Polycyclic Aromatic Hydrocarbon-DNA Adducts in Human Lung and Cancer Susceptibility Genes P

(CANCER RESEARCH 53, 3486-3492, August 1. 19931 Polycyclic Aromatic Hydrocarbon-DNA Adducts in Human Lung and Cancer Susceptibility Genes P. G. Shields,1 E. D. Bowman, A. M. Harrington, V. T. Doan, and A. Weston

Laboratory- of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute. Bethesda, Mailand 20892

ABSTRACT tency in laboratory animal studies exists (9). Human studies have associated PAH exposure, which typically occurs as complex mix Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA ad- tures, with skin and lung cancers (7). Increased sister chromatid ex ducts, genetic predisposition to cancer, and their interrelationships are change frequency also occurs (10). PAHs form DNA adducts via a under study in numerous laboratories. This report describes a modified â€¢¿"P-postlabelingassayfor the detection of polycyclic aromatic hydrocar complex metabolic activation pathway that includes CYP1A1, ep- bon-DNA adducts that uses immunoaffmity chromatography to enhance oxide hydroxylase, CYP3A4, and other enzymes (11). However, in chemical specificity and quantitative reliability. The assay incorporates termediate metabolites can be detoxified through pathways beginning internal standards to determine direct molar ratios of adducts to unmod with GST/x. Thus, the measurement of PAH-DNA adducts would ified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. provide indications of exposure as well as the net success of compet High performance liquid chromatography is used to assure adequacy of ing metabolic and detoxification pathways. DNA enzymatic digestion. The assay was validated using radiolabeled The 32P-postlabeling assay is considered to be one of the most benzoloIpyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby sensitive procedures for the detection of carcinogen-DNA adducts assessing all variables from enzymatic digestion to detection. Thirty-eight (12). This assay typically removes vast excesses of unmodified nu human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide- cleotides by either TLC (12), nuclease P, enzymatic treatment (13), or butanol extraction (14). The main advantage of the 32P-postlabeling DNA adducts by immunoaffmity chromatography, high performance liq uid chromatography, and synchronous fluorescence spectroscopy. A high assay over other conventional methods is that the chemical identity of correlation between the two assays was found (P = 0.006). The lung putative adducts is not required, making it a useful screening proce samples were then analyzed by the polymerase chain reaction for the dure. Nonetheless, the method also is limited because human DNA presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione analysis yields complex TLC elution maps and adducts cannot be S-transferase // (GST//) genes. A positive association was identified for chemically identified (frequently described as diagonal radioactive adduci levels and (.SI// null genotypes (/' = 0.038). No correlation was zones). Micropreparative techniques such as HPLC (15-18) and IAC found between polycyclic aromatic hydrocarbon-adduct levels and (19) have been combined with the 32P-postlabeling assay to enhance CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related chemical specificity and quantitative accuracy. These assays, how to adduct levels. Multivariate analysis indicated that only the GST// genotype was associated with polycyclic aromatic hydrocarbon-DNA ever, sacrifice sensitivity for specificity and are relatively labor inten adduct levels. This work demonstrates that the -"P-postlabeling assay can sive. Other methods exist for PAH-DNA adduct detection that rely be modified for chemically specific adduct detection and that it can be upon micropreparative techniques such as mass spectroscopy (20, 21) used in the assessment of potentially important genetic factors for cancer and SFS (22-24), but these require larger amounts of DNA. Immu- risk. The absence of a functional GST// gene in humans is likely one such noassays for PAH-DNA adduct detection (25-30) do not necessarily factor. require micropreparative techniques but they cannot distinguish indi vidual adducts of a given chemical class because of antibody cross- INTRODUCTION reactivity (31-35). Genetic polymorphisms in the CYP1 Al and GSTju genes might be Environmental carcinogen exposure, DNA damage such as DNA markers for PAH cancer risk. A deletion of the GST/x gene (36) has adducts, and genetic predisposition to cancer may be important cancer been associated with lung cancer (37, 38), sister chromatid exchanges risk factors. Host factors govern DNA adduct levels through carcin (39), and mutations in the Salmonella typhimurium mutation assay ogen metabolic activation and detoxification. Both of these are inher (40). Separately, CYP1A1 activity is higher in peripheral WBC of itable traits that can vary widely from individual to individual (1), as evidenced by differences in metabolic capacity (2) or DNA-adduct cancer patients (41, 42) and has been correlated with sister chromatid levels ( 1, 3-6). Cancer risk assessments might therefore include DNA exchanges (39, 43) and also with DNA adduct levels measured by the 32P-postlabeling assay without micropreparative techniques (40). A adduct measurements in order to evaluate recent carcinogen exposure, as well as provide a marker for the body's reaction to that exposure. mutation in the catalytic region of CYP1A1 (exon 7) is associated with an Msp\ restriction fragment length polymorphism that is found However, risk assessments also might include a direct determination more frequently in Japanese lung cancer patients compared to controls of inheritable traits (e.g., metabolic capacity) in order to provide an (44, 45). The Japanese data (46) further demonstrated that the com estimation of an integrated lifetime response to carcinogenic expo bined presence of the CYP 1A1 exon 7 mutation and deletion of GST/x sures. Sufficient data implicate environmentally ubiquitous PAHs2 as po increased lung cancer risk. It may be hypothesized, therefore, that the tential human carcinogens (7, 8), although varying carcinogenic po- relationship of CYP1A1 activity and GST/x to carcinogenesis in vivo would be demonstrable through correlations with PAH-DNA adduct levels. We report herein the development of a chemically specific Received 1/7/93; accepted 5/25/93. The costs of publication of this article were defrayed in part by the payment of page assay for PAH-DNA adduct detection and the investigation of charges. This article must therefore be hereby marked advenisemenl in accordance with CYP1A1 and GST/i genetic polymorphisms as determinants of adduct 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Laboratory of Human Car- formation. cinogenesis. Division of Cancer Etiology, National Cancer Institute, Building 37, Room 2C16. Bethesda, MD 20892. 2 The abbreviations used are: PAH. polycyclic aromatic hydrocarbon; dGp, 2'-deoxyguanosine-3'-monophosphate; AHH, aryl hydrocarbon hydroxylase; BPDE, benzo(a)- polymerase chain reaction; PNK, polynucleotide kinase; BPDE, r-7,/-8-dihydroxy-i-9.10- pyrene-diol-epoxide; CYP, cytochrome P-450; GST/i, glutathione 5-transferase n: HPLC. epoxy-7,8,9,10-tetrahydroben/o(Â«(pyrene; SFS, synchronous fluorescence spectroscopy; high performance liquid chromatography; IAC, Â¡mmunoaffmily chromatography; PCR, TLC, thin layer chromatography. 3486

MATERIALS AND METHODS

Overall Strategy for PAH-DNA Adduct Detection. The protocol for com bining micropreparative techniques and the 12P-posllabeling assay is shown in Fig. 1. Following enzymatic digestion, adducts from DNA samples are purified by IAC and then recombined with a specific amount of dGp, which serves as an internal control for labeling efficiency and determination of direct molar ratios. The adduci and dGp mixtures are subjected to the 32P-posllabeiing assay i and resolved by multidimensional TLC on the same TLC plate. Separately, the BPDE-dGp eluates from IAC not containing adducts are subjected to HPLC in order to I confirm that enzymatic digestion was complete and to quantitate the amount of dGp present in the DNA sample. Reagents for Micropreparative Techniques and the 32P-Postlabeling Assay. BPDE was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository (Midwest Research Institute, Kan sas City, MO). Authentic BPDE-dGp was prepared as described previously Fig. 2. Multidimensional development of TLC plates begins by locating the origin 1 cm from the edge and 1 cm from the midline (A). Following Dl, which allows unmodified (47). Micrococcal nuclease and nuclease-Pl were purchased from Sigma (St. nucleotides and unused ATP to migrate to one side, the plate is cut in half with scissors. Louis, MO). Calf-spleen phosphodiesterase was obtained from Boehringer- Each side is then developed separately as described in the text. The overall scheme is Mannheim (Indianapolis, IN). T4-polynucleotide kinase was procured from shown and the solvent conditions indicated in the text. This process yields separate New England Nuclear (Boston, MA). [y-32P]ATP (specific activity, >5000 Chromatographie maps for dGp (B) and the adduci (C). Ci/mMole) was procured from Amersham (Arlington Heights, IL). Trielhy- lamine acetate was purchased from Applied Biosystems (Foster City, CA). All estimated by the area under the curve and compared to known amounts of dGp. solvents were reagent or HPLC grade. Adequacy of digestion was assessed by the demonstration of predicted Calf thymus DNA was modified at low levels with [3H]BPDE ( 1.48 Ci/ amounts of each nucleotide. mmol) and extracted as described previously (18, 32). The level of adduction Methanol eluates containing the adducts were dried in a vacuum, redis solved in water (4 /j.1),and subjected to the 32P-posllabeling assay. Unmodified was determined by liquid scintillation counting. Immunoaffinity Purification of PAH-DNA Adducts and the "P-Postla- dGp was added to the adduci fraction (0.7 pmol in 1 /j.1) prior to the 32P- beling Assay. For development and validation of the assay, BPDE-dGp and postlabeling assay. The postlabeling buffer (2 Â¿il;0.1 Mbicine-10 mm spermi- dine-O.I MMgCl2, pH 9.5), 0.1 Mdithiothreitol (1.0 /j.1), 0.33 JIM [7-32P]ATP dGp mixtures (0.07-700 fmol of adduci and 0.7 pmol of dGp) or enzymatic digests of DNA were subjected to IAC. The modified BPDE-DNA or human (2 fil; total amount of ATP, 4 pmol), and PNK (2 ju.1;5 units/jx!) were added lung DNA (100 /Â¿g;1 mg/ml) was enzymatically digested (4 h; 37Â°C)with separately. The samples were then incubated at 37Â°Cfor 30 min. The sample micrococcal nuclease (5 units) and desalted calf spleen phosphodiesterase was resolved by TLC as described previously (47). Polyethyleneimine cellu (0.08 unit; filter purification by Centricon 100 microconcentrators; Centricon, lose TLC plates (Machery Nagel, Germany; 20 x 20 cm), spotted with UV detectable (254 nm) quantities of nonradiolabeled 2'-deoxyguanosine-3',5'- Beverly, MA) in 100 ITIMsodium succinate buffer (pH 6.0) and 50 IÃ•IMcalcium bisphosphate (Pharmacia, Piscataway, NJ) and the "P-postlabeled sample (12 chloride. IAC columns were prepared with monoclonal antibodies against PAH-DNA adducts (kindly provided by Dr. Regina Santella. Columbia Uni ju.1;PCR Pipettes; Drummond Scientific Co., Brogmull, PA) were developed as versity, New York, NY) (31) immobilized on Affinica protein A-agarose described in Fig. 2. Plates were prewashed by immersion in water (60 min) and (Schleicher and Schuell. Keene, NH), according to the manufacturer's instruc then dried by cool air. Samples were spotted 1 cm from the edge of the plate tions. The matrix (10 ITIMTris-HCl, pH 7.5; 0.5 ml) was placed in a 1-ml and 1 cm from the midline. Dl (1.25 M lithium chloride) causes the dGp and syringe and equilibrated with Tris-HCI (10 MM, pH 7.5). The nucleotide unreacted ATP to migrate to the left side of the plate, while the PAH-DNA mixtures and enzymatic DNA digests were then applied to the column (0.3 ml). adducts remain at the origin. The plate is then washed by immersion in Unbound materials were eluted with water ( 1 ml) and then PAH-DNA adducts methanol (20 min), dried by cool air, and cut in half with scissors, ensuring that were eluted with methanol (100%; 1 ml). all the dGp is to the left by visualizing the UV marker. The left side of the plate Water eluates from the IAC containing unmodified nucleotides were dried containing unmodified nucleotides was then developed in D2a (saturated am in a vacuum, redissolved in water (100 jxl), and analyzed by HPLC (47). monium sulfate/isopropyl alcohol/1 Msodium acetate, 80/2/18, v/v) and radio Ion-pair reverse-phase chromatography (Beckman Altex Ultrasphere ion pair active cpm for dGp were determined by radioanalytical scanning (Ambis column, 5 fxm, 4.6 mm x 25 cm) was utilized isocratically [0.1 Mtriethylamine Model 100; Ambis, Anaheim, CA). The portion of the plate containing adducts acetate (pH 7.0) and 2% acetonitrile at 1 ml/min for 15 min] followed by was developed overnight in D2b (2.3 Msodium phosphate, pH 6) after attach gradient elution (2 to 95% acetonitrile over 65 min). The quantity of dGp was ing a wick (Whatman No. 3MM CHR) with staples. Following removal of the wick, the plates were washed in water (20 min) and developed in D3 (3.5 M lithium formate/8.5 M urea, pH 3.5) and D4 (1.2 M lithium chloride/0.5 M Genomic DNA Tris-HCl/8.5 M urea, pH 8.0). The cpm of the adduci was determined by Phosphodiesterase radioanalytical detection, minus the mean of 2 background sites near the Micrococcal Nuclease adduci and of equal size. Direct molar ratios determined by nel cpm for PAH-DNA adduci levels to dGp were Ihen calculated using previously deter 3'-Monophosphate Nucleotides mined calibralion curves. .Immunoaffinrty Immunoaffinity Chromatography and Synchronous Fluorescence Spec- Chromatography troscopy for Polycyclic Aromatic Hydrocarbon-DNA Adducts. Human dGp DNA samples were enzymatically digested and purified by IAC as described previously (23). Materials eluling from IAC in melhanol were dried in a vacuum, redissolved in 1 ml 0.1 N HC1, and heated al 90Â°Cfor 3h. Acid Unmodified 32P-postlabeling 3'-Monophosphate hydrolysales were then subjected lo HPLC on ocladecasilane (Vydac) columns Assay Nucleotides wilh a waler-melhanol gradient (30-60%; 20 min; flow rate, 1 ml/min) and

HPLC analyzed by SFS (MPF-66 spectropholofluorimeler; Perkin-Elmer, Rockville, TLC MD). Fluorescence speclra were generated by driving the excitation and emis sion monochromalors synchronously with a wavelength difference of 34 nm dGp Quantitiation (UV) Adduct Quantitation (AA.34 nm). Samples conlaining ihe pyrene fluorophore were characterized by Radioisotope Scanning the presence of a fluorescence emission peak at 379 nm. Second derivative Fig. 1. Combined microipreparative techniques and the ^-postlabeling assay for the spectra were generated from zeroth order speclra using ihe Saviisky-Golay detection of PAH-DNA adducts in human tissues. algorilhm (48). The limil of deleclion for mis assay is 6.25 pg of BP-telrol in 3487

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1993 American Association for Cancer Research. PAH-DNA ADDUCTS IN HUMAN LUNG AND GENETIC SUSCEPTIBILITY water (500 /j.1in a microcuvette; 5x5 mm). Therefore this assay can measure 1 BP udduct in IO7 unmodified nucleotides when 1 mg of DNA is available for analysis (50 fmolAtil or 0.050 fmol BPDE/p,g DNA). Genotyping Methods. The GST/u, genetic polymorphism was determined by PCR according to the method of Comstock (49). Reaction mixture products .1-1 were analyzed by gel electrophoresis (1.8% agarose) for the presence of a 273-base pair product, which was indicative of the GSTfi gene. The lack of an - .01 Adduct Standard amplification product is consistent with the null genotype. Amplifications were performed in duplicate and analyzed separately to enhance quality control. Polymerase Chain Reaction for the Detection of the CYP1A1 Exon 7 .01 10 Mutation. The presence of the CYP1A1 exon 7 mutation, which codes for valine rather than isoleucine (50), was determined by PCR and restriction enzyme digestion. Here, a primer was designed so that a one base substitution results in a Ncol restriction enzyme site encompassing the mutation. DNA (0.4 Â¿ig)was amplified in the presence of primers (5'-GAACTGCCACTTCAGCT- GTCT-.V and 5'-CCAGGAAGAGAAAGACCTCCCAGCGGGCCA-3'; base substitution underlined; 0.4 ^M), buffer (10 min Tris-HCl, pH 8.3-50 mm kCl-2 DMA imi MgCli), nucleoside 3'-triphosphates (Pharmacia; 1.875 ITIM),and Taq .01 polyinerase (Perkin-Elmer, Norwalk. CT; 2.5 units). The reaction began at 94Â°Cfor 4 min, followed by 35 cycles of 94Â°Cfor 1 min and 68Â°Cfor 1 min, and ended with a single final step at 72Â°Cfor 4 min. Amplified products (195 Known Molar Ratio (x10~5) base pairs; 17.5 p.1) were subjected to Ncol restriction fragment length diges BPDE-dGp/dGp tion (New England Biolabs, Beverly, MA; 2.5 units; 37Â°C;2 h). PCR in the Fig. 3. To calibrate the assay, the level of synthesized adduci standards determined by presence of 1 HIMdithiothreitol, MgC12 (final concentration, 10 HIM),Tris-HCl UV and SFS is correlated to the level determined by IAC and 12P-postlabeling, expressed (final concentration, 20 HIM),and Ncol enzyme. Analysis by gel electrophoresis as a ratio to dGp (A). For assessment of all factors involved in determining adduct levels (1.8% agarose) revealed 163- and 32-base pair fragments for wild type alÃeles from digestion to detection, [3H]BPDE-treated DNA is used and the calibration curve is (isoleucine) or a single 195-base pair fragment when the mutation (valine) was determined (ÃŸ). present. Determination of Serum Cotinine Levels. Radioimmunoassay using enzymatic digestion for human lung samples, but no effect was found polyclonal antibodies was performed as described previously (51, 52). (data not shown). Separately, different amounts of one human DNA Preparation of Human DNA Samples. Parenchymal lung tissue was ob sample (50-300 fig) known to be positive by IAC and 32P-postlabel- tained from autopsy donors (adult and infant) within 12 h of death and stored at -70Â°C. Tissues were pulverized in liquid nitrogen and then subjected to ing assay were assayed and revealed the expected proportional in RNase A (50 /xg/ml; 0.6% sodium dodecyl sulfate-10 min EDTA-10 mm Tris- creases in adduct levels (data not shown). HCl) and proteinase K treatment (200 /xg/ml; 37Â°C;16 h). The samples then Thirty-eight human parenchymal lung samples were analyzed by underwent extraction with equal volumes of phenol, phenol and chloroform the combined micropreparative techniques (IAC and HPLC) and the 32P-postlabeling assay (Fig. 4). Seven samples were found to have (1:1), and chloroform. The DNA was precipitated with ethanol (2.5 volumes) and NaCl (100 nui). Samples were dried in a vacuum and redissolved in water detectable adduct levels (Table 1). Analyses were performed in trip at a concentration of 2 /xg/fJ.1. licate. In all positive cases, adducts only were detected in a single area Statistical Analyses. Analyses were performed using the Statistical Anal which coeluted with the BPDE-dGp adduct standard. DNA samples ysis System (SAS Institute, Cary, NC). Pearson correlation coefficients using from 23 of the above individuals also were analyzed by SFS. Six log transformed data and first order regression equations were used for deter samples were found to be positive by both the 32P-postlabeling assay mination of calibration curves. Spearman's rank correlation was used accord and SFS (the seventh positive by 32P-postlabeling assay was not ing to standard methods for analysis of positive samples from corroborative assays (53). All samples for the corrobative assays were assessed in a 2 X 2 analyzed by SFS) (Table 1). The limit of detection for the two assays, contingency table with a x2 analysis. General linear regression, Fisher's exact because the SFS used 10-fold more DNA, was similar. Analysis of test, and Wilcoxon rank sum scores were performed for analysis of genetic susceptibilities and DNA adduci levels, according to standard procedures (53). B

RESULTS The combined IAC/HPLC and 32P-postlabeling assay for the de tection of PAH-DNA adducts was optimized and calibrated using authentic BPDE-dGp adducts. Known concentrations of BPDE-dGp and dGp were subjected to the 32P-postlabeling assay (Figs. 2 and 3). Labeling efficiency by PNK was optimized for incubation time and ATP concentration at concentrations of 1 adduci/IO6 dGp (data not 0.2 0.4 0.6 OB 1.0 shown). The IAC was optimized to allow for maximal purification Â»PostlabelingAssay without adduci loss resulting from too low or too high antibody (fmol/n9) affinity. The limit of detection for the combined procedure was 1 adduci/IO7 dGp for 100 jig of DNA (7 fmol absolute value). The assay was calibrated using calf thymus DNA modified with [3H]BPDE, which accounted for total potential adduci loss from di Fig. 4. Autoradiograph detecting PAH-DNA adducts in a human lung sample (A ). This radiograph is from a 45-year-old African American female who had 5 PAH-DNA adducts/ gestion to detection (Fig. 3). The overall recovery of adducts was IO7dGp. B, radiograph of a sample where adducts were noi detected. Results of combined approximately 50%. HPLC analysis of DNA enzymatic digests indi micropreparative techniques and the 32P-postlabeling assay versus SFS were determined for 23 samples. Nine samples were positive by SFS while six were positive by 12P- cated a greater than 95% recovery of unadducted nucleotides. Human postlabeling assay (C). The results were highly correlated (Fisher's exact test; P = 0.006). lung DNA also was treated with [3H]BPDE to assess the effects of For the positive samples in either assay, as shown in the figure, the correlation coefficient complex adduci mixtures upon labeling and possible differences in was 0.67, P = 0.047). 3488

Table I Pol\cyciic aromatic hvdrocarhon-DNA adduci in hitman lunga DISCUSSION Sample1069112811321143106411771577158216061585"P-postlabeling''0.400.260.390.56ND'NDND0.741.140.37SFS*0.120.080.460.060.060.060.040.280.39NTThis study represents the first use of a PAH-specific 32P-postlabeling assay for human DNA analysis, corroborated by multiple detection methods, and finds that increased adduct levels in parenchymal lung tissue are associated with the null GSTju. genotype. There was no correlation with the presence of a mutation in CYP1A1 that affects PAH metabolic activation. The combined IAC and 32P-postlabeling assay relies upon micropreparative techniques that establish chemical class specificity. Au " Only positive samples are included. 32P-postlabeling assay and synchronous fluo thentic BPDE-dGp adduct standards were used to optimize all steps of rescence spectroscopy results are correlated (P = 0.047 for only the positive samples and the assay and to assess PNK enzymatic labeling efficiency. Internal P = 0.001 for all samples). standards (dGp) also were used to confirm that the labeling reaction ' ND, nondetected: NT. not tested (insufficient DNAI. was complete and allowed for a direct determination of molar ratios (adducts to unmodified dGp). Calibration curves and regression equa tions, comparing determined versus known radiolabeled adducts, as positive samples in either assay, as continuous variables (Fig. 4), was highly correlated (Spearman's ranked correlation; r = 0.67; P = certain overall adduct recovery from digestion to detection. Several features of this assay resolve some limitations of the classical 32P- 0.047). Categorization of all adduct analyses as either positive or postlabeling assay while introducing others. The introduction of IAC nondetected for the 32P-postlabeling assay and SFS also was highly associated (Fisher's exact test; P = 0.006). and HPLC results in lower sensitivity, additional labor requirements, and increased cost. Substantially more DNA is required because of the The DNA samples were analyzed for the CYP1 Al exon 7 mutation lower sensitivity. However, the specificity of the assay is significantly and GST/i genetic polymorphisms (Fig. 5). Smoking history was not enhanced due to the multiple Chromatographie steps (IAC and TLC). available but serum cotinine levels at the time of death were available The limit of detection for the assay is 1 adduct in IO7 dGp, which is for 30 cases. Levels ranged from nondetected to 1021 ng/ml. There suitable for some human lung samples but not all. were 31 males and 7 females. Twelve persons were African Americans PAH-DNA adducts were detected in lung samples from 7 of 38 and 26 were Caucasian Americans. The causes of death varied and persons by the 32P-postlabeling assay. The levels of PAH-DNA ad included coronary artery disease (n = 7), drowning (n = 1), electro ducts ranged from nondetected to 1.14 fmol/jng of DNA, which were cution (n = 1), gunshot wounds (n = 3), hanging (n = 2), motor highly correlated with SFS, and had been validated earlier by gas vehicle accident (n = 8), pneumonia (n = 1), sudden infant death syndrome (3), and trauma (1). Death data were not available for the remainder. Adduct levels by the 32P-postlabeling assay were categorized as present or nondetected. Wilcoxon rank sum scores did not reveal a relationship with serum cotinine levels (P = 0.38) or age (P = 0.61). The mean age was 36 Â±12.6 (SD) for positive samples and 41 Â±17.0 for nondetectable samples. Fisher's exact test failed to identify a statistically significant relationship for PAH-DNA adduct levels and gender (P = 1.0), race (P = 1.0), or serum cotinine, when categorized by high and low levels (median, 100; P = 0.86). The positive samples included 6 men and 1 woman while the nondetectable samples in cluded 25 men and 6 women. There were 4 African Americans and 3 Caucasians for the positives and 8 African Americans and 23 Cauca sians for the nondetectables. There were 20 persons with GSTpi and 18 who were null. When categorized by positive or non-detected PAH-DNA adduct levels by the 32P-postlabeling assay, a statistically significant relationship with GSTfi genotype was found (Fisher's Exact Test; P = 0.038), as shown in Table 2. Six of seven persons with positive PAH-DNA adduct levels were null GSTfi genotype. The mean PAH-DNA adduct level for persons who had a GST/o. null genotype was 0.20 fmoliYig while it was 0.038 fmol/^g for individuals who had GSTji present (Wilcoxon rank sum test; P = 0.023). The GST/j. genotype was not associated with any other variable (serum cotinine, age, race, sex, or CYP1A1 genotype). For the CYP1A1 genotypes, there were 32 homozygote wild types, 2 hÃ©tÃ©rozygotes,and4 homozygote mutants. When cate gorized by positive or nondetected, PAH-DNA adduct levels were not Fig. 5. The polymerase chain reaction also is used to amplify a 273-base pair fragment from the GST/Â¿gene (A}. Individuals with the null genotype cannot be amplified. The associated with the presence of a mutant CYP1A1 exon 7 alÃele (Fisher's exact test; P = 0.56). The results are presented in Table 2. detection of an amplified product indicating the presence of GST/i (Lanes 2 and 3) while the absence indicating the GSTji null genotype (Lanes 1 and 4). The polymerase chain A combined general linear regression model for PAH-DNA adducts reaction is used to amplify 195-base pair fragments flanking the locus for a mutation of exon 7 in CYP1A1 (ÃŸ).Lanes 1, 3, and 5 show PCR fragments prior to enzymatic and available variables was performed. Data were categorized for digestion while Lanes 2. 4, and 6 show fragments after Nco\ restriction enzyme digestion. serum cotinine content and log transformed for age. Only the null Wild type alÃelesare identified by the presence of a restriction site yielding a 32- and a GSTju. genotype was associated with PAH-DNA adduct levels (Table 163-base pair fragment. Lanes I and 2 demonstrate a homozygote wild-type individual, Lanes 3 and 4 demonstrate a hÃ©tÃ©rozygote,whileLanes 5 shown and 6 demonstrate a 3). homozygote mutant. 3489

Table 2 PolvcvcHc aromatic h\drocarbon-DNA adduci levels, GYPÃŒAIgenoi\pes and increased catalylic acliviiy.4 In a separate study of normal volunteers, glulafhione-S-transferase y genotypes the CYP1A1 Msp\ genolype, which is associated wilh ihe exon 7 mutalion, was related lo increased elhoxyresorufin-O-deethylase ac- PAH-DNAadduci"Present Null1 typeMutant6 tivily in peripheral lymphocytes.5 Other investigators have found lhal 6 026 AHH activity was associated wilh DNA-adducl levels measured by NondetectedGSV'Present 19 12CYP1A1'Wild 6 ihe classical 32P-posllabeling assay (40, 63). However, ihere was no " Detected by IAC/12P-postlabeling assay. Limit of detection is I adduct/107 dGp. relationship of PAH-DNA adduci levels lo ihe CYP1A1 exon 7 mu ''Fisher's exact test (2-tailed); P = 0.038. " Fisher's exact test (2-tailed); P - 0.562. tation. One explanation for Ihe conlrasl is lhat AHH activily and adduci levels mighi be independeni markers of tobacco use, confound Table 3 General linear regression model for PAH-DNA adduci levels" ing a relationship between ihe iwo. Adducls ideniified by the 32P- postlabeling assay used previously would detect numerous non-PAH- VariableCSVCYPIA1ConnineAgeGenderRaceF7.140.260.250.010.930.76/'0.010.770.620.920.390.39 DNA adducts resulting from tobacco use. AHH activity is a reflection of induction. Thus, while both would increase with tobacco exposure, they may not be directly related. Aliernalively, ihe increased calalylic aclivity of the CYP1A1 exon 7 mutalion mighl be less importanl lhan AHH induclion (2, 40, 64, 65) or olher delerminanls of PAHs-DNA " PAH-DNA adduci levels and age were log transformed for normalization. adduci formalion and persistence including epoxide hydroxylase, CYP3A4, and DNA repair. Serum connine was noi associated with PAH-DNA adduci levels chromatography and mass spectroscopy (20). Adduci levels in human tissues detected by the 12P-postlabeling assay without microprepara which might be due to the relatively short half-life of cotinine com tive techniques typically range from 0.1 to 5 in IO7 bases in human pared to DNA adducls or slrong effecls of hosl faclors lhal influence lung tissues (3, 4, 54, 55), similar to our findings. The results reported adduci levels following internal absorplion. Alternatively, cotinine herein also are consistent with PAH-DNA adduci levels reported by levels does not reflect tolal PAH exposure, such as mighl occur immunoassays in lung (28) and other tissues (5, 29, 56, 57) as is the through occupation, air pollulion, or diet. The work reported herein demonstrates thai PAH-DNA adducls can relative proportion of positive samples (approximately 20%) above 1 be measured in human lung tissue using modifications of the 32P- adduct/107 bases using other detection methods (3, 4, 55, 58). The 32P-postlabeling assay for the positive human samples revealed posllabeling assay lhal enhance chemical specificily. This approach a TLC elevation profile with radioactivity in only one area which exemplifies the use of such an assay for assessing the determinanls of coelutes with the BPDE-dGp standard. This finding would have been DNA adduci formation and persistence. The deleclion of DNA adducts predicted for an assay that is optimized for BPDE-dGp detection. reflects the biologically effeclive dose and provides an indicalion of While the antibodies used in 1AC have been shown to cross-react with ihe number of polenlial promutagenic lesions. However, DNA adduci levels reflect only short-term exposure. Alternatively, genotyping in some PAH-DNA adducts in immunoassays, their affinity in a com petitive assay (not IAC) for most PAH-DNA adducts other than BP dividuals for the ability to metabolically adivate and detoxify carcin ogens provides informalion about an individual's lifetime response lo DE-dGp are substantially lower.1 Affinity for aromatic amines is carcinogenic exposures. Correlalions of adduci levels, environmenlal essentially nonexistent and three of four PAHs had a lower affinity than BPDE-dGp (only benzo(fc)fluoranthene had a greater affinity). exposures, and genolypes are an importanl approach in determining Separately, using the procedure described here, we have previously genetic susceptibilitÃ©s to cancer. observed that other PAH adducts have up to 100-fold lower labeling efficiencies (47) so that these adducts would not be detected [benzo- ACKNOWLEDGMENTS (i(:)fluoranthene has a 100-fold lower labeling efficiency]. The combined IAC and 32P-posilabeling assay for PAH-DNA ad The authors would like to thank Dr. Curtis Harris for his support in these ongoing studies and his insightfulness in data analysis. We also would like to ducts allows for an assessment of how environmental exposures in thank Dorothea Dudek for her skillful editorial assistance. The help and co teract with genetic factors for carcinogen metabolism and detoxifica operation of the following Baltimore hospitals are greatly appreciated: Uni tion. Persons who were GST/u null had a greater likelihood of having versity of Maryland, Loch Raven Veterans Administration, Union Memorial, detectable PAH-DNA adducts. GST/x is polymorphic in human pop St. Agnes, Sinai, Harbor Center. Mercy Medical Center, and Northwest Med ulations (36), is present in lung tissue (59-61), and contributes to the ical Center. detoxification of reactive benzo(a)pyrene metabolites (60, 62). 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