fish and meat consumption, and some occupational exposures. Epidemiologic .... Activation of aromatic and heterocyclic amines typically were 15 females and ...
Carcinogenesis vol.18 no.5 pp.1085–1092, 1997
Metabolic activation of aromatic amines by human pancreas
Kristin E.Anderson1,9, George J.Hammons2, Fred F.Kadlubar2, John D.Potter3, Keith R.Kaderlik2, Kenneth F.Ilett4, Rodney F.Minchin4, Candee H.Teitel2, Hsien-Chang Chou2, Martha V.Martin5, F.Peter Guengerich5, Gary W.Barone6, Nicholas P.Lang6,7 and Lisa A.Peterson8 1Division
of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, MN 55454, 2Division of Molecular Epidemiology, National Center for Toxicological Research, Jefferson, AR 72079, 3Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA, 4Department of Pharmacology, University of Western Australia, Nedlands, Western Australia 6009, 5Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232, 6Department of Surgery, University of Arkansas for Medical Sciences, Little Rock, AR 72205, 7Department of Surgery, John L.McClellan Veterans’ Administration Hospital, Little Rock, AR 72205 and 8Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, NY 10595, USA 9To
whom correspondence should be addressed at: 1300 South Second Street, Suite 300, University of Minnesota, Minneapolis, MN 55454, USA
Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas. Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens. Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine Ndemethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethylamine N-demethylation (P450 2E1); lauric acid ωhydroxylation (P450 4A1); and 4-(methylnitrosamino)-1(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) α-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/ 4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were ,1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [3H]Nhydroxy-ABP) of pancreatic cytosols was high, about twothirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using 32P*Abbreviations: IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQx, 2amino-3,8-dimethylimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine; AαC, 2-amino-α-carboline; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL, 4-(methylnitros-amino)-1(3-pyridyl)-1-butanol; HPLC, high-performance liquid chromatography. © Oxford University Press
post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.
Introduction Cancer of the pancreas is the fifth leading cause of cancer mortality in the United States, with ~27,800 deaths in 1996 (1). Cigarette smoking is the most consistently reported risk factor (2). Risk estimates from smoking are ~two-fold, with some as high as six-fold in association with high exposure categories. Other risk factors include high levels of cooked fish and meat consumption, and some occupational exposures (2). However, the primary etiology of human pancreatic cancer is still poorly understood. A common link among these risk factors may be a means of exposure to aromatic amines and nitroaromatic hydrocarbons, and it has been hypothesized that these agents may be carcinogenic to human pancreas (3,4). About 30 aromatic amines, including 2-naphthylamine and 4-aminobiphenyl have been detected in nanogram quantities in mainstream cigarette smoke and in even higher levels in sidestream smoke (5). Aromatic amines are also found in coal- and shale-derived oils (6) and in agricultural chemicals (7) and they are used in a variety of industrial processes (8). The carcinogenicity of aromatic amines, such as ABP (4aminobiphenyl),2-naphthylamine and benzidine, has long been established in both humans and experimental animals (9). More recently, a variety of highly mutagenic heterocyclic amines have been found in cooked foods (10); the most abundant include 2-amino-3-methylimidazo[4,5-f]quinoline (IQ*) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-α-carboline (AαC). PhIP and IQ are also present in mainstream cigarette smoke (11). These chemicals are potent mutagens and are known to be carcinogens in rodents, inducing tumors in several organs (12). IQ is also a powerful hepatocarcinogen in a non-human primate (13). Nitroaromatic hydrocarbons are widespread environmental contaminants (14) and have been detected in food (15). Some of these agents are both very potent bacterial mutagens and animal carcinogens (14). Although the importance of nitroaromatic hydrocarbons in the induction of human cancer is presently unknown, truck drivers exposed to diesel emission, a particularly rich source of these compounds, are reported to be at increased risk for the development of urinary bladder cancer (16). Nitrosamines are present in cigarette smoke and in the human diet and have been suggested as human carcinogens 1085
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(17). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific nitrosamine that is formed from nicotine during the curing process of tobacco (18). Pancreatic tumors are induced in F344 rats when NNK or its metabolite 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are given in the drinking water (19). NNK is also a potent transplacental inducer of pancreatic tumors in hamsters when combined with ethanol (20). As with most chemical carcinogens, aromatic and heterocyclic amines, nitroaromatic hydrocarbons, and nitrosamines need to be metabolized to reactive electrophiles that react with DNA in order to exert their carcinogenic effects (21,22). Activation of aromatic and heterocyclic amines typically involves an initial N-oxidation to form N-hydroxy derivatives, which in human and animal models is mediated primarily by cytochrome P450 1A2. Other P450 enzymes may contribute, but generally only to a minor extent. The initial activation of nitroaromatic hydrocarbons is likewise through the formation of an N-hydroxy arylamine, which involves the enzymatic reduction of the nitro group. Nitroreductase activity is associated with a number of enzymes including DT-diaphorase, xanthine oxidase, NADPH-cytochrome c reductase, aldehyde oxidase, and alcohol dehydrogenase (23,24). Further activation of the N-hydroxy arylamine formed by either of these two pathways can then occur through enzymatic esterification, involving sulfonylation or acetylation catalysed by sulfotransferases or acetyltransferases, respectively. In addition to these activation pathways via nitrogen oxidation and reduction reactions, certain aromatic amines and nitroaromatic hydrocarbons can also be converted into reactive intermediates via oxidation of phenolic arylamine metabolites to electrophilic iminoquinones; this reaction can be catalysed by extrahepatic peroxidases (e.g., prostaglandin H-synthase). NNK and NNAL are believed to be activated to potential DNA alkylating species via α-carbon hydroxylation reactions catalysed by cytochrome P450. The primary products of these pathways have been observed in microsomal incubations (25–29). Cancers of several human tissues are associated with risk factors similar to those of pancreatic cancer. Smoking has been conclusively linked with lung cancer (2). Urinary bladder cancer has two very important risk factors: smoking and aromatic amine exposure (30,31); and the role of diet has come under increasing scrutiny as a major risk factor in colorectal cancer (32). In each of these target tissues, the presence of DNA adducts of carcinogenic arylamines and the enzymes capable of metabolically activating them have been demonstrated, suggesting their role as etiological agents in cancer induction (33). In the present study, we examined pancreatic tissues from organ donors for their metabolic capacity to activate aromatic and heterocyclic amines and nitroaromatic hydrocarbons and for the presence of DNAadducts of arylamines. We also examined this organ for the ability to activate the tobacco-specific nitrosamines NNK and its metabolite NNAL because of their known role in pancreatic carcinogenesis in animals and their putative role in human carcinogenesis (22,34). Materials and methods Chemicals [2,29-3H]ABP (55 mCi/mmol), [2,29-3H]4-nitrobiphenyl (55 mCi/mmol), [5-3H]4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; 2100 mCi/ mmol) and [2,29-3H]benzidine (64 mCi/mmol) were purchased from Chemsyn Science Laboratories (Lenexa, KS). [5-3H]4-(Methylnitrosamino)-1-(3-pyri-
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dyl-1-butanol) (NNAL; 2100 mCi/mmol) was prepared from [5-3H]NNK according to published procedures (35). Calf thymus DNA (Type I), PABA (p-aminobenzoic acid), AcCoA (acetyl coenzyme A), hypoxanthine, N9methylnicotinamide, 7-ethoxyresorufin, 7-pentoxyresorufin, resorufin, aminopyrine, ethylmorpine and p-nitrophenol were purchased from Sigma Chemical Co. (St Louis, MO). The protease inhibitors, pepstatin and 4-(2-aminoethyl)benzenesulfonylfluoride, were obtained from Calbiochem Corp. (La Jolla, CA). [1-14C]Lauric acid (53 mCi/mmol) was purchased from Amersham Corp. (Arlington Heights, IL). N-Nitrosodimethylamine was purchased from Aldrich Chemical Co. (Milwaukee, WI). [2,29-3H]N-Hydroxy-ABP (55 mCi/mmol) was synthesized from [2,29-3H]4-nitrobiphenyl according to published procedures (36). Other reagents were analytical grade. Pancreatic tissues Tissues were obtained from donated organs made available for research (16 non-smokers, i.e. never and past smokers, and 13 current smokers). There were 15 females and 13 males among the donors (sex information was missing for one). There were eight females, four males, and one individual for whom sex information was missing among smokers and nine males and seven females among the non-smokers. The age range was 5–65 years (for two, age information was missing). The average age for smokers and non-smokers was 33.7 years and 30.3 years, respectively. The number of samples used in particular assays varied due to the amount of tissue available. After procurement, the organs were kept in UW (University of Wisconsin) cold storage solution for pancreas preservation (37) on ice, from 5 to 30 h post-crossclamp time. Organs preserved in this manner are used for human transplantation up to 30 h post-cross-clamp time (38). Tissues were then trimmed of fat, rinsed in saline, drained, wrapped in aluminum foil, snap frozen in liquid nitrogen, and stored at 280°C until used for preparation of subcellular fractions. Preparation of tissue microsomes and cytosols Pancreatic tissues were thawed and minced at 5°C in 50 mM sodium pyrophosphate buffer (pH 7) containing 1 mM dithiothreitol and 0.1 mM 4(2-aminoethyl)-benzenesulfonylfluoride. The tissues were homogenized with a polytron, followed by a Potter-Elvehjem homogenizer, as described by Flammang et al. (39) in 10 mM Tris-HCl buffer (pH 7.8), containing 0.1 mM EDTA, 0.25 M sucrose, 0.1 mM dithiothreitol, 20 µM butylated hydroxytoluene, 0.1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride and 5 µg/ ml pepstatin. The homogenate was centrifuged, and microsomal and cytosolic fractions were prepared as previously described (39,40). Protein concentration was measured by the method of Bradford (41). Microsomal enzyme assays Incubation mixtures (1 ml), used to measure the microsomal N-hydroxylation of ABP or the reduction of 4-nitrobiphenyl, contained 100 mM potassium phosphate buffer (pH 7.5), 0.5 mM EDTA, 5 mM glucose 6-phosphate, 0.4 mM NADP1, 0.3 mM NAD1, glucose 6-phosphate dehydrogenase (1 unit/ ml), 1.0–2.0 mg/ml microsomal protein, and 0.1 mM of either radiolabelled ABP or 4-nitrobiphenyl. The N-oxidation assays were carried out in open 10-ml vials in a shaking water bath, while nitroreductase assays were carried out in 163125-mm test tubes with stoppers under argon. After a 3-min preincubation at 37°C, the reactions were initiated by the addition of substrate and were continued for up to 20 min. Reactions were terminated by extraction of the assay mixture with water-saturated ethyl acetate and aliquots were analysed by high-performance liquid chromatography (HPLC) as previously described (42). NADPH-cytochrome P450 reductase activity was assayed with cytochrome c as substrate (43). Change in absorbance at 550 nm was measured, and the concentration of reduced cytochrome c was calculated using an extinction coefficient of 21 cm/mM. The metabolic assays of NNK and NNAL were conducted as previously described (29). Prostaglandin H synthase activity was measured as previously described (44). Assays for a variety of P450 activities were conducted (with substrate concentrations indicated) as referenced: aminopyrine N-demethylation (8.33 mM) and ethylmorphine Ndemethylation (10 mM) (45,46); ethoxyresorufin O-de-ethylation (1.7 µM) and pentoxyresorufin O-dealkylation (10 µM) (47); p-nitrophenol hydroxylation (0.2 mM) (48); N-nitrosodimethylamine N-demethylation (1 and 100 mM) (46,49); and lauric acid ω-hydroxylation (100 µM) (50,51). Cytosolic enzyme assays Incubation mixtures to measure cytosolic nitroreductase activity were carried out with [3H]4-nitrobiphenyl, as described above for the microsomal activity, but using 0.5–2.0 mg/ml cytosol protein. To measure nitroreductase activity catalyzed by xanthine oxidase or aldehyde oxidase, the NADH/NADHgenerating system was replaced with 0.5 mM hypoxanthine or 0.5 mM N9methylnicotinamide, respectively. O-Acetyltransferase activity (NAT1 and NAT2) was measured by the AcCoA-dependent binding of [3H]N-hydroxyABP to calf thymus DNA in a manner similar to that described by Flammang et al. (39). The assays were performed at 37°C in argon-saturated, 50 mM
Metabolism and DNA adducts in pancreas sodium pyrophosphate buffer (pH 7.0) containing 1 mM dithiothreitol, 2 mg/ ml calf thymus DNA, 0.25–1.0 mg/ml cytosolic protein, 1 mM AcCoA, and 100 µM radiolabeled N-hydroxy-ABP. The DNA binding reactions were terminated after 15 min by the addition of 2 volumes of water-saturated n-butanol. The DNA was isolated by multiple solvent extractions and precipitations and the extent of AcCoA-dependent covalent binding of N-OH-ABP to DNA was estimated by liquid scintillation counting. The DNA binding reactions were pseudo first-order and were linear with time for up to 15 min. Sulfotransferase activity was measured in a manner similar to the 39phosphoadenosine-59-phosphosulfate-dependent covalent binding of N-OHABP to DNA as previously described (52). PABA N-acetyltransferase activity (NAT1) was measured in two ways, first by the disappearance of substrate as determined by the Bratton-Marshall colorimetric procedure (53). Second, PABA N-acetyltransferase, in addition to SMZ (sulfamethazine) N-acetyltransferase activities were determined using HPLC. Reactions were carried out using 1 mM dithriothreitol, 500 µM AcCoA, and 100 µM PABA or 500 µM SMZ, at 37°C under conditions, which were linear with respect to time (15 min) and protein concentration (0.5–1 mg/ml), and were terminated by the addition of cold trichloroacetic acid (final concentration 4%). AcCoA was omitted from the blanks. Acetylated PABA and SMZ were quantified by HPLC as previously described (54,55). There was no association observed between enzyme activity level and length of post-cross-clamp time. Gel electrophoresis and immunoblotting Immunochemical staining for P450 protein levels was performed as previously described (56). Microsomal protein (100 µg) from human pancreas were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli (57) using a 4–15% gradient slab gel. Protein bands were then electrophoretically transferred onto nitrocellulose membranes by the Western immunoblotting technique as described by Towbin et al. (58) and modified by Guengerich et al. (56). Polyclonal antibodies were obtained against the human enzymes specified below as previously described: P450 1A2 (59); epoxide hydrolase (60); P450 2E1 (61); P450 3A4 (62); P450 2D6 (rat 2D1) (63); P450 2C (all) (64) and P450 2A6 (65). N-Acetyltransferase genotyping DNA was isolated from nuclear pellets obtained during differential centrifugation as described above by the method of Gupta (66). Polymorphisms in the human N-acetyltransferase gene, NAT2, were determined as described by Blum et al. (67). Briefly, DNA extracted from tissue samples was amplified by PCR (polymerase chain reaction) using specific oligonucleotides primers and then analyzed for the presence of three mutant alleles known to be predominant in the US population (68). The DNA was subjected to 25 cycles of allele-specific polymerase-chain reaction in a Perkin Elmer Cetus Thermal Cycler (final vol, 25 µl). The allele-specific method of Blum et al. (67) was used for NAT2*5A, NAT2*6A, NAT2*7A, and their respective mutants. This was followed by electrophoresis on a 1.5% agarose gel (NuSieve 3:1; FMC BioProducts, Rockland, ME) containing ethidium bromide and visualization of the PCR products with UV trans-illumination. Primers used were as follows: NAT2*5A-NAT2*4, CTGATTTGGTCCAG; NAT2*5A-mutant, CTGATTTGGTCCAA; NAT2*6A-NAT2*4, TTTACGCTTGAACCTCG: NAT2*6Amutant, TTTACGCTTGAACCTCA; NAT2*7A-NAT2*4, AATAGTAAGGGATC; NAT2*7A-mutant, AATAGTAAGGGATT; reverse primer for NAT2*5 and NAT2*7A; AATTAGTCACACGAGGA; and reverse primer for NAT2*6A, TCTAGCATGAATCACTCTGC. 32P-post-labeling A [3H]ABP-DNA adduct standard used for quantitation of 32P-post-labeling analyses was prepared by reacting [3H]N-OH-ABP with calf thymus DNA as described previously (69,70). The 32P-post-labeling assay was carried out essentially as described previously (71,72). Briefly, 5 µg of each DNA sample was hydrolysed to 39-nucleotides with 1.25 units of micrococcal nuclease and 0.125 µ units of spleen phosphodiesterase at 37°C for 3 h. Adducted nucleotides were enriched by extraction into n-butanol (73) and then [5932P]phosphorylated using 3 units of polynucleotide kinase and 300 µCi of [γ32P]ATP at 37°C for 40 min. The 32P-labelled nucleotides were then spotted on PEI-cellulose plates and developed as previously described (72). Adducts were visualized as dark spots on X-ray film after subjecting the thin-layer plates to autoradiography. The corresponding region on the thin-layer plate was then excised and radioactivity determined by scintillation counting. Putative ABP-adducts were identified by chromatographic comparison with the ABP-adduct standard and quantitated by comparing the radioactivity in the sample spot with the corresponding spot observed for the adduct standard.
Results Oxidative metabolism by human pancreas microsomes Pancreatic tissue microsomes were examined for their ability to carry out a variety of metabolic reactions that are known
Table I. Summary of oxidative metabolism by human pancreatic microsomes Substrate activity
ABP N-oxidationa Aminopyrine N-demethylationa Ethylmorphine N-demethylationa Ethoxyresorufin O-deethylationa Pentoxyresorufin O-dealkylationa p-Nitrophenol hydroxylationa N-nitrosodimethylamine demethylationa Lauric acid ω-hydroxylationa NNK, NNAL α-oxidationa Benzidine peroxidationb
Major Rates enzyme associated Smokers (n) with activity P450 P450 P450 P450 P450 P450 P450
1A2 3A4 3A4 1A1 2B6 2E1 2E1
,1 (7) ,500 (7) ,500 ,50 ,50 ,200 ,500
P450 4A1 ,5 P450 1A2 ,0.02 (6) PHSc 1.6 6 1.5 (8)
Non-smokers (n) ,1 (10) ,500 (10) ,500 ,50 ,50 ,500 ,500 ,5 ,0.02 (4) 1.5 6 1.0 (10)
aRates
are expressed in pmol/min/mg protein. are expressed as pmol substrate bound/mg DNA/50 µg protein/5 min 6 SD. cPHS, prostaglandin H-synthase. bResults
to be catalysed by the specific P450s that are prevalent in human liver and in extrahepatic tissues (Table I). P450 1A2 activity was initially examined, since the enzyme is known to catalyse the N-oxidation of most carcinogenic arylamines and, thereby, plays a major role in their metabolic activation. However, N-oxidation activity for ABP was not detected in microsomal preparations from any of the samples examined (n 5 17; 10 non-smokers, seven smokers, including two alcoholics). The rate in both smokers and non-smokers was ,1 pmol/min/mg protein; by comparison, the rate of ABP Noxidation in human liver microsomes (n 5 21) was 574 6 416 pmol/min/mg protein (47). We also examined these microsomal preparations for activities associated with P450s that metabolize several other classes of xenobiotics. The activities were below the limits of detection (Table I) for each of the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethylamine N-demethylation (P450 2E1); and lauric acid ω-hydroxylation (P450 4A11/12). Assays for the metabolism of the tobacco-specific nitrosamines, NNK and NNAL gave similar negative results (n 5 10, six smokers, four non-smokers). No α-oxidation products were observed in either NNK or NNAL incubation mixtures. The only significant metabolite of NNK detected was the carbonyl reduction product, NNAL. The rate of reduction ranged from 0.01–1.0 pmol/min/mg protein [smokers (n 5 6) 0.51 6 0.26 pmol/min/mg protein versus non-smokers (n 5 3) 0.26 6 0.08 pmol/min/mg protein]. By comparison, the rate of NNK α-hydroxylation and carbonyl reduction in human liver microsomes was 0.5 and 5.1 pmol/min/mg protein, respectively. Microsomal prostaglandin H-synthase activity (n 5 18; 8 smokers, 10 non-smokers) was measured using either [3H]ABP or [3H]benzidine as substrate (Table I). The results showed only a very low level of activity with benzidine (1–3 pmol ´ of the activity bound/mg DNA), which corresponds to ~3–10% observed in human urinary bladder (39). The presence of P450s, as well as epoxide hydrolase, was 1087
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Table II. Reductase activity of human pancreatic microsomes and cytosols Substrate activity
Cell fraction
Rates 6 SD
Table III. Summary of metabolic esterification by human pancreatic cytosols Substrate activity
Smokers (n) 4-Nitrobiphenyl nitroreductiona NADPH-cyt. c (P450) reductionb 4-Nitrobiphenyl nitroreductiona,c
Microsomes
92 6 48 (12)
138 6 48 (15)
Microsomes
6.93 6 1.03 (12) 112, 114 (2)
7.06 6 1.49 (15) 57 6 30 (6)
Cytosol
aRates bRates
are expressed in pmol/min/mg protein. are expressed in nmol/min/mg protein. cSubstitution of 0.5 mM hypoxanthine or 0.5 mM N9-methylnicotinamide for the NADPH/NADH-generating system did not result in detectable rate of nitroreduction (,0.01 nmol/min/mg protein).
also investigated through immunological methods. Antibodies to epoxide hydrolase and to cytochromes P450 1A1/1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/4/5/7 were used in immunoblots of pancreatic microsomes (n 5 10, six smokers, four non-smokers). Of these, only epoxide hydrolase was detected at low levels; ~1% of liver (data not shown); in all samples tested, the levels of P450s were ,1% of liver. 4-Nitrobiphenyl reductase activity of pancreas microsomes and cytosols Exposure to the carcinogenic metabolites of nitroaromatic hydrocarbons may occur through nitroreduction of these agents to their corresponding N-hydroxy aromatic amine derivatives (23). Thus, it was of interest to examine the tissue preparations for nitroreductase activity (Table II). Microsomal 4-nitrobiphenyl reductase activity was found in all the pancreatic tissues examined (n 5 27, 12 smokers, 15 non-smokers) and at high levels, similar to that found in human lung and liver (74). HPLC analyses showed the formation of both N-hydroxy-ABP (5–20%) and ABP (80–95%) as reduction products, the relative yields of which varied with different samples. We then explored the possibility that the NADPH-cytochrome P450 reductase might be the enzyme responsible for the 4-nitrobiphenyl reductase activity, since Hall et al. (75) had detected this protein in human pancreatic ductal cells (but not exocrine cells), using an immunostaining technique. Accordingly, we examined P450 reductase activity via a photometric assay; however, the detected activity levels of P450 reductase showed no significant correlation with the levels of 4-nitrobiphenyl reductase activity. Cytosolic preparations were also assayed for 4-nitrobiphenyl reductase (n 5 8, two smokers, six non-smokers) and appreciable activity was detected, at levels that were similar to those found in the microsomes. 4-Nitrobiphenyl reduction did not occur when hypoxanthine or N1-methyl-nicotinamide were substituted for the NADPH/NADH-generating system, suggesting that neither xanthine oxidase nor aldehyde oxidase participate in the reduction of this nitro compound. Metabolic esterification by human pancreas cytosols O-Acetyltransferase activity of pancreatic cytosols (Table III), as measured by AcCoA-dependent DNA-binding of [3H]NOH-ABP, was at high levels, comparable to approximately two-thirds the levels seen in human colon (76). All 29 samples showed significant activity. Since both the NAT1 and NAT2 can carry out the O-acetylation of N-hydroxy aromatic amines 1088
Enzyme
Non-smokers (n) N-hydroxy-ABP NAT1 1 O-Acetyltransferasea NAT2 b PABA N-acetyltransferase NAT1 SMZ N-acetyltransferaseb N-hydroxy-ABP sulfotransferasea aRates
NAT2 ST1A3c
Rate 6 SD Smokers (n)
Non-smokers (n)
1.45 6 0.90 (13) 2423 6 1325 (7) 23, 20 (2/7) ,0.01 (2)
1.24 6 0.72 (16) 1619 6 1376 (14) 23 6 12.0 (7/14) 0.02 6 0.02 (6)
are expressed in pmol bound/min/mg DNA. in pmol/min/mg protein.
bRates are expressed cTowbin et al. (57).
(77), we sought to determine whether or not both enzymes were present in this tissue. Using PABA as substrate, under assay conditions that have shown it to be a selective substrate for NAT1 (78), we detected activity for NAT1 (Table III) in all the cytosols assayed (n 5 21, seven smokers, 14 nonsmokers). Using SMZ as substrate, under assay conditions that have been shown to be selective for NAT2 (79), we detected very low activity in 9/21 samples; however, the activity detected was ,5% of that reported for human colon (80); and, upon NAT2 genotyping of these samples, there was no association between SMZ activity and the presence of the rapid acetylator allele (NAT2*4) (data not shown). Sulfotransferase activity for N-hydroxy-ABP (Table III), which is catalysed in humans selectively by the thermostable phenol sulfotransferases (53), was also measured in pancreatic cytosols (n 5 8, two smokers, six non-smokers) and was found only at low levels (2–4% of liver activity). DNA adducts Post-labeling analysis of pancreatic DNA (n 5 29, 13 smokers, 16 non-smokers) for putative aromatic amine-DNA adducts, revealed a number of radiolabelled spots, but there was no apparent association with current smoking status. In eight of 29 tissue DNA samples, we observed the presence of an adduct (Figure 1) that was chromatographically identical to the major ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. This adduct was detected in DNA from both smokers (5/13) and nonsmokers (3/16) and was not observed using the nuclease P1 enhancement method, which is known to cause selective 39dephosphorylation of guanine-C8-substituted aromatic amineDNA adducts and subsequent inactivity for 32P-post-labeling. The overall adduct levels were relatively low, in the range of 0.2 to 1.1 adducts per 108 nucleotides. Discussion Pancreatic cancer is associated with several risk factors that make plausible the involvement of carcinogenic aromatic amines and nitroaromatic hydrocarbons in the etiology of this disease (2,4). These chemical agents, however, require activation to DNA-reactive species in order to exert their carcinogenic effect (21). This study was undertaken to investigate the capacity of human pancreas to metabolically activate arylamines and other carcinogens, and to examine whether or not aromatic amine-DNA adducts are present in this tissue. Nitrosamines have also been suggested as human carcinogens. These compounds are present in the human diet (17)
Metabolism and DNA adducts in pancreas
Fig. 1. Autoradiogram of 32P-post-labeled DNA from human pancreas using the butanol extraction method. The arrow designates the chromatographic mobility of N-deoxyguanosin-8-yl-ABP 39,59-bis(phosphate).
Fig. 2. A hypothesis for the metabolic activation of aromatic amines and nitroaromatic hydrocarbons by human pancreas; AA, aromatic amines; nitro-PAHs, nitro-polycyclic aromatic hydrocarbons.
and at high levels in cigarette smoke (17). The Surgeon General’s report of 1989 (34) suggested NNK as the putative human pancreatic carcinogen in cigarette smoke. Moreover, nitrosamines induce pancreatic cancers in several animal models (81,82). Thus, the metabolism of tobacco-specific nitrosamines were of interest in this tissue analysis. There is evidence from human tissues that metabolism of NNK is carried out by several P450s, including 1A2, 2A6, 2B6, 2E1, 2F1, 3A5 and to a small extent, by other enzymes as well (83). Our data indicate that, unlike rodents, enzymes which activate these carcinogens are not present in the human pancreas. This does not rule out a role for these compounds in pancreatic carcinogenesis, for example, via activation in the liver. However, the presence of enzymes involved in arylamine metabolism in the pancreas favors an alternative hypothesis in the human. Several enzyme systems are known to be involved in the metabolic activation of carcinogenic aromatic amines (21). P450 1A2, which catalyzes the initial N-oxidation of these agents, could not be detected, either by enzyme assay or by immunochemical analysis, in any of the pancreatic tissue samples (including those from smokers) examined in this study. Tissue samples from smokers and non-smokers were included because this enzyme is known to be induced in the liver by cigarette smoking (84,85). The absence of this pathway in the human pancreas is consistent with data that have failed to detect P450 1A2 from many human extrahepatic tissues (86). Attempts to detect other P450s involved in carcinogen or drug metabolism in the same tissue samples also were unsuccessful. Our negative findings are inconsistent with those of Foster et al. (87) who reported the immunohistochemical detection of P450s in pancreatic tissue from organ donors. Levels of these enzymes were higher in pancreas samples from patients with chronic pancreatitis and pancreatic cancer. The reasons for this difference between our findings and theirs are uncertain, but may reflect a lower limit of detection with cytochemical methods. We also examined the tissue samples for the presence of prostaglandin H-synthase because an alternative pathway for aromatic amine metabolism involves peroxidative activation in the target tissue, as has been demonstrated in human urinary bladder and prostate tissues (39,88). However, this pathway appears to be of negligible importance in human pancreas. The metabolic activation of nitroaromatic hydrocarbons involves conversion to the N-hydroxy derivative through reduction of the nitro group (23). In contrast to the absence of Noxidation, nitroreductase activity was found in all tissue samples. Rates for the reduction of 4-nitrobiphenyl were comparable with those of microsomal and cytosolic preparations from human lung and liver tissues (74). These results demonstrate the capacity of pancreas to catalyse the reductive metabolism of carcinogenic nitroaromatic hydrocarbons to their N-hydroxy derivatives, thus providing a critical pathway for their metabolic activation. Although there is no strong epidemiologic evidence associating nitroaromatic hydrocarbons with pancreatic cancer, such capacity may underlie elevated risks associated with certain exposures, such as combustion products (89), jet fuel exhaust (90) and coke oven emissions (91). The identity of the nitroreductase activities remain unclear. Several enzymes can catalyse the reduction of nitroaromatics, including microsomal NADPH-cytochrome P450 reductase, cytosolic DT-diaphorase (quinone reductase) and aldehyde oxidase (80). Activity for the former was found 1089
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to be present in all of the tissue samples examined; however, there was no correlation between this activity and the total nitroreductase activity, which suggests that multiple enzymes are likely to be involved in the pancreas. Activation of aromatic and heterocyclic amines and nitroaromatic hydrocarbons also involves esterification of their N-hydroxy derivatives, which can generate more reactive electrophiles (21). O-Acetyltransferase activity, which can be catalyzed by NAT1 and NAT2, was found in all of the tissue samples examined. However, NAT1 appears to play a predominant role in the pancreas, since appreciable activity could be detected only for the NAT1-selective substrate, PABA, and not for SMZ, an NAT2-selective substrate. Detection of NAT1 activity in the human pancreas is a novel finding and suggests a pathway for metabolic activation of arylamines and nitroaromatics in the pancreas similar to that proposed for human urinary bladder (92,93) and human peripheral lung (74). Although aromatic amine N-oxidation did not occur in the pancreas, these compounds could undergo N-oxidation in the liver by P450 1A2 and be transported through the circulation to the pancreas, where they could be O-acetylated by NAT1 present in pancreatic tissue. A similar metabolic activation pathway has been described for the heterocyclic amine, PhIP, in animal studies. Oral administration of radiolabelled PhIP in rats and dogs resulted in high levels of radioactivity bound to DNA in many organs; and the levels of DNA adducts were highest in the pancreas in both animal species (94). In addition, i.v. injection of radiolabelled N-hydroxy-PhIP or N-acetoxyPhIP, the putative proximate and ultimate carcinogenic forms of PhIP, into rats results in high levels of DNA-PhIP adducts in the pancreas (94,95). The apparent presence of the major ABP-DNA adduct, N(deoxyguanosin-8-yl)-ABP, in several of the organ donor tissue samples further suggests that the human pancreas may also be a target organ for carcinogenic aromatic amines and nitroaromatic hydrocarbons. Our data do not provide evidence that cigarette smoke is the primary source of these DNA adducts. These data are preliminary and further analyses are required to confirm these findings. Although P450 activities have been detected in pancreatic tissue in the rat and hamster animal models, the limited ability of these enzymes within the rodent pancreas to metabolize xenobiotics has also suggested the possibility that other organs may play a role in the activation of rodent pancreatic procarcinogens (81). NAT1 activity was found in all tissue samples examined. Recently, a genetic polymorphism in NAT1 has been shown to result in differential expression of NAT1 activity in both human urinary bladder and colon mucosa (92). Furthermore, the rapid acetylator genotype (NAT*10) has been associated with an increased risk of bladder cancer among smokers (96) and an increased risk of colon cancer (smoking status could not be assessed independently (97). Accordingly, there may also be a significant role for NAT1 in metabolic activation in the human pancreas; and appropriate molecular epidemiological studies will be needed to test this hypothesis. In conclusion, the carcinogen-metabolizing activities of human pancreas provide a plausible pathway for the metabolic activation of arylamines and nitroaromatic hydrocarbons (Figure 2). Involvement of these agents in the etiology of pancreatic cancer is further suggested by the presence of putative aromatic amine-DNA adducts in pancreatic tissues. These findings may help to explain the etiology of this disease 1090
in smokers and also in those individuals with certain dietary and environmental exposures. Acknowledgements The authors would like to thank the following individuals for their generous assistance in obtaining tissue samples: Dr David Sutherland, Dr David Scharp, Dr Paul Lacy, Dr Paul Gores, Mr William Minor, Ms Jane Field, Ms J.J.Loveras, Ms Barbara Olack, Ms Joyce Massengill, Ms Micky Dunning, Mr Ed Van Bergen and the staff of LifeSource (Upper Midwest Organ Procurement Organization, Inc.), Minneapolis, MN. The authors are indebted to Pat Thomas for her excellent assistance in preparation of this manuscript, and to Drs Fred Beland and Sandra Culp for their helpful comments. This work is supported in part by grants from the USPHS to K.Anderson (5T32 CA09607 and R01CA58697), to F.Guengerich (CA44353 and ES00267) and to S.Hecht (L.Peterson) (CA 44377).
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