Deuterium Isotope Effect on the Carcinogenicity of Dimethylnitrosamine in Rat Liver
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Keefer, Larry K.; Lijinsky, William; Astiazarán-García, Humberto

doi:10.1093/jnci/51.1.299

Cited by 75 publications

(44 citation statements)

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“…Steady-state Kinetic Parameters-In rat liver microsomes, deuterium substitution of P450 2E1 substrates usually results in a 3-5-fold increase in K m without any effect on k cat (9,11,12,19,20,50). Recombinant human cytochrome P450 2E1 was reconstituted with NADPH-P450 reductase and b 5 and incubated with a variety of substrates and their deuterated analogs to determine the effects of deuterium substitution on the steady-state kinetic parameters ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Oxidation of substrates by P450 2E1 involves a C-H bond breaking step, but deuterium substitution has been shown by several investigators to have little effect on k cat . Rather, there is an intermolecular isotope effect on K m for many substrates of P450 2E1 (9,11,12,19,20,50).…”

Section: Discussionmentioning

confidence: 99%

“…nitrosamines and alkenes) associated with human cancers, and the reactivity of products with DNA has been demonstrated (7,8). DNA alkylating ability and carcinogenicity were shown to be decreased upon deuterium substitution of N-nitrosodimethylamine, now known to be a substrate of P450 2E1 (9). When rat liver microsomes were examined, deuteration was found to increase K m for these reactions ϳ5-fold, but the k cat (V max ) remained unaffected by deuterium substitution (10 -12).…”

Section: Microsomal Cytochromes P450mentioning

confidence: 99%

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Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1

Bell¹,

Guengerich

1997

Journal of Biological Chemistry

108

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) even in the absence of substrate. These results indicate that the basis for the increase in K m is in the latter portion of the catalytic cycle. The intrinsic isotope effect ( D k) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Presteady-state studies showed a burst of product formation (k ‫؍‬ 410 min ؊1 ), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the k cat approximates the rate of product release, and K m is an expression in which the denominator is dominated by the rate of C-H bond breaking. Microsomal cytochromes P4501 (also termed heme-thiolate protein P450 by the Enzyme Commission, EC 1.14.14.1) (1) catalyze a variety of mixed-function monooxygenase reactions that often result in detoxication of drugs and other xenobiotics (2-4). Occasionally, oxidation results in the bioactivation of potentially potent carcinogens, particularly with substrates metabolized by P450 2E1 (5, 6). P450 2E1 is active in the oxidation of many low molecular weight organic compounds (e.g. nitrosamines and alkenes) associated with human cancers, and the reactivity of products with DNA has been demonstrated (7,8). DNA alkylating ability and carcinogenicity were shown to be decreased upon deuterium substitution of N-nitrosodimethylamine, now known to be a substrate of P450 2E1 (9). When rat liver microsomes were examined, deuteration was found to increase K m for these reactions ϳ5-fold, but the k cat (V max ) remained unaffected by deuterium substitution (10 -12).Most P450s are considered to operate according to a general scheme ( Fig. 1) (13). Following substrate binding (step 1), ferric P450 receives 1 electron via NADPH-P450 reductase (step 2). Steps 1a and 2a represent a potential pathway by which Fe 3ϩ is reduced to Fe 2ϩ in the absence of substrate and suggests a possibility for later entry of the substrate into the catalytic cycle. The ferrous form of the heme binds O 2 (step 3) before undergoing a second 1-electron reduction to begin O 2 activation (step 4). Although this second electron originates from NADPH-P450 reductase, the accessory protein cytochrome b 5 (b 5 , EC 4.4.2 group) appears to play some role in the delivery of the electron to the P450 (14). Insertion of the activated oxygen into the substrate is believed to occur by way of C-H bond cleavage (step 6) followed by rapid oxygen rebound to form product (step 7) (15).Step 8 is release of the product from the enzyme active site. Within the context of this scheme, the reduction (steps 2 and 4) and chemistry (step 6) are generally considered to be rate-limiting (13). Steps 9 and 10 reflect the potential for the Fe 3ϩ ROH complex to receive an electron from NADPH-P450 reductase, possibly leading to a second cycl...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Microsomal Cytochromes P450mentioning

confidence: 99%

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Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1

Bell¹,

Guengerich

1997

Journal of Biological Chemistry

108

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“…N-Nitrosodimethylamine (NDMA) is a hepatocarcinogen in rats, and Keefer et al (1973) found that overall tumor incidence in NDMA-treated rats was approximately 3-fold greater than in NDMA-d 6 -treated rats at low doses, with only one hepatoma found in the NDMA-d 6 -treated group (29 rats) versus eight hepatomas in the NDMA-treated group (30 rats). At 5-fold higher doses, 18 hepatomas were detected in the NDMA-d 6 -treated group and 24 hepatomas in the NDMA-treated group.…”

Section: Nelson and Tragermentioning

confidence: 99%

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

Nelson¹,

Trager²

2003

Drug Metab Dispos

142

121

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Critical elements from studies that have led to our current understanding of the factors that cause the observed primary deuterium isotope effect, (k H /k D )obs, of most enzymatically mediated reactions to be much smaller than the "true" or intrinsic primary deuterium isotope effect, k H /k D , for the reaction are presented. This new understanding has provided a unique and powerful tool for probing the catalytic and active site properties of enzymes, particularly the cytochromes P450 (P450). Examples are presented that illustrate how the technique has been used to determine k H /k D , and properties such as the catalytic nature of the reactive oxenoid intermediate, prochiral selectivity, the chemical and enzymatic mechanisms of cytochrome P450-catalyzed reactions, and the relative active site size of different P450 isoforms. Examples are also presented of how deuterium isotope effects have been used to probe mechanisms of the formation of reactive metabolites that can cause toxic effects.Historically, the determination of deuterium isotope effects has been a powerful tool to help unravel the intricacies of carbon-hydrogen bond cleavage and define the mechanism of specific chemical reactions. The intrinsic primary isotope effect, k H /k D , for a reaction is the magnitude of the isotope effect on the rate constant for the bond-breaking step (C-H versus C-D) of the reaction and is related to the symmetry of the transition state for that step. The larger the isotope effect, the more symmetrical the transition state, with the theoretical limit being 9 at 37°C in the absence of tunneling effects (Bell, 1974). The chemical events leading to the transition state and subsequent formation of products are the descriptors of a chemical mechanism. It is the relationship of k H /k D to transition state that provides mechanistic insight. Thus, k H /k D is the quantity that needs to be known, and for homogenous chemical reactions, the experimentally observed deuterium isotope effect, (k H /k D )obs, where (k H /k D )obs is defined as the ratio of a kinetic parameter such as V max or V max /K m obtained from a nondeuterated substrate to a deuterated substrate, is identical to k H /k D . This is generally not true of enzymatically mediated reactions and is the reason for the much less successful application of deuterium isotope effects to such reactions until the system was better understood. The experimentally observed isotope effect, (k H /k D )obs, was invariably found to be much smaller than k H /k D , the intrinsic isotope effect for that reaction, thereby obscuring both meaning and mechanism. Even more perplexing was the observation that (k H /k D

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“…Esters of a-hydroxylated nitrosamines, which can be obtained without having to prepare the alchohols themselves (22)(23)(24)(25)(26), have been shown to be direct-acting carcinogens (27), requiring only esterase-catalyzed hydrolysis. A compelling result, which indicates the importance of a-hydroxylation in carcinogenesis, is that replacement of the hydrogens of dimethylnitrosamine by deuterium drastically lowers carcinogenic potency (28) and that nitrosomorpholine is significantly more carcinogenic than its deuterated analog, 3,3,5,5-tetradeuteronitrosomorpholine (29).…”

Section: Resultsmentioning

confidence: 99%

alpha-Hydroxylation pathway in the in vitro metabolism of carcinogenic nitrosamines: N-nitrosodimethylamine and N-nitroso-N-methylaniline.

Kroeger-Koepke¹,

Koepke²,

McClusky³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Evolution of 5N2-labeled molecular nitrogen was used to gauge the extent of a-hydroxylation during rat liver homogenate metabolism of doubly '5N-labeled N-nitrosodimethylamine (DMN) and N-nitrosomethylaniline (NMA). These measurements were correlated with the extent of total metabolism as measured by the Except for certain special cases, N-nitrosamines must be activated metabolically before their carcinogenic or mutagenic potential can be expressed. In the most widely held hypothesis, the metabolic activation involves the hydroxylation of the acarbon (1) (Fig. 1). The initially formed a-hydroxymethyl (or alkyl, in the general case) nitrosamine is unstable and undergoes a fragmentation reaction to the diazonium ion and formaldehyde. The former reacts with any adventitious nucleophiles to give alkylation products and 1 equivalent ofmolecular nitrogen. The formation ofmolecular nitrogen in principle should provide a convenient probe of the extent of a-hydroxylation.The determination of nitrogen as a measure of a-hydroxylation has been investigated previously. The published reports, however, are dramatically contradictory. Cottrell et aL (2) reported that less than 5% of the metabolism of N-nitrosodimethylamine (DMN) by postmitochondrial rat liver homogenates (S9 fraction) led to the formation of molecular nitrogen. These workers used doubly '5N-labeled DMN. On the other hand, Milstein and Guttenplan (3) used unlabeled DMN and microsomes and found that essentially all ofthe nitrosamine was metabolized by the a-hydroxylation pathway.The extent of a-hydroxylation in the metabolism of two nitrosamines, DMN and N-nitroso-N-methylaniline (NMA), by rat S9 fraction is reported here.

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Deuterium Isotope Effect on the Carcinogenicity of Dimethylnitrosamine in Rat Liver 2

Cited by 75 publications

References 0 publications

Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1

Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

alpha-Hydroxylation pathway in the in vitro metabolism of carcinogenic nitrosamines: N-nitrosodimethylamine and N-nitroso-N-methylaniline.

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