In vitro bioactivation of N-hydroxy-2-amino-{alpha}-carboline

Roberta S. King1, Candee H. Teitel and Fred F. Kadlubar2

Division of Molecular Epidemiology, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079-9502, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2-Amino-{alpha}-carboline (A{alpha}C) is a mutagenic and carcinogenic heterocyclic amine present in foods cooked at high temperature and in cigarette smoke. The mutagenic activity of A{alpha}C is dependent upon metabolic activation to N-hydroxy-A{alpha}C (N-OH-A{alpha}C); however, the metabolism of N-OH-A{alpha}C has not been studied. We have synthesized 2-nitro-{alpha}-carboline and N-OH-A{alpha}C and have examined in vitro bioactivation of N-OH-A{alpha}C by human and rodent liver cytosolic sulfotransferase(s) and acetyltransferase(s) and by recombinant human N-acetyltransferases, NAT1 and NAT2. The sulfotransferase-dependent bioactivation of N-OH-A{alpha}C by human liver cytosol exhibited large inter-individual variation (0.5–75, n = 14) and was significantly higher than bioactivation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). Correlation and inhibition studies suggested that the isoform of sulfotransferase primarily responsible for bioactivation of N-OH-A{alpha}C in human liver cytosol is SULT1A1. O-Acetyltransferase-dependent bioactivation of N-OH-A{alpha}C by human liver cytosol also exhibited large inter-individual variation (16–192, n = 18). In contrast to other N-hydroxy heterocyclic amines, which are primarily substrates only for NAT2, both NAT1 and NAT2 catalyzed bioactivation of N-OH-A{alpha}C. The rate of bioactivation of N-OH-A{alpha}C by both NAT1 and NAT2 was significantly higher than that for N-OH-PhIP. In rat and mouse liver cytosols, the level of sulfotransferase-dependent bioactivation of N-OH-A{alpha}C was similar to the level in the high sulfotransferase activity human liver cytosol. The level of O-acetyltransferase-dependent bioactivation of N-OH-A{alpha}C in rat liver cytosol was also comparable with that in the high acetyltransferase activity human liver cytosol. However, the level of O-acetyltransferase-dependent bioactivation of N-OH-A{alpha}C in mouse liver cytosol was comparable with that in the low acetyltransferase activity human liver cytosol. In contrast to N-OH-PhIP, bioactivation of N-OH-A{alpha}C was not inhibited by glutathione S-transferase activity; however, DNA binding of N-acetoxy-A{alpha}C was inhibited 20% in the presence of GSH. These results suggest that bioactivation of N-OH-A{alpha}C may be a significant source of DNA damage in human tissues after dietary exposure to A{alpha}C and that the relative contribution of each pathway to bioactivation or detoxification of N-OH-A{alpha}C differs significantly from other N-hydroxy heterocyclic or aromatic amines.

Abbreviations: A{alpha}C, 2-amino-{alpha}-carboline, 2-amino-9H-pyrido[2,3-b]indole; ABP, 4-aminobiphenyl; AcCoA; acetyl coenzyme A; AF, 2-aminofluorene; DCNP, 2,4-dichloronitrophenol; DTT, dithiothreitol; GSH, glutathione (reduced); GST, glutathione S-transferase; IQ, 2-amino-3-methylimidazo [4,5-f]quinoline; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NAT, N-acetyltransferase; N-OAc, N-acetoxy; N-OH, N-hydroxy; NO2{alpha}C, 2-nitro-{alpha}-carboline; PABA, p-aminobenzoic acid; PAPS, 3'-phosphoadenosine 5'-monophosphate; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; SMZ, sulfamethazine; THF, tetrahydrofuran.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2-Amino-{alpha}-carboline (A{alpha}C) is a mutagenic and carcinogenic heterocyclic amine that is present in fried or broiled fish, beef and chicken (14), in cigarette smoke condensate (5) and in diesel exhaust particles (6). Dietary intake of A{alpha}C in the US population has been estimated to be 5 ng/kg/day, the second most mass-abundant of the five heterocyclic amines examined (2). Oral administration of A{alpha}C induces lymphomas and liver tumors in CDF1 mice (7), mutations in the colon of Big BlueTM mice (8), preneoplastic lesions in the colon of C57BL/6N mice (9) and liver nodules in neonatal B6C3F1 mice (10). A{alpha}C also induces preneoplastic lesions in the livers of male F344 rats, although carcinogenicity of A{alpha}C has not been reported in the rat (11). In primary hepatocytes from mice, rats and hamsters, A{alpha}C induced unscheduled DNA repair synthesis, indicating DNA damage (12). We (13) and others (14,15) have measured and characterized the A{alpha}C–DNA adducts formed after oral administration of A{alpha}C to mice and rats and the major adduct was determined to be bound to guanosine (13,15) at the C8 position (15). Mutagenic activity of A{alpha}C in the presence of rat liver S9 mix has been demonstrated in a number of bacterial systems, including Salmonella typhimurium TA98 and TA100, lysogenic Escherichia coli K12 and Bacillus subtilis (1). A{alpha}C is also mutagenic in insect and mammalian systems, including Drosophila melanogaster, Chinese hamster lung cells in the presence and absence of S9 and in human lymphoblastoid cells in the presence of S9 (1). Thus, chronic low level human exposure to A{alpha}C may be a significant risk for human health.

The mutagenic activity of A{alpha}C is dependent upon N-oxidation to form N-hydroxy-A{alpha}C (N-OH-A{alpha}C) (1,16). This N-oxidation is catalyzed by CYP1A2 in human and rodent liver microsomes and extrahepatically by CYP1A1 and CYP2C9/10 (17). We have previously discussed the potential significance of A{alpha}C N-oxidation in larynx, lung and colorectal carcinogenesis; however, the enzymatic pathways for subsequent bioactivation of N-OH-A{alpha}C have not been described. Many N-hydroxy heterocyclic amines are further metabolized to highly reactive N-acetoxy (N-OAc) and N-sulfonyloxy esters that bind covalently to DNA and are presumed to initiate the neoplastic process (1820). Formation of N-OAc esters of heterocyclic and aromatic amines is catalyzed by one or both isoforms of human N-acetyltransferase, NAT1 or NAT2 (19), and both NAT1 and NAT2 exhibit a polymorphic distribution in the human population. Similarly, the thermostable form of phenol sulfotransferase (SULT1A1, formerly ST1A3), which is the isoform primarily responsible for bioactivation of N-OH-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (20), is polymorphically distributed in humans. Thus, we sought to elucidate the pathways for bioactivation of N-OH-A{alpha}C by studying the metabolism of N-OH-A{alpha}C by human and rodent liver cytosolic and recombinant enzymes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Instrumentation
HPLC analyses were performed with a Waters (Milford, MA) Alliance 2690 separations module with in-line connection of a Waters 996 photodiode detector, a Waters 470 scanning fluorescent detector and a Packard Radiomatic Flo-one series 500TR radiodetector. Scintillation counting was performed on a Packard Tri-Carb 1600TR liquid scintillation counter. UV-Vis spectral readings were made with a Beckman DU640 spectrophotometer.

Chemicals and reagents
2-Nitro-{alpha}-carboline (NO2{alpha}C) was synthesized as described below or was prepared by Bionetics Corporation (Jefferson, AR) and was radiolabeled (6.5 Ci/mmol) by Chemsyn Science Laboratories (Lenexa, KS). This high specific activity [G-3H]NO2{alpha}C was radiochemically diluted to 311 mCi/mmol (99.1% chemical purity, 99.5% radiochemical purity) before synthesis of [G-3H]N-OH-A{alpha}C. A{alpha}C was purchased from Toronto Research Chemicals (Toronto, Canada). Hydrazine hydrate (80%) and 10% palladium on charcoal (Pd/C) were purchased from Fluka (Buchs, Switzerland). 3'-Phosphoadenosine 5'-monophosphate (PAPS) (80–90%) was purchased from Sigma Chemical Co. (St Louis, MO) and was used without purification. DNA (calf thymus type 1, highly polymerized) was purchased from Sigma. [G-3H]N-OH-4-aminobiphenyl (ABP) (48 mCi/mmol), [G-3H]N-OH-2-aminofluorene (AF) (200 mCi/mmol) and [G-3H]N-OH-PhIP (100 mCi/mmol) were synthesized from their nitro derivatives according to published procedures (18). The purity of the N-hydroxy derivatives was assessed by HPLC with photodiode array and radioactive flow detection and by comparison of specific activity with that of the nitro derivatives.

Syntheses
Dimethyldioxirane was synthesized by modification of a published method (21). A 1 l three-necked flask (containing 60 ml water, 36 ml acetone, 72 g sodium bicarbonate and a large magnetic stirring bar) was fitted with: (i) an air condenser; (ii) a solid addition funnel containing 150 g oxone (Aldrich, St. Louis MO); and (iii) a pressure equalized dropping funnel containing a mixture of 60 ml water and 42 ml acetone. The air condenser was attached in series to a cold-finger condenser containing dry ice/acetone and a condenser chilled with methanol/liquid nitrogen slush. The reaction mixture was stirred vigorously and the oxone and water/acetone mixture was added over ~30 min at room temperature. Helium was passed gently through the reaction flask to carry the product to the condensers and the dimethyldioxirane product in acetone was collected in a receiving flask attached to the cold-finger condenser. The condensed solution was dried over magnesium sulfate and the concentration of dimethyldioxirane was determined by absorbance at 335 nm ({varepsilon}335 = 10 M–1) (22). These reaction conditions typically produced 25–35 ml of dimethyldioxirane at a concentration of 0.08–0.10 M. The dimethyldioxirane was used immediately or after storage overnight at –20°C.

NO2{alpha}C was synthesized by direct oxidation of A{alpha}C with dimethyldioxirane by modification of a method described for primary aromatic amines (23). A{alpha}C (65 mg, 0.36 mmol) was dissolved in 15 ml acetone and was added over 5 min at room temperature to a stirred solution of dimethyldioxirane in acetone (35 ml, 0.09 M, 3.15 mmol). After 30 min the excess dimethyldioxirane and acetone were removed by rotary evaporation in vacuo. The crude product contained NO2{alpha}C (97%), A{alpha}C (1%) and an unknown product (2%) as judged by HPLC with photodiode array detection. The crude NO2{alpha}C was purified on basic alumina (AG10, 100–200 mesh, activity grade I; Bio-Rad) with elution with tetrahydrofuran (THF). The NO2{alpha}C was identified by its characteristic UV spectrum and by comparison with an authentic standard and purity was assessed by HPLC. The isolated yield of pure NO2{alpha}C was 55–65% as determined by absorbance at 338 nm in methanol ({varepsilon}338 = 9830 M–1 cm–1; Bionetics Corporation).

[G-3H]N-OH-A{alpha}C was synthesized from [3H]NO2{alpha}C by modification of a method utilized previously (17). Dry, peroxide-free conditions were essential for success of the reduction. Briefly, 4 mg of NO2{alpha}C (0.02 mmol) were dissolved in 4 ml of dry and peroxide-free THF (freshly distilled from LiAlH4). The solution was bubbled gently with argon and chilled to 4°C in an ice/water bath for 5 min, after which Pd/C (1.5 mg, 10%) was added. Hydrazine hydrate (80%, 15 µl, 0.375 mmol) was diluted with 700 µl of THF and chilled to 4°C. While continuously bubbling the NO2{alpha}C solution with a gentle stream of argon, the diluted hydrazine was added in 50 µl aliquots over 15 min (50 µl/min) and the reaction was stirred at 4°C for an additional 15–30 min. The charcoal was removed by filtration through a cotton plugged disposable glass pipette directly into a plastic syringe (containing 60 ml argon-saturated, ice-cold 0.1 mM EDTA) fitted with a C18 solid phase extraction cartridge (360 mg C18 Sep-Pak; Waters). The diluted solution was applied to the cartridge and the cartridge was washed with 50 ml argon-saturated, ice-cold 0.1 mM EDTA as quickly as possible. The product was immediately eluted with ice-cold, argon-saturated DMSO/ethanol (4:1). The solution was flushed gently with argon and stored in liquid nitrogen. The concentration of [3H]N-OH-A{alpha}C in solution was determined by reducing equivalents (24). Purity of [3H]N-OH-A{alpha}C was assessed by HPLC with photodiode array and radioactive flow detection and by comparison of specific activity with that of [3H]NO2{alpha}C. The isolated yield was 95%. The radiochemical purity of the [3H]N-OH-A{alpha}C was 90% or above, with A{alpha}C as the only impurity.

N-OAc-A{alpha}C was synthesized immediately prior to use by the addition of acetic anhydride (0.8 µl, 8.5 µmol) to N-OH-A{alpha}C [10 µl, 5 mM in DMSO/ethanol (4:1), 50 nmol] at 4°C. HPLC with spectral and radioactive flow analyses showed that 15 min at 4°C was optimal for formation of N-OAc-A{alpha}C with minimal formation of non-polar decomposition products. Nonetheless, only 18% of the starting material was converted to N-OAc-A{alpha}C, the remainder being unreacted N-OH-A{alpha}C (77%), N-acetyl-A{alpha}C (1.5%) and an unknown product (3.5%). In assays utilizing synthetic N-OAc-A{alpha}C, the concentration cited is calculated from the 18% conversion of N-OH-A{alpha}C to N-OAc-A{alpha}C.

HPLC analyses
HPLC separations were conducted on a Waters µBondapak column (5 µm, 3.9x300 mm) with elution at 1.0 ml/min with one of the following linear gradients over 30 min: (i) from 30% methanol in 20 mM diethylamine–acetic acid (pH 6.0) to 100% methanol; (ii) from 25% acetonitrile in 20 mM ammonium hydroxide–formic acid (pH 3.5) to 100% acetonitrile.

Tissue fractionation and enzyme preparation
Human liver surgical samples were obtained from the US Cooperative Tissue Network (Columbus, OH), the John L.McClellan Memorial Veteran's Hospital (Little Rock, AR) or from the International Institute for the Advancement of Medicine (Exton, PA). Tissues were snap frozen within 1 h of removal, shipped on dry ice and stored at –80°C until preparation of homogenates. Adult B6C3F1 mice and F344 rats were obtained from the NCTR breeding colony. The animals were killed by CO2 inhalation and the liver tissue was removed and homogenized immediately (18). Human and rodent liver cytosol fractions (100 000 g supernatant) were prepared at 4°C by differential centrifugation after homogenization in 4 vol of a solution containing 0.25 M sucrose, 50 mM Tris–HCl, pH 7.8, 0.5 mM EDTA, 20 µM butylated hydroxytoluene and 0.1 mM dithiothreitol. Protein concentrations were determined by Biuret assay (Total Protein Reagent; Sigma Diagnostics, St Louis, MO). Cytosol fractions were stored in 1 ml aliquots at –80°C and, after thawing, were kept at 4°C. Recombinant human N-acetyltransferases NAT1 and NAT2 were a generous gift of Dr David W.Hein (University of Louisville, Louisville, KY).

Non-enzymatic DNA binding of N-OH-A{alpha}C
Non-enzymatic covalent binding of [3H]N-OH-A{alpha}C and [3H]N-OAc-A{alpha}C was determined in a 0.5-ml reaction containing 100 µM N-OH-A{alpha}C or 18 µM N-OAc-A{alpha}C, 1 mg DNA, 1 mM EDTA, and 10 mM potassium citrate buffer at pH 5.0 or 7.0. After 2.5 h at 37°C, the reaction was terminated by addition of 1 ml of water-saturated 1-butanol. The DNA was isolated by sequential 1-butanol (three times) and phenol (twice) extractions and precipitation from ethanol and from ethanol/acetone (1:1). After reconstitution of the DNA in water, the level of covalent binding was determined by liquid scintillation counting and the concentration of DNA recovered was determined by a modified diphenylamine assay (25).

Cytosolic bioactivation and detoxification assays
In vitro bioactivation of N-OH-A{alpha}C was measured as the cofactor- or enzyme-dependent covalent binding of [3H]N-OH-A{alpha}C to calf thymus DNA. Reaction conditions were adjusted for each assay so that the reactions were saturating with N-OH-A{alpha}C substrate, saturating with cofactor, proportional to protein concentration and linear with time throughout the incubation at 37°C. The assay mixtures were saturated with argon prior to adding cytosol (or recombinant enzyme) and the reactions were started by addition of substrate. Careful argon saturation of the reaction mixture prior to addition of substrate was important to minimize non-enzymatic binding of the N-OH substrate. The enzyme reactions were terminated by addition of 2 vol of water-saturated 1-butanol with mixing. The DNA was isolated and the extent of covalent binding was determined as described above.

Assays for O-acetyltransferase-dependent bioactivation [acetyl coenzyme A (AcCoA)- or enzyme-dependent] were carried out similarly to those described previously (18). The assay mixture (0.5 ml) contained 0.5 mg cytosolic protein, 1 mg calf thymus DNA, 2 mM AcCoA, 1 mM dithiothreitol and 25 mM sodium pyrophosphate buffer, pH 7.0. Control reactions were conducted without AcCoA or in the absence of enzyme. The reaction with human liver cytosols was found to be saturating at 100 µM N-OH-A{alpha}C, linear with time up to 20 min at 37°C and linear with cytosolic protein concentration up to 1.5 mg/ml. Reactions with recombinant NAT1 or NAT2 contained 50 µg cellular protein/0.5 ml assay. The reaction was found to be saturating at 100 µM N-OH-A{alpha}C and linear with protein concentration up to 64 µg NAT1/0.5 ml assay and up to 160 µg NAT2/0.5 ml assay. The reaction with mouse liver cytosol was found to be saturating at 10 µM N-OH-A{alpha}C. With rat liver cytosol, substrate inhibition was exhibited above 10 µM N-OH-A{alpha}C.

To determine if glutathione S-transferase (GST) activity could protect against the cytosolic O-acetyltransferase-catalyzed covalent binding of N-OH-A{alpha}C to DNA (as it can for N-OH-PhIP; 26), 3 mM reduced glutathione (GSH) was included in a reaction with human liver cytosol. In addition, the GST inhibitor triethyltin sulfate (5 µM) was added to some incubations. Partially purified rat liver GST1a was also tested for its ability to protect against the binding of N-OAc-A{alpha}C to DNA in a 0.5-ml reaction containing 18 µM N-OAc-A{alpha}C, 5 mM GSH, 0.1 mM EDTA, 1 mg DNA, 0.1 M potassium phosphate, pH 7.5, and 75 µg partially purified rat liver GST1a.

Sulfotransferase-dependent bioactivation assays (0.5 ml) were conducted with 50 mM potassium phosphate buffer, pH 7.5, 5 mM magnesium chloride, 0.5 mM EDTA, 0.2 mM PAPS, 1 mg calf thymus DNA and 0.5 mg cytosolic protein. Control reactions were conducted without PAPS or in the absence of enzyme. Substrate inhibition was observed above 10 µM N-OH-A{alpha}C with human and rodent liver cytosols, thus 5 or 10 µM N-OH-A{alpha}C was used for most reactions. Reaction rates were linear with time up to 30 min at 37°C and were linear with protein concentration up to 0.75 mg/ml. Some reactions contained the phenol sulfotransferase inhibitor 2,4-dichloronitrophenol (DCNP) (10 µM).

The potential bioactivation of N-OH-A{alpha}C (100 µM) by cytosolic ATP-dependent kinase(s) was investigated in 0.5 ml reactions containing 0.5 M bicine, pH 8.0, 0.05 M magnesium acetate, 10 mM dithiothreitol (DTT), 10 mM ATP, 1 mg DNA and 0.5 mg cytosolic protein.

Assay with selective substrates for NAT1 or NAT2
Assays for NAT1 activity in human liver cytosol was conducted in 0.15-ml reactions containing 250 µM p-aminobenzoic acid (PABA), 1 mM AcCoA, 1 mM DTT, 0.1 M Tris–HCl, pH 7.4, and 0.2 or 0.8 mg cytosolic protein, and incubated for 10 min at 37°C. Assays for NAT2 activity were conducted under identical conditions, except with 500 µM sulfamethazine (SMZ) and 0.2 mg cytosolic protein. Under these conditions the reactions were saturating with substrate and cofactor, linear with protein, and linear with time. Reactions were stopped by addition of 25 µl of 25% (w/v) trichloroacetic acid to precipitate the protein, vortexed, then immediately neutralized with 25 µl of 1.5 N NaOH to prevent acid hydrolysis of N-acetyl-PABA or N-acetyl-SMZ. Rates of formation of N-acetyl-SMZ were determined after separation by HPLC with detection at 260 nm (27). Similarly, rates of formation of N-acetyl-PABA were determined after separation by HPLC with detection at 280 nm (28).

NAT2 genotype analysis
The primers and procedures used for PCR amplification of human NAT2 alleles were as published by Doll et al. (29). Restriction fragment length polymorphism analysis was carried out after digesting the PCR-amplified human NAT2 with the following combinations of restriction enzymes: MspI/KpnI, TaqI/BamHI and DdeI (29). Digested samples were separated on a 3% Metaphor agarose gel and visualized by ethidium bromide staining with UV transillumination.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Synthesis of NO2{alpha}C
This paper reports a clean and reliable method of synthesis of NO2{alpha}C from A{alpha}C utilizing dimethyldioxirane as the oxidizing agent. The isolated yield of the reaction was 55–65%. This yield could likely be improved with modification of the purification method, as the yield in the reaction mixture was 97%. Another method has been published recently for synthesis of NO2{alpha}C from A{alpha}C, but the reported yield was only 20–37% (30). We attempted to synthesize nitro-PhIP from PhIP by the dimethyldioxirane method; however, only unidentified products were formed. Thus, while oxidation of A{alpha}C with dimethyldioxirane should become the synthesis method of choice for NO2{alpha}C, it may not be widely applicable to other heterocyclic amines.

In vitro metabolic activation of N-OH-A{alpha}C
O-Acetyltransferase-dependent bioactivation of N-OH-A{alpha}C was determined with human liver cytosols (Table IGo) and the correlation of the O-acetyltransferase-dependent DNA binding of N-OH-A{alpha}C and three other N-OH substrates was examined. As shown in Table IIGo, the bioactivation of N-OH-A{alpha}C correlated with bioactivation of N-OH-PhIP (r = 0.94) and N-OH-ABP (r = 0.88) and not with bioactivation of N-OH-AF. This result suggests that the AcCoA-dependent bioactivation of N-OH-A{alpha}C is catalyzed by the same enzyme(s) in human liver cytosol as that for N-OH-PhIP and N-OH-ABP. The values presented here for AcCoA-dependent bioactivation of N-OH-ABP and N-OH-PhIP compare favorably with those observed in previous studies (18,31).


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Table I. AcCoA-dependent bioactivation of N-OH-A{alpha}C by human liver cytosol: comparison with other N-OH aromatic amines and with selective substrates for NAT2 or NAT1
 

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Table II. Correlationa of N-OH-A{alpha}C acetyltransferase activities in human liver cytosol (n = 9 or 10) with other N-OH aromatic amines and with selective substrates for NAT2 or NAT1
 
In humans, O-acetylation of N-OH aromatic and heterocyclic amines can be catalyzed by one or both isoforms of N-acetyltransferase, NAT1 and NAT2 (19). Thus, the substrate specificity of recombinant human NAT1 and NAT2 was investigated with N-OH-A{alpha}C and compared with that of other N-OH aromatic and heterocyclic amines. As shown in Table IIIGo, both NAT1 and NAT2 catalyzed bioactivation of N-OH-A{alpha}C. This is in contrast to the other substrates tested herein and previously (19), which exhibited selectivity for either NAT1 or NAT2. N-OH-A{alpha}C is the first N-OH heterocyclic amine to be shown to be highly activated by NAT1.


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Table III. AcCoA-dependent bioactivation of N-OH-A{alpha}C by recombinant human NAT1 and NAT2: comparison with other N-OH aromatic amines and with selective substrates for NAT2 or NAT1
 
We determined the NAT2 genotype of each sample for which the DNA was available (IIAM-25–IIAM-34). The method utilized could distinguish the presence or absence of four alleles (NAT2*4, *5, *6 and *7) (29). Samples containing at least one copy of the NAT2*4 allele were predicted to have the NAT2 `rapid' phenotype; all other samples were predicted to have the NAT2 `slow' phenotype (32). As shown in Table IVGo, the observed NAT2 phenotype matched that predicted from the genotype in nine of 10 samples. However, one sample, IIAM-34, contained the `rapid' NAT2*4/*6 genotype and showed low SMZ N-acetylation activity.


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Table IV. Genotype and phenotype for NAT2
 
Previous studies with N-OH-PhIP have shown that human and rodent GSTs and GSH could inhibit the DNA binding of N-OAc-PhIP (26). Thus, similar determinations were made for N-OH-A{alpha}C. In contrast to N-OH-PhIP, human cytosolic GST had no effect on the O-acetyltransferase-catalyzed covalent binding of N-OH-A{alpha}C to DNA (t-test, P = 0.96, n = 8; Table VGo). Similarly, partially purified rat GST1a had no effect on covalent binding of N-acetoxy-A{alpha}C to DNA (t-test, P = 0.25; Table VIGo). However, GSH decreased the DNA binding of N-OAc-A{alpha}C by 20% (t-test, P = 0.002; Table VGo) and decreased the in situ DNA binding of N-OH-A{alpha}C by 21% (P = 0.002, n = 8; Table VGo). This exhibition of GSH- but not GST-dependent inhibition of binding of N-OAc-A{alpha}C to DNA is similar to that shown previously for metabolically generated N-OAc-2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-OAc-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (26).


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Table V. Effect of GSH and human liver cytosolic GST on the acetyltransferase-mediated DNA binding of N-OH-A{alpha}C
 

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Table VI. Effect of GSH and rat GST1a on DNA binding of N-OAc-A{alpha}C and N-OAc-PhIPa
 
Sulfotransferase-dependent bioactivation of N-OH-A{alpha}C was determined with human liver cytosols and compared with that of other N-OH aromatic and heterocyclic amines (Table VIIGo). PAPS-dependent binding of N-OH-A{alpha}C with human liver cytosols was significantly greater than bioactivation of N-OH-PhIP (4.9-fold difference in mean, P < 0.001, n = 13). The correlation of sulfotransferase-dependent DNA binding of N-OH-A{alpha}C and the other substrates was also examined. As shown in Table VIIIGo, bioactivation of N-OH-A{alpha}C correlated with bioactivation of N-OH-PhIP (r = 0.96), N-OH-ABP (r = 0.81) and N-OH-AF (r = 0.76). This result suggests that PAPS-dependent bioactivation of N-OH-A{alpha}C is catalyzed by the same enzyme(s) in human liver cytosol as that for N-OH-PhIP. Previous results (20) have shown that the isoform of sulfotransferase primarily responsible for hepatic bioactivation of N-OH-PhIP is the thermostable phenol sulfotransferase (designated SULT1A1, formerly ST1A3). In addition, the N-OH-A{alpha}C sulfotransferase activity resembles SULT1A1 in that it is highly susceptible to inhibition by 10 µM DCNP (Table VIIGo). The values presented in Table VIIGo for PAPS-dependent bioactivation of N-OH-ABP, N-OH-PhIP and N-OH-AF compare favorably with those observed previously (20,33).


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Table VII. PAPS-dependent bioactivation of N-OH-A{alpha}C by human liver cytosol: comparison with other N-OH aromatic amines and inhibition by DCNPa
 

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Table VIII. Correlationa of N-OH amine sulfotransferase activities in human liver cytosols (n = 13)
 
Levels of AcCoA-dependent and PAPS-dependent DNA binding of N-OH-A{alpha}C by rodent liver cytosols was also examined. As shown in Table IXGo, levels of acetyltransferase-dependent bioactivation of N-OH-A{alpha}C in mouse liver cytosol were comparable to those in the low activity human liver cytosol (Table IGo). In contrast, levels of acetyltransferase-dependent bioactivation of N-OH-A{alpha}C in rat liver cytosol were comparable to those in the high activity human liver cytosol. Sulfotransferase-dependent bioactivation of N-OH-A{alpha}C by both rat and mice liver cytosols was comparable to that in the high sulfotransferase activity human liver cytosol (Tables VII and IXGoGo).


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Table IX. Bioactivation of N-OH-A{alpha}C by rat and mice liver cytosola
 
Levels of ATP kinase-dependent binding of N-OH-A{alpha}C were only slightly greater than the background non-enzymatic binding of N-OH-A{alpha}C to DNA (8–32 pmol bound/mg DNA/20 min in the presence of ATP and 5–22 pmol bound/mg DNA/20 min in the absence of ATP) for both human (n = 8) and rodent (n = 4) liver cytosols.

Reactivity of N-OH-A{alpha}C and synthetic N-OAc-A{alpha}C with DNA
N-OH-A{alpha}C itself was not highly reactive with DNA, even at slightly acidic pH (Table XGo). However, the N-OAc ester of N-OH-A{alpha}C was very reactive with calf thymus DNA (Tables VI and XGoGo). This synthetic N-OAc-A{alpha}C exhibited much greater binding to DNA than synthetic N-OAc-PhIP, especially considering that the initial concentration of N-OAc-A{alpha}C was 2.8-fold lower than N-OAc-PhIP (Table VIGo).


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Table X. Non-enzymatic covalent binding of N-OH-A{alpha}C and N-OAc-A{alpha}C to DNAa
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
N-OH-A{alpha}C was metabolized to reactive intermediates by enzymes present in human and rodent liver cytosols. Bioactivation of N-OH-A{alpha}C by phase II conjugations in human liver tissue, specifically by O-acetyltransferase and sulfotransferase, was found to be higher than the corresponding bioactivation of another N-OH heterocyclic amine, N-OH-PhIP, and the bicyclic aromatic amines N-OH-ABP and N-OH-AF (Tables I and VIIGoGo). Both sulfotransferase-dependent and O-acetyltransferase-dependent bioactivation of N-OH-A{alpha}C in human liver cytosols appear to exhibit a polymorphic distribution, although the small sample size prevents definitive probit analysis. The level of bioactivation of N-OH-A{alpha}C by rat liver cytosol was similar to the highest activity human liver cytosol for both sulfotransferase and O-acetyltransferase. This relatively high rate of bioactivation of N-OH-A{alpha}C by rat liver cytosol corresponds well with the observation in vivo in rats that the liver is the major site of adduct formation (13). In Sprague–Dawley rats fed A{alpha}C (single dose, 75 mg/kg), male rats formed detectable levels of A{alpha}C–DNA adducts only in the liver, while female rats formed the majority of A{alpha}C–DNA adducts in the liver. Low A{alpha}C–DNA adduct levels were also found extrahepatically in female rats in stomach, small intestine, colon, kidney and mammary gland. Another study detected a relatively high A{alpha}C–DNA adduct level in the liver of transgenic mice as compared with other heterocyclic amines (14). O-Acetyltransferase-dependent bioactivation of N-OH-A{alpha}C in the B6C3F1 mouse liver cytosol examined in the current study was comparable with the low activity human liver cytosol, while sulfotransferase-dependent bioactivation of N-OH-A{alpha}C in the mouse was comparable with the high activity human liver cytosol.

In addition to SULT1A1, human liver contains at least three other isoforms of sulfotransferase, thermolabile phenol sulfotransferase (SULT1A3), estrogen sulfotransferase (SULT1E1) and hydroxysteroid sulfotransferase (SULT2A1). Previous studies have shown that SULT1A1 is the isoform primarily responsible for bioactivation of N-OH-ABP, N-OH-PhIP, N-OH-AF and N-OH-2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (20,33). The correlation and inhibition studies reported herein also suggest that SULT1A1 is the isoform primarily responsible for bioactivation of N-OH-A{alpha}C in human liver cytosols. SULT1A1 exhibits a polymorphic distribution among individuals (34) and we observed a similar distribution in the sulfotransferase-dependent bioactivation of N-OH-A{alpha}C, varying widely among 14 individuals. Studies with a model SULT1A1 substrate, p-nitrophenol, have found SULT1A1 activity in many tissues, including liver, lung, brain, small intestine, kidney, platelets, colon, pancreas, larynx and bladder (20,33,35). Thus, SULT1A1-dependent bioactivation of N-OH-A{alpha}C could occur in both hepatic and extrahepatic tissues.

N-OAc-A{alpha}C is much more reactive with DNA than N-OH-A{alpha}C (Table XGo). N-OAc-A{alpha}C also seems much more reactive than N-OAc-PhIP (Table VIGo). Previous studies have shown that N-OAc-PhIP, while reactive with DNA, is stable enough to be transported through the blood stream of rats to form adducts in all tissues analyzed. It remains to be determined if N-OAc-A{alpha}C is stable enough to be transported systemically. However, even if N-OAc-A{alpha}C is not stable enough for transport, it could be formed from N-OH-A{alpha}C in extrahepatic tissues containing NAT1 or NAT2. Accordingly, tumor studies in CDF1 mice fed A{alpha}C found in both males and females a high incidence of liver tumors (39 and 97%, respectively) and blood vessel tumors (53 and 18%, respectively). No such tumors were found in control mice and no other tumor types were increased in the A{alpha}C-treated mice over control mice (7,36).

Although NAT1 is present only in low amounts in human liver, many extrahepatic tissues, including mammary gland, small intestine, colon, esophagus, bladder, stomach, lung, placenta, erythrocytes and leukocytes, contain NAT1 (3744). As seen in Table IIIGo, NAT1-catalyzed bioactivation of N-OH-A{alpha}C was 47-fold greater than that of N-OH-PhIP. Thus, the contribution of NAT1 to bioactivation of N-OH-A{alpha}C should be more substantial than for N-OH-PhIP. Prior studies have discounted a role of NAT1 in bioactivation of N-OH heterocyclic amines based on the low mutagenicity of N-OH-PhIP, N-OH-IQ, N-OH-MeIQx and several other N-OH heterocyclic amines in bacterial strains expressing only NAT1 (45) and on the relatively low bioactivation of N-OH-PhIP, N-OH-IQ and N-OH-MeIQx by NAT1 expressed in COS-1 cells (19). In contrast, the current study suggests that NAT1 may play a role in the carcinogenicity of A{alpha}C. In light of the observation of a genetic NAT1 polymorphism in humans these findings may provide the rationale for the higher prevalence of rapid NAT1*10 alleles among colon, bladder and oral cancer cases as compared with controls (4649).

NAT2 is predominantly expressed in the liver and epithelial cells of the intestine (39) and, to a lesser extent, in the pancreas (50), lung (40,51), esophagus (52) and placenta (42). The role of NAT2 in bioactivation of N-OH heterocyclic amines has been acknowledged for many years (19) and a few studies have found an increased risk of colon cancer with exposure to heterocyclic amines through consumption of well-done meats and NAT2 genotype (47,53). As seen in Table IIIGo, recombinant NAT2-catalyzed bioactivation of N-OH-A{alpha}C was significantly greater than that of N-OH-PhIP (4.6-fold difference in mean, n = 3, P = 0.002). Thus, tissues with relatively high NAT2 expression may be especially suceptible to DNA damage from N-OH-A{alpha}C.

It is important to note that NATs catalyze both detoxification and bioactivation of aromatic amines by N-acetylation and O-acetylation, respectively. However, N-acetylation of heterocyclic amines in humans is very low or non-existent (18,19). Another pathway, N-sulfamation, constitutes a major detoxification pathway of IQ and MeIQx in humans and rats, however, it is unknown if A{alpha}C can undergo this N-sulfamation detoxification. Certainly, N-sulfamation of heterocyclic amines is not universal, as PhIP-N-sulfamate has not been detected in mice, rats or humans.

This work, combined with that published previously (17), provides a sound basis for studying the role of CYP1A2, N-acetyltransferase and sulfotransferase polymorphisms and A{alpha}C exposure in individual cancer susceptibility. Determination of the role of these enzymatic pathways in bioactivation and detoxification of A{alpha}C will allow future molecular epidemiological studies to evaluate the impact of polymorphic variability and exposure on individual susceptibility to human cancers in which A{alpha}C may be implicated. In light of the ability of N-OH-A{alpha}C to be bioactivated by both NAT2 and NAT1, the high reactivity of N-OAc-A{alpha}C and the relatively high bioactivation of N-OH-A{alpha}C by SULT1A1, N-OH-A{alpha}C may be a significant source of DNA damage in human tissues after dietary exposure to A{alpha}C.


    Notes
 
1 Present address: Department of Biomedical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA Back

2 To whom correspondence should be addressed Email: fkadlubar{at}nctr.fda.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 16, 1999; revised March 6, 2000; accepted March 15, 2000.