Chem. Res. Toxicol. 1989, 2, 436-441
436
Metabolism of N-Nitrosodimethyl- and N-Nitrosodiethylamine by Rat Hepatocytes: Effects of Pretreatment with Ethanol Lee D. Gorskyt and Paul F. Hollenberg* Departments of Pathology and Molecular Biology and Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, and Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201 Received December 14, 1988
Hepatocytes were isolated from the livers of ethanol-pretreated rats, and the relationship between the generation of COP and the loss of N-nitrosodimethylamine (NDMA) and Nnitrosodiethylamine (NDEA) from the incubation mixtures was examined. The evolution of COPby hepatocytes isolatad from untreated, control rats was compared with the evolution of COPby hepatocytes isolated from rats treated with 10% EtOH in their drinking water. The C 0 2generated from either NDMA or NDEA represented only a fraction of the parent compound that was metabolized during the incubation period. Therefore, the measurement of C02evolution as an indication of the metabolism of these simple dialkylnitrosamines is inadequate, and the actual loss of the parent compound must be measured directly when utilizing isolated hepatocytes as a model system to study the metabolism of nitrosamines. The liver microsomal metabolism of NDMA and NDEA was also examined. Pretreatment of the rats with ethanol resulted in a marked increase in the microsomal metabolism of NDMA but had a relatively small effect on NDEA metabolism. Phenobarbital pretreatment did not result in any increase in NDMA metabolism whereas there was a very significant (&fold) increase in NDEA metabolism. These results suggest that different isozymes of cytochrome P-450 may be primarily responsible for the metabolism of the two nitrosamines. The inhibition patterns observed when an antibody inhibitory to cytochrome P-45Oj was added to microsomes derived from control and ethanoland phenobarbital-pretreated rats conclusively demonstrate that NDMA and NDEA are preferentially metabolized by distinct isozymes of cytochrome P-450.
Introduction Suspensions of isolated hepatocytes are being utilized more and more frequently in order to relate studies on the metabolism of chemical carcinogens and other xenobiotics using isolated subcellular fractions (e.g., S-9, microsomes, etc.) with whole animal studies. Since hepatocytes retain essentially all of their structural and enzymic integrity upon isolation, it is possible for the substrates to be completely metabolized to the level of C02. Thus, numerous investigators have used the evolution of C 0 2 as a measure of drug metabolism by hepatocytes (1-5). One aim of this study was to examine the relationship between C 0 2 evolution and the metabolism of NDMA' or NDEA in order to determine the usefulness of measuring C 0 2 evolution as an indicator of the metabolism of short chain dialkylnitrosamines. Since EtOH administration increases the rate of NDMA demethylation (6-lo), we have used hepatocytes isolated from EtOH-pretreated rats as well as hepatocytes isolated from untreated animals. EtOH increases the concentration of a specific isozyme of cytochrome P-450 in rabbit, rat, and man (10-12). This isozyme of P-450 has been shown to be induced by a wide variety of compounds and to be very effective in catalyzing the demethylation of NDMA (7-9,131. The metabolism of NDEA, on the other hand, has not been studied in great detail. Since NDMA and NDEA differ chemically only by the addition of a meth~
~
ylene group to each of the alkyl side chains of the nitrosamine, it might be expected that the metabolism of these two compounds would be very similar. In the present study, the evolution of C 0 2 from NDMA and NDEA as a result of metabolism by hepatocytes isolated from control and EtOH-pretreated rats has been determined and compared with substrate utilization. The microsomal metabolism of NDMA and NDEA has also been determined in hepatic microsomes isolated from control and EtOH-pretreated rats to examine differences that may exist in the metabolic fates of these two very closely chemically related environmental carcinogens. The present study demonstrates that (1) EtOH pretreatment differentially affects the metabolism of NDMA and NDEA by microsomes and by suspensions of freshly isolated hepatocytes and (2) the evolution of C 0 2 is not an accurate measure of the rate of metabolism of the parent nitrosamine. Materials and Methods Materials. [14C]NDMA (54 mCi/mmol) and [14C]ureawere purchased from New England Nuclear (Boston, MA). ['*C]NDEA (57 mCi/mmol) and NaH[14C]03were purchased from Amenham (Chicago, IL). Unlabeled NDMA and NDEA were from Aldrich (Milwaukee, WI). Collagenase was purchased from Cooper Biomedical (Freehold, NJ). All other chemicals and solvents used were of the highest purity available from commercial suppliers. Animals. Male Fischer 344 rats (150-200 g) were obtained from Harlan Industries, Indianapolis, IN. All animals were fed
~~
*To whom correspondence should be addressed at the Department of Pharmacology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. 'Present address: Abbott Laboratories, Abbott Park, IL 60064. 0893-228~ I89 /2702-0436$01.50 I O
Abbreviations: NDMA, N-nitrosodimethylamine (dimethylnitrosamine);NDEA, N-nitrosodiethylamine (diethylnitrosamine);Pb, phenobarbital; HPLC,high-performance liquid chromatography. 0 1989 American Chemical Societv
Nitrosamine Metabolism by Isolated Hepatocytes a standard rat chow diet and water ad libitum. EtOH-pretreated rats were given 10% EtOH in their drinking water for 2-3 weeks prior to the isolation of hepatocytes. Preparation of Hepatocytes. Hepatocytes were isolated by the two-step Seglen modification (14) of the Berry and Friend method (15). Pentobarbital (0.65 mg/g body weight) was used as the anesthetic. Pentobarbital was chosen as the anesthetic rather than ether because ether has been shown to strongly inhibit the metabolism of NDMA in F344 rats (16) and this inhibition was observed for up to 3 h after the initial exposure. Spiegelhandler et al. (I 7) have also reported the inhibitory effect of ether anesthesia on the metabolism of NDMA and N-nitrosomorpholine. On the other hand, when pentobarbital was used as the anesthetic in F344 rats, the activities of several Phase I and Phase I1 enzymes were not significantly different in the liver when compared just prior to perfusion, in freshly dissociated hepatocytes, or in hepatocytes after 2 h in culture (18). Preparation of Microsomes. Microsomes were prepared from the livers of F344 rats according to the procedure described by Coon et al. (19). Control microsomes were prepared from untreated animals. Microsomes from ethanol-treated animals were prepared from rats given 10% EtOH in their drinking water for 2 weeks, and microsomes from phenobarbital-treated animals were prepared from rats that received 0.1% Pb in their drinking water for 1 week. All animals received rat chow ad libitum. Preparation of Antibody. Antibody to the EtOH-inducible rabbit liver cytochrome P-450 isozyme 3a was prepared as described (20) and kindly provided by Dr. Dennis R. Koop of Case-Western Reserve University. Measurement of COz Evolution. The production of 14C02 was measured by using the method of Hauber et al. ( 4 ) with slight modification. The hepatocytes were placed into scintillation vials, and the vials were sealed with caps containing a Whatman glass fiber filter that had been treated with 175 pL of 5 N NaOH. At the appropriate times, perchloric acid (0.2 mM final concentration) was added and the caps were quickly replaced. The vials were then left for 2 h at 25 "C, after which the filters were removed and dried overnight at 50 "C to remove volatile compounds, leaving the 14C02bound to the filter as Na214C03.After drying, the filters were placed in 2 mL of water to dissolved the Na24C03, 10 mL of scintillation fluid was added, and the amount of radioactivity bound to the filter was determined by liquid scintillation counting. Metabolism of NDMA and NDEA by Hepatocytes. The loss of NDMA or NDEA from the incubation medium was measured by using reverse-phase C18 HPLC. The isolated hepatocytes (4 x lo6) were incubated in 1mL of Hams F10 medium with 25 mM Hepes, pH 7.4, with shaking at 37 OC. The reactions were initiated by the addition of either NDMA or NDEA. After termintion of the reaction with perchloric acid (100 p L of 0.6 N) and addition of the internal standard (p-nitroanisole, 25 nmol), the incubation mixture was centrifuged for 5 min at 15000g. The supernatant, containing unmetabolized parent compound and acid-soluble metabolites, was filtered through a 0.2-pm Acro LC 13 filter (Gelman), and an aliquot of the filtrate (80 pL) was analyzed by HPLC. The reaction mixtures were separated on an Altex 5-pm Ultrasphere CIScolumn with a gradient program starting with 5% acetonitrile/water for 1.0 min followed by a linear gradient to 75% acetonitrile/water over 15 min. The flow rate was 2 mL/min, and the eluate was monitored at 220 nm by using a Beckman 164 detector. Fractions were collected at 15-9intervals, and the radioactivity of each fraction was determined by liquid scintillation counting after the addition of 10 mL of scintillation fluid. Under these conditions the retention times of NDMA and NDEA were 2.8 and 8.3 min, respectively. Microsomal Metabolism of NDMA and NDEA. Reactions were carried out in 150 mM potassium phosphate buffer, pH 7.4, at 37 "C. The reactions were initiated by the addition of 1.0 mM NADPH and terminated by the addition of 100 pL of 0.6 N perchloric acid. Formaldehyde formed from the demethylation of NDMA was measured by the Nash reaction (21). Acetaldehyde formed from the deethylation of NDEA was measured by gas chromatography as described (20). Measurement of Alkylation. The acid-precipitated material was extracted exhaustively with 95% EtOH until no counts could be detected in the wash solvent. The pellets were then dissolved
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 437
-30
-20
-IO
IO
20
30
40
I/[NDMA], pM-' (~10')
Figure 1. Production of 14C02by hepatocytes isolated from untreated rats as a function of NDMA concentration. Hepatocytes were isolated as described under Materials and Methods and incubated with the concentrations of NDMA indicated. The incubations contained between 4 X lo6 and 6 x lo6 cells/mL in Hams F10 medium. Reactions were kept at 37 "C in a water bath with constant shaking. The data are plotted as velocity versus the concentration of NDMA (A) and in the double-reciprocal form (B). in 100 pL of BTS-450 (Beckman). The samples were neutralized with glacial acetic acid, decolorized with 30% hydrogen peroxide, and counted after the addition of 10 mL of 3a70b scintillation fluid.
Results C 0 2 Evolution from NDMA and NDEA by Hepa-
tocytes Isolated from Control and EtOH-Pretreated Rats. Figure 1 illustrates t h e concentration dependence for the evolution of C 0 2from NDMA by hepatocytes from untreated, control rats. T h e r a t e of C 0 2 evolution was measured as described under Materials and Methods. The data for t h e rates of COz evolution were taken from t h e linear portion of t h e time dependence curve (not shown). T h e evolution of C 0 2 showed a distinct lag period for approximately t h e first 30 min after nitrosamine addition followed by a linear generation of C 0 2 for at least 3 h, which was when d a t a collection was terminated. A plot of t h e data in t h e double-reciprocal form (Figure 1B) indicates that t h e C 0 2 evolution was biphasic with respect t o NDMA concentration.2 When t h e rates of C 0 2 evolu-
438 Chem. Res. Toxicol., Vol. 2, No. 6, 1989
Gorsky and Hollenberg 100 I
-40-30-20-10
1
IO 20 30 40 50 60 70 80 90 I00
I/[NDMA]
, pM-' ( x I 0 3 )
Figure 2. Production of 14C02 by hepatocytes isolated from EtOH-treated rats as a function of NDMA concentration. The conditions were as described in Figure 1except that EtOH-treated rats were used as the source of the hepatocytes. The data are plotted as velocity versus the concentration of NDMA (A) and in the double-reciprocal form (B).
tion were plotted as a function of NDMA concentration added to isolated hepatocytes from EtOH-pretreated rats, a concentration dependence was observed which was very different from that with hepatocytes from control rats. NDMA metabolism by hepatocytes from EtOH-pretreated rats resulted in a monophasic production of COz which was maximal at about 100 pM NDMA (Figure 2A) and did not increase further when the NDMA concentration was increased up to 1.32 mM (data not shown). In fact, a slight decrease in the rate of C 0 2 production was observed at higher concentrations of NDMA, which may be indicative of the substrate inhibition reported by Yang et al. (6) for the in vitro metabolism of NDMA. A double-reciprocal plot of the data (Figure 2B) indicated a monophasic dependence on NDMA concentration. The rate of CO, evolution was then measured with NDEA as the substrate. In this case, double-reciprocal plots of the data indicate that C 0 2 production exhibited a biphasic dependence on NDEA concentration for metabolism by hepatocytes isolated from both untreated rats (Figure 3B) and EtOH-pretreated rats (Figure 3C). Relationship between Substrate Consumption and COz Evolution from NDMA and NDEA by Hepatocytes Isolated from Control and EtOH-Pretreated Rats. The metabolism of NDMA and NDEA by hepatocytes from EtOH-pretreated rats was measured by folThe purpose of utilizing the double-reciprocalanalysis of the velocity versua concentration data was not to derive kinetic constants; rather, it is used soley as a means of comparing the data from the different experiments. The complexity of the hepatocyte precludes the existence of simple Michaelis-Menten conditions.
I/[NOq , p Y - ' ( x I O 3 )
Figure 3. Production of I4CO2 by hepatocytes isolated from untreated and EtOH-treated rats as a function of NDEA concentration. The conditions were as described in Figure 1except that NDEA was substituted for the NDMA. The hepatocytes were from untreated- (A)or EtOH-treated ( 0 )rats. Data are plotted as velocity versus concentration of NDEA (A) and in the double-reciprocal forms for untreated (B) and EtOH-treated (C) rats.
lowing the loss of the parent compound as described under Materials and Methods. The data for CO, evolution illustrated in Figures 1-3 suggested that the rate of metabolism of the parent compound, as measured by substrate loss, was such that the decrease in the substrate present at the end of 1 h would represent only a small percentage of the total substrate in the incubation mixture. However, as shown in Table I, when COPevolution and nitrosamine depletion were measured under identical conditions, the loss of either NDMA or NDEA was found to be much greater than the production of COP. The data in Table I were determined after 1-h incubations and represent the total amounts of substrate consumed and C 0 2 generated. It can be seen that for both substrates
Nitrosamine Metabolism by Isolated Hepatocytes Table I. Comparison of Substrate Utilization and "CO2 Generation concn of nitrosamine, wMn NDMA 10 47 75 159 300 1332 NDEA 10 41 82 166 331 1324
substrate consumed,b nmol/106 cells
COZ produced,b nmol/106 cells
1.5 5.3 10.3 16.6 14.6 17.8
0.07 0.11 0.18 0.23 0.33 0.62
0.85 3.48 4.20 6.03 7.86 12.19
0.002 0.007 0.010 0.016 0.026 0.074
Hepatocytes isolated from EtOH-treated rats were incubated with the indicated concentration of NDMA or NDEA. The loss of substrate and the generation of l4COZwere determined as described under Materials and Methods. These values represent total amounts of substrate consumed or C02 produced in a 1-h incubation.
*
there was a marked discrepancy between COz production and substrate consumption throughout the range of nitrosamine concentrations studied. In experiments not shown, the production of COzfrom NDMA varied between 1% and 20% of the loss of parent compound while the production of COz from NDEA varied between 0.2% and 1.0% of total substrate consumed. Control Experiments for COPEvolution. In order to assure that the measurements of COz evolution were accurate and that the discrepancies between COz evolution and substrate utilization were not artifactual, the following experiments were performed. In the first experiment, 172.5 nmol of NaH14C03 was added to 1.0 mL of Hams F-10 medium and '%O2 was measured after the addition of acid as described under Materials and Methods. The radioactivity recovered from COz evolution under these experimental conditions was compared with the amount of radioactivity obtained when 172.5 nmol of NaH14C03was counted directly. This comparison indicated that essentially 100% of the NaH14C03 added to the incubation medium was detectable as l4CO2. In the second experiment, l4CO2evolution was measured in the absence and the presence of 2 mM methionine. (The concentration of methionine in the Hams F10 medium is 0.03 mM.) Methionine has been reported to increase COz production in certain systems by supplying the substrate required for the one-carbon tetrahydrofolate pathway (2,22,23). When this experiment was performed with 1.3 mM NDMA, the rates of COz evolution in the absence or the presence of added methionine (2 mM) were identical, indicating that the pathway for one-carbon metabolism was not a limiting factor in the production of C02 under the conditions of this study. These control experiments validate the marked difference between the loss of NDMA or NDEA from the incubation medium and the production of COz. In order to further investigate the basis for this discrepancy, mass balance experiments were performed with both NDMA and NDEA. Hepatocytes were incubated for 2 h with either NDMA or NDEA in scintillation vials with screw top caps into which Whatman glass fiber filters pretreated with NaOH were inserted. In this experiment the filters were not heated to dryness to remove non-COz radioactivity so that all of the volatile radioactivity could be accounted for. Therefore, any radioactivity adhering to the
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 439 Table 11. Mass Balance for NDMA and NDEA Metabolism in Isolated Hepatocytes substrate recovery of consumed or substrate product consumed, nitrosamine added" recovered, nmol % NDMA (a) unmetabolized substrate 6.9 (b) substrate consumed 90.1 (c) acid-soluble products 34.3 38 (d) volatile compounds 51.2 57 (e) alkylated macromolecules 1.3 1 (f) c + d + e 86.8 96 NDEA (a) unmetabolized substrate 2.3 (b) substrate consumed 64.3 (c) acid-soluble products 57.1 89 (d) volatile compounds 6.4 10 (e) alkylated macromolecules 1.5 2 (f)c+d+e 65.0 101 Hepatocytes isolated from EtOH-treated rat were incubated with NDMA (95 nmol) or NDEA (79nmol) as indicated. See text for details.
filter was considered to be a volatile compound and probably included some of the parent compound as well as COz and the related alcohols and aldehydes. At the end of the incubation, the substrate remaining in the incubation medium was separated from the more polar metabolites by reverse-phase HPLC as described under Materials and Methods. Alkylation of the cellular macromolecules was measured as described under Materials and Methods. The results of these mass balance experiments are presented in Table 11. These results indicate that essentially all of the radioactivity initially present as either NDMA or NDEA could be accounted for as either COz, volatile and polar metabolites, alkylated macromolecules, or unmetabolized substrate. One of the key functions of the liver is the production and export of urea in order to maintain the nitrogen balance and pH homeostasis of the body (24). The formation of urea from HC03- and ammonia is catalyzed in the liver. Therefore, we investigated the possibility that C02produced during the metabolism of both NDMA and NDEA was not detected due to its incorporation into urea. However, there was no difference in COzproduction in the absence or presence of urease (data not shown), indicating that volatile COz was not being lost due to incorporation into urea. Liver Microsomal Metabolism of NDMA a n d NDEA. The a-hydroxylation of NDMA and NDEA by liver microsomes isolated from untreated, EtOH-pretreated, and Pb-pretreated Fischer F344 rats was determined. Figure 4 illustrates the effect of antibody raised against the EtOH-inducible rabbit cytochrome P-450 isozyme 3a on NDMA and NDEA a-hydroxylation in microsomes derived from an EtOH-pretreated rat. This antibody (anti-3a) has been shown to cross-react with, and be inhibitory toward, the homologous EtOH-inducible P-450 isozyme present in rat liver, cytochrome P-45Oj (8, 25). As shown in Figure 4, maximum inhibition of the P-450j-dependent3a-hydroxylation of NDMA and NDEA was observed at an antibody:microsomal protein ratio of 2.0. Since the highest specific content of cytochrome P-45Oj (nmol of P-450j/mg of protein) is found in EtOHpretreated animals, an antib0dy:microsomal protein ratio of 2.0 was used for the subsequent inhibition studies. We refer to the EtOH-inducible P-450of the F344 rat as P-45Oj although we are aware that strain differences may exist as shown by Ryan et al. (28) for P-450b purified from Holtzman and Long-Evans rata.
Gorsky and Hollenberg
440 Chem. Res. Toxicol., Vol. 2, No.6,1989
EtOH-pretreated rats while 66% was inhibited in microsomes from control rats. Thus, the P-45Oj-dependent rate of NDEA deethylation was 0.92 nmol/ (min-mgof protein) in control microsomes and 1.39 nmol/(min.mg of protein) in microsomes from EtOH-treated animals. When the a-hydroxylation of NDMA and NDEA by microsomes isolated from Pb-pretreated rats was measured, an unexpected result was obtained. The rate of NDMA demethylation in these microsomes was decreased to 82% of the rate in control microsomes whereas the rate of NDEA deethylation was increased to 616% of the rate in control microsomes. Furthermore, the anti-3a IgG inhibited 70% of the NDMA demethylase in Pb microsomes, but it did not inhibit the NDEA deethylase at all.
100 90 80
70 60 L
0 E 5 0
40
30 20 10
0
0.4
0.8
1.2
1.6
2.0
IgG ( mg per mg Microsomal Protein )
Figure 4. Inhibition of liver microsomal metabolism of NDMA and NDEA by antibodies to cytochrome P-450form 3a. The reaction mixture (1.0 mL) contained 0.5 mg of microsomal protein, 100 mM potassium phosphate buffer (pH 7.4), and 1.36 mM NDMA (A)or 1.84 mM NDEA (M). Total IgG concentrations were kept constant at 1.0 mg by the addition of preimmune sheep IgG to supplementthe concentrations of the antibodies to form 3a. The reactions were initiated with 1mM NADPH and incubated at 37 O C for 20 min. The reactions were linear with respect to both time and protein concentration. Table 111. Microsomal Metabolism of NDMA and NDEA rate of formaldehyde or acetaldehyde formation,' nmol/(min.mg of protein) pretreat-
menta control EtOH Pb control EtOH
substrate no Ab Ab added NDMA 0.87 0.35 NDMA 1.59 0.08 0.71 0.21 NDMA
NDEA NDEA
1.40
0.48 0.35 8.62
inhibited by Ab
0.52 1.51 0.50 0.92
% inhibn
60 95 70 66 80 0
1.39 8.62 0.00 Pb NDEA 'The reaction mixtures (1.0 mL) contained 0.5 mg of microsomal protein derived from control or EtOH- or phenobarbitaltreated F344 rats, 100 mM potassium phosphate buffer (pH 7.4), 1.36 mM NDMA or 1.84 mM NDEA, and 1.0 mg of anti-3a IgG. 1.74
The reactions were initiated by the addition of 1 mM NADPH and were incubated at 37 O C for 20 min. The reactions were terminated by the addition of perchloric acid, and formaldehyde formation was determined as described under Materials and Methods.
The results of a series of inhibition experiments using anti-3a IgG are shown in Table 111. In microsomes from control rats, 60% of the NDMA demethylase activity was inhibited by anti-3a IgG whereas greater than 95% of the NDMA demethylase activity was inhibited by the antibody in microsomes from EtOH-pretreated rats. Thus, the P-450j-dependent rate of NDMA demethylation was 0.52 nmol/(min.mg of protein) in control microsomes and 1.51 nmol/ (mimmg of protein) in microsomes from EtOH-induced rata. The induction of P-45Oj as determined by the antibody-inhibitable rates was 2.9-fold whereas the total rate of NDMA demethylation was increased only 1.8-fold. This result illustrates two important points: (1)Fisher F344 rata treated with EtOH as described under Materials and Methods show induction of the EtOH-inducible P-45Oj and (2) essentially all of the NDMA demethylase activity in the microsomes from EtOH-treated rats is P-450j-dependent, in agreement with the reports of Levin et al. (26) and Yang et al. (8). As shown in Table 111, the anti-3a IgG inhibited 80% of the NDEA deethylase activity in microsomes from
Discussion The generation of C 0 2 has been used as a measure of the metabolism of NDMA ( 3 , 4 )and NDEA (3,5)as well as nitrosopyrrolidine (I). Hauber et al. ( 4 ) demonstrated that the C 0 2 evolution was markedly delayed relative to NDMA degradation in guinea pig hepatocytes. The results obtained in the present study confirm this discrepancy for rat hepatocytes and extend the observations to hepatocytes from ethanol-induced animals and to the next symmetrical dialkylnitrosamine, NDEA. The data presented in this study illustrate that the generation of C 0 2from NDMA as a function of the NDMA concentration is biphasic in hepatocytes from control animals but is monophasic in hepatocytes from EtOH-treated animals. The generation of C 0 2 from NDEA, on the other hand, is biphasic with respect to the nitrosamine concentration in hepatoctyes isolated from both control and EtOH-treated rats. Several hypotheses may be advanced to explain the discrepancy between the rates of substrate consumption and C 0 2 evolution. One hypothesis is that the rate of C02 formation from the initial product of the nitrosamine metabolism is very slow relative to the rate of the nitrosamine oxidation. The alternative hypothesis is that the rate of COPformation from thp initial product of nitrosamine metabolism is relatively rapid yet only a small fraction of the metabolism leads to C 0 2 . The second hypothesis is favored over the first because of the substrate dependence for the evolution of C 0 2 observed with both NDMA and NDEA. If a very slow step occurred between the oxidation of the nitrosamine and the evolution of C 0 2 and it was responsible for the discrepancy, a substrate dependence would not be observed. The data for the metabolism of NDMA by microsomes demonstrate that at least two isozymes of P-450 carry out the demethylation of NDMA in the untreated rat. The microsomes from the EtOH-treated rat, on the other hand, appear to catalyze the demethylation of NDMA with only a single isozyme. In addition, the evolution of C 0 2 by control hepatocytes is biphasic, and that of hepatocytes from EtOH-pretreated rats is monophasic. Thus, this observation can be interpreted as arising from the same isozyme alterations that are observable when the incubations are performed with microsomes. However, it is also possible that metabolic changes distinct from changes in the levels of the various isozymes of cytochrome P-450 may play a role in altering the apparent number of phases for the generation of C 0 2 from dialkylnitrosamines. Our results demonstrate that the measurement of C02 evolution is not a reliable method for determining the rate of the metabolism of the parent nitrosamine. In addition, the difference between substrate consumption and C 0 2 evolution is much greater for NDEA than for NDMA. This may be due to the fact that for substrates undergoing
Nitrosamine Metabolism by Isolated Hepatocytes
demethylation the pathway to COz production is more direct than for substrates undergoing deethylation. For deethylation, the product would be acetaldehyde which could then be incorporated into intermediary metabolism at the level of acetate. This more indirect route would result in a much greater dilution of the isotope and give rise to a decreased rate of appearance of labeled COP The results of these studies suggest that for larger alkyl side chains the divergence between substrate consumption and COz evolution would be even greater. The data in Table 111show that induction with phenobarbital results in a decrease in the metabolism of NDMA while the metabolism of NDEA is greatly increased. In addition, NDMA demethylation in microsomes from Pbtreated rats was inhibited 70% by the anti-3a antibody while NDEA deethylation in Pb microsomes was not affected at all by this antibody. Although NDMA and NDEA differ by only a single methylene group on each alkyl side chain, they are apparently metabolized most efficiently by distinct isozymes of P-450. This observation is consistent with a report by Mizrahi and Emmelot (27) that administration of cysteine to rats resulted in decreased demethylation of NDMA whereas NDEA deethylation was unaffected, suggesting the involvement of different enzymes in the metabolism of the two compounds. It is curious that a relatively small change in the structure of the nitrosamine will give rise to such a pronounced difference in the identity of the isozymes responsible for its metabolism. This result was unexpected since the structural difference between NDMA and NDEA is relatively small, especially for the P-450 family of isozymes. In this regard, it would be interesting to explore the isozyme specificity for a series of symmetrical dialkylnitrosamines.
Acknowledgment. This work was supported in part by Grant CA 16954 from the National Cancer Institute, USPHS, DHHS.
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