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Rat Liver Cytosol Catalyzes a Reaction Involving Activated N-Nitrosodimethylamine and a Carbohydrate from the Pentose Phosphate Pathway James R. Reed, Michael D. Kraft, and Paul F. Hollenberg* Department of Pharmacology, Medical Science Research Building III, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0632 Received August 17, 1999
N-Nitrosodimethylamine is a liver toxin and mutagen following activation by cytochrome P450. The role of the cytosol in N-nitrosodimethylamine metabolism is not well understood. The effect of cytosol on N-nitrosodimethylamine metabolism was investigated using microsomes and cytosol from rat liver in in vitro reactions with N-nitrosodimethylamine and an NADPH generating system. Studies in which [14C]-N-nitrosodimethylamine and calf thymus DNA were used indicated that the addition of cytosol to the microsomal reaction mixture resulted in >200% enhancement of the radioactivity associated with DNA after the DNA was isolated from the reaction mixture by phenol extraction followed by ethanol precipitation. This stimulatory effect was associated with a cytosolic protein and was found to be dependent on both the microsomes and the carbohydrate used in the glucose-6-phosphate dehydrogenase system for the generation of NADPH. The carbohydrate requirement was found to be specific for intermediates of the pentose phosphate pathway, and maximum stimulation occurred with ribulose 5-phosphate. Most of the counts from [14C]-N-nitrosodimethylamine which were isolated with DNA after the addition of cytosol to reaction mixtures were not covalently bound to the DNA. HPLC analysis identified four radiolabeled metabolites derived from [14C]-N-nitrosodimethylamine following the in vitro incubations. One of the four products was formed only when both cytosol and ribulose 5-phosphate were added to the enzymatic incubations. This product also formed from [14C]-R-acetoxy nitrosodimethylamine in the absence of microsomes, only when cytosol and ribulose 5-phosphate were added to the reaction mixtures. Thus, these data demonstrate that an enzyme in the cytosol catalyzes a reaction involving a metabolite of N-nitrosodimethylamine (which is formed following cytochrome P450-mediated activation) and a carbohydrate related to the pentose phosphate pathway. A similar reaction also occurs with N-diethylnitrosamine but not with N-dipropylnitrosamine or N-dibutylnitrosamine.
Introduction N-Nitrosodimethylamine (NDMA)1 is mutagenic, carcinogenic, and hepatotoxic (1-4). NDMA requires metabolic activation, primarily by cytochrome P450 2E1, to exert these deleterious effects (5, 6). Cytochrome P450 (P450)-catalyzed activation of NDMA results in the formation of R-hydroxy NDMA, which, in turn, breaks down nonenzymatically to form the methyldiazonium ion (7). This metabolite alkylates DNA and other cellular macromolecules. The methylation of DNA by the methyldiazonium ion has been implicated as the major factor in the mutagenesis and carcinogenesis caused by NDMA (8). Various reactions catalyzed by cytosolic enzymes may modulate the simple sequence of events described above (9-12). In addition, several studies have described a poorly understood enhancement in the association of * To whom correspondence should be addressed. E-mail: phollen@ umich.edu. Fax: (734) 763-5387. Phone: (734) 764-8166. 1 Abbreviations: GNGS, glucose-6-phosphate dehydrogenase-dependent NADPH generating system; INGS, isocitrate dehydrogenasedependent NADPH generating system; NDMA, N-nitrosodimethylamine; NDEA, N-nitrosodiethylamine; NDBA, N-nitrosodibutylamine; NDPA, N-nitrosodipropylamine; PPP, pentose phosphate pathway; AANDMA, R-acetoxy NDMA.
counts from [14C]dialkylnitrosamines with calf thymus DNA following the addition of cytosol to in vitro incubations with microsomes and a source of NADPH (13, 14). Part of this enhancement is attributed to the cytosolic stimulation of P450-catalyzed N-dealkylation reactions which occurs by an unknown mechanism (15; reviewed in ref 16). The stimulation of NDMA metabolism by the cytosol does not appear to be sufficient to account for the total increase in counts from [14C]NDMA which become associated with DNA as a result of adding cytosol to the incubations (13). In contrast to these studies that implicate a role for the cytosol in the metabolism of NDMA, other investigators have not observed any effect of cytosol on the alkylation of DNA by NDMA or in enhancing the activation of NDMA (17, 18). Our lab previously studied this cytosol-related increase in counts from [14C]NDMA associated with calf thymus DNA following in vitro incubations with microsomes and an NADPH generating system (19). Four major observations were made. First, the cytosolic enhancement was dependent on microsomal activation of NDMA. Second, the effect was not due to a cytosol-related change in the Km for the metabolism of NDMA, because the cytosolic stimulation of the association of counts with DNA was constant at all NDMA concentrations that were tested,
10.1021/tx990151j CCC: $19.00 © 2000 American Chemical Society Published on Web 02/01/2000
Cytosol and N-Nitrosodimethylamine Metabolism
up to 240 µM. Third, the cytosolic effect was proteindependent. Fourth, the cytosol also exhibited this effect when added to incubations using a NADPH generating system and a purified, reconstituted system consisting of P450 2E1, P450 reductase, and dilauryl phosphatidylcholine. Thus, the cytosolic enhancement involves a metabolite of NDMA formed by P450, and this effect requires no other microsomal enzymes (19). This report presents evidence that the dramatic increase in the association of counts from [14C]NDMA with calf thymus DNA following cytosol addition to in vitro incubations is not due to covalent binding of NDMA metabolites to DNA, as was previously considered to be the case (19). Rather, this increase is due to the formation of a small (55 °C). Effect of Cytosol and Ribulose 5-Phosphate on the Metabolism of Other N-Dialkylnitrosamines. The metabolites formed from reactions with microsomes and 14C-labeled NDEA, NDPA, and NDBA in the presence and absence of ribulose 5-phosphate, cytosolic protein, and sodium pyrophosphate were examined by HPLC. There did not appear to be any hydrophilic metabolites formed from NDPA and NDBA (when each nitrosamine was tested at 1 mM) that specifically required the presence of either ribulose 5-phosphate or cytosol (data not shown). Rat liver microsomes from both phenobarbital- and pyridine-induced rats were tested with these nitrosamines. In contrast, the metabolism of NDEA by both pyridineand phenobarbital-induced rat liver microsomes did result in the formation of a product that was only observed when both ribulose 5-phosphate and cytosol were added to the incubations (Figure 3A). Higher levels of this compound were observed with the microsomes from pyridine-induced rats. This metabolite of NDEA had a slightly shorter retention time on normal phase HPLC and was formed in much lower amounts than the NDMArelated product (Figure 3B). When the two nitrosamines were compared at equal concentrations (320 µM), 602 and 95 pmol of these products were formed from NDMA and NDEA, respectively. For the metabolism of NDEA, the addition of sodium pyrophosphate buffer resulted in the formation of two additional peaks, in contrast to the one metabolite (compound 4) formed during the metabolism of NDMA.
Discussion This study provides additional information regarding a previously unknown cytosolic reaction involving an intermediate formed during the microsomal metabolism of NDMA. This reaction is unusual because it requires
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Figure 3. HPLC profiles comparing the products formed from a reaction with [14C]NDEA to those with [14C]NDMA (panels A and B, respectively). The reactions were performed with an INGS, and products were extracted and analyzed as described in Materials and Methods. For both reactions, we used the nitrosamine substrate (320 µM), 5 mM ribulose 5-phosphate, 50 M sodium pyrophosphate (pH 7.4), 0.21 mg of PEG-cytosolic protein, and microsomes from pyridine-induced rats containing 0.4 nmol of P450.
carbohydrate. Ascorbate and sucrose, which interact with a number of nitrosamines, increase the mutagenicity of some nitrosamines in Salmonella typhimurium assays (25, 26). However, the reaction described here exhibits a specific requirement for carbohydrates in the PPP. Ribulose 5-phosphate resulted in the greatest increase in the extent of the association of counts from [14C]NDMA with DNA. Because the level of activation increased with proximity of the intermediates to ribulose 5-phosphate within the pathway (Table 2), we believe that the reaction is specific for ribulose 5-phosphate. Of course, the only way to prove this assumption is to purify and characterize the catalytic properties of the cytosolic enzyme responsible for the observed effect. HPLC analysis shows that cytosol produces an additional metabolite from both AANDMA and activated NDMA in the presence of ribulose 5-phosphate (compound 3 in Figures 2 and 3). A similar reaction occurred to a lesser extent with NDEA (Figure 3) but not with NDPA and NDBA (data not shown). All of the compounds resolved by HPLC must be very polar because they were extracted with DNA. Subsequent experiments with NDMA and all of the studies with AANDMA were conducted in the absence of DNA; thus, DNA is not required for formation of these compounds in these reactions. Because of the possible contamination of DNA preparations by these metabolites, caution should be used when interpreting the results of earlier studies involving the effect of cytosol on the P450-catalyzed alkylation of DNA by NDMA (15, 16). We believe that some or all of these compounds may not have been identified in previous studies because they would not be isolated by commonly used reverse phase chromatographic techniques. The carbohydrate-dependent cytosolic reaction was dramatically enhanced by the presence of polyanions
Reed et al.
(Table 3). The addition of 50 mM sodium pyrophosphate resulted in an even larger stimulation of polar metabolites (Table 3). Tables 4 and 5 show that compounds 2 and 4 did not form in the absence of potassium phosphate and sodium pyrophosphate, respectively. Thus, the polyanion-associated increase in the production of unbound polar metabolites in the absence of cytosol is attributed to the formation of either compound 2 or 4. Furthermore, compounds 2 and 4 apparently represent the reaction of phosphate and pyrophosphate, respectively, with a metabolite of NDMA. Compound 4 was also formed from [14C]diazomethane in pyrophosphate buffer. Compounds 2 and 4 may represent the reaction of the same metabolite of NDMA with phosphate and pyrophosphate, respectively. Because NDMA degradation is irreversible, the possible NDMA degradation products that may react to form compound 4 are the methyldiazonium ion, diazomethane, and methyl carbocation. These data seem to preclude the possibility that compound 4 represents a phosphate ester of R-hydroxy NDMA (29). However, since we did not test whether diazomethane reacted with phosphate to form compound 2, we cannot rule out the possibility that compound 2 may be a phosphate ester of R-hydroxy NDMA. As alluded to in the above discussion, the ribulose 5-phosphate-dependent cytosolic reaction does not appear to be related to the other ways which have been proposed for the cytosol to participate in the metabolism of NDMA. NAD exhibited inhibitory effects on the carbohydraterelated reaction (Table 1). Thus, the alcohol dehydrogenase which was invoked to affect the mutagenicity of NDMA (11, 12) may be in competition with the enzyme related to the reaction described in this study. Similarly, as shown in the previous study from our lab (19), acetylCoA, PAPS, UDP-glucuronic acid, and glutathione either inhibited or had no effect; thus, the reaction does not appear to involve acetylation, sulfation, glucuronidation, or conjugation with glutathione. In addition, the product does not appear to be related to formaldehyde metabolism (Table 1). The catalytic reaction described here possesses a number of similarities to the nonenzymatic Maillard reaction. The latter involves the reaction of amine groups with the reducing carbons of carbohydrates and has been implicated in the deleterious effects of aging and Alzheimer’s disease pathology (30, 31). Nitrosated derivatives of this reaction have been shown to be mutagenic (32). An early product of the Maillard reaction is a Schiff base known as the Amadori compound (33). This compound is unstable at acidic and basic pH and can rearrange to ultimately form highly oxidized and reactive products. The instability of the product from the reaction described herein is consistent with it being similar to an Amadori compound. Furthermore, the Maillard reaction also has been shown to be activated by polyanions (34). As a result, we suspect that this may represent the first example of an enzymatic Maillard reaction. Work is in progress to isolate and identify the compound(s) formed from activated NDMA by the cytosol in the presence of PPP carbohydrate and phosphate. At this point, both the instability and the hydrophilic nature of the carbohydrate-related compound have complicated its isolation and analysis. We also will attempt to identify the enzyme which catalyzes this reaction.
Cytosol and N-Nitrosodimethylamine Metabolism
Acknowledgment. We thank Dr. Fred Guengerich (Vanderbilt University, Nashville, TN) for providing human liver samples. We also thank Dr. Imad Hanna and John F. Teiber for helpful discussion. This work was supported in part by U.S. Public Health Service Grants T32-ES-07062 and CA 16954.
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