Int. J . Cancer: 48, 485-492 (1991) 0 1991 Wiley-Liss, Inc.

Publication of t h e International Union Aaainst Cancer Publication de I‘Union lnternationale Contre 1e Cancer

DNA ADDUCTS IN BRONCHIAL BIOPSIES Bruce P. DuNN’,~, Sverre VEDAL’,Richard H.C. S A N ’ . Wan-Fung ~, KWAN’,Bill NELEMS~, Donald A. EN ARSON^ and Hans F. STICH’ ‘British Columbia Cancer Research Centre, Vancouver, BC; 2Department of Medicine and 3Department of Surgery, Vancouver General Hospital, University of British Columbia, Vancouver, BC; and 4Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. To investigate the feasibility of measuring DNA-carcinogen adducts in the lungs of non-surgical patients, endobronchial biopsies were obtained from 78 patients undergoing routine diagnostic bronchoscopy. Lung cancer was present in 37 (47%) of the patients. D N A was isolated from the tissues and analyzed by HPLC- or nuclease-Pl-enriched 32P-postlabelling, using procedures selective for aromatic adducts. Chromatograms from all 28 current smokers showed a distinctive diagonal adduct zone which was present in only 24 of 40 exsmokers and 4 of 10 lifetime non-smokers. Adduct levels and chromatographic patterns were similar in bronchial tissue from different lobes of the lung, in bronchial and alveolar tissue, and in tumor and non-tumor bronchial tissue taken from the same subject. Bronchial D N A adduct levels were strongly associated with cigarette smoking status and dropped rapidly after smoking ceased. Higher levels of D N A adducts seen in the lung-cancer patients were mainly due to cigarette smoking. Frequent alcohol intake was the only dietary factor associated with higher levels of bronchial D N A adducts. We conclude that the level of bronchial D N A adducts is strongly associated with cigarette-smokinghistory and with alcohol intake, but is not associated with lung cancer independently from its relation to smoking. The results indicate the feasibility of using 32P-postlabellingto detect and quantitate genetic damage in bronchial biopsy specimens.

1988). Previous studies of adducts in smokers have utilized either adduct intensification (Everson et al., 1986, 1988; Randerath et al., 1986) or nuclease-PI enrichment (Cuzick et al., 1990; Phillips et al., 1988; Randerath et al., 1989). In our study, we used both the nuclease-P1 procedure and the HPLC procedure in parallel and compared the results of the two assays. MATERIAL AND METHODS

Tissue sampling Patients undergoing diagnostic bronchoscopy at the Vancouver General Hospital gave informed consent to allow endobronchial biopsies to be obtained from normal-appearing bronchi. An interviewer-administered questionnaire was completed by these patients to gather information on cigarettesmoking habit, diet, occupational history, and medication use. Questions on diet were: How often do you eat fresh or frozen vegetables? How often do you have fresh fruit or fruit juice? Are you taking any regular vitamin supplement? In the last 12 months, how often have you taken at least one drink of liquor, one bottle of beer, or one glass of wine? Possible responses ranged from “ 2 or more times a day” to “not at all in the last 12 months”. In addition to the biopsies that might have been The measurement of co-valent DNA and protein adducts of required for diagnostic purposes, 3 to 5 endobronchial biopsies carcinogens in human subjects shows considerable promise in were obtained through a flexible fiberoptic bronchoscope from investigating the relationship between exposure to environmen- sites which appeared grossly normal. Biopsies were approxital carcinogens and cancer risk (Harris et al., 1987; Perera, mately 1 to 2 mm in diameter and consisted mainly of mucosal 1987, 1988). A number of new techniques have been devel- and a small amount of submucosal tissue. In some cases, reoped for detecting such adducts (Santella, 1988). 32P-post- sected lung tissue was obtained after surgery for primary lung labelling of DNA-carcinogen adducts has been shown to be cancer. particularly sensitive for measurements of low levels of aro- 32P-postlabelling matic DNA-carcinogen adducts in cells in culture or animals DNA was extracted from tissues, and 2.5-p,g samples of exposed in the laboratory to carcinogens (Watson, 1987). The application of this procedure to individuals who smoke may DNA were assayed for aromatic adducts as previously deprove of benefit in understanding the nature of the initiation scribed (Dunn and San, 1988), using nuclease-P1 digestion to enrich adducts prior to post-labelling analysis (Reddy and Ranprocess in tobacco carcinogenesis. 1986). Most of the samples were analyzed in parallel Several recent studies have detected smoking-related aro- derath, an alternate version of the post-labelling assay which uses matic DNA-carcinogen adducts in human surgical specimens by HPLC on reverse-phase columns to selectively enrich hydroor autopsy material (Cuzick et al., 1990; Everson et a l . , 1986, phobic adducts before post-labelling (Dunn and San, 1988). 1988; Randerath et al., 1986, 1989; Phillips et al., 1988; Wil- The radioactivity in zones and spots on chromatograms was son et a l . , 1989). If adduct measurements were proven to have quantitated by scintillation counting. Radioactivity in backpredictive value in assessing the risk of future lung cancer, then ground spots (Fig. 1 and associated discussion) were excluded measurements on living persons would be highly desirable. For from this analysis. These values were to moles of this reason, the current study explores the possibility of per- adduct using the specific activity of theconverted 32P-ATP used in the forming DNA-adduct measurements on very small amounts of biopsy tissue obtained during diagnostic bronchoscopy of labelling reaction. For both the nuclease-P1 and HPLC versmokers, ex-smokers and non-smokers. Recent refinements of 32P-post-labellingprocedures have increased the sensitivity of the procedures, either by “adduct 5Present address: Microbiological Associates Inc., 9900 Blackwell intensification” (Randerath et al., 1985), which involves se- Road, Rockville, MD 20850, USA. lective labelling of adducts under ATP-deficient conditions, or by selectively enriching carcinogen-DNA adducts in DNA di6To whom corresoondence and reorint reauests should be addressed. at gests before the post-labelling assay. Enrichment has been B.C. Cancer Reseakh Centre, 601 ‘West 16th Avenue, Vancouver, BC, done by selective extraction of hydrophobic adducts into bu- V5Z 1L3 Canada. tanol (Gupta, 1985), by selective destruction of normal nucleotides by nuclease P1 (Reddy and Randerath, 1986), and by Received: September 26, 1990 and in revised form January 31, 1991. high-pressure liquid chromatography (HPLC) (Dunn and San,

486

D U N N ET AL.

were directly calculated for each assay by dividing adduct amounts by total nucleotide amounts. To avoid any possible bias in analyses should the assay procedures change from week to week, samples were assayed in groups randomly composed of smokers, ex-smokers and non-smokers, in the order in which the samples were obtained. Chromatograms in this report are oriented with the origin to the lower left. Solvent D3 was run from bottom to top, while D4 was run from left to right. Autoradiography of human samples was carried out for 12 to 24 hr at -70°C. For comparison purposes, postlabelling assays were performed with calf thymus DNA which had been reacted in vitro with ( + )-anti-benzo(a)pyrene diol epoxide to give an approximate modification level of 200 nmoles/mole. This material was a gift of Dr. A. Weston, and was prepared in a manner similar to that previously reported (Manchester et a l . , 1988). In this case, autoradiography was performed for 6 hr at room temperature. Statistical analysis A patient was defined as a current cigarette smoker if at least one cigarette on average was smoked daily and smoking occurred in the preceding month; an ex-smoker if no cigarettes were smoked in the preceding month but at least one cigarette on average per day had been smoked for at least one year; and as a non-smoker otherwise. The frequency distribution of bronchial DNA adducts was skewed with a tail at the higher values. A natural logarithmic transformation of DNA adducts produced a near-normal distribution. The log-transformed data were therefore used in the statistical analyses comparing group means. However, for the linear regression analyses, the untransformed data were used, since analysis of residuals from the regression models using the untransformed data showed that the variance of the residuals was more uniform (homoscedastic) than when the logtransformed data were used (Kleinbaum and Kupper, 1978). Comparisons of adduct level among groups were performed using Student’s t-test and analysis of variance (ANOVA). Two-way ANOVA was used to compare groups crossclassified by another factor. Simple linear regression was used to analyze the association between adduct level and continuously distributed variables. Multiple linear regression techniques were used to analyze the associations between adduct level (the dependent variable) and an independent variable while controlling for the potential influence of other variables. Indicator variables were included in the regression models to evaluate the influence of discrete variables. For example, for alcohol intake the indicator variable was 1 if one or more drinks were taken daily, and 0 otherwise (Kleinbaum and Kupper, 1978). FIGURE1 - Nuclease-P 1 - and HPLC-enhanced 32P-post-labelling analysis of human bronchial biopsies. DNA was analyzed as described in “Material and Methods”, using either nuclease-P1 or HPLC enrichment of adducts before post-labelling. Arrows on the first 4 panels indicate the presence of assay background spots. Numbers refer to individual patients, with their smoking status indicated by CS for current smoker, ES for ex-smoker and NS for lifetime non-smokers. Patient characteristics are: # 12 CS: 59-year-old male, smoking for 29 years, currently smoking 60 cigarettedday; #15 CS: 66-year-old female, smoking for 50 years, currently smoking 20 cigarettedday; 6 3 4 ES: 58-year-old male, smoked 20 cigarettedday for 40 years, quit approximately 1 month; #60 ES: 66-year-old female, smoked 20 cigarettes/day for 40 years, quit 8 years; #09 NS: 67-year-old male, lifetime non-smoker; #3 1 NS: 77-year-old male, lifetime nonsmoker.

sions of the assay, the amount of DNA nucleotides associated with each chromatogram was independently determined by HPLC with UV absorption detection (Dunn and San, 1988). Finally, the adduct levels present in individual DNA samples

RESULTS

Nuclease-PI - and HPLC-enhanced 32P-post-labelling Bronchial biopsies were obtained from 90 patients undergoing routine bronchoscopy . DNA yields (estimated by ultraviolet spectrophotometry) were less than 5 p,g for 20% of samples, 5-10 p,g for 51% of samples, 10-15 pg for 22% of samples, and more than 15 pg for 7% of samples. Postlabelling analysis was attempted on at least one sample from 83 of the 90 patients, the main reason for excluding patients being insufficient DNA. After assaying, a further 5 patients were excluded from the analysis due to inadequate DNA (quantitated during the assay). Descriptive data on the 78 patients are presented in Table I. Of these, all had adduct levels measured by nuclease-P1-enhanced 32P-post-labelling, and 69 were in addition measured by HPLC-enhanced post-labelling . Figure 1 shows typical chromatographic patterns for nuclease-P1- and HPLC-enhanced post-labelling analysis of

BRONCHIAL D N A ADDUCTS

487

TABLE I - DESCRIFTIVE DATA ON 78 PATIENTS WHO HAD DNA ADDUCTS MEASURED IN BRONCHIAL BIOPSIES Nonsmokers

Number Male/female Number with lung cancer (%) Age (mean SD) Years since quitting smoking (mean 2 SD) Cigarettes per day (mean SD) Pack-years (mean SD)

*

10

Exsmokers

40

515 0 (0%)

25/15 22 (55%)

63

65

f

13.0

* *

Current smokers

28 15/13 15 (54%)

* 12.9 * 16.6

63

15

* 9.1

22

f

17.7

26

* 12.4

43

2

42.4

56

5

23.3

DNA derived from bronchial biopsies from 2 current smokers (CS), 2 ex-smokers (ES), and 2 lifetime non-smokers (NS). Two background spots appearing in chromatograms of samples analyzed by HPLC enhancement and one background spot appearing in samples analyzed by the nuclease-P1 procedure are indicated with arrows in the first 4 panels of the figure. These spots consistently appear in DNA from a variety of sources, including freshly synthesized DNA from mammalian cells in culture. The precursors of these spots are very hydrophobic, chromatographing on a reverse-phase column later than the principal benzo(a)pyrene-DNA adduct (Dunn and San, 1988). While the origin of these background spots is not known, they appear to be artifacts unrelated to the origin of the DNA being analyzed. Gupta (1989) has shown that similar spots can be spontaneously generated by room-temperature incubation of DNA or 3’ nucleotides in aqueous solution in plastic tubes. Many chromatograms exhibited a strong diagonal radioactive zone running from near the origin at the lower left of the chromatograms towards the upper right. In most cases, the diagonal zone appeared as a smooth smear without internal structure, while in occasional cases the zone contained discrete spots. In addition to the diagonal radioactive zone, a number of samples exhibited one to many discrete spots elsewhere on the chromatograms. In some samples, discrete spots were combined with a diffuse diagonal radioactive zone. For simplicity, radioactive spots and zones other than the background spots noted above will be referred to in this report as adducts, although definitive proof of their chemical nature is lacking. In general, the pattern of adducts seen by nuclease-P1 enhancement and by HPLC enrichment of adducts prior to postlabelling was very similar (Fig. 1 and bottom 4 panels of Fig. 3). Adduct levels as measured by the two procedures were strongly correlated (r = 0.795; n = 69), with levels determined by nuclease-P1 enhancement averaging approximately 25% higher.

FIGURE2 - Post-labelling analysis of multiple bronchial biopsies from a single patient. Bronchial biopsies from different lung lobes were obtained from a 76-year-old female, currently smoking 20 cigaretteslday, and were analyzed by nuclease-P1 enrichment of adducts prior to post-labelling. For comparison, the panel labelled BPDE shows the adduct resulting from nuclease-P1-enhanced post-labelling analysis of calf thymus DNA reacted in vitro with (+)-antibenzo(a)pyrene diol epoxide. Letters indicate lobe; values shown are adduct levels in nmole/mole. LU = left upper, 67; LL = left lower, 73; RU = right upper, 78; RM = right medial, 65; RL = right lower, 92.

HPLC versions of the assay. Chromatographic patterns (Fig. 3 ) and adduct levels (Table 11) were very similar in bronchial and alveolar tissue, and in samples analyzed by the nuclease-P1and the HPLC-enrichment variations of the post-labelling assay. The most notable discrepancy between the two procedures was with patient M3, who had a very unusual pattern, consisting largely of discrete spots. In this case, certain adducts in the upper part of the chromatogram were present only in samples processed by the HPLC-enrichment version of the assay. From 4 ex-smokers and one smoker, biopsies of both tumor tissue and normal tissue were available, and were analyzed using the nuclease-P1 procedure (Fig. 4). The chromatographic pattern in tumor and normal tissue was generally similar, although in 2 of the 5 cases (patients 97 and 108, both exsmokers) adduct levels were substantially higher in tumors. Adducts and smoking behavior

Adducts in tissues from digerent sites

For one smoking patient, endobronchial biopsies were available from 5 different sites in the lung, and were analyzed by the nuclease-P1 version of the assay. Figure 2 shows that the overall adduct pattern was very similar in all samples, as were quantitative measurements of diagonal zone adduct levels (Fig. 2). For comparative purposes, the bottom right-hand panel of Figure 2 shows the adduct pattern resulting from post-labelling analysis of DNA reacted in vitro with benzo(a)pyrene diol epoxide. For this standard, the background spot seen in other samples is not visible due to the limited autoradiography exposure. For one ex-smoker and 2 current smokers, resected lung tissue was available after surgery for lung cancer. For individual lobes of the lung, both bronchial and alveolar tissue were analyzed by post-labelling , using both the nuclease-P1 and

Adduct levels in diagonal radioactive zones (or in corresponding areas of the chromatograms where the zones were not visible) were quantitated by scintillation counting. Quantitation included any discrete spots present in the area of the diagonal zone, but excluded assay-related background spots. Adduct levels from duplicate analysis on the same sample (if any) and from multiple lung samples taken from an individual during a single bronchoscopy were averaged. Because a number of samples were analyzed only by the nuclease-P1 procedure, only adduct levels from this procedure were employed in these comparisons. Figure 5 shows adduct levels and chromatographic patterns in lung biopsy DNA samples from non-smokers, ex-smokers and smokers. The diagonal zone was present in all 28 current smokers, 24 of 40 ex-smokers, and only 4 out of 10 lifetime non-smokers. The ex-smoker group included patients who had

488

DUNN ET A1 TABLE 11 - ADDUCTS IN RESECTED LUNG TISSUE Turd1 adductc

Patient ~

~

Lobe'

Tissue

-~

MI M1 MI M1 M1 M1 MI MI M2 M2 M2 M2 M2 M2 M33 M33

(nmole/mule) Nuclease PI

Anterior LUL Anterior LUL Apical-post. LUL Apical-post. LUL Superior LIN Superior LIN Inferior LIN Inferior LIN Apical RUL Apical RUL Anterior RUL

Anterior RUL Posterior RUL Posterior RUL Anterior LUL Apical-post. LUL

bronchial alveolar bronchial alveolar bronchial alveolar bronchial alveolar bronchial alveolar bronchial alveolar bronchial alveolar bronchial alveolar

36 40 292

p

51 28 24 27 1002 92* 28 87 152 35

HPL C

59 47 41 39 51 44

17

40 117 101 47 71 103

6

z2 m2 2502 m2

'LUL, left upper lobe; RUL, nght upper lobe; LIN, lingula.-2Underlined values indicate that chromatograms correspinding to these samples are reproduced in Figure 4.-3Bronchial tissue from apical-posterior LUL and alveolar tissue from anterior LUL not available for comparison.-Patient M1: male ex-smoker, age 64.formerly smoking 90 cigarettesiday, quit 7 years ago; patient M2: male smoker, age 63, 20 cigarettesiday; patient M3. male smuker. age 7 7 , 30 cigarettesiday

smoking more than 4 ycars previously had a geometric mean level of DNA adducts comparable to the non-smokers. An intermediate level of DNA adducts was present in the exsmokers who stopped smoking between 1 and 4 years previously. The association between years since quitting smoking and level of DNA adducts was statistically significant both FIGURE 3 - Post-labelling analysis of bronchial and alveolar tissue when analyzed by ANOVA, comparing the 3 ex-smoking from individual patients. Samples were analyzed by either the nu- groups 0) = 0.01), and when analyzed by simple linear reclease-P1 (first three assay pairs) or HPLC version of the post- gression, expressing years since quitting as a continuous varilabelling assay. See Table I1 for further information on samples and able (p = 0.05). adduct levels. Patient M1: male ex-smoker, age 64, formerly smoking Of the 5 patients who had quit smoking between one and 12 90 cigarettedday, quit 7 years ago; patient M2: male smoker, age 63, 20 cigarettedday; patient M3: male smoker, age 77, 30 cigarettedday. months before bronchoscopy, 2 had a level of DNA adducts outside the range seen in the 10 non-smoking patients (Fig. 5 ) . The patients with levcls of 194 and 131 nmoles of adducts per ceased cigarette smoking from a few wceks to 50 years before mole of nucleotidc had stopped smoking one month and 8 the biopsy. In this group, there appeared to be a tendency for months before the bronchoscopy, respectively. One of the 10 higher adduct levels and a greater prevalence of chromato- patients who had quit smoking between one and 4 years before grams with diagonal adduct zones in those who had recently the bronchoscopy had a level outside the non-smoking range, ceased smoking. Of the three populations, current smokers had and 2 of the 25 who had quit 5 or more years before were the highest adduct levels and the highest proportion of chro- outside the range. Twelve of the 28 currently smoking patients matograms exhibiting a diagonal zone. had levels outside the range seen in the non-smokers. In the currently smoking patients there appeared to be an Predictors of bronchial DNA adduct levels association between the intensity of cigarette smoking, in Among the 78 patients, lung cancer was present in 37 packs per day (ppd), and the level of DNA adducts, with those (47%), of whom 22 (59%) had stopped smoking cigarettes at smoking more intensely having the higher adduct levels (Table least one month before the bronchoscopy was performed and 111). However, this association was not statistically significant 15 (41%) were still smoking (Table 111). There were no lifelong (p = 0.22 by ANOVA, comparing groups of less than 1 ppd, non-smokers in the group of patients with lung cancer. In the 1 to 1.5 ppd, and greater than 1.5 ppd; p = 0.07 by linear patients without lung cancer, 10 (24%) were lifelong non- regression, expressing ppd as a continuous variable). There smokers, 18 (44%) were former smokers, and 13 (32%) were was no association between smoking intensity and DNA adstill smoking cigarettes. Thirty-three (42%) were women and ducts in the former cigarette smokers (data not shown). the mean age was 64.1 2 10.3 years. Neither sex distribution Lung-cancer patients had a higher geometric mean level of nor age varied significantly by smoking category. DNA adducts (p = 0.04) than patients without lung cancer Cigarette-smoking status was strongly associated with level (Table 111). Even when stratified by cigarette-smoking categoof DNA adducts (Table 111, Fig. 5 ) ; current smokers had the ries, lung-cancer patients uniformly had higher levels of DNA highest level, former smokers an intermediate level, and life- adducts, except in the most heavily smoking current smokers long non-smokers the lowest level (p < 0.001 by ANOVA). (Table 111). However, in none of the cigarette-smoking strata Among ex-smokers, there was an association between the was the difference in DNA adducts between cancer and nonnumber of years since smoking stopped and level of DNA cancer patients statistically significant. Similarly, there was no adducts (Table 111, Fig. 5 ) . Those ex-smokers who stopped statistically significant difference between cancer and non-

489

Pu'"'ICHIAL DNA ADDUCTS

.

0

SMOKER - M-SMOKW (YEARSWTI SMOKER FIGURE5 - Smoking habits vs. bronchial DNA adduct levels: open circles, no clear diagonal adduct zone apparent on chromatograms; partly closed circles, weak or inconclusive diagonal zone present; closed circles, clear diagonal zone present.

FIGURE4 - Post-labelling analysis of tumor and non-tumor tissue from bronchial biopsies of individual patients. Samples were analyzed by the nuclease-PI version of the assay. Patient 97: female, age 61, ex-smoker (20/day),quit 13 years; patient 100: male, age 57, smoker (20/day); patient 104: male, age 82, ex-smoker (Wday), quit 15 years; patient 108: male, age 67, ex-smoker (lO/day), quit 15 years; patient 115: female, age 52, ex-smoker (30/day),quit less than 1 year.

cancer patients after accounting for the differences in cigarette smoking using a two-way ANOVA (p = 0.10). The difference between former smokers and current smokers, however, remained highly significant (p < 0.001) after accounting for the differences due to lung cancer. The 40 lung-cancer cases comprised 12 with squamous-cell carcinoma, 11 with adenocarcinoma, 6 with oat-cell carcinoma and 8 with other cell types of cancer. Adduct levels did not vary according to cell type, even after adjustment for cigarette smoking. Likewise, DNA adduct levels were not different in men and women (data not shown). Level of DNA adducts tended to be higher in patients drinking alcohol at least daily compared with those drinking alcohol less frequently (p = 0.09, Table IV). Patients who reported having fresh fruit or fruit juice less than twice weekly also had a higher geometric mean level of DNA adducts than those having fruit or juice more frequently (p < 0.01, Table IV). There was no association between reported vegetable intake or supplemental vitamin use and levels of DNA adducts.

Multiple linear regression models, regressing level of DNA adducts on combinations of independent variables, were generated in order to identify the variables that were independently associated with adduct level (Table V). As compared with non-smokers and former smokers who had stopped smoking more than 4 years previously, cigarette smokers in all categories had higher adduct levels. After accounting for the associations with cigarette smoking, there was no significant association between lung cancer and DNA adduct level. The association of adduct level with fruit or fruit-juice intake was weakened after accounting for associations with cigarette smoking, but the association with high alcohol intake persisted. This model accounted for 47% of the variability in adduct levels (rz = 0.47). The cigarette-smoking associations were largely unaffected after accounting for the associations with diet, except that the level of adducts in former smokers who had stopped smoking between one and 4 years previously was no longer significantly elevated. DISCUSSION

Using adduct intensification procedures, Everson et al. (1986, 1988) and Randerath et al. (1986) reported the presence of cigarette-smoking-related adducts in human placental DNA, and in DNA from bronchi and aryepiglottic fold. In these studies, both discrete-adduct spots and diagonal-adduct zones were seen. Using the generally more sensitive nuclease-PI procedure, Phillips et al. (1988) and Randerath et al. (1989) found a strong smoking-associated diagonal zone in human lung DNA. The zone was accompanied by discrete spots which were generally less prominent than when samples were analyzed by the intensification procedure. In a series of 17 smokers, Phillips et al. (1988) found a linear relationship between lung-adduct levels and smoking levels, while Randerath et al. (1989) showed the presence of smoking-related adducts not only in lung and bronchus, but also in heart, liver, kidney, aorta, bladder and esophagus. In the latter study, changes in chromatography procedures also revealed the presence of a second fainter diagonal zone migrating more rapidly in the f i s t chromatography direction than the main adduct zone. Cuzick et al. (1990), using nuclease-P1-enhanced post-labelling procedures, found diagonal-zone smoking-related adducts in lung, bladder and pancreatic tissue, but not in breast tissue.

490

DUNN ET AL.

-

TABLE 111 GEOMETRIC MEAN LEVELS OF AROMATIC BRONCHIAL DNA ADDUCTS (NMOLESIMOLE NUCLEOTIDE) CLASSIFIED BY LUNG CANCER AND CIGARETTE SMOKING Years since

stopping smoking

Average packs p"r day

__

Non-smokers Former smokers

58 (1%194) 52 (21-263) 24 (5-108) 36 (5-263) 92 (74-114) 82 (26-185) 98 (40-232) 88 (26-232) 52 (5-263)

0-1 1 4 >4 Total

Current smokers

N o cancer

Cancer

Level (range)

1.5

Total Total

n

0 5 6 11 22 2 9 4 15 37

Level (range)

25 (4-88)

-_

42 (16-68) 18 (4-129) 22 (4-129) 49 (32-63) 69 (32-128) 124 (117-132) 73 (32-132) 33 (4-132)

Total n

Level (range)

n

10 0 4 14 18 3 7 3 13 41

25 (4-88) 58 (19-194) 47 (16-263) 20 (4129) 29 (4-263) 63 (32-114) 76 (26-185) 109 (49-232) 81 (26-232) 41 (4263)

10 5 10 25 40 5 16 7 28 78

TABLE IV - GEOMETRIC MEAN LEVELS OF BRONCHIAL AROMATIC-DNA ADDUCTS (NANOMOLESIMOLE NUCLEOTIDE) CLASSIFIED BY DIETARY FACTORS Dietary item

Frequency

Level (range)

Fresh fruit or fruit juice

Daily At least twice weekly Less than twice weekly At least once daily Less than once daily Daily At least twice weekly Less than twice weekly Some None

35 (4-194) 30 (7-185) 91 (32-263) 54 (7-194) 35 (4-232) 42 (4-263) 44 (7-185) 33 (5-194) 42 (11-185) 40 (4-263)

Alcohol2 Vegetables Vitamin supplements

p value'

0.004

0.09 0.68

0.87

n

56 9 13 21 56 54 11 13 20 58

'Statistical significance of test for group differences in natural logarithm of adduct levels.-*l patient had missing data on alcohol intake.

v - ESTIMATED INCREASES' IN LEVEL OF AROMATIC BRONCHIAL DNA ADDUCTS ASSOCIATED WITH CIGARETTE SMOKING, DIET AND

TABLE

LIJNG CANCER

Adduct level

Independent variables

Current cigarette smoker

(n

value\

Packstday 30.0 (0.11) 5 1-1.5 51.2 (1.5 88.6 (

DNA adducts in bronchial biopsies.

To investigate the feasibility of measuring DNA-carcinogen adducts in the lungs of non-surgical patients, endobronchial biopsies were obtained from 78...
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