Chem. Res. Tonicol. 1990, 3, 587-594
587
Characterization of S- [2- ( N '-Aden y l)ethy Ilglutathione as an Adduct Formed in RNA and DNA from 1,2-Dibromoethane D o n g - H y u n Kim,'
W. Griffith H u m p h r e y s , a n d F. P e t e r Guengerich*
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received July 27, 1990 T h e major DNA adduct derived from 1,2-dibromoethane is known to be S-[2-(N"-guanyl)ethyllglutathione; minor nucleic acid DNA adducts were characterized in view of the possibility that some might be unusually persistent or biologically active. RNA was modified in vitro by treatment with 1,2-dibromoethane and glutathione in the presence of rat liver cytosol, and bases were released by mild acid hydrolysis, which liberated >99% of the bound radioactivity. One of the minor adducts was identified as S-[2-(Nl-adeny1)ethyllglutathione on the basis of its UV, mass, and NMR spectra. This adduct could be synthesized by reaction of S-(2-chloroethyl)glutathione with adenosine. The material was desulfurized by treatment with Raney Ni to give Wethyladenine in low yield. The Raney Ni reaction was accompanied by considerable formation of the corresponding NG-adenine derivative via Dimroth rearrangement. Another adduct was identified as S- [ 2-(ZV-guanyl)ethyl]cysteinylglycineby its UV, mass, and NMR spectra, but the material was demonstrated to be formed from the major DNA adduct, S-[2-(N"-guanyl)ethyllglutathione under conditions of mild acid hydrolysis. T h e imidazole ring opened derivative of S-[2-(ZV-guanyl)ethyl]glutathionewas synthesized and found not to be formed in DNA in vitro or in vivo. T h e two remaining minor adducts account for 1-270 of the total binding, but insufficient quantities were recovered to allow for structure determination; however, neither of these (uncharacterized) minor products are seen after the reaction of S-(2-chloroethyl)glutathione with guanosine or adenosine. S-[2-(Nl-Adeny1)ethyljglutathione was formed in rat liver RNA and DNA. Rates of loss of the minor adducts from rat liver DNA are very similar to the rate of loss of S-[2-(N7-guanyl)ethyl]glutathione(tllP 100 h), and no evidence for unusual persistence was found.
-
1,2-Dibromoethane (ethylene dibromide) is toxic, and its use as a pesticide has been restricted (1,2). While the cancer risk to humans is not clear, the compound causes tumors at a variety of sites in rodents (3-7) and the hepatocarcinogenicity can be quite dramatic when disulfiram is used as a cocarcinogen to block detoxication (8). The apparent mechanism of activation of 1,2-dibromoethane and other uic-dihaloalkanes to their genotoxic forms differs from those seen with many other procarcinogens in that GSH' conjugation is the key step in the generation of an electrophile (Scheme I). This pathway has been documented in the formation of S-[2-(N'-guanyl)ethyl]GSH, the major DNA adduct derived from 1,2-dihromoethane (9-1 I). While S-[2-(N'-guanyl)ethyl]GSHis clearly the major adduct formed in DNA and RNA, the possibility must be considered that minor adducts may show unusual persistence or be capable of producing mutations or other genotoxic responses. Such considerations have been important in the study of adducts derived from methylating agents, where the importance of 06-alkylguanine and 0alkylpyrimidines is known (12-14). In this work searches were made for the identities of the minor nucleic acid adducts. The adduct S-[2-(N1adeny1)ethylJGSH was characterized in detail, and the kinetics of removal of the minor adducts are also reported.
Experimental Section Chemicals. 1,2-Dibromo[1,2-14C]ethanewas purchased from New England Nuclear-Du Pont (Boston, MA), and its radiochemical purity was judged to be >99% by radio-HPLC using 'Present address: Korea Institute of Science and Technology, P.O. Box 131, Chongyangni, Seoul, South Korea. 0893-228x/90/2703-05a7$02.50/0
a C18 column and an HzO to CH30H gradient (15). We have found that the most satisfactory method of long-term storage of concentrated stock solutions is in a 1:l mixture of poly(ethy1ene glycol) 400 and acetone-impurities are formed during long-term storage in (CH3)zS0. "-Methyladenine, OB-methyladenine, fl-methyladenine, N-methyladenine, and GSH were purchased from the Sigma Chemical Co. (St. Louis, MO). "-Ethyladenine was prepared by treatment of 0.6 g of adenine with 2.3 g (4.4 equiv) of ethyl methanesulfonate in 3 mL of HCOPH (16). The products were separated by HPLC (9),and the N1ethyladenine peak was identified by its characteristic UV spectra (17, 18). S-(2-Chloroethyl)GSH was prepared as described elsewhere (19). The half-mustard (10 equiv) was mixed with adenosine in H20/(CH3),S0 (1:l) for 2 h a t 37 "C, and the resulting solution was heated a t 100 "C for 30 min a t pH 1. The product, S-[2(N'-adenyl)ethyl]GSH, was separated by HPLC using the procedures described below for the isolation of the adduct formed in RNA. The yield was 12%: NMR (2Hz0, ppm relative to sodium 4,4-dimethyl-4-silapentane-l-sulfonate) b 8.46 (s, 1 H, Ha), 8.27 (s, 1 H, H2), 4.52 (m, 3 H, CHzCHzS + Cys a ) , 3.72 (m, 3 H, Gly a Glu a ) , 3.07 (m, 3 H, CHzCHzS+ Cys p Hb), 2.85 (dd, 1 H, J = 14.1, 8.4, Cys /.3 Haj, 2.45 (t, 2 H, J = 7.6, Glu T), 2.08 (m, 2 H , Glu 0). S-[2-(N6-Adenyljethyl]GSH was prepared by incubation of S-[2-(N1-adenyl)ethy1]GSH a t pH 10 and 37 "C for 5 days. This resulted in a 50% conversion to product, which was purified in the same manner as the N' derivative: NMR (2Hz0,ppm relative
+
' Abbreviations: GSH, glutathione;FAB-MS, fast atom bombardment
mass spectrometry; HPLC, high-performance liquid chromatography; SCX, strong cation exchange (HPLC); SAX, strong anion exchange (HPLC); C18, octadecylsilyl (HPLC); P-450, microsomal cytochrome P-450, Tris, tris(hydroxymethy1)aminomethane;GSCH,CH,-FAPY I, S-[ 2- [N-formyl-N-(2,6-diamino-4-0~0-3,4-dihydropyrimidin-5-yl)amino]-
ethyllglutathione; GSCH,CH,-FAPY 11, S-[2-[(2-amino-6-formamido-4oxo-3,4-dihydropyrimidin-5-yl)amino]ethyl]glutathione,
0 1990 American Chemical Society
588
Chem. Res. Toxicol., Vol. 3, No. 6, 1990
Kim et al.
Scheme I. General Scheme Depicting Reactions Involved in the Biotransformation of 1,2-Dibromoethane GSH -GSCH*CHO
NADH
GSCH2CHzOH
Alcohol dehydrogenase
BrCH2CH2Br
0
\
GSH\ GSH S-Transferase
GSCH2CH2Br L
I ,dRib,
\ NH
Other
I
to sodium 4,4-dimethyl-4-silapentane-l-sulfonate) 6 8.24 (s, 1 H , Ha), 8.14 (s, 1 H, H2), 4.52 (dd, 1 H, J = 8.9, 5.0, Cys a),3.82 (m, 2 H, CH2CH2S),3.74 (d, 1 H, J = 17.3, Gly CY Hb), 3.69 (t, 1 H, J = 6.4, Glu n ) , 3.68 (d, 1 H, J = 17.3, Gly (Y Ha), 3.11 (dd, 1 H, J = 14.1, 5.0, CYSp Hb), 2.94 (t, 2 H , J = 6.3, CHpCHpS), 2.87 (dd, 1 H, J = 14.1,9.0, Cys /3 Ha), 2.41 (m, 2 H, Glu y), 2.05 (m, 2 H, Glu p). S-[2-(h"-Guanyl)ethyl]GSH was synthesized as described elsewhere ( 1 I ) . Treatment with 0.1 N NaOH a t 23 "C for 60 min quantitatively converted the material to the imidazole ring opened form. The adduct was then deribosylated by treatment a t 100 "C for 30 min a t pH 1 to give a mixture of GSCH2CH2-FAPYI and GSCH,CH,-FAPY I1 in nearly quantitative yield: FAB-MS (negative ion, glycerol) m / z 501 (M - H)-; UV (HpO,pH 5.1) A, 270 nm; NMR (,H,O, ppm relative to sodium 4,4-dimethyl-4silapentane-1-sulfonate)d 8.24 (s, NCHO), 7.95 (s, NCHO), 4.57 (m, 1 H, Cys ( t ) , 3.77 (s, 1 H , Gly N Hb), 3.76 (s, 1 H, Gly N Ha), 3.73 (m, 3 H, CH2CHpS+ Glu a ) , 3.08 (dd, 1 H, Cys (3 Hb), 2.89 (dd, 1 H, Cys /j Ha), 2.77 (m, 2 H, CH,CH2S), 2.52 (m, 2 H , Glu y ) , 2.13 (m, 2 H, Glu @). Assays. U V spectra were obtained by using a Cary 210 spectrophotometer (Varian, Walnut Creek, CAI in the automatic base-line correction mode-all measurements were made in aqueous solutions. All 'H NMR spectra were recorded on a Bruker AM-400 instrument (Bruker, Billerica, MA) in ,H,O in the Vanderbilt University facility. The peak corresponding to the water resonance was first measured relative to sodium 4,4-dimethyl-4-silapentane-l-sulfonate, and then the residual water peak in each spectrum was used as internal standard. Mass spectra were obtained from samples injected from a probe into a VG 70-250 instrument (VG, Manchester, U.K.), generally utilizing FAB-MS as detailed elsewhere (10) with glycerol as the matrix. All rats used in these studies were male Sprague-Dawley (-200 g) obtained from Harlan Industries (Indianapolis, !N). DNA concentrations were estimated by using A,,, measurements (Ehz = 200 cm-l). Protein concentrations were measured by using the bincinchonic acid (BCA) system (Pierce Chemical Co., Rockford, IL). Radioactivity measurements were made on a Beckman LS7000 liquid scintillation counter (Beckman, Irvine, CA). Nucleic acids were usually isolated from in vitro incubation systems by extraction of other materials with a mixture of phenol/isoamyl alcohol/CHC13, 24:1:25 v/v/v, and precipitation of the nucleic acids in the resulting aqueous phase with C2HjOH. In the case of in vivo studies hydroxylapatite chromatography was also done (20). Purification of Adducts (Scheme 11). In a large-scale preparation, yeast-soluhle RNA (4mg mL-') was incubated with rat liver cytosol (4 mg of protein mL-'1, 5 mM GSH, and 2 mM l,%dibromoethane in 600 mL of 0.1 M Tris-HC1 buffer (pH 7.7) for 2 h a t 37 "C. The mixture was washed with an equal volume of a mixture of phenol. isoamyl alcohol, and CHCI3 (vide supra), and the RNA was precipitated by the addition of CzHjOH and dried. The recovered RNA was dissolved in 0.1 N HCl and heated
Scheme 11. Flow Diagram Describing Isolation of 1,2-Dibromoethane-DerivedAdducts from RNA Incubate 2 g 01 yeaSI.So1Uble RNA in the presence 01 rat lhver cytos01, GSH. and 1.2-61bromoethanetor 2 h
.1 .1 J.
Isolate RNA by solvent extraction and C2H5OH precipitation
Acid hydrolySis (0 1 5 N HCI, at 1 0 0 r C lor30 mln)
HPLC pwtication
4
Semlprep Cl8 column (10x 250 mm) (25 mM NHdHzPOq. PH 2 51 5.30% C H 3 0 H gradient Over 30 min
.1
Semlprep Ct8 column (10x 250 mm) (50 mM NHdOAC, pH 5 1) 5.25% C H 3 0 H gradient Over 25 min
peak n PropyI-NH2 Column (4.6 x 250 mm)
200 mM KHzPOI.
8-760mM NH40Ac in 80% C H 3 0 H
pH 4 3
3% CH3CN
over40 min
.1
SCX column (4.0 x 200 mm)
0 5 M NP'qH2P04. pV 2
,
peak b SAX column (4.6 x 250 mm)
1
3% C H j C N ~ s o c ~ a t ~ c
peak d Propyl-NH? column (4.6x 250 mml 8-760mM NH40Ac 8"
80% CH30H over 40 min
.1
Cl8column (4.6 x 250 mm)
C18 column (4.6x 250 mm)
50 mM N b O A c . pH 5 1
50 mM NHqOAc pH 5 1
3.25% C H 3 0 H gradient
3.25% CHJOH gradlent
over 20 m r
Over 20 min
for 30 min a t 100 "C. Experiments done on a smaller scale with 1,2-dibromo[1,2-14C]ethaneindicated that this procedure released >99% of the radioactivity from the RNA. HPLC of an aliquot of the hydrolysate is shown in Figure 1. In order to facilitate monitoring of the purification (Scheme II), radioactive adducts derived from in vivo and smaller scale in vitro experiments were added to the preparative-scale mixture. The hydrolysate was concentrated in vacuo, and aliquots were applied to a 10 x 250 mm Beckman Semi-prep C18 HPLC column (Beckman, San Ramon, CA). The column was eluted with a 5 4 0 % CH30H (v/v) gradient in 25 mM NH4H2P04(pH 2.5) over 30 min (flow rate 3 mL min-'). The partially resolved radioactive peaks were further resolved by HPLC using the same column eluted with a 3-25% CH30H (v/v) gradient in 50 mM NH,CH3CO2 (pH 5.1) over 20 min (Figure 1) (flow rate 3 mL min-I). [In subsequent work NH4CH3C02was removed from fractions by lyophilization prior to further HPLC, and a t the end of the purification sequence (see Scheme 11) samples were applied to C18 columns and eluted with volatile buffers to remove phosphate.] Peak a was applied to a 4.6 X 250 mm Beckman propylamino HPLC column and eluted with an 8-760 mM gradient of NH4CH3C0, (all in 80% CH30H) over a period of 40 min (flow rate 1.0 mL min-'). The radioactive peak was applied to a 4.0 X 200 mm Macherey-Nagel Nucleosil 5 SA (SCX) column (Rainin,
1,2-Dibromoethane DNA Adducts
Chem. Res. Toxicol., Vol. 3, No. 6, 1990 589
hydrolysis (10,21). More than 99% is released by mild acid treatment (0.1 N HC1,lOO "C, 30 min). In either case >95% of the adducts can be accounted for as S-[2-(Wguany1)ethyllGSH (9, 10,21). Radioactivity eluted near the void volume of C18 HPLC columns consists primarily of S-(2-hydroxyethy1)GSH and S,S-bis(ethy1ene)GSH (9),which appear to resist complete separation from DNA. The possibility that the alcohol might have been bonded to a phosphate or other moiety of the DNA cannot be dismissed, but no evidence in support of such a hypothesis is available, and the radioactivity eluted from the column prior to peak a in Figure 1 is not considered to result from a DNA adduct. In previous in vivo studies it was found that more 1,2dibromoethane adducts were formed in RNA than DNA (22). Higher adduct levels were obtained in vitro with RNA than DNA in systems using GSH (Table I), and RNA was used as a target for the preparation of adducts for structure determination. In confirmation of previous results with 1,2-dichloroethane (24), only traces of DNA adducts are formed from 1,Zdibromoethaneby microsomal oxidation, although a significant amount of RNA adducts are formed (Table I). Modification of RNA using GSH and 1,Zdibromoethane yielded an adduct pattern similar to those typically seen with DNA samples, except that the levels of the minor peaks (a, b, and d in Figure 1)were relatively higher. The individual adducts were isolated according to the procedures described under Experimental Section. S-[2-(N1-Adeny1)ethyllGSH.Peak a (Figure 1)was isolated as described and identified as S-[2-(N-adenyl)ethyl]GSH. The negative ion mass spectrum clearly showed the expected M - H ion and a sodium adduct (Figure 2), consistent with a structure containing the elements of GSH and adenine linked by an ethylene bridge. The 'H NMR spectra of the isolated material showed the N- and S-linked methylene hydrogens of the ethylene bridge, all GSH protons, and hydrogens assigned to C-2 and C-8 of adenine (Figure 3). The position of linkage to adenine was established primarily by UV spectroscopy. The UV spectra of the various N-alkyl adenine derivatives are known to be quite distinct, particularly when considered as a function of pH (17,18,25). When N-, W - ,N6-,and N7-methyladenine
.-C
E . c
d
200
3 0
.-.-s > c L
0
g
a
100
b
6 0:
5
10
15
20
25
Elution time, min
Figure 1. HPLC of adducts released by mild acid hydrolysis from RNA modified by incubation with GSH, rat liver cytosol, and 1,2-dibromo[1,2-14C]ethane. Yeast-soluble RNA was modified by incubation with rat liver cytosol, GSH, and 1,2-dibromo[l,214C]ethane as described under Experimental Section, and an aliquot of' the acid-hydrolyzed sample was analyzed by HPLC using a 4.6 X 250 mm Beckman C18 column with a gradient of 5-25% CHROH(in 50 mM NH4CH3C02,pH 5.1) applied over 25 min (flow rate 1.0 mL min-l). Subsequent work showed that peak peak c is S-[2-(N'-guanyl)a is S-[2-(N'-adenyl)ethyl]GSH, ethyl]GSH, peak d is S-[2-(N'-guanyl)ethyl]cysteinylglycine.
Woburn, MA) and eluted with a mixture of 3% CH3CN (v/v) in 0.5 M NH4H2PO4 (pH 2.1). Peak b (Figure 1)was applied to a 4.6 X 250 mm Partisil SAX column (Altex, Berkeley, CA) and eluted with a mixture of 3% CH3CN (v/v) in 0.2 M KH2P04(pH 4.3). The peak was applied to a 4.6 X 250 mm Beckman C18 column and eluted with a 5-25% (v/v) gradient of CH30H in 5 mM NH4CH3CO2(pH 5.1). Peak d (Figure 1)was purified by chromatography on a propylamino column in the same way as peak a and on the last C18 column in the manner described directly above for peak b.
Results Preliminary Experiments. In samples of DNA modified with 1,2-dibromo[1,2-14C]ethaneand GSH in vitro or in vivo, we have consistently found that >95% of the bound radioisotope can be released by neutral thermal
(M-H)-
100
-
467
> ctn
z W
k
50.
2 la -I
(M-H +N a ) 489
W
a
100
200
300
400
500
MIZ
Figure 2. FAB mass spectrum of S-[2-(Nl-adenyl)ethyl]GSHisolated from RNA (unknown peak 1). The spectrum was obtained in the negative ion mode, and the contribution of the matrix (glycerol) was not subtracted.
590 Chem. Res. Toxicol., Vol. 3, No. 6, 1990
Kim et al. Nbnethyladenine
Table I. In Vitro Binding of Radioactivity from 1,2-Dibromo[1,2-"C]ethane to RNA and DNA in the Presence of Microsomal and Cytosolic Enzyme Systems nmol bound (mn of nucleic acid)-' DNA RNA microsomes, NADPH 0.03* 0.89 f 0.31 cytosol, GSH 1.20 f 0.14 4.56 f 0.37 I
"All incuhations were done for 60 min a t 37 "C and included 0.1 M Tris-HCI buffer and, as indicated, calf thymus DNA (2 mg mL-') or yeast-soluble RNA (2 mg mL-') and either rat liver microsomes (2 mg of protein mL-') plus an NADPH-generating system (23) or rat liver cytosol (2.2 mg of protein mL-') plus 2 mM GSH and 2.2 mM 1,2-dibromo[1,2-'4C]ethane( 5 mCi mmol-'). Nucleic acids were isolated by phenol extraction and hydroxylapatite chromatography (20), and radioactivity was estimated. *Single determination-separate experiments have yielded similar results.
'.---__ ~
240
280
320
240
280
320
WAVELENGTH (nm)
WAVELENGTH (MI)
Figure 4. UV spectra of authentic Nl-methyladenine (left) and S-[2-(N1-adenyl)ethy1]GSH isolated from RNA (right). Spectra were measured at pH 5.1 (-) or in 0.1 N HCI (---) or 0.1 N NaOH
A
(-. -). HOD HE
NCH
+
cy$ a
8.0
9.0
6.0
7.0
5.0
3.0
4.0
2.0
I
'
5
10
15
0
4
8
12
Elution time, min
Figure 5. Cochromatography of radioactivity in peak a with synthetic S-[2-(iV-adenyl)ethyl]GSH. The C18 and SCX columns are those described in the isolation of peak a in Scheme 11.
B
9,'O
0
810
'
7,'O
'
6:O
'
5:O
'
4,'O
.
3:O
'
2.'0
Chemical Shin (ppm)
Figure 3. 'H NMR spectra of synthetically prepared S-[2(M-adenyl)ethyl]GSH (part A) and compound isolated from RNA (part B, unknown peak 1). Spectrum B was acquired by using approximately 20 fig of sample (5572 transients). T h e solvent in both cases was 2H20. Assignments are indicated in the figure. The broad band in spectrum B centered a t 6 3.55 and unassigned peaks are considered to be impurities.
were compared, only Nl-methyladenine showed UV behavior similar to that of the purified compound identified as peak a (Figure 4). A sample of S-[2-(IV-adenyl)ethyl]GSH was synthesized (in low yield) by treatment of adenosine with S-(2chloroethy1)GSH. Although several products were formed in the reaction, the major compound formed was the N'
derivative. Following mild acid hydrolysis, the product was found to have UV and mass spectra identical with those presented in Figures 2 and 3. The NMR spectra for the sample along with the compound isolated from RNA are shown in Figure 4. Cochromatography of the radioactivity in the sample of peak a derived from RNA with an excess of synthetic material is shown in Figure 5. Further characterization of synthetic S-[2-(N1adenyl)ethyl]GSH was obtained upon conversion of the material to Wethyladenine. Treatment of the thioether adduct under typical desulfurization conditions (9, 26) yielded N'-ethyladenine in 7% yield (Figure 6). The major products were adenine, IV-ethyladenine (identified by HPLC and UV comparison with synthetic material), and S-[2-(NG-adenyl)ethyl]GSH, identified by its mass and NMR spectra (see Experimental Section) and the change in its UV spectrum from that of an N1-alkyladenine derivative to that of an NG-alkyladenine, This transformation (Nl-alkyl- to NG-alkyladenine) is known as a Dimroth rearrangement, reported to be base-catalyzed (27, 28) (Scheme 111). Treatment of S-[2-(IV-adenosyl)ethyl]GSH at pH 10 and 37 "C for 20 min (followed by neutralization prior to HPLC) resulted in an essentially quantitative yield of the NG-adenosine adduct. The same reaction occurred with the deribosylated adduct [S-[2-(N1-adenyl)ethyl]GSH], but at a greatly decreased rate. The NMR spectrum of the NG-adenyl adduct shows the resonance due to the N-linked methylene protons shifted from 6 4.52 to 3.82,
Chem. Res. Toxicol., Vol. 3, NO. 6, 1990 591
1,2-Dibromoethane DNA Adducts
o.26
7
0.24 A270 0.22
0.20
0.18
PH
Figure 7. pH titrations of S-[2-(N1-adenyl)ethylJGSH, monitored by UV absorbance. See Figure 4. Elution time, min
Figure 6. HPLC of S-[2-(N1-adenyl)ethyl]GSH isolated from RNA after treatment with Raney Ni. The HPLC system is the first one described in Scheme I1 (see Experimental Section for details). The indicated peaks are assigned as follows: (a) S[ % W a d e n 1)ethylIGSH;(b) N-ethyladenine; (c) adenine; and (d) S - [ 2 - ( dadeny1)ethylJGSH.
N-CH2 g'ya
Scheme 111. Reactions of S-[2-(N1-Adenyl)ethy1]GSH: Reduction with Raney Ni (a) and Dimroth Rearrangement to S-[2-(N6-Adenyl)ethyl]GSH (b)O
~
Remy Ni (bank)
I I1 ST
6.0
8.0
4.0
-
2.0
Chemical shift, ppm N '
N '
..-.5$ Ny)
Figure 8. 'H NMR spectrum of S-[2-(N'-guanyl)ethyl]cysteinylglycine isolated from RNA. The spectrum was recorded in 2H20with approximately 40 pg of material. Assigned peaks (cf. ref 10) are indicated, and all others, including the broad band centered at 6 3.55 (cf. Figure 4), are considered to be impurities. The peak corresponding to the Cys proton is not visible in this spectrum. (Y
OS*NH
4,
Dlmrolh OH
rearrangement
Scheme IV. Tautomers of S-[2-(N1-Adenyl)ethyI]GSHa
c
N '
See text for discussion of rearrangements.
as might be expected after the rearrangement to an exocyclic nitrogen. The remainder of the resonances are quite similar to those of the M-adenyl adduct. No evidence for formation of the @-adenyl adduct (without base treatment) has been seen in DNA or RNA samples. It is unclear why S-[2-(NG-adenyl)ethyl]GSH is not reduced by Raney Ni. Several tautomers of the S-[2-(N1-adenyl)ethy1]GSH adduct are possible (Scheme IV). pH titration of the adduct showed a pK, value of 6.9 (Figure 7). On the basis of precedents in the literature (25),the imino structure (a) in Scheme IV is postulated to predominate at pH < 6.9. S-[2-(N7-Guanyl)ethyl]cysteinylglycine. Peak d (Figure 1) was purified as described. Negative ion FABMS yielded a peak at m / z 354 (apparent M - H), consistent with a structure containing guanine, cysteine, and glycine moieties linked by an ethylene bridge. The 'H NMR spectrum was nearly identical with that seen with S-[2-(A"-guanyl)ethyl]GSH(10)except that the glutamate hydrogens were missing (Figure 8). The UV spectra were typical of an N"-alkylguanine, with A,, 253 nm in acid and A, 284 nm i n base (9). Treatment of authentic S-[2-(N'-guanyl)ethyl]GSH under the conditions used to hydrolyze the RNA (0.1 N
a
'I
b
C
a
Forms a and b are postulated to predominate,with a pK, of 6.9
(Figure 7).
HC1, 100 "C, 30 min) yielded S-[2-(N'-guanyl)ethyl]cysteinylglycine in 2% yield, a percentage similar to that seen in the chromatograms (Figure 1). Evidence against Formation of Imidazole Ring Opened S -[2- (N7-Guany1)ethyl]GSH. Imidazole ring opening is relatively facile in certain W-guanyl DNA adducts, such as that derived from aflatoxin B1 (27). Investigations of several different series of W-alkylguanines have found that electron-withdrawing entities favor such base-catalyzed hydrolysis (28, 29). The ring-opened de-
592 Chem. Res. Toxicol., Vol. 3, No. 6, 1990
Kim et al.
Scheme V. Base-Catalyzed Opening of the Imidazole Ring of S-[2-(N7-Guanosyl)ethyl]GSHand Postulated Equilibration of Congeners
Table 11. Formation of Hepatic Nucleic Acid Adducts following Administration of 1.2-Dibromoethane to Rats" pmol adduct (mg of nucleic acid)-' S-[2-(M-guanyl)S-[2-(N1-adenyl)ethyl]GSH ethyllGSH
SG
I
DNA
RNA
455 664
14 60
'Rats were administered a single dose of 37 mg of 1,2-dibromo[ 1,2-14C]ethanekg-' (specific radioactivity 10 mCi mmol-I). After 4 h livers were isolated (from 2 rats) and the nucleic acids were iso-
ddib
lated by using phenol extraction and hydroxylapatite chromatography. The nucleic acids were hydrolyzed by treatment with 0.1 N HCl a t 100 "C for 30 min, and the individual adducts were isolated by HPLC (Figure 1, Scheme 11) for liquid scintillation counting.
OH'
S-[2-(Nl-adenyl)ethyl]GSH, but these have not been
7" 1
ddib
ddib
I
I
0.1 N HCI
Deribosylated adducts
rivative of S - [2-(N'-guanyl)ethyl]GSH was prepared by alkaline hydrolysis and characterized. Apparently the two possible formyl derivatives interconvert (Scheme V), as postulated in other cases (32, 33). A single HPLC peak was observed, but two formyl hydrogens of equal intensity were seen in the NMR spectrum. Thus the two forms are present at nearly equal concentrations and apparently are free to interconvert, but do so a t a rate that is slow on the NMR time scale. An alternative possibility is that the two NMR peaks are the result of rotamers of a single isomer, but further studies will be required to address this possibility. The ring-opened adduct eluted between the first radioactive peaks and peak a in the HPLC system shown in Figure 1. No evidence for the presence of a detectable level of such an adduct was obtained with this particular RNA sample or a DNA sample that had been modified with 1,2-dibromoethane (and GSH and GSH S-transferase) and incubated for up to 12 days a t 37 "C. In in vivo work no evidence for the presence of a detectable ring-opened adduct was seen in rat liver DNA even 21 days after treatment (vide infra). Other Nucleic Acid Adducts. In addition to the peaks shown in Figure 1, a small peak (-0.5% total adducts) was seen eluting a t about twice the tR of S-[2(W-guany1)ethyllGSH (not shown). This peak and peak b (Figure 1) were both purified to apparent homogeneity using HPLC, but only very small amounts of material were recovered (-0.1 A,,, unit seen during elution from HPLC in a typical cell). Efforts to obtain NMR and mass spectra were unsuccessful. The UV spectra of peak b showed A,, a t 260 and 285 nm at pH 1.5 and 5.1 and at 266 nm at pH 12. The late-eluting, very minor adduct showed A,, at 260 nm a t acidic, neutral, and basic pH with an increased absorbance peak a t 230 nm in acid. Neither compound appeared to be fluorescent. In an effort to identify these trace materials, S-(khloroethyl)GSHwas incubated with adenosine or guanosine and the bases were released by mild acid hydrolysis. A number of minor products were obtained in addition to S-[2-(N'-guanyl)ethyl]GSH and
characterized. None of these products were eluted from HPLC at the same tR as either of the materials in question. In Vivo Formation and Loss of Adducts. Rats were treated with 1,2-dibromoethane, and both S-[2-(Wguany1)ethyllGSH and S- [2-(Nl-adeny1)ethyllGSH were detected in hepatic DNA (Table 11). The level of adducts was higher in RNA than DNA, being 4-fold higher (per milligram of nucleic acid) in the case of S-[2-(N1adeny1)e thyl] GSH. The kinetics of removal of the various adducts in hepatic DNA were considered. T h e removal of S-[2-(Wguanyl)ethyl]GSH, S-[2-(N1-adenyl)ethyI]GSH, and the unidentified peak b proceeded with an initial tlI2of -100 h. A second, slower phase may be occurring, but there was no obvious difference in the various decay curves. Similar rates have been estimated earlier for the loss of total (unidentified) DNA adducts from the livers, kidneys, lungs, and stomachs of 1,2-dibromoethane-treated rats (21).
Discussion In a number of cases the significance of what are relatively minor DNA adducts has been appreciated. With 1,2-dibromoethane the major adduct (>95%) in DNA is clearly S-[2-(iV'-guanyl)ethyl]GSH,but it is not yet possible to conclude that this is necessarily the lesion most relevant to carcinogenesis. A study was undertaken with the purpose of characterizing the minor adducts and measuring their persistence in rat liver. RNA was modified in vitro, as a source of adducts, because it provided higher levels of the minor adducts. Mild acid hydrolysis was used in order to release all purine adducts. The assignment of the structure S-[2-(N1-adenyl)ethyl]GSH to one of the adducts would appear to be unambiguous on the basis of the NMR, mass, and UV spectra (Figures 2-4) and its spectral and chromatographic comparison with the synthetic adduct. The synthetic adduct could be reduced to the characterized Nl-ethyladenine. S - [2-(Nl-Adenyl)ethyl]GSH also undergoes Dimroth rearrangement to the W-adenyl derivative, but this adduct does not appear to be formed in DNA. The characterization of S- [2-(N'-guanyl)ethyl]cysteinylglycine is also unambiguous, but the presence of this compound appears to be an artifact of the isolation procedure. Two other minor adducts account for
