Chem. Res. Toxicol. 1994, 7, 82-88
82
Application of Capillary Liquid Chromatography Coupled with Tandem Mass Spectrometric Methods to the Rapid Screening of Adducts Formed by the Reaction of N-Acetoxy-N-acetyl-2-aminofluorene with Calf Thymus DNA Susan M. Wolf and Paul Vouros* Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 Received July 19, 199P
Capillary liquid chromatography-continuous-flow fast atom bombardment mass spectrometry is applied to the detection of deoxynucleoside adducts of N-acetoxy-N-acetyl-2-aminofluorene. In such a configuration, normal scan and tandem mass spectrometric techniques are shown to provide useful structural information for an N-acetyl-N-(deoxyguanosin-8-yl)-2-aminofluorene adduct standard for low- nanogram (low-picomole) amounts sampled. In addition, multiple reaction monitoring gives limits of detection below 25 pg (50 fmol) for this adduct, suggesting the potential for routine screening of intact deoxynucleoside adducts formed below the 1:106 level from as little as 1mg of DNA. When applied to the analysis of the products of an in vitro these techniques are reaction of calf thymus DNA with N-acetoxy-N-acetyl-2-aminofluorene, readily able to detect and supply structural data for the N2 and C8 deoxyguanosine adducts formed.
Introduction DNA adducts formed by the covalent attachment of carcinogenic species to DNA have long been considered to play a major role in the process of carcinogenesis. When DNA has been exposed to carcinogens (whether in vitro or in vivo), a mixture of adduct types normally results. In order to elucidate the biochemistry of the carcinogenDNA interaction and to assess exposure, it is necessary to characterize as many adducts in a given mixture as possible; however, this task is not always trivial. For instance, it has previously been established ( I ) that the in vitro products resulting from the reaction of calf thymus DNA with N-acetoxy-N-acetyl-2-aminofluorene (AAAF)include primarily N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (C8-AAF-dGuo)l and 3-(deoxyguanosin-W-y1)-2(acety1amino)fluorene (N2-AAF-dGuo);the structures of these adducts are shown in Figure 1. However, the formation of several minor AAF adducts including a possible deoxyadenosine adduct, some of which have yet to be definitively characterized, has been noted (2,3).In addition to minor adducts, other species may arise which warrant characterization. For instance, recent reports have discussed the appearance of adduct derivatives resulting from adverse chemical or enzymatic conditions employed in adduct isolation (4-6). An on-line separationlstructural characterization approach to analyzing the products of these reactions could contribute to the rapid analysis of typical as well as unusual or trace adducts and would make rapid screeningfor such compounds feasiblewhile avoiding published in Advance ACS Abstracts, January 1, 1994. C8-AAF-dGuo, N-(deoxyguanosin-6-yl)-N-acetyl-2aminofluorene;W-AAF-dGuo,3-(deoxyguanoein-Wyl)-2-(acetylamino)fluorene; AAAF, N-acetoxy-N-acetyl-2-aminofluorene; dGuo, deoxyguanosine; FAB, fast atom bombardment; LC-CFFAB-MS,liquid chromatography-continuow flow fast atom bombardment mass spectrometry; CID, collision-induceddissociation;MRM, multiple reaction monitoring; CNL, constant neutral loss. a Abstract
1 Abbreviations:
Q
0
II
I I
C=O NH CH3 I
“Ic?i
NZ-AAF-dW O
C8-AAFdWO
Figure 1. Structures of N-(deoxyguanosin-&yl)-2-(acetylamino)fluorene (C8-AAF-dGuo)and 3-(deoxyguanosin-N2-y1)-2-(acety1amino)fluorene (N2-AAF-dGuo).
the inevitable sample losses associated with peak collection and off-line characterization. While a variety of mass spectrometric techniques have been employed for the characterization of nucleic acid adducts (7)) it has been fast atom bombardment (FAB) ionization, combined with tandem mass spectrometric methods, which has been used most widely for structural characterization of intact polycyclic aromatic hydrocarbons (PAH)-deoxynucleoside adducts and similar compounds (8-18). In studies where high-sensitivity static FAB characterization has been emphasized, chemical derivatization of isolated materials has typically been
o~93-22a~19412101-0082~o4.5010 0 1994 American Chemical Society
LC-CFFAB-MSIMS Analysis of PAH-DNA Adducts
Chem. Res. Toxicol., Vol. 7,No. 1, 1994 83
Teflon tubing connector
outfitted with a cesium ion gun. Collision-induced dissociation (CID) at 50 eV utilizing argon as the collision gas was conducted at pressures determined to provide optimal sensitivity for the C&AAF-dGuo standard. Preparation of t h e Adducts. Adducts were generated by the reaction of 1.6 mg of calf thymus DNA (in 2400 pL of 10 mM 600 pL of ethanol) with 2.5 mg of citrate buffer, pH 6.0, N-acetoxy-N-acetyl-2-aminofluorene (dissolved in 300 pL of ethanol, and added in three aliquots over 30 h) at 37 "C. After extraction of the reaction mixture with ether (3 X 3000 pL), the DNA was precipitated in NaCl and cold ethanol, centrifuged at 12000g for 20 min, and redissolved in 3000 pL of 5mM Bis-Tris, pH 7.1. Enzymatic digestion to the deoxynucleoside level was accomplished by the addition of 300 pg of DNase I (4 h, 37 "C), followed by 0.3 unit of snake venom phosphodiesterase and 6 units of alkaline phosphatase (18 h, 37 "C). The DNase I was added from a 1 mg/mL solution which also contained 0.15 M NaCl and 0.010 M MgC12. As discussed below, adducts were separated from the enzymatic digest by solid-phase extraction (SPE) in addition to the more commonly employed partitioning into water-saturated 1-butanol. It was found that this additional purification step provided a cleaner adduct extract containing fewer insoluble materials. Calf thymus DNA (type I), DNase I (type 11),snake venom phosphodiesterase (type VII), alkaline phosphatase (type 111),EDTA, reagent-grade sodium citrate, and Bis-Tris were obtained from Sigma (St.Louis,MO). Preparations of all buffers contained 0.1 mM EDTA. An adduct standard, CS-AAF-dGuo,was prepared according to the method of Heflich et al. by Mohamed Itani (ref 24 and references therein), Department of Medicinal Chemistry, Northeastern University, was generBoston, MA. N-Acetoxy-N-acetyl-2-aminofluorene ously provided by Dr. Frederick Beland, National Center for Toxicological Research in Jefferson, AR. Isolation of the Adducts. 1-Butanol extractions were conducted by adding avolume of water-saturated 1-butanolequal to the volume of the reaction mix to the reaction vial with mixing on a rotating extractor for 15 min. The extraction was repeated three times, and all 1-butanol fractions were combined and dried in a vacuum centrifuge. 1-Butanol (99+ % ) used for extractions was purchased from Aldrich (Milwaukee, WI). Bio-Beads SM-2 100-200-mesh polystyrene divinylbenzene packing (Bio-Rad, Richmond, CA) was employed as the SPE solid phase. This packing had previously been shown to give selective retention of benzo[a]pyrene diol epoxide adducts in DNA reaction mixtures.2 Materials isolated by 1-butanol extraction were dissolved in 10% methanol and loaded onto the SPE column (containing '350 mg of solid phase). The column was then washed with 1000 pL of 10% MeOH, which was discarded. Adducts were eluted with 500 pL of 50% MeOH followed by 2250 p L of MeOH. The adduct-containing eluates were combined and dried by vacuum centrifugation.
Capillary Column
I Syringe Pump
I
IU V e c t o r
CFFAB Probe
Figure 2. Liquid chromatographic-mass spectrometric (LCMS) system which consisted of a Carlo Erba Phoenix 20 syringe pump and MicroUVIS 20 ultraviolet detector, an LC Packings 3-pm C18 320 pm X 15 cm column and a Valco C14W internal volume injector outfitted with a 0.5-pL volume. A 90-cm length of 50-pm i.d. 375-pm 0.d. polyimide-coated fused silica capillary connected to the outlet of the capillary column with a piece of Teflon tubing transferred materials eluted from the column to the CFFAB tip in the ion source.
employed (10, 11). With the advent of continuous flow FAB techniques (19, 20), the low-level analysis of FABamenable compounds under conditions which allow for interfacing with liquid chromatography has become routine for a variety of biomedical and other applications (21). In addition, the availability of capillary liquid chromatographic columns which operate at flows compatible with continuous-flow fast atom bombardment (CFFAB) flow rates ( - 5 pL/min) has made it possible to conduct separations on-line with mass spectrometric detection without the need for splitting of the eluate. This report details the application of the combined capillary LC-CFFAB-MS technique to the analysis of intact, nonderivatized deoxynucleosideadducts generated by the in vitro reaction of AAAF with DNA. This particular reaction system was chosen for its ability to serve as a well-characterized standard against which the merits of the proposed methodology could be judged. Concurrent with the presentation of these results at a recent meeting (22), Quilliam (23) reported that the use of electrospray ionization in conjunction with microbore and capillary liquid chromatography can provide similar information for the analysis of PAH-DNA adducts.
Experimental Section Instrumental. The capillary LC-CFFAB-MS system employed for adduct analysis is depicted in Figure 2. It consisted of a Carlo Erba (Danvers, MA) Phoenix 20 syringe pump and MicroUVIS 20 ultraviolet detector, an LC Packings (San Francisco, CA) 3-pm C18 320 pm X 15 cm column, and a Valco (Houston, TX) C14W internal volume injector outfitted with a 0.5-pL volume. A 90-cm length of 50-pm i.d. 375-pm 0.d. polyimide-coatedfused silica capillary (PolymicroTechnologies, Phoenix, AZ) connected to the outlet of the capillary column with a piece of Teflon tubing transferred materials eluted from the column to the CFFAB tip in the ion source. Ultraviolet detection was conducted at a distance of 15 cm from the outlet end of the LC column. All solvents used were HPLC grade and were purchased from J. T. Baker (Medford, MA). All mobile phases contained 1% glycerol to assist FAB ionization. Comparison of the LC-UV and LC-MS profiles indicated a small loss of chromatographic efficiency, which can be attributed to the dead volume mixing at the screen tip of the CFFAB probe and which tended to increase with increased sample amount. Continuous-flow fast atom bombardment mass spectrometry was conducted on a VG Quattro triple quadrupole instrument
+
Results Tandem MS Techniques. Before proceeding with the actual discussion of the results, it is important to review the strategy employed for the analysis of DNA adducts in complex mixtures. This strategy depended on the judicious use of the appropriate modes available in the tandem MS combination which, in turn, was formulated on the basis of the mass spectral fragmentation features displayed by PAH-DNA adducts in general. Clearly, operation of the MS in the normal full-scan mode provides for the acquisition of complete FAB mass spectra of the analytes present in a liquid chromatogram. It was apparent, however, that more selective andlor sensitive approaches were necessary for the detection of trace amounts of either unknown or target adducts. To that effect, the scanning C. Norwood and E. Jackim, personal communication.
Wolf and Vouros
84 Chem. Res. Toxicol., Vol. 7, No. 1, 1994
techniques of constant neutral loss (CNL) and multiple reaction monitoring (MRM) were found to be particularly useful. For the analysis of a class of compounds, the CNL mode of MSIMS operation takes advantage of a common fragmentation process for the selective detection of the individual constituents in the mixture during LC-MS elution. As indicated in the following paragraphs, in the case of deoxynucleosides,the loss of the deoxyribose group (116 daltons) provides for the selective detection of all deoxynucleoside-type species (11). When used in combination with a separation method such as LC or capillary zone electrophoresis (CZE), the CNL mode can target adducts in complex separation profiles and establish the molecular weights of these compounds. Alternatively, in cases where an adduct is known or suspected to be present, a more sensitive scan type, multiple reaction monitoring (MRM),may be employed. This mode of scanning involves the selective passage of a known precursor ion@)through the first quadrupole followed by fragmentation in an rfonly hexapole collision cell and the selective passage of a known product ion through the final mass analyzer. A more detailed discussion of the application of these principles to the analysis of the DNA adducts from an in vitro reaction is presented below. Sensitivity of Standard. Before examining the DNA adducts formed in the in vitro reaction, the detection capabilities of the LC-CFFAB-MSIMS system were established using a reference adduct. Toward that end, C8AAF-dGuowas utilized and its detectability was evaluated using normal full-scan FAB spectra as well as the tandem MS techniques of constant neutral loss (CNL) scanning and multiple reaction monitoring (MRM). Figure 3ashows the full-scan FAB spectrum of C8-AAF-dGuo obtained from 12.5 ng (25 pmol) of the adduct injected into the LC-CFFAB-MSsystem described in Figure 2. In addition to strong signals for the MH+ and AH2+ ([MH - deoxyribose + HI+) ions, the spectrum of C8-AAF-dGuo also exhibits prominent losses of CH&O and CHzCO from MH+ and AH2+ to yield the ions at mlz 4461447 and 3301 331 (10,17,25). As shown in the discussion relating to the analysis of the in vitro reaction mixture, these and other source-produced ions may then be subjected to collisioninduced dissociation (CID) to produce a wealth of further structure-specific fragmentations which can be used for adduct characterization. The CNL spectrum for the loss of deoxyribose (116 daltons) obtained for a 5-ng injection of the C8-AAF-dGuo standard is shown in Figure 3b. Featured in the spectrum is a strong peak for the MH+ at m/z 489. The signals at mlz 4461447 arise from the fragmentation of mlz 489 in the ion source to give mlz 4461447 which may then lose 116 daltons in the collision cell. A further improvement in detectability is realized through the use of the MRM scanning mode. As an example, the signals resulting from the monitoring (MRM) of the mlz 489 (MH+) mlz 373 (AH2+)transition for picogram (femtomolel-level injections of the standard C8-AAF-dGuoadduct are illustrated in the inset of Figure 3b. The use of the three aforementioned scanning modes in conjunction with one another is potentially sufficient to target and partially characterize unknown adducts in a mixture. This is illustrated in the following discussion, which considers their application to the analysis of the adducts formed in an in vitro reaction.
-
a AHl+
330
b
Figure 3. (a) Normal-scan spectrum obtained from a 12.5-ng injection of the C8-AAF-dGuo standard through the LC-MS system. (b) Constant neutralloss spectrum obtained from a 5-ng injection of the C8-AAF-dGuostandard.Inset: Signals obtained from replicate injections of 25-250 pg amounts of a C8-AAFdGuo standard detected by reaction monitoring of the m/z 489 mlz 373 transition. r = 0.9998.
-
Analysis of Reaction Mixture. Adducts isolated from the DNA reaction were reconstituted in 30% MeOH. The HPLC-UV chromatogram resulting from an injection of a 0.5-1L aliquot of this solution is shown in Figure 4. Constant neutral loss scanning over the course of the separation was employed to selectively target compounds which could lose the 116 mass units associated with the deoxyribose group. The spectrum obtained from summation of all the scans acquired over the chromatographic run gave a spectrum similar to that shown in Figure 3b in which prominent ions at mlz 252,268,4461447, and 489 were observed. The ion chromatograms for these mlz values are depicted in Figure 5. The signals arising from mlz 252 and 268 correspond to the MH+of deoxyadenosine and deoxyguanosine, respectively; this assignment is consistent with the void volume elution of these compounds observed under these chromatographic conditions. The mlz 489 trace shows two peaks which correspond in retention time to peaks A and C in the LC-UV chromatogram in Figure 4 and indicate the presence of the two expected deoxyguanosine adducts of acetylaminofluorene. The doublet at mlz 4461447 also shows the 116dalton loss for reasons discussed above. Notably, there was no signal corresponding in retention time to peak B in the LC-UV chromatogram, suggesting that it was
Chem. Res. Toxicol., Vol. 7,No. 1, 1994 85
LC-CFFAB-MSIMS Analysis of PAH-DNA Adducts
331
Figure 4. LC-UV chromatogram obtained from a 0.5-pL injection of the adduct isolate. Mobile phase: 1% glycerol and 49% methanol in water at a flow rate = 4 pL/min. Detection: ultraviolet at 214 nm. C c
m/z 489
A
'i'
"'
'
'
i
'00 '
4
loo
'
6
-_ m
sod
r
~
o
'
14'00' o
'
16'00
I
TI-
Figure 5. Mass chromatograms for various ions determined by constant neutral loss analysis to lose 116 daltons.
1
MRM MH+-+AH,+ (m/z489+m/z373) DNA Reaction Mix
::$
22.
I
5
1.6
2.0.5
0
Figure 7. Normal-scan spectra obtained from .the peaks corresponding to peaks (a) A, (b) B, and (c) C in the LC-UV chromatogram in Figure 4.
Figure 6. Reaction monitoring of the mlz 489 transition for an injection of the adduct extract.
-
m/z 313
something other than a deoxynucleoside-related species. Better definition of the chromatographic profiles of especially some of the trace components was obtained via the use of the MRM mode of detection. As shown in Figure 6, monitoring of the mlz 489 mlz 373 transition gives a greatly intensified signal for peaks A and C. In addition to the two major adducts, two low-intensity signals appearing before peak A suggest the likely presence of other minor deoxyguanosine adducts.
-
The normal full-scan spectra obtained for peaks A-C denoted in the chromatogram of Figure 4 are given in Figure 7. All three spectra show quite similar features. As the C8-AAF-dGuostandard displayed the same retention time as peak C and the spectrum in Figure 7c matched that of the standard shown in Figure 3a, peak C was identified as the C8-AAF-dGuo adduct. Given that the normal-scan spectrum (Figure 7a) for peak A also gave a spectrum containing an MH+ ion at mlz 489 and the aglycon AH2+ion at mlz 373, peak A was confirmed as the expected N2-AAF-dGuo adduct. The normal-scan spectrum for peak B, shown in Figure 7b, was virtually identical to the spectrum for C8-AAF-dGuo from mlz 150 to mlz 400 (Figure 712). However, the 16-dalton shift in MH+ to mlz 505 for this species (along with the shift of mlz 4461
86 Chem. Res. Toxicol., Vol. 7, No. 1, 1994
Wolf and Vouros
,
'1 1.
165
I
>'I Figure 9. Spectrum obtained from the collision-induced dissociation of the mlz 331 ion of NZ-AAF-dGuo. 3 1E7
1'
mlr473
--m/zasi
i
_JcC-JU(/ 5 DO
Figure 8. Spectra obtained from the collision induced dissociation of the mlz 331ion from (a)peak B and (b)peak C indicated in the LC-UV chromatogram in Figure 4. 447 to mlz 4621463) suggested that the deoxyriboseportion of the molecule contained an extra 16 daltons. A rea-
10'00
IS"
29 00
25'00
39 0 0
35 00
40 00
d o 0
11-
Figure 10. Reaction monitoring of the mlz 489 mlz 373 and mlz 473 mlz 357 transitions for a different adduct isolate. HPLC conditions: same as in Figure 4 except 5-pm C18 320 pm X 30 cm column (LC Packings, San Francisco, CA).
be noted that this reaction was conducted several times in order to optimize various analytical parameters. The data obtained for the dGuo adducts were consistent in each case. Interestingly, in some of the isolates, signals arising from the loss of 116 daltons from mlz 473, consistent with detection of AAF-dAdo adducts, were observed in both the MRM (Figure 10) and CNL modes. Unfortunately, due to the low level at which these species were present, it was not possible to obtain a definitive confirmation of the identity of these compounds.
sonable explanation for this observation is that the compound is a C8 RNA guanosine adduct of AAF, presumably arising from RNA contamination of the DNA used in the reaction. In order to further substantiate the structural assignments made above, CID spectra of selected ions were taken. Figure 8 shows the CID spectra arising from the mlz 331 ion from peak B and peak C. These spectra match that obtained from CID analysis of mlz 331 from the C8-AAFdGuo standard (data not shown). This result further Discussion confirms the identity of peak C as C8-AAF-dGuo and supports the assignment of the RNA adduct as a C8-AAFThe results presented above illustrate the utility of Guo species. Figure 9 shows the CID spectrum obtained continuous-flow fast atom bombardment tandem mass from mlz 331 of the N2 adduct. A comparison of this spectrometric methods coupled to capillary LC for the spectrum with those pictured in Figure 8 illustrates the determination of deoxynucleoside adduct identities and power of the CID experiment in differentiating adducts for low-level target adduct detection. As the necessary which differ only by the site of adduction. sample amounts fall well below those required for NMR The amount of C8-AAF-dGuopresent in the isolate was analysis, these techniques can make a significant contriquantitated by LC-UV at 274 nm using the C8-AAF-dGuo bution toward the structure elucidation of adducb formed standard and was found to be 14 pg. N2-AAF-dGuoand in small amounts from DNA reactions. Also, aside from C8-AAF-Guo, for which no standards were available, are a simple liquid or solid-phase extraction, no further estimated to have been present at approximately 3 and isolation or derivatization step is required prior to adduct 1.5 pg, respectively (assuming t c & m - d G u o = ~ N P - A A F - ~ G ~ ~ ) .mixture analysis by LC-CFFAB-MS. Sensitive-scan types On the basis of this estimation, the spectra in Figures like reaction monitoring can be used to target suspected 7a,b, 8a, and 9 for the N2-AAF-dGuoandC8-AAF-Guowere adducts and to confirm the presence of known trace collected at a level of 130 ng injected. adducts present at the picogram level in mixtures. For 1 mg of isolated DNA, the MRM detection limits should be While all of the above data were generated from the below 1 adduct:106 normal nucleotides. These levels of analysis of a single reaction of DNA with AAAF, it should
LC-CFFAB-MSIMS Analysis of PAH-DNA Adducts
detection should certainly permit the application of the method to studies of adducts generated in cell cultures and some animal tissues. Normal and CID spectra can provide a wealth of information about the structures of these compounds. Useful features of the normal-scan spectra common to the adducts examined here include prominent MH+and AHz+ ions as well as peaks at mlz 330,331,446, and 447 due to losses of CHzCO and CHBCO.from AHz+ and MH+, indicating the presence of an acetyl group in the adducts. Also of interest are the many lower intensity peaks which differ for the two positional isomers, indicating that the features of the normal-scan spectra are structurally diagnostic for this class of adducts. Many of these peaks were also observed in the CID spectra discussed below, although a few are unique to the normal-scan spectra and should be noted. These include a peak at m l z 224 in the C8-AAF-dGuo spectrum corresponding to [AAF + HI+ (the intensity of this peak varied due to the irreproducible background subtraction of a very high-intensity background peak at mlz 224.9 likely assignable to a [Cs + glycerol]+cluster ion). Also present in the spectra of both AAF-dGuo adducts was a peak at mlz 152 assignable to [guanine + HI+. Facile losses of HzO from mlz 373 and NH3 from mlz 331 to give peaks at mlz 355 and mlz 314, respectively, are unique to the N2-AAF-dGuo spectrum and may be distinguishing features for adducts of this general structure. Many of the other low-intensity ions are easily generated by CID of the AHz+ or other related precursor ions and have been found to carry useful structural information. (CID spectra of MH+ for amino-PAH adducts of dGuo tend to be less useful as they are typically dominated by the deoxyribose loss.) Certain of the pertinent peaks appearing in the CID spectra in Figures 8 and 9 are assigned in structures 1 (CBAAF-dGuo) and 2 (N2-AAF-dGuo) below. The fragmentation of the mlz 331 peak from C8AAF-dGuo has been discussed previously (10, 11), and a preliminary report detailing the fragmentation of mlz 331 and 373 from N2-AAF-dGuoanalyzed by static FAB as its trimethylsilyl derivative has also appeared (26). During the course of this work, CID spectra were also obtained for the mlz 373 ions of both the C8- and N2-AAF-dGuo adducts and were found to compare well with spectra (26). previously obtained on standards (data not We are currently confirming and further elucidating these fragmentations in a study involving isotopically-labeled materials. Used judiciously, such spectra can provide such information as the site of adduction, the mass of the PAH, and the identity of the adducted base. We (10) and others (11)are currently exploring CID scanning to generate data bases of structural information for known amino-PAH and PAH adducts in an effort to facilitate the identification of similar unknown adducts in mixtures. While absolute conclusions cannot be drawn about the formation of dA adducts in this system without the benefit of further data, the results obtained support previous speculations concerning the formation of these compounds ( 3 , 6 ) . Whether or not difficulties in reproducibly detecting these species were due to their formation at low levels, the inability of the enzyme system to liberate these compounds from DNA, or spurious interference from isobaric impurities is not presently known. A scaling up of the reaction 3 S.
M. Wolf and P. Vouros, unpublished results.
Chem. Res. Toxicol., Vol. 7, No. 1, 1994 87 -mlz
mlz 140-
1
+H
4
192
: im/Z180
mlz 207
1
+H m/z 110
T
i
NH2 m/z 180
J
I
H
I
I j
2
mlz 2 2 2 2
in order to acquire detailed CID data is warranted to confirm the presence and structure of these compounds. Future plans include the application of these methodologies to the characterization of unknown adducts from DNA modified in vitro or in vivo and to the investigation of the various degradation and isolation procedures utilized for adduct isolation from DNA. As a logical extension of this work, we are currently also looking into the utility of electrospray ionization for the analysis of DNA adducts.
Acknowledgment. This work was supported by agrant from the US. Environmental Protection Agency (Grant No. R820113-01-0). We are indebted to Dr. Frederick Beland of the National Center for Toxicological Research (Jefferson, AR) for his generous donation of the N-acetoxyN-acetyl-Zaminofluorene and to both Dr. Beland (NCTR) and Curt Norwood (U.S. Environmental Protection Agency, Narragansett, RI) for their helpful advice regarding the DNA reaction. In addition, we thank Mohamed Itani for synthesizing the C8-AAF-dGuo adduct. We also gratefully acknowledge a fellowship from the Arthur D. Little Foundation and support from the NIH through an instrument grant (lSlORR05602-01). This is Contribution No. 597 from the Barnett Institute. References (1) Kriek, E. (1979) Effect of pH on the Ratio of Substitution Producta in DNA after Reaction with the Carcinogen N-acetoxy-2-acetylaminofluorene Cancer Lett. 7, 141-146. (2) Gupta, R. C., and Dighe, N. R. (1984) Formation and Removal of DNA Adducts in Rat Liver Treated with N-hydroxy Derivatives of 2-Acetylaminofluorene,4-Acetylaminobiphenyl,and 2-Acetylaminophenanthrene. Carcinogenesis 5,343-349. (3) Harvan, D.J., Hass, J. R., and Lieberman, M. W. (1977) Adduct Formationbetween the CarcinogenN-acetoxy-2-acetylaminofluorene and Synthetic Polydeoxyribonucleotides.Chem.-Biol Interact. 17, 203-210. (4) Shibutani, S., Gentles, R. G., Iden, C. R., and Johnson, F. (1990) Facile Aerial Oxidation of the DNA-Base Adduct N-(2’-deoxyguanosin-8-yl)-2-aminofluorene [dG(CS)AF].J. Am. Chem. SOC. 112, 5667-5668. (5) Declos, K. B., Miller, D. W., Lay, J. O., Cesciano, D. A., Walker, R. P., Fu, P. P., and Kadlubar, F. F. (1987)Identification of CS-Modified Deoxyinosine and N*- and C8 Modified Deoxyguanosine88 Major Products of the In Vitro Reaction of N-hydroxy-6-aminochrysene with DNA and the Formation of these Adducta in Isolated Rat Hepatocytes Treated with 6-Nitrochrysene and 6-Aminochrysene. Carcinogenesis 8, 1703-1709.
88 Chem. Res. Toxicol., Vol. 7, No. 1, 1994 (6) Gupta, R. C. (1984) Nonrandom Binding of the Carcinogen N-hydroxy-2-acetylaminofluoreneto the Repetitive Sequences of Rat Liver DNA In Vivo. Proc. Natl. Acad. Sci. US.A. 81,69433947. (7) Crain P. F. (1990) Mass Spectrometric Techniques in Nucleic Acid Research. Mass Spectrom. Reu. 9, 505-554. (8) RamaKrishna, N. V. S., Gao, F., Padmavathi, N. S., Cavalieri, E. L., Rogan, E. G., Cerny, R. L., and Gross, M. L. (1992) Model Adducts of Benzo[alpyrene and Nucleosides Formed from its Radical Cation and Diol Epoxide. Chem. Res. Toxicol. 5, 293-302. (9) Wickramanayake, P. P., Arbogast, B. L., Buhler, D. R., Deinzer, M. L., and Burlingame, A. L. (1985) Alkylation of Nucleosides and Nucleotides by Dehydroretronecine; Characterization of Covalent Adducts by Liquid Secondary Ion Mass Spectrometry. J.Am. Chem. SOC. 107, 2485-2488. (10) Annan, R. S., Giese, R. W., and Vouros, P. (1990) Detection and Structural Characterization of Amino Polyaromatic HydrocarbonDeoxynucleoside Adducts Using Fast Atom Bombardment and Tandem Mass Spectrometry. Anal. Biochem. 191,86-95. (11) Bryant, M. &,Lay, J. O., Jr., and Chiarelli, M. P. (1992)Development of Fast Atom Bombardment Mass Spectral Methods for the Identificationof Carcinogen-Nucleoside Adducts. J.Am. SOC.Mass Spectrom. 3, 360-371. (12) Dietrich, A. M., Guenat, C. R., Tomer, K. B., and Ball, L. M. (1988) Identification and Characterization of the Major DNA Adduct Formed Chemicallyand In Vitro from the EnvironmentalGenotoxin 3-Nitrofluoranthene. Carcinogenesis 9, 2113-2119. (13) Rogan, E. G., Cavalieri, E. L., Tibbels, S. R., Cremonesi, P., Warner, C.D., Nagel, D.L., Tomer, K. B., Cerny, R. L., and Gross, M. L. (1988) Synthesis and Identification of Benzo[a]pyrene-Guanine Nucleoside Adducts Formed by Electrochemical Oxidation and by Horseradish Peroxidase Catalyzed Reaction of Benzo[alpyrene with DNA J. Am. Chem. SOC. 110, 4023-4029. (14) Tomer, K. B., Gross, M. L., and Deinzer, M. L. (1986) Fast Atom Bombardment and Tandem Mass Spectrometry of Covalently Modified Nucleosides and Nucleotides: Adducts of Pyrrolizidine Alkaloid Metabolites. Anal. Chem. 58, 2527-2534. (15) Tondeur, Y., Moschel, R. C., Dipple, A., and Koepke, S. R. (1986) Fast Atom Bombardment and Collisional Activation Mass Spectrometry as Probes for the Identification of Positional Isomers in a Series of Benzylated Guanosines. Anal. Chem. 58, 1316-1324. (16) Pogracz, K., Kaur, S., Burlingame, A. L., and Bodell, W. J. (1992) Identification of NWubstituted 2’-Deoxyguanosine-3’-phosphate Adducts Detected by 32P-postlabelingof Styrene-oxide-treated DNA. Carcinogenesis 13, 315-319.
Wolf and Vouros (17) Mitchum, R. K., Evans, F. E., Freeman, J. P., and Roach, D. (1983) Fast Atom Bombardment Mass Spectrometry of Nucleoside and Nucleotide Adductaof ChemicalCarcinogena.Int.J.Mass Spectrom. Ion Proc. 46,383-386. (18) Dolnikowski,G. G., Gross, M. L., and Cavalieri, E. L. (1991) Isomer Differentiation in 7,12-Dimethylbenz[aIanthracene-PyridineAdducts by Fast Atom Bombardment Tandem Mass Spectrometry.J. Am. SOC.Mass Spectrom. 2, 256-258. (19) Ito, Y., Takeuchi, T., Ishii, D., and Goto, M.(1985) Direct Coupling of MicroHigh-Performance Liquid ChromatographywithFastAtom Bombardment Mass Spectrometry. J. Chromatogr. 346, 161-166. (20) Caprioli, R. M., Fan, T., and Cottrell, J. S. (1986) Continuous-flow Sample Probe for Fast Atom Bombardment Mass Spectrometry. Anal. Chem. 58, 2949-2954. (21) For a review of CFFAB techniques and applications, see: Caprioli, R. M., Ed. (1990) Continuous-flow Fast Atom Bombardment Mass Spectrometry, John Wiley and Sons, Ltd., New York. (22) Wolf,& M.,Norwood,C., Jackim,E.,andVouros,P. (1992)Capillary Liquid Chromatography/ContinuousFlow Fast Atom Bombardment MassSpectrometric AnalysisofDNA Adducts from InVitro Reaction Mixtures. Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics, 1992, pp 388-389, Washington, D.C. (23) Quilliam, M. A. (1992) DNA-Carcinogen Adduct Analysis by Ionspray Liquid Chromatography-Mass Spectrometry. Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics, 1992, pp 430-431, Washington, D.C. (24) Heflich, R. H., Djuric, Z., Zhou, Z., Fullerton, N. F., Casciano, D. A,, and Beland, F. A. (1988) Metabolism of 2-Acetylaminofluorene in the Chinese Hamster Ovary Cell Mutation Assay. Enuiron. Mol. Mutagen. 11, 167-181. (25) Wolf, S. M., Annan, R. S., Vouros, P., and Giese, R. W. (1992) Characterization of Amino Polyaromatic Hydrocarbon-DNA Adducts Using Continuous-flow Fast Atom Bombardment and Collision-induced Dissociation: Positive and Negative Ion Spectra. Biol. Mass Spectrom. 21, 647-654. (26) Chiarelli, M. P., Bryant, M. S., and Lay, J. 0. (1992) Structure Elucidation of Isomeric 2-Acetylaminofluorene-Deoxyguanosine Adducts by FAB MS/MS.Proceedings oftheloth ASMSConference on Mass Spectrometry and Allied Topics, 1992, pp 531-532, Washington, D.C.
