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The binding of ethyl carbamate to DNA of mouse liver in vivo: The nature of the bound molecule and the site of binding
Chem.-Biol. Interactions, 14 (1976) 149-163 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands THE BINDING OF ETHYL CARBAMATE TO DNA OF MOUSE LIVER IN VIVO: THE NATURE OF THE BOUND MOLECULE AND THE SITE OF BINDING
A.W. POUND, F. FRANKE
* and T.A. LAWSON
Department of Pathology, University of Queensland, Herston, Queensland (Australia, 4006) (Recr ived September 30th, 1975) (AccLpted October 20th, 1975) --
~--__-.-
..-. _~.~__.
SUMMARY
Ethyl carbamate, labelled at C1 with 14C, bound in vivo to liver DNA of intact and partially hepatectomised mice. Isotope (‘“0) enrichment was not detected in the oxygen of liver DNA of mice injected with [I801 ethyl carbamate, C&l&-18 O-CO-NH2. This suggests that it was the ethyl group and not the ethoxy group which bound to DNA. Chromatographic analysis of acid hydrolysates of liver DNA from mice treated with [l-14C] ethyl carbamate provided no evidence of alkylation or other form of binding to purine or pyrimidine bases. On relatively mild acid hydrolysis the alkyl group remained bound to the “apurinic acid” fraction, while more vigorous hydrolysis lead progressively first to its separation as highly ionisable hydrophilic non-volatile compounds and then to its loss as a volatile compound. DNAase I followed by phosphodiesterase hydrolysis also split off the 14Ccontaining group as a volatile compound. The volatile compound was identified as ethanol. These results suggest that the alkyl group was bound as an ester to a phosphate group in the DNA chain. Results with DNA from partially hepatectomised mice did not differ from those with DNA from intact mice.
II%TRODUCTION
Ethyl carbamate is an active carcinogen in mice, particularly for lung, liver and, as an initiating agent, for skin [ 11. Its mono-l\r-substituted derivatives are also active [2-6] but the other simple homologous alkyl carbamates are * Present address: School 4111).
of Science,
Griffith University,
Nathan, Queensland
(Australia,
149
not or are very weakly so. When the target tissue is induced to proliferate at the time of its administration more tumours are produced, e.g. in skin after chemical stimulation [ 7-101 and in liver after partial hepatectomy [ 11-141. Many, perhaps all, chemical carcinogens or their metabolites [15 ] form covalent linkages with cell macromolecules in vivo, commonly through interactions with the bases of DNA, although RNA and cell proteins are also involved [16] . The role of alkylation in the carcinogenic process is undecided [ 16,17 ] . Esterification of the free phosphate groups occurs to a lesser extent during the interaction of alkylating agents with DNA in vitro [18-211 but consideration of its role has been overshadowed by the more efficient alkylation of the bases. It has only recently been detected in vivo 1221 A metabolite of ethyl carbamate (urethane) bound to liver DNA in intact and in partially hepatectomised mice [23,24 1. It also bound to epidermal and lung DNA. The bound molecule involved either an ethyl or an ethoxy group, but not the carbonyl group, of urethane [25]. A similar result was found in rats [26] in which animal urethane is carcinogenic only for lungs [ 271. In this laboratory it was found, in mice, that the binding occurred predominantly to DNA in liver [23] and skin (unpublished data) and that there was relatively little binding to RNA or cell proteins. The latter therefore have not been examined further by us. Qther workers have reported that the carbo-ethoxy group bound to RNA as the ethyl ester of cytosinedcarboxylic acid [28,29] but that only smaller amounts of binding occurred to DNA, the nature of which was not examined in detail. There is therefore no agreement on the site and nature of the binding of ethyl carbamate to macromolecules in cells. The present work was performed firstly, to determine whether the ethyl or the ethoxy group of urethane bound to DNA. Mice were given ethyl carbarn&e, C2H5-180-CO-NH2, and the I80 content of the resulting DNA measured by determination of the 1zC’60’60/‘2C’60’60 ratio of the carbon dioxide obtained by its decomposition. Secondly it was desired to determine the nature of the binding of the ethyl or the ethoxy group by giving 14Clabelled ethyl carbamate and identifying the bound molecule in hydrolysates of DNA by chromatographic examination. Thirdly it was necessary to determine if the nature and site of binding were the same in intact and hepatectomised mice. l
MATERIALS AND METHODS
Animals
Random bred, male Crackenbush mice, 6-8 weeks old, weighing 35’! 40 g were used. Diet and conditions of animal housing were the same as before [23,14]. Chemicals
Ethyl carbamate (urethane) was obtained from B.D.H. Ltd., Poole, Great 150
Britain. [‘80] Ethyl
~~~a~,
(59.0 mCi/mmole) Great Britain and nucleosiiles were oh Dose schedules
o~tirn~ [ 25]. Extraction
of DNA
DNA was extracted tact and two-thirds hep The ‘~~~I60 ratios were dete carbon dioxide. The 8 spectrometrically. The t (I) Guanidine h y~r~~h mg) and ~~idine hydro~lorj outgw+ed at 80°C for 30 min maintained at 390” C for 2 h.
that described in frozen. The vol
as described above An MS 10 mass tions were rn~i~~n~d i of CC&the 46 and 44
average 46/44 ratio. The measurement alternated with that of a “Standard”
of the ratio for COz from DNA was CO2 of normal isotopic composition.
Hydrolysk of DNA (i) Acid hydrolysis: Acid hydrolysis was carried out in sealed Pyrex glass tubes under five sets of conditions - (a) HCOOH (98%; v/v) at 175°C for 30 min; (b) HCOOH (90%; v/v) at 175°C for 30 min; (c) 1 N HCl at 100°C for 1.5 h; (d) 6 N HCl at 100°C for 1 h; and (e) 6 N HCl at 125°C for 2 h. (ii) Enzymatic hydrolysis: 15 mg DNA, dissolved in 0.1 M NH4HCOJ and 0.015 M MgClz, 3 ml, was incubated with 50 pl(250 pg) DNAase I at 37°C for 2 h. The pH was adjusted to 8.5 with dilute ammonia and 75 ~1 (375 pg) Crotalus adamanteus phosphodiesterase was added. Incubation was continued at 37” C for a further 2 h. Chromatography of hydrolysates Unless otherwise stated, the products of the acid hydrolyses were, after evaporation to dryness in a stream of nitrogen, taken up in 0.1 N HCl, applied to Dowex 50 (H+) columns (50 cm X 1 cm) and eluted with a linear gradient of 0.1 to 4.0 N HCl. 5 ml fractions were collected. The products of enzymatic hydrolysis were chromatographed on a Dowex l-XH (formate form) column without previous evaporation to dryness and were eluted with a linear gradient of 0 to 4 N HCOOH followed by 1 N HCOONH4. The Ezs4 of the eluates was monitored continuously. The radioactivity was determined by adding 10 ml of Bray’s scintillation fluid [343 to 1 ml aliquots of the neutralised fractions and counting their activity in a Packard Scintillation Spectrometer. The positions of the four major bases, thymine, cytosine, adenine and guanine, were identified by co-chromatography with pure authentic samples. In the case of acid hydrolysis by method (d), fractions containing radioactivity were examined after prior concentration by descending chromatography on Whatman No. 1 paper using propan-2-01 : Hz0 : concentrated HCl (68 : 16.4 : 15.6, v/v/v) as a solvent. The chromatograms were developed for 9 h after which the spots were visualised on the dried papers by their UVabsorption at 254 nm. The distribution of labelled material was determined by cutting the dried paper into small equal sized strips, immersing them directly into the scintillation fluid and measuring their radioactivity. Measurement of distribution coefficients Distribution coefficients (K := concentration in organic phase/concentration in aqueous phase) of radioactive products between aqueous and organic phases were determined, Aliquots of the material under examination were (1) left acid as obtained, (2) neutralised with solid NaHC03, and (3) made alkaline with solid NaOH and equilibrated by shaking with ether, isobutanol or butan-l-01. Aliquots of the two phases were withdrawn and their radioactivity was determined after neutralisation.
152
RESULTS
Nature of the bound molecule
Experiments in mice on the in vivo binding of [1-14C]ethyl carbamate to liver DNA showed that 1 mg of DNA contained about 8.0 - lo-’ mg of urethane, or the equivalent of its metabolite, 12 h after a dose of 10 mg [25]. If the DNA contained the oxygen [I801 originally present in this dose of [ lsO]ethyl carbamate and if, during the thermal decomposition to C02, this became randomly mixed with the other oxygen of normal isotopic composition in the DNA, then the 46/44 ratio for this COz should be about 15 * 10e5 greater than the /6/44 ratio for CO2 of “normal” isotopic composition. This calculation is based on the assumption that DNA contains 31 wt. % of oxygen, derived from the recorded base composition of .mouse DNA [35] . In point of fact, the dose of [ *8O]ethyl carbamate given was 20 mg. Since the extent of binding is dose-dependent [24] the increase in the 46/44 ratio should be greater than this. COz, slightly enriched in ‘*O, was prepared with a 46/44 ratio about 10 10” in excess of the 46/44 ratio of COz of normal isotopic composition. This excess is of the same order as the excess ratio expected if the I80 in the labelled ethyl carbamate were bound to the DNA. Samples of this I80 enriched CO* were put through the guanidine and also the mercuric chloride isolation techniques to establish whether the procedures involved any isotopic dilution. The guanidine technique showed no isotopic dilution but the mercuric chloride technique did introduce some dilution whereby the 46/44 ratio decreased by 2 - 10”. The isotopic ratios (46/44) of COz obtained from liver DNA of intact and partially hepateetomised mice that had been given [ ‘8Q]ethyl carbamate, and from normal untreated mice are shown in Table I. The results obtained with both the guanidine hydrochloride and mercuric chloride methods are shown. Within each group there are no significant differences in the isotope ratios from those obtained with standard pure COZ. No measurable isotope enrichment had occurred in any of the samples. l
Site of binding Formic acid hydrolysis:
Liver DNA (10 mg) from intact mice given [ 1-14C]ethyl carbamate (6 PCi; 10 mg; i.p.) was hydrolysed with 98% (v/v) formic acid at 175°C for 1 h (method a). Elution of the evaporated hydrolysate from a Dowex 50 (H+ form) column with HCl (0.1 to 4 N) produced eleven UV-absorbing fractions, only one of which, adenine, was identified. .No radioactivity was detected in any of the fractions. The radioactive component or components must therefore have been liberated as a volatile material, possibly ethanol. On less vigorous hydrolysis, i.e. with 90% formic acid (method b), the radioactivity was recovered quantitatively in the. first UV-absorbing peak eluted from the columns, and was therefore not present in this case as a volatile compound. 153
TABLE I ISOTOPIC RATIOS (46/44) OF CO1 OBTAINED GIVEN [‘so ] ETHYL CARBAMATE, 20 mg, i.p. Treatment of mice from which liver DNA was obtained
FROM LIVER DNA OF MALE MICE
10s X Isotopic
ratios a
-_-
Guanidine HCl method
Mercuric chloride method
Intact mice + urethane Standard CO2 b Intact mice + uerthane Standard CO*
449.6 448.6 448.6 448.4
447.3 448.9 446.4 446.6
Hepatectomised Standard CO* Hepatectomised Standard CO*
449.2 448.2 449.2 449.2
445.4 446.6 446.7 449.8
446.2 448.5 448.4 447.5
446.3 447.6 -
Untreated Standard Untreated Standard
mice CO* mice COz
mice + urethane mice + urethane
a Each value is the average from 6 measurements with a maximum deviation of 1.1 - lo-‘. The values have been adjusted to a standard output voltage of 30 V for the m/e = 44 beam from an experimentally determined graph. b Measurements of standard CO2, normal isotopic composition, were alternai.ed with CO2 from experimental animals.
Hydrochloric acid hydrolysis. Liver DNA (15 mg) from intact mice given [l-14C] et.hyl carbamate (6 PCi; 10 mg; i.p.) was hydrolysed with 1 N HCl at 100°C for 1.5 h (method c). The chromatographic behaviour of the hydrolysate, after prior evaporation to dryness, on a Dowex 50 column is shown in Fig. 1. Thymine (peak I) and cytosine (peak II) in small amounts, guanine, (peak III) and adenine (Peak IV) in large amounts were identified, by cochromatography with authentic samples, together with four o:her unidentified UV-absorbing compounds (presumably nucleosides and nucleotides). The first strongly UV-absorbing fractions (peak V) to be eiuted from the column contained 311the radioactivity present in the original material. The ratio of the radioactivity to the UV-absorbance was nearly ccnstant through all the fractions proving that the radioactive compound was still linked to the UV-absorbing substance, that is, the apurinic acid fraction. More vigorous hydrolysis (12 mg DNA), with 6 N HCl at 100°C for 1 h (method d), and chromatography of the evaporated hydrolysate on Dowex 50 columns produced the pattern shown in Fig. 2. The four major bases and several smaller UV-absorbing compounds were present. Compared with the hydrolysate from method c (Fig. l), thymine (peak I) and cytosine (peak II) were now relatively increased in amount, guanine (peak III) and adenine (peak IV) were about the same in amount, and some unidentified products appeared ahead of cytosine but none of these fractions contained any radio-
154
Fraction
No.
Fig. 1. Ion exchange chromatography on Dowex 50 (H+ form) of hydrolysate of liver DNA from mouse given [1-‘4C]ethyl carbamate, 10 mg, 6 PCi, 12 h previously. Ezs4 of each 5 ml fraction; - - - - - -, Radioactivity dpm of each 5 ml fraction. 15 mg DNA hydrolysed by method c, 1 N HCI, lOO”C, 1.5 h. Peak I, thymine; peal II, cytosine; peak III, guanine; peak IV, adenine; peak V, “apurinic acid” fraction.
activity. Most of the radioactivity was recovered in the first WV-absorbing peak (peak V) which appeared to be similar in behaviour to peak V in Fig. 1, but the ratio of radioactivity to W-absorbance in the different fractions was no longer constant. It is also evident that some radioactivity was lost during the evaporation to dryness. The same UVabsorbing pattern was obtained in similar hydrolysates of DNA from untreT&ed mice.
4”
a0
Froct ion
RD
NO.
on Rowcx 50 (W” form) af Fig. 2. Ion cxchnnge chromatography carbamste. 10 mg, 6 PCi. 12 DNA from mouse given [ 1 I ‘“C]ethyl dpm of each 5 ml Ez~4 of each 5 ml fraction ; - - - - . -, Radioactivity hydrolysed by method d, 6 N HCI, lOO’C, 1 h. Peaks 1, II, lil, IV, V
hydrolyeate
of liver
h previously. ..----fraction. 12 m as rn Fig. 1.
155
The first UV-absorbing fractions containing most of the radioactivity were examined by paper chromatography. Five UV-absorbing spots were found, with RF values 0.35,0.61,0.78,0.92 and 0.98; the 0.35 and 0.61 spots were present in very small amounts. Radioactivity was distributed throughout the paper but there was a maximum at RF 0.58, Fig. 3. This coincided fairly closely with one of the UV-absorbing spots which however was only present in very small amount. None of the UV-absorbing spots was identified, but it is evident that the first UV-absorbing material comprises a mixture of compounds, and that only some of these could be associated with UV-absorbing components. The nature of the radioactive compounds was further investigated by measuring the distribution between aqueous and organic phases in neutral, alkaline and acidic conditions. The distribution coefficients obtained are given in Table II. Although these values do not represent well-defined thermodynamic quantities because of the variety of compounds involved, they demonstrate their remarkably hydrophilic character. The independence of the distribution coefficients from the pH of the aqueous phase as well as the very low affinity of the compounds for the organic phase indicate that the compounds contain a completely ionized group, most likely a phosphate group. Increasing the temperature and duration of hydrolysis (10 mg DNA), 6 N HCl at 125°C for 2 h (method e), and chromatography on Dowex 50 columns under the same conditions as before, produced the pattern shown in Fig. 4. In this case the hydrolysate was chromatographed without prior evaporation to dryness. The pyrimidine bases (peaks I and II) were now present in quantity. Guanine (peak III) was reduced and adenine (peak IV)
0
04
na
OR
Rf Fig. 3. Distribution of radioactivity on paper chromatogram (Whatman filter mobile phase propan-2+l : water : HCl(68 : 16.4 : 15.6 v/v/v).
156
wwr No.1;
Fraction
Wo
virtually absent; it is known that these compounds are d these conditions. The fast W-absorbing peak (peak V) 2 were not found, but there was now an unidentified absorbing material which contained no radioactivity thymine (peak I). The first few ml eluted off the colum
was
during the hydrolysis. TABLE II DISTRIBUTION
COEFFICIENT, K, HYDROLYSIS OF DNA, METHOD d
OF LABELLED PRODUCT 0 Mu&e from DNA of ---~lll-
Intact mice EtherlwrtW
Butnn.14 -
0.03 0.633 0.036
Neutral Acid AhliRf?
a”-=---
C___M_C 0.032 at333 0.037
‘w&or Acid Alkalins
_-_
.__
Enzymatic hydrolysis
Liver DNA (15 mg) from mice treated with [lJ4C]ethyl carbamate (6 PCi; 10 mg; i.p.) was hydrolysed with pancreatic deoxyribonuclease I followed by Crotalus adamanteus phosphodiesterase. The hydrolysate was chromatographed on a Dowex 1 column when all the radioactivity was found in the first UV-absorbing peak in the first few ml of eluate. On evaporation of the eluate to dryness all the radioactivity was lost. No radioactivity was found in association with the other hydrolysis products. Comparison of binding to liver DNA Sn intact and in hepa tectomised mice
Acid hydrolysis of DNA samples from mice given [ 1J4C] ethyl carbamate 18 h after partial hepatectomy failed to provide any evidence of a difference in the nature or site of binding of the ethyl group to DNA from intact mice. The chromatographic behaviour of the hydrolysates was identical in both cases. Distribution coefficients of the compounds formed by hydrolysis with 6 N HCl 100°C for 1 h (method d) were the same for intact as well as hepatectomised mice, Table II. Identification of volatile product The volatile compound split off during vigorous hydrolysis appeared to be
ethanol. Since the minute quantities could not be isolated, characterisation through the distribution coefficients seemed useful. These were determined on the first fractions, peak VII, in Fig. 4, obtained by chromatography of the hydrolysate from method e. They were found to be Ketherlwater= 0.32 and Kbutan-1-al/water = 0.83. Values determined for [*4C]ethanol by the same procedure were found to be Kether,water = 0.24, Kbutan.i.ol,water = 0.72, and K.lsobutanollwater = 1.03. These values are in good agreement with established values of K for ethanol, KetherIwater= 0.26, and Klsabutanol,water= 1.0 [36]. DISCUSSION
After administration of [l-“Cl ethyl carbamate to mice, the ethyl group bound to liver DNA [25] to an extent of about 8.0 - 10” mg per mg DNA. The carbonyl group did not bind [25] . If “0 replaced the ethoxy oxygen and bound to DNA, it should be expected to be incorporated to the same extent as the labelled alkyl group. However, no isotopic enrichment of the DNA oxygen was found. Consequently it must be assumed that it was the ethyl group and not the ethoxy group which bound to DNA. Under mild conditions of acid hydrolysis of the DNA, the purine bases were hydrolysed off leaving the “apurinic acid” fraction, which under the conditions of more vigorous hydrolysis lost some of the pyrimidines. However, none of the liberated purine or pyrimidine bases contained any of the radioactivity from the [1J4C]ethyl carbamate. Since the radioactivity was quantitatively recovered linked to the first “apurinic acid” fractions, this is not due to dealkylation of labelled bases unless it is postulated that a labelled base was less prone to hydrolysis from the DNA and remained with the 158
“apurinic acid”. However, this postulate seems improbable because in this event, as the conditions of hydrolysis became more s~gent, either the labelled base would be f&d or, if it decomposed, the labelled alkyl group would split off as ethanol or other possible products and not as a group of strongly ionizing hyd~~hili~ ~om~unds as was found. In the conditions of hydrolysis used, desertion of pyrimidine b does not appear to have occurred to a signifieant extent. IIence it must be concluded that no significant binding of the alkyl group to the purine or pyrimidine bases occurred. It is of some interest that the ethyl groups appeared main bound to the ‘“apurinic acid” fraction after removal of the purine Clearly the possibility that the bedim oecurwd ~~f~~nt~y tu p of the DNA chain containing the pyrimidines needs invest&&ion. After the most severe acid hy~lys~ (Fig. 4) the mdioactive component was completely dissociated from other compounds in a volatile form. The only compound likely to be liberated in this manner is ethanol and this is subs~n~~ly nonfat by me~~ement of the partition ~oeff~~ien~ of the labelled compound between aqueous and organic solvents. Since, apart from the py~midine and purine bases, the only groups in DNA available for binding of ethanol are the phosphate groups, the evidence is that the alkyl group is bound to the DNA as a phosphate ester. This is perhaps supported by the results of enzymatic hydrolysis with DNAase foliowed by C~otaius adammfeus phosph~i~~~. Such an ethyl ester of DNA would give rise to three strongly ionising phosphates containing the ethyl group in the course of acid hydrolysis, viz. deoxyribose-3-phosphoethyl ester, deoxyriboseGphosphoethy1 ester and the mono-ethyl phosphate among other compounds still linked to pyramidines, the proportions de~ndin~ on the reaction kinetics of each bond broken. Moderately severe acid hydrolysis (method c) of DNA from urethane treated mice did in fact lead to the pr~uc~o~ of a mixture of highly ioni~ble hydrophilic non-volatile compounds, some associated with W-absorbing ~y~midines~ which eluted in the earliest fractions of Dowex 50 columns. None of these however were identity, but since they gave rise under more severe acid hydrolysis to ethanol, it is likely that they represent the expected compounds. Skill ~om~unds have been found in hydroly~t~s of DNA alkylated in vitro with ethyl rne~~es~~honate [ZO] or alkylated in vivo with dime~ylnit~s~ina IZ%] and id~ntifi~ as ethyl and methyl phosphate~ ~s~~~tively* lt has been report These results do conflict with those of other of ethyl ~~barna~ that, in mice, both the alkyl and the carbon re acid, and that there 1 not [ZS) . The nature of the the hyd~lysis the In ~~ti~ul~ the ureth~e bound to the rapidly RNA [29] . Other workers have however, found in rats that the group was not inwolved in the bindin 371 4 Both sets of workers e amounts of isotopic label and small tal doses of urethane. The l
159
present authors have used a relatively small (10 NCi), but adequate, dose of l*C. The dose of [‘“C] ethyl carbamate was 10 mg, which is a carcinogenic dose, albeit at the lower level of the effective range. The carcinogenic effect of ethyl carbamate in all its target tissues was dose dependent [1,6 f as was the level of binding [ 251. In our experiments [23-251 the major site of binding has been to DNA. There was relatively little binding to RNA and cell proteins. We can therefore find no relevant basis on which to compare these results with those of different workers. It is possible to speculate on a mechanism of such binding, If hydrolysis of the ethyl carbamate preceded its interaction with cell constituents, ethoxy oxygen-carbonyl carbon fission would take place [38) and the ethoxy oxygen would transfer with the alkyl group, but the data suggest that this does not occur. Con~quently, ure~~e must react with fission of the ethyl-oxygen bond, in some form of nucleophilic displacement ma&ion at tie carbon-l centre, NHIT--O as the leaving group, the attacking nucleophile 0 being either oxygen or nitrogen of a base of DNA or, as the experiments suggest, the oxygen of a phosphate group. A possible mechanic is suggested below where B represents a nucleophilic site of attachment of the ethyl group, either in the DNA molecule or in a transferring molecule or enzyme.
P
H.$I-C-O-C-
I I CY
t
:B
-
H 2-N
i!%‘C’. -
*B
L
-
P
H2&-C-6
+ -C:
I
B f CH3
However, the specificity of the carcinogenic action of the ethyl carbag mates and the effects of substitution of the amide hydrogen [5,6] would need further elaboration. Although methyl and propyl carbamates also bound to some extent to DNA [25] it is possible that they combined in a different manner or, for example, the stability of the resulting products varied. Alkylations of DNA in vitro by methyl methanesulphonate and ethyl methanesulphonate resulted in alkylation of the purine bases and esterification of the free phosphate groups [20]. The total amount of alkyl group bound was similar in both instances. However, in the case of the methylation, phosphate groups were involved only to an extent of about l%, whereas, in the case of the ethylation, 15% was through the phosphate group. It was suggested that the alkylation of the diester phosphate required an alkylating agent that can ionise and react by an SN1 mechanism f 391. Such a mechanism seems unlikely in the present instance since the highly reactive carbonium ions produced would tend to bind to any ~tentia~ nu~leophil~ and the limitation of the binding to DNA would not have been found. Lastly it is significant that the nature of the binding to DNA of regenerating liver did not differ from that of normal liver, since it has been shown that mice with regenerating live: giver. ethyl carbamate develop more liver tumours [ll-141. The extent of badging appeared to be incre~ed f14] but hardly in proportion to the increase in tumour yields. 160
It has been suggested from time to time that &erification of phosphate groups might be a significant step in chemical carcinogenesis [18,20] but, because of the greater extent of alkylation of the bases, attention has largely been confined to this aspect of the problem. However, the esterification of phosphate groups by a variety of alkylating agents in vitro is well documented [20,21]. Recently attention has been directed to the formation of phosphate esters by dimethylnitrosamine [ 221 and AL-methyl-N-nitrosourea [ 211 in vivo in addition to alkylations at 0 and N sites on the bases. The formation of such esters might be expected to have a variety of effects in cells, related to base pairing, transcription, the action of endonucleases and the formation of linkages with proteins; all of which are involved in one way or another in proliferating cells. The relative significance of the different types of binding are likely to be difficult to sort out. Ethyl carbamate appears to bind predominantly to the phosphate groups and emphasises the need for review of the situation. ACKNOWLEDGEMENTS
This work was supported by grants from the Queensland Cancer Fund, the University of Queensland Cancer Research Fund, and the National Health and Medical Research Council of Australia. The authors thank Professor I. Lauder, Department of Chemistry, University of Queensland, for his advice and for providing the mass spectrometric facilities. REFERENCES 1 S.S. Mir\ ish, The carcinogenic action and metabolism of urethan and N-hpdroxyurethan, 4dv. Cancer I&., 11 (1968) 1. 2 C.D. Lar :cn, Evaluation of carcinogenicity of a series of esters of carbamic acid, J. Natl. Carter Inst., 8 (1947) 99. 3 C.D. Larsen, Pulmonary-tumor induction with alkylated urethans, J. Natl. Cancer Inst., 9 (: 948) 35. 4 I. Berenblum, D. Ben-Ishai, N. Haran-Ghera, A. Lapidot, E. Simon and N. Trainin, Skin initiating action and lung carcinogenesis by derivatives of urethane (ethyl carbamate) and related compounds, Biochem. Pharmacol., 2 (1959) 168. 5 A.W. Pound, The initiation of skin tumours in mice by homologues and N-substituted derivatives of ethyl carbamate, Aust. J. Exp. B3ol. Med. Sci., 45 (1967) 507. 6 A.W. Pound, The initiation of skin tumour formation in mice by N-hydroxycarbamates, Pathology, 1 (1969) 27. 7 A.W. Pound and JR. Bell, The influence of croton oil stimulation on tumour initiation by urethane in mice, Br. J. Cancer, 16 (1962) 690. 8 A.W. Pound and H.R. Withers, The influence of some irritant chemicals and scarification on tumour initiation by urethane in mice, Br. J. Cancer, 17 (1963) 460. 9 A.W. Pound, Further observations concerning the influence of preliminary stimulation by croton oil and acetic acid on the initiation of skin tumours in mice by ure thane, Br. J. Cancer, 20 (1966) 385. 10 H. Hennings, D. Michael and E. Pattercnn, Enhancement of skin tumorigenesis by a single application of croton oil before or soon after initiation by urethan, Cancer Res., 33 (1973) 3130. 11 A.W. Pound, Carcinogenesis and cell proliferation, N.Z. Med. J. (Special Issue), 67 (1968) 88.
161
12 M. Lane, A. Liebelt, J. Calvert and R.A. Liebelt, Effect of partial hepatectomy on tumor incidence in BALBlc mice treated with urethan, Cancer Res., 30 (1970) 1812. 13 I.N. Chernozemski and G.P. Warwick, Liver regeneration and induction of hepatomas in B6AFi mice by urethan, Cancer Res., 30 (1970) 2685. 14 A.W. Pound and T.A. Lawson, Effects of partial hepatectomy on carcinogenicity, metabolism, and binding to DNA of ethyl carbamate, J. Natl. Cancer Inst., 53 (1974) 423. 15 J.A. Miller, Carcinogenesis by chemicals: an overview, Cancer Res., 30 (1970) 559. 16 E.C. Miller and J.A. Miller, Approaches to the mechanisms and control of chemical carcinogenesis, in Environment and Cancer, Williams and Wilkins, Baltimore, 1972, pp. 5-39. 17 P.N. Magee, V.M. Craddock and P.F. Swann, The possible significance of alkylation of nucleic acids in carcinogenesis of the liver and other organs, in Carcinogenesis: A Broad Critique, Williams and Wilkins, Baltimore, 1967, pp. 421-439. 18 D.T. Elmore, J.M. Gulland, D.O. Jordan and H.F.W. Taylor, Reaction of nucleic acids with mustard gas, Biochem. J., 42 (1948) 308. 19 P.D. Lawley and P. Brookes, Further studies on the alkylation of nucleic acids and their constituent nucleotides, Biochem. J., 89 (1963) 127. 20 P. Bannon and W. Verly, Alkylation of phosphates and stability of phosphate triesters in DNA, Europ. J. Biochem., 31 (1972) 103. 21 P.D. Lawley, Reaction of N-methyl nitrosourea (MNUA) with 32P-labelled DNA: evidence for formation of phosphotriesters, Chem.-Biol. Interact., 7 (1973) 127. 22 P.J. O’Connor, G.P. Margison and A.W. Craig, Phosphotriesters in rat liver deoxyribonucleic acid after the administration of the carcinogen N-dimethylnitrosamine in vivo, Biochem. J., 145 (1975) 475. 23 T.A. Lawson and A.W. Pound, The reaction of urethane with mouse liver nucleic acids in vivo, Pathology, 3 (1971) 323. 24 T.A. Lawson and A.W. Pound, The interaction of [ 3H]ethyl carbamate with nucleic acids of regenerating mouse liver, Chem:biol. Interact., 4 (1971/72) 329. 25 T.A. Lawson and A.W. Pound, The interaction of carbon-ll-labelled alkyl carbamates, labelled in the alkyl and carbonyl positions, with DNA in vivo, Chem.-Biol. Interact., 6 (1973) 99. 26 S.V. Bhide, E. Premkumar, M.A. Siddiqui and P.M. Bhargava, Preliminary observations on the binding of urethan to DNA of several tissues of mice and rats, Ind. J. Cancer, 8 (1971) 172. 27 M.B. Shimkin, Pulmonary tumors in experimental animals, Adv. Cancer Res., 3 (1955) 223. 28 E. Boyland and K. Williams, Reaction of urethane with nucleic acids in vivo, Biothem. J., 111 (1969) 121. 29 K. Williams, W. Kunz, K. Petersen and B. Schneiders, Changes in mouse liver RNA induced by ethyl carbamate (urethane) and methyl carbamate, 2. Krebsforsch., 76 (1971) 69. 30 J.H, Parish and KS. Kirby, An extension of naphthalene disulphonate method for mammalian nucleic acids, Biochim. Biophys. Acta, 42 (1967) 273. 31 P.D. Boy.er, D.J. Graves, C.H. Suelter and M.E. Dempsey, Simple procedure for conversion of oxygen of orthophosphate or water to carbon dioxide for oxygen-18 determination, Anal. Chem., 33 (1961) 1906. 32 D. Rittenberg and L. Ponticorvo, A method for the determination of the Oia concentration of the oxygen or organic compounds, Int. J. App. Radiation and Isotopes, 1 (1956) 208. 33 H. Dahn, H. Moll and R. Menas& Eine neue Methode zur Bestimmung von Sauerstoffisotopen in organischen verbindungen, Helv. Chim. Acta. 42 (1959) 1226. 34 G.A. Bray, A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter, Anal. Biochem., 1 (1960) 279. ,35 In C. Long (Ed.), Biochemists Handbook, Spon, London, 1961, p. 199.
162
36 R. Collander, The distribution of organic compounds between iso-butsnol and water, Acta Chem. &and., 4 (1950) 1085. 37 G. Prodi, P. Rocchi and S. Grilli, in vivo interaction of urethan with nucleic acids and proteins, Cancer I&s., 30 (1970) 2887. 38 P. Adams and F.A. Baron, Esters of carbamic acid, Cbem. Ftev., 65 (1965) 567. 39 W.G. Verly, Monofunctional alkylating agents end apurinic sites in DNA, Biochem. Pharmacol., 23 (1974) 3.
A.W. POUND, F. FRANKE
* and T.A. LAWSON
Department of Pathology, University of Queensland, Herston, Queensland (Australia, 4006) (Recr ived September 30th, 1975) (AccLpted October 20th, 1975) --
~--__-.-
..-. _~.~__.
SUMMARY
Ethyl carbamate, labelled at C1 with 14C, bound in vivo to liver DNA of intact and partially hepatectomised mice. Isotope (‘“0) enrichment was not detected in the oxygen of liver DNA of mice injected with [I801 ethyl carbamate, C&l&-18 O-CO-NH2. This suggests that it was the ethyl group and not the ethoxy group which bound to DNA. Chromatographic analysis of acid hydrolysates of liver DNA from mice treated with [l-14C] ethyl carbamate provided no evidence of alkylation or other form of binding to purine or pyrimidine bases. On relatively mild acid hydrolysis the alkyl group remained bound to the “apurinic acid” fraction, while more vigorous hydrolysis lead progressively first to its separation as highly ionisable hydrophilic non-volatile compounds and then to its loss as a volatile compound. DNAase I followed by phosphodiesterase hydrolysis also split off the 14Ccontaining group as a volatile compound. The volatile compound was identified as ethanol. These results suggest that the alkyl group was bound as an ester to a phosphate group in the DNA chain. Results with DNA from partially hepatectomised mice did not differ from those with DNA from intact mice.
II%TRODUCTION
Ethyl carbamate is an active carcinogen in mice, particularly for lung, liver and, as an initiating agent, for skin [ 11. Its mono-l\r-substituted derivatives are also active [2-6] but the other simple homologous alkyl carbamates are * Present address: School 4111).
of Science,
Griffith University,
Nathan, Queensland
(Australia,
149
not or are very weakly so. When the target tissue is induced to proliferate at the time of its administration more tumours are produced, e.g. in skin after chemical stimulation [ 7-101 and in liver after partial hepatectomy [ 11-141. Many, perhaps all, chemical carcinogens or their metabolites [15 ] form covalent linkages with cell macromolecules in vivo, commonly through interactions with the bases of DNA, although RNA and cell proteins are also involved [16] . The role of alkylation in the carcinogenic process is undecided [ 16,17 ] . Esterification of the free phosphate groups occurs to a lesser extent during the interaction of alkylating agents with DNA in vitro [18-211 but consideration of its role has been overshadowed by the more efficient alkylation of the bases. It has only recently been detected in vivo 1221 A metabolite of ethyl carbamate (urethane) bound to liver DNA in intact and in partially hepatectomised mice [23,24 1. It also bound to epidermal and lung DNA. The bound molecule involved either an ethyl or an ethoxy group, but not the carbonyl group, of urethane [25]. A similar result was found in rats [26] in which animal urethane is carcinogenic only for lungs [ 271. In this laboratory it was found, in mice, that the binding occurred predominantly to DNA in liver [23] and skin (unpublished data) and that there was relatively little binding to RNA or cell proteins. The latter therefore have not been examined further by us. Qther workers have reported that the carbo-ethoxy group bound to RNA as the ethyl ester of cytosinedcarboxylic acid [28,29] but that only smaller amounts of binding occurred to DNA, the nature of which was not examined in detail. There is therefore no agreement on the site and nature of the binding of ethyl carbamate to macromolecules in cells. The present work was performed firstly, to determine whether the ethyl or the ethoxy group of urethane bound to DNA. Mice were given ethyl carbarn&e, C2H5-180-CO-NH2, and the I80 content of the resulting DNA measured by determination of the 1zC’60’60/‘2C’60’60 ratio of the carbon dioxide obtained by its decomposition. Secondly it was desired to determine the nature of the binding of the ethyl or the ethoxy group by giving 14Clabelled ethyl carbamate and identifying the bound molecule in hydrolysates of DNA by chromatographic examination. Thirdly it was necessary to determine if the nature and site of binding were the same in intact and hepatectomised mice. l
MATERIALS AND METHODS
Animals
Random bred, male Crackenbush mice, 6-8 weeks old, weighing 35’! 40 g were used. Diet and conditions of animal housing were the same as before [23,14]. Chemicals
Ethyl carbamate (urethane) was obtained from B.D.H. Ltd., Poole, Great 150
Britain. [‘80] Ethyl
~~~a~,
(59.0 mCi/mmole) Great Britain and nucleosiiles were oh Dose schedules
o~tirn~ [ 25]. Extraction
of DNA
DNA was extracted tact and two-thirds hep The ‘~~~I60 ratios were dete carbon dioxide. The 8 spectrometrically. The t (I) Guanidine h y~r~~h mg) and ~~idine hydro~lorj outgw+ed at 80°C for 30 min maintained at 390” C for 2 h.
that described in frozen. The vol
as described above An MS 10 mass tions were rn~i~~n~d i of CC&the 46 and 44
average 46/44 ratio. The measurement alternated with that of a “Standard”
of the ratio for COz from DNA was CO2 of normal isotopic composition.
Hydrolysk of DNA (i) Acid hydrolysis: Acid hydrolysis was carried out in sealed Pyrex glass tubes under five sets of conditions - (a) HCOOH (98%; v/v) at 175°C for 30 min; (b) HCOOH (90%; v/v) at 175°C for 30 min; (c) 1 N HCl at 100°C for 1.5 h; (d) 6 N HCl at 100°C for 1 h; and (e) 6 N HCl at 125°C for 2 h. (ii) Enzymatic hydrolysis: 15 mg DNA, dissolved in 0.1 M NH4HCOJ and 0.015 M MgClz, 3 ml, was incubated with 50 pl(250 pg) DNAase I at 37°C for 2 h. The pH was adjusted to 8.5 with dilute ammonia and 75 ~1 (375 pg) Crotalus adamanteus phosphodiesterase was added. Incubation was continued at 37” C for a further 2 h. Chromatography of hydrolysates Unless otherwise stated, the products of the acid hydrolyses were, after evaporation to dryness in a stream of nitrogen, taken up in 0.1 N HCl, applied to Dowex 50 (H+) columns (50 cm X 1 cm) and eluted with a linear gradient of 0.1 to 4.0 N HCl. 5 ml fractions were collected. The products of enzymatic hydrolysis were chromatographed on a Dowex l-XH (formate form) column without previous evaporation to dryness and were eluted with a linear gradient of 0 to 4 N HCOOH followed by 1 N HCOONH4. The Ezs4 of the eluates was monitored continuously. The radioactivity was determined by adding 10 ml of Bray’s scintillation fluid [343 to 1 ml aliquots of the neutralised fractions and counting their activity in a Packard Scintillation Spectrometer. The positions of the four major bases, thymine, cytosine, adenine and guanine, were identified by co-chromatography with pure authentic samples. In the case of acid hydrolysis by method (d), fractions containing radioactivity were examined after prior concentration by descending chromatography on Whatman No. 1 paper using propan-2-01 : Hz0 : concentrated HCl (68 : 16.4 : 15.6, v/v/v) as a solvent. The chromatograms were developed for 9 h after which the spots were visualised on the dried papers by their UVabsorption at 254 nm. The distribution of labelled material was determined by cutting the dried paper into small equal sized strips, immersing them directly into the scintillation fluid and measuring their radioactivity. Measurement of distribution coefficients Distribution coefficients (K := concentration in organic phase/concentration in aqueous phase) of radioactive products between aqueous and organic phases were determined, Aliquots of the material under examination were (1) left acid as obtained, (2) neutralised with solid NaHC03, and (3) made alkaline with solid NaOH and equilibrated by shaking with ether, isobutanol or butan-l-01. Aliquots of the two phases were withdrawn and their radioactivity was determined after neutralisation.
152
RESULTS
Nature of the bound molecule
Experiments in mice on the in vivo binding of [1-14C]ethyl carbamate to liver DNA showed that 1 mg of DNA contained about 8.0 - lo-’ mg of urethane, or the equivalent of its metabolite, 12 h after a dose of 10 mg [25]. If the DNA contained the oxygen [I801 originally present in this dose of [ lsO]ethyl carbamate and if, during the thermal decomposition to C02, this became randomly mixed with the other oxygen of normal isotopic composition in the DNA, then the 46/44 ratio for this COz should be about 15 * 10e5 greater than the /6/44 ratio for CO2 of “normal” isotopic composition. This calculation is based on the assumption that DNA contains 31 wt. % of oxygen, derived from the recorded base composition of .mouse DNA [35] . In point of fact, the dose of [ *8O]ethyl carbamate given was 20 mg. Since the extent of binding is dose-dependent [24] the increase in the 46/44 ratio should be greater than this. COz, slightly enriched in ‘*O, was prepared with a 46/44 ratio about 10 10” in excess of the 46/44 ratio of COz of normal isotopic composition. This excess is of the same order as the excess ratio expected if the I80 in the labelled ethyl carbamate were bound to the DNA. Samples of this I80 enriched CO* were put through the guanidine and also the mercuric chloride isolation techniques to establish whether the procedures involved any isotopic dilution. The guanidine technique showed no isotopic dilution but the mercuric chloride technique did introduce some dilution whereby the 46/44 ratio decreased by 2 - 10”. The isotopic ratios (46/44) of COz obtained from liver DNA of intact and partially hepateetomised mice that had been given [ ‘8Q]ethyl carbamate, and from normal untreated mice are shown in Table I. The results obtained with both the guanidine hydrochloride and mercuric chloride methods are shown. Within each group there are no significant differences in the isotope ratios from those obtained with standard pure COZ. No measurable isotope enrichment had occurred in any of the samples. l
Site of binding Formic acid hydrolysis:
Liver DNA (10 mg) from intact mice given [ 1-14C]ethyl carbamate (6 PCi; 10 mg; i.p.) was hydrolysed with 98% (v/v) formic acid at 175°C for 1 h (method a). Elution of the evaporated hydrolysate from a Dowex 50 (H+ form) column with HCl (0.1 to 4 N) produced eleven UV-absorbing fractions, only one of which, adenine, was identified. .No radioactivity was detected in any of the fractions. The radioactive component or components must therefore have been liberated as a volatile material, possibly ethanol. On less vigorous hydrolysis, i.e. with 90% formic acid (method b), the radioactivity was recovered quantitatively in the. first UV-absorbing peak eluted from the columns, and was therefore not present in this case as a volatile compound. 153
TABLE I ISOTOPIC RATIOS (46/44) OF CO1 OBTAINED GIVEN [‘so ] ETHYL CARBAMATE, 20 mg, i.p. Treatment of mice from which liver DNA was obtained
FROM LIVER DNA OF MALE MICE
10s X Isotopic
ratios a
-_-
Guanidine HCl method
Mercuric chloride method
Intact mice + urethane Standard CO2 b Intact mice + uerthane Standard CO*
449.6 448.6 448.6 448.4
447.3 448.9 446.4 446.6
Hepatectomised Standard CO* Hepatectomised Standard CO*
449.2 448.2 449.2 449.2
445.4 446.6 446.7 449.8
446.2 448.5 448.4 447.5
446.3 447.6 -
Untreated Standard Untreated Standard
mice CO* mice COz
mice + urethane mice + urethane
a Each value is the average from 6 measurements with a maximum deviation of 1.1 - lo-‘. The values have been adjusted to a standard output voltage of 30 V for the m/e = 44 beam from an experimentally determined graph. b Measurements of standard CO2, normal isotopic composition, were alternai.ed with CO2 from experimental animals.
Hydrochloric acid hydrolysis. Liver DNA (15 mg) from intact mice given [l-14C] et.hyl carbamate (6 PCi; 10 mg; i.p.) was hydrolysed with 1 N HCl at 100°C for 1.5 h (method c). The chromatographic behaviour of the hydrolysate, after prior evaporation to dryness, on a Dowex 50 column is shown in Fig. 1. Thymine (peak I) and cytosine (peak II) in small amounts, guanine, (peak III) and adenine (Peak IV) in large amounts were identified, by cochromatography with authentic samples, together with four o:her unidentified UV-absorbing compounds (presumably nucleosides and nucleotides). The first strongly UV-absorbing fractions (peak V) to be eiuted from the column contained 311the radioactivity present in the original material. The ratio of the radioactivity to the UV-absorbance was nearly ccnstant through all the fractions proving that the radioactive compound was still linked to the UV-absorbing substance, that is, the apurinic acid fraction. More vigorous hydrolysis (12 mg DNA), with 6 N HCl at 100°C for 1 h (method d), and chromatography of the evaporated hydrolysate on Dowex 50 columns produced the pattern shown in Fig. 2. The four major bases and several smaller UV-absorbing compounds were present. Compared with the hydrolysate from method c (Fig. l), thymine (peak I) and cytosine (peak II) were now relatively increased in amount, guanine (peak III) and adenine (peak IV) were about the same in amount, and some unidentified products appeared ahead of cytosine but none of these fractions contained any radio-
154
Fraction
No.
Fig. 1. Ion exchange chromatography on Dowex 50 (H+ form) of hydrolysate of liver DNA from mouse given [1-‘4C]ethyl carbamate, 10 mg, 6 PCi, 12 h previously. Ezs4 of each 5 ml fraction; - - - - - -, Radioactivity dpm of each 5 ml fraction. 15 mg DNA hydrolysed by method c, 1 N HCI, lOO”C, 1.5 h. Peak I, thymine; peal II, cytosine; peak III, guanine; peak IV, adenine; peak V, “apurinic acid” fraction.
activity. Most of the radioactivity was recovered in the first WV-absorbing peak (peak V) which appeared to be similar in behaviour to peak V in Fig. 1, but the ratio of radioactivity to W-absorbance in the different fractions was no longer constant. It is also evident that some radioactivity was lost during the evaporation to dryness. The same UVabsorbing pattern was obtained in similar hydrolysates of DNA from untreT&ed mice.
4”
a0
Froct ion
RD
NO.
on Rowcx 50 (W” form) af Fig. 2. Ion cxchnnge chromatography carbamste. 10 mg, 6 PCi. 12 DNA from mouse given [ 1 I ‘“C]ethyl dpm of each 5 ml Ez~4 of each 5 ml fraction ; - - - - . -, Radioactivity hydrolysed by method d, 6 N HCI, lOO’C, 1 h. Peaks 1, II, lil, IV, V
hydrolyeate
of liver
h previously. ..----fraction. 12 m as rn Fig. 1.
155
The first UV-absorbing fractions containing most of the radioactivity were examined by paper chromatography. Five UV-absorbing spots were found, with RF values 0.35,0.61,0.78,0.92 and 0.98; the 0.35 and 0.61 spots were present in very small amounts. Radioactivity was distributed throughout the paper but there was a maximum at RF 0.58, Fig. 3. This coincided fairly closely with one of the UV-absorbing spots which however was only present in very small amount. None of the UV-absorbing spots was identified, but it is evident that the first UV-absorbing material comprises a mixture of compounds, and that only some of these could be associated with UV-absorbing components. The nature of the radioactive compounds was further investigated by measuring the distribution between aqueous and organic phases in neutral, alkaline and acidic conditions. The distribution coefficients obtained are given in Table II. Although these values do not represent well-defined thermodynamic quantities because of the variety of compounds involved, they demonstrate their remarkably hydrophilic character. The independence of the distribution coefficients from the pH of the aqueous phase as well as the very low affinity of the compounds for the organic phase indicate that the compounds contain a completely ionized group, most likely a phosphate group. Increasing the temperature and duration of hydrolysis (10 mg DNA), 6 N HCl at 125°C for 2 h (method e), and chromatography on Dowex 50 columns under the same conditions as before, produced the pattern shown in Fig. 4. In this case the hydrolysate was chromatographed without prior evaporation to dryness. The pyrimidine bases (peaks I and II) were now present in quantity. Guanine (peak III) was reduced and adenine (peak IV)
0
04
na
OR
Rf Fig. 3. Distribution of radioactivity on paper chromatogram (Whatman filter mobile phase propan-2+l : water : HCl(68 : 16.4 : 15.6 v/v/v).
156
wwr No.1;
Fraction
Wo
virtually absent; it is known that these compounds are d these conditions. The fast W-absorbing peak (peak V) 2 were not found, but there was now an unidentified absorbing material which contained no radioactivity thymine (peak I). The first few ml eluted off the colum
was
during the hydrolysis. TABLE II DISTRIBUTION
COEFFICIENT, K, HYDROLYSIS OF DNA, METHOD d
OF LABELLED PRODUCT 0 Mu&e from DNA of ---~lll-
Intact mice EtherlwrtW
Butnn.14 -
0.03 0.633 0.036
Neutral Acid AhliRf?
a”-=---
C___M_C 0.032 at333 0.037
‘w&or Acid Alkalins
_-_
.__
Enzymatic hydrolysis
Liver DNA (15 mg) from mice treated with [lJ4C]ethyl carbamate (6 PCi; 10 mg; i.p.) was hydrolysed with pancreatic deoxyribonuclease I followed by Crotalus adamanteus phosphodiesterase. The hydrolysate was chromatographed on a Dowex 1 column when all the radioactivity was found in the first UV-absorbing peak in the first few ml of eluate. On evaporation of the eluate to dryness all the radioactivity was lost. No radioactivity was found in association with the other hydrolysis products. Comparison of binding to liver DNA Sn intact and in hepa tectomised mice
Acid hydrolysis of DNA samples from mice given [ 1J4C] ethyl carbamate 18 h after partial hepatectomy failed to provide any evidence of a difference in the nature or site of binding of the ethyl group to DNA from intact mice. The chromatographic behaviour of the hydrolysates was identical in both cases. Distribution coefficients of the compounds formed by hydrolysis with 6 N HCl 100°C for 1 h (method d) were the same for intact as well as hepatectomised mice, Table II. Identification of volatile product The volatile compound split off during vigorous hydrolysis appeared to be
ethanol. Since the minute quantities could not be isolated, characterisation through the distribution coefficients seemed useful. These were determined on the first fractions, peak VII, in Fig. 4, obtained by chromatography of the hydrolysate from method e. They were found to be Ketherlwater= 0.32 and Kbutan-1-al/water = 0.83. Values determined for [*4C]ethanol by the same procedure were found to be Kether,water = 0.24, Kbutan.i.ol,water = 0.72, and K.lsobutanollwater = 1.03. These values are in good agreement with established values of K for ethanol, KetherIwater= 0.26, and Klsabutanol,water= 1.0 [36]. DISCUSSION
After administration of [l-“Cl ethyl carbamate to mice, the ethyl group bound to liver DNA [25] to an extent of about 8.0 - 10” mg per mg DNA. The carbonyl group did not bind [25] . If “0 replaced the ethoxy oxygen and bound to DNA, it should be expected to be incorporated to the same extent as the labelled alkyl group. However, no isotopic enrichment of the DNA oxygen was found. Consequently it must be assumed that it was the ethyl group and not the ethoxy group which bound to DNA. Under mild conditions of acid hydrolysis of the DNA, the purine bases were hydrolysed off leaving the “apurinic acid” fraction, which under the conditions of more vigorous hydrolysis lost some of the pyrimidines. However, none of the liberated purine or pyrimidine bases contained any of the radioactivity from the [1J4C]ethyl carbamate. Since the radioactivity was quantitatively recovered linked to the first “apurinic acid” fractions, this is not due to dealkylation of labelled bases unless it is postulated that a labelled base was less prone to hydrolysis from the DNA and remained with the 158
“apurinic acid”. However, this postulate seems improbable because in this event, as the conditions of hydrolysis became more s~gent, either the labelled base would be f&d or, if it decomposed, the labelled alkyl group would split off as ethanol or other possible products and not as a group of strongly ionizing hyd~~hili~ ~om~unds as was found. In the conditions of hydrolysis used, desertion of pyrimidine b does not appear to have occurred to a signifieant extent. IIence it must be concluded that no significant binding of the alkyl group to the purine or pyrimidine bases occurred. It is of some interest that the ethyl groups appeared main bound to the ‘“apurinic acid” fraction after removal of the purine Clearly the possibility that the bedim oecurwd ~~f~~nt~y tu p of the DNA chain containing the pyrimidines needs invest&&ion. After the most severe acid hy~lys~ (Fig. 4) the mdioactive component was completely dissociated from other compounds in a volatile form. The only compound likely to be liberated in this manner is ethanol and this is subs~n~~ly nonfat by me~~ement of the partition ~oeff~~ien~ of the labelled compound between aqueous and organic solvents. Since, apart from the py~midine and purine bases, the only groups in DNA available for binding of ethanol are the phosphate groups, the evidence is that the alkyl group is bound to the DNA as a phosphate ester. This is perhaps supported by the results of enzymatic hydrolysis with DNAase foliowed by C~otaius adammfeus phosph~i~~~. Such an ethyl ester of DNA would give rise to three strongly ionising phosphates containing the ethyl group in the course of acid hydrolysis, viz. deoxyribose-3-phosphoethyl ester, deoxyriboseGphosphoethy1 ester and the mono-ethyl phosphate among other compounds still linked to pyramidines, the proportions de~ndin~ on the reaction kinetics of each bond broken. Moderately severe acid hydrolysis (method c) of DNA from urethane treated mice did in fact lead to the pr~uc~o~ of a mixture of highly ioni~ble hydrophilic non-volatile compounds, some associated with W-absorbing ~y~midines~ which eluted in the earliest fractions of Dowex 50 columns. None of these however were identity, but since they gave rise under more severe acid hydrolysis to ethanol, it is likely that they represent the expected compounds. Skill ~om~unds have been found in hydroly~t~s of DNA alkylated in vitro with ethyl rne~~es~~honate [ZO] or alkylated in vivo with dime~ylnit~s~ina IZ%] and id~ntifi~ as ethyl and methyl phosphate~ ~s~~~tively* lt has been report These results do conflict with those of other of ethyl ~~barna~ that, in mice, both the alkyl and the carbon re acid, and that there 1 not [ZS) . The nature of the the hyd~lysis the In ~~ti~ul~ the ureth~e bound to the rapidly RNA [29] . Other workers have however, found in rats that the group was not inwolved in the bindin 371 4 Both sets of workers e amounts of isotopic label and small tal doses of urethane. The l
159
present authors have used a relatively small (10 NCi), but adequate, dose of l*C. The dose of [‘“C] ethyl carbamate was 10 mg, which is a carcinogenic dose, albeit at the lower level of the effective range. The carcinogenic effect of ethyl carbamate in all its target tissues was dose dependent [1,6 f as was the level of binding [ 251. In our experiments [23-251 the major site of binding has been to DNA. There was relatively little binding to RNA and cell proteins. We can therefore find no relevant basis on which to compare these results with those of different workers. It is possible to speculate on a mechanism of such binding, If hydrolysis of the ethyl carbamate preceded its interaction with cell constituents, ethoxy oxygen-carbonyl carbon fission would take place [38) and the ethoxy oxygen would transfer with the alkyl group, but the data suggest that this does not occur. Con~quently, ure~~e must react with fission of the ethyl-oxygen bond, in some form of nucleophilic displacement ma&ion at tie carbon-l centre, NHIT--O as the leaving group, the attacking nucleophile 0 being either oxygen or nitrogen of a base of DNA or, as the experiments suggest, the oxygen of a phosphate group. A possible mechanic is suggested below where B represents a nucleophilic site of attachment of the ethyl group, either in the DNA molecule or in a transferring molecule or enzyme.
P
H.$I-C-O-C-
I I CY
t
:B
-
H 2-N
i!%‘C’. -
*B
L
-
P
H2&-C-6
+ -C:
I
B f CH3
However, the specificity of the carcinogenic action of the ethyl carbag mates and the effects of substitution of the amide hydrogen [5,6] would need further elaboration. Although methyl and propyl carbamates also bound to some extent to DNA [25] it is possible that they combined in a different manner or, for example, the stability of the resulting products varied. Alkylations of DNA in vitro by methyl methanesulphonate and ethyl methanesulphonate resulted in alkylation of the purine bases and esterification of the free phosphate groups [20]. The total amount of alkyl group bound was similar in both instances. However, in the case of the methylation, phosphate groups were involved only to an extent of about l%, whereas, in the case of the ethylation, 15% was through the phosphate group. It was suggested that the alkylation of the diester phosphate required an alkylating agent that can ionise and react by an SN1 mechanism f 391. Such a mechanism seems unlikely in the present instance since the highly reactive carbonium ions produced would tend to bind to any ~tentia~ nu~leophil~ and the limitation of the binding to DNA would not have been found. Lastly it is significant that the nature of the binding to DNA of regenerating liver did not differ from that of normal liver, since it has been shown that mice with regenerating live: giver. ethyl carbamate develop more liver tumours [ll-141. The extent of badging appeared to be incre~ed f14] but hardly in proportion to the increase in tumour yields. 160
It has been suggested from time to time that &erification of phosphate groups might be a significant step in chemical carcinogenesis [18,20] but, because of the greater extent of alkylation of the bases, attention has largely been confined to this aspect of the problem. However, the esterification of phosphate groups by a variety of alkylating agents in vitro is well documented [20,21]. Recently attention has been directed to the formation of phosphate esters by dimethylnitrosamine [ 221 and AL-methyl-N-nitrosourea [ 211 in vivo in addition to alkylations at 0 and N sites on the bases. The formation of such esters might be expected to have a variety of effects in cells, related to base pairing, transcription, the action of endonucleases and the formation of linkages with proteins; all of which are involved in one way or another in proliferating cells. The relative significance of the different types of binding are likely to be difficult to sort out. Ethyl carbamate appears to bind predominantly to the phosphate groups and emphasises the need for review of the situation. ACKNOWLEDGEMENTS
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