metabolism of nitrosamines by purified rabbit liver ... · 17,18, 20, 26, 32). (b) studies with...

[CANCER RESEARCH 45,1140-1145, March 1985]

Metabolism of Nitrosamines by Purified Rabbit Liver Cytochrome P-450Isozymes1

Chung S. Yang,2 Yityoong Y. Tu, Dennis R. Koop, and Minor J. Coon

Department of Biochemistry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103 [C. S. Y., Y. Y. T.], andDepartment of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, Michigan 48109 [D. R. K., M. J. C.J

ABSTRACT

The metabolism of nitrosamines by microsomal cytochromeP-450 (P-450) isozymes was studied in a reconstituted monoox-ygenase system. P-450 LM2, LM^, LMa, and LMac, LM4, andLMB were purified, respectively, from the livers of phenobarbital-treated, ethanol-treated, untreated, isosafrole-treated, and imida-zole-treated rabbits. Of these isozymes, LMaÂ»had the highestW-nitrosodimethylamine demethylase (NDMAd) activity with a Km

of 2.9 row and VmÂ«of 9.3 nmol/min/nmol. LM2, LM4, and LM6exhibited NDMAd activity only at high A/-nitrosodimethylamine

concentrations, and isozymes LMsb and LMac had poor activityeven at the highest substrate concentrations examined. LM2,however, was more active than LMo, in the metabolism of N-

nitrosomethylaniline. With each isozyme (LM3aor LM4), only oneKm for NDMAd was observed, whereas with rabbit liver micro-

somes, multiple Km of 0.07, 0.27, and 36.8 mw were obtained.P-450 isozymes also catalyzed the denitrosation of nitrosaminesat rates comparable to or lower than the demethylation, and theratio of these two reactions was different with different nitrosamines. 2-Phenylethylamine and 3-amino-1,2,4-triazole, which

were believed previously to affect NDMAd by mechanisms independent of P-450, were shown to be potent inhibitors of P-450-dependent NDMAd. These results further establish the roleof P-450 isozymes in the metabolism of nitrosamines and indicatethat LMaa is apparently responsible for the increased A/-nitroso-

dimethylamine metabolism associated with ethanol treatment.

INTRODUCTION

The metabolism of nitrosamines has been a subject of extensive investigation because of its importance in understanding thecarcinogenicity and toxicity of this group of compounds. OxygÃ©nationat the a-carbon (Â«-hydroxylation) is believed to be a

key step in the metabolic activation of many nitrosamines (6,17,24, 28). There is evidence indicating that NDMA3 is metabolized

by a P-450-dependent monooxygenase system (5, 9, 10). The

efficiency and the importance of this system in the metabolismof nitrosamines, however, have been questioned, and alternativemechanisms of metabolism have been postulated (18-21, 31-

34). The following observations concerning NDMA metabolismby rat liver microsomes have previously puzzled many investigators, (a) NDMAd actvity was not induced by classical inducers,such as phenobarbitol, 3-methylcholanthrene, jS-naphthoflavone,Aroclor 1254, and pregnenolone-16a-carbonitrile, especially

1This work was supported by Grants CA-16788, CA-37037, and AM-10339

from the NIH.2To whom requests for reprints should be addressed.3The abbreviations used are: NDMA, W-nitrosodimethylamine; NDMAd, nitro-

sodimethylamine demethylase; P-450, cytochrome P-450. Different liver microsomal P-450 isozymes are designated as LM2, LMa., LMÂ»,etc.

Received June 18, 1984; accepted November 6,1984.

when the activity was assayed with 1 to 4 mw NDMA (2, 3, 7,17,18, 20, 26, 32). (b) Studies with inducers led to the proposalof the existence of NDMAd I and II (17,18). In fact, more than 2Km values can be observed for NDMAd even in hepatic micro-somes of untreated rats (17-19, 38). Lake eÃal. (19) reported

the existence of 3 Kmvalues: 0.32; 1.5; and 35 ITIM.In addition,Tu and Yang (38) reported recently the existence of a lower Km(0.07 mu) NDMAd in control rat liver microsomes. (c) NDMAdwas not significantly inhibited by classical monooxygenase inhibitors, such as SKF-525A and metyrapone, but was inhibited by

compounds which are not known to be strong inhibitors of thecommonly studied monooxygenase reactions, such as 3-amino-1,2,4-triazole and 2-phenylethylamine (20, 21,31,33,34).

More recent work, however, is beginning to elucidate the roleof P-450 isozymes in the metabolism of nitrosamines. The he

patic NDMAd activity in the rat liver was shown to exist predominately in the microsomes and to be P-450 dependent (Ref. 39;

Footnote 4). Furthermore, the activity is inducible by pretreatment with acetone, isopropanol, ethanol, or pyrazole and byfasting and diabetes (29, 30, 36-39). A unique P-450 isozyme,LMaa,was isolated recently from ethanol-treated rabbits (14,25),

and the identical isozyme was shown to be inducible in rabbitsby Â¡midazole (13), a known inducer of hepatic microsomalNDMAd in rabbits (11). In the present work, we have studied themetabolism of NDMA by P-450 LMaa and by 5 other P-450

isozymes purified from rabbit liver microsomes. In addition, thespecificity of the 6 isozymes in the demethylation and denitrosation of several other nitrosamines was examined.

MATERIALS AND METHODS

Chemicals. NDMA was purchased from Aldrich Chemical Co., Milwaukee, Wl. W-Nitroso-W-methylethylamine, N-nitroso-Af-methylbutylam-ine, rV-nitroso-W-methylaniline, 2-phenylethylamine, 3-amino-1,2,4-tria-zote, rV-1-naphthylethylenediamine, NADP, and NADPH generatingreagents were obtained from Sigma Chemical Co., St. Louis, MO. N-Nitroso-A/-methylbenzylamine was from Ash Stevens, Inc., Detroit, Ml.

Dilauroylphosphatidylcholine was purchased from Serdary ResearchLaboratories, London, Ontario, Canada.

Microsomes and Microsomal Enzymes. Hepatic microsomes wereprepared from control or ethanol-treated adult male New Zealand rabbits

(2.0 to 2.5 kg) according to previous procedures (14, 15). The controland ethand-induced microsomes contained 2.16 and 2.30 nmol P-450per mg protein, respectively. The P-450 isozymes were purified from

rabbits which had been subjected to the following treatments: LM2,phÃ©nobarbital; LMa,, ethanol; LMÂ», no treatment; LM^, no treatment;LMÂ«,Â¡sosafrole;and LM6, imidazote (13-15). These P-450 preparations

were electrophoretically homogenous, and the specific contents, expressed as nmol P-450 per mg protein, were 18.2, 19.0, 17.4, 13.5,

19.5, and 16.2 for LM2, LM*, LMÂ», LM*. LM4, and LM6, respectively.Electrophoretically pure NADPH: cytochrome P-450 reducÃase was pre-

4J. Hong and C. S. Yang, manuscript in preparation.

CANCER RESEARCH VOL.45 MARCH 1985

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NITROSAMINE METABOLISM BY CYTOCHROMES P-450

pared from rabbit liver microsomes as described previously (8). Thespecific activity of the reductase was 54 units (which catalyzed theNADPH-dependent reduction of 54 Â¿imolcytochrome c per min) per mgprotein. The microsomes and enzymes were stored at -86Â° prior to use.

Metabolism of Nitrosamines. The demethylation and denitrosationreactions were assayed at 37Â°by previously described procedures (22,

38) with some modifications. To reconstitute the monooxygenase activity, 0.2 to 0.3 nmol of P-450,1.2 to 2.0 units of NADPH: P-450 reductase,

and 15 ng of dilauroylphosphatidylcholine were mixed in a glass testtube at room temperature. Buffer, water, and the NADPH-generating

system were then added to obtain an incubation mixture containing 50rriM tris-HCI (pH 7.4), 10 mw MgCI2,150 mw KCI, 0.4 rriM NADP, 10 mM

isocitrate, and 0.15 unit of isocitrate dehydrogenase in 0.5 ml (38).Standards and 2 sets of blanks were run concomitantly: in one set, thenitrosamine substrate was omitted; and in the other, the NADPH-generating system was omitted. After preincubation at 37Â° for 2 min, the

reaction was initiated by the addition of the nitrosamine substrate. Insome experiments, the nitrosamines were added before the preincubation, and the reaction was initiated by the addition of the NADPH-generating mixture. After a 20-min incubation, the reaction was termi

nated by the addition of 0.05 ml each of 25% ZnSO4 and saturatedBa(OH}2. The mixture was centrifuged, and 2 aliquots of the supernatantwere transferred to glass test tubes: 0.2 ml for formaldehyde determination and 0.2 ml for nitrite determination. To the former, 0.1 ml of aconcentrated Nash reagent (5 g ammonium acetate and 0.1 ml acetyl-

acetone in 6 ml 3% acetic acid) was added, and the mixture wasincubated at 55Â°for 10 min. The mixture was cooled to room tempera

ture, and absorbance at 412 nm was measured for formaldehyde determination (27, 39). For the determination of nitrite, 0.1 ml of a reagentcontaining 1.65% sulfanilic acid, 10% acetic acid, and 0.05% /V-1-

naphthylethylenediamine was added. After 20 min of color developmentat room temperature, absorbance at 545 nm was measured (22). Resultsare expressed as nmol HCHO (or NO2~) formed per min per nmol P-450

or per mg microsomal protein. When the nitrite determination was notneeded, the reaction mixture was scaled down to one half the volume tosave the purified enzymes. The assay was carried out in duplicate, andthe differences between the duplicate tubes were < 10%. Each experiment was repeated at least once, and the results presented are representative data.

RESULTS

Metabolism of NDMA by P-450 Isozymes. Upon mixing P-450 LM3a with purified NADPH: P-450 reductase and dilauroyl

phosphatidylcholine, the reconstituted monooxygenase systemefficiently catalyzed the demethylation of NDMA (Table 1). No orvery low activity was observed when any of the components, P-

450, reductase, or the phospholipid, was omitted from the reconstituted system. The reaction was linear within at least 30min, and the activity was proportional to the quantity of P-450present, with a turnover number of 5.5/min. Other P-450 iso-

TabtelConditions for the reconstitution of NDMAd

The complete reaction system contained 0.1 nmol P-450 LMâ€ž,0.63 unitNADPH:P-450 reductase, 7.5 >ig dilauroylphosphatidylcholine, and 4 mM NDMA ina final volume of 0.25 ml.

Conditions nmol HCHO/min/nmol P-450

Complete system-P-450- Reductase- Dilauroylphosphatidylcholine

Complete system10-min incubation20-min incubation30-min incubation

5.10<0.03<0.03

0.16

5.505.455.45

zymes, however, were less effective in catalyzing the demethylation of NDMA (Table 2). When assayed with 4 HIM NDMA, thedemethylase activity of LM6 was only 5% that of LMa., and theactivity of other P-450 isozymes was even lower. At a substrate

concentration of 100 mw, however, substantial NDMAd activitywas observed with LM6, LM2, and LMÂ«which displayed 65, 33,and 30% of the activity of LMs., respectively. The 2 constitutiveforms, LM3b and LM:i,, had low activity even at high substrateconcentrations.

Substrate Specificities of P-450 Isozymes. P-450 LM;i;1,LM;,

and LM4 were selected in this study to investigate whether othernitrosamines are also metabolized by P-450 isozymes (Table 3).For the convenience of the assay, only A/-methyl-containing

nitrosamines were used, and the demethylase activity was assayed at substrate concentrations of 4 and 40 mw. With P-450LMsa, the activity with nitrosomethylaniline was about one halfthe rate with NDMA, and the activity was much lower when theother /v-alkyl group was ethyl, butyl, or benzyl. With nitroso-

methylbutylamine and nitrosomethylbenzylamine, the demethylase activity was elevated 4- to 5-fold when the substrate con

centration was increased from 4 to 40 mw. In contrast, a similarincrease in substrate concentration produced only a 45 to 69%rise in activity with the other 3 substrates. The substrate specificity of LM2 was quite different from that of LMa. in its highactivity toward nitrosomethylaniline, showing a demethylase ratemore than twice that of LM3;1.LM2 also catalyzed the demethylation of mtrosomethylbutylamine. and the activity decreasedwhen the W-butyl moiety was substituted by benzyl, ethyl, or

methyl. When the substrate concentration was increased from 4to 40 mM, a large increase in LM2-dependent demethylase activ

ity was observed with NDMA and nitrosomethylethylamine butnot with the other 3 substrates. P-450 LM4 was not effective in

catalyzing the metabolism of most of the nitrosamines tested,and appreciable activity was seen only with nitrosomethylaniline.

In addition to the demethylase reaction, the rate of nitrosaminedenitrosation was measured concomitantly (Table 4). Becauseof the high rate of the nonenzymatic denitrosation of nitroso-

Tabte2NDMAd activity of different P-450 isozymes

The reaction mixture contained 0.1 nmol P-450 isozymes, 0.63 unit NADPH:P-450 reductase, 7.5 >Â¡gdilauroylphosphatidylcholine, and different concentrations ofNDMA in a final volume of 0.25 ml.

NDMA(mM)0.1

4.010

100P-450

isozymes (nmol HCHO/min/nmolP-450)LM2<0.03

0.100.362.21LM*0.25

5.896.686.71LMÂ»<0.03

0.030.171.45LMae<0.03

0.080.220.79LM.<0.03

<0.030.182.01LM.<0.03

0.310.724.35

TablesSubstrate specificity of P-450 isozymes in the demethylation of nitrosamines

The experimental conditions were the same as those for Tabte 2, except that 4and 40 mM nitrosamines were used as substrates.

P-450 isozymes (nmol HCHO/min/nmolP-450)SubstrateNitrosodimethylamine

NitrosomethylethylamineNitrosomethylbutylamineNitrosomethylbenzylamineNitrosomethylanilineLM,4

mM 40mM0.13

1.430.07 1.391.73 1.910.50 1.085.57 7.36U4

mM5.01

0.940.230.282.51fc.40

mM7.89

1.591.171.173.65LM.4

mM 40mm<0.03

0.510.07 0.350.10 0.320.08 0.221.57 2.46

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methylaniline, the enzymatic rate was not accurately measuredand not included in Table 4. As was shown previously with ratliver microsomes (22), the ratio of the demethylation and denitro-sation rates (HCHO:N02~ ratio) was different with different sub

strates. With LMaa, the ratio was 6 to 7 for NDMA and rangedfrom 0.6 to 1.6 for the other substrates. Thus, even though LMa,had higher NDMAd than nitrosomethylethylamine demethylaseactivity, it catalyzed the denitrosation of these 2 nitrosamines atabout equal rates, which were higher than the denitrosation ratesof substrates with an N-butyl or N-benzyl group. Denitrosation

of nitrosamines was also catalyzed by LM2 and LM4, but thepossible error in measuring low quantities of NO2~ in some of

the data points prevented a more thorough analysis of theresults. Nevertheless, parallelism between the denitrosation anddemethylation reactions was observed. In comparison to LM3a,LM2 was less effective in catalyzing the denitrosation of NDMAand nitrosomethylethylamine but more effective with nitroso-

methylbutylamine and nitrosomethylbenzylamine. With LM2, thedenitrosation rates for NDMA and nitrosomethylethylamine werealso increased greatly upon changing the substrate concentration from 4 to 40 mw, analogous to the demethylase activity. Inreactions catalyzed by LM2, the HCHO:NO2~ ratio for NDMA

was higher than that for other nitrosamines. The data, however,cannot distinguish with certainty whether the ratios produced byLM2 were different from those produced by LM3a.LM4 which hadlow demethylase activities also was ineffective in catalyzing thedenitrosation of the nitrosamines studied herein.

Kinetics of NDMAd in Microsomes and Reconstituted Systems. Analogous to rat liver microsomes (38), rabbit liver micro-

Tabte4Substrate specificity of P-450 isozymes in the denitrosation of nitrosamines

The results were obtained concomitantly with those shown in Table 3.

P-450 isozymes (nmol NOr/min/nmol P-450)

LM4

Substrate 4 rriM 40 mu 4 mw 40 DIM 4 mM 40 HIM

NitrosodimethylamineNitrosomethylethylamineNitrosomethylbutylamineNitrosomethylbenzylamine

0.05 0.40 0.81 1.040.04 1.03 1.09 1.130.64 1.02 0.38 0.740.62 0.96 0.42 0.87

<0.02 0.110.02 0.210.10 0.360.16 0.22

somal NDMAd also displayed a complex set of kinetic parameters (Chart 1); the data were analyzed similarly (38). With controlrabbits, the microsomal demethylase activity increased with increasing substrate concentrations, and saturation was not observed even at 200 ITIM NDMA (Chart 1/\). With ethanol-pre-treated rabbits, the activity was high in the low substrate concentration range (<1 mM), decreased in the range of 4 to 10 mw,and increased further in the range of 30 to 200 HIM NDMA. In adouble-reciprocal plot, at least 3 apparent Km values were ob

served for the NDMAd of control rabbit hepatic microsomes.They were 0.07, 0.27, and 36.8 mw with corresponding Vâ„¢Â»values of 0.65, 0.96, and 3.24 nmol/min/mg protein (Chart 1B).With ethanol-induced microsomes, the predominant NDMAd had

an apparent Km of 0.11 mw and a Vâ„¢Â»of 2.66 nmol/min/mg(Chart 1fl). In addition, an estimated Kmof 33.9 mM and Vâ„¢Â«of4.74 nmol/min/mg were also observed. In a second experimentwith ethanol-induced microsomes, Km values of 0.08 and 42.3

mM with corresponding VmÂ«values of 1.73 and 3.84 nmol/min/mg were obtained.

In the reconstituted system with LM3a,a single Kmof 2.91 mMand a Vâ„¢Â«of 9.33 nmol/min/nmol were observed (Chart 2). In asecond experiment, the corresponding values were 3.06 mM and9.42 nmol/min/nmol, respectively. With LM4, an estimated Kmof456 mM and Vâ„¢Â«of 7.60 nmol/min/nmol were obtained (Chart3), and values of 602 mM and 9.35 nmol/min/nmol, respectively,were observed in a second experiment.

Inhibition of P-450-dependent NDMAd. The effects of several

inhibitors on the NDMAd of microsomes and the reconstitutedsystem were examined (Table 5). 2-Phenylethylamine, a com

pound known to be a substrate of monoamine oxidase and toinhibit NDMA metabolism by rat tissues (31, 34), was a potentinhibitor of NDMAd of the microsomes and of the LMa. and LM4reconstituted systems. Since the reconstituted system had essentially no monoamine oxidase activity, the results were clearlydue to inhibition of the P-450-dependent monooxygenase activity. The P-450-dependent NDMAd was also inhibited by 3-amino-1,2,4-triazole and pyrazole at concentrations similar to thoseused in previous studies (20, 31, 33). 3-Amino~1,2,4-triazoleappeared to be more inhibitory toward the LM3a-dependentsystem than microsomes, whereas pyrazole was less inhibitory

8 10 3OS.mM

150 210 25 50 751/[S.mM]

100 125

Chart 1. Substrate dependence of microsomal NDMAd. The reaction mixture contained control microsomes (â€¢)or ethanol-induced microsomes (A) corresponding to0.95 and 0.30 mg protein, respectively, in an incubation mixture of 0.5 ml. The results are shown as velocity (V) (nmol HCHO per min per mg protein) versus substrateconcentration (S) plots (A) and double reciprocal plots (6). In B, the data obtained with control microsomes are fitted by 3 linear regression lines with corresponding Kmvalues of 0.07 mw (4 points with correlation coefficient, r = 0.983), 0.27 mM (3 points, r = 0.995), and 36.80 mw (5 points, r = 0.993). With ethanol-induced microsomes,the data are fitted by 2 lines with corresponding Kmvalues of 0.10 mM (6 points, r = 0.994) and 33.90 mM (4 points, r = 0.973). The data points of the high Kmare notshown due to space limitation.


1142




06

04

0.2

0 8 16 24S/nM

0.5 1.0 1.5 20

Chart 2. Substrate dependence of NDMAd in the reconstituted system with P-450 LMj.. Data are shown as a double-reciprocal plot and, in the inset, a velocity(V) versus substrate concentration (S) plot. The regression line represents a Km of2.91 mu (r = 0.999).

20 4O 60 80 100

Chart 3. Substrate dependence of NDMAd in the reconstituted system with P-450 LMÂ».Data are shown as a double-reciprocal plot and, in the inset, a velocity(V) versus substrate concentration (S) plot. The regression line represents a Kmof435 mm (r = 0.999).

TablesInhibition of reconstituted and microsomal NDMAd

The reaction mixture contained 0.1 nmol of P-450 isozymes in the reconstitutedsystem as described Â¡n'Materials and Methods", or control or ethanol-induced

microsomes (0.48 and 0.18 mg protein, respectively) in an incubation mixture of0.25 ml. The substrate concentration was 4 mM NDMA, except for the experimentswith P-450 LM4, in which 200 mM NDMA was used. Inhibitors were added as

aqueous solutions.

% of inhibition of NDMAdactivityInhibitorNone

2-Phenytethylamine (1 mM)3-Amino-1,2,4-triazole(10

mm)Pyrazote (1 HIM)P-450

LMa.0

(5.58f918840P-450

LM40(3.11)

443020Control

micro-somes0

(0.78)903970Ethanol-

inducedmicrosomes0(2.21)

835580

* Numbers in parentheses, the NDMAd activity in the absence of inhibitor. Theactivity is expressed as nmol HCHO per min per nmol P-450 for the reconstituted

systems and as nmol HCHO per min per mg protein for the microsomes.

in the reconstituted system. In comparison to the LMaa-depend-ent activity, the LM4-dependent one seemed less susceptible to

the inhibitors, possibly due to the fact that the activity wasassayed with a substrate concentration of 200 mM NDMA.

DISCUSSION

When multiple forms of liver microsomal P-450 were first

separated and characterized, the conclusion was reached thatthese cytochromes have the ability to bind many or all of thepotential substrates but differ in their relative efficiency in thehydroxylation of such compounds (12, 23). The present workclearly provides another example of this principle, since all of theP-450 isozymes were found to be active with all of the nitros-amines examined and to differ only in the rates of catalysis. Inthe intact organism, however, such differences could determinethe extent of toxicity and carcinogenicity when a particular animalspecies is exposed to varying levels of nitrosamines along withagents which induce the individual cytochromes. The resultsobtained in the present study show that LM3,â€žthe ethanol-

induced form from rabbits, is highly efficient in catalyzing themetabolism of NDMA, whereas the other forms are less efficient.The latter group of P-450 isozymes, however, can metabolize

NDMA when assayed at high substrate concentrations (Table2), suggesting that they function with a Kmvalue higher than thatexhibited by LM3a. In the case of LM4, an apparent Kâ€ž,value ofover 400 mM is seen (Chart 3).

The metabolism of nitrosomethylethylamine follows the samepattern as NDMA, except that the demethylase rate is lower.LM2, the phenobarbital-induced form, on the other hand, is moreactive than LM3i, in metabolizing nitrosomethylaniline and nitro-somethylbutylamine. With W-dialkyl nitrosamines, both the A/methyl and the other N-alkyl (ethyl, butyl, or benzyl) groups are

the targets of the oxygÃ©nation.Studies with microsomes haveindicated that the other alkyl groups are usually more readilyoxidized than the A/-methyl group (1, 4). It is possible that the

relative oxygÃ©nationrate of the 2 alkyl groups in a nitrosamineis also determined by the substrate specificity of P-450, but thispoint remains to be substantiated. In comparison to other P-450isozymes, LM3a is also more active in the p-hydroxylation of

aniline and the oxidation of ethanol to acetaldehyde (14). Theparallelism in the metabolism of NDMA and these 2 substrateswas also observed in different types of rat liver microsomes.4

Denitrosation of nitrosamines has been studied previously withrat liver microsomes and P-450 isozymes purified from pheno-barbital-treated rats (22). The present work demonstrates thatother P-450 isozymes, particularly LM3a, can efficiently catalyze

the oxidative denitrosation of nitrosamines. The relative rates ofHCHO and NO2~ formation are different with different nitros

amines. Concerning a specified nitrosamine substrate, however,the rates of these 2 reactions roughly parallel each other. This isconsistent with the previous conclusion based on studies withrat liver microsomes (23). Although closely related to the de-

methylation, denitrosation is distinguished from the former reaction in its response to Superoxide dismutase. In a reconstitutedsystem consisting of P-450 isozyme purified from ethanol-treated

rats, Superoxide dismutase inhibited the denitrosation by 73%,but it inhibited demethylation only to a maximum of 20%.5

Previous investigators have attempted to measure the quantityof N2 formation upon oxidative metabolism of NDMA, and discrepancies exist between nitrosamine metabolized and N2 recovered (16). The nitrite formation, as described herein, mayaccount for some of the discrepancies. The mechanism of thedenitrosation and the biological consequence of the reaction

* Y. Y. Tu and C. S. Yang, manuscript submitted for publication.

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remain to be investigated.Because different P-450 isozymes have different affinities for

NDMA, it is understandable that multiple Kmvalues are observedin microsomes. Depending on the number and type of P-450

isozymes present, many Kmvalues with possible overlap may beexpected. Our present approach of resolving them into 2 or 3major Kâ„¢values without correction for overlap is a treatment byapproximation. The 3 apparent Kmvalues for control rabbit livermicrosomes are similar to those observed in rat liver microsomes(38). The kinetics of the ethanol-induced rabbit microsomes is

also similar to that observed with rats in the concentration rangesstudied (30). The increased NDMAd activity is mainly dueto the induction of a low Kâ„¢form of this enzyme. The presentdata, however, do not distinguish whether or not the low KmNDMAd activities in control and ethanol-induced microsomes aredue to the same P-450 isozyme. The observation that P-450

LMaa and LM4 each have a single and distinct Kâ„¢value forNDMAd is consistent with the concept that the multiple Kmvaluesin microsomes are due to the multiple P-450 isozymes present.It was unexpected, however, that the Kâ„¢for LMaa, 2.95 mM, ismuch higher than the low Km, 0.07 to 0.10 mM, observed inmicrosomes. Antibodies prepared against LMaahave been shownto inhibit the low KmNDMAd in microsomes.6 It appears that the

kinetic constant is different when the cytochrome is in themembrane or in the reconstituted system.

Based on studies with inhibitors, some investigators (31, 33,34) have previously doubted the involvement of P450 in themetabolism of NDMA and postulated that other enzymes, suchas monoamine oxidase, might be involved in catalyzing theoxidation of NDMA. The present work with the reconstitutedmonooxygenase system demonstrates clearly that compounds,such as 2-phenylethylamine, 3-aminc-1,2,4-triazole, and pyra-zole, are potent inhibitors of P-450-dependent NDMAd activity.Similar results were also obtained in studies with rat liver micro-somal P-450 (39). These compounds were not known previouslyto be inhibitors of P-450-dependent reactions, because they donot effectively inhibit monooxygenase reactions with classicaldrug substrates (20, 39). The results of the present study suggest that many of the previous observations (20,31, 33, 34) canbe interpreted on the basis of inhibition of P-450-dependentNDMAd and thus further establish the role of P-450 isozymes in

the metabolism of nitrosamines.

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C""*

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1985;45:1140-1145. Cancer Res Chung S. Yang, Yityoong Y. Tu, Dennis R. Koop, et al. Cytochrome P-450 IsozymesMetabolism of Nitrosamines by Purified Rabbit Liver

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