Chem. Res. Toxicol. 1991,4, 77-81
77
DNA Methylation in Various Rat Tissues by the Esophageal Carcinogen N-Nitrosomethyl-n-amylamine and Six of Its Positional Isomers Chuan Ji,tJ Barbara I. Ludeke,f Paul Kleihues,*?t and Manfred Wiesslers Laboratory of Neuropathology, Institute of Pathology, University Hospital, CH-8091 Zurich, Switzerland, and Institute of Toxicology and Chemotherapy, German Cancer Research Center, 0-6900 Heidelberg, Germany Received June 20, 1990
The major pathway for the bioactivation of asymmetric N-nitrosomethylalkylaminesinvolves cytochrome P-450catalyzed a-C hydroxylation of the alkyl moiety opposite the methyl group, leading to the formation of a methanediazonium ion as the ultimate carcinogen. In the present study we have investigated the effect of the steric configuration of the pentyl chain on the a potent bioactivation of N-nitrosomethylpentylamines in vivo. N-Nitrosomethyl-n-amylamine, esophageal carcinogen, and six of its positional isomers were synthesized by nitrosation of the precursor amines. Overall yields and proportions of the respective 2- and E-isomers reflected the steric hindrance at the a-carbon of the pentyl groups. The extent of DNA methylation in various rat tissues after a single dose (0.1 mmol/kg; 6-h survival) of the isomers was determined by cation-exchange HPLC with fluorescence detection. (1)Among extrahepatic tissues, methylpurine concentrations were highest in esophagus, followed by the nasal and tracheal mucosa and lung. In these tissues, which are phylogenetically derived from the rat ventral entoderm, the relative extent of DNA methylation followed the same pattern for all isomers. (2) In the esophagus, formation of 06-methylguanine (06-meG) was observed only for isomers with an -n-amylunsubstituted a-methylene in the pentyl moiety, i.e., N-nitrosomethylisoamylamine, amine, -(2-methylbutyl)amine, and -(2,2-dimethylpropyl)amine. (3) In trachea, methylated purines were also detectable after administration of the a-substituted isomer, N-nitrosomethyl( 1-methylbuty1)amine. (4) Only nasal mucosa and lung were capable of bioactivating N-nitrosomethyl(1,2-dimethylpropyl)amine.( 5 ) Significant levels of hepatic DNA methylation were obtained only with isomers bearing unsubstituted a- and 0-methylene groups, i.e., N nitrosomethyl-n-amylamine and 4soamylamine. Of particular biological interest is N-nitrosomethy1(2,2-dimethylpropyl)amine. This isomer produced high levels of alkylation in esophagus, in nasal and tracheal mucosa, and in lung, but not in liver. Our results strongly indicate that the bioactivation of asymmetric N-nitrosomethylalkylaminesis mediated by a family of cytochrome P-450isozymes with distinct substrate specificities and that some of these are selectively expressed in extrahepatic tissues.
Introduction Asymmetric N-nitrosdialkylamineswith a methyl group as one of the substituents have been shown to be among the most potent and often highly selective esophageal carcinogens in rats, largely irrespective of the route of administration (I). The major bioactivation pathway for these compounds has been postulated to involve a-C hydroxylation of the alkyl chain opposite the methyl moiety by cytochrome P-450isozymes, leading to a methylating intermediate as the ultimate carcinogen (2-4). Systematic structure-activity analyses of N-nitrosomethyl-n-alkylamines have revealed that DNA methylation and carcinogenic potency in the target tissue are most extensive with those homologues that contain an alkyl chain with three to five carbon atoms, i.e., N-nitrosomethylpropylamine, -butylamine, and -amylamine (5). In the present study, we have investigated whether, i n addition to the number of carbon atoms, the steric configuration of the alkyl chain
* To whom correspondence should be addressed.
also affects the pattern of bioactivation in target and nontarget tissues. The results obtained with N-nitrosomethyl-n-amylamine (NMAA)' and six of its positional isomers clearly indicate that there is a strong influence of the position of side-chain substitutions on a-C hydroxylation and that for some of these isomers enzymatic activation is restricted to extrahepatic tissues.
Materials and Methods Materials. RNase TI from Aspergillus oryzae and proteinase K were obtained from Boehringer-Mannheim AG, CH-6343 Rotkreuz (Switzerland). RNase A from bovine pancreas and calf thymus DNA were purchased from Sigma Chemie, D-8024 Deisenhofen (FRG). DNA purification grade lysis buffer, 70% phenol/water/chloroform,and phenol/chloroform reagents were from Applied Biosystems, Inc., Foster City, CA 94404. All commercial chemicals were of analytical grade or higher. Synthesis of Methylpentylamine Hydrochlorides. Methylpentylamine hydrochlorides were synthesized by two methods. Methyl(3-methylbuty1)aminehydrochloride, methyl-n-amylamine hydrochloride, methyl(2-methylbuty1)aminehydrochloride, and
'University Hospital, Zurich.
* Visiting scientist from the Cancer Institute, Chinese Academy
of Medical Sciences, Beijing, People's Republic of China. German Cancer Research Center.
Abbreviations: NMAA, N-nitrosomethyl-n-amylamine; P-meG, 06-methylguanine;7-meG, 7-methylguanine.
0893-228x/91/2704-0077$02.50/00 1991 American Chemical Society
Ji et al.
78 Chem. Res. Toxicol., Vol. 4, No. 1, 1991 Table I. Physicochemical Data of Isomeric N-Nitrosomethylpemtylamines HPLC retention time, min 'H NMR (proportion, %) % N-nitrosomethyl- yield E-isomer Z-isomer E-isomer Z-isomer 16.75 (84) 18.3 (16) -isoamylamine (11) 65 0.97 ( 6 H, d), 1.40 (1 H, m), 0.93 (6 H, d), 3.60 (2 H, t), 3.73 (3 H, 8) (approx 20%) 1.63 (2 H.m). 3.03 (3 . H. . s), . . 4.15 (2 H; t).' 16.66 (87) 18.3 (13) 3.60 (2 H, t), 3.77 (3 H, s) -amylamine (I) 75 0.90 (3 H, t), 1.33 (4 H, m), (approx 15%) 1.75 (2 H, m), 3.07 (3 H, s), 4.15 (2 H, t) 15.77 (88) 17.61 (12) 0.80 (3 H, d), 0.95 (3 H, t), -(a-methylbutyl)- 65 0.90 (3 H, d), 0.95 (3 H, t), 1.37 (2 H, m), 1.85 (1 H, m), 1.37 (2 H, m), 1.85 (1 H, m), amine (111) 3.47 (2 H, d), 3.77 (3 H, s) 3.03 (3 H, s), 3.97 (2 H, d) (approx 15%) 13.76 (91) 17.36 (9) 0.93 (9 H, s), 3.45 (2 H, s), -(2,2-dimethyl80 1.00 (9 H, s), 3.13 (3 H, s), 3.85 (3 H, s) (approx 7%) 3.95 (2 H, s) propy1)amine
(IV) 41-methylbuty1)amine (V)
65
- (1,2-dimethyl-
25
propy1)amine (VI) 41-ethylpropy1)amine (VU) ~
~I
30
3.63 (3 H, s) (approx 7%) 0.93 (3 H, t), 1.25 (2 H,m), 1.37 (3 H, d), 1.67 (2 H, m), 2.95 (3 H, s), 4.70 (1H, m) 3.67 (3 H, s) (approx 5 % ) 0.85 (3 H, d), 1.03 (3 H, d), 1.40 (3 H, d), 1.90 (1 H, m), 2.97 (3 H, s), 4.35 (1 H, m) 3.60 (3 H, s) (approx 5%) 0.88 (6 H, t), 1.77 (4 H, m), 2.95 (3 H, s), 4.33 (1 H, m)
12.93 (93) 14.99 (7)
mass spectra' m / e (re1 intensity) 130 (2), 113 (22), 74 (64), 73 (64), 70 (15)
130 (21), 113 (45),84 (7), 74 (35), 73 (78),70 (13) 130 (24), 87 (6), 83 (4), 73 (58), 71 (14), 70 (53) 131 (17), 130 (881, 115 (291, 85 (341, 84 (15), 74 (26), 73 (59), 70 (64) 130 (55), 113 (21), 101 (27), 88 (12), 87 (49), 71 (76), 70 (71)
10.28 (94) 11.92 (6)
130 (221, 88 (41, 87 (80),71 (16),70 (48)
11.29 (96) 13.01 (4)
130 (64), 113 (la), 101 (591, 72 (42), 71 (871, 69 (85)
70 eV, 0 "C.
methyl(l,2-dimethylpropyl)aminehydrochloride were prepared by condensation of the respective C5 aldehyde or ketone with methylamine to the corresponding imine, followed by hydrogenation (PtO,/EtOH). Methyl(2,2-dimethylpropyl)aminehydrochloride, methyl(1-methylbuty1)amine hydrochloride, and methyl( 1-ethylpropy1)amine hydrochloride were obtained after condensation of the respective C5 amine with formaldehyde to the corresponding hexahydrotriazines and subsequent hydrogenation (PtOz/EtOH). The amine hydrochlorides were purified by recrystallization. Nitrosation of Amines. Amine hydrochlorides (1.95 g, 0.015 mol) were dissolved in 15 mL of water and reacted with 1.8 g (0.030 mol) of sodium nitrite and 0.7 mL of 2 N H2S04for 1 h. Reaction mixtures were extracted twice with ether, and the combined ether extracts were dried with Na2SO4. After filtration, the ether was carefully evaporated, and the residues were distilled a t high vacuum. HPLC analyses were done on a Hewlett Packard HP1090, equipped with a Spherisorb Cl8/2 5-pm column (4.6 X 250 mm). Nitrosamines were eluted with isocratic HzO/ acetonitrile (7525) at a flow rate of 1.0 mL/min and detected by absorption a t 235 nm. Chemical structures were confirmed by 'H NMR spectroscopy, recorded on a Bruker HX 90 in CDCl, as solvent, and by mass spectroscopy, using a Finnigan MAT 711 (70 eV; 0 "C). Animal Treatment. Young male Fischer 344 rats (120-140 g body weight) were obtained from Charles River Wiga GmbH, D-8741 Sulzfeld (FRG) and maintained on a standard laboratory diet with tap water ad libitum for 1-2 days prior to the experiment. Each of the isomers was dissolved in saline and administered by ip injection (0.5mL/kg) to 10 rats at a dose of 0.1 mmol/kg. After a survival time of 6 h, the animals were killed by exsanguination during ether anesthesia. Tissues were pooled (esophagus, trachea, lung, liver, kidney) or collected individually (liver), rapidly frozen in liquid nitrogen, and stored at -70 "C.Material from the nasal cavity was obtained by curettage after median splitting of the head, but was otherwise processed in the same way. DNA Isolation. DNA was isolated by automated phenolic extraction using a Model 340A nucleic acid extractor (Applied Biosystems, Inc.). Liver, lung, and kidney tissue (0.5-1.0 g) was pulverized in liquid nitrogen, homogenized in 10 mL of PBS (phosphate-buffered saline; 140 mM NaCl, 2.7 mM KCl, 8.1 mM Na,HPO,, 1.5 mM KH2P04,pH 7.4) with 10-12 passages in a Potter-Elvehjem homogenizer, and filtered through two to four layers of prewet gauze. Cell nuclei were collected by centrifugation for 5 min a t lOOOg, resuspended in 0.2-0.4 mL of PBS, and transferred to 5-mL Potter-Elvehjem homogenization vessels. Following the addition of 1 mL of lysis buffer, the mixtures were homogenized by hand (three passages) and transferred to 8 mL of lysis buffer. Crushed esophagi, tracheas, and nasal cavity
scrapings (0.5-1.0 g) were homogenized directly in 10 mL of lysis buffer and filtered through gauze. The crude nuclei or whole tissue homogenates were predigested with RNase A and T1(400 units/g of tissue of each) are 37 "C for 1 h. After adding proteinase K (85-170 units/g of tissue), digestion was continued for an additional 2 h a t 37 "C and overnight at 4 "C. Samples were transferred to the 30-mL vessels of the DNA extractor and extracted twice with with an equal volume of phenol/chloroform (1:l v/v; crude nuclei) or 70% phenol/chloroform/water (tissue homogenates) for 10 min a t room temperature. Residual phenol was removed by a single extraction with chloroform (8 min). All phase volume separations were carried out a t 60 "C. After adding of 3 M sodium acetate, pH 5.5, DNA was precipitated with ethanol, dried for 1-2 h at room temperature, and stored a t -80 "C until analysis. Aliquots were dissolved in 10 mM Tris-HC1, pH 7.8, containing 1 mM EDTA, and analyzed by agarose gel electrophoresis and UV spectroscopy (6);no contamination with RNA or protein was detected (not shown). Determination of OB-Methylguanine( 06-meG) and 7Methylguanine (7-meG). Following mild acid hydrolysis (0.1 M HCl a t 37 "C for 20 h), the amounts of 06-meG and 7-meG were determined by HPLC using a modification of the procedure described previously (7,8). Briefly, purine bases were separated isocratically on a strong cation-exchange column (Whatman Partisill0 SCX, 4.6 X 250 mm). For the determination of 7-meG, the column was eluted at 2 mL/min with 50 mM NH4H2P04,pH 2. Retention times were 3.5 and 5.4 min for guanine and adenine, respectively, and 8.1 min for 7-meG. The same buffer containing 3% acetonitrile was used to assay for 06-meG. Under these conditions, guanine and adenine eluted a t 3.2 and 4.6 min, respectively, and 06-meGa t 8.3 min. Adenine and guanine were quantitated by absorbance a t 254 nm with a Shimadzu S4 UV detector. Quantitation of methylpurines was carried out with a Shimadzu spectrofluorophotometer (RF-540), set at 295 nm for excitation and 370 nm for emission. Calibration of the fluorescence signal was performed by injecting standard solutions which had been quantitated by UV absorption using published extinction coefficients (9,lO). The limits of quantitation for 06-meG and 7-meG were 1 pmol(2.5 pmol/mol of guanine) and 36 pmol (100 pmol/mol), respectively. The limits of detection were approximately 0.6 pmol/mol for OB-meGand 25 pmol/mol for 7-meG.
Results The yields of the respective syntheses and characterizations of the N-nitrosomethylpentylamines a r e detailed in Table I. Our data agree well with a previous report (12) on the s y n t h e s i s of N-nitrosomethyl(2,2-dimethylpropy1)amine (IV). The low yields of N-nitrosomethyl-
D N A Methylation by N - Nitrosomethylpentylamines
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 79
Table 11. DNA Methylation in Vivo by N-Nitrosomethylamylamineand Six Isomers" esophagus nasal mucosa trachea lung N-nitrosomethyl7-meG OB-meG 7-meG 06-meG 7-meG OB-meG 7-meG OB-meG 4soamylamine (11)
/CH3 CHsNCH&H&H
I
NO
-amylamine (I)
liver 7-meG OB-meG
897b
141
571
87
589
95
200
26 f 1 270 f 1
22 f 2
502
80
501
64
387
65
126
19 f 1 324 f 28
28 f 1
496
87
359
55
327
51
5100
12f 1
nd'
12
219
31
122
18
211
26
1100
10f1
nd
nd
nd
nd
1100
7
nd
2
nd
4 f 1 nd
nd
nd
nd
1100
8
nd
nd
nd
4f 1
nd
nd
nd
nd
nd
4
nd
nd
nd
nd
nd
'cH3
CHsNCH&H&H&H&H3
I
NO
42-methylbuty1)amine (111)
CHsNCH&HCH&Hs
-(2,2-dimethylpropyl)-
C , H3 CHaNCH&XY
amine (IV)
I
I
NO CH3
bo -(l-methylbuty1)amine (V)
bH3
CH3
I
CHsNCH&H&H&Hs
I
NO
-( 1,2-dimethylpropyl)-
amine (VI)
9'3 /CH3 CHsNCHCH
bo -(1-ethylpropy1)amine
(VII)
bH3
/CH2CH3
nd
CHsNCH bObH2CH3
"Male Fischer 344 rats received a single ip injection of one of seven isomers of N-nitrosomethylpentylamineat a dose of 0.1 mmol/kg (13 mg/kg) and were killed 6 h later. Methylpurines were determined by HPLC as described under Materials and Methods. Results are expressed as pmol/mol of guanine. bSingle determination of pooled tissues or, where indicated, mean f standard error duplicate (lung) or triplicate (liver) measurements. nd, not detected.
Table 111. ComDarison between Concentrations of Methylated Purines Determined in Individual and Pooled Liver Samples individual livers pooled (10 livers) 1 2 3 7-meG 06-meG 7-meG OB-meG 7-meG 06-meG 7-meG 06-meG compund 324 f 28" 339 f 18 I 28 f 1 29 f 1 255 23 f 0 308 24 I1 270 f 20 22 f 2 260 f 26 26 f 4 228 20 f 1 aResults are expressed as pmol/mol of guanine f standard error (triplicate measurements). On animal 3, only one determination was carried out.
(1,2-dimethylpropyl)amine (compound VI) and N nitrosomethyl(1-ethylpropyl)amine (VII) clearly illustrate the increase in steric hindrance by substituents at the a-carbon of the pentyl chain. The assignments of the respective 2- and E-isomers on the basis of the 'H NMR data were confirmed by HPLC. Quantitation of the proportion of 2-isomers, however, was much more precise with HPLC data. Furthermore, the E-isomer of a given nitrosamine consistently exhibited a shorter retention time than the 2-isomer under the conditions of these HPLC analyses irrespective of the configuration of the pentyl moiety. Levels of methylated guanines formed in various rat tissues 6 h after a single dose of 0.1 mmol/kg N-nitroson-amylamine or one of six of its isomers are shown in Table 11. Highest concentrations of 06-meG and 7-meG were observed in esophageal DNA for nitrosamines with an unsubstituted a-methylene group. The order of the extent of methylation in this tissue was N-nitrosomethylisoamylamine (11) > N-nitrosomethyl-n-amylamine(I), N nitrosomethyl(2-methylbuty1)amine (111) > N-nitrosomethyl(2,2-dimethylpropyl)amine(IV). No methylation was detected in esophageal DNA with isomers in which the a-methylene group carries a methyl or ethyl substituent. In contrast, low but significant levels of DNA methylation by all three a-substituted N-nitrosomethylpentylamines were found in nasal epithelia, the only tissue in which DNA methylation by all seven isomers was observed. In tracheal and pulmonary DNA, methylation by
N-nitrosomethyl( 1-methylbuty1)amine (V) was detected in addition to methylation by compounds I-IV. In liver, methylated purines were formed to a significant extent (I) and N-nitrosoonly by N-nitrosomethyl-n-amylamine methylisoamylamine(11). Trace amounts of 06-meG were also observed in hepatic DNA after administration of N-nitrosomethyl(2-methylbuty1)amine (111). No DNA methylation was detected in kidney with any of the compounds (not shown). Due to the small volume of some of the tissues (e.g., esophagus, trachea, nasal mucosa), single determinations had to be performed on pooled tissue samples from 10 animals. The standard error of the HPLC determinations amounted to less than 10% of the mean. Interindividual variation in the formation of 06-meG and 7-meG was determined for liver (Table 111).
Discussion Conversion of asymmetric N-nitrosomethylalkylamines to methylating intermediates is initiated by enzymatic hydroxylation at the a-carbon of the alkyl moiety opposite the methyl group (2-4). In the present study we have investigated the effects of configurational changes in the pentyl group on this reaction in N-nitrosomethylpentylamines. We determined the biological end point, i.e., the in vivo reaction of the ultimate metabolite with nuclear DNA. This approach has the advantage over in vitro studies of allowing a direct correlation with carcinogenicity
80 Chem. Res. Toxicol., Vol. 4, No. 1, 1991
in chronic bioassay studies. Formation of methylated purines was assessed in DNA in various rat tissues after a single dose of NMAA or one of six positional isomers. The parent compound, NMAA, is a potent esophageal carcinogen in rats (1). Occasional target tissues include the nasal cavity and trachea (13),but not liver or lung. DNA methylation was chosen as a measure of carcinogen bioactivation since previous studies have shown that the formation of methylated purines by NMAA correlated well with tumor induction in extrahepatic tissues (5, 12). Furthermore, deuteration of the a-methylene of N nitrosomethyl-n-butylamine, which decreases the rate of enzymatic a-C hydroxylation of the butyl group and hence the formation of a methylating species, has been found to significantly reduce the carcinogenicity of this closely related nitrosamine (3). The extent of formation of methylated purines following a single dose of one of seven isomers of N-nitrosomethylpentylamine differed considerably among the tissues investigated. However, an observation common to all was a general decrease in bioactivation with increasingly bulky pentyl groups (Table 11). Similarly, the relative amount of the Z-isomer at equilibrium in the synthesis of a given N-nitrosomethylpentylamineis a sensitive indicator of steric hindrance in the pentyl group. Both the extent of DNA methylation in extrahepatic organs and the relative concentration of the respective Z-isomers were highest for isomers bearing unsubstituted methylene groups a t positions 1 and 2, followed by isomers with a methyl group a t position 2, and were lowest for isomers with a methyl or an ethyl group at the a-substituted carbon. This order of a-C hydroxylation would be expected if the active sites of the P-450 isozymes involved encompass rather narrow grooves, into which substrates with a stretch of flexible, unbranched alkyl chain adjacent to the nitroso group fit more easily than those with a bulkier configuration. Also, steric hindrance a t the a-C, e.g., by a methyl or ethyl moiety as in compounds V-VII, may render this carbon less readily accessible to enzymatic attack. Alternatively, our observations could indicate that binding of these nitrosamines with the pentyl rather than the methyl group oriented toward the oxygenation site may occur preferentially with the respective Z-isomers. A model for the selective positioning of nitrosamines at the active site has recently been proposed for the demethylation of N nitrosomethyl-n-butylamine by hepatic cytochrome P450IIE1 in which the butyl chain was suggested to bind to a hydrophobic pocket, enabling oxidation of the methyl group (14). Overall, there were four types of alkylation profiles. The simplest type was observed for liver, which showed significant activity only toward those isomers in which neither the a- nor the &methylene group was substituted (compounds I and 11). This is consistent with the lack of hepatic tumor induction by compound IV in chronic carcinogenicity studies (15). However, significant rates of depentylation of this nitrosamine by isolated microsomes in vitro have been reported (16). The bioactivation profiles in extrahepatic tissues were strikingly different, the most prominent feature being the fact that highest levels of DNA methylation were obtained with compound I1 rather than with NMAA (I). This finding suggests that compound I1 may be more effective than NMAA as an esophageal carcinogen in rats. This nitrosamine may also be implicated in the etiology of human esophageal cancer since in vivo formation from isoamylamine by fungi, both isolated from foods in the high-risk area of Linxian County (China), in the presence of sodium nitrate has recently
Ji et al. been demonstrated (17). Purified hepatic cytochrome P-450IIB1 has been shown to hydroxylate NMAA a t all carbon atoms of the pentyl chain, with the most extensive reactions taking place at the a-C and a t position 4 (18). Similarly, pretreatment with 3-methylcholanthrene induced both the formation of pentanal and N-nitrosomethyl(3-hydroxyamy1)amine from NMAA in rat liver microsomes (18). Conceivably, extrahepatic P-450 isozymes exhibit similar polyreactivities. It has been proposed that a single enzyme could catalyze hydroxylation reactons at various carbon atoms of the same substrate if the bound substrate is able to rapidly move within the active site (18). This mechanism could explain our results with compounds I and I1 if the methyl group a t position 3 of compound I1 serves to anchor the nitrosamine with the a-C close to the site of hydroxylation. In the esophagus, all isomers bearing an unsubstituted a-methylene (compounds I-IV) were efficiently metabolized to a methylating intermediate. Compound IV has been reported to be nearly as potent an inducer of esophageal neoplasms as NMAA (15). Similar results were obtained in the respiratory tract; however, in contrast to the esophagus, DNA methylation by compound I was slightly higher than by compound I11 in these tissues. In trachea, the simplest a-substituted isomer (V) was also a-hydroxylated, albeit to a very low extent. In the fourth type of methylation profile, which was observed in nasal mucosa and lung, concentrations of methylpurines formed from a-substituted isomers amounted to approximately 50% of the levels produced by compound IV. It is very likely that methylation by compound VI1 was not detected in DNA extracted from whole lungs since its metabolism may be restricted to bronchiolar epithelia, as is that of NMAA (13)and its close analogues, including N-nitrosomethyl- and N-nitrosoethyl-n-butylamine (19) and the tobacco-specific nitrosamine, 4-(N-methyl-N-nitrosoamino)-l-(3-pyridyl)-l-butanone(NNK) (20). This could have resulted in concentrations of methylated purines in total DNA below the limits of detection by HPLC. A similarly sharp distinction between liver and those extrahepatic tissues which are derived from the rat ventral entoderm has been found with respect to the formation of stable hydroxy metabolites of NMAA (21,22). In the uninduced rat liver, the most predominant of these metabolites is N-nitrosomethyl(4-hydroxyamy1)amine.In extrahepatic tissues, significant levels of hydroxylation were also observed at carbon atoms 2-5 of the amyl chain. The organ-specific bioactivation patterns of the present investigation do not, however, completely agree with the metabolism profiles observed in those experiments. Esophageal metabolism was clearly distinguishable from that of the tissues of the respiratory tract in both studies. The relative formation of hydroxy metabolites was similar in nasal cavity, trachea, and lung, but in the latter tissue an exceptionally high level of o-hydroxylation was also detected. In contrast, nasal mucosa was much more similar to lung than to trachea in its ability to hydroxylate substituted a-methylene carbons. The present data on DNA methylation by isomers of N-nitrosomethylamylaminesupport earlier results from our laboratory suggesting the existence of at least three extrahepatic P-450 isozymes with high activity toward aliphatic asymmetric dialkylnitrosamines (19). In those experiments we compared the effects of various modulators of nitrosamine metabolism on methylation by N-nitrosomethyl-n-butylamine and ethylation by N-nitrosoethyln-butylamine. In esophagus, lung, and nasal cavity, DNA methylation was increased severalfold in rats pretreated
D N A Methylation by N-Nitrosomethylpentylamines
with disulfiram whereas the increase in ethylation was marginal. In contrast, an acute dose of ethanol, administered 20 min prior to the nitrosamine, led to increased DNA alkylation by both compounds in esophagus and lung but almost completely abolished nitrosamine bioactivation in nasal mucosa. In conclusion, there is growing evidence that the cytochrome P-450s that are expressed in tissues derived from the rat ventral entoderm comprise a family of closely related but nevertheless distinct isozymes, some of which are exclusively expressed in extrahepatic tissues. Acknowledgment. This work was supported in part by a fellowship from the Union International Contre le Cancer (UICC) to C.J., and by the Swiss National Science Foundation. Registry No. N-Nitrosomethylamylamine,13256-07-0; Nnitrosomethylisoamylamine, 35606-38-3; N-nitrosomethyl(2methylbutyl)amine, 130787-81-4; N-nitrosomethyl(2,2-dimethylpropyl)amine, 31820-22-1; N-nitrosomethyl(1-methylbutyl)amine, 130985-76-1; N-nitrosomethyl(l,2-dimethylpropyl)amine, 130985-77-2;N-nitrosomethyl( 1-ethylpropyl)amine, 130985-78-3; methyl(3-methylbuty1)aminehydrochloride, 241959-2; methyl-n-amylamine hydrochloride, 74109-19-6; methyl(2methylbuty1)amine hydrochloride, 130985-79-4; methyl( 1,2-dimethylpropy1)amine hydrochloride, 2738-05-8; methyl(2,2-dimethylpropy1)amine hydrochloride, 31820-19-6; methyl(1methylbuty1)amine hydrochloride, 130985-80-7;methyl( l-ethylpropy1)amine hydrochloride, 130985-81-8; 7-methylguanine, 578-76-7; 06-methylguanine, 20535-83-5.
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