Carcinogenesis vol.12 no.l pp.127—131, 1991
Metabolism of azoxymethane, methylazoxymethanol and iV-nitrosodimethylamine by cytochrome P450HE1
Ock Soon Sohn, Hiroyuki Ishizaki1, Chung S.Yang1 and Emerich S.Fiala2 American Health Foundation, Valhalla, NY 10595 and 'College of Pharmacy, Rutgers University, Piscataway, NJ 08855, USA 2
To whom reprint requests should be sent
Introduction Azoxymethane (AOM*), principally a colon carcinogen in rodents (1-4), is metabolically derived from 1,2-dimethylhydrazine via azomethane as an intermediate (1,5,6). AOM is activated by methyl group hydroxylation to methylazoxymethanol (MAM), a compound that can yield alkylating species, probably methyldiazonium ion or methyldiazohydroxide, either spontaneously (7) or by enzyme-catalyzed reactions (8-10). Structurally, AOM may be considered an isomer of A'-nitrosodimethylamine (NDMA), a liver and kidney carcinogen, which is also activated by methyl group hydroxylation (11). However, a basic difference between AOM and NDMA, which bears on their organotropism, is in the stability of their respective hydroxylated metabolites. While a-hydroxy-NDMA decomposes to alkylating species with a half-life estimated in the order of seconds (12), MAM, the proximate metabolite of AOM, has a half-life of ~ 12 h under physiological conditions (8). Because of its relatively higher stability, MAM can be readily transported to extrahepatic organs such as the colon via the blood stream for further activation. Alternatively, MAM may also be metabolized in the liver, the major site of its production, resulting •Abbreviations: AOM, azoxymethane; MAM, methylazoxymethanol; NDMA, A'-nitrosodimethylamine. © Oxford University Press
Materials and methods Chemicals AOM was purchased from Ash Stevens Inc., Detroit, MI. NADP + , glucose 6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co., St Louis, MO. MAM-acetate and NDMA were obtained from Aldrich Chemical Co., Milwaukee, WI. A',Ar-Di['4C]methylnitrosamine (9.3 mCi/mmol) was purchased from Amersham Corp., Arlington Heights, IL. [Dimethyl-14C]AOM (3.99 mCi/mmol) and [l,2- 14 C]MAM-acetate (2.07 mCi/mmol) were obtained from NEN Research Products, Boston, MA. [1,2-I4C]MAM was prepared by hydrolyzing [l,2-14C]MAM-acetate with porcine liver esterase (Sigma Chemical Co., St Louis, MO) followed by HPLC purification as described previously (9). [I4C]Methylamine HCI, [l4C]formaldehyde, [14C]methanol and [l4C]formic acid, purchased from NEN Research Products, were used as standards for the identification of metabolic products. [l4C]Methylphosphate was produced by refluxing a mixture of phosphoric acid and ['"C]methanol, followed by purification by HPLC in the system described below. Its identity as the trimethylsilane derivative was verified by MS (mlz 73, 24%; 133, 24%; 163, 18%; 211, 15%; 241, 100%; 256, 5%). Preparation of microsomes, enzymes and antibodies Acetone-induced microsomes were isolated from young male Sprague—Dawley rats (body wt 85-100 g) which had been treated orally with 5 ml/kg acetone 20 h before they were killed (24). Cytochrome P450HEI (P450ac), NADPHcytochrome P450 reductase, and cytochrome b5 were prepared as described previously (24). The preparation of monoclonal antibodies against cytochrome P450IIE1 (MAb 1-91-3) and control ascites fluid (HyHel-9) was also previously described (23). Incubation conditions and HPLC analysis of metabolites For the determination of microsomal activities, incubation mixtures consisted of an NADPH-generating system (3.5 mM glucose 6-phosphate, 5 U glucose6-phosphate dehydrogenase, 1.5 mM NADP + , 3.5 mM MgCl2, 0.1 M potassium phosphate (pH 7.0), 1 mM l4C-labeled substrate (sp. act. —2000 d.p.m./nmol) and 1 mg microsomal protein in a total volume of 0.5 ml (13,14).
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The metabolism of azoxymethane (AOM), methylazoxymethanol (MAM) and A'-nitrosodimethylamine (NDMA) by liver microsomes from acetone-induced rats as well as by a reconstituted system containing purified cytochrome P450DE1 was examined. The products consisted of MAM from AOM; methanol and formic acid from MAM; and methylamine, formaldehyde, methanol, methylphosphate and formic acid from NDMA. Compared to liver microsomes from untreated rats, the metabolic activity of acetone-induced microsomes was ~ 4 times higher for all three carcinogens. Using the reconstituted system, the enzyme activities (nmol substrate metabolized/nmol P450/min) for AOM, MAM and NDMA were 2.88 ± 1.14,2.87 ± 0.59 and 9.47 ± 2.24 respectively. Incubations carried out in the presence of a monoclonal antibody to cytochrome P450IIE1 resulted in a 85-90% inhibition of all three reactions in this system. These results provide conclusive evidence that AOM, MAM and NDMA are metabolized by the same form of rat liver cytochrome P450. In addition, the stoichiometry of NDMA products formed in these reactions indicates that denitrosation, a presumed detoxication process, and a-hydroxylation, an activation reaction, are also catalyzed by the same cytochrome P450 isozyme.
in decreased amounts reaching the colon. It is evident diat the rates of liver metabolism of AOM and of MAM play a significant role in determining colon carcinogenicity, and it is important to characterize the enzymes involved. Previous studies have shown strong similarities in the effects of chronic ethanol administration to rats on die metabolism of AOM, MAM and NDMA by liver microsomes, leading to the suggestion that all three carcinogens may be activated by the same enzyme (13,14). Recently, ethanol was shown to be a strong inducer of liver cytochrome P450IIE1 (P450j or P450.,,. from rat liver microsomes, or P450LM3a from rabbit liver microsomes) (15—18). This isozyme, which is also induced by treatment widi acetone, imidazole, pyrazole and isoniazid, and by diabetes and fasting (18-23), catalyzes the hydroxylation of NDMA (18,24,25). In the present study, we examined the metabolism of AOM, MAM and NDMA by acetone-induced rat liver microsomes and by a reconstituted system containing cytochrome P450IIE1 that had been purified from acetone-induced rat liver microsomes. We also examined the inhibitory effects of a monoclonal antibody to cytochrome P450IIE1 on these reactions. In agreement with previous suggestions (13,14), the results indicate that AOM and MAM are activated by a form of cytochrome P450 isozyme (IIE1) that is also responsible for the a-hydroxylation of NDMA (18,24,25).
O.S.Sohn et at. The mixture was incubated at 37°C for 15 min with liver microsomes from untreated rats and for 10 min with liver microsomes from acetone-induced rats. To determine the activities of reconstituted enzyme systems, 0.16 nmol of purified P450IIE1, purified NADPH-cytochrome P450 reductase (1900 U/0.1 nmol cytochrome P450), purified cytochrome b5 (0.2 nmol/0.1 nmol cytochrome P450) and dilauroylphosphatidylcholine (28.1 /ig/0.1 nmol cytochrome P450) were mixed in a glass tube at room temperature. Immediately thereafter, 0.1 M potassium phosphate (pH 7.0), 3.5 mM MgCl2, 1 mM l4C-labeled substrate and the NADPH-generating system were added in a total volume of 0.5 ml (23), and incubation was carried out at 37°C for 15 min. In experiments involving antibody inhibition, purified P450IIE1 was first exposed to 200 /ig of monoclonal antibody (1-91-3) or control ascites fluid (HyHel-9) for 5 min at room temperature prior to the addition of other components of the reconstituted system (26,27). After incubation at 37°C, reaction vessels were quickly chilled to 0°C, and the suspensions were deproteinized by centrifugal ultrafiltration (600 g, 30—60 min, 0 - 4 ° C ) using Centnfree Micropartition tubes (Amicon, Danvers, MA). Ultrafiltrates were submitted to HPLC using two Whatman ODS-3 (0.46 x 25 cm) columns in series, preceded by a 0.7 x 5 cm column packed with Aminex A-29 anion exchange resin (Bio-Rad Labs, Rockville Centre, NY) in the phosphate form as described previously (13,14).
Metabolism by a reconstituted system with purified cytochrome P450IIE1 and inhibition by monoclonal antibody As shown in Table II, a reconstituted system, consisting of purified P450IIE1, NADPH-P450-reductase, lipids, cytochrome b5 and an NADPH-generating system, was capable of metabolizing not only NDMA but also AOM and MAM. Moreover, the ratios of enzyme activities using the three substrates in this system were similar to those obtained when liver microsomes from control or acetone-induced rats were used. HPLC analysis of the incubation mixtures confirmed that the \0M
400
MAM
300 200 100 0
o o
I
I Control microsomes
I
1 Acetone indxuced
HC00H
0£
microsomes
a
methyl phosphate
7
8 9 10 1112 13 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ELUTION VOLUME, ml Fig. 1. Typical HPLC profiles of metabolites resulting from incubation of 14C-labeled AOM (top), MAM (middle) and NDMA (bottom) in the presence of control or acetone-induced rat liver microsomes. See Materials and methods for conditions of incubation and HPLC.
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Results Microsomal metabolism of AOM, MAM and NDMA: stimulation by acetone pretreatment HPLC profiles of metabolites obtained using AOM, MAM and NDMA as substrates in the presence of the control or the acetoneinduced microsomes are shown in Figure 1. As previously observed (13), MAM is the only product of the microsomal metabolism of AOM, and methanol and formic acid are the major products of MAM metabolism (14). In the case of NDMA, the HPLC system resolves methylamine, formaldehyde, methanol, methylphosphate and formic acid as the reaction products. The I4C peaks eluting between 19 and 24 ml, also observed previously (13), were shown to be methylphosphate (19—20 ml) and formic acid (22 - 2 4 ml) by co-chromatography with standard compounds. Methylphosphate probably represents the reaction
product of a methylating species, derived from labeled NDMA, with phosphate in the incubation medium; this product is not detected when incubations are carried out in Tris buffer (28). The [14C] formic acid most likely originates from the further microsomal oxidation of the primary NDMA metabolite, formaldehyde. A preliminary experiment using 12 ;*M [I4C]formaldehyde as substrate showed that ~ 8% of the substrate was converted to [l4C]formic acid when incubated for 30 min in the presence of rat liver microsomes and an NADPH-generating system. The activities of liver microsomes from untreated (control) or acetone-induced rats with respect to the metabolism of l4 C-labeled AOM, MAM and NDMA are shown in Table I. Control microsomes were capable of metabolizing AOM and MAM as well as NDMA; the enzyme activity with NDMA was 3- to 4-fold higher than with AOM or MAM. In the case of acetone-induced liver microsomes, metabolism of all three carcinogens was stimulated ~ 4-fold over the control microsomes; the ratio of activities for the three substrates was the same as that obtained using the control microsomes. Whether or not acetone treatment also led to the induction of other liver cytochromes P450 capable of catalyzing the metabolism of the three carcinogens was not examined in these experiments.
Metabolism of AOM, MAM and NDMA
metabolites of the carcinogens obtained with the reconstituted cytochrome P450 system were identical to those produced during incubations with rat liver microsomes (induced or uninduced). The reconstituted enzyme system required the presence of cytochrome Z»5 for maximum activity; conversion to metabolites was reduced significantly when cytochrome b5 was omitted from the incubation mixture. Such stimulatory effects of cytochrome b5 on NDMA metabolism have been noted previously (24,25). Table I . In vitro metabolism of 14C-labeled AOM, MAM and NDMA by control and acetone-induced irat liver microsomes a nmol converted/mg protein/minb
AOM MAM NDMA
Control
Acetone-induced
0.4 (0.37, 0.44) 0.27 (0.21, 0.32) 1.22 (1.09, 1.35)
1.63 (1.61, 1.65) 1.31 (1.32, 1.30) 4.94 (5.24, 4.63)
Table II. Metabolism of l4C-labeled AOM, MAM and NDMA by a reconstituted system with purified P450IIE1 and inhibition by monoclonal antibody nmol converted/nmo! P450/mina AOM
MAM
2.88 ± (100) 0.69 ± (24) 0.03 ± (1) 3.0 ± (104) 0.44 ± (15)
Complete systemb Complete Complete minus P450IIE1 Complete plus control ascitesc Complete plus MAbc
1.14 0.12 0.04 0.82 0.28
2.87 ± (100) 0.25 ± (9) 0.23 ± (8) 2.76 ± (96) 0.34 ± (12)
NDMA 0.59 0.25 0.2 0.6 0.34
9.47 (100) 1.60 (17) 0.09 (1) 11.52 (122) 1.18 (12)
± 2.24 ± 0.53 ± 0.07 ± 1.09 ± 0.35
"Values represent means ± SD of three or four determinations. Numbers in parentheses show percentage activity compared to the complete system. "Complete incubations consisted of 0.16 nmol cytochrome P450IIE1, 3040 U NADPH-cytochrome P450 reductase, 45 ^g dilauroylphosphatidylcholine, 0.32 nmol cytochrome b5, 1 mM substrate and the NADPH-generating system in a final volume of 0.5 ml. c Control ascites fluid (HyHel-9) or monoclonal antibody (MAb 1-91-3) was added to a level of 200 ^g protein per incubation.
Table III. Stoichiometry of NDMA metabolite formation by reconstituted purified cytochrome P450IIE1 systema nmol metabolite produced/min/nmol cytochrome P450IIE1 CH3NH2 Complete system 1.23 Complete 0.1 minus b5 Complete 0.02 minus P450IIE1 Complete plus 1.36 control ascites Complete plus 0.18 MAb
CH3OH
CH3H2PO4
HCOOH
8.96 1.58
7.1 1.03
1.34 0.3
0.45 0.16
0.08
0.03
0
0.05
8.24
1.53
0.5
0.89
0.28
0.16
HCHO
11.4 1.05
T h e conditions are the same as described in Table II.
Discussion Acetone and ethanol both induce the same form of cytochrome P450 in rat liver (17,22). Previous studies (13,14) indicated strong similarities in the metabolism of NDMA, AOM and MAM by rat liver microsomes in that all three reactions were stimulated by ethanol pretreatment. Not surprisingly, the present data in Table I show that the microsomal metabolism of AOM and MAM is also stimulated by acetone pretreatment. Also, the magnitude of the stimulation of AOM and MAM metabolism by acetone administration is similar to that observed for the metabolism of NDMA. These considerations, together with the demonstration that a reconstituted purified cytochrome P450HE1 system is capable of metabolizing not only NDMA but also AOM and MAM (Table II), lead to the conclusion that the same cytochrome P450 isozyme can catalyze the metabolic activation of all three carcinogens. Whereas the activation of both AOM and NDMA involves methyl group hydroxylation, the activation of MAM depends on the oxidation of its alcohol function to yield the extremely unstable methylazoxyformaldehyde (8) (see Figure 2). The oxidation of MAM can be catalyzed by alcohol dehydrogenase (8,33), and this enzyme probably plays a major role in the activation of MAM in the rat colon mucosa, a tissue relatively deficient in cytochromes P450 (34). In analogy to the oxidation of ethanol to acetaldehyde (35), the oxidation of MAM to methylazoxyformaldehyde can also be catalyzed by cytochrome P450HE1, as shown in this work. At present, it is not clear whether alcohol dehydrogenase or cytochromes P450, or both, catalyze the 129
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a Incubation system consisted of 1 mM substrate, 1 mg microsomal protein and an NADPH-generating system in a final volume of 0.5 ml. Values were obtained by determining the products detected by HPLC (see Figure 1). ""Values represent the mean of two determinations. Individual values are shown in parentheses.
Table II also shows the effects of monoclonal antibody to P450IIE1 on the metabolism of AOM, MAM and NDMA by reconstituted cytochrome P450HE1 system. While addition of control ascites fluid (HyHel-9) to the complete system did not significantly affect the metabolism of the carcinogens, preincubation of monoclonal antibody (1-91-3) with purified P450IIE1 resulted in an inhibition of AOM, MAM and NDMA metabolism by 85-90%. Stoichiometry of the formation of NDMA metabolites by reconstituted purified cytochrome P450IIE1 system Unlike most other studies on the in vitro metabolism of NDMA which utilize the Nash reaction (29) for determination of HCHO production, the HPLC separation method used in the present experiments allows quantitation of individual NDMA metabolites [recently, Heur et al. (30) have reported a further improvement of this method which affords higher resolution]. As shown in Table HI, the major products of NDMA metabolism detected following incubation in the reconstituted cytochrome P450IIE1 system are HCHO (47% of total) and CH3OH (37.2%), while CH3NH2 (6.4%), CH3H2PO4 (7.0%) and HCOOH (2.3%) represent minor products. According to the scheme proposed by Keefer etal. (31), formaldehyde, methanol, formic acid (probably a further oxidation product of formaldehyde) and methylphosphate (derived from the reaction of methyldiazohydroxide with phosphate buffering the system) are formed during the demethylation of NDMA, an activation reaction, whereas methylamine is formed from NDMA denitrosation, a presumed detoxication reaction. Table IE shows that the extent of inhibition of the activation reaction and of the denitrosation reaction, for instance by omitting cytochrome b5, is approximately the same. This may be regarded as further evidence that both demethylation and denitrosation are catalyzed by the same enzyme, as indicated by the studies of Patten et al. (24) and Wade et al. (32).
O.S.Sohn et at.
.CH,
CH 3
O=N-N CH 3
0
CHi
CH3NH2 + CH 2 O + NO
NDMA
AOM
a — hydroxylation
1)
.CH,
CH 3 0
5)
\
O=N-N' CH, HO'
CH, C
//
HO MAM
CH 2 0
[CH 3 -N=N] o'
c
CH 3 X 4)
HCOO
'OH
Met hylazoxy formaldehyde Fig. 2. Comparison of metabolism of AOM and NDMA. Cytochrome P450IIEI-catalyzed activation reactions 1, 2 and 3 result in the formation of a reactive species such as the methyldiazonium ion. Reactions of the latter with water or phosphate will yield methanol or methylphosphate. Denitrosation reaction 5, likewise catalyzed by cytochrome P450IIE1, results in the detoxication of NDMA (31).
metabolism of MAM in rat liver under normal conditions. This question is not easily answered because inhibitors of alcohol dehydrogenase also inhibit cytochrome P450-mediated microsomal metabolism of MAM (10). Following chronic ethanol administration to rats, the in vivo metabolism of MAM is significantly increased (14). Because this effect is accompanied by enhanced liver microsomal metabolism of the carcinogen in vitro, it is possible that cytochrome P450-mediated metabolism of MAM may predominate in ethanol-induced rat liver. This seems likely, since rat liver alcohol dehydrogenase does not appear to be induced by ethanol (35). If both AOM and MAM are mainly metabolized by the same cytochrome P450 isozyme in rat liver, it is interesting to consider that the parent compound (AOM) may inhibit competitively the further metabolism of its product (MAM) by hepatic cytochrome P450HE1. Such inhibition would make more MAM available to the colon. Although the biological significance of such inhibition is difficult to assess because data on the kinetics of these reactions are not yet available, a possible consequence might be a greater systemic persistence of MAM after the administration of AOM than after the administration of MAM-acetate. Thus, AOM might be a more effective carcinogen for the colon than would be anticipated from an AOM — MAM precursor—product metabolic relationship. In conclusion, we have provided further evidence that AOM, MAM and NDMA are metabolically activated by the same liver cytochrome P450 isozyme in vitro. The relevance of cytochrome P450 mediated metabolism of AOM and MAM to the carcinogenicity and organotropism of these compounds is currently under investigation. Acknowledgements This work was supported by grants nos. CA31012 (E.S.F.) and CA37O37 (C.S.Y.) from the National Cancer Institute.
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Metabolism of azoxymethane, methylazoxymethanol and iV-nitrosodimethylamine by cytochrome P450HE1
Ock Soon Sohn, Hiroyuki Ishizaki1, Chung S.Yang1 and Emerich S.Fiala2 American Health Foundation, Valhalla, NY 10595 and 'College of Pharmacy, Rutgers University, Piscataway, NJ 08855, USA 2
To whom reprint requests should be sent
Introduction Azoxymethane (AOM*), principally a colon carcinogen in rodents (1-4), is metabolically derived from 1,2-dimethylhydrazine via azomethane as an intermediate (1,5,6). AOM is activated by methyl group hydroxylation to methylazoxymethanol (MAM), a compound that can yield alkylating species, probably methyldiazonium ion or methyldiazohydroxide, either spontaneously (7) or by enzyme-catalyzed reactions (8-10). Structurally, AOM may be considered an isomer of A'-nitrosodimethylamine (NDMA), a liver and kidney carcinogen, which is also activated by methyl group hydroxylation (11). However, a basic difference between AOM and NDMA, which bears on their organotropism, is in the stability of their respective hydroxylated metabolites. While a-hydroxy-NDMA decomposes to alkylating species with a half-life estimated in the order of seconds (12), MAM, the proximate metabolite of AOM, has a half-life of ~ 12 h under physiological conditions (8). Because of its relatively higher stability, MAM can be readily transported to extrahepatic organs such as the colon via the blood stream for further activation. Alternatively, MAM may also be metabolized in the liver, the major site of its production, resulting •Abbreviations: AOM, azoxymethane; MAM, methylazoxymethanol; NDMA, A'-nitrosodimethylamine. © Oxford University Press
Materials and methods Chemicals AOM was purchased from Ash Stevens Inc., Detroit, MI. NADP + , glucose 6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co., St Louis, MO. MAM-acetate and NDMA were obtained from Aldrich Chemical Co., Milwaukee, WI. A',Ar-Di['4C]methylnitrosamine (9.3 mCi/mmol) was purchased from Amersham Corp., Arlington Heights, IL. [Dimethyl-14C]AOM (3.99 mCi/mmol) and [l,2- 14 C]MAM-acetate (2.07 mCi/mmol) were obtained from NEN Research Products, Boston, MA. [1,2-I4C]MAM was prepared by hydrolyzing [l,2-14C]MAM-acetate with porcine liver esterase (Sigma Chemical Co., St Louis, MO) followed by HPLC purification as described previously (9). [I4C]Methylamine HCI, [l4C]formaldehyde, [14C]methanol and [l4C]formic acid, purchased from NEN Research Products, were used as standards for the identification of metabolic products. [l4C]Methylphosphate was produced by refluxing a mixture of phosphoric acid and ['"C]methanol, followed by purification by HPLC in the system described below. Its identity as the trimethylsilane derivative was verified by MS (mlz 73, 24%; 133, 24%; 163, 18%; 211, 15%; 241, 100%; 256, 5%). Preparation of microsomes, enzymes and antibodies Acetone-induced microsomes were isolated from young male Sprague—Dawley rats (body wt 85-100 g) which had been treated orally with 5 ml/kg acetone 20 h before they were killed (24). Cytochrome P450HEI (P450ac), NADPHcytochrome P450 reductase, and cytochrome b5 were prepared as described previously (24). The preparation of monoclonal antibodies against cytochrome P450IIE1 (MAb 1-91-3) and control ascites fluid (HyHel-9) was also previously described (23). Incubation conditions and HPLC analysis of metabolites For the determination of microsomal activities, incubation mixtures consisted of an NADPH-generating system (3.5 mM glucose 6-phosphate, 5 U glucose6-phosphate dehydrogenase, 1.5 mM NADP + , 3.5 mM MgCl2, 0.1 M potassium phosphate (pH 7.0), 1 mM l4C-labeled substrate (sp. act. —2000 d.p.m./nmol) and 1 mg microsomal protein in a total volume of 0.5 ml (13,14).
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The metabolism of azoxymethane (AOM), methylazoxymethanol (MAM) and A'-nitrosodimethylamine (NDMA) by liver microsomes from acetone-induced rats as well as by a reconstituted system containing purified cytochrome P450DE1 was examined. The products consisted of MAM from AOM; methanol and formic acid from MAM; and methylamine, formaldehyde, methanol, methylphosphate and formic acid from NDMA. Compared to liver microsomes from untreated rats, the metabolic activity of acetone-induced microsomes was ~ 4 times higher for all three carcinogens. Using the reconstituted system, the enzyme activities (nmol substrate metabolized/nmol P450/min) for AOM, MAM and NDMA were 2.88 ± 1.14,2.87 ± 0.59 and 9.47 ± 2.24 respectively. Incubations carried out in the presence of a monoclonal antibody to cytochrome P450IIE1 resulted in a 85-90% inhibition of all three reactions in this system. These results provide conclusive evidence that AOM, MAM and NDMA are metabolized by the same form of rat liver cytochrome P450. In addition, the stoichiometry of NDMA products formed in these reactions indicates that denitrosation, a presumed detoxication process, and a-hydroxylation, an activation reaction, are also catalyzed by the same cytochrome P450 isozyme.
in decreased amounts reaching the colon. It is evident diat the rates of liver metabolism of AOM and of MAM play a significant role in determining colon carcinogenicity, and it is important to characterize the enzymes involved. Previous studies have shown strong similarities in the effects of chronic ethanol administration to rats on die metabolism of AOM, MAM and NDMA by liver microsomes, leading to the suggestion that all three carcinogens may be activated by the same enzyme (13,14). Recently, ethanol was shown to be a strong inducer of liver cytochrome P450IIE1 (P450j or P450.,,. from rat liver microsomes, or P450LM3a from rabbit liver microsomes) (15—18). This isozyme, which is also induced by treatment widi acetone, imidazole, pyrazole and isoniazid, and by diabetes and fasting (18-23), catalyzes the hydroxylation of NDMA (18,24,25). In the present study, we examined the metabolism of AOM, MAM and NDMA by acetone-induced rat liver microsomes and by a reconstituted system containing cytochrome P450IIE1 that had been purified from acetone-induced rat liver microsomes. We also examined the inhibitory effects of a monoclonal antibody to cytochrome P450IIE1 on these reactions. In agreement with previous suggestions (13,14), the results indicate that AOM and MAM are activated by a form of cytochrome P450 isozyme (IIE1) that is also responsible for the a-hydroxylation of NDMA (18,24,25).
O.S.Sohn et at. The mixture was incubated at 37°C for 15 min with liver microsomes from untreated rats and for 10 min with liver microsomes from acetone-induced rats. To determine the activities of reconstituted enzyme systems, 0.16 nmol of purified P450IIE1, purified NADPH-cytochrome P450 reductase (1900 U/0.1 nmol cytochrome P450), purified cytochrome b5 (0.2 nmol/0.1 nmol cytochrome P450) and dilauroylphosphatidylcholine (28.1 /ig/0.1 nmol cytochrome P450) were mixed in a glass tube at room temperature. Immediately thereafter, 0.1 M potassium phosphate (pH 7.0), 3.5 mM MgCl2, 1 mM l4C-labeled substrate and the NADPH-generating system were added in a total volume of 0.5 ml (23), and incubation was carried out at 37°C for 15 min. In experiments involving antibody inhibition, purified P450IIE1 was first exposed to 200 /ig of monoclonal antibody (1-91-3) or control ascites fluid (HyHel-9) for 5 min at room temperature prior to the addition of other components of the reconstituted system (26,27). After incubation at 37°C, reaction vessels were quickly chilled to 0°C, and the suspensions were deproteinized by centrifugal ultrafiltration (600 g, 30—60 min, 0 - 4 ° C ) using Centnfree Micropartition tubes (Amicon, Danvers, MA). Ultrafiltrates were submitted to HPLC using two Whatman ODS-3 (0.46 x 25 cm) columns in series, preceded by a 0.7 x 5 cm column packed with Aminex A-29 anion exchange resin (Bio-Rad Labs, Rockville Centre, NY) in the phosphate form as described previously (13,14).
Metabolism by a reconstituted system with purified cytochrome P450IIE1 and inhibition by monoclonal antibody As shown in Table II, a reconstituted system, consisting of purified P450IIE1, NADPH-P450-reductase, lipids, cytochrome b5 and an NADPH-generating system, was capable of metabolizing not only NDMA but also AOM and MAM. Moreover, the ratios of enzyme activities using the three substrates in this system were similar to those obtained when liver microsomes from control or acetone-induced rats were used. HPLC analysis of the incubation mixtures confirmed that the \0M
400
MAM
300 200 100 0
o o
I
I Control microsomes
I
1 Acetone indxuced
HC00H
0£
microsomes
a
methyl phosphate
7
8 9 10 1112 13 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ELUTION VOLUME, ml Fig. 1. Typical HPLC profiles of metabolites resulting from incubation of 14C-labeled AOM (top), MAM (middle) and NDMA (bottom) in the presence of control or acetone-induced rat liver microsomes. See Materials and methods for conditions of incubation and HPLC.
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Results Microsomal metabolism of AOM, MAM and NDMA: stimulation by acetone pretreatment HPLC profiles of metabolites obtained using AOM, MAM and NDMA as substrates in the presence of the control or the acetoneinduced microsomes are shown in Figure 1. As previously observed (13), MAM is the only product of the microsomal metabolism of AOM, and methanol and formic acid are the major products of MAM metabolism (14). In the case of NDMA, the HPLC system resolves methylamine, formaldehyde, methanol, methylphosphate and formic acid as the reaction products. The I4C peaks eluting between 19 and 24 ml, also observed previously (13), were shown to be methylphosphate (19—20 ml) and formic acid (22 - 2 4 ml) by co-chromatography with standard compounds. Methylphosphate probably represents the reaction
product of a methylating species, derived from labeled NDMA, with phosphate in the incubation medium; this product is not detected when incubations are carried out in Tris buffer (28). The [14C] formic acid most likely originates from the further microsomal oxidation of the primary NDMA metabolite, formaldehyde. A preliminary experiment using 12 ;*M [I4C]formaldehyde as substrate showed that ~ 8% of the substrate was converted to [l4C]formic acid when incubated for 30 min in the presence of rat liver microsomes and an NADPH-generating system. The activities of liver microsomes from untreated (control) or acetone-induced rats with respect to the metabolism of l4 C-labeled AOM, MAM and NDMA are shown in Table I. Control microsomes were capable of metabolizing AOM and MAM as well as NDMA; the enzyme activity with NDMA was 3- to 4-fold higher than with AOM or MAM. In the case of acetone-induced liver microsomes, metabolism of all three carcinogens was stimulated ~ 4-fold over the control microsomes; the ratio of activities for the three substrates was the same as that obtained using the control microsomes. Whether or not acetone treatment also led to the induction of other liver cytochromes P450 capable of catalyzing the metabolism of the three carcinogens was not examined in these experiments.
Metabolism of AOM, MAM and NDMA
metabolites of the carcinogens obtained with the reconstituted cytochrome P450 system were identical to those produced during incubations with rat liver microsomes (induced or uninduced). The reconstituted enzyme system required the presence of cytochrome Z»5 for maximum activity; conversion to metabolites was reduced significantly when cytochrome b5 was omitted from the incubation mixture. Such stimulatory effects of cytochrome b5 on NDMA metabolism have been noted previously (24,25). Table I . In vitro metabolism of 14C-labeled AOM, MAM and NDMA by control and acetone-induced irat liver microsomes a nmol converted/mg protein/minb
AOM MAM NDMA
Control
Acetone-induced
0.4 (0.37, 0.44) 0.27 (0.21, 0.32) 1.22 (1.09, 1.35)
1.63 (1.61, 1.65) 1.31 (1.32, 1.30) 4.94 (5.24, 4.63)
Table II. Metabolism of l4C-labeled AOM, MAM and NDMA by a reconstituted system with purified P450IIE1 and inhibition by monoclonal antibody nmol converted/nmo! P450/mina AOM
MAM
2.88 ± (100) 0.69 ± (24) 0.03 ± (1) 3.0 ± (104) 0.44 ± (15)
Complete systemb Complete Complete minus P450IIE1 Complete plus control ascitesc Complete plus MAbc
1.14 0.12 0.04 0.82 0.28
2.87 ± (100) 0.25 ± (9) 0.23 ± (8) 2.76 ± (96) 0.34 ± (12)
NDMA 0.59 0.25 0.2 0.6 0.34
9.47 (100) 1.60 (17) 0.09 (1) 11.52 (122) 1.18 (12)
± 2.24 ± 0.53 ± 0.07 ± 1.09 ± 0.35
"Values represent means ± SD of three or four determinations. Numbers in parentheses show percentage activity compared to the complete system. "Complete incubations consisted of 0.16 nmol cytochrome P450IIE1, 3040 U NADPH-cytochrome P450 reductase, 45 ^g dilauroylphosphatidylcholine, 0.32 nmol cytochrome b5, 1 mM substrate and the NADPH-generating system in a final volume of 0.5 ml. c Control ascites fluid (HyHel-9) or monoclonal antibody (MAb 1-91-3) was added to a level of 200 ^g protein per incubation.
Table III. Stoichiometry of NDMA metabolite formation by reconstituted purified cytochrome P450IIE1 systema nmol metabolite produced/min/nmol cytochrome P450IIE1 CH3NH2 Complete system 1.23 Complete 0.1 minus b5 Complete 0.02 minus P450IIE1 Complete plus 1.36 control ascites Complete plus 0.18 MAb
CH3OH
CH3H2PO4
HCOOH
8.96 1.58
7.1 1.03
1.34 0.3
0.45 0.16
0.08
0.03
0
0.05
8.24
1.53
0.5
0.89
0.28
0.16
HCHO
11.4 1.05
T h e conditions are the same as described in Table II.
Discussion Acetone and ethanol both induce the same form of cytochrome P450 in rat liver (17,22). Previous studies (13,14) indicated strong similarities in the metabolism of NDMA, AOM and MAM by rat liver microsomes in that all three reactions were stimulated by ethanol pretreatment. Not surprisingly, the present data in Table I show that the microsomal metabolism of AOM and MAM is also stimulated by acetone pretreatment. Also, the magnitude of the stimulation of AOM and MAM metabolism by acetone administration is similar to that observed for the metabolism of NDMA. These considerations, together with the demonstration that a reconstituted purified cytochrome P450HE1 system is capable of metabolizing not only NDMA but also AOM and MAM (Table II), lead to the conclusion that the same cytochrome P450 isozyme can catalyze the metabolic activation of all three carcinogens. Whereas the activation of both AOM and NDMA involves methyl group hydroxylation, the activation of MAM depends on the oxidation of its alcohol function to yield the extremely unstable methylazoxyformaldehyde (8) (see Figure 2). The oxidation of MAM can be catalyzed by alcohol dehydrogenase (8,33), and this enzyme probably plays a major role in the activation of MAM in the rat colon mucosa, a tissue relatively deficient in cytochromes P450 (34). In analogy to the oxidation of ethanol to acetaldehyde (35), the oxidation of MAM to methylazoxyformaldehyde can also be catalyzed by cytochrome P450HE1, as shown in this work. At present, it is not clear whether alcohol dehydrogenase or cytochromes P450, or both, catalyze the 129
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a Incubation system consisted of 1 mM substrate, 1 mg microsomal protein and an NADPH-generating system in a final volume of 0.5 ml. Values were obtained by determining the products detected by HPLC (see Figure 1). ""Values represent the mean of two determinations. Individual values are shown in parentheses.
Table II also shows the effects of monoclonal antibody to P450IIE1 on the metabolism of AOM, MAM and NDMA by reconstituted cytochrome P450HE1 system. While addition of control ascites fluid (HyHel-9) to the complete system did not significantly affect the metabolism of the carcinogens, preincubation of monoclonal antibody (1-91-3) with purified P450IIE1 resulted in an inhibition of AOM, MAM and NDMA metabolism by 85-90%. Stoichiometry of the formation of NDMA metabolites by reconstituted purified cytochrome P450IIE1 system Unlike most other studies on the in vitro metabolism of NDMA which utilize the Nash reaction (29) for determination of HCHO production, the HPLC separation method used in the present experiments allows quantitation of individual NDMA metabolites [recently, Heur et al. (30) have reported a further improvement of this method which affords higher resolution]. As shown in Table HI, the major products of NDMA metabolism detected following incubation in the reconstituted cytochrome P450IIE1 system are HCHO (47% of total) and CH3OH (37.2%), while CH3NH2 (6.4%), CH3H2PO4 (7.0%) and HCOOH (2.3%) represent minor products. According to the scheme proposed by Keefer etal. (31), formaldehyde, methanol, formic acid (probably a further oxidation product of formaldehyde) and methylphosphate (derived from the reaction of methyldiazohydroxide with phosphate buffering the system) are formed during the demethylation of NDMA, an activation reaction, whereas methylamine is formed from NDMA denitrosation, a presumed detoxication reaction. Table IE shows that the extent of inhibition of the activation reaction and of the denitrosation reaction, for instance by omitting cytochrome b5, is approximately the same. This may be regarded as further evidence that both demethylation and denitrosation are catalyzed by the same enzyme, as indicated by the studies of Patten et al. (24) and Wade et al. (32).
O.S.Sohn et at.
.CH,
CH 3
O=N-N CH 3
0
CHi
CH3NH2 + CH 2 O + NO
NDMA
AOM
a — hydroxylation
1)
.CH,
CH 3 0
5)
\
O=N-N' CH, HO'
CH, C
//
HO MAM
CH 2 0
[CH 3 -N=N] o'
c
CH 3 X 4)
HCOO
'OH
Met hylazoxy formaldehyde Fig. 2. Comparison of metabolism of AOM and NDMA. Cytochrome P450IIEI-catalyzed activation reactions 1, 2 and 3 result in the formation of a reactive species such as the methyldiazonium ion. Reactions of the latter with water or phosphate will yield methanol or methylphosphate. Denitrosation reaction 5, likewise catalyzed by cytochrome P450IIE1, results in the detoxication of NDMA (31).
metabolism of MAM in rat liver under normal conditions. This question is not easily answered because inhibitors of alcohol dehydrogenase also inhibit cytochrome P450-mediated microsomal metabolism of MAM (10). Following chronic ethanol administration to rats, the in vivo metabolism of MAM is significantly increased (14). Because this effect is accompanied by enhanced liver microsomal metabolism of the carcinogen in vitro, it is possible that cytochrome P450-mediated metabolism of MAM may predominate in ethanol-induced rat liver. This seems likely, since rat liver alcohol dehydrogenase does not appear to be induced by ethanol (35). If both AOM and MAM are mainly metabolized by the same cytochrome P450 isozyme in rat liver, it is interesting to consider that the parent compound (AOM) may inhibit competitively the further metabolism of its product (MAM) by hepatic cytochrome P450HE1. Such inhibition would make more MAM available to the colon. Although the biological significance of such inhibition is difficult to assess because data on the kinetics of these reactions are not yet available, a possible consequence might be a greater systemic persistence of MAM after the administration of AOM than after the administration of MAM-acetate. Thus, AOM might be a more effective carcinogen for the colon than would be anticipated from an AOM — MAM precursor—product metabolic relationship. In conclusion, we have provided further evidence that AOM, MAM and NDMA are metabolically activated by the same liver cytochrome P450 isozyme in vitro. The relevance of cytochrome P450 mediated metabolism of AOM and MAM to the carcinogenicity and organotropism of these compounds is currently under investigation. Acknowledgements This work was supported by grants nos. CA31012 (E.S.F.) and CA37O37 (C.S.Y.) from the National Cancer Institute.
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