Concurrent Generation of Methylamine and Nitrite During Denitrosation of Jv-Nitrosodimethylamine by Rat Liver Microsomes1

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[CANCER RESEARCH 47, 447-452, January 15, 1987] Concurrent Generation of Methylamine and Nitrite during Denitrosation of jV-Nitrosodimethylamine by Rat Liver Microsomes1

Larry K. Keefer,2 Takako Anjo, David Wade, Tianyuan Wang, and Chung S. Yang

Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701 Â¡L.K. K., T. A.]; and Department of Biochemistry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103 ID. W., T. W., C. S. YJ

ABSTRACT basic organic products. Two limiting cases, one reductive and one oxidative, served as working hypotheses; these are illus With the goal of identifying the organic amine produces) of enzymatic trated in Fig. \. paths b ande, respectively. The reductive route /V-nitrosodinifth> lamine (NOMA) denitrosation, 4 HIMNDMA was in was considered because it is well established that some A- cubated with liver microsomes from ethanol-treated rats. The concentra tions of dimethylamine and methylamine were determined by derivatiza- nitroso compounds [for example, the carcinostatic nitrosoureas tion with 2,4-dinitrufluorobenzcncfollowed by gas chromatography-mass (41)] are metabolized in this way. The resulting Nâ€”Nbond cleavage initially produces nitric oxide (NO-), which is easily spectrometry. There was no net increase in the concentration of dimeth ylamine during incubation, but the yield of methylamine was equimolar converted to nitrite by ambient oxidizing agents. If this is the with that of nitrite. Additional incubations of NDMA using acetone- mechanism of NDMA denitrosation, dimethylamine would be induced microsomes, 1 imi and 0.1 HIMsubstrate, gave methylamine/ the by-product of nitrite generation, as shown in Fig. l,path b. nitrite ratios of 0.9 and 0.7, respectively, confirming the quantitative On the other hand, our previous results have suggested that linkage between these two products. Control incubations conducted with denitrosation may be accomplished via an initial oxidative pure methylamine or dimethylamine indicated that the secondary amine pathway that is closely related to the a-oxidation of NDMA is not a significant intermediate in the metabolic generationof the primary (26-28). Such a pathway could result in fragmentation of amine. Experiments with "N-labeled NDMA showed that the methyl NDMA to the imine, CH3â€”N=CH2, which should in turn amine nitrogen came from the amino moiety of the nitrosamine. The results suggest that NDMA metabolism is best viewed as a competition hydrolyze rapidly at physiological pH to give formaldehyde and between at least two importantpathways, demethylation and the presum methylamine as the organic denitrosation products, as shown ably deactivating denitrosation route, a formulation which seems to in Fig. I,path c. account for the previously reported detection of methylamine as a urinary In the work described below, we have assessed the relative metabolite of NDMA and for the production of less than theoretical importance ofpaths b and c in the overall mechanism of NDMA yields of labeled dinitrogen gas during NDMA metabolism. denitrosation /// vitro by measuring the quantities of dimethyl amine and methylamine produced compared with that of nitrite.

INTRODUCTION MATERIALS AND METHODS Growing evidence that the potent carcinogen, NDMA,3 may be produced in the human body (1-11) makes a thorough Chemicals. NDMA, methylamine hydrochloride, and dimethylamine hydrochloride were purchased from Aldrich Chemical Co. (Milwaukee, knowledge of its metabolic fate in vivo especially important. WI). NADPH-generating reagents were obtained from Sigma Chemical While there is general agreement (12-16) that NDMA is acti Co. (St. Louis, MO) and |:11,]nn-ili\ lamine hydrochloride was supplied vated by rapid metabolism to formaldehyde, dinitrogen gas, by KOR Isotopes (Cambridge, MA). Methyl([2H3]methyl)amine hydro- and a methylating agent presumed to be the ultimately carcin chloride and methyl['5N]amine hydrochloride were purchased from ogenic form, as illustrated in Fig. 1 (path a), there is evidence Merck Sharpe & Dohme Isotopes (Montreal, Quebec). Alumina Woelm that at least one other clearance mechanism must be operative B-Super 1was obtained from ICN Nutritional Biochemicals (Cleveland, in vivo. For example, yields of labeled dinitrogen gas recovered OH). 2,4-Dinitrofluorobenzene, purchased from Pierce Chemical Co. after administration of 15N-labeledNDMA to rats were only (Rockford, IL), was reacted with the five amine hydrochlorides men tioned above as previously described (43) to produce the A inclini, 52-90% of theoretical (16, 17), and labeled methylamine has N,N-dimethyl, A42H3]methyl, and Ar-methyl-AL[2H3]methyl derivatives been isolated in significant quantity from the urine of rats dosed of 2,4-dinitroaniline as well as A'-(2,4-dinitrophenyl)-methyl[l5N]amine with MC-labeledNDMA (18). for use as GC standards. ("NJNDMA was prepared by nitrosating We have been interested in the possibility suggested several dimethyl['5Njamine hydrochloride (KOR Isotopes) in acidified nitrite times in the past that enzymatic denitrosation (16, 19-42), a solution (44). nitrosamine-metabolizing pathway previously shown in vitro to Microsome Preparation and Incubations. Male Sprague-Dawley rats be capable of cleaving NDMA to nitrite, might be the missing (body weight, 85-100 g) were treated with either acetone (one intragas- clearance route, the existence of which the above-mentioned tric dose of 5 ml/kg, 20 h before sacrifice) or ethanol (15% in drinking experiments (16-18) seem to imply. The present work was water for 3 days) and microsomes were prepared as previously described (45, 46). For the NDMA metabolism studies described in Table 1, undertaken to characterize the NDMA denitrosation mecha ethanol-induced microsomes (10 mg of protein) were incubated with nism more fully, in particular by identifying and quantifying its 40 /imol of NDMA and an NADPH generating system (4 Mmol of NAD1' , 0.1 mmol of isocitrate, and 5 units of isocitrate dehydrogenase) Received 7/22/86; revised 10/8/86; accepted 10/16/86. The costs of publication of this article were defrayed in part by the payment in 10 ml of a pH 7.4 buffer containing 50 HIMTris, 10 IHMmagnesium of page charges. This article must therefore be hereby marked advertisement in chloride, and 150 HIMpotassium chloride. Similar conditions were used accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by Grant CA-37037 from the National to collect data for Table 2 and Fig. 4, except that 1 Â¿imolof NDMA Cancer Institute. and different concentrations of dimethylamine, respectively, were in 2To whom requests for reprints should be addressed, at National Cancer cubated with acetone-induced microsomes. Incubation was conducted Institute, Frederick Cancer Research Facility, Building 538, Frederick, MD for 30 min (or 20 min for the experiments of Table 2) at 37Â°C, 21701. 3The abbreviations used are: NDMA, Ar-nitrosodimethylamine; [I5N]NDMA, whereupon the reaction was cooled on ice and terminated by adding 1 A'-nitrosodimethyl[15N]amine; GC, gas chromatography; MS, mass spectrometry; ml of 25% zinc sulfate and 1 ml of saturated barium hydroxide. The in/:, ionic mass to charge ratio. mixture was then centrifuged at 3000 rpm for 7 min in a closed tube, 447

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1987 American Association for Cancer Research. MICROSOMAL DENITROSATION OF NDMA and the supernatant was transferred to a new set of tubes. An aliquot alternative. Aliquots containing 0.5-5 ml of the supernatant described totaling 0.7 ml was taken for formaldehyde and nitrite determinations above were diluted with water as necessary to 5 ml and mixed with 0.05 as previously described (26). For the other incubations described in ml of an internal standard solution containing 3 nmol of [2H3]methyl- Table 1, the NDMA was replaced by 0.02 mM dimothylamini- or amine in water. A mixture containing 0.5 ml of water, 12.5 mg of methylamine hydrochloride. Each incubation with active microsomes borax, 0.08 ml of 2,4-dinitrofluorobenzene, and 0.92 ml of dioxane was was accompanied by a control incubation using heat-inactivated micro- added. The resulting mixture was heated at 100Â°Cfor 15 min, cooled, somes. Each type of incubation was conducted in replicate sets of two and mixed well with 1.5 ml of 2 M sodium hydroxide solution. After or more. Conditions similar to the above were used for the [15N]NDMA standing at room temperature for 60 min, the mixture was extracted studies, except that some parameters were changed as follows: acetone- with 5 ml of ethyl acetate. The organic layer was washed with 5 ml of induced microsomes (two different preparations), 0.8 mg protein per 0.1 M sodium hydroxide and dried over anhydrous sodium sulfate. A ml; substrate, 1 mM NDMA; incubation time, 30 or 10 min, as indi 3.5-ml portion of the ethyl acetate solution was concentrated to 0.5 ml cated. in a gentle stream of dinitrogen gas, then passed through a column of Amine Analysis. The method of Koga et al. (43) was employed, 3 g of basic alumina by elution with five I nil portions of ethyl acetate. except that a capillary GC column was used; this provided much better The eluate was concentrated to dryness. The residue was taken up in resolution of the constituents of interest than the packed column 1(1(1idof ethyl acetate and 2-u\ aliquots were injected on-column onto a Durabond DB-1701 fused silica capillary GC column (Alltech Asso ciates, Inc., Deerfield, IL) with a film thickness of 0.25 tan. The helium MeX + N2 + CH2O + OH- flow rate was adjusted so that the solvent peak emerged from the 30-m (a nucleophile) x 0.32-mm inner diameter column in 50 Â±5 s at 70'C; this linear velocity afforded maximum efficiency. The column was held at 70Â°C until the solvent was purged, then heated at 20Â°C/minto 220Â°C,2Â°C/ min to 240Â°C,and 25Â°C/minto 280Â°Cina gas Chromatograph (5790A; Hewlett-Packard, Palo Alto, CA). In some earlier samples (Tables 1 path and 2), large quantities of 2,4-dinitrophenol and unreacted 2,4-dinitro fluorobenzene overloaded the column and caused pronounced tailing of the monitored peaks; for these cases the program was changed to 70Â°Cfor 1 min, 25'C/min to 200Â°C,4Â°C/minto 240Â°C,and 25Â°C/min palh .. Mev /Me Me*. /Me \/ MeN /M to 280Â°C.While the tailing did not usually affect quantitation due to N ^-^ â€” I A the resolution (3000) of the mass spectrometer, it did diminish capillary o-/ column life by blackening the mass spectrometer end and forcing periodic removal of the last 0.5 m of column to affect partial rejuvena tion. This problem was overcome in the later experiments (["N]NDMA studies) by decreasing the quantity of 2,4-dinitrofluorobenzene used for derivatization 10-fold. Quantification of both methylamine and dimethylamine in the in cubation mixtures was achieved by GC/MS. Of several similar protocols used in these studies, the following is typical. The derivatized incubation mixture containing the internal standard was introduced via the Dura- NO2 CH2O?) bond column into a VG 70-250,7070 EQ, or 7070 H mass spectrometer (VG Analytical Limited, Manchester, UK) operated at a resolution of Fig. 1. Pathways of NDMA metabolism considered in this work. Path a is the 3000 (10% valley definition) in the Selected Ion Recording mode and accepted (see Refs. 12-16) route of metabolic activation, in which NDMA is set to monitor masses m/z 197.04 for C7H7N3O4, m/z 200.06 for converted by n-hydroxylation to a methylating agent capable of reacting with DNA. I'utli h is the route enzymatic denitrosation would take if NDMA were C7H4D3N3O4,and m/z 211.06 for C8H<>N3O4.Toverify that exchange loss of deuterium from the internal standard was not compromising cleaved by the reductive mechanism shown to be operative in the denitrosation of certain other iV-nitroso compounds (see, for example, Ref. 41). Path c shows the the quantitation, the ions at m/z 198.05 (C7H6DN3O4)and m/z 199.06 stoichiometry of enzymatic NDMA denitrosation inferred from the present study. were also monitored. Acquisition time was 100 ms;

Table I Concentrations (Â¡Ã•M)ofamines, nitrite, and formaldehyde after incubating NDMA, dimethylamine, or methylamine with ethanol-induced microsomes Substrate4000 MMNDMA Â±1.8 Â±0.7 Â±0.2 Â±5.5 4000 MMNDMA20 Boiled (control) 9.8 Â±3.1 5.4 Â±3.03.7 0 0 DifferenceActive 19.80.0016)0.2 Â±3.7 (P = NSC18.7 20.3 Â±0.2ND 222.4 Â±5.57.2

MMDimethylamine Â±0.2 Â±4.018.2 Â±0.2 20 MMDimethylamine20 Boiled (control) 0.3 Â±0.3 Â±4.8 NDND 0 DifferenceActive -0.1NS17.0 0.5NS1.2 7.2 Â±0.2'ND

MMMethylamine Â±0.8 Â±0.4 20 MMMethylamineMicrosomesActiveBoiled (control) 16.6 Â±1.0 1.1 Â±0.5 NDFormaldehyde*222.4ND DifferenceMethylamine"29.6 0.4 NSDimethylamine'9.1 0.1 NSNitrite*20.3 " Mean Â±SE of three to 16 determinations: One to five replicate GC/MS injections of each of one to eight replicate derivatizations of each of one to three replicate incubations. For each determination, the intensity for the molecular ion (m/z 197 for methylamine, m/z 211 for dimethylamine) of the 2,4-dinitroaniline derivative was divided by that for the 3 nmol of |'II ,|metli\ lamim- hydrochloride (molecular ion of 2,4-dinitroaniline derivative, m/z 200) added as internal standard before derivatization. The resulting intensity ratio (/) was converted to the concentration value (c) through the equation <â€¢-(/- a)b after calculating the linear least-squares fit, / = a + be, of the (c. /) graph obtained for a set of standard aqueous amine solutions by adding deuterated internal standard, derivatizing, and measuring the intensity ratios by GC/MS as above. For methylamine, the slope was Â¿>=0.542 Â±0.014 (SE) MM~'and the intercept was a = 0.39 Â±0.33 (SE), while the values for dimethylamine were 0.444 Â±0.022 <;\i ' and 0.51 Â±0.53, respectively. Standard solutions were prepared by dissolving weighed amounts of the dried amine hydrochloride in water at concentrations of 50, 25, 20, 10, 5, and 0 MM;seven to 18 determinations were made at each concentration (one to four injections for each of four to 10 replicate derivatizations). Typical coefficients of variation were: 9% interinjection; 34% interderivatization; and 19% interincubation. * Mean of single determinations for duplicate incubations Â±deviation from mean. ' NS, not significantly different from 0 (P > 0.05) by analysis of variance; ND, not determined. d The origin of this significant difference is not clear, but may be due to the relative imprecision of the optical density readings observed. The difference does not, however, affect our conclusions regarding nitrite and methylamine production. 448

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1987 American Association for Cancer Research. MICROSOMAL DENITROSATION OF NDMA Table 2 Metabolite concentrations (UM)after incubating NDMA with acetone-induced microsomes Substrate100 MMlOOuMMicrosomesActive Â±0.2 Â±0.2 Â±0.3 Â±0.3 Boiled (control) 1.3 Â±0.1 1.1 Â±0.3 0 0 DifferenceMethylamine"9.1 7.8 Â±0.2 (P< 0.00001)Dimethylamine"0.7-0.4 NSCNitrite*11.5 11.5 + 0.3Formaldehyde*73.373.3 Â±0.3 " Mean Â±SEof seven to nine determinations: One to three replicate GC/MS injections of each of two replicate derivatizations of each of two replicate incubations. Replicate derivatizations for the incubation mixture were run simultaneously with those for the four corresponding calibration standards, which contained the equivalent of 0, 5, 10, and 20 nM concentrations of each amine analyte. The amines were quantified using a procedure similar to that of Table 1. * Mean of single determinations for each of duplicate incubations Â±deviation from mean. c NS, not significantly different from O(P > 0.05) by analysis of variance. settling time was 30 ms. A VG 11-250 data system was used for data significantly different from that of nitrite, 20.3 MM. These acquisition and manipulation. Amine concentrations in the incubation results are consistent with those of Grilli and Prodi (30), who mixtures were inferred from the resulting peak areas as determined in the footnote to Table 1. Quantification in the [I5N]NDMA studies was identified methylamine but not dimethylamine among the prod similarly achieved by GC/MS, except that methyl([2H3]methyl)amine ucts of NDMA metabolism in vitro. was used as internal standard and the peaks at m/z 198.04 (C7H7[I4N2] To determine whether the methylamine observed might have [15N]O4)and 214.08 (CgF^Da^O.,) were integrated to determine the been produced by rapid demethylation of dimethylamine relative proportions of methyl["N]amine and standard, respectively; formed as the initial denitrosation product, dimethylamine was calibration curves were constructed from intensity ratio data from incubated with the same ethanol-induced microsomes at an solutions containing either 0, 1,2, and 3 or 0, 5, 10, and IS nmol of initial concentration (20 MM)similar to that of the methylamine authentic methyl[15N]amine with a constant 3 nmol of internal stand generated in the NDMA incubation. No significant change in ard. amine concentration was observed when the boiled versus active The following steps were taken to validate the above analytical microsome incubations were compared, as can be seen in Table procedure for the desired application. The initial question to be resolved 1. The stability of methylamine under these conditions was also was whether compounds as volatile as methylamine and dimethylamine, which are gases at room temperature, might disappear from the 37Â°C studied. Incubation of this substrate at a concentration of 20 MMalso resulted in no significant change with respect to the incubation mixture as soon as they are generated. This was investigated by adding 14C-labeled methylamine and dimethylamine to typical in boiled-microsome control. cubation mixtures and incubating them in parallel for 30 min. Radio To look more closely at the question of the possible involve activity was retained to the extent of at least 99% in both cases. We ment of dimethylamine as an intermediate in the production of concluded that the amines are not volatile under these conditions, the methylamine observed in NDMA incubation, the secondary presumably because they are strongly basic and thus almost completely amine was separately incubated with acetone-induced micro ionized at pH 7.4. We then spiked an unincubated microsomal mixture somes at several concentrations and the extent of formaldehyde with authentic methylamine and dimethylamine and derivati/ed it production in each was followed colorimetrically. The results without addition of the deuterated internal standard. The extracts were are summarized in Fig. 4. Some conversion of dimethylamine analyzed using a flame ionization detector instead of MS. The resulting to formaldehyde was seen at high concentrations, with Km = chromatogram is shown at the top of Fig. 2. Of the many peaks 7.2 HIM and Fmax= 0.45 nmol/min/mg, but these constants observed, two corresponded in retention time to the 2,4-dinitrophenyl derivatives of methylamine and dimethylamine; these are designated as indicate that the demethylation of dimethylamine is too slow M and D, respectively, in Fig. 2. When the same procedure was applied at low concentrations to make a significant contribution to the to an incubation mixture in which 4 HIMNDMA was exposed for 30 generation of methylamine during incubation of NDMA. We min at 37Â°Ctothe action of ethanol-induced rat liver microsomes, both conclude, therefore, that methylamine is produced directly in peaks were again found but the M peak was many times as intense as the denitrosation reaction, as indicated in Fig. 1, path c. the D, as is shown in the lower chromatogram of Fig. 2. A chromato To examine the quantitative relationship between the yields gram run after incubating NDMA with heat-inactivated microsomes of methylamine and nitrite under a rather different set of followed by identical derivatization-GC (data not shown) continued to conditions, microsomes from animals induced with acetone exhibit M and D peaks, but both were similar in size to the D peak in instead of ethanol were incubated with NDMA at a substrate the lower trace of Fig. 2. concentration of 0.1 HIM rather than 4 mM. The results are That the M peak in the chromatogram of the NDMA incubation mixture truly reflected the presence of methylamine was confirmed by given in Table 2. Again, the dimethylamine concentration did MS. Its full scan spectrum obtained using a VG Micromass ZAB-2F not increase significantly after incubation with active versus mass spectrometer was practically identical to that of authentic N- boiled microsomes, while the mean increase in methylamine methyl-2,4-dinitroaniline, as shown in Fig. 3. For this experiment, the concentration relative to the boiled microsome control was incubation mixture was worked up as above with the [2H3]methylamine similar to, though significantly smaller than, that of nitrite (7.8 internal standard being omitted from the derivatization procedure. The versus 11.5 MM,respectively). effluent from the GC column corresponding in retention time to au Another experiment investigated the source of the nitrogen thentic W-methyl-2,4-dinitroaniline was analyzed in the electron ioni atom of the methylamine produced in denitrosation. Grilli et zation mode (70 eV). The elemental composition of the molecular ion al. (47) had shown that methylamine can be formed when the in the mass spectrum of the derivatized methylamine from the incuba alkylating agent generated on a-oxidation of NDMA as in Fig. tion mixture was confirmed by accurate mass measurements collected at high resolution (>4000) under full scan conditions. I,path a, reacts with a nucleophilic nitrogen center such as that at the 7-position of a guanine residue with subsequent hydrol ysis of the resulting methylated adduci to the corresponding RESULTS primary amine. In addition, Barrows (48) has recently demon Data for our first amine determinations in NDMA incubation strated that up to 30% of the 7-[14C]methylguanine formed in mixtures are summarized in Table 1. They show that no di liver DNA when carbon-14-labeled NDMA was administered methylamine was formed when 4 HIM NDMA was incubated to rats at doses of 20 mg/kg derived its methyl carbon atom with ethanol-induced microsomes, but that methylamine was from the one-carbon pool rather than from the methyldiazo- produced to the extent of 19.8 MM. This quantity was not nium ion. To study the possible role of such indirect processes 449

100 106 197 M-1 From Standard 80 Mixture 5?

Â» 60 i

Standard

IL 90 130 170 210 250 290 330 370 m/z

IUU80gX75

60"e1 Incubation Mixture

40toÂ£20n.-104-!Ã•IL105197J

l L Tl. ,From 130 170 210 290 330 370 m/z Fig. 3. Mass spectra of Ar-methyl-2,4-dinitroaniline: top, authentic reference From Incubation standard; bottom, isolated from the incubation of 4 HIMNDMA with acetone- Mixture induced rat liver microsomes after subsequent derivatization of the methylamine produced therein. The exact mass observed for the m/z 197 peak in the lower trace was 197.0441 atomic mass units; calculated for C7H7N3O4, 197.0437 atomic mass units.

0.5 ?!o â€” Â°-4

Is2 tt 0.3

0.2 OEH Ã®l Â« Â«.' K

20 40 60 80 100 Dimethylamine Concentration (mM) Fig. 4. Initial rate of formaldehyde formation from dimethylamine as a func tion of substrate concentration in the presence of acetone-induced rat liver microsomes (0.8 mg of protein in a 1.0-ml incubation mixture).

D derivatized methylamine isolated from this incubation mixture JO displayed a molecular ion at m/z 198 rather than 197, indicating v I that the nitrogen atom of the methylamine produced during o 10 15 20 NDMA metabolism in vitro comes from the substrate and not from met hylatimi of nitrogenous species in the medium. Time (min) The quantitative outcome of this experiment again indicated Fig. 2. Capillary gas chromatograms of derivatized methylamine (M) and dimethylamine (Â£>):lower trace, from the incubation of 4 mm NDMA with a roughly equimolar correspondence between methylamine and ethanol-induced rat liver microsomes; upper trace, from an unincubated micro- nitrite as denitrosation products. In this case, acetone-induced somal mixture spiked with 80 MMmethylamine and 40 ;/\i dimethylamine. microsomes were used with a substrate concentration that was 10-fold greater than that in Table 2 (i.e., 1 HIM).The methyl- in the present experiments, NDMA having I5N in the amino [I5N]amine concentrations after incubation with active versus moiety was incubated for 30 min at a concentration of 1 mM boiled microsomes were 4.3 and 2.2 ^M, respectively, for a net with acetone-induced microsomes. The mass spectrum of the production of 2.1 //M. This agrees well with the observed nitrite 450

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1987 American Association for Cancer Research. MICROSOMAL DENITROSATION OF NDMA yield of 2.0 MM. Replication of this experiment with freshly to yield the secondary amine (or urea, etc.) as the organic prepared microsomes and an incubation time of 10 min gave a product (reviewed in Ref. 14). net methyl[15N]amine yield of 2.6 MMcompared to an observed Further research on the stoichiometry, mechanism, and in nitrite production of 3.3 MM, i.e., the observed CHj[I5N]H2/ vivo course of denitrosation, that we hope will provide impor NO2" ratio was 0.8. tant new insights into the detailed fate of NDMA under con ditions relevant to determining the toxicological consequences of its synthesis in the human body, is currently in progress. DISCUSSION The present experiments show that methylamine is generated ACKNOWLEDGMENTS during the enzymatic denitrosation of NDMA by rat liver We thank D. Harvan, J. R. Hass, C. Metral, S. Missler, J. Roman, microsomes in amounts similar to those of nitrite. This finding and Y. Tondeur for performing the mass spectrometric investigations; provides a plausible mechanistic explanation for the appearance J. Hrabie for synthesizing the [15N]NDMA; and C. Riggs for statistical of labeled methylamine in the urine of rats given [I4C]NDMA evaluation of the results. (18). Quantitatively, the extent of NDMA metabolism not proceeding by path a (Fig. 1) to product dinitrogen gas in vivo, 10-48% (16, 17), roughly corresponds to the extent of denitro REFERENCES sation as measured in the nitrite/formaldehyde ratios observed 1. Brooks, J. B., Cherry, W. B., Thacker, L., and Alley, C. C. Analysis by gas during NDMA metabolism in vitro (9-15% in Tables 1 and 2). chromatography of amines and nitrosamines produced in vivoand in vitro by Proteus mirabilis. J. Infect. Dis., 126:143-153, 1972. These considerations suggest that metabolism of NDMA is 2. Fine, D. II.. Ross, R., Rounbehler, D. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1987 American Association for Cancer Research. Concurrent Generation of Methylamine and Nitrite during Denitrosation of N-Nitrosodimethylamine by Rat Liver Microsomes

Larry K. Keefer, Takako Anjo, David Wade, et al.

Cancer Res 1987;47:447-452.

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