Chem. Res. Toxicol. 1994, 7,9-14
9
The Effect of Cytosol on Liver Microsomal Metabolic Activation and Demethylation of N-Nitrosodimethylamine Susan S. M a t t a n o t and Paul F. Hollenberg* Department of Pharmacology, School of Medicine, Wayne State University, 540 East Canfield, Detroit, Michigan 48201 Received January 7,1993”
The role of rat liver cytosol in the demethylation and metabolic activation of N-nitrosodimethylamine (NDMA) was examined. Addition of cytosol to liver microsomes from pyridinepretreated rats enhanced DNA alkylation by NDMA 10- to 14-fold over microsomes alone, while cytosol alone had little DNA alkylating activity. The cytosolic activity responsible for the enhancement of DNA alkylation was heat labile, required NADPH, and was not a general protein effect. Addition of cytosol to purified rabbit liver cytochrome P450 2E1 in a reconstituted system consisting of NADPH-cytochrome P450 reductase, 2E1, and phospholipid produced an 18-fold increase in DNA alkylation over that observed with the reconstituted system alone. The cytosolic activity responsible for the enhancement of DNA alkylation did not work by inhibition of lipid peroxidation, nor did the addition of cytosol affect the level of NADPH present in the reaction mixtures. Attempts to identify the cytosolic component(s) responsible for the DNA alkylation enhancing activity demonstrated no evidence for the involvement of sulfhydryldependent enzymes, a flavoprotein, or conjugating enzymes. Studies with semicarbazide and phenylhydrazine suggest that carbonyl groups may be involved in the cytosolic activity. Measurements of NDMA demethylation demonstrated that cytosol addition led to a significant decrease in formaldehyde production, indicating that cytosol was not enhancing the activation of NDMA to a DNA alkylating species by facilitating the cytochrome P450-catalyzed demethylation reaction, aqd suggested that a cytosolic reaction might be occurring at the expense of formaldehyde formation.
Introduction N-Nitrosodimethylamine (NDMA),l the simplest member of the dialkylnitrosamine family, is hepatotoxic, mutagenic, and carcinogenic and requires metabolic activation to an unstable intermediate capable of alkylating cellular macromolecules (I,2). Following in vivo administration, NDMA is extensively and almost exclusively metabolized by the liver (3). The initial, rate-limiting step in NDMA bioactivation is generally accepted to be hydroxylation of the a carbon, catalyzed by cytochrome P450 2E1 (P450 2E1) (2,4,5). Subsequent demethylation results in the generation of formaldehyde and dinitrogen gas and produces the methylating species which alkylates DNA and proteins. Formaldehyde production has often been used as a measure of metabolic activation of NDMA, but while some investigators have found a good correlation (6-8),others have not (9-13). Denitrosation, a detoxification pathway for NDMA, also appears to be catalyzed by P450 2E1 and was estimated to account for 14-20% of the elimination of NDMA in Fischer rats in vivo (14-16).
* To whom correspondence should be addressed. Phone: (313)5771580; Fax: (313)577-6739. t Presentaddress: Genetic Toxicology, Co., 301 Henrietta -. The Upjohn .. St., Kalamazoo, MI 49001. 0 Abstract published in Advance ACS Abstracts, November 15,1993. 1 Abbreviations: NDMA, N-nitrosodimethylamine; NDEA, N-nitrosodiethylamine;NMEHA, N-nitrosomethyl(2-hydroxyethy1)amine; P450, cytochrome P450; S9, liver postmitochondrial fraction; GGPD, glucoseBphosphatadehydrogenase;Os-MeG,Os-methylguanine;SOD,superoxide dismutase; IAA, iodoacetamide; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); PAPS, 3’-phoephoadenosine 5’-phosphosulfae TE, Tris-EDTA buffer; UDPGA, uridine 5’-diphosphoglucuronic acid; AcCoA, acetyl coenzyme A; M A , rat serum albumin; DLPC, dilauroylphosphatidylcholine;TCA, trichloroacetic acid;SDS, sodium dodecyl sulfate;S9, liver postmitochondrial fraction.
The denitrosation reaction products are monomethylamine, nitrite, and formaldehyde, underscoring the difficulty of using formaldehyde production strictly as a measure of NDMA metabolic activation. In studies of NDMA metabolism and metabolic activation, a controversy exists over a role for additional, cytosolic activities. For example, Lai et al. (17) reported a 2.2-fold increase in DNA alkylation by NDMA when cytosol was added to microsomal incubation mixtures. Prival and Mitchell found that cytosol was required for mutagenesis by NDMA in a Salmonella typhimurium plate incorporation assay and that the cytosolic activity was trypsin sensitive and not dialyzable (9). Yo0 and Yang, on the other hand, reported that microsomal and liver postmitochondrial (S9) fractions were equally effective in producing NDMA-induced mutation when added to Chinese hamster V79 cells (6). In addition, Hong and Yang saw no effect of added cytosol on either NDMA demethylation by acetone-induced microsomes (measured by formaldehyde production), or on the levels of 06methylguanine (06-MeG)formation in DNA (7).Recently, Guttenplan reported microsomal-mediated NDMA mutagenesis in S. typhimurium strains TAlOO and TA104 to be differentially affected by the addition of cytosol, suggesting that a cytosolic reaction may lead to the production of a metabolic intermediate, and subsequent DNA alkylation product, different from that produced by microsomes alone (18). In the present work, we report further studies of the effects of cytosol on DNA alkylation by NDMA and demethylation of NDMA as well as investigations aimed at characterization of the nature of the cytosolic activity involved in stimulating the metabolic activation of NDMA.
O893-22S~/94/27O7-OOO9~Q4.5QlQ 0 1994 American Chemical Society
10 Chem. Res. Toxicol., Vol. 7, No. 1, 1994
Materials and Methods Caution: NDMA is a carcinogen and should be handled and disposed of according t o NIH guidelines (19). Chemicals. NDMA, NADP, glucose 6-phosphate (GGPD), and calf thymus DNA were purchased from the Sigma Chemical Co. (St.Louis, MO). [14C]NDMA(17.1 mCi/mmol) was obtained from the Amersham Corp. (Arlington Heights, IL). All other chemicals and solvents used were of the highest purity available from commercial suppliers. Enzyme Sources. Male Fischer 344 rats (86-140 g) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). All animals were fed a standard rat chow diet and water ad libitum. Pyridine-pretreated rata were given 150 mg of pyridine/kg body weight/day (ip) for 3 days prior to isolation of microsomes. Acetone-pretreated rats were given 1% acetone in the drinking water for 12-14 days. Phenobarbital (0.1% ) was administered in the drinking water for 8days. All animals were fasted overnight and sacrificed on the following day. Microsomes were prepared from the livers of the F344 rats according to the procedure described by Coon et al. (20). Microsomes were resuspended in 100 mM potassium phosphate buffer (pH 7.4) containing 1mM EDTA and 10% glycerol. The P450 contents of the microsomes were determined from the reduced CO difference spectra (21). Protein was determined using the method of Lowry et al. (22). Bovine serum albumin was used to produce standard curves. Cytochrome P450 contents were as follows: microsomes from untreated animals, 0.84 nmol of P450/mg of protein; microsomes from acetone-pretreated rats, 0.97 nmol of P450/mg of protein; microsomes from pyridine-pretreated rats, 1.88 nmol of P450/ mg of protein; and microsomes from phenobarbital-pretreated induced rata, 2.67 nmol of P450/mg of protein. Rat NADPHcytochrome P450 reductase and rabbit cytochromes P450 2B4 and P450 1A2 were purified as previously described (20,23,24). Rabbit cytochrome P450 2E1 was a gift from Professor M. J. Coon, (University of Michigan, Ann Arbor, MI). The cytosolic fraction was obtained by collecting the supernatant from the centrifugation of liver homogenate5 from untreated male F344 rats for 75 min a t 105000g. The cytosol was filtered through cheesecloth to remove particulate matter. Unless otherwise indicated, a volume of 10 pL of cytosol was routinely added to the assay systems. NDMA Demethylation Assay. The demethylation of NDMA was determined by measuring formaldehyde formation using the method of Nash (25). Reaction mixtures (0.5 mL) contained 50 mM Tris buffer (pH 7.3), 150 mM KCl, 10 mM MgC12,and a NADPH-regenerating system consisting of 10 mM glucose 6-phosphate, 0.4 mM NADP, and 0.75 unit of G6PD [lyophilized, resuspended in 5 mM citrate buffer (pH 7.5) to a concentration of 0.75 unit/mL], Reaction mixtures were incubated in capped polypropylene microcentrifuge tubes a t 37 "C, and the reactions were stopped by the addition of 100 pL of 60% trichloroacetic acid (TCA). Unless indicated otherwise, incubations were for 30 min. Under the conditionsused, formaldehyde formation was linear with time for a t least 45 min. The protein was precipitated on ice and pelleted by centrifugation a t 12000g for 1 min. For formaldehyde determination, 408 pL of the supernatant was added to 157 pL of concentrated Nash reagent (15g of ammonium acetate and 210 pL of acetylacetone in 20 mL of 3 % acetic acid) and incubated a t 50 "C for 30 min. The absorbance of the samples was measured a t 412 nm, and the quantity of formaldehyde formed was determined by comparison with standards prepared with identical amounts of microsomes and/or cytosol without the NADPH-generatingsystem or without DMN. DNA Alkylation by NDMA. The alkylation of DNA by NDMA was determined by incubating microsomes or purified enzymes and [WINDMA with 12 pg of calf thymus DNA in a total volume of 120 pL containing 50 mM Tris (pH 7.3), 150 mM KCl, 10 mM MgCls, and the NADPH-generatingsystem described above in capped polypropylene microfuge tubes a t 37 "C. Reactions were terminated by the addition of 6 pL of 10 % sodium
Mattano and Hollenberg Table 1. Effect of Cytosol on Alkylation of DNA by NDMA in Vitrd addition
-NADPH
+NADPH
+NADPH +1%ethanol
microsomes 0.019 f 0.002 0.163 f 0.029 0.029 f 0.004 cytosolic protein 0.020 f 0.005 0.380 f 0.063 0.348 f 0.024 microsomes + cytosol 0.029 f 0.009 2.30 f 0.24 0.380 f 0.058 =Activities are expressed as pmol of [l4C1 bound per minute. Values represent mean f SD of duplicate samples from 3 experiments. Samples contained 0.1 mM [WINDMA, 12 pg of calf thymus DNA, and 0.4 nmol of microsomal P450 (0.25 mg of protein) from pyridinepretreated rats, in a total volume of 120 pL. Cytosol (0.21 mg) was added when indicated. Incubations were performed and labeling was determined as described in Materials and Methods. dodecyl sulfate (SDS) and 1pL of 500 mM EDTA (pH 8). Unless indicated otherwise, incubations were for 30 min. Reactions were linear with time for a t least 45 min. DNA was extracted with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1, pH 8). The phenol mixture was re-extracted with an equal volume of Tris-EDTA buffer (TE) consisting of 10 mM Tris (pH 7.5) and 1 mM EDTA, and the DNA was precipitated from the combined aqueous extracts with 150 mM NaCl and 400 mM sodium acetate (pH 5.5) a t 70 "C for 20 min followed by centrifugation for 15 min a t 12000g. The DNA pellet was washed once with 70 % ethanol, resuspended in 50pL of TE, and subjected to liquid scinillation counting in 5 mL of Safety-Solve (Research Products International Corp., Mount Prospect, IL).
Determination of GGPD Activity and NADPH Levels. G6PD activity of cytosol was determined by measuring NADP reduction a t 340 nm using 0.15 mM NADP and 2.5 mM glucose 6-phosphate. Commercial G6PD was used for the assay control. NADPH levels were determined by measuring the absorbance a t 340 nm using an extinction coefficient of 6.2 mmol-1 cm-l.
Results Effect of Liver Cytosol on DNA Alkylation by NDMA. The addition of liver cytosol from untreated rats to microsomes from pyridine-pretreated rats increased the alkylation of DNA by NDMA 14-fold over that seen with microsomes alone (Table 1). This effect was dependent on the presence of NADPH and was inhibited by the addition of 1% ethanol, a concentration sufficient to inhibit P450 2E1activity by approximately 95 % (data not shown). Cytosol addition increased the velocity of the microsomal NDMA DNA alkylation reaction at all NDMA concentrations examined (6.25-225 pM, Figure 1). Although it is not apparent in Figure 1because of the compression of the scale at lower concentrations of NDMA, the ratio of DNA alkylation in the presence of cytosol to that in the absence ranged from approximately 10 (at 6.25pM NDMA) to 7 (at 225 pM NDMA). Therefore, the increase in DNA alkylation in the presence of cytosol does not appear to be due to a change in the K, for DNA alkylation. As shown in Table I, cytosol alone showed some ability to activate NDMA. This activity was NADPH-dependent and was not inhibited with 1% ethanol. No DNA alkylation activity was observed when NADH (1mM) was substituted for NADPH, either with cytosol alone or in the combined microsome/cytosolsystem (data not shown). G6PD activity in the cytosol was determined to be 0.4 milliunit/pL, corresponding to an addition of 4 milliunits of G6PD per reaction mixture containing cytosol. Incubation of the microsomes alone with 5 or 10units of GGPD in the NADPH-regenerating system did not enhance the alkylation of DNA by NDMA over that observed with the
Chem. Res. Toxicol., Vol. 7, No. 1, 1994 11
Cytosolic Stimulation of Microsomal NDMA Activation
B l21
T I
Table 3. DNA Alkylation by NDMA Catalyzed by Liver Microsomes from Rats Treated with Different Inducing Agents with a n d without Cytosol. pmol of [14Cl bound/ (nmol of P450.min) 0.63 k 0.09 2.43 f 0.18 0.39 0.01 3.85 f 0.26 0.81 f 0.06 4.10 f 0.40 0.14 0.02 1.50 f 0.11
treatment untreated control +cytosol pyridine +cytosol acetone +cytosol phenobarbital +cytosol -
0
40
120
80
160
200
240
NDMA (pM)
Figure 1. Initial velocity pattern for DNA alkylation by NDMA catalyzed by microsomes. Samples contained microsomes from pyridine-pretreated rats (0.4 nmol of P450), 12 pg of calf thymus DNA, an NADPH-generating system, and no further additions (0) or 0.21 mg of cytosolic protein (0) in a total volume of 120 pL. Incubations were performed and labeling was determined as described in Materials and Methods. Numbers represent the mean SD of duplicate samples from 3 experiments.
*
Table 2. Effect of Boiled Cytosol a n d Rat Serum Albumin on DNA Alkylation by NDMA. addition microsomes Cytosolic protein microsomes + cytosol microsomes + RSA microsomes + boiled cytosol ~
Dmol of P4Cl bound/min 0.35 f 0.06 0.09 0.01 1.83 f 0.26 0.53 f 0.07 0.69 k 0.18
a Samples contained 0.1 mM [WINDMA, 12 pg of calf thymus DNA, 0.4 nmol of microsomal P450 (0.25 mg of protein) and 0.21 mg of cytosolic protein or rat serum albumin (RSA) when applicable, and an NADPH-generating system in a total volume of 120 pL. Incubationswere performed and labeling was determined as described in Materials and Methods. Values represent the mean f SD of duplicate samples from 3 experiments.
0.75 unit routinely used (data not shown). The addition of cytosol to the microsomal system did not enhance the levels of NADPH in the assay system (data not shown). The specificity of the cytosolic enhancement of DNA alkylation by NDMA catalyzed by microsomes from pyridine-pretreated rata was examined (Table 2). Addition of an equal amount of rat serum albumin, on a milligram protein basis, did not enhance the DNA alkylating activity of the microsomes. In addition, boiling the cytosol abolished the enhancing activity. The molecular weight of the cytosolic component(s) responsible for enhancing DNA alkylation was shown to be greater than 30 000 since essentially all activity was retained after filtration through a Centricon 30 microfiltration device (data not shown). The alkylation of DNA by NDMA catalyzed by liver microsomes from rats pretreated with pyridine, acetone, or phenobarbital or from untreated rats was compared with and without added cytosol from untreated rats (Table 3). In the absence of cytosol, the greatest amount of DNA alkylation was observed with microsomes from acetonepretreated rats followed by microsomes from untreated rats, microsomes from pyridine-pretreated rats, and microsomesfrom phenobarbital-pretreated rats. Stimulation of alkylation by cytosol addition was seen in all cases, with nearly 4- and 5-fold enhancements of DNA alkylation with microsomes from untreated and acetone-pretreated rats, respectively, and 10- and 11-fold enhancements with microsomes f r o m pyridine- and phenobarbital-pretreated rats, respectively. Addition of cytosol to purified rabbit
*
a Samples contained 0.033 mM [''CINDMA, 12 pg of calf thymus DNA, 0.4 nmol of microsomal P450 corresponding to 0.25 mg of protein for microsomes from pyridine-treated rats, 0.40 mg of protein for microsomes from acetone-treated rata, 0.14 mg of protein for microsomes from phenobarbital-treated rats, 0.48 mg of protein for microsomes from untreated rats, or 0.21 mg of cytosolic protein from untreated rats when applicable, and an NADPH- generating system in a total volume of 120 pL. In each case, the total amount of P450 was 0.4 nmol. Incubations were performed and labeling was determined as described in Materials and Methods. Values represent means i SD of duplicate samples from 3 experiments.
Table 4. Effects of Cytosol on DNA Alkylation by NDMA Catalyzed by Purified Forms of P450. addition
pmol of P C I bound/ (nmol of P450,min)
rabbit liver P450 2El +cytosolic protein +RSA rabbit liver P450 2B4 +cytosolic protein rabbit liver P450 1A2 +cytosolic protein
0.41 0.11 7.49 f 0.66 0.71 f 0.12 NDb 0.67 f 0.17 ND 0.49 k 0.06
a Samples contained 0.1 mM [WINDMA, 12 pg of calf thymus DNA, 0.1 nmol of purified P450 protein, 0.2 nmol of P450 reductaae, 30 pg of DLPC, the NADPH-generating system, and 0.21 mg of cytosolic protein or RSA where applicable, in a total volume of 120 pL. Incubations were performed and labeling was determined as described in Materials and Methods. Values represent means h SD of triplicate samples from 2 experiments. ND, not determined.
Table 5. Effect of Liver Cytosol on NDMA Demethylation by Microsomes from Pyridine-Treated Rats. cytosolic protein added (mg)
microsomal protein added (mg)
nmol of HCHO producedlmin
0.25 0.25 0.25 0.25
0.60 0.30 0.50 f 0.15 1.13 & 0.18 2.64 f 0.15 2.96 i 0.55 1.84 0.12 1.90 0.26
0.2 1.0 2.0 0.2 1.0 2.0
*
Samples contained 0.07 mM NDMA and an NADPH-generating system in a total volume of 0.5 mL. Incubations were performed and formaldehyde was measured as described in Materials and Methods. Numbers represent mean SD of triplicate samples from 2 experiments.
*
liver P450 2E1 enhanced DNA alkylation 18-fold (Table 41, and this enhancement was not observed with addition of equal amounts of rat serum albumin. The combination of cytosol and purified P450 2B4 or P450 1A2 gave low levels of DNA alkylation by NDMA. Effect of Cytosol on Demethylation of NDMA. Demethylation of NDMA by microsomes from pyridinepretreated rats, cytosol alone, or the two in combination was examined (Table 5). Some demethylation activity was seen with cytosol alone, particularly with cytosol concentrations 10-fold greater than that used in the DNA
12 Chem. Res. Toxicol., Vol. 7, No. 1, 1994
Mattano and Hollenberg
Table 6. Effect of Inhibitors of Lipid Peroxidation on DNA Alkylation by NDMA Catalyzed by Microsomes from Pyridine-Treated Rats. addition pmol of [1*Cl bound/min microsomes 0.23 f 0.03 +cytosolic protein (0.21 mg) 1.98 f 0.24 0.22 f 0.04 +catalase (1pg) 0.29 f 0.04 +SOD (80 units) 0.23 f 0.01 +SOD (80 units) and catalase (1pg) 0.21 f 0.03 +desferoxamine (0.5 mM) +EDTA (1.0 mM) 0.24 f 0.03 a Reaction mixtures consisted of 0.1 mM [14C]NDMA,0.4 nmol of microsomal P450 (0.25 mg of protein), 12 pg of calf thymus DNA, and an NADPH-generating system in a total volume of 120 pL. Incubations were performed and the labeling was determined as described in Materials and Methods. Values represent mean f SD of duplicate samples from 2 experiments.
Table 7. Effects of Various Agents on the Alkylation of DNA by NDMA in the Presence of Microsomes, Cytosol, or Both.
treatment
cytosol alone
none IAAb (1 mM) DTNB (0.1 mM) phenylhydrazine (10 mM) semicarbazide (10 mM) paraquat (0.1 mM) quinacrine (0.1 mM) FAD (0.1 mM) FMN (0.1 mM) PAPS (0.5 mM) AcCoA (1mM) UDPGA (3 mM) GSH (5 mM) GSH (10 mM)
100 NDc ND ND 9.8 100 ND 107 94.0 ND ND ND 77.0 60.0
% of control microsomes cytosol + alone microsomes
100 ND ND ND 29.7 89.0 ND 158 146 96.6 ND 74.6 90.0 51.5
100 116 112 1.8 3.9 89.1 94.6 81.9 73.8 94.9 97.3 48.0 64.7 21.5
0 Samples contained 0.1 mM [WINDMA, 12 pg of calf thymus DNA, 0.4 nmol of microsomal P450 (0.25 mg of protein) and/or 0.21 mg of cytosolicprotein, and an NADPH-generating system in a total volume of 120pL. Incubations were performed and the labeling was determined as described in Materials and Methods. Values represent the average of least duplicate samples from 2 experiments (n 1 4). b Abbrevations: IAA,iodoacetic acid; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide;PAPS, 3’-phosphoadenosine5‘phosphosulfate; AcCoA, acetyl coenzyme A; and UDPGA, uridine 5’-diphosphoglucuronicacid. ND, not determined.
alkylation studies described above. However, in combination with microsomes there was no enhancement of demethylation. In fact, there was a slight decrease in demethylation with increasing cytosol concentrations. Studies on the Mechanism of Cytosolic Enhancement of DNA Alkylation by NDMA. To establish whether the cytosolic stimulation of microsomal activity could be due to suppression of activated oxygen formation and/or lipid peroxidation, the effect of adding cytosol was compared with that resulting from adding catalase, superoxide dismutase (SOD), desferoxamine mesylate, or EDTA to microsomes from pyridine-pretreated rats (Table 6). None of these treatments were able tomimic the effect of added cytosol. A variety of enzyme inhibitors and cofactors were examined in attempts to identify the factor responsible for the cytosolic enhancement of DNA alkylation by NDMA (Table 7). The cytosolic stimulatory “activity” exhibited no requirement for sulfhydryl groups, as demonstrated by the lack of inhibition by iodoacetic acid (IAA) and 5,5’-dithiobis(2-nitrobenzoicacid) (DTNB). Carbonyl groups may be involved in the cytosolic activity, as
suggested by the inhibition with semicarbazide and by the inhibition of the combined microsomal and cytosolic activity with semicarbazide or phenylhydrazine. The addition of the electron donor paraquat did not affect the cytosolicactivity or the combined cytosolicand microsomal activity. Quinacrine, an inhibitor of several flavoenzymes, did not inhibit the combined activity. The addition of flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) did not have any significant effect on the combined system even though they both showed marked stimulation of the microsomes alone. Addition of the cofactors 3’-phosphoadenosine 5’-phosphosulfate (PAPS) for sulfation or acetylcoenzyme A (AcCoA)for acetylation activities also had no effect. Uridine 5’-diphosphoglucuronic acid (UDPGA) inhibited alkylation of DNA by NDMA in the combined system by 52%, suggesting that one of the metabolites may form a glucuronide conjugate. The addition of glutathione inhibited both the cytosolic activity and the activity of the combined system, possibly through trapping of the reactive intermediate@).
Discussion Our studies support previous findings of a role for extramicrosomal factors in the metabolic activation of NDMA. We have observed a heat-labile activity present in liver cytosol that enhances DNA alkylation by NDMA catalyzed by microsomes from pyridine-treated rats by 10-to 14-fold (Tables 1and 3) and that enhances alkylation catalyzed by purified rabbit liver P450 2E1 in a reconstituted system by 18-fold (Table 4). While the role of hepatic cytosolic reactions in the metabolism and activation of NDMA in vivo is not clear, reports of differences in the disposition of deuterated NDMA in microsomes and in vivo could reflect a significant contribution of extramicrosomal metabolism (15, 16). Denitrosation, a detoxification reaction, has been estimated to account for 14-20% of the elimination of NDMA in rats in vivo (16), similar to the estimate of 9-15 % obtained with microsomes from acetone-treated rats (14). When deuterated NDMA was administered in vivo, a shift in metabolism to 4048% denitrosation occurred (16), while the balance of demethylation and denitrosation was not affected in microsomes from acetone-treated rats (15). This could be consistent with an isotope effect on a cytosolic NDMA activation reaction, since a decreased incidence of hepatic tumors was seen following administration of deuterated NDMA in comparison with the nondeuterated compound (26).
The metabolic activation of NDMA is believed to involve an initial P450-catalyzed hydroxylation to give the a-hydroxynitrosamine, which has a half-life at physiological pH of approximately 1min. However, this intermediate is much more stable in organic solvents such as methylene chloride and is presumed to be more stable in lipophilic environments such as those provided by some proteins. Decomposition of the a-hydroxynitrosamine yields the N-hydroxydiazine or diazohydroxide. This intermediate ionizes rapidly to form the methanediazonium ion, which then alkylates DNA. Under physiological conditions the half-life of the methanediazonium ion, which decomposes by a very facile s N 2 reaction, is about 350 ms. Due to their lack of stability, none of the above-mentioned intermediates (thea-hydroxynitrosamine, the diazohydroxide,and the methanediazonium ion) have been isolated from i n vitro metabolism experiments. However, their existence
Cytosolic Stimulation of Microsomal N D M A Activation
as intermediates is supported by a variety of different types of evidence including isotope effect studies and the identity of the products generated as a result of metabolism of NDMA. Thus, a factor which could stabilize any of the intermediates might be expected to result in an increase in the amount of DNA alkylation. There have been several reports of NDMA demethylase activity in hepatic cytosol, and this activity was suggested to account for cytosoliccontributions to NDMA activation (27-29). Hong and Yang, however, found cytosol to contribute little to the generation of formaldehyde from NDMA, or to its activation (7). Our studies did not show a good correlation between the amount of enhancement of NDMA metabolic activation by added cytosol and formaldehyde formation (Table 5). With increased amounts of added cytosol,we saw decreased formaldehyde production, consistent with the observations of Hong and Yang (7). There is a precedent for the involvement of cytosolic proteins in enhancing reactions catalyzed by cytochrome P450, with proposed mechanisms including suppression of lipid peroxidation or facilitation of the P450 reactions through enhancing substrate binding, participating in electron transfer, or stabilizing components of the P450 complex (30-33). We have found no evidence for a role for lipid peroxidation (Table 7). In addition, if the cytosolic activity facilitated the P450 reaction, formaldehyde production would be expected to increase along with DNA alkylation activity, as was clearly not the case. In this system, it appears that the cytosolic activity is further metabolizing a product of the P450 2E1 reaction, since little DNA alkylation activity was observed with cytosol alone (Table 11, inhibitors of P450 2E1 inhibited the cytosolic stimulation of DNA binding (Tables 1 and 7), and relatively little alkylation of DNA was observed with P450 isoforms lacking NDMA substrate specificity (34)even in the presence of cytosol (Table 4). Thus, we may speculate that a-hydroxyl NDMA could be the substrate for a cytosolic activity which converts it to a more reactive species while diverting it away from formaldehyde formation. Several different cytosolic enzyme systems have been suggested to be involved in the metabolic activation of a-hydroxylated nitrosamines. Kroeger-Koepke et al. have identified formation of sulfate conjugates of N-nitrosomethyl(2-hydroxyethy1)amine (NMEHA) in vivo and have proposed that sulfation is the activating step in NMEHA carcinogenicity (35). Their studies with NDMA, however, showed no effect of inhibitors of sulfation on NDMA 7-MeG or 06-MeGadduct formation (35),and we observed no effect of PAPS addition on NDMA DNA alkylation by microsomes and cytosol in the present studies (Table 7). Similarly, Wiench et al. have proposed glucuronidation of N-nitrosomethyl-n-pentylamine to be a step in its metabolic activation and to account for extrahepatic carcinogenicity of the more complex nitrosamines, since the glucuronide conjugates could be transported to other organs where cleavage by glucuronidase could free the reactive nitrosamines (36). While the more lipophilic, longer chain nitrosamines were more extensively conjugated, they found relatively low levels of glucuronic acid conjugate formation in vivo with NDMA and NDEA. In our studies, addition of UDPGA inhibited NDMA DNA alkylation activity in the presence of cytosol by 52 % (Table 7), indicating that glucuronide conjugation of NDMA can occur in vitro, but the conjugate has decreased reactivity
Chem. Res. Toxicol., Vol. 7,No. 1, 1994 13
with DNA in this system. Yamazaki e t a1 demonstrated increased mutagenicity of NDMA in a bacterial system expressinghigh O-acetyltransferaseactivity in the presence of microsomes from isoniazid-treated rats, and with purified rat P450 2E1 (37). They suggest the alkanediazohydroxide formed by tautomerization of the a-hydroxynitrosamine could be a substrate for O-acetylation. Loss of the acetyl group would produce the reactive diazonium ion which alkylates DNA. In our system, the addition of 1mM AcCoA did not affect NDMA alkylation, and more detailed studies will be necessary to investigate the role of O-acetyltransferase activity in our system. In studies of NDMA activation to a mutagen in S. typhimurium, Guttenplan found evidence that adding cytosol to microsomes resulted in production of a different reactive intermediate than microsomes alone (17). With microsomes alone, NDMA was more mutagenic in S. typhimurium strain TAlOO than TA104. With the S9 fraction, however, NDMA showed increased mutagenicity in TA104 but not in TA100. Since TA104 detects mutagenesis at AT base pairs, this could account for the discrepancy between his study and that of Hong and Yang, in which no enhancing effect of cytosol was seen on the formation of 06-MeG adducts by NDMA (7). Our assay detects all DNA alkylation products formed, and our observations are consistent with Guttenplan’s conclusions. We have not yet determined whether cytosol addition affects the DNA alkylation spectrum in our system. In subsequent studies, Guttenplan reported that cytosol in conjuction with microsomes generated an NDMA metabolite with a different mutational specificity in TA104 than the methanediazonium ion produced by spontaneous decomposition of a-OH-NDMA (38). He proposed the reactive metabolite to be N-nitroso-N-methylformamide, formed by oxidation of a-OH-NDMA by a cytosolic alcohol dehydrogenase, because NAD, a cofactor for alcohol dehydrogenases, enhanced the mutagenic activity of the cytosolic fraction. Elespuru and Lijinsky have recently to be more mureported N-nitroso-N-methylformamide tagenic than NDMA and to yield similar mutation frequency ratios at several loci compared with NDMA (39),as may be expected of a proximate mutagen. In our studies, the carbonyl reagents phenylhydrazine and semicarbazide inhibited DNA alkylation extensively (Table 7). While semicarbazide is a competitive inhibitor of P450 2E1 (40), DNA alkylation by microsomes alone was inhibited 70%, compared with 96% inhibition seen with microsomes plus cytosol, suggesting a carbonyl derivative may be involved in the cytosolic activity observed here. Further studies will be necessary to establish a role for an a-carbonyl derivative in the mutagenicity of NDMA, and what its biological significance may be. These questions will be important in furthering our understanding of the metabolic activation of NDMA and its carcinogenicity.
Acknowledgment. This work was supported in part by NCI Grant CA 16954. The authors would like to thank Professor M. J. Coon for providing purified rabbit cytochrome P450 2E1, Dave Putt for preparation of purified P450 enzymes, reductase, and microsomes and for many helpful discussions, and Jan Crowley for valuable discussions.
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