Carcinogenesis vol 11 no. 12 pp.2239-2243, 1990
dealkylaMomi amd demiitrosaitnoini Roles of cytoclirome P450IEE1 in of N-Mtrosodimethylamtoe and N- •mtorosodietlhyllamme m rat liver mkrosomes
Jeong-Sook H.Yoo, Hiroyuki Ishizaki and Chung S.Yang1 Laboratory for Cancer Research, Department of Chemical Biology and Pharmacogrtosy, College of Pharmacy, Rutgers University, Piscataway, NJ 08855-0789, USA 'To whom correspondence should be addressed
Introduction A'-Nitrosodimethylamine (NDMA*) and A'-nitrosodiethylamine (NDEA), widely occurring carcinogenic compounds in the environment, require metabolic activation to exert their cytotoxic and carcinogenic actions (1). The key metabolic activation step is believed to be the oxygenation of a-carbon, which leads to demethylation or deethylation and the formation of a methylating or ethylating species. The metabolism of NDMA is catalyzed by a cytochrome P450 (P450)-dependent enzyme system commonly known as NDMA demethylase (NDMAd) (2). Early studies, as reviewed by Lai and Arcos, revealed multiple Km values for this enzyme system in hepatic microsomes: NDMAd I, 0.2-0.3 mM and NDMAd U, 44-51 mM (2). Using rat, •Abbreviations: NDMA, A'-nitrosodimethylamine; NDEA, A'-nitrosodiethylamine; P450, cytochrome P450; NDMAd, NDMA demethylase; NDEAd, NDEA deethylase; P450IIE1 is an acetone-inducible isozyme in rats, which has been referred to as P450ac by Patten et al. (6), P45OJ by Ryan el al. (7) and P450LM3a by Koop el al. (8). © Oxford University Press
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A'-Nitrosodimethylamine (NDMA) and A'-nitrosodiethylamine (NDEA) are widely occurring nitrosamines and require enzyme-catalyzed activation for their carcinogenic actions. The low K,,, forms of the enzyme are generally considered to be important in the activation of environmental carcinogens. In this work we examined the role of cytochrome P450HE1—a constitutive enzyme that is also inducible by acetone, ethanol, fasting and other factors—in catalyzing the dealkylation and denitrosation of these two carcinogens. The experimentally determined Km value of NDMA demethylase depended upon the experimental conditions and was lower when lower protein concentrations were used. Low F^ values of 15-20 /xM were observed for NDMA demethylase with different preparations of mkrosomes. In the deethylation of NDEA, a low A^, of —40 /*M was observed for both control and acetone-induced mkrosomes. Immunoinhibition studies indicated that P450IEE1 was responsible for almost all the low An, NDMA demethylase activity in acetoneinduced mkrosomes and >80% in control microsomes. This enzyme was also responsible for about three-quarters of the low A^, NDEA deethylase activity in acetone-induced mkrosomes and about half in control microsomes. The denitrosation of NDMA and NDEA was inhibited to approximately the same extents as the dealkylation reactions under different experimental conditions, suggesting the involvement of the same enzyme and perhaps a common initial intermediate in these two types of reactions. The relevance of this work and its relationship to related information in the literature are discussed.
hamster and human liver microsomes, we observed a low apparent Km value of 60-70 /iM (3-5) in addition to the Km values of the previously reported NDMAd I and II (2). The low Km form has been shown to be inducible several-fold by factors such as acetone, ethanol, isopropanol, isoniazid, fasting and diabetes, and has been demonstrated to be due to the catalytic activity of P450IIE1 (9,10). Nevertheless, even much lower Km values than 60—70 yM have been reported for NDMA clearance or metabolism by isolated perfused rat liver (8.3 /tM), rat liver slices (25 /iM) and isolated guinea-pig liver cells (10-20 yM) (11-13). This raised a question as to whether this low Km form of NDMA metabolizing enzyme is the same as the low Km NDMAd studied in our laboratory (3-5,14). It was found previously that the presence of inhibitors in the assay system could increase the Km value. When the previously used NADPH-generating system, consisting of NADP, isocitrate and isocitrate dehydrogenase (3,4), was replaced by a new system consisting of NADP, glucose 6-phosphate and glucose-6-phosphate dehydrogenase, the observed low ^ m value was decreased to 40—50/iM (14). However, this Km value was still higher than the reported low Km values of 10-20 yM observed in isolated liver slices and cells (11-13). Due to its structural similarity to NDMA, the metabolism of NDEA is thought to be catalyzed by the same enzyme that mediates the metabolism of NDMA. In human microsomes, the NDEA deethylase (NDEAd) activities in different samples (when assayed with 0.2 mM NDEA) were proportional to the NDMAd activities (5). In rat liver microsomes, the rate for microsomal NDEA metabolism was enhanced by treatment of rats with acetone (15). Puccini et al. (16) also observed a similar result with rats and suggested that P450IIE1 was responsible for the low Km (0.43 mM) form of NDEAd. This Km value is, however, several-fold higher than the low Km value for NDMAd. The role of P45011E1 in NDEA metabolism requires further examination. In addition to the dealkylation reactions, A/-nitrosodialkylamines also undergo denitrosation reaction leading to the formation of nitrite (17,18). The mechanism of such reactions, however, is a subject of debate. Our previous results suggest that at low substrate concentrations, the denitrosation and demethylation reactions are most probably catalyzed by the same enzyme and share an initial common intermediate (a-nitrosamino radical) in an oxidative pathway (18,19). In contrast, Appel et al. proposed that the denitrosation of nitrosamines was due to a reductive pathway and the denitrosation of NDMA behaved differently from the demethylation of NDMA in terms of P450 isozyme specificity (17,20,21). The microsomal enzymes responsible for the denitrosation of NDEA also require investigation. In the present communication, we further studied the enzymology of NDMA and NDEA metabolism in rat liver microsomes by (i) investigating the nature of the Michaelis-Menten constant for the demethylation of low concentrations of NDMA, with special attention paid to the experimental conditions and methods of calculating the results;
J.-S.H.Yoo, H.Ishizaki and C.S.Yang
(ii) characterizing the low Km form of NDEAd; and (iii) examining the roles of P450HE1 in catalyzing the dealkylation and denitrosation of NDMA and NDEA, as well as the relationship between these two types of reactions by immunoinhibition studies. Materials and methods Chemicals NDEA, NADP, glucose 6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co. (St Louis, MO). NDMA was from AJdrich Chemical Co. (Milwaukee, WI). All other chemicals were of reagent grade. Microsomes and antibodies Male Sprague-Dawley rats from Taconic, Inc. (Germantown, NY), with body wts of 90-100 g, were subjected to one of the following treatments: no treatment (control), an intragastnc administration of acetone at a dosage of 5 ml/kg body wt 20 h before the animal was killed, and fasting for 48 h. Microsomes were prepared by differential centnfugation (4), and microsomal protein was determined by a modification of the Lowry method (14) Monoclonal antibodies against P450IIE1 (MAb 1-91-3) and control ascites fluid (HyHel - 9 ) were prepared as descnbed previously and the specificity of the antibodies toward P450IIE1 have been characterized (22).
NDEA deethylation and denitrosation assays The assay conditions were similar to those for the metabolism of NDMA, except that the tubes were capped to minimize the loss of acetaldehyde through evaporation. For A"m determinations, NDEA was present at concentrations of 10, 20, 40, 160 and 320 /iM After an incubation for 10 min, the reaction was terminated by the injection of a 0 1 ml mixture of 17% ZnSO4 and 0.55 mM semicarbazide which serves to trap the acetaldehyde. The mixture was deproCeinized and acetaldehyde was analyzed as the 2,4-dinitrophenylhydrazone derivative by HPLC based on the method of Farrelly (25) with conditions as described previously (5). The denitrosaUon assays were canned out separately in the absence of semicarbazide, which interferes with the denitrosation assay Imunochemical inhibition studies Antibodies against P450IIE1 (MAb 1-91-3) were mixed with microsomes for 5 min before other reagents were added to the reaction mixture, and the assays were conducted as described above. Control ascites fluid (HyHel -9) containing an equal amount of protein was used in the control tubes. Data calculation The Km value was calculated by the use of a computerized non-linear regression analysis (EnzFitter; Elsevier BIOSOFT, Cambridge, UK) or by the Eadie-Hofstee plot. The mean substrate concentration was used in the calculation to correct for the substrate utilization during the incubation period (26).
Results Nature of the low Km value of NDMAd Using acetone-induced microsomal protein in the range 0 . 1 - 0 . 8 mg/ml, apparent Km values of 15-22 ^M for NDMAd were obtained as estimated by the non-linear regression analysis (Table I), showing a dependency of Km on the 2240
Microsomal protein (mg/ml) 0.1 02 03 0.4 0.5 0.8 Microsomal protein versus Km
Non-linear regression
Eadie —Hofstee
15 ± 3' 16 ± 3 19 ± 1 21 ± 6 22C
16 16 19 21 21 24
r = 0 889 (P < 0 05)
r = 0.960 (P < 0.01)
2|
b
± 2 ± 3 ± 1 ± 6
"Mean ± SD of three determinations in duplicate ^Average of two determinations in duplicate. c Data of a single determination in duplicate.
and Vmu( values of NDMAd in different
Table II. Comparison of tht types of microsomes" Microsomal source
Non-linear regression
Control 1 Control 2 Acetone-induced 1 Acetone-induced 2 Fasting (48 h)
22 20 24 15 15
Eadie — Hofstee
Krot
*m ± ± ± ± ±
1 4 2 3 2
1.52 1.60 8.16 751 4.13
± ± ± ± ±
0.10 0 12 0 29 0.66 0.19
22 20 24 15 15
± ± ± ± ±
1 3 2 3 2
l. 51 l. 59 8 16 7. 51 4. 13
± ± ± ± ±
009 0.12 0.28 0.65 0.20
"Values are expressed as mean ± SD of three determinations in duplicate with 0.5 mg microsomal protein/ml incubation.
microsomal protein concentration (r = 0.889, P < 0.05). When extrapolated to zero protein concentration, a Km value of 15 jtM was obtained. Gillette (27) reported that the Km value for the metabolism of imipramine was a function of the enzyme (microsomal protein) concentration, but was independent of the enzyme concentration if the aqueous (unbound) substrate concentration was used to estimate the Km value. The presently observed dependency of the apparent Km on protein concentration remained, however, even when the aqueous substrate concentration, estimated from the oil to water partition coefficient of Kp = 0.03 for NDMA (28), was used for the calculation (data not shown). The result obtained by the Eadie-Hofstee plot did not differ from that by the non-linear regression analysis: Km values ranged from 16 to 24 pM with a dependency of Km on the amounts of protein (r = 0.960, P < 0.01) with an extrapolated Km value of 15 fiM at zero protein concentration. In order to determine whether the Km value varies with different microsomal preparations, the Km values obtained with five different microsomal preparations were compared. As shown in Table n, the Km values estimated by the non-linear regression analysis ranged from 15 to 24 /iM (median = 20 /tM), which were not significantly different as analyzed by the Newman — Keuls multiple comparison test (29). The Km values estimated by the Eadie-Hofstee plot again did not differ from those by the non-linear regression analysis. The values from 15 to 24 yM are probably due to experimental variabilities as well as some subtle unknown differences in the microsomes. Inhibition of NDMA demethylation and denitrosation by antibodies against P450IIE1 A substrate concentration of 0.2 mM was used for assaying the low Km form of NDMAd, and 4 mM, a concentration used in
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NDMA demelhylation and denitrosation assays The assays were based on the methods of Nash (23) and Appel and Graf (24). All reactions were run in duplicate in borosilicate glass test tubes In brief, the assay mixture (0 5 ml) contained 50 mM Tns-HCl (pH 7.4), 10 mM MgCl2, 150 mM KC1, an NADPH-generating system (0.4 mM NADP, 10 mM glucose 6-phosphate and 0.2 unit glucose-6-phosphate dehydrogenase), microsomes at the amounts indicated, and NDMA. For Km determinations, NDMA concentrations of 40, 80, 160, 320 and 640 ^M were used. The components were preincubated at 37°C for 2 nun and the reaction was initiated by the addition of the NADPH-generating mixture. After an incubation period of 10 min the reaction was terminated by the addition of 0.05 ml each of 25% ZnSO4 and saturated Ba(OH>2, and the mixture was centrifuged. For the determination of HCHO, 0.35 ml of supernatant was mixed with 0.15 ml of a concentrated Nash reagent (5 g ammonium sulfate and 0.1 ml of acetylacetone in 6 ml of 3 % acetic acid, made fresh). After the mixture is incubated at 50°C for 30 min, absorbance at 412 nm was measured. In some experiments, the supernatant from 1 ml assay mixture was split into two 0.35 ml aliquots for the determination of HCHO and nitrite. For the nitrite determination, the 0.35 ml aliquot was mixed with 75 jtl of 100 mM sulfanilamide (in 3 N HC1) and after 5 min, 75 pi of 1 mM N(l-naphthyl)ethylenediamine dihydrochlonde (in 3 N HCI) were added. The mixture was kept at room temperature for 10 min and absorbance at 546 nm was measured. The differences between the duplicated assays were < 10%.
Table I. Effect of microsomal protein concentration on the Km value of NDMAd
Metabolism of NDMA and NDEA by P450UE1
1 Table III. Inhibition of NDMA metabolism by antibodies against P450IIE1
Microsomes
Antibodies
0«g) Acetone-induced Acetone-induced Acetone-induced Control Control
40 80 200 40 80
% inhibition 0.2 mM NDMA Demethylation
Denitrosation
4 mM NDMA Demethylation
Denitrosation
74 93 98 73 81
68 83 95 65 83
92 52 62
87 39 53
'Antibodies (expressed in /ig protein) were added to either acetone-induced rat liver microsomes (0.12 mg protein) or control microsomes (0.3 mg) in 0.5 ml and the incubation was carried out for 10 min. The demethylase activities (nmol/min/mg) in the presence of control ascites fluid were (i) for acetone-induced microsomes, 6.0 and 6.1 when assayed with 0.2 and 4 mM NDMA respectively; and (ii) for control microsomes, the corresponding values were 1.2 and 1 6. The corresponding denitrosation rates were (i) 0.56 and 0.59, and (ii) 0 14 and 0.15. ''Not determined.
Low Km values for NDEAd The substrate dependency of NDEAd activity was studied with both control and acetone-induced microsomes (Figure 1). At these low substrate concentrations used, in three determinations, an apparent Km of 40.0 ± 3.3 ^M and K ^ of 0.63 ± 0.17 nmoL/min/mg protein were observed with control microsomes; the corresponding values with acetone-induced microsomes were 42.2 ± 8.3 /iM and 4.77 ± 0.88 nmol/ min/mg. At higher substrate concentrations, the control microsomes had higher activities than estimated from the above Km using the Michaelis — Menten equation, suggesting the presence of higher Km forms of NDEAd. However, this form was not fully characterized. The results indicate that a low Km form of NDEAd exists in both control and acetone-induced microsomes and it is induced several-fold by acetone treatment, suggesting that P450IIE1 is mainly responsible for this activity. Inhibition of NDEA metabolism by antibodies against P450IIEJ Similar to NDMA metabolism, the deethylation and denitrosation of NDEA were inhibited by antibodies against P450HE1 (Table IV). However, the extent of inhibition was lower than that for NDMA. With acetone-induced microsomes, 200 \i% of antibodies inhibited the deethylation reaction 77 and 44% respectively when assayed with 0.16 and 4 mM of NDEA. With control microsomes, the corresponding extents of inhibition were 47 and 23 %. According to the results from Table HI and other studies, this amount of antibodies should inhibit P450IIE] activity almost completely. It appears that both types of reactions were inhibited to a lesser extent in control microsomes than acetone-induced microsomes, and when assayed at 4 mM than 0.16 mM NDEA. Although there were more variabilities in the denitrosation data due to the rather low denitrosation activity,
150
200
350
[NDEA] Fig. 1. Substrate dependence of NDEA metabolism by rat liver microsomes. The incubation mixture contained either acetone-induced (0.075 mg) or control (0.4 mg) microsomes and NDEA (10-320 /tM) in 0.5 ml. The incubation was carried out for 10 nun at 37°C Km and l/!mx were calculated by non-linear regression analysis. Km and Vm^ for acetoneinduced microsomes were 49.4 /iM and 5.27 nmol/min/mg respectively. The corresponding values for control microsomes were 37.8 and 0.683.
the results in Table IV are consistent with the concept that the denitrosation and deethylation reactions were inhibited by similar magnitudes. Discussion The present work demonstrates that rat liver microsomes displayed apparent Km values of 14—24 /tM and these values are close to the values of 10—25 yM observed with liver slices and liver cells (12,13). Recent work employing a radiometric assay for NDMAd with a slight modification from Yoo et al. (14), using 10-100/iM NDMA and 10-40 /tg microsomal protein (in 0.1 ml incubation), also yielded Km values of 15—20/tM. The difference between the presently observed values and our previously reported values is not fully understood. It is possible that our currently used NADPH-generating system (especially NADP and glucose-6-phosphate dehydrogenase) contained less inhibitors than that used previously. In addition, the correction of substrate utilization may also have contributed to the difference. For example, our previously reported low Km values of 40—50 /*M, calculated using initial substrate concentrations (14), were decreased to 34-42 pM when mean substrate concentrations were used for the calculation. The use of higher amounts of protein in general resulted in higher Km values. The use of plastic tubes for the incubation also resulted in higher Km values than with glass tubes. Although the possible bias of the 2241
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many previous studies, was also used. When antibodies against P450IIE1 were added to the incubation mixture containing acetone-induced microsomes, both the demethylation and denitrosation of NDMA were inhibited at a dose-dependent manner (Table III). With 200 /tg antibodies (protein), both reactions were inhibited ~95 and - 9 0 % when assayed with 0.2 and 4 mM NDMA respectively. With control microsomes, antibodies (80 /*g) inhibited both the demethylation and denitrosation of NDMA —80 and —60% when assayed with 0.2 and 4 mM NDMA respectively. The extent of inhibition was generally higher with acetone-induced microsomes than with control microsomes when the lower substrate concentration was used. Under each set of experimental conditions, the demethylation and denitrosation were inhibited by similar magnitudes.
J.-S.H.Yoo, H.Ishizaki and C.S.Yang
Tab4e IV. Inhibition of NDEA metabolism by antibodies against P450IIE1" Microsomes
Acetone-induced Acetone-induced Control Control
Antibodies G*g)
40 200 40 200
% inhibition 0.16 mM NDEA Deethylation
Denitrosation
4mM NDEA Deethylation
Denitrosation
69 77 31 47
71 93 29 65
35 44 7 23
40 42 13 30
"Antibodies (expressed in /ig procein) were added to either acetone-induced rat liver microsomes (0.12 mg protein) or control microsomes (0.7 mg) in 0.5 ml and the incubation was earned out for 10 mm. The deethylase activities (nmol/min/mg) in the presence of control ascites fluid were (i) for acetone-induced microsomes, 2.3 and 2.3 when assayed with 0.16 and 4 mM NDEA respectively, and (ii) for control microsomes, the corresponding values were 0.58 and 0.87. The corresponding denitrosation rates were (i) 0.44 and 0.41, and (ii) 0 12 and 0.22.
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of NDMA and NDEA were inhibited to similar extents by antibodies against P450HE1, illustrating the close relationship between these two types of reactions. This concept is different from the thesis advanced by Amelizad et al. (21), suggesting that the demethylation and denitrosation of NDMA are mediated by different P450 forms and via different mechanisms. Using antiserum against P450 PB3a (UBI) these authors observed an 80% inhibition of the demethylation, but not the denitrosation, of 3 mM NDMA in ethanol-induced microsomes. This result suggests that P450IIB1, not HE1, is responsible for most of the NDMAd activity. Since ethanol can induce P450IIE1 to the same level as acetone (31,32), this result also contradicts our results in Table m. The reason for the difference is not known, but may be related to the experimental conditions and the specificity of the antibodies used. It is noted that their uninhibited NDMAd activity, 35 nmol/mg (per 30 min), was only - 1 5 - 1 6 % those observed in our laboratory (31). In addition, P450HB1 is known to be expressed at very low levels, if at all, in rat liver and may be induced by ethanol to a low level (33); it is unlikely to account for 80% of the NDMA demethylase. In conclusion, the present results demonstrate the role of P450IIE1 in the metabolism of low concentrations of NDMA and NDEA, and most likely is responsible for the low apparent Km values, 15-20 /*M for NDMAd and - 4 0 /tM for NDEAd, in microsomes. Since cells are seldom exposed to high concentrations of these carcinogens, this low Km form of enzyme is believed to be more important in carcinogen activation than higher Km forms. The denitrosation of NDMA and NDEA is shown to accompany closely the dealkylation reactions under different experimental conditions. The role of P450IIE1 and the low Km form of NDMAd in the metabolic activation of NDMA has been demonstrated both in vitro and in vivo (4,34 — 36). The present work points out that P450HE1 also plays an important role in the metabolic activation of NDEA, although the role is not as prominent as is for NDMA. Acknowledgements We thank Mr M.J.Lee for capable technical help and Ms Dorothy Wong for excellent secretarial assistance in the preparation of this manuscript. This work was supported by grams from the National Institutes of Health CA37037, ES03938, and GM38336.
References 1. Magee.P.N. and BamesJ.M. (1967) Carcinogenic nitroso compounds Ath' Cancer Res.. 10. 163-246. 2. Lai,D.Y. and ArcosJ.C. (1980) Miiureview. dialkylmtrosamine bioactivation and carcinogenesis. Life 5a'., 27, 2149 — 2165. 3. Tu.Y.Y. and Yang.C.S. (1983) High-affinity nitrosamine dealkylase system in rat liver microsomes and its induction by fasting Cancer Res.. 43. 623-629.
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Eadie—Hofstee plot has been discussed (30), the present results indicate that similar Km values were observed with both methods when the data points were not scattered and the previously observed higher Km values (40—50 /*M) were not due to the use of the Eadie-Hofstee plot. The presently observed apparent Km values for NDEAd, ~ 40 /iM was much lower than the 430 /iM reported by Puccini et al. (16). The reason for this discrepancy is not clear. Judging from the statement that these authors could not detect the acetaldehyde formed from 1 mM NDEA in incubations with control microsomes, our method, which affords the detection of acetaldehyde in incubations with 10 and 20 /tM NDEA (Figure 1), appears to be more sensitive. The use of lower substrate concentrations enabled us to characterize the presently observed low Km form of NDEAd. If we use datum points obtained in incubations with 0.16, 0.32, 1 and 4 mM NDEA in a doublereciprocal plot, a much higher Km value would be obtained (data not shown). The possible presence of inhibitors in the assay system for Puccini et al. (16), though not apparent from their report, may also be a factor. We and other investigators have experienced such problems in studying the low Km form of NDMAd (14). The role of P450ITE1 in the microsomal metabolism of NDMA and NDEA is demonstrated by the immunoinhibition studies (Tables HI and IV). Consistent with the idea that P450IIE1 is responsible for the low Km form of NDMAd, almost all the activity in acetone-induced microsomes was inhibited when assayed with 0.2 mM NDMA. At 4 mM NDMA, only ~ 10% of the NDMAd activity was not inhibited, possibly due to the contribution by other P450 isozymes. The contribution by other P450 isozymes appears to be more in control microsomes, especially when assayed with 4 mM NDMA. Although P450IIE1 is still responsible for most of the low Km NDEAd activity in acetone-induced microsomes, other P450 isozymes may be responsible for about half of the activity with 4 mM NDEA. In control microsomes, the relative importance of P450IIE1 decreased, especially when assayed with 4 mM NDEA. The results again suggest that P450HE1 is responsible for the low Km form of NDEAd and other P450 isozymes also catalyze the oxidation of NDEA, especially when present at higher concentrations. The concept is consistent with previous studies with human microsomes (5) and is important in understanding previously published results, usually derived from studies using high concentrations (millimolar rather than micromolar) of NDEA. Because of the low sensitivity of the denitrosation assay, we did not conduct thorough determinations on the low Km values for the denitrosation reactions. It is apparent from the results in Tables HI and IV that the denitrosation and dealkylation reactions
Metabolism of NDMA and NDEA by P450HE1 29. Dayton,CM. (1970) The Design of Educational Experiments. McGraw-Hill, New York. 30. Eisenthal.R. and Comish-Bowden.A. (1974) The direct linear plot. Biochem. J., 139, 715-720. 31. Peng,R Tu,Y.Y. and Yang.C.S. (1982) The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamtne demethylase by ethanol Carcinogenesis, 3, 1457-1461. 32. Tu,Y.Y. Peng.R. Chang,Z.-F. and Yang.C.S. (1983) Induction of a high affinity nitrosamine demethylase in rat liver microsomes by acetone and isopropanol. Chem.-Biol. Interactions, 44, 247—260. 33. Johansson.I., Ekstrom.G., Schofte.B., Puzycki.D., Jomvall.H. and IngelmanSundberg.M. (1988) Ethanol-, fasting-, acetone-inducible cytochromes P-450 in rat liver: regulation and characteristics of enzymes belonging to the HB and IIE gene subfamilies. Biochemistry, 27, 1925-1934. 34. Lorr.N.A., Miller,K.W., Chung, H.R.'and Yang.C.S. (1984) Potentiation of the hepatoxicity of A'-nitrosodimethylamine by fasting, diabetes, acetone, and isopropanol. Toxicol. Appl. Pharmacol., 73, 423—431. 35. Hong.J. and Yang.C.S. (1985) The nature of microsomal A'nitrosodimethylamine demethylase and its role in carcinogen activation. Carcinogenesis, 6, 1805-1809. 36. Yoo.J.-S.H. and Yang.C.S. (1985) Enzyme specificity in the metabolic activation of A'-nitrosodimethylamine to a mutagen for Chinese hamster V79 cells. Cancer Res., 45, 5569-5574. Downloaded from http://carcin.oxfordjournals.org/ at Yale University on May 15, 2015
4. YooJ.-S.H., Ning.S.M , Patten.C.J. and Yang,C S. (1987) Metabolism and activation of A'-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals. Cancer Res., 47, 992-998. 5.YooJ.-S.H., Guengerich.F.P. and Yang.C.S. (1988) Metabolism of Nnitrosodialkylamines by human liver microsomes. Cancer Res., 48, 1499-1504. 6 Patten,C.J.,Ning,S.M.,Lu,A.Y.H. and Yang.C.S (1986) Acetone-inducible cytochrome P-450: purification, catalytic activity and interaction with cytochrome bi. Arch. Biochem. Biophys., 251, 629-638. 7. Ryan.D.E., Ramanathan.L., Iida,S. Thomas,P.E., Haniu.M., Shively.J.E., Ueber,C.S. and Levin,W. (1985) Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoiuazid. J Biol. Oxem., 260, 6385-6393. 8. Koop.D.R., Morgan.E.T., Tarr,G.E. and Coon,M J. (1982) Purification and characterization of a unique isozyme of cytochrome P^50 from liver microsomes of ethanol-treated rabbits. J. Biol Chem., 257, 8472-8480. 9. Yang.C.S. and YooJ.-S.H. (1988) Dietary effects on drug metabolism by the mixed-function oxidase system. Pharmacol. Ther., 38, 53-72. tO. Yang.C.S., Y00J.-S.H., Ishizaki.H. and Hong,J. (1990) Cytochrome P450IIE1: roles in nitrosamine metabolism and mechanisms of regulation. Drug. Metab. Rev., 22, 147-159. 11. Skipper,P.L., Tomera,J.F., Wishnok,J.S., Brunengraber.H. and Tannenbaum.S.R. (1983) Pharmacokinetic model for A'-nitrosodimethylamine based on Michaelis—Menten constants determined with the isolated perfused rat liver. Cancer Res., 43, 4786-4790 12. Swann.P.F. (1984) Effect of ethanol on nitrosamine metabolism and distribution. Implications for the role of nitrosamines in human cancer and for the influence of alcohol consumption on cancer incidence. In O'Neill.I.K., von Borstel.R.C, Miller.C.T., Long,J. and Bartch.H. (eds), N-Mtroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer, IARC Scientific Publication. IARC Lyon, pp. 501-512. 13.Hauber,G., Frommberger.R., Remmer.H. and Schwenk.M. (1984) Metabolism of low concentrations of A'-nitrosodirnethylamine in isolated liver cells of the guinea pig. Cancer Res., 44, 1343-1346. 14. Yoo.J.-S.H., Cheung.R.J., Patten.C.J , Wade.D. and Yang.C.S (1987) Nature of A'-nhrosodimethylamine demethylase and its inhibitors. Cancer Res., 47, 3378-3383. 15. Lee.M., Ishizaki,H., Brady,J.F. and Yang.C.S. (1989) Substrate specificity and alkyl group selectivity in the metabolism of A'-nhrosodialkylamines. Cancer Res., 49, 1470-1474. 16 Puccini,P., Fiorio.R., Longo.V. and Gervasi,P.G. (1989) High affinity diethylnitrosamine-deethylase in liver microsomes from acetone-induced rats. Carcmogenesis, 10, 1629-1634. 17. Appel.K.E., Schrenk.D., Schwarz.M. Mahr.B. and Kunz.W. (1980) Denitrosation of A'-nitrosomorpholine by liver microsomes; possible role of cytochrome P^50. Cancer Lett., 9, 13-20. 18. Lorr.N.A., Tu.Y.Y. and Yang.C.S. (1982) The nature of nitrosamine denitrosation by rat liver microsomes. Caranogenesis, 3, 1039-1043. 19. Wade.D , Yang.C.S., Metral.C.J., RomanJ.M., HrabieJ.A., Riggs.C.W , Anjo.T., Keefer,L.K. and Mico.B.A. (1987) Deuterium isotope effect on denitrosation and demethylation of A'-nitrosodimethylamine by rat liver microsomes. Cancer Res., 47, 3373-3377. 20 Appel.K.E., Ruhl.C.S. and Hildebrandt.A.G (1985) Oxidative dealkylation and reductive denitrosation of nitrosomethylaniline in vitro Chem.-Biol. Interactions, 53, 69-76. 21 Amehzad.Z., Apple.K.E., Oesch.F. and Hildebrandt.A.G. (1988) Effect of antibodies against cytochrome P-450 on demethylation and denitrosation of A'-nitrosodimethylamine and A'-nitrosomethylaniline. J. Cancer Res. CUn. Oncol., 114, 380-384. 22. Ko,I.-Y., Park.S.S., Song.B.J., Patten.C, Tan.Y., Hah,Y.C. Yang.C.S. and Gelboin.H.V (1987) Monoclonal antibodies to ethanol-induced rat liver cytochrome P^50 that metabolizes aniline and nitrosamines Cancer Res. ,47, 3101-3109. 23. Nash.T. (1953) The colonmetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J., 55, 416-421. 24. Appel,K.E. and Graf,H. (1982) Metabolic nitrite formation from Nnitrosamines: evidence for a cytochrome P-450 dependent reaction. Caranogenesis, 3, 293-296. 25. FarrellyJ.G. (1980) A new assay for the microsomal metabolism of nitrosamines. Cancer Res., 40, 3241-3244. 26. Segel.I.W. (1975) Enzyme Kinetics. John Wiley, New York 27. Gillette.J.R. (1983) Metabolism of drugs and other foreign compounds by enzymatic mechanisms. Prog. Drug. Res., 6, 11-73. 28. Dnickrey.H., Preussmann.R , Ivankovic.S. and Schmahl,D. (1967) Organotrope carcinogene Wirkung bei 65 verschiedenen A'-Nitroso-Verbindungen an BD-Ratten. S. Krebsforsch., 69, 103-201.
Received on July 30, 1990; accepted on September 6, 1990
2243
dealkylaMomi amd demiitrosaitnoini Roles of cytoclirome P450IEE1 in of N-Mtrosodimethylamtoe and N- •mtorosodietlhyllamme m rat liver mkrosomes
Jeong-Sook H.Yoo, Hiroyuki Ishizaki and Chung S.Yang1 Laboratory for Cancer Research, Department of Chemical Biology and Pharmacogrtosy, College of Pharmacy, Rutgers University, Piscataway, NJ 08855-0789, USA 'To whom correspondence should be addressed
Introduction A'-Nitrosodimethylamine (NDMA*) and A'-nitrosodiethylamine (NDEA), widely occurring carcinogenic compounds in the environment, require metabolic activation to exert their cytotoxic and carcinogenic actions (1). The key metabolic activation step is believed to be the oxygenation of a-carbon, which leads to demethylation or deethylation and the formation of a methylating or ethylating species. The metabolism of NDMA is catalyzed by a cytochrome P450 (P450)-dependent enzyme system commonly known as NDMA demethylase (NDMAd) (2). Early studies, as reviewed by Lai and Arcos, revealed multiple Km values for this enzyme system in hepatic microsomes: NDMAd I, 0.2-0.3 mM and NDMAd U, 44-51 mM (2). Using rat, •Abbreviations: NDMA, A'-nitrosodimethylamine; NDEA, A'-nitrosodiethylamine; P450, cytochrome P450; NDMAd, NDMA demethylase; NDEAd, NDEA deethylase; P450IIE1 is an acetone-inducible isozyme in rats, which has been referred to as P450ac by Patten et al. (6), P45OJ by Ryan el al. (7) and P450LM3a by Koop el al. (8). © Oxford University Press
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A'-Nitrosodimethylamine (NDMA) and A'-nitrosodiethylamine (NDEA) are widely occurring nitrosamines and require enzyme-catalyzed activation for their carcinogenic actions. The low K,,, forms of the enzyme are generally considered to be important in the activation of environmental carcinogens. In this work we examined the role of cytochrome P450HE1—a constitutive enzyme that is also inducible by acetone, ethanol, fasting and other factors—in catalyzing the dealkylation and denitrosation of these two carcinogens. The experimentally determined Km value of NDMA demethylase depended upon the experimental conditions and was lower when lower protein concentrations were used. Low F^ values of 15-20 /xM were observed for NDMA demethylase with different preparations of mkrosomes. In the deethylation of NDEA, a low A^, of —40 /*M was observed for both control and acetone-induced mkrosomes. Immunoinhibition studies indicated that P450IEE1 was responsible for almost all the low An, NDMA demethylase activity in acetoneinduced mkrosomes and >80% in control microsomes. This enzyme was also responsible for about three-quarters of the low A^, NDEA deethylase activity in acetone-induced mkrosomes and about half in control microsomes. The denitrosation of NDMA and NDEA was inhibited to approximately the same extents as the dealkylation reactions under different experimental conditions, suggesting the involvement of the same enzyme and perhaps a common initial intermediate in these two types of reactions. The relevance of this work and its relationship to related information in the literature are discussed.
hamster and human liver microsomes, we observed a low apparent Km value of 60-70 /iM (3-5) in addition to the Km values of the previously reported NDMAd I and II (2). The low Km form has been shown to be inducible several-fold by factors such as acetone, ethanol, isopropanol, isoniazid, fasting and diabetes, and has been demonstrated to be due to the catalytic activity of P450IIE1 (9,10). Nevertheless, even much lower Km values than 60—70 yM have been reported for NDMA clearance or metabolism by isolated perfused rat liver (8.3 /tM), rat liver slices (25 /iM) and isolated guinea-pig liver cells (10-20 yM) (11-13). This raised a question as to whether this low Km form of NDMA metabolizing enzyme is the same as the low Km NDMAd studied in our laboratory (3-5,14). It was found previously that the presence of inhibitors in the assay system could increase the Km value. When the previously used NADPH-generating system, consisting of NADP, isocitrate and isocitrate dehydrogenase (3,4), was replaced by a new system consisting of NADP, glucose 6-phosphate and glucose-6-phosphate dehydrogenase, the observed low ^ m value was decreased to 40—50/iM (14). However, this Km value was still higher than the reported low Km values of 10-20 yM observed in isolated liver slices and cells (11-13). Due to its structural similarity to NDMA, the metabolism of NDEA is thought to be catalyzed by the same enzyme that mediates the metabolism of NDMA. In human microsomes, the NDEA deethylase (NDEAd) activities in different samples (when assayed with 0.2 mM NDEA) were proportional to the NDMAd activities (5). In rat liver microsomes, the rate for microsomal NDEA metabolism was enhanced by treatment of rats with acetone (15). Puccini et al. (16) also observed a similar result with rats and suggested that P450IIE1 was responsible for the low Km (0.43 mM) form of NDEAd. This Km value is, however, several-fold higher than the low Km value for NDMAd. The role of P45011E1 in NDEA metabolism requires further examination. In addition to the dealkylation reactions, A/-nitrosodialkylamines also undergo denitrosation reaction leading to the formation of nitrite (17,18). The mechanism of such reactions, however, is a subject of debate. Our previous results suggest that at low substrate concentrations, the denitrosation and demethylation reactions are most probably catalyzed by the same enzyme and share an initial common intermediate (a-nitrosamino radical) in an oxidative pathway (18,19). In contrast, Appel et al. proposed that the denitrosation of nitrosamines was due to a reductive pathway and the denitrosation of NDMA behaved differently from the demethylation of NDMA in terms of P450 isozyme specificity (17,20,21). The microsomal enzymes responsible for the denitrosation of NDEA also require investigation. In the present communication, we further studied the enzymology of NDMA and NDEA metabolism in rat liver microsomes by (i) investigating the nature of the Michaelis-Menten constant for the demethylation of low concentrations of NDMA, with special attention paid to the experimental conditions and methods of calculating the results;
J.-S.H.Yoo, H.Ishizaki and C.S.Yang
(ii) characterizing the low Km form of NDEAd; and (iii) examining the roles of P450HE1 in catalyzing the dealkylation and denitrosation of NDMA and NDEA, as well as the relationship between these two types of reactions by immunoinhibition studies. Materials and methods Chemicals NDEA, NADP, glucose 6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co. (St Louis, MO). NDMA was from AJdrich Chemical Co. (Milwaukee, WI). All other chemicals were of reagent grade. Microsomes and antibodies Male Sprague-Dawley rats from Taconic, Inc. (Germantown, NY), with body wts of 90-100 g, were subjected to one of the following treatments: no treatment (control), an intragastnc administration of acetone at a dosage of 5 ml/kg body wt 20 h before the animal was killed, and fasting for 48 h. Microsomes were prepared by differential centnfugation (4), and microsomal protein was determined by a modification of the Lowry method (14) Monoclonal antibodies against P450IIE1 (MAb 1-91-3) and control ascites fluid (HyHel - 9 ) were prepared as descnbed previously and the specificity of the antibodies toward P450IIE1 have been characterized (22).
NDEA deethylation and denitrosation assays The assay conditions were similar to those for the metabolism of NDMA, except that the tubes were capped to minimize the loss of acetaldehyde through evaporation. For A"m determinations, NDEA was present at concentrations of 10, 20, 40, 160 and 320 /iM After an incubation for 10 min, the reaction was terminated by the injection of a 0 1 ml mixture of 17% ZnSO4 and 0.55 mM semicarbazide which serves to trap the acetaldehyde. The mixture was deproCeinized and acetaldehyde was analyzed as the 2,4-dinitrophenylhydrazone derivative by HPLC based on the method of Farrelly (25) with conditions as described previously (5). The denitrosaUon assays were canned out separately in the absence of semicarbazide, which interferes with the denitrosation assay Imunochemical inhibition studies Antibodies against P450IIE1 (MAb 1-91-3) were mixed with microsomes for 5 min before other reagents were added to the reaction mixture, and the assays were conducted as described above. Control ascites fluid (HyHel -9) containing an equal amount of protein was used in the control tubes. Data calculation The Km value was calculated by the use of a computerized non-linear regression analysis (EnzFitter; Elsevier BIOSOFT, Cambridge, UK) or by the Eadie-Hofstee plot. The mean substrate concentration was used in the calculation to correct for the substrate utilization during the incubation period (26).
Results Nature of the low Km value of NDMAd Using acetone-induced microsomal protein in the range 0 . 1 - 0 . 8 mg/ml, apparent Km values of 15-22 ^M for NDMAd were obtained as estimated by the non-linear regression analysis (Table I), showing a dependency of Km on the 2240
Microsomal protein (mg/ml) 0.1 02 03 0.4 0.5 0.8 Microsomal protein versus Km
Non-linear regression
Eadie —Hofstee
15 ± 3' 16 ± 3 19 ± 1 21 ± 6 22C
16 16 19 21 21 24
r = 0 889 (P < 0 05)
r = 0.960 (P < 0.01)
2|
b
± 2 ± 3 ± 1 ± 6
"Mean ± SD of three determinations in duplicate ^Average of two determinations in duplicate. c Data of a single determination in duplicate.
and Vmu( values of NDMAd in different
Table II. Comparison of tht types of microsomes" Microsomal source
Non-linear regression
Control 1 Control 2 Acetone-induced 1 Acetone-induced 2 Fasting (48 h)
22 20 24 15 15
Eadie — Hofstee
Krot
*m ± ± ± ± ±
1 4 2 3 2
1.52 1.60 8.16 751 4.13
± ± ± ± ±
0.10 0 12 0 29 0.66 0.19
22 20 24 15 15
± ± ± ± ±
1 3 2 3 2
l. 51 l. 59 8 16 7. 51 4. 13
± ± ± ± ±
009 0.12 0.28 0.65 0.20
"Values are expressed as mean ± SD of three determinations in duplicate with 0.5 mg microsomal protein/ml incubation.
microsomal protein concentration (r = 0.889, P < 0.05). When extrapolated to zero protein concentration, a Km value of 15 jtM was obtained. Gillette (27) reported that the Km value for the metabolism of imipramine was a function of the enzyme (microsomal protein) concentration, but was independent of the enzyme concentration if the aqueous (unbound) substrate concentration was used to estimate the Km value. The presently observed dependency of the apparent Km on protein concentration remained, however, even when the aqueous substrate concentration, estimated from the oil to water partition coefficient of Kp = 0.03 for NDMA (28), was used for the calculation (data not shown). The result obtained by the Eadie-Hofstee plot did not differ from that by the non-linear regression analysis: Km values ranged from 16 to 24 pM with a dependency of Km on the amounts of protein (r = 0.960, P < 0.01) with an extrapolated Km value of 15 fiM at zero protein concentration. In order to determine whether the Km value varies with different microsomal preparations, the Km values obtained with five different microsomal preparations were compared. As shown in Table n, the Km values estimated by the non-linear regression analysis ranged from 15 to 24 /iM (median = 20 /tM), which were not significantly different as analyzed by the Newman — Keuls multiple comparison test (29). The Km values estimated by the Eadie-Hofstee plot again did not differ from those by the non-linear regression analysis. The values from 15 to 24 yM are probably due to experimental variabilities as well as some subtle unknown differences in the microsomes. Inhibition of NDMA demethylation and denitrosation by antibodies against P450IIE1 A substrate concentration of 0.2 mM was used for assaying the low Km form of NDMAd, and 4 mM, a concentration used in
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NDMA demelhylation and denitrosation assays The assays were based on the methods of Nash (23) and Appel and Graf (24). All reactions were run in duplicate in borosilicate glass test tubes In brief, the assay mixture (0 5 ml) contained 50 mM Tns-HCl (pH 7.4), 10 mM MgCl2, 150 mM KC1, an NADPH-generating system (0.4 mM NADP, 10 mM glucose 6-phosphate and 0.2 unit glucose-6-phosphate dehydrogenase), microsomes at the amounts indicated, and NDMA. For Km determinations, NDMA concentrations of 40, 80, 160, 320 and 640 ^M were used. The components were preincubated at 37°C for 2 nun and the reaction was initiated by the addition of the NADPH-generating mixture. After an incubation period of 10 min the reaction was terminated by the addition of 0.05 ml each of 25% ZnSO4 and saturated Ba(OH>2, and the mixture was centrifuged. For the determination of HCHO, 0.35 ml of supernatant was mixed with 0.15 ml of a concentrated Nash reagent (5 g ammonium sulfate and 0.1 ml of acetylacetone in 6 ml of 3 % acetic acid, made fresh). After the mixture is incubated at 50°C for 30 min, absorbance at 412 nm was measured. In some experiments, the supernatant from 1 ml assay mixture was split into two 0.35 ml aliquots for the determination of HCHO and nitrite. For the nitrite determination, the 0.35 ml aliquot was mixed with 75 jtl of 100 mM sulfanilamide (in 3 N HC1) and after 5 min, 75 pi of 1 mM N(l-naphthyl)ethylenediamine dihydrochlonde (in 3 N HCI) were added. The mixture was kept at room temperature for 10 min and absorbance at 546 nm was measured. The differences between the duplicated assays were < 10%.
Table I. Effect of microsomal protein concentration on the Km value of NDMAd
Metabolism of NDMA and NDEA by P450UE1
1 Table III. Inhibition of NDMA metabolism by antibodies against P450IIE1
Microsomes
Antibodies
0«g) Acetone-induced Acetone-induced Acetone-induced Control Control
40 80 200 40 80
% inhibition 0.2 mM NDMA Demethylation
Denitrosation
4 mM NDMA Demethylation
Denitrosation
74 93 98 73 81
68 83 95 65 83
92 52 62
87 39 53
'Antibodies (expressed in /ig protein) were added to either acetone-induced rat liver microsomes (0.12 mg protein) or control microsomes (0.3 mg) in 0.5 ml and the incubation was carried out for 10 min. The demethylase activities (nmol/min/mg) in the presence of control ascites fluid were (i) for acetone-induced microsomes, 6.0 and 6.1 when assayed with 0.2 and 4 mM NDMA respectively; and (ii) for control microsomes, the corresponding values were 1.2 and 1 6. The corresponding denitrosation rates were (i) 0.56 and 0.59, and (ii) 0 14 and 0.15. ''Not determined.
Low Km values for NDEAd The substrate dependency of NDEAd activity was studied with both control and acetone-induced microsomes (Figure 1). At these low substrate concentrations used, in three determinations, an apparent Km of 40.0 ± 3.3 ^M and K ^ of 0.63 ± 0.17 nmoL/min/mg protein were observed with control microsomes; the corresponding values with acetone-induced microsomes were 42.2 ± 8.3 /iM and 4.77 ± 0.88 nmol/ min/mg. At higher substrate concentrations, the control microsomes had higher activities than estimated from the above Km using the Michaelis — Menten equation, suggesting the presence of higher Km forms of NDEAd. However, this form was not fully characterized. The results indicate that a low Km form of NDEAd exists in both control and acetone-induced microsomes and it is induced several-fold by acetone treatment, suggesting that P450IIE1 is mainly responsible for this activity. Inhibition of NDEA metabolism by antibodies against P450IIEJ Similar to NDMA metabolism, the deethylation and denitrosation of NDEA were inhibited by antibodies against P450HE1 (Table IV). However, the extent of inhibition was lower than that for NDMA. With acetone-induced microsomes, 200 \i% of antibodies inhibited the deethylation reaction 77 and 44% respectively when assayed with 0.16 and 4 mM of NDEA. With control microsomes, the corresponding extents of inhibition were 47 and 23 %. According to the results from Table HI and other studies, this amount of antibodies should inhibit P450IIE] activity almost completely. It appears that both types of reactions were inhibited to a lesser extent in control microsomes than acetone-induced microsomes, and when assayed at 4 mM than 0.16 mM NDEA. Although there were more variabilities in the denitrosation data due to the rather low denitrosation activity,
150
200
350
[NDEA] Fig. 1. Substrate dependence of NDEA metabolism by rat liver microsomes. The incubation mixture contained either acetone-induced (0.075 mg) or control (0.4 mg) microsomes and NDEA (10-320 /tM) in 0.5 ml. The incubation was carried out for 10 nun at 37°C Km and l/!mx were calculated by non-linear regression analysis. Km and Vm^ for acetoneinduced microsomes were 49.4 /iM and 5.27 nmol/min/mg respectively. The corresponding values for control microsomes were 37.8 and 0.683.
the results in Table IV are consistent with the concept that the denitrosation and deethylation reactions were inhibited by similar magnitudes. Discussion The present work demonstrates that rat liver microsomes displayed apparent Km values of 14—24 /tM and these values are close to the values of 10—25 yM observed with liver slices and liver cells (12,13). Recent work employing a radiometric assay for NDMAd with a slight modification from Yoo et al. (14), using 10-100/iM NDMA and 10-40 /tg microsomal protein (in 0.1 ml incubation), also yielded Km values of 15—20/tM. The difference between the presently observed values and our previously reported values is not fully understood. It is possible that our currently used NADPH-generating system (especially NADP and glucose-6-phosphate dehydrogenase) contained less inhibitors than that used previously. In addition, the correction of substrate utilization may also have contributed to the difference. For example, our previously reported low Km values of 40—50 /*M, calculated using initial substrate concentrations (14), were decreased to 34-42 pM when mean substrate concentrations were used for the calculation. The use of higher amounts of protein in general resulted in higher Km values. The use of plastic tubes for the incubation also resulted in higher Km values than with glass tubes. Although the possible bias of the 2241
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many previous studies, was also used. When antibodies against P450IIE1 were added to the incubation mixture containing acetone-induced microsomes, both the demethylation and denitrosation of NDMA were inhibited at a dose-dependent manner (Table III). With 200 /tg antibodies (protein), both reactions were inhibited ~95 and - 9 0 % when assayed with 0.2 and 4 mM NDMA respectively. With control microsomes, antibodies (80 /*g) inhibited both the demethylation and denitrosation of NDMA —80 and —60% when assayed with 0.2 and 4 mM NDMA respectively. The extent of inhibition was generally higher with acetone-induced microsomes than with control microsomes when the lower substrate concentration was used. Under each set of experimental conditions, the demethylation and denitrosation were inhibited by similar magnitudes.
J.-S.H.Yoo, H.Ishizaki and C.S.Yang
Tab4e IV. Inhibition of NDEA metabolism by antibodies against P450IIE1" Microsomes
Acetone-induced Acetone-induced Control Control
Antibodies G*g)
40 200 40 200
% inhibition 0.16 mM NDEA Deethylation
Denitrosation
4mM NDEA Deethylation
Denitrosation
69 77 31 47
71 93 29 65
35 44 7 23
40 42 13 30
"Antibodies (expressed in /ig procein) were added to either acetone-induced rat liver microsomes (0.12 mg protein) or control microsomes (0.7 mg) in 0.5 ml and the incubation was earned out for 10 mm. The deethylase activities (nmol/min/mg) in the presence of control ascites fluid were (i) for acetone-induced microsomes, 2.3 and 2.3 when assayed with 0.16 and 4 mM NDEA respectively, and (ii) for control microsomes, the corresponding values were 0.58 and 0.87. The corresponding denitrosation rates were (i) 0.44 and 0.41, and (ii) 0 12 and 0.22.
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of NDMA and NDEA were inhibited to similar extents by antibodies against P450HE1, illustrating the close relationship between these two types of reactions. This concept is different from the thesis advanced by Amelizad et al. (21), suggesting that the demethylation and denitrosation of NDMA are mediated by different P450 forms and via different mechanisms. Using antiserum against P450 PB3a (UBI) these authors observed an 80% inhibition of the demethylation, but not the denitrosation, of 3 mM NDMA in ethanol-induced microsomes. This result suggests that P450IIB1, not HE1, is responsible for most of the NDMAd activity. Since ethanol can induce P450IIE1 to the same level as acetone (31,32), this result also contradicts our results in Table m. The reason for the difference is not known, but may be related to the experimental conditions and the specificity of the antibodies used. It is noted that their uninhibited NDMAd activity, 35 nmol/mg (per 30 min), was only - 1 5 - 1 6 % those observed in our laboratory (31). In addition, P450HB1 is known to be expressed at very low levels, if at all, in rat liver and may be induced by ethanol to a low level (33); it is unlikely to account for 80% of the NDMA demethylase. In conclusion, the present results demonstrate the role of P450IIE1 in the metabolism of low concentrations of NDMA and NDEA, and most likely is responsible for the low apparent Km values, 15-20 /*M for NDMAd and - 4 0 /tM for NDEAd, in microsomes. Since cells are seldom exposed to high concentrations of these carcinogens, this low Km form of enzyme is believed to be more important in carcinogen activation than higher Km forms. The denitrosation of NDMA and NDEA is shown to accompany closely the dealkylation reactions under different experimental conditions. The role of P450IIE1 and the low Km form of NDMAd in the metabolic activation of NDMA has been demonstrated both in vitro and in vivo (4,34 — 36). The present work points out that P450HE1 also plays an important role in the metabolic activation of NDEA, although the role is not as prominent as is for NDMA. Acknowledgements We thank Mr M.J.Lee for capable technical help and Ms Dorothy Wong for excellent secretarial assistance in the preparation of this manuscript. This work was supported by grams from the National Institutes of Health CA37037, ES03938, and GM38336.
References 1. Magee.P.N. and BamesJ.M. (1967) Carcinogenic nitroso compounds Ath' Cancer Res.. 10. 163-246. 2. Lai,D.Y. and ArcosJ.C. (1980) Miiureview. dialkylmtrosamine bioactivation and carcinogenesis. Life 5a'., 27, 2149 — 2165. 3. Tu.Y.Y. and Yang.C.S. (1983) High-affinity nitrosamine dealkylase system in rat liver microsomes and its induction by fasting Cancer Res.. 43. 623-629.
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Eadie—Hofstee plot has been discussed (30), the present results indicate that similar Km values were observed with both methods when the data points were not scattered and the previously observed higher Km values (40—50 /*M) were not due to the use of the Eadie-Hofstee plot. The presently observed apparent Km values for NDEAd, ~ 40 /iM was much lower than the 430 /iM reported by Puccini et al. (16). The reason for this discrepancy is not clear. Judging from the statement that these authors could not detect the acetaldehyde formed from 1 mM NDEA in incubations with control microsomes, our method, which affords the detection of acetaldehyde in incubations with 10 and 20 /tM NDEA (Figure 1), appears to be more sensitive. The use of lower substrate concentrations enabled us to characterize the presently observed low Km form of NDEAd. If we use datum points obtained in incubations with 0.16, 0.32, 1 and 4 mM NDEA in a doublereciprocal plot, a much higher Km value would be obtained (data not shown). The possible presence of inhibitors in the assay system for Puccini et al. (16), though not apparent from their report, may also be a factor. We and other investigators have experienced such problems in studying the low Km form of NDMAd (14). The role of P450ITE1 in the microsomal metabolism of NDMA and NDEA is demonstrated by the immunoinhibition studies (Tables HI and IV). Consistent with the idea that P450IIE1 is responsible for the low Km form of NDMAd, almost all the activity in acetone-induced microsomes was inhibited when assayed with 0.2 mM NDMA. At 4 mM NDMA, only ~ 10% of the NDMAd activity was not inhibited, possibly due to the contribution by other P450 isozymes. The contribution by other P450 isozymes appears to be more in control microsomes, especially when assayed with 4 mM NDMA. Although P450IIE1 is still responsible for most of the low Km NDEAd activity in acetone-induced microsomes, other P450 isozymes may be responsible for about half of the activity with 4 mM NDEA. In control microsomes, the relative importance of P450IIE1 decreased, especially when assayed with 4 mM NDEA. The results again suggest that P450HE1 is responsible for the low Km form of NDEAd and other P450 isozymes also catalyze the oxidation of NDEA, especially when present at higher concentrations. The concept is consistent with previous studies with human microsomes (5) and is important in understanding previously published results, usually derived from studies using high concentrations (millimolar rather than micromolar) of NDEA. Because of the low sensitivity of the denitrosation assay, we did not conduct thorough determinations on the low Km values for the denitrosation reactions. It is apparent from the results in Tables HI and IV that the denitrosation and dealkylation reactions
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Received on July 30, 1990; accepted on September 6, 1990
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