Esophageal and Hepatic Microsomal Metabolism of Ammitrosomethyl- Benzylamine and JV-Nitrosodimethylamine in the Rat1

Home , Benzylamine

[CANCER RESEARCH 42, 3181-3186, August 1982] 0008-5472/82/0042-0000$02.00 Esophageal and Hepatic Microsomal Metabolism of AMMitrosomethyl- benzylamine and JV-Nitrosodimethylamine in the Rat1

George E. Labuc and Michael C. Archer2

Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, Toronto, Ontario, Canada M4X 1K9

ABSTRACT as esophageal slices (2) or cultured expiants (23), providing indirect evidence for the metabolic activation of these com The metabolism of the rat esophageal carcinogen A/-nitro- pounds in the esophagus. Rat and hamster esophageal slices somethylbenzylamine (NMBzA) was studied using microsomes have also been reported to oxidize NDMA3 and /V-nitrosodi- prepared from liver and esophageal mucosa of untreated male ethylamine to CO2 (14). However, direct studies on the forma Sprague-Dawley rats. NMBzA was extensively metabolized to tion of nitrosamine metabolites using cell-free systems have benzaldehyde, benzyl alcohol, and formaldehyde by hepatic provided contradictory results. Whereas one report indicated microsomes. The rate of metabolism at the benzyl moiety was a higher rate of nitrosamine metabolism by esophageal micro 10-fold higher than that at the methyl moiety. Mucosal micro somes than by hepatic microsomes (22), another study re somes metabolized NMBzA to benzaldehyde and formaldehyde ported little if any activity in the esophagus (21). at rates one-fifth and one-sixtieth of those in the liver, respec NMBzA is a potent esophageal carcinogen and toxin in the tively; benzyl alcohol formation was undetectable. Esophageal rat but produces no liver tumors (1 ); NDMA is a potent hepa metabolism of NMBzA was exclusively located in the mucosa, tocarcinogen that gives no esophageal tumors in the rat (1, preferentially in the microsomal fraction, was reduced nicotin- 11). The aim of the present study was to investigate the amide adenine dinucleotide phosphate dependent, and was metabolism of NMBzA and NDMA in both target and nontarget inhibited by CO and 2-diethylaminoethyl-2,2-diphenylvalerate. tissues. The predominant pathway for nitrosamines is consid A low level of cytochrome P-450 was detected in the mucosal ered to be hydroxylation at the a-carbon atom, an activation microsomes. Whereas hepatic metabolism of NMBzA was in- step which ultimately yields an alkylating agent and an alde ducible by phenobarbitone pretreatment, mucosal metabolism hyde in stoichiometric amounts (9). The alkylating agent can was not altered by either phenobarbitone or 3-methylcholan- react with tissue nucleophiles or with water to form the corre threne pretreatment. The hepatocarcinogen A/-nitrosodimethyl- sponding alcohol. Thus, NDMA is known to yield formaldehyde amine was extensively metabolized by hepatic microsomes to and a methylating agent (9). NMBzA is expected to yield formaldehyde, but demethylation was not detected in the mi formaldehyde and a benzylating agent from hydroxylation at crosomes from esophageal mucosa, a nontarget tissue. The the methyl carbon, or benzaldehyde and a methylating agent results indicate that rat esophageal mucosa contains an en from hydroxylation at the mÃ©thylÃ¨necarbon of the benzyl zyme system which metabolizes NMBzA at a high rate and moiety. NMBzA was found to be metabolized by esophageal exhibits properties typical of cytochrome P-450. This enzyme microsomes via a cytochrome P-450 pathway almost exclu may play a role in determining which compounds induce tumors sively at the mÃ©thylÃ¨necarbon. In contrast, hepatic microsomal in rat esophagus. metabolism of NMBzA occurred at both a-carbons. NDMA metabolism was detected only in the liver. INTRODUCTION MATERIALS AND METHODS The rat esophagus is a primary target organ for many carci nogenic /V-nitrosamines (1, 11). High incidences of basal cell Chemicals. NMBzA (b.p. 60-62Â°/0.01 mm), synthesized by the and squamous cell tumors are induced by these nitrosamines, method of Druckrey ef al. (1), was >99% pure as determined by gas independent of the route of administration (1, 11). Apart from chromatography and high-performance liquid chromatography. NDMA the nitrosamines and some nitrosamides, few other chemicals was purchased from Eastman Kodak Co., Rochester, N. Y. [methyl- have been reported to induce esophageal tumors (6, 20). I4C]NMBzA (5.46 mCi/mmol) and [mef/7y/-14C]NDMA (54.2 mCi/mmol) However, the mechanism of this remarkable sensitivity of the were obtained from New England Nuclear, Boston, Mass, and [meth- y/ene-14C]NMBzA (30 mCi/mmol) was obtained from Amersham-Searle rat esophagus to nitrosamines is not known. Corp., Arlington Heights, III. NADP* (sodium), G-6-P (sodium), G-6-P In general, tissues that are sensitive to tumor induction by dehydrogenase (type XV), /8-NAD+ (Grade III), arachidonic acid (type indirect acting chemicals exhibit the capacity to metabolize I), phenazine ethosulfate, and bovine serum albumin (Fraction V) were such compounds into electrophilic intermediates, which are purchased from Sigma Chemical Co., St. Louis, Mo. SKF 525-A was a capable of interacting with tissue macromolecules (9, 13). generous gift from Smith, Kline & French Laboratories, Mississauga, However, little is known concerning the metabolism of nitros Ontario. amines by the rat esophagus. A number of studies have dem Animals. Male Sprague-Dawley rats (21 to 23 days old) were pur onstrated the alkylation of esophageal macromolecules when chased from Charles River Canada, Inc., Laprairie, Quebec, Canada. nitrosamines are incubated with intact cell preparations, such Rats were maintained on Teklad 6% fat rat-mouse diet (Teklad Mills, Iowa) ad libitum for 5 to 10 days prior to sacrifice. Where specified, ' This work was supported by the Ontario Cancer Treatment and Research Foundation and Grant MT7025 from the Medical Research Council of Canada. 3 The abbreviations used are: NDMA, N-nitroso-N,N-dimethylamine; NMBzA, 2 To whom requests for reprints should be addressed. N-nitroso-N-methyl-N-benzylamine; G-6-P, glucose 6-phosphate; SKF 525-A, 2- Received November 18, 1981; accepted April 1, 1982. (diethylamino)ethyl-2,2-diphenylvalerate HCI; 3-MC, 3-methylcholanthrene.

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animals were pretreated with phenobarbitone (0.1% in drinking water zone was treated with HNO2 (3) to generate free benzaldehyde, which for 5 days) or either corn oil (0.8 ml/100 g body weight) or 3-MC (80 was then extracted into dichloromethane. mg/kg body weight) in corn oil, injected i.p. 40 hr prior to sacrifice. In some studies, incubations containing [mef/>y/-14C]NMBzA or Enzyme Preparation. Rats were sacrificed by a blow to the head. [mef/7y/ene-'4C]NMBzA were deproteinized and subjected to chroma Liver and esophagus were excised and rinsed in cold homogenizing tography as above. Fractions (0.5 min) were collected and counted for buffer [1.15% (w/v) KCI-50 mM Tris-HCI, pH 7.40]. All subsequent 14C in 10 ml ACS (Amersham). steps were carried out at 4Â°. Esophageal mucosa was obtained by Incubation mixtures containing [mefhy/-'4C]NMBzA or -NDMA were physically shearing the outer muscle-submucosa layers away from the analyzed for [14C]formaldehyde production by quantitative precipitation mucosa. The whole esophagus was gripped centrally with 2 pairs of as the dimedone derivative by the method of Paik and Kim (19), except forceps; holding one pair firmly in place, the second was gently moved that the deproteinized supernatants were extracted 3 times with dichlo towards one end of the tissue, such that the outher muscle-submucosa romethane (1.0 ml) prior to dimedone formation in order to remove peeled away from the mucosa. This process was repeated at the other residual substrate. end. The resulting mucosa was completely intact and contained no Controls were mixtures incubated at 37Â°in the absence of tissue or muscle or submucosa contamination, as determined by routine histol mixtures incubated at 0Â°in the presence of tissue. Both gave similar ogy. The mucosa was slit longitudinally, blotted dry, weighed, and cut background levels. Low levels of benzaldehyde (0.005%) and benzoic into small pieces. Mucosae from an appropriate number of rats (typi acid (0.05%) were present as contaminants in the NMBzA, and [14C] cally 7 to 10) to give the required amount of microsomal protein were formaldehyde (0.01 and 2.3%) was detected in the [metf7y/-14C]NMBzA combined and homogenized in 9 volumes of homogenizing buffer, with and [mef/7y/-14C]NDMA, respectively. 30 passes of a motor-driven Duali all-glass homogenizer (20-sec stop Electron Microscopy. The 100,000 x g pellet obtained from esoph- pages after each 10 passes). Where specified, whole esophagus or ageal mucosa was fixed using 3.5% glutaraldehyde in cacodylate the muscle-submucosa was similarly homogenized. Liver was homog buffer, followed by 1% osmium tetroxide. The pellet was dehydrated in enized in 3 volumes of homogenizing buffer with 8 passes of a motor- acetone and then embedded in Epon. Thin sections were stained with driven Potter Elvehjem homogenizer with Teflon pestle. Homogenates uranyl acetate and lead citrate and examined in an Elmiskop 1 electron were routinely centrifuged at 9,000 x g for 20 min, and the postmito- microscope. chondrial supernatants were centrifuged at 100,000 x g for 60 min Other Analyses. Cytochrome P-450 was determined spectrophoto- (an initial spin at 2,000 x g for 10 min was undertaken for the detailed metrically by the method of Jakobsson and Cinti (7), using dithionite as subcellular fractionation study). The 100,000 x g pellet, referred to the reducing agent. Protein was analyzed by the method of Lowry et henceforth as the microsomal fraction, was resuspended to a final al., as described by Munro and Fleck (15), using bovine serum albumin concentration of approximately 5 mg protein per ml in homogenizing as standard. Statistical analyses were carried out using 2-tailed Stu dent's f tests. buffer for metabolism studies. A yield of 4.49 Â±0.17 (S.E.) mg of microsomal protein was obtained per g mucosa, each mucosa weighing 40 to 50 mg. For cytochrome P-450 assays, the microsomes were washed with 1.15% KCI and resuspended in 50 mw Tris-HCI (pH 7.40). RESULTS Incubation Conditions. Unless stated otherwise, incubation mix tures contained 5 mw substrate (NMBzA; [mefhy/-14C]NMBzA, 0.33 Microsomes prepared from either liver or esophageal mu mCi/mmol; [mef/7y/ene-'4C]NMBzA, 0.33 mCi/mmol; or [mef/?y/-14C] cosa metabolized NMBzA to benzaldehyde and formaldehyde NDMA, 0.33 mCi/mmol), 20 mM semicarbazide, 2 mw NADP+, 10 mM 0.08 G-6-P, G-6-P dehydrogenase, 2 units/ml, 10 mw MgCI2, 50 mM Tris NMBzA (pH 7.40), and 0.1 ml of microsomal suspension (approximately 0.5 mg of protein) in a final volume of 0.6 ml. Reactions were started by Cofoctors the addition of microsomes to the other components which had been preincubated at 37Â°for 5 min. Samples were incubated at 37Â°under air with shaking for 20 min (NMBzA metabolism) or for 40 min (NDMA Â¿CHO-SC metabolism). Reactions were terminated by placing the mixtures on ice. Under these conditions, reaction rates were linear with respect to both incubation time and protein concentration. Formation of benzyl Â°-05 alcohol from benzaldehyde as substrate (16.7 JUM)was measured using identical incubation conditions. Metabolite Analyses. Incubation mixtures containing unlabeled NMBzA were processed for determination of benzaldehyde, benzyl alcohol, and benzoic acid. Samples were deproteinized with 0.3 ml of 20% (w/v) ZnSCv7H2O and 0.3 ml of saturated Ba(OH)2 solution. Following centrifugation of the precipitates, supernatants were stored in the dark at -20Â° overnight. Freezing was essential for minimizing i nonenzymatic production of benzaldehyde prior to analysis. Superna tants were thawed, and immediately chromatographed on a Ci8-/iBon- J dapak liquid chromatography column (Waters Associates, Inc., Milford, Mass.), eluted with 15% (v/v) methanol-3% (v/v) acetic acid, pH 4.20, at a flow rate of 2.0 ml/min. The eluate was monitored at 260 nm, and metabolites were quantitated by peak height analysis. A typical chro- matogram is shown in Chart 1. Under these conditions, benzaldehyde semicarbazone exists in equilibrium with smaJI quantities of free ben 10 20 30 zaldehyde. The identities of the metabolites that cochromatographed Time (min) with authentic benzyl alcohol and benzaldehyde semicarbazone were confirmed by gas chromatography-mass spectroscopy. The fraction Chart 1. High-performance liquid chromatography profile obtained following incubation of NMBzA in the presence of hepatic microsomes as described in the containing benzyl alcohol was collected and extracted directly into text. CH2OH,benzyl alcohol; <)>COOH.benzoic acid; CHO,benzaldehyde; dichloromethane. The fraction containing benzaldehyde semicarba 0CHO-SC, benzaldehyde semicarbazone.

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(Table 1). In addition, benzyl alcohol was a major hepatic Table 2 metabolite but was not detectable in incubations containing Tissue localization of NMBzA debenzylase in rat esophagus mucosal microsomes (Table 1). Benzyl alcohol formation from Benzaldehyde production the reaction of a benzylating intermediate with water theoreti Tissue layer (pmol/min/esophagus) 81.0 Â± 9.8a cally cannot exceed the rate of formaldehyde formation. How Whole esophagus Mucosa 111.1 Â±17.4 ever, hepatic microsomes extensively reduced benzaldehyde Muscle-submucosa 2.2 Â± 1.1 to benzyl alcohol (0.73 nmol/min/mg protein), using a ben Mean Â±S.E. of 4 replicate assays. zaldehyde concentration (16.7 JUM)typically measured in the NMBzA incubation mixtures. Thus, the high rate of benzyl 0.60 alcohol formation from NMBzA by hepatic microsomes results primarily from benzaldehyde reduction. Preliminary experi S ments demonstrated that 20 mw semicarbazide in the incuba 0.50 tion medium minimized benzaldehyde reduction; higher con centrations lacked further effect. Since semicarbazide did not alter nitrosamine metabolism, it was routinely included in all 0.40 subsequent assays. In contrast to hepatic microsomes, mu cosal microsomes lacked benzaldehyde reductase activity (<0.14 nmol/min/mg protein). In neither microsomal fraction 0.30 was oxidation of benzaldehyde to benzoic acid detectable, although benzoic acid was present as a minor contaminant of 0.20 the NMBzA. Furthermore, formaldehyde was stable with both tissues under the incubation conditions, indicating minimal underestimation of NMBzA demethylase activities. Taking O.IO these factors into account, it was calculated that microsomes from esophageal mucosa metabolized NMBzA at the mÃ©thylÃ¨ne carbon of the benzyl moiety to yield benzaldehyde, at about 20 40 60 80 IOO one-fifth the rate of hepatic microsomal metabolism (allowing Percent total protein/g mucosa in this case for reduction of benzaldehyde). In contrast, mu Chart 2. Subcellular distribution of NMBzA metabolizing activity in esophageal cosal metabolism of NMBzA at the methyl carbon to yield mucosa. Mucosal homogenate was subjected to differential centrifugaron, the formaldehyde occurred at a rate 60 times lower than the rate resultant fractions were each incubated with NMBzA, and benzaldehyde produc tion was measured as described in the text. Columns, mean values of 3 replicate of hepatic metabolism. assays; bars, S.E. N, "nuclear" 2,000 x g pellet; Mt, "mitochondria!" 9,000 x g pellet; C, "cytosol" 100,000 x g supernatant; Me, "microsomal" 100,000 x Radioactivity profiles of the chromatograms revealed the absence of metabolites of [mef/7y/-14C]- or [mefriy/ene-14C]- g pellet. NMBzA other than those described above in either hepatic or agus, mucosa, or residual muscle-submucosa indicated that mucosal incubations. In particular, enzymatic formation of the mucosa contained the entire esophageal metabolizing ac methylbenzylamine (eluting at 3.4 min) was not detected tivity, as determining by benzaldehyde production (Table 2). (<0.08 nmol/min/mg protein), indicating an absence of deni- Microsomes prepared from whole esophagus exhibited lower trosation. activity per esophagus compared to those prepared from the Under incubation conditions identical to those used for mucosa, although the difference was not significant. The lower NMBzA metabolism, NOMA was extensively metabolized to activity probably reflects a lower efficiency of homogenization formaldehyde by hepatic microsomes (Table 1). In contrast, of whole esophagus, resulting in a lower yield of microsomes. microsomes prepared from esophageal mucosa lacked detect An identical tissue distribution of NMBzA metabolism was ob able NDMA demethylase activity (Table 1). The high detection tained when using whole homogenates rather than micro limit (0.03 nmol/min/mg protein) was due to the presence of somes. relatively high levels of [14C]formaldehyde as a contaminant of Subcellular fractionation of the mucosa was carried out using the [meiAiy/-14C]NDMA. a differential centrifugation method commonly used for hepatic The characteristics of the metabolizing system in the mucosa subcellular fractionation. Although the "nuclear" (2,000 x g were then examined. Incubation of NMBzA in the presence of pellet) and "mitochondrial" (9,000 x g pellet) fractions con microsomes (100,000 x g pellet) obtained from whole esoph- tained some capacity to metabolize NMBzA to benzaldehyde, the microsomal (100,000 x g pellet) fraction was markedly Table 1 enriched in this activity compared to the other fractions (Chart NMBzA and NDMA metabolism by microsomes from rat liver and esophageal 2) and contained approximately 40% of total homogenate activity. No activity was detected in the "cytosol" (100,000 microsomes x g supernatant). Electron microscopy of the microsomes from microsomes (nmol/min/mg esophageal mucosa indicated the presence of some endoplas- SubstrateNMBzA (nmol/min/mgprotein)0.978 protein)0.549 Â±0.108a(4)6 Â±0.055 (18) mic reticulum vesicles, with masses of free ribosomes and low Benzyl alcohol 2.0 Â±0.3 (4) Â£0.1 levels of contaminating fragments of other organelles (Fig. 1). FormaldehydeFormaldehydeHepatic(4)0.8710.289 Â±0.087 (6)Â£0.030.005 Â±0.001 Thus, based on the preferential localization of NMBzA metabo NDMAMetaboliteBenzaldehyde Â±0.102 (4)Mucosal lizing activity and the electron microscopic appearance, micro Mean Â±S.E. somes prepared from esophageal mucosa appear to be similar ' Numbers in parentheses, number of replicate assays. to hepatic microsomes.

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Table 3 Tissue and cofactor requirements and effects of inhibitors on NMBzf metabolism to benzaldehyde by microsomes from esophageal mucosa Control incubations contained mucosal microsomes and the NADPH-generating system. Where specified, microsomes were boiled for 5 min prior to incubation, the NADPH-generating system was omitted, 0.6 mM SKF 525-A was added, or incubations were carried out under CO. Benzaldehyde production Incubation mixture {% of control) Control 100.0 Â±22.3 Boiled microsomes 0.7 Â± 0.56' Minus NADPH-generating system 0.7 Â± 0.9C Plus 0.6 HIM SKF 525-A 30.8 Â± 3.6C Plus CO 3.4 Â± 0.5C 0.610 Â±0.136 nmol/min/mg protein. 6 Mean Â±S.E. of 3 replicate assays. c Significantly different from control, p < 0.05.

O.IO -

Fig. 1. Electron microscopic appearance of microsomes isolated from esoph ageal mucosa, x 70,000. NMBzA metabolism to benzaldehyde by mucosal micro I somes appeared to be enzymatic in nature. Metabolism was 0.05 dependent on both the presence of tissue and incubation at 37Â°,whereas boiling the microsomes for 5 min prior to incu bation completely abolished activity (Table 3). Furthermore, I this metabolizing enzyme appears to be a typical cytochrome P-450-dependent system. Metabolism was dependent on the presence of a NADPH-generating system, and it was markedly inhibited by the classical cytochrome P-450 inhibitors, CO or 0.6 mWSKF (525-A (Table 3). Metabolism of NMBzA was not detected when arachidonic acid (0.18 jitmol in 2.7 jiil ethanol) was used in place of the NADPH-generating system. 0.00 Analogous studies examining NMBzA metabolism to formal dehyde also indicated this metabolic pathway to be preferen II tially located in the mucosal microsomes and to be cytochrome 4IO 450 500 P-450 mediated. To further examine the cytochrome P-450 dependency of Wavelength (nm) NMBzA metabolism, rats were pretreated with either pheno- Chart 3. Reduced CO difference spectrum of microsomes prepared from esophageal mucosa, as described by Jakobsson and Cinti (7), with dithionite as barbitone or 3-MC. Phenobarbitone pretreatment greatly in reducing agent. creased hepatic microsomal metabolism of NMBzA to benzal dehyde (3.4-fold), benzyl alcohol (6.0-fold), and formaldehyde 450 nm (Chart 3). The peak at 423 nm is likely to represent (4.2-fold), and a significant production of benzoic acid was cytochrome t>5,which would also exhibit a difference spectrum detected (0.40 nmol/min/mg protein). In contrast, phenobar- under these conditions (7). Incubation in the presence of ascor- bitone pretreatment did not alter NMBzA metabolism by micro bate and phenazine ethosulfate did not alter the height of the somes from esophageal mucosa. 3-MC pretreatment did not peak at 423 nm, indicating a lack of heme contamination (8). alter NMBzA metabolism by either hepatic or mucosal micro Using an extinction coefficient of 91 m.M~1cm"1 (18), the level somes. of cytochrome P-450 in the mucosal microsomes was calcu Cytochrome P-450 could not be detected in the mucosal lated to be 0.096 Â±0.012 nmol/mg protein (n = 4), approxi microsomes using the conventional reduced CO difference mately 15% of that detected in hepatic microsomes (0.66 Â± spectrum method of Omura and Sato (18) due to the presence 0.10 nmol/mg protein, n = 4). of contaminating cytochrome oxidase. However, when the method of Jakobsson and Cinti (7) was used, which involved DISCUSSION preincubating the microsomes with succinate to remove mitochondrial interference, reduced CO difference spectroscopy The present results demonstrate that both rat esophageal revealed a typical cytochrome P-450 spectrum, with \ma)

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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1982 American Association for Cancer Research. NMBzA and NOMA Metabolism by Rat Esophagus and Liver carcinogen NMBzA. However, whereas hepatic microsomes of NMBzA. oxidized NMBzA at the mÃ©thylÃ¨necarbon at a rate approxi Two important differences between hepatic- and esophageal mately 10-fold higher than at the methyl carbon, this differential mucosa-mediated metabolism of NMBzA were noted in this was 100-fold in the case of mucosal metabolism. Schweinsberg study, (a) The ratio of oxidation at the 2 a-carbons of NMBzA and Kouros (22) have reported similarly a high rate of NMBzA differs by 10-fold in the 2 tissues. (D) Hepatic metabolism of metabolism to benzaldehyde by microsomes prepared from the NMBzA at both a-carbons is induced by phenobarbitone pre whole esophagus of the rat, whereas formaldehyde was not treatment, in contrast to mucosal metabolism which is not detected. In that study (22), formaldehyde was assayed by the altered. Since the liver contains multiple forms of cytochrome colorimetrie method of Nash (16), which is much less sensitive P-450, each having different, although broad, substrate spec than is the dimedone assay (19). In contrast to the present ificities (10), the results of the present study suggest that the results, Schweinsberg and Kouros (22) did not detect hepatic esophageal mucosa may contain only a subset of hepatic metabolism of NMBzA to formaldehyde and reported that the cytochromes P-450, different ratios of forms, or different specific activity of the debenzylase in the whole esophageal form(s) altogether. Attempts to examine this question by sub microsomes was greater than that in the hepatic microsomes. jecting the microsomes to sodium dodecyl sulfate-polyacryl- Schweinsberg and Kouros (22), however, did not use an al amide gel electrophoresis and staining for heme-containing dehyde-trapping agent, such as semicarbazide, in their incu proteins have been unsuccessful to date, due to the low levels bation mixtures nor did they measure formation of benzyl of cytochrome P-450 in the mucosa. alcohol. Under the present conditions, both formaldehyde and The present study thus demonstrates that rat esophageal benzaldehyde were further metabolized when they were incu mucosa contains an enzyme which can metabolize the esoph bated with hepatic microsomes in the absence of a trapping ageal carcinogen NMBzA at a high rate and shows properties agent, but they were considerably more stable in the presence typical of a cytochrome P-450. In contrast, NDMA, which is of mucosal microsomes. Thus, the hepatic metabolism of not an esophageal carcinogen, is a poor substrate for this NMBzA in the study of Schweinsberg and Kouros (22) is enzyme. Furthermore, the esophagus is deficient in other typ probably underestimated. ical carcinogen-metabolizing enzymes, such as aryl hydrocar In contrast to the present findings and those of Schweinsberg bon hydroxylase (25, 26) and epoxide hydrase (17). The and Kouros (22) that indicate a high level of NMBzA metabolism enzyme system in the mucosa may thus play a crucial role in by rat esophageal microsomes, Scanlan ef al. (21) were unable determining which chemicals are carcinogenic in the rat esoph to detect metabolism of another esophageal carcinogen, 2,6- agus. Since rat liver can metabolize NMBzA and NDMA to yield dimethyldinitrosopiperazine, by microsomes and cytosol ob methylating agents at similar rates (present study; Ref. 2), tained from the combined esophagus and forestomach of the other factors must be responsible for the inability of NMBzA to rat. However, A/-nitrosopyrrolidine, which is not an esophageal induce liver tumors. carcinogen, was metabolized at a low rate (21). The reason for the lack of metabolism of 2,6-dimethyldinitrosopiperazine by ACKNOWLEDGMENTS the esophagus is not known at this stage. Since the esophageal mucosa metabolizes NMBzA to ben The authors thank Dr. A. Howatson tor performing the electron microscopy, zaldehyde at approximately 100 times the rate of metabolism Dr. K. Jain tor assistance with the gel electrophoresis, and Kwan Leung and Louis Marai for performing the gas chromatography-mass spectroscopy. to formaldehyde, formation of a methylating intermediate is expected to occur similarly at a 100-fold faster rate than is formation of the benzylating intermediate. Studies by Hodgson REFERENCES ef al. (4, 5) have in fact demonstrated that, following a single 1. Druckrey, H., Preussmann, R.. Ivankovic, S., and SchmÃ¤hl, D. Organotrope i.v. dose of NMBzA to rats, high levels of esophageal DNA carcinogene Wirkungen bei 65 verschiedenen N-nitroso-Verbindungen an methylation, including the promutagenic adduct O6-methyl- BD-Ratten. Z. Krebsforsch., 69. 103-201. 1967. guanine, are produced, whereas benzylation of DNA is unde- 2. Fong. L. Y., Lin, H. J., and Lee, C. L. Methylation of DNA in target and nontarget organs of the rat with methylbenzylnitrosamine and dimethylnitrosa- tectable. Thus, although other factors such as differences in mine. Int. J. Cancer, 23: 679-682, 1979. stability or nucleophile affinities (24) may exist between the 3. Goldschmidt, S., and Veer, W. L. A simple method for the fission of semi- methylating and benzylating intermediates, the present study carbazones. Reel. Trav. Chim. Pays-Bas Belg., 65. 796-798, 1946. 4. Hodgson, R. M., Kleihues, P., Wiessler, M., and Schweinsberg, F. Mecha suggests that the relative lack of esophageal DNA benzylation nism of esophageal tumor induction by methylbenzylnitrosamine. Proc. Am. is primarily due to a low rate of formation of benzylating Assoc. Cancer Res., 22. 103, 1981. 5. Hodgson, R. M., Wiessler, M., and Kleihues, P. Preferential methylation of intermediate. target organ DNA by the oesophageal carcinogen N-nitroso-methylbenzyl- The metabolism of NMBzA within the esophageal mucosa amine. Carcinogenesis, 7: 861-866, 1980. appears to involve a typical cytochrome P-450 pathway. Like 6. Ito, N. In vivo Carcinogenesis of 4-nitroquinoline 1-oxide and related com typical mixed-function oxidases in the liver, NMBzA metabolism pounds, in. T. Sugimura (ed.), Carcinogenesis, Vol. 6, pp. 117-153. New York: Raven Press, 1981. in the mucosa is NADPH dependent, is preferentially located in 7. Jakobsson, S. V., and Cinti, D. L. Studies on the cytochrome P-450-con- the microsomal fraction, and is inhibited by CO and SKF 525- taining mono-oxygenase system in human kidney cortex microsomes. J. Pharmacol. Exp. Ther.. 785. 226-234, 1973. A. A low level of cytochrome P-450 is also detectable in the 8. Johannesen, K. A., and DePierre, J. W. Measurement of cytochrome P-450 mucosal microsomes. A number of studies have demonstrated in the presence of large amounts of contaminating hemoglobin and methe- moglobin. Anal. Biochem., 86: 725-732, 1978. that extrahepatic xenobiotic metabolism may be mediated via 9. Lai, D. Y., and Arcos, J. C. 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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1982 American Association for Cancer Research. Esophageal and Hepatic Microsomal Metabolism of N -Nitrosomethylbenzylamine and N-Nitrosodimethylamine in the Rat

George E. Labuc and Michael C. Archer

Cancer Res 1982;42:3181-3186.

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