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Cigarette smoking has been associated with various cancers including bladder and pancreas. 4-Aminobiphenyl has been isolated as a constituent of cigarette smoke and has been established as a carcinogen in various animal models and humans. In rodents and humans, 4-aminobiphenyl is N-hydroxylated and forms adducts to DNA, the predominant one being N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). In this study, we report a micro-electrospray mass spectrometric (μESI-MS) isotope dilution method for the detection and quantification of dG-C8-ABP in human pancreatic tissue. A reverse phase capillary column (320 μm ID) was connected to a triple quadrupole mass spectrometer via a commercially available micro-ESI source. The system was operated in the selected reaction monitoring mode transmitting the [M + H]⁺ → [M + H − 116]⁺ transitions for both the analyte and the isotopically labeled internal standard. Twelve human pancreas samples were analyzed, where six were current smokers (three male and three female) and six were considered nonsmokers (three female and three male). Of the samples analyzed, six showed dG-C8-ABP levels above the limit of quantification for the method, five were considered to have levels that were undetectable, and one was discarded due to inconsistent internal standard signal. The age of the human subjects ranged from 17 to 63, and, in samples where adduct was present, levels ranged anywhere from 1 to 60/10⁸ nucleotides. Although no correlation between smoking preference, age, or gender was proven with this particular sample pool, this report demonstrates that capillary LC-μESI-MS can provide a sensitive and definitive method for DNA adduct analysis in human tissue.

Lactoperoxidase, an enzyme secreted from the human mammary gland, plays a host defensive role through antimicrobial activity. It has been implicated in mutagenic and carcinogenic activation in the human mammary gland. The potential role of heterocyclic and aromatic amines in the etiology of breast cancer led us to examination of the lactoperoxidase-catalyzed activation of the most commonly studied arylamine carcinogens:  2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), benzidine, 4-aminobiphenyl (ABP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). In vitro activation was performed with lactoperoxidase (partially purified from bovine milk or human milk) in the presence of hydrogen peroxide and calf thymus DNA. Products formed during enzymatic activation were monitored by HPLC with ultraviolet and radiometric detection. Two of these products were characterized as hydrazo and azo derivatives by means of mass spectrometry. The DNA binding level of ³H- and ¹⁴C-radiolabeled amines after peroxidase-catalyzed activation was dependent on the hydrogen peroxide concentration, and the highest levels of carcinogen binding to DNA were observed at 100 μM H2O2. Carcinogen activation and the level of binding to DNA were in the order of benzidine > ABP > IQ > MeIQx > PhIP. One of the ABP adducts was identified, and the level at which it is formed was estimated to be six adducts/10⁵ nucleotides. The susceptibility of aromatic and heterocyclic amines for lactoperoxidase-catalyzed activation and the binding levels of activated products to DNA suggest a potential role of lactoperoxidase-catalyzed activation of carcinogens in the etiology of breast cancer.

Cytochrome P450 (P450) 1A2 and NADPH-P450 reductase (NPR) catalyzed the oxidation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), with consumption of NADPH. The oxidation rate of NADPH by P450 1A2/NPR increased with time in the presence of IQ until depletion of NADPH. This unusual autocatalytic pattern of NADPH oxidation could be rationalized by formation of a nitroso derivative (IQ-N&dbd;O) and the subsequent reduction of the hydroxylamine (IQ-NHOH) and IQ-N&dbd;O, which would consume more NADPH. The formation of IQ-NHOH and IQ-N&dbd;O from IQ was confirmed using HPLC/MS. Reduction of IQ-NHOH and IQ-N&dbd;O was NPR-dependent but did not require P450. Autocatalytic NADPH oxidation was also observed in the oxidation of other heterocyclic and arylamines. However, the N-hydroxyl and nitroso oxidation products of 2-aminofluorene and 4-aminobiphenyl were reduced nonenzymatically by NADPH, and NPR did not catalyze the reactions. We simulated the enzymatic kinetic model for possible pathways for IQ metabolism, which included the formation of IQ-N&dbd;O, using some kinetic parameters obtained from the experimental results. In the kinetic model, we could reproduce the similar curvature for NADPH oxidation and the formation of IQ-N&dbd;O, and the reduction of IQ-NHOH and IQ-N&dbd;O is required to explain the observed results for NADPH oxidation. Our results support a role for nitroso derivatives of HAAs in the unusual autocatalytic NADPH oxidation and may have relevance in terms of possible toxicities of the nitroso derivatives. Both IQ-NHOH and IQ-N&dbd;O were mutagenic in a bacterial tester system devoid of P450 and NPR; the mutagenicity of both was decreased by expression of NPR, consistent with the reduction of these compounds observed with purified NPR.

A recent epidemiological study suggested that aromatic amines present in hair dyes may contribute to an increased risk of bladder cancer (Gago-Dominguez, et al. (2003) Carcinogenesis 24, 483−489). Moreover, a preliminary study linked frequent hair dye usage with elevated levels of DNA adducts of 4-aminobiphenyl (4-ABP) in human epithelial breast cells (Gorlewska, et al. Proc. Am. Assoc. Cancer Res. 43, 1018−1019). Therefore, we sought to determine if 4-ABP, a recognized human urinary bladder carcinogen, is present in commercial hair dyes. 4-ABP was isolated from dyes by solvent extraction with hexane, followed by silica gel chromatography, either with or without chemical treatment of the extract with Zinc/HCl, and a final purification with a mixed cation exchange reversed-phase resin. The identity of 4-ABP was confirmed by both HPLC with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS) following chemical derivatization with pentafluoropropionic anhydride (PFPA). The levels of 4-ABP ranged from not detectable (<0.29 parts per billion (ppb)) up to 12.8 ppb. The noncarcinogenic isomer 2-aminobiphenyl (2-ABP) was also found at quantities up to 310 ppb. 4-ABP was detected in eight of the 11 hair dyes and found in black, red, and blonde hair dyes but not in brown hair dyes. 1,4-Phenylenediamine (PPD) is a key constituent for color development of many permanent hair dyes. Some batches of chemical research grade PPD were contaminated with 4-ABP (up to 500 ppb) and 2-ABP (up to 70 parts per million) and may be a source of ABP contamination in hair dyes. These analytical data demonstrate that 4-ABP is present in some hair dyes. Studies on dermal absorption and bioavailability of 4-ABP from hair dyes are required to determine if this aromatic amine contributes to the increased risk of bladder cancer reported in frequent users of hair dyes.

Riddelliine, a widespread naturally occurring genotoxic pyrrolizidine alkaloid, induced liver tumors in rats and mice in an NTP 2-year carcinogenicity bioassay. We have determined that riddelliine induces liver tumors in rats through a genotoxic mechanism involving the formation of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which reacts with DNA to form a set of eight DNA adducts. To determine the relevance to humans of the results obtained in experimental animals, the metabolism of riddelliine was conducted using human liver microsomes. As with rat liver microsomes, DHP and riddelliine N-oxide were major metabolites in incubations conducted with human liver microsomes. The levels of DHP and riddelliine N-oxide were 0.20−0.62 and 0.03−0.15 nmol/min/mg protein, respectively, which are comparable to those obtained from rat liver microsomal metabolism. When metabolism was conducted in the presence of calf thymus DNA, the same set of eight DHP-derived DNA adducts was formed. Both the metabolism pattern and DNA adduct profile were very similar to those obtained from rat liver microsomes. When metabolism was conducted in the presence of the P450 3A4 enzyme inhibitor triacetyleandomycin, the formation of DHP and riddelliine N-oxide was reduced 84 and 92%, respectively. For DHP formation, the Km values were determined to be 0.37 ± 0.05 and 0.66 ± 0.08 mM from female rats and female humans; the Vmax values from female rat and human liver microsomal metabolism were 0.48 ± 0.03 and 1.70 ± 0.09 nmol/min/mg protein, respectively. These results strongly indicate the mechanistic data on liver tumor induction obtained for riddelliine in laboratory rodents is highly relevant to humans.

The activation of carcinogenic aromatic and heterocyclic amines and benzo[a]pyrene-7,8-diol to intracellular electrophiles by prostaglandin H synthase (COX) is well documented for ovine sources of this enzyme. Here, the arachidonic acid-dependent activation of substrates by human (h)COX-1 and-2 is examined, utilizing recombinant enzymes. The COX-dependent activation of benzidine (BZ), 4-aminobiphenyl, (+)benzo[a]pyrene-7,8-diol, (+)benzo[a]pyrene-7,8-diol, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), and 4,4'-methylenebis(2-chloroaniline) (MOCA) is assessed by means of COX-catalyzed, covalent DNA binding. The hCOX isozymes activated all substrates tested, activation varied from barely detectable for IQ (0.76 and 1.52 pmol bound/mg DNA for COX-1 and -2, respectively) to a high of 65 and 117 pmol bound/mg DNA for COX-1 and -2, respectively, for the activation of MOCA. BZ, which is an excellent peroxidase substrate, did not exhibit high DNA binding levels in hCOX assays and this phenomenon was found to be due to high levels of binding to protein, which effectively competed with the DNA for binding in the assay. The demonstrated ability of the COX enzymes to activate a variety of environmental and dietary carcinogens indicates a potential role for COX in the activation pathway of aromatic and heterocyclic amines and polycyclic hydrocarbons at extra-hepatic sites during early or late stages of carcinogenesis.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics presented at the joint meeting of the American Society for Biochemistry and Molecular Biology and the American Society for Pharmacology and Experimental Therapeutics, June 4-8, Boston, Massachusetts. The presentations focused on the pharmacogenetics of the NAT1 and NAT2 arylamine N-acetyltransferases, including developmental regulation, structure-function relationships, and their possible role in susceptibility to breast, colon, and pancreatic cancers. The symposium honored Wendell W. Weber for over 35 years of leadership and scientific advancement in pharmacogenetics and was highlighted by his overview of the historical development of the field.

A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M(-1)) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.

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 AalphaC 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.

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods. These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA. To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters. In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [(3)H]N-OH-IQ to DNA. ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and approximately 50% of that seen with acetyl coenzyme A (AcCoA). In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites. With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein. The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Arylamines are mutagens and carcinogens and are thought to initiate tumors by forming adducts with DNA. The major adducts are C⁸-guanyl, and we have previously suggested a role for guanyl-N⁷ intermediates in the formation process. N⁷-Aminoguanosine (Guo) was synthesized and characterized, with the position of the NH2 at N7 established by two-dimensional rotating frame Overhauser enhancement NMR spectroscopy. In DMF, N⁷-NH2Guo formed C⁸-NH2Guo and the cyclic product C⁸:5‘-O-cycloGuo. In aqueous media, these products were formed along with 8-oxo-7,8-dihydroGuo, N⁷-NH2guanine, and a product characterized as a purine 8,9-ring-opened derivative (N-aminoformamidopyrimidine). The rate of aqueous decomposition of N⁷-NH2Guo increased with pH, with a t1/2 of 10 h at pH 7 and a t1/2 of 2 h at pH 9. The rate of migration of NH2 from N7 to C8 is fast enough to explain the formation of C⁸-NH2Guo from the reaction of 2,4-dinitrophenoxyamine with Guo but not the formation of C⁸-(arylamino)Guo in the reaction of Guo with aryl hydroxylamine esters; however, the fluorenyl moiety may facilitate the proposed rearrangement by stabilizing an incipient negative charge in the transfer. In the reaction of Guo with N-hydroxy-2-aminofluorene and acetylsalicylic acid, a peak with the mass spectrum expected for N⁷-(2-aminofluorenyl)Guo was detected early in the reaction and was distinguished from C⁸-(2-aminofluorenyl)Guo. NMR experiments with [8-¹³C]Guo also provided some additional support for transient formation of N⁷-(2-aminofluorenyl)Guo. We conclude that a guanyl-N⁷ intermediate is reasonable in the reaction of activated arylamines with nucleic acids, although an exact rate of transfer of an N⁷-arylamine group to the C8 position has not yet been quantified. The results provide an explanation for the numerous products associated with modification of DNA by activated arylamines. However, the contribution of “direct” reaction at the guanine C8 atom cannot be excluded.