[CANCER RESEARCH 47, 3373-3377, July 1, 1987]

Deuterium Isotope Effect on Denitrosation and Demethylation ofW-Nitrosodimethylamine by Rat Liver Microsomes1

David Wade, Chung S. Yang,2 Climaco J. Metral, John M. Roman, Joseph A. Hrabie, Charles W. Riggs,

Takako Anjo, Larry K. Keefer, and Bruce A. MicoDepartment of Biochemistry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103 [D. W., C. S. YJ; ProgramResources, Inc. fC. J. M., J. M. R., J. A. H.J, Information Management Services, Inc. [C. W. R.J, and Chemistry Section, Laboratory of Comparative Carcinogenesis,National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701 [T. A., L. K. K.J; and Department of Drug Metabolism, Smith, Kline &French Laboratories, Philadelphia, Pennsylvania 19101 [B. A, M.]

ABSTRACT

In an attempt to elucidate the molecular basis for the decrease in ratliver carcinogenicity and DNA-alkylating ability that accompanies deu-teration of Ar-nitrosodimethylamine (NDMA), NDMA and its fully deu-terated analogue (|2H«]NDMA)were incubated with acetone-induced rat

liver microsomes. Rates for the competing metabolic routes, denitrosationand dcmcthylation, were determined from colorimetrie data on nitrite andformaldehyde generation, respectively. The F,,,»,calculated for demeth-ylation of NDMA was 7.9 nmol/min/mg, while that for dénitrosa tion was0.83 nmol/min/mg. Deuteration of NDMA did not significantly changethe V„.Kfor either pathway, but it did increase the A",,,for demethylation

from 0.06 to 0.3 niM. The Ä„,for denitrosation was also increased from0.06 to 0.3 HIMon deuteration, as determined by incubating an equimolarmixture of amino-"N-labeled NDMA with [2II6|M)M A and measuringthe methyl[l5N]amine:[2H3]methylaniine ratio by derivatization-gas chro-matography-mass spectrometry. The fact that the A',,,values for denitro

sation were so similar to those for demethylation suggested that the twopathways were catalyzed by the same enzyme. The isotope effects calculated from these data [ K„„H/Km„D«1 and (Kmx/ÕTm)H/(JW¿rm)D~

5]show that microsomal metabolism of NDMA is not significantly shiftedfrom demethylation to denitrosation on deuteration of substrate and mayindicate a low commitment to catalysis for the enzyme. The results areconsistent with the view that the metabolism of NDMA is initiated byformation of an a-nitrosamino radical which either combines with ahydroxyl radical to form the a-hydroxynitrosamine as the initial productof the demethylation pathway or fragments to nitric oxide and /V-meth-ylformaldimine as the first products of denitrosation.

INTRODUCTION

NDMA3 has been shown to be a more potent carcinogen andDNA-alkylating agent in rat liver than its fully deuteratedanalogue, [2H6]NDMA (1, 2). A possible explanation for these

differences is that there is more than one significant eliminationroute available to NDMA in vivo (3) and that the greaterstability of the C—D versus C—H bond (4) slows the rate ofthe pathway responsible for alkylation and tumor induction,thereby increasing the extent of a competing deactivation mechanism.

To date, two competing mechanisms of NDMA metabolismhave been identified as important in rat liver microsomes. Oneis the demethylation route (reviewed in Ref. 5) considered toproduce the ultimately carcinogenic form. It is already knownto be associated with a significant kinetic isotope effect (6-8),

Received 12/15/86; revised 3/10/87; accepted 3/20/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Research was supported in part by National Cancer Institute Grant CA-37037 and Contract N01-CO-23910. A portion of this work was presented at theInternational Agency for Research on Cancer's Ninth International Meeting onyV-Nitroso Compounds: Relevance to Human Cancer, September 1-5, 1986,Baden, Austria (33).

2To whom requests for reprints should be addressed.3The abbreviations used are: NDMA, JV-nitrosodimethylamine; [2H«]NDMA,

Ar-nitrosodi([2H3]methyl)amine; ["NJNDMA, /V-nitrosodimethyl[l5N]amine; GC,gas chromatography: MS, mass spectrometry; m/z, ionic mass:charge ratio; [2H3]NDMA, Ar-nitrosomethyl([2H3]methyl)amine.

but the details of its origins remain to be fully elucidated. Theother metabolic pathway is enzymatic denitrosation (Refs. 9-14 and references therein), presumed to be a deactivating mechanism because it cleaves NDMA to methylamine and nitrite(14).

The purposes of the present work were to examine themolecular basis for the influence of deuteration upon the metabolism of NDMA, to measure the isotope effect on denitrosation, and to determine whether deuteration increases theproportion of total NDMA metabolism that proceeds by thedenitrosation pathway. Acetone-induced rat liver microsomeswere used as a source of enzyme in these studies, and the effectsof deuteration were monitored by examining the demethylationand denitrosation of NDMA in the same incubation. Treatmentof rats with acetone has been shown to induce a cytochrome P-450 dependent low-ATmform of NDMA demethylase (11). Thisactivity, which also exists in untreated animals, is believed tobe important in the activation of NDMA (15, 16). The simultaneous study of demethylation and denitrosation also allowsus to present evidence on the mechanistic relationship betweenthese two reactions in the metabolism of NDMA.

MATERIALS AND METHODS

Chemicals. Ar-Nitrosomethyl([2H3]methyl)amine was prepared by reacting [2H3]methyl iodide with thallium(I) E-methanediazotate as separately described.4 [2H6]NDMA was synthesized as detailed earlier (1).

All other materials were obtained from the sources indicated in aprevious paper (14), except that the commercial [2H3]methylamine

hydrochloride had to be recrystallized from absolute ethanol to removesubstantial quantities of ammonium chloride impurity. After severalrecrystallizations the melting range had increased to 221-225"C; al

though this was still significantly different from that of commercialmethylamine hydrochloride (m.p. 228-230°C), the material gave sat

isfactory combustion analysis data (Galbraith Laboratories, Inc., Knox-ville. TN) and was used as such for construction of the calibrationcurves.

Microsome Preparation, Incubations, and Analytical Methods. Acetone-induced rat liver microsomes were prepared and incubated asdescribed previously (14) at a level of 0.8 mg of protein/ml of reactionmixture for 10 to 20 min with 0.04, 0.08, 0.10, 0.20, 0.33, 0.50, 1.00,or 1.5 mM NDMA or [2H6]NDMA or for 10 min with equimolarmixtures of [2H6]NDMA and [15N]NDMA (1 or 0.3 mM each). Each

incubation with active microsomes was conducted in parallel with aboiled microsome control so that amounts of the various analytesproduced by enzymatic action could be calculated by subtracting theconcentration in the latter from that in the former. Formaldehyde andnitrite were determined co lorimet r¡cally (14) and amines were assayedby spiking with 3 nmol of Ar-methyl-A'-[2H3]methylamine hydrochloridefollowed by derivatization with 2,4-dinitrofluorobenzene and capillaryGC introduction into a mass spectrometer operating in the SelectedIon Recording mode; all these operations were as described previously

4 L. K. Keefer, S-M. Wang, T. Anjo, J. C. Fanning, and C. S. Day. Preparationof thall inni(I) diazotate. Structure, physicochemical characterization, and conversion to novel ,V-nitroso compounds, manuscript in preparation.

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ISOTOPE EFFECTS ON NDMA METABOLISM IN VITRO

(14), except that only one-tenth as much 2.4 -dinitrofluoroben/ene was

used in the present work (i.e., 8 ¿il/derivatization) to improve GCseparations and column life. Methyl['5N]amine and [2H3]methylamine

were quantified by normalizing the intensities of the m/z 198 and 200peaks, respectively, to that of the m/z 214 peak due to the internalstandard. The resulting intensity ratios were converted to concentrations via calibration curves prepared by similar derivatization-GC/MSof standard mixtures of authentic methyl['sN]amine, [•H.i|methylaminc,and ¿V-methyl-A42H3]methylaminehydrochlorides. This procedure waspatterned after that detailed previously (14). Unreacted [2H«]NDMAand [15N]NDMA were determined in the incubation mixtures by spiking1-ml aliquots with 10 M!of 5 mM [2H3]NDMA, extracting with 1 ml of

dichloromethane, drying the organic layer with anhydrous sodiumsulfate, and injecting I-/:<!aliquots on-column onto a 0.32 mm- x 30-mfused silica capillary column of DB-WAX (J&W Scientific, Palo Alto,CA) at 35'C in a Hewlett-Packard 5790A gas Chromatograph (Hewlett-

Packard, Palo Alto, CA). After a 75-s solvent delay, the temperaturewas programmed to 70°Cat 8°C/min.The carrier gas was helium at a

linear flow rate of 50 cm/s. The transfer line to the mass spectrometerwas maintained at 250°C.The spectrometer, a VG 70-250 instrument

(VG Analytical Ltd., Manchester, United Kingdom), was tuned tomonitor the molecular ions of [I5N]NDMA, [2H3]NDMA, and [2H6]

NDMA at m/z 75.0450, 77.0668, and 80.0856, respectively, at 70 eVand a resolving power of 2000 in the Selected Ion Recording modewith a 250°Csource temperature, a 0.1-mA trap current, a dwell time

for each ion of 0.1 s, and a reset time of 30 IDS.The intensities of them/z 80 and 75 peaks were converted to concentrations of [2H6]NDMAand [I5N]NDMA, respectively, by normalizing each to the intensity of

the m/z 77 peak and computing the corresponding concentrations fromcalibration curves constructed from intensity ratios for standard mixtures of the three isotopie variants of NDMA.

RESULTS

The selection of acetone-induced rat liver microsomes, inwhich a low-An, (0.06 mM) NDMA demethylase is the predominant form of this enzyme, and low substrate concentrations(from 0.04 to 1.50 HIM) for the present study allowed us tofocus on this form of enzyme with little interference from thehigh-A"mNDMA demethylase activities. Eadie-Hofstee plots in

which the means of data points from different sets of experiments were graphed as in Fig. 1 were used to obtain kineticconstants for the metabolism of NDMA. Apparent Km valuesof 0.06 HIMwere observed for both demethylation and denitro-sation of NDMA whereas apparent Fmaxvalues of 7.8 and 0.81nmol/min/mg were observed for these two reactions, respectively. With deuterated substrate, the Vm^ values were notsignificantly altered but the Kmvalue was increased 3- to 5-fold.A similar conclusion was also reached with the Km and Kmaxvalues obtained from Lineweaver-Burk plots. In order to checkwhether there was any artifact caused by averaging data pointsfrom different sets of experiments, a second approach to datatreatment was used. In this approach, the individual data fromeach set of experiments were graphed as Eadie-Hofstee plots.

Y - -0.06X + 7.84Km - 0.06 mM NDMAVm - 7.84 nmol HCHO/min-mgr - -0.98

10 20 30 40nmol HCHO/mln-mg prot.-mM NDMA

80

0.9

QJt

£ 0.7

0.6

oE 0.5

0.4

Y - -0.06X + 0.81Km « 0.06 mM NDMAVm = 0.81 nmol N02~/rnIn-mg

r = -0.98

1234nmol N02~/mln-mg prot.-mM NDMA

«L4SÉ— 2

Y - -0.31 x + 7.41Km - 0.31 mM [2Hg]NDMA

Vm - 7.41 nmol HCHO/m!n-mgr - -0.98

io.

0.4

I«

0.0

Y - -0.20X + 0.82Km = 0.20 mM [2Hg]NDMA

Vm » 0.82 nmol HO^/mìn-mqr = -0.98

O 4 8 12 18 20nmol HCHO/mln-mg prot.-mM NDMA nmol N02~/mln-mg prot.-mM NDMA

Fig. I. Eadie-Hofstee plots of microsomal demethylation and denitrosation of NDMA and [2H6)NDMA. The reaction mixture contained acetone-induced rat livermicrosomes (0.8 mg protein) and 0.04, 0.08, 0.10, 0.20, 0.33, 0.50, 1.00, or 1.50 mM NDMA or [2H<¡]NDMAin 1 ml of total volume. The means ±SE from ninesets of experiments are shown. Upper left, demethylation of NDMA; upper right, denitrosation of NDMA; lower left, demethylation of [2H,JNDMA; lower right,denitrosation of [2H6JNDMA.

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ISOTOPE EFFECTS ON NOMA METABOLISM IN VITRO

Table 1 Kinetic constants for metabolism of NOMA and pHJNDMA byacetone-induced rat liver microsomes in noncompetitive incubations"

Incubation mixtures contained 0.8 mg/ml protein and either 0.04, 0.08, 0.10,0.20, 0.33, 0.50, 1.00, or 1.50 mM NDMA or pHJNDMA. Rates of demethyla-tion and denitrosation were determined by assaying colorimetrically for formaldehyde and nitrite, respectively, at the end of the 10- or 20-min incubation.

Demethylation Denitrosation

Substrate (nmol/min/mg) (mM)4 (nmol/min/mg) (HIM)*

NDMA[2H«]NDMA

7.92 ±0.677.73 ±0.44

0.06 ±0.010.30 ±0.03

0.83 ±0.060.83 ±0.15

0.06 ±0.020.19 ±0.06

" Data are given as mean t SD for 9 replicate incubations except that for

demethylation of NDMA, which was for 14 replicates.* The means in this column differed significantly (P < 0.001).

The means ±SD of the kinetic parameters determined in thisway are summarized in Table 1. The results were very similarto those of the first approach. It should be noted that in thisexperiment, because of the sensitivity limit of the assay, trueinitial velocities were not measured in incubations with verylow substrate concentrations. With these concentrations, thesubstrate utilization was as high as 38% at the end of theincubation, and the reported data points represent underestimates of the true initial rates. However, this did not lead to asignificant overestimation of the A',,,values, nor did it change

the interpretation of the results; similar Kmvalues were obtainedwhen we omitted these data from our calculations.

The observed ÄVs for demethylation and denitrosation of[2H6]NDMA (0.30 and 0.19 HIM, respectively) appeared to be

slightly different; however, because of the considerable uncertainty in the latter value arising from the low nitrite concentrations generated in several of the [2H6]NDMA incubations, it

was not clear whether or not this Kmdifference was significant.As an alternative means of determining the exact relationship

between the Km values for denitrosation and demethylation of[2H6]NDMA, a competitive experiment in which this substrate

was incubated in the presence of equimolar NDMA was conducted. Since neither substrate can truly saturate the metabolizing enzymes in the presence of the other, the ratio of initialdenitrosation rates as measured in the accumulation of meth-ylamine and [2H3]methylamine at very low substrate conversion

is a direct measure of VmKi/Kmfor one substrate divided by thatfor the other (17, 18). Because the F„,axvalues for the twosubstrates are known with some precision to be equal (as shownin Table 1), the indicated division provides a value for the Kmratio that does not depend on nitrite determinations at theborderline of significance for the colorimetrie method. Accordingly, a mixture of 1 HIM[15N]NDMA with 1 HIM[2H6]NDMA

was incubated for 10 min and the amines produced were analyzed by conversion to their 2,4-dinitroaniline derivatives followed by GC/MS. After subtraction of the concentration (0.04-0.12 MM)of each amine found in the control incubation withheat-inactivated microsomes, the concentrations of methyl[15N]amine and | 'H >|methylamine generated during microsomal me

tabolism were found to be 1.81 and 0.35 MM,respectively, for aKmD/Km" ratio of 5.2. The extent of total metabolism was

estimated from the formaldehyde production (43 MM)in the 2mM nitrosamine solution as 2%, indicating that substrate conversion was small; this conclusion was confirmed by analyzingthe nitrosamine extracted from the completed reaction mixture,GC/MS showing that the concentrations of [15N]NDMA and[2H6]NDMA did not differ significantly after incubation. Rep

etition of this competitive isotope effect experiment undervarious conditions produced additional values for the A'mratio.

These data are summarized in Table 2. The mean observed forthe KmD/Km"isotope effect for the denitrosation of NDMA was

Table 2 Isotope effect on Kmfor denitrosation of NDMA in a competitiveincubation

Incubations were performed for IO min in the presence of 0.8 mg of acetone-induced rat liver microsomes in 1 ml of total volume.

Substrate concentration(mM)

Amine concentration produced

[I5N]NDMA1

110.3[2H6]NDMA1

110.3CH315NH22.34

2.341.200.96CD3NH20.27

0.400.300.22(KaD/Kn»)"8.6

5.84.1

4.4

Mean (±SE) = 5.7 (±1.0)" Determined by subtracting the concentration of amine found in a control

incubation conducted with heat-inactivated microsomes from that observed in asimultaneously run incubation with active microsomes. Each entry was obtainedfrom four to six determinations, two to four replicate GC/MS injections of eachof one or two replicate derivatizations of each of two replicate incubations. Foreach determination, the intenstiy for the molecular ion of the 2,4-dinitroanilinederivative (m/z 198 for CH3"NH2, m/z 200 for CD3NH2) was divided by that forthe 3 nmol of methyl([2H3]methyl)amine hydrochloride (molecular ion of 2,4-dinitroaniline derivative, m/z 214) added as internal standard before derivatiza-tion. The resulting intensity ratio (/) was converted to the concentration value(Q through the equation C = (1 - a)/b after calculating the linear least-squaresfit / = a + bC of the (C, I) graph obtained for a set of aqueous standard solutionscontaining known amounts of CH3"NH2 and CD3NH2 after adding internalstandard, derivatizing, and measuring the intensity ratios by GC/MS as above.For CH3"NH2, the slope was * = 0.3947 ±0.0133 (SE) ^M"1 and the interceptwas a = -0.0297 ±0.0409 (SE), while the values for (2H,)methylaminc were0.4373 ±0.0145 »iM'1and -0.0435 ±0.0444, respectively. Other details are

similar to those given in Ref. 14.* Calculated by dividing the concentration of CH3"NH2 produced by that of

CD3NH2.

5.7 ±1.0 (SE), not significantly different from the corresponding value for demethylation calculated from the data of Table1 (0.30 mM -s-0.06 HIM = 5). We consider the Km calculatedfrom the former data [KmD= (Km°/Km")Km"= 5.7 x 0.06 «

0.3 HIM]to be a better estimate of the true Kmfor denitrosationof [2H6]NDMA than the value of 0.19 mM calculated in Table

1 from the data for the parallel incubations.Since denitrosation of [2H6]NDMA was quantified in this

experiment by integrating the ion current due to the parentpeak at m/z 200 in the Selected Ion Recording mode for the N-[2H3]methyl-2,4-dinitroaniline produced on derivatizing the denitrosation product with 2,4-dinitrofluorobenzene, any denitrosation accompanied by exchange loss of deuterium would notbe detected and the resulting Km°/KmHratio would be artifac-

tually high. To prove that this was not the case, the lowresolution spectrum of the derivatized supernatant from incubation of 1 mM [2H6]NDMA alone for 10 min with acetone-

induced microsomes was determined. It was very similar to thatobtained on derivatization-GC/MS of authentic [2H3]methyl-

amine. The spectra are shown in Fig. 2. The lack of a significantpeak at m/z 199 indicated that the metabolic product wasessentially completely deuterated. By contrast, the presence ofsuch a peak in the reference spectrum showed that the commercial standard was of rather low isotopie purity (94%).

To confirm our previous conclusion (14) that methylamineand not dimethylamine is the stoichiometric by-product ofNDMA denitrosation by rat liver microsomes, both bases werequantified in the supernatant from the incubation of [2H6]

NDMA. As expected from the earlier results, the concentrations of deuterated dimethylamine after exposure to activeversus heat-inactivated microsomes did not differ significantly,while [2H3]methylamine was found to be produced to the extent

of 2.6 MM.The latter value was similar, as expected, to theamount of nitrite found at the conclusion of incubation, 2.5MM.Similarly, the mean total methylamine concentration (±SD) produced in the four experiments with equimolar [I5N]NDMA/[2H6]NDMA summarized in Table 2 was 2.0 ±0.8 MM,

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ISOTOPE EFFECTS ON NDMA METABOLISM IN VITRO

100

90

70

60

40

30

20

IIs

108

168

90-

80

220

Fig. 2. Mass spectra of derivatized [2H3]methylamine (bottom) prepared from

commercially available reference standard and (top) isolated from the microsomaldenitrosation of [ÕI,,|M >MA. Because of the low signal level in the latterspectrum, background substraction eliminated the weak peak at m/z 199 butallowed impurity peaks such as those at m/z 91 and 168 to remain.

which was again similar to the mean (±SD) nitrite generated,2.7 ±0.8 MM.

DISCUSSION

Previous isotope effect studies have shown that the majorpart (79-92%) of a small NDMA dose p.o. is metabolized onfirst pass through the liver of the rat whether the nitrosamineis deuterated or not (3). Even the small amount that escapesinto the general circulation is expected eventually to be eliminated predominantly by hepatic metabolism, because the liverhas long been recognized as the primary site of metabolism forthis nitrosamine (19). For these reasons, the extent of metabol-ically induced liver DNA alkylation following p.o. doses of[2H6]NDMA was expected to be essentially the same as that

produced by undeuterated NDMA under identical conditions(3). This was not the case. Rats receiving [2H6]NDMA had onlytwo-thirds as many 7-methylguanine residues in their hepaticDNA as those given equimolar doses of NDMA (2). Onepossible explanation is that metabolism in liver by the established activation pathway that leads to alkylation (5) is not theonly elimination route available to NDMA in vivo; if there werea competing elimination route that had a significantly smallerkinetic isotope effect than the activation pathway and thuswould be increased in extent at the latter's expense on deuter-

ation of the substrate, the earlier isotope effect data could be

H3C CH3

N

S,

H3CX

HCHO+ H3CNH2

N02

[Fev=0]

•P-450

[Felv-OH]

H3C XCH3 H3C CH2Nt ^N\~T \

Fig. 3. Possible mechanisms of NDMA metabolism, as adapted from Ref. 23and supported in the present work. Path a is the postulated production of the a-nitrosamino radical as the key intermediate in both demethylation (path b) anddenitrosation (path c). X' represents a cellular nucleophile, e.g., the If positionof a DNA guanine residue. The .V-nitroso compounds indicated as the firstproducts of paths a and b are drawn as single IÕ./7.conformers for convenienceonly; this should not be construed as rejection of the opposite conformeràinvolvement (nitroso oxygen syn to the methyl group) in either case.

explained. Denitrosation seemed in many ways to fit the description of the hypothetical missing elimination route: it isvery probably a deactivation pathway because in cleaving thenitrogen-nitrogen bond it prohibits methylation of DNA viathe diazonium ion considered to be the ultimate carcinogen (9-13); it accounts for a significant proportion of NDMA metabolism in vitro (9-11); and there is growing evidence that itoccurs in vivo (20-22). However, the present results suggestthat metabolic switching from demethylation to denitrosationdoes not occur when fully deuterated [2He]NDMA is substituted

for the undeuterated substrate in the metabolism of NDMA byliver microsomes, because the deuterium isotope effects for thetwo processes were very similar in magnitude. We are thus leftfor the time being without an explanation for the isotope effectson alkylation and carcinogenesis by NDMA. It is possible thatexcretion or exhalation of the unmetabolized nitrosamine maybe at least partly responsible, but this point remains to besubstantiated.

Nevertheless, the data have several implications regardingthe mechanisms of NDMA action. For example, the similarityof Km values for denitrosation and demethylation and of theisotope effects thereon seems to suggest that the two pathwaysare catalyzed by the same enzyme and share at least onecommon intermediate in the initial steps. A mechanism thatappears to be capable of rationalizing these and a variety ofother findings regarding the metabolism of NDMA has recentlybeen advanced by Haussmann and Werringloer (23), who suggested that the a-nitrosamino radical may be a common intermediate in both processes. If this radical is produced, it appearsto have two fates. Recombination with -OH would lead to thea-nitrosamino alcohol considered to be the proximate carcinogen and DNA-alkylating agent (5). Alternatively, loss of nitricoxide would produce an imine that would be expected to hydro-lyze under physiological conditions to methylamine and formaldehyde, both of which are observed products of in vitroNDMA metabolism; the nitric oxide would undergo rapidoxidation to nitrite, as observed previously (23). This mechanism is depicted in Fig. 3. It is consistent with the considerableparallelism noted previously between denitrosation and demethylation (9-11, 13, 14) and also with the fact that the freea-nitrosamino alcohol generated on hydrolysis of W-nitroso-methyl(acetoxymethyl)amine does not decompose to significantmethylamine5 and nitrite (12), the latter finding having been

taken as evidence that the nitrosamino alcohol is not an intermediate in denitrosation (12).

5L. K. Keefer, T. Anjo, T. Wang, and C. S. Yang, unpublished results.

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It is not yet clear whether the postulated a-nitrosaminoradical is produced directly by enzymatic hydrogen atom abstraction or indirectly by proton loss from an initially formedcation radical (Fig. 3). The hydrogen abstraction mechanism,generally displaying a large intrinsic isotope effect, has beenproposed for aliphatic hydroxylation and some /V-dealkylationreactions catalyzed by peroxidases, whereas the 2-step mechanism (1-electron oxidation followed by deprotonation) generallydisplays a small kinetic isotope effect and is being proposed formany A^-dealkylation reactions catalyzed by cytochrome P-450(24-30). On the basis of the presently observed isotope effecton Vmxi/Kmand the fact that this value may be a lower limit ofthe intrinsic kinetic isotope effect, one may propose that theoxidation of NDMA involves an initial hydrogen abstractionstep. On the other hand, the 2-step mechanism is possible if weassume that the severalfold increase in Km associated withdeuteration is mainly due to the difference in the reversiblebinding of NDMA and [2H6]NDMA to the enzyme.

The observation of a negligible deuterium isotope effect onKnaxbut a 5-fold effect on Km, implying that the isotope effecton Vmitx/Kmalso has a value of 5, may indicate that the commitment to catalysis associated with the binding of NDMA tothe metabolizing enzyme is low (17, 31). This conclusion appears to fit well with that of Swann et al. (2), who interpretedthe sigmoidal plots of extent of [2H6]NDMA metabolism versustime when this substrate was given in mixtures with undeuter-ated NDMA to rats as implying that the complex betweenNDMA and the principal enzyme metabolizing it in vivo freelyequilibrates with unbound substrate.

The isotope effects on the two metabolic routes studied invitro were not only similar to each other, but they were alsosimilar in size to the ratio of intrinsic clearances for NDMAversus [2H6]NDMA after p.o. bolus administration in male

Fischer rats (3). In addition, the small Kmaxbut large VmKl/Kmisotope effects found in the present experiments agree withprevious in vivo observations demonstrating a large isotopeeffect when low doses of NDMA and [2H6]NDM A were admin

istered separately or when they were given in mixtures of anytotal dose size (Vmn/Km conditions) (2, 3) but a small isotopeeffect when large doses were given separately (Vmi*conditions)(2, 3). The results support the conclusion (15) that the low-/fmform of NDMA demethylase in the microsomes is responsiblefor most of the NDMA metabolism that occurs in the liver invivo. Future work will focus particularly on the course ofNDMA denitrosation in vivo, since this pathway may constitutea means of detoxifying the otherwise potent carcinogen evenwhen it is endogenously produced (32).

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