic acid (PAS) (337 ( 12 µmol/mg protein/min). The specific activity of the AcCoA-dependent N-acetylation of SMZ in HeLa cytosol was 0.18 ( 0.02 nmol/mg protein/min, which was 4% of the rate of the cytosolic N-acetylation of both PABA (4.40 nmol/mg protein/min) and PAS (4.08 ( 0.58 nmol/mg protein/ min). The consistent relationships between the specific activities determined in HeLa cell cytosol and those obtained with recombinant NAT1 and NAT2 indicate that the concentrations of NAT1 and NAT2 in HeLa cells are similar and that the apparent predominance of NAT1 activity is primarily a reflection of the lower efficiency of the NAT2 catalyzed N-acetylation of the probe substrate, SMZ, rather than being due to a paucity of functional NAT2 protein. This conclusion is supported by the observation that the specific activity of SMZ N-acetylation by HeLa cell cytosolic protein is 0.001% of the specific activity of N-acetylation of SMZ by homogeneous recombinant NAT2. The results described in this paper and in our previous report demonstrate that both NAT1 and NAT2 are rapidly inactivated by nitrosoarene metabolites of arylamines having structural characteristics that facilitate N-acetylation by the NATs (34). Exposure to nitrosoarenes occurs after oxidation of N-arylhydroxylamines, which are formed in mammalian tissues by either cytochrome P450-catalyzed N-hydroxylation of arylamines (Scheme 1) or by metabolic reduction of nitroarenes (51-53). The impairment of NAT function by nitrosoarenes would be expected to alter the metabolic disposition and biological effects of xenobiotics by decreasing cellular capacity for both detoxification by N-acetylation of arylamines and bioactivation through O-acetylation of N-arylhydroxylamines (4). The apparent high sensitivity of intracellular NAT1 to the inhibitory effects of low concentrations of nitrosoarenes may have markedly broad toxicological implications. The ubiquitous distribution of NAT1 among human tissues is well-documented and includes sites of xenobiotic exposure such as lung, skin, stomach, and intestine (49, 54). It was demonstrated recently that NAT1 catalyzes the intradermal N-acetylation of arylamines following topical exposure (55). NAT1 also catalyzes N-acetylation of 4-aminobenzoylglutamic acid, the endogenous oxidative catabolite

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of folic acid, and may influence the regulation of cellular folate levels (56, 57). Human NAT1 is expressed early in development, and epidemiological data suggest that expression of NAT1 variants is associated with enhanced risk of birth defects, such as cleft palate, spina bifida, and limb deficiencies (58). Mouse model investigations of Nat2, the murine orthologue of human NAT1, have supported the relevance of NAT1 to development. Wakefield et al. found that the progeny of transgenic mice having at least one Nat2-null allele were more likely to exhibit ocular defects than offspring of mice in which neither parent carried the null allele (59). It was suggested that an imbalance in the expression of the Nat2 protein may contribute to the occurrence of the ocular defects. Thus, it is reasonable to anticipate that exposure to environmental arylamines, such as 4-ABP in cigarette smoke, may result in tissue levels of nitrosoarenes that can impair both the biotransformation functions and the endogenous functions of NAT1. Acknowledgment. The mass spectrometry data were obtained with the assistance of Drs. Lorraine Anderson, Lee Ann Higgins, and Sean Murray of the Center for Mass Spectrometry and Proteomics, University of Minnesota. This research was supported in part by a Developmental Grant for Drug Design and Discovery from the Department of Medicinal Chemistry, University of Minnesota. Supporting Information Available: Effect of AcCoA on N-OH-AAF and N-OH-4-AABP inactivation of NAT1 and NAT2 in HeLa cell cytosol (Table S1); time- and concentrationdependent inactivation of recombinant human NAT2 by N-OH4-AABP (Figure S1); segments of the MALDI Q-TOF mass spectra of pepsin digests of native human NAT1 and N-OHAAF-inactivated NAT1 (Figure S2); MALDI Q-TOF tandem mass spectra of the 1590.78 Da peptide obtained by pepsin digestion of N-OH-AAF-inactivated NAT1 and of the 1727.87 Da peptide obtained by pepsin digestion of N-OH-AAFinactivated NAT1 (Figure S3); segments of the MALDI Q-TOF mass spectra of pepsin digests of native human NAT2, N-OH4-AABP-inactivated NAT2, 4-NO-BP-inactivated NAT2, NOH-AAF-inactivated NAT2, and 2-NO-F-inactivated NAT2 (Figure S4); MALDI Q-TOF tandem mass spectra of the 1613.8 Da peptide obtained by pepsin digestion of N-OH-4-AABPinactivated NAT2, the 1613.80 Da peptide obtained by pepsin digestion of 4-NO-BP-inactivated NAT2, the 1613.81 Da peptide obtained by pepsin digestion of N-OH-AAF-inactivated NAT2, and the 1613.81 Da peptide obtained by pepsin digestion of 2-NO-F-inactivated NAT2 (Figure S5); time- and concentration-dependent inactivation of human NAT2 by 4-NO-BP, 2-NO-F, NO-B, and 2-NO-T (Figure S6); and deconvoluted nano-ESI-Q-TOF mass spectra of 4-NO-BP-inactivated NAT2 and 2-NO-F-inactivated NAT2 (Figure S7). This material is available free of charge via the Internet at http://pubs.acs.org.

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TX9002676

Isoform-Selective Inactivation of Human Arylamine N

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