In vitro metabolism and activation of carcinogenic aromatic amines by subcellular fractions of human liver.

In vitro metabolism and metabolic activation of 2-acetylaminofluorene (AAF), 2-aminofluorene, and 2,4-diaminoanisole to mutagenic (Salmonella test system) and covalently proteinbound intermediates were evaluated in subcellular fractions from seven human livers. The cytochrome P-450 content, aryl hydrocarbon hydroxylase activity, and sodium dodecyl sulfate: polyacrylamide gel electrophoresis of the human liver microsomes were concomitantly studied. AAF was extensively me tabolized (both Cand A/-hydroxylated) by all of the human liver microsomal samples, but severalfold variations between the individual metabolites were observed among the different mi crosomal fractions. Similar variation was also observed for aryl hydrocarbon hydroxylase activity. Electrophoresis of the mi crosomal fractions revealed several polypeptides with a molec ular weight range of 40,000 to 60,000. Two of these polypep tides, with molecular weights of approximately 54,000 and 55,000, correspond to those seen in rat liver microsomes following pretreatment with 3-methylcholanthrene. The in vitro mutagenicity of AAF with the individual samples corresponded well with those of 2-aminofluorene and 2,4-diaminoanisole, as did the degree of AAF /V-hydroxylation. A/-Hydroxy-2-acetylaminofluorene was converted to mutagen(s) by both human liver microsomal and cytosol fractions, presumably via deacetylation. A poor association between the extent of covalent binding of AAF to liver microsomal proteins and the degree of mutagenicity in the Salmonella system was observed among the samples, possibly indicating that the reactive metabolite(s) arylating the protein differs from that causing the frame-shift mutation in the Salmonella. The results from the . present study on AAF and benzo(a)pyrene hydroxylation; the metabolic activation of AAF, 2-aminofluorene, and 2,4-diaminoanisole; as well as the electrophoretic characterization of hepatic cytochrome P-450 in dicate great qualitative similarities between the subcellular fractions from human liver and those from the rat, the mouse, and the rhesus monkey. INTRODUCTION Aromatic amines, and in particular AAF,2 have been used extensively as model compounds in studies on chemical carcinogenesis and mutagenesis (1, 16, 29, 33). From these ' This study was supported in part by Grant SRG/13 from the NATO Special Programme Panel on Eco-Sciences Grant 04P-4933 from the Swedish Medical Research Council and a grant from the Swedish Cancer Society. 2 The abbreviations used are: AAF, 2-acetylaminofluorene; N-OH-AAF. Nhydroxy-2-acetylaminofluorene; AF, 2-aminofluorene; 2.4-DAA, 2,4-diaminoani sole; AHH, aryl hydrocarbon (benzo(a)pyrene] hydroxylase; 7-OH-AAF, 7-hydroxy-2-acetylaminofluorene. Received March 5. 1979; accepted July 3. 1979. studies, it is apparent that the carcinogenicity of AAF differs greatly among animal species and even among strains of the same species (17, 33). AAF is a procarcinogen and requires, as do many other chemical carcinogens, metabolic activation before its carcinogenic potential is expressed. The metabolic activation of AAF involves at least 2 steps, the first being a cytochrome P-450-dependent /V-hydroxylation resulting in the formation of N-OH-AAF, which becomes a substrate for several enzymes each known to further activate N-OH-AAF to a reac tive intermediate(s) capable of covalent interaction with cellular macromolecules. The cytosolic sulfotransferase and A/-O-acyltransferase as well as the membrane-bound deacetylase and UDP-glucuronyltransferase have all been implicated in forming the ultimate carcinogenic and mutagenic species of AAF (4, 17, 31 ). Since AAF requires metabolic activation, explanations for the carcinogenicity or lack thereof in the various animal species have been sought, and in some instances found, in the metabolism of AAF in the exposed animals. The resistance to AAF carcinogenesis in guinea pigs appears, for example, to be due to lack of /V-hydroxylation (16, 30). However, studies in other resistant animals such as rhesus monkey, cotton rat, and X/Gf mouse indicate a more complex relationship between AAF metabolism and carcinogenesis (7,11,16, 24, 28). In previous studies with subcellular fractions from mouse and rat liver, we have shown that the mutagenic activation of AAF in the Salmonella system (1) requires 2 activation steps, .namely, A/-hydroxylation and a subsequent deacetylation by either the membrane-bound deacetylase or the soluble A/-Oacyltransferase (9, 23-25). In this paper, we have examined the in vitro metabolism and activation of AAF, AF, and 2,4-DAA to mutagenic and covalently protein-bound intermediates in subcellular human liver fractions. MATERIALS AND METHODS Materials. [9-14C]AAF (46.16 mCi/mmol) was obtained from New England Nuclear Chemicals (Dreieichenhain, Federal Re public of Germany). The [9-'4C]AAF was more than 99.9% pure [thin-layer chromatography with silica gel and chloroform: methanol (97;3)]. AAF and AF were purchased from Koch-Light Laboratories (Colnbrook, England); 2,4-DAA was from ICN Pharmaceuticals (Plainview, N. Y.); benzo(a)pyrene was from Eastman Organic Chemicals (Rochester, N. Y.). N-OH-AAF and 9-hydroxy-2-acetylaminofluorene were generous gifts of Dr. Elizabeth K. Weisburger, National Cancer Institute (Bethesda, Md.), and Dr. Duane D. Miller, Ohio State University (Columbus, Ohio), respectively. S. typhimuhum TA98 was kindly provided by Dr. Bruce N. Ames, University of California (Berkeley, Calif.). Human Liver Samples. Samples of human liver were ob tained from 7 patients with total cerebral infarction who served 4206 CANCER RESEARCH VOL. 39 on June 8, 2017. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from AAF Metabolism in Human Microsomes as kidney transplant donors at the Huddinge University Hospi tal, Huddinge, Sweden. Consent to the experimental use of the livers was given by the Swedish National Board of Health and Welfare. Information on the donors is presented in Table 1, but regrettably no information on smoking habits has been availa ble. The patients were given 250 mg chlorpromazine and 25,000 IU heparin i.v. 15 min before liver extirpation. The livers were perfused with cold Perfadex for about 5 min. Thereafter, the livers were cut into small pieces and immediately placed in liquid nitrogen. About 2 hr later, the pieces were transferred to a freezer and stored at —80° until used. Preparation of Liver Subfractions. Ten-g pieces of frozen liver were thawed, minced in 2 volumes of sterile, ice-cold 20 HIM Tris buffer, pH 7.4, containing 1.15% KCI, with an UltraTurrax homogenizer for 2 sec and subsequently homogenized with 5 strokes of a Tefloniglass homogenizer. Liver 9,000 x g supernatant fractions, washed microsomes, and 105,000 x g supernatant fractions were prepared as previously described (5, 25). Sodium Dodecyl Sulfate:Polyacrylamide Gel Electrophoresis. Slab gels (1.5 mm thick) with 1-cm tracks were prepared as described by Laemmli (14), and 30 fig of microsomal protein were applied per track. The mixture of standards run alongside, with their subunit molecular weight, included aldolase (40,000), ovalbumin (43,000), glutamate dehydrogenase (53,000), and bovine serum albumin (68,000). Gels were stained for 1 hr in methanoliglacial acetic acid:water (5:1:4) containing 2 g of Coomassie Brilliant Blue R per liter and then destained over night in methanohglacial acetic acid:water (10:3:27). Assays. The in vitro mutagenesis was assayed in the Sal monella test system of Ames ef al. (1 ) as described previously (6, 25). Total microsomal cytochrome P-450 content was measured according to the method of Omura and Sato (20), and protein concentrations were measured by the method of Lowry ef al. (15). AHH activity was determined fluorometrically by reference to the fluorescence of recrystallized 3-hydroxybenzo(a)pyrene as standard (19) and using the benzo(a)pyrene and microsomal protein concentrations of 80 ¿tgand 0.6 mg/ml in the incubation mixture, respectively. The NADPHdependent covalent binding of 0.6 HIMfCJAAF to microsomal protein was determined as previously described (5). The oxidative metabolism of AAF was assayed, and the separation of metabolites was achieved using high-pressure liquid chromatography according to the method of Thorgeirsson and Nelson Table 1 Individual patient data Patient Sex Age Cause of death Known drug intake last week before death Regular drug intake and smoking and drinking habits Other relevant information 1 M 22 Drug intoxication. 2 F 68 Cerebral aneurysm. 3 F 28 Cerebral contusion, road accident. 27 Cerebral contusion, head trauma. 69 Cerebral infarction. 6 F 52 Meningioma. Postoperative infarction 7 F 59 Cerebral aneurysm. Intoxication with Doleron natt (acetylsalicylic acid, dextropropoxyfen, antipyrine, diethylaminocarboxylphenothiazine, vinbarbital). After admission to hospital, sin gle doses of epinephrine and atropine at admission: a few days treatment with dexamethasone. furosemide. ampicillin. Mannitol, dextran. a few doses of betamethazone and furo semide. At operation 1-2 days before death; atropine, succinylcholine, droperidol, fentanyl, tubocurarine. Mannitol. dextran, furosemide, a few doses of betametha zone. benzylpenicillin. guaifenesin. A few doses of betamethasone and diazepam. One dose of dexamethasone and meperidine. Barbiturate, fentanyl, pancuronium. tubocurarine at opera tion 5 and 3 days before death. Betamethazone, a few doses of furosemide, paracetamol, dihydroergotamine, metoclopramide. None Alcohol intake at intoxication. Drugs and smoking habits un known.