Chem. Res. Toxicol. 1992,5, 870-875
870
A Thermospray Liquid Chromatography/Mass Spectrometry Method for Analysis of Human Urine for the Major Malondialdehyde-Guanine Adduct Hemant K. Jajoo, Philip C. Burcham, Yukihiro Goda,+Ian A. Blair,l and Lawrence J. Marnett' The A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Departments of Biochemistry and Pharmacology, and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received May 15, 1992
A method is described for detection and quantitation of the major malondialdehyde-guanine adduct (M1G) based on thermospray liquid chromatography/massspectrometry. A stable isotope analog of M1G ([2H21M1G)was used as an internal standard. Thermospray mass spectra of MIG and [2H2]M1G showed intense protonated molecular (MH+) ions that were suitable for use in quantitation of M1G. M1G was purified from human urine and reduced with NaBH4 to a dihydro derivative that was cleanly separated from the contaminants in the urine. The detection limit of reduced M1G by thermospray liquid chromatography/mass spectrometry in the selected ion monitoring mode was 250 fmol on column. Six human urine samples were analyzed, and the concentrations of M1G were below the limit of detection of the assay (500 fmol/mL). Introduction
Chemical modification of DNA by oxidation or adduction with carcinogens can cause mutations in genes and contribute to various stages in the process of cancer development (I,2). Exposure to carcinogens may occur from endogenoussources (3-6)due to various physiological processes, or from exogenous sources such as environmental (7)or dietary exposure (8). There are many reports in the literature confirming the chemical modification of DNA bases by alkylating agents (4-11), reactive oxygen species (12-151, and other carcinogens (9,16-18). MDA1is a product of oxidative lipid metabolism, arising as a byproduct of prostaglandin biosynthesis and an end product of lipid peroxidation (14-22). It is mutagenic in bacterial and mammalian test systems and carcinogenic in rodents (23,24). Thus, MDA is an example of a naturally occurring, endogenously produced carcinogen that may contribute to the genesis of human cancer. MDA reacts with deoxynucleosides in vitro to produce a variety of adducts (25-28). The order of reactivity of MDA with DNA bases is guanine > adenine > cytosine > thymine (25). If the reactivity of MDA with DNA bases present in intact DNA is similar to that observed with deoxynucleosides, it would be expected that the MDAguanine adduct (M1G; eq 1) would be a major product following treatment of DNA with MDA. In keeping with this prediction, hydrolysates of MDA-treated DNA have been found to contain M1G (2!2-31). If M1G is, in fact, formed in cellular DNA, it is possible that an endogenous DNA repair enzyme could excise it
* Address correspondence to this author at the Department of Biochemistry, Vanderbilt University, Nashville, T N 37232-0146. Phone: 615-343-7329;FAX: 615-343-7534. t Present address: National Institute of Hygienic Sciences, Kamiyoga 1-chrome, Setagaya-ku, Tokyo 158, Japan. 3 Department of Pharmacology. 1 Abbreviations: malondialdehyde (MDA), electrochemical detection (ECD), liquid chromatogaphy/mass spectrometry (LC/MS), dihydroMIG (DHMlG), solid-phase extraction (SPE), collisionally induced dissociation (CID), dideuterated MIG ([2HzlM1G).
MDA
Mi0
from the genome, raising the likelihood that the liberated base is excreted in urine. Quantification of urinary M1G would then provide an index of exposure to MDA. The recent report by Hadley and Draper that M1G is present in rat and human urine seems consistent with this scenario (32). Preliminary results from our laboratory using HPLC with ECD (29) indicated that M1G may be present in human urine at concentrations as high as 5 pmol/mL. In order to determine whether the compound detected by HPLC/ECD was in fact MIG, we have developed a method for its analysis using thermospray liquid chromatography/ mass spectrometry. The combination of highly efficient chromatographic separation with the selectivity of a mass spectrometer allows the analysis to be carried out on low adduct concentrations and provides structural characterization of the adduct. We report herein the development of this method and its application to the analysis of M1G in human urine. Materials and Methods Chemicals. MIG was synthesized by reaction of MDA with guanine as describedelsewhere (25)and purified by HPLC (>98 % pure). [2H2]MDAwas synthesized as described elsewhere (33) and used in the synthesis of [ZHzlMlG (25). All other chemicals were reagent grade, and solvents were HPLC grade. The Cls SPE columns (SupelcleanLC-18,6-mLcolumns)were purchased from Supelco (Bellefonte, PA). DHMlG was synthesized by reaction of M1G with N&Hd at 37 O C for 2 h (29). Urine Samples. The urine samples used in this study (100200 mL) were collected from six healthy volunteers (4 male and 2 female). The samples were stored in a refrigeratorat 4 O C until analyzed. Solid-Phase Extraction. The CISSPE columns were washed with 5 mL of methanol followed by 5 mL of water prior to use.
0893-228~/92/2705-087~~03.00/00 1992 American Chemical Society
Quantitation of MIGby Thermospray LCIMS Urine samples (10 mL) were loaded onto the prewashed SPE columns which were then washed with water (5mL) and eluted with methanol (5 mL). High-Performance Liquid Chromatography. The HPLC system consisted of a Hitachi L-6200 intelligent pump, a Valco C6W injector, an LDC Spectromonitor 3000 variable-wavelength UV detector, and a Hitachi D-2500 Chromato-integrator. The column was a Supelcosil LC-18 (4.5 mm X 25 cm; 5 pm, Supelco), and detection was by UV at 254 nm. Three solvent systems were used. HPLC system A used methanoV0.05M ammonium acetate (7:93 v/v; pH 6.5) at a flow rate of 1mL/min. HPLC system B used methanol/0.05 M ammonium acetate (1090 v/v; pH 6.5) at a flow rate of 1.5 mL/min. HPLC system C used methanol/0.05 M ammonium acetate (20:80 v/v; pH 6.5) at a flow rate of 1.5 mL/min. HPLC/ECD. A modified version of the method reported previously from our laboratory for quantification of M1G by HPLC with ECD (29) was used for analysis of human urine samples. The method involves the reduction of MIG by NaBH4 to DHMIG,which is electrochemicallymore active than the parent compound. HPLC was carried out using an Altex Ultrasphere ODS column (5pm, 4.6 mm X 250 mm) and a Varian 9010 pump equipped with a Shimadzu L-ECD-6A detector. The electrode potential of the Shimadzu detector was +800 mV for all determinations. The electrochemical cell, column, and guard column were housed in a Shimadzu CTO-6A oven maintained at 40 OC. The mobile phase was 1%methanol in 50 mM potassium phosphate (pH 5.5) and was maintained at a flow rate of 1mL/ min. Thermospray Mass Spectrometry. Mass spectral data were obtained on a Nermag R30-10 triple quadrupole mass spectrometer. The spectra were collected and processed using a SIDAR datasystem. Thermospray ionizationmass spectra were obtained using a thermospray interface based on the Vestec design (34). Thermospray conditions were optimized for M1G as follows: capillary temperature (250 "C), source temperature (265 OC), and voltage of the repeller electrode (250 V). The photomultiplier detector of the third quadrupole was set at 600 V. Selected ion monitoring was carried out for the MH+ ions of DHMlG (m/z 190) and [2H2]DHM1G ( m / z 192). Tandem Mass Spectrometry. Tandem mass spectrometric data were obtained by selection of the protonated molecular ion (MH+)of MIG through the first quadrupole, and allowing it to undergo CID with argon in the collision cell, which is an rf-only quadrupole. The ions from the collision cell were mass analyzed by the third quadrupole and were detected on the final photomultiplier detector at 600 V. The collision gas pressure (2.0 E-6 Torr) and collision energy (30 eV) were optimized to obtain maximum sensitivity for the fragment ions. Collision conditions and transmission through the quadrupoles were optimized for MIG using argon as the collision gas.
Results Thermospray and Tandem Mass Spectrometry. M1G chromatographed on a reverse-phase HPLC column eluted with solvent system C as a symmetrical peak with a retention time of 6.8 min. The thermospray mass spectrum of M1G showed a dominant protonated molecular ion at mlz 188 (Figure la). CID of the MH+ ion of MIG (mlz 188)with argon resulted in the formation of daughter ions at mlz 79 and 106 arising from the fragmentation reactions illustrated in Figure lb. [2H21M1G was synthesized for use as an internal standard (25, 33). The thermospray mass spectrum of f2HzlM1Gshowed a dominant protonated molecular ion at mlz 190. CID of the MH+ ion for f2H21M1G( m l z 190) resulted in the formation of daughter ions at mlz 81 and 108, confirming the assignment of the fragment ions obtained for nondeuterated M1G. A series of dilution
Chem. Res. Toxicol., Vol. 5, No. 6,1992 871
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experiments established that the detection limit for M1G using the selected ion monitoring mode was 250 fmol of M1G injected on column (signallnoise 5:l). It has been shown that reduction of M1G with NaBH4 yields a dihydro product (DHM1G) that is electrochemically more active than M1G and DNA bases (eq 2) (29). 0
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Reduction of M1G with NaBH4 also changes the retention time on HPLC system C from 6.8 to 12.5min. This change in retention time upon reduction proved useful for separation of M1G from interfering substances present in the biological matrix. DHMlG was obtained by reaction of M1G with NaBH4 at 37 OC for 2 h. The thermospray mass spectrum of reduced M1G showed a dominant ion at mlz 190 (Figure 2a), corresponding to the protonated molecular ion. CID of the MH+ ion of DHMlG (mlz 190) resulted in the formation of daughter ions at mlz 56 and 135(Figure 2b). The thermospray mass spectrum of [2H2]DHMlG showed a dominant ion a t mlz 192, corresponding to the protonated molecular ion of the molecule. CID of the MH+ ion of f2H21DHM1G(mlz 192) resulted in the formation of the expected daughter ions at mlz 58 and 135, thereby confirming the assignment of the fragment ions. Isolation of MIG from Urine Samples. The protocol employed for the isolation and analysis of M1G from human urine is shown in Figure 3. Ten-milliliter volumes of urine were spiked with 30 ng of internal standard before they were extracted by means of a Cl8 SPE column as described in the Materials and Methods section. It was established that the recovery of M1G during the extraction step was 80%. The fractions were concentrated and then injected
872 Chem. Res. Toxicol., Vol. 5, No. 6,1992
Jajoo et al.
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Figure 4. UV (254-nm)chromatogramsfor (a) 20 ng of synthetic MIG and (b) a 10-mL urine sample after solid-phase extraction. The chromatography was carried out on a Supelcosil LC-18 column (4.6 mm i.d. X 25 cm; 5 pm). The mobile phase was methanol/0.05M ammonium acetate (7:93 v/v) at a flow rate of 1.0 mL/min. (c) The urine fraction after reduction with NaBH4. The mobile phase was methanol/0.05 M ammonium acetate (15: 85 v/v) at a flow rate of 1.0 mL/min. The retention time for DHMIG was 19.3 min.
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Figure 3. Scheme for isolation and analysis of MIG from human urine samples. onto a reverse-phase HPLC system. The HPLC chromatograms for urine extracts analyzed at this stage (Figure 4b) were very complex due to the presence of a number of interfering substances in the samples which exhibited similar retention times to authentic M1G (12.6 min, Figure 4a). Fortunately, this difficulty of identifying the M1G peak among the interfering compounds could be ameliorated by exploiting the change in chromatographic be-
havior of M1G upon reduction to a dihydro compound. Thus, the fraction eluting from 11to 14 min was reduced with NaBH4 prior to analysis by HPLC (Figure 412). Most of the interfering peaks from the biological matrix eluted early in the chromatogram (6-12 min) whereas the DHMlG eluted at 19.2 min. The peak that eluted with a retention time of 19.2 min was collected and evaporated to dryness before it was subjected to analysis by thermospray LC/ MS. The recovery of M1G through all the steps of sample preparation was approximately 30%. In a separate experiment, a urine sample was spiked with 10 ng/mL each of M1G and [2H21MlG,and the sample was purified as described above. Analysis by Thermospray LC/MS. The sample isolated in the previous step was analyzed by thermospray LClMS in the selected ion monitoring mode. The selected ion current profiles obtained for ions at m/z190 and 192 for the urine sample spiked with 10 ng/mL each of M1G and [2H21M1G are shown in Figure 5 , panels a and b, respectively. The peak a t retention time 8.1 min in Figure 5a corresponds to DHMlG formed as a result of the reduction of MlG, whereas the peak at retention time 8.1 min in Figure 5b corresponds to reduced [2H21M1G. The urine samples with and without internal standard were analyzed by thermospray LC/MS in the selected ion monitoring model. The selected ion current profiles for ions at mlz 190 and 192 for the urine sample without addition of internal standard showed no peaks in the chromatograms at the retention time of DHMlG. The selected ion current profiles obtained for ions at mlz 190 and 192 for the urine sample with addition of internal standard are shown in Figure 6, panels aand b, respectively. The chromatogram for mlz 192 (Figure 6b)showed a peak at retention time 8.1 min from the internal standard. The peak in the chromatogram for mlz 190 at 8.1 min (Figure 6a) was due to the contribution of the protium of the internal standard. For all six urine samples analyzed, the peak in the chromatogram for mlz 190 was less than or equal to the protium contribution of the internal standard. These results indicated that the levels of M1G in the human urine samples were below the limit of detection of the thermospray LC/MS method (500 fmol/mL).
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 873
Quantitation of MIG by Thermospray LCIMS
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Figure 7. HPLC/ECD chromatogram for a 10-mLhuman urine sample prior to analysis by thermospray LC/MS. Experimental conditions are described in the Materials and Methods section. The retention time for DHMlG was 43.6 min under these conditions as indicated by the arrow.
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sample to which 100 ng each of M1G and [2H2]M1G had been added. (a) m/z 190 for MH+ for DHMlG and (b)mlz 192 for the MH+ ion for reduced [2Hz]MlG. Chromatography was carried out on a Supelcosil LC-18 column (4.6mm i.d. X 25 cm; 5 Hm). The mobile phase was methanol/0.05 M ammonium acetate (20 80 v/v) at a flow rate of 1.5 mL/min.
fmol/mL, the urine samples were reanalyzed by HPLC/ ECD as described in the Materials and Methods section. The urine samples were prepared for HPLC/ECD analysis in exactly the same manner in which they were processed prior to thermospray LC/MS. The HPLC/ECD chromatogram of the human urine extract is shown in Figure 7. The retention time for reduced M1G was 46.2 min under these conditions. No peak was evident a t the.retention time of reduced M1G in the human urine extract. Coinjection of M1G with the human urine extract showed that reduced M1G could be satisfactorily resolved from the adjacent large peak.
Discussion
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Figure 6. Selected ion current profiles for a 10-mLhuman urine sample to which 30 ng of 12H21M1Ghad been added. (a) mlz 190 for MH+ for DHMIG and (b) m/z for the MH+ ion for reduced [2H2]M1G. Chromatographic conditions were the same as for Figure 5. Analysis of Urine Sample by HPLC/ECD. Initial experiments using HPLC/ECD had suggested that the levels of M1G in human urine were as high as 5 pmol/mL (data not shown). Since LC/MS analysis of the urine samples indicated that the levels of MIG were below 500
This report is the first description of the use of LC/MS for analysis of a carcinogen-purine adduct in human urine. Thermospray LC/MS was chosen for the analysis of M1G because it introduces the effluent from analytical-scale HPLC directly into the ion source of the mass spectrometer. Derivatization is not necessary, and volatility is not a requirement for sample introduction. Thermospray LC/ MS is a soft ionization technique and provides intense protonated molecular ions (MH+) in the mass spectra of nucleosides, nucleic acid bases, and a variety of other compounds (35-38). This makes it suitable for quantitation by selected ion monitoring. Thermospray analysis of M1G revealed an abundant protonated molecular ion a t mlz 188. A dideuterated analog of M1G that demonstrated the expected molecular ion at m/z 190 was synthesized for use as an internal standard. During HPLC purification of human urine, M1G coeluted with large amounts of interfering substances. In order to circumvent this problem, urine extracts were reduced with NaBH4, which allowed the separation of DHMIG from contaminants that were not reduced. Quantitative analysisof the human urine extracta indicated that the levels of M1G were below the limit of detection of the assay (500 fmol/mL). In the light of our inability to detect M1G in human urine by thermospray LC/MS, the HPLC/ECD method that had originally detected urinary DHMlG was reexamined. It was found that, by decreasing the solvent polarity, DHMlG could be separated from a coeluting electrochemically-active interfering substance present in urine. The concentrations of DHMlG were found to be below the limit of detection of the HPLC/ECD method.
874 Chem. Res. Toxicol., Vol. 5,No.6,1992
Thus, two different analytical techniques, thermospray LC/MS and HPLC/ECD, establish an upper limit for M1G in human urine of 500 fmol/mL. Given the complexity of the urine samples (Figure 4a), it seems likely that the material assigned as M1G in the report of Hadley and Draper (32)was, in fact, a coeluting contaminant. However, this conclusion must be considered tentative because the analytical procedure employed, its limit of detection, and the concentration of M1G were not described in their report. Thermospray LC/MS appears to be a promising method for quantitating modified bases in urine. Derivatization and volatization of the sample is not required, molecular ions are abundant peaks in the mass spectra, and the use of stable isotope dilution with selected ion monitoring verifiesthe identities of the analyte. The limit of detection of MIG by this technique (500 fmol/mL) is in the range necessary for quantitation of alkylated or oxidized purines. For example, methylated and ethylated bases are present in human urine at concentrations of 800 fmol/mL to 1.5 nmol/mL (39);the aflatoxin-Wguanine adduct is present in the urine of exposed individuals at levels of 100-1OOO fmol/mL (40);and 8-oxodeoxyguanosine is present in normal human urine at a concentration of 10 pmol/mL (41).(The level of the base 8-oxoguanine in urine appears to be comparable to the level of 8-oxodeoxyguanosine,but it varies dramatically with dietary factors.) Thus, LC/ MS should be applicable to a wide range of structurally diverse, adducted bases. Our inability to detect M1G in normal human urine begs the question of whether it is formed in human genomic DNA. MDA may not react extensively with DNA in intact cells, or M1G may not be repaired by a glycosylase. The likelihood of the former awaits the determination of levels of MIG in cellular DNA. As regards the latter possibility, we have been unable to detect enzyme activities in human liver extracts that nick MDA-modified DNA, which suggests the absence of an M1G glycosylase/AP endonuclease activity.2 Furthermore, Fedtke et al. have estimated that the half-life of the N2,3-ethenoguanine adduct in rat liver DNA is greater than 30 days (16). Since W,3ethenoguanine is structurally similar to M1G this finding suggests that M1G may also be poorly repaired and, thus, may persist in genomic DNA. Acknowledgment. This work was supported by NIH Grants CA47479,ES00267, and RR05697. We are grateful to Udai Singh for preparation of [2HzlM~G. References (1) Harris,C. C. (1991)Chemicalandphysicalcarcinogenesis:Advances and perspectives for the 1990s. Cancer Res 51, 50238-5044s. (2) Yuspa, S. H., andPoirer, M. C. (1988)Chemical carcinogenesisfrom animal models to molecular models in one decade. Adu. Cancer Res. 50, 25-70. (3) Davis, D. D. (1989) Natural anticarcinogens, carcinogens and changing patterns in cancer: Some speculation. Enuiron. Res. 50, 322-340. (4) Ames B. N., and Gold, L. S. (1991) Endogenous mutagens and the causes of aging and cancer. Mutat. Res. 250, 3-16. (5) Lutz, W. K. (1990) Endogenous genotoxic agents and processes as a basis of spontaneous carcinogenesis. Mutat. Res. 238, 287-295. (6) Glatt, H. (1990) Endogenous mutagens derived from amino acids. Mutat. Res. 238, 235-243. (7) Sugimura, T. (1986)What is the role of environmental mutagens for human cancer development? Prog. Clin. Biol. Res. 209A, 105-128.
* P. Burcham, L. J. Marnett, and R. S. Lloyd, unpublished results.
Jajoo et al. (8) Sugimura,T.,andWakabayashi,K. (1990)Mutagensandcarcinogens in food. Prog. Clin. Biol. Res. 347, 1-18. (9) Singer, B. (1985) In vivo formation and persistence of modified nucleosides resulting from alkylating agents. Enuiron. Health Perspect. 62,41-48. (10) Maher, V. M., Domoradzki,J.,Bhattacharyya, N. P., Tsujimura, T., Corner, R. C., and McCormick, J. J. (1990)Alkylation damage, DNA repair and mutagenesis in human cells. Mutat.Res. 233,235-245. (11) Hall, J., and Montesano, R. (1990) DNA alkylation damage: Consequences,and relevanceto tumor production. Mutat. Res. 233, 247-252. (12) Lewis, J. G., and Adams, D. 0. (1987) Inflammation, oxidative DNA damage and carcinogenesis. Enuiron. Health Perspect. 76, 19-27. (13) Aruoma, 0. I., Halliwell, B., Gajewski, E., and Dizdaroglu,M. (1989) Damage to DNA bases induced by hydrogen peroxide and ferric ion chelates. J. Biol. Chem. 264, 20509-20512. (14) Reddy, J. K., and Rao, M. S. (1989) Oxidative DNA damage caused by persistent peroxisome proliferation. Its role in hepatocarcinogenesis. Mutat. Res. 214, 63-68. (15) Breimer, L. H. (1990) Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis. The role of DNA base damage. Mol. Carcinog. 3, 188-197. (16) Fedtke, N., Boucheron, J. A., Walker, V. E., and Swenberg, J. A. (1990) Vinyl chloride-induced DNA adducts 11. Formation and persistence of 7-(2'-oxoethyl)guanineand N2,3-ethenoguaninein rat tissue DNA. Carcinogenesis 11,1287-1292. (17) Beland, F. A., and Kadlubar, F. F. (1985)Formation and persistence of arylamine DNA adducts in vivo. Enuiron. Heath Perspect. 62, 19-30. (18) Hecht, S. S.,and Hoffmann,D. (1988)Tobaccospecificnitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9, 875-884. (19) Bernheim, F., Bernheim, M. L. C., and Wilbur, K. M. (1948) The reaction between thiobarbituric acid and the oxidation products of certain lipids. J. Biol. Chem. 174, 257-264. (20) Pryor, W. A., and Stanley, J. P. (1975) A suggested mechanism for the production of malondialdehyde during the autoxidation of polyunsaturated fatty acids. Nonenzymatic production of prostaglandin endoperoxides during autoxidation. J. Org. Chem. 40, 3615-3617. (21) Schauenstein, E., Esterbauer, H., and Zollner, H. (1977) Malondialdehyde. In Aldehydes in BiologicalSystem (Schauenstein, E., Esterbauer, H., and Zollner, H., Eds.) pp 133-140, Pion Limited, London. (22) Spalding, J. W. (1988) Toxicology and carcinogenesis studies of malondialdehyde sodium salt (3-hydroxy-2-propenal,sodium salt) in F344/N rata and B6C3F1 mice. NTP Tech. Rep. 331,513. (23) Mukai, F. H., and Goldstein, B. D. (1976) Mutagenicity of malondialdehyde, a decompositionproduct of peroxidid polyunsaturated fatty acids. Science 191, 868-869. (24) Basu, A. K., and Marnett, L. J. (1983) Unequivocal demonstration that malondialdehyde is a mutagen. Carcinogenesis 4, 331-333. (25) Basu, A. K., O'Hara, S. M., Valladier, P., Stone, K., Mob, O., and Marnett, L. J. (1988) Identification of adducts formed by reaction of guanine nucleosideswith malondialdehydeand structually related aldehydes. Chem. Res. Toxicol. 1, 53-59. (26) Nair, V., Turner, G. A., and Offerman, R. J. (1984) Novel adducts from the modification of nucleic acid bases by malondialdehyde. J. Am. Chem. SOC.106,3370-3371. (27) Stone, K., Uzieblo, A., and Marnett, L. J. (1990) Studies on the reaction of malondialdehyde with cytosine nucleosides. Chem. Res. Toxicol. 3, 467-472. (28) Stone, K., Ksebati, M. B., and Marnett, L. J. (1990) Investigation of the adducts formed by reaction of malondialdehyde with adenosine. Chem. Res. Toxicol. 3, 33-38. (29) Goda, Y., and Marnett, L. J. (1991) High-performance liquid chromatography with electrochemical detection for determination of the major malondialdehyde-guanine adduct.. Chem. Res. Toxicol. 4,520-524. (30) Seto, H., Okuda, T., Takesue, T., and Ikemura, T. (1983) Reaction of malondialdehyde with nucleic acid. I. Formation of fluorescent pyrimido[l,2-alpurin-10(3H)-onenucleosides. Bull. Chem. Soc.Jpn. 56, 1799-1802. (31) Seto, H., Seto, T., Takesue, T., and Ikemura, T. (1986) Reaction of malondialdehyde with nucleic acid. 111. Studies of the fluorescent substances released by enzymatic digestion of nucleicacids modified with malondialdehyde. Chem. Pharm. Bull. 34,5079-5085. (32) Hadley, M., and Draper, H. H. (1990) Isolation of a guanine malondialdehyde adduct from rat and human urine. Lipids 25,8285. (33) Basu, A. K., andMarnett,L. J. (1985)Synthesisof malondialdehydeJ. Labelled Compd. Radio1-ZH and mal0ndialdehyde-l,3-~H2. pharm. 22, 1175-1179.
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Quantitation of MIG by Thermospray LCIMS (34) Vestal, M. L., and Fergusson, G. J. (1985) Thermospray liquid
(39) Shuker, D. E. G., and Farmer, P. B. (1992) Relevance of urinary
chromatograph/mass spectrometer interface with direct electrical heating of the capillary. Anal. Chem. 57, 2373-2378. (35) Crain, P. F. (1990) Mass spectrometric techniques in nucleic acid research. Mass Spectrom. Rev. 9, 505-554. (36) Pomerantz, S. C., and McCloskey, J. A. (1990) Analysis of RNA hydrolysates by liquid chromatographymaas spectrometry. Methods Enzymol. 193, 796824. (37) Tomer, K. B., and Parker, C. E. (1989) Biochemical applications of liquidchromatography/massspectrometry. J.Chromatogr. 492,189-
DNA adducts as markers of carcinogen exposure. Chem. Res. Toxicol. 5, 450-460. (40) Groopman, J. D., Jiaqi, Z., Donahue, P. R., Pikul, A., Lisheng, Z., Jun-shi, C., and Wogan, G. N. (1992) Molecular dosimetry of urinary aflatoxin-DNA adducts in people living in Guangxi autonomous region, People's Republic of China. Cancer Res. 62,4552. (41) Park, E.-M., Shigenaga, M. K., Degan, P., Korn, T. S., Kitzler, J. W., Wehr, C. M., Kolachana, P., and Ames, B. N. (1992) Assay of excised oxidative DNA lesions: Isolation of 8-oxoguanine and ita nucleoside derivatives from biologicalfluids with a monoclonal antibodycolumn. h o c . Natl. Acad. Sci. U.S.A. 89. 3375-3379.
221. (38) Kim, H. Y., Yergey, J. A., and Salem, N., Jr. (1987) Determination
of eicosanoids,phospholipids and related compounds by thermospray liquid chromatography/madd spectrometry. J. Chromatogr. 394, 155-170.
Registry No. MIG, 103408-45-3.
A d d it ions and Correct ions Examination of Diols and Diol Epoxides of Polycyclic Aromatic Hydrocarbons as Substrates for Rat Liver Dihydrodiol Dehydrogenase [Volume 5, Number 4, July/August 1992,pp 576-583 I LYNNFLOWERS-GEARY, RONALDG. HARVEY,and TREVOR M. PENNING' Page 576. The first sentence of the Introduction should read as follows: Polycyclic aromatic hydrocarbons (PAH)' are environmental pollutants that require metabolic activation to exert their carcinogenic effects (1).
