Carcinogeoesis vol.13 no.6 pp.987-993, 1992
Polycyclic aromatic hydrocarbon—DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung
F.J.van Schooten1, M.J.X.Hillebrand, F.E.van Leeuwen2, N.van Zandwijk3, H.M.Jansen4, L.den Engelse and E.Kriek5 Division of Molecular Carcinogenesis, 2Division of Psychosocial Research and Epidemiology and 'Division of Clinical Oncology, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), 121 Plesmanlaan, 1066 CX Amsterdam and 4Department of Pulmonology, University Hospital of Amsterdam, Academic Medical Center, 15 Meibergdreef, 1105 AZ Amsterdam, The Netherlands 'Present address: Department of Health Risk Analysis, University of Limburg, Becldsnijdersdreef 101, 6200 MD Maastricht, The Netherlands 'To whom correspondence should be addressed
Smokers of cigarettes are exposed to a number of carcinogens, including polycylic aromatic hydrocarbons (PAHs), and are at a high risk for lung cancer. PAHs exert their carcinogenic activity after metabolic activation to reactive intermediates that can damage DNA through adduct formation. Measuring DNA adducts in peripheral white blood cells (WBC) could serve as a means of monitoring human exposure to genotoxic agents and subsequently risk assessment. In this study, DNA from WBC obtained from 39 lung cancer patients was examined for PAH-DNA adducts both in an ELISA using a polyclonal antibody against benzo[a]pyrene 7,8-diol-9,10epoxide (BPDE)-DNA and the ^P-post-labeling technique. The ELISA results showed BPDE-DNA antigenidty in WBC DNA from 12/38 (32%) patients and adduct levels ranged from 1.5 to > 150 adducts in 108 nucleotides. The autoradiographs of chromatograms of ^P-post-labeled digests of WBC DNA from the 38 patients showed a variety of adduct spots; relative adduct labeling (RAL) values ranged from 0.3 to 407 adducts in 108 nucleotides. In 18 of the 38 (47%) persons an adduct spot was detected that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG); RAL values ranged from 0.03 to 382 adducts in lO8 nucleotides. Correlations were not significant between the adduct levels in WBC and smoking habits, age or sex. From 20 patients of the same group lung tissue was collected at surgery and examined for PAH-DNA adducts by ELISA and %-post-labeling assay. No significant correlation was found between DNA adduct levels in blood and lung. This finding stresses the limitations of the use of WBC as a surrogate for adduct levels in the target organ.
Introduction Tobacco smoking is the most important and well-documented cause of lung cancer currently known (1). It has been well established that the risk of lung cancer increases with the number •Abbreviations: PAH, polycyclic aromatic hydrocarbon; B[a]P, benzo(a)pyTene; WBC, white blood cells; BPDE, (±)/ra/is-7,8-dihydroxy-150
ND ND 382.3
2.0 ± 0.7 3.9 ± 1.3 407.4 ± 19.7
34.3 ± 10.9 8.2 ± 2.7 21.1 ± 10.8
10.8 ± 2.7 >150 4.8 ± 0.9 >150 ND >150 ND ND ND 1.5 ± 0.2 ND ND ND 1.5 ± 0.2 ND ND ND ND ND >150 ND ND ND ND ND ND ND 150 ± 50.7 ND >150 ND >150 ND >150 ND 1.5 ± 0.3 ND ND ND ND ND
1.2 ± 79.8 ± 3.2 ± 57.8 ± ND 123.4 ± 0.2 ± ND ND 0.15 ± ND ND ND 0.6 ± ND 0.1 0.3 ± ND ND 40.2 ± ND ND ND ND ND ND ND 9.8 ± ND 45.9 ± 0.2 ± 70.6 ± 0.03 ± 127.8 ± 0.15 ± 0.33 ± ND ND ND ND ND
ELISAC
Last year"
AC ACr ACh
51 68 59
M M F
0 0 10
* 5 10
no no no
AC
42
M
0
23
yes
SC1
58
M
18
18
no
SC AC SC SC AC LC
75 53 54 69 56 43
M F M M M M
10 0 0 1 75 18
10 4 0 41 55
yes yes no yes no no
LC SC SC SC SC * SC SC SC SC AC SC *
73 72 64 77 68 53 69 73 54 78 83 49 58
M M M M M M M M F M M M F
0 0 20 0 8 0 0 0 0 * 0 10 0
*
LC * SC AC Sc Sc SC SC * * LC * Sc *
79 40 54 57 71 65 71 62 32 54 39 57 63 63
M M M F M M M M M F M M M M
0 * 0 0 0 0 0 7 * 11 0 0 0 0
•
0 20 0 8 25 0 0 13 * 0 25 25 0 •
26 0 0 0 21 4 * 11 0 0 8 8
yes yes no no no no no no yes * no no yes no * no no no no no no * yes no * no no
32
± 4.2 0.1 9.2 1.3 9.9 34 0.1
0.1
0.2
0.1
9.3
2.0 23 0.06 2.2 0.03 52 0.03 0.03
6.2 89.1 16.6 74.7 3.2 134.8 2.7 1.7 1.3 0.2 38.2 65.7 1.4 0.6 3.5 4.5 0.3 2.2 1.7 52.3 1.9 0.6 12.3 0.3 2.2 6.7 6.2 12.4 1.3 48.1 3.8 5.3 1.1 135.0 2.4 3.6 0.8 1.6 1.4 1.6 1.0
± 0.5 ± 7.7 ± 7.5 ± 11.7 ± 0.8 ± 36.6 ± 0.1 ± 0.3 ± 0.6 ± 0.03 ± 5.6 ± 11.7 ± 0.9 ± 0.2 ± 1.6 ± 1.5 ± 0.03 ± 1.0 ± 0.2 ± 9.6 ± 0.5 ± 0.2 ± 3.2 ± 0.1 ± 1.0 ± 2.0 ± 0.5 ± 4.5 ± 0.3 ± 25.5 ± 1.35 ± 1.5 ± 0.5 ± 55.9 ± 0.7 ± 0.8 ± 0.1 ± 0.9 0.6 ± 0.2 ± 0.2
2.3 ±
0.7
19.9 ±
4.7
6.7 5.7 19.6 4.3 11.0 20.0
± ± ± ± ± ±
0.7 1.9 5.4 1.9 0.1 1.5
4.9 3.7 11.9 20.7 5.2 12.8 1.9 2.5 9.3
± ± ± ± ± ± ± ± ±
0.2 0.4 4.2 3.1 0.8 4.1 0.7 0.4 2.4
AC, adenocarcinoma. SC, squamous cell carcinoma. LC, large cell carcinoma; *unknown. ND, non detectable. "Average number smoked during last month. b Average number smoked during last year. c Expressed as equivalents of BPDE-DNA adducts/108 nucleotides, mean ± SD, n = 3. d Expressed as adducts/108 nucleotides calculated from RAL values, mean ± SD, n = 2 - 3 . c Data from Van Schooten el al. (28). f Primary rectum carcinoma. 'Sampled 1 week before surgery. h Primary mammacarcinoma. 'Sampled on the day of surgery. JSampled 3 weeks before surgery. 'Sampled 2 days after surgery. •"Sampled 1 day before surgery. "Sampled 2 weeks after surgery. "Sampled 12 days after sample 1.
described elsewhere (28) and will be summarized here only. The ELISA results showed BPDE-DNA antigenicity in lung DNA from 6/20 (30%) patients and adduct levels ranged beween 2 and
134 adducts in 108 nucleotides. For all 20 patients the autoradiographs of chromatograms of 32P-post-labeling digests of DNA from non-tumorous lung tissue showed a strong diagonal 989
FJ.van Schooten et at.
Fig. 1. Representative autoradiograms of chromatograms of ^P-post-labeled DNA digests from WBC from lung cancer patients: (a) patient no. 5; (b) patient no. 5, sampled 1 week later; (c) patient no. 4; (d) patient no. 4, sampled 3 weeks and 2 days later; (e) patient no. 3; (f) patient no. 3, sampled 1 week later; (g) patient no. 11; (h) patient no. 11, sampled 2 weeks and 1 day later. Spot 1 co-chromatographcd with the major BPDE-DNA adduct (BPDE-dG). The origin is located at the bottom left-hand corner of each chromatogram and excised before autoradiography. The autoradiography was carried out at -80°C for 18 h.
radioactive zone (DRZ). The level of DRZ adducts ranged from 1.9 to 34 adducts in 108 nucleotides. This DRZ was generally absent in tumorous tissue. DNA samples that were positive in the ELJSA contained a dominant spot within the DRZ that cochromatographed with BPDE-dG (see also Figure 2). The quantities of the BPDE—dG spots ranged from 0.2 to 4 adducts in 108 nucleotides. Adduct levels obtained by 32P-post-labeling were 5 — 10 times lower than, but correlated well with, those found in the ELJSA (Kendall W = 1; P < 0.01). Correlation between blood and lung adduct levels The relation between adduct levels in lung and WBC from the 20 patients examined is illustrated in Figure 3. Although a positive correlation was found for eight current smokers (r = 0.515, P = 0.19) and a negative correlation for 12 ex-smokers (r = —0.388, P = 0.21), these correlations were not significant. Possible reasons may be the small number of persons examined and the use of WBC for adduct determination (see Discussion). A significant correlation was seen for the presence of the prominent spot that co-chromatographed with BPDE—dG in blood and lung samples (Kendall W = 0.64; P = 0.003), but no such relationship was seen for the ELISA results (Kendall W = 0.02; P = 0.48). Analysis of variance and multiple regression models showed that putative BPDE-DNA adduct levels in WBC (irrespective of the analysis method) were not significantly related to smoking habits, i.e. the number of cigarettes per day in the year or month before diagnosis, use of filters, time elapsed since the patient stopped smoking, age or sex. A slightly better relationship was found when smoking habits were compared with total PAH—DNA adduct levels by analysis of covariance and multiple regression models. Variation was best explained in a simple model where last year's and last month's smoking habits were entered as categorial variables. Smoking in the year before diagnosis (yes/no), smoking in the month before diagnosis 990
(yes/no), number of years smoked and age, together explain 26% of variation in PAH—DNA adduct levels (Table II). Inclusion of the number of cigarettes smoked did not improve the fit of the model, nor did inclusion of PAH, job or leisure time exposure, alcohol, sex or urban/rural living. The results in Table II, though statistically non-significant, indicate some effect of recent smoking and increasing age on PAH—DNA adduct levels in WBC. Correlation between smoking and DNA adduct levels in lung was poor (28). We observed significantly lower DNA adduct levels when filter cigarettes were used. Other parameters like age, diet, medication, alcohol consumption, occupational exposure to PAH and habitation (urban or rural) were not related with adduct levels in the lung. The high adduct levels we found in WBC DNA from a number of patients prompted us to have these samples examined in another laboratory. The results are compared to Table III. Generally, there is reasonably good agreement between our data and those found by Dr Phillips, with the exception of one sample. At present we have no explanation for this discrepancy. Discussion We have examined the formation of PAH-DNA adducts in WBC from 38 lung cancer patients. The adduct profiles obtained by 32P-post-labeling of WBC DNA digests suggest the presence of a variety of adducts with rather similar chromatographic behavior. We could identify a spot in WBC of 18/38 (47%) patients which co-chromatographed with the major B[a]P—DNA adduct, BPDE—dG. Although physico-chemical evidence (e.g. LC -MS) is still needed to establish whether substantial amounts of BPDE—dG are present, our evidence for die presence of BPDE-DG in human lung tissue reported previously (28) was confirmed by Weston and Bowman (50), who described fluorescence detection and identification of BPDE—DNA adducts
PAH-DNA adducts in lung cancer ceUs
a 'BPDE-DNA
107
1
b"
1
• C
P5
Table D. Multiple regression model for variables predictive of PAH-DNA adduct levels in WBC of lung cancer patients (n = 38)
'Sir'
A ' e P5
WBC
Regression coefficient*
SEb
P value
Intercept Smoking last month (yes/no)c Smoking last year (yes/no)d No. of years smoked Age (years)
-1.760 0.865 0.702 -0.028 0.748
— 0.451 0.436 0.014 0.289
— 0.07 0.07 0.06 0.02
I? = 0.26, P = 0.13. •Regression coefficient indicates log change in DNA adducts (frnol/jig DNA) per unit change in variable. °SE, standard error. c Average number of cigarettes smoked during last month. d Average number of cigarettes smoked during last year.
d
LUNG
Variable
TaWe m . Total PAH-DNA adduct levels in WBC analyzed in two laboratories by the nuclcase PI enhanced 32P-post-labeling assay from nine lung cancer patients Patient no.
Mean level of adducts/108 nucleotides ( ± SD) Van Schooten et al.* Phillips1"
3/1 6 33/2 32 19 28
407.4 (19.7) 134.8 (36.6) 486.7 (76.4) 135.0 (55.9) 52.3 ( 5.3) 48.1 (25.5) 65.7 (11.7) 0.6 ( 0.2) 0.8 ( 0.1)
f
• •
< *
I*
\m
21 37
Fig. 2. Comparison of autoradiograms of chromatograms of ^P-postlabeling DNA digests of: (a, b) BPDE-modified DNA; (c,d) lung tissue from patient no. 5; (e,f) WBC from patient no. 5. For (b), (d) and (f) the chromatography in the D3 development was performed with 0.72 M sodium phosphate, 0.45 M Tris-HCl, 7.6 M urea, pH 8.2. Filter paper wicks were used in D3 and D4 to maneuver spots further from the origin. Autoradiography was carried out at -80°C for 20 h. O
1OO
Adducts/10* nucleot. (Lung) Fig. 3. Relation between the total PAH-DNA adduct levels as determined by 32P-postlabeling in lung and WBC from 20 lung cancer patients: ( • ) current smokers, (A) ex-smokers. The WBC data used were from blood samples collected 2.7 ± 4.1 days before thoracic surgery.
in human lung. According to the ELJSA, 12/38 (32%) patients had BPDE-dG antigenicity in their WBC DNA. The lower percentage of BPDE—dG-positive samples in the ELJSA compared with the post-labeling (32 versus 47%) is thought to be related to the higher sensitivity of the ^P-post-labeling assay.
119.9 95.2 467.2 107.6 34.7 40.4 4.4 0.8 2.8
(36.4) ( 3.0) (57.5 (15.6) ( 5.3) ( 8.2) ( 0.8) ( 0.1) ( 0.4)
"Data from this study (Table I). •"Data obtained by DT D.H.Phillips (Haddow Laboratories, Sutton, UK).
Quantification of the putative BPDE-DNA adducts by ELJSA and by 32P-post-labeling showed a difference in absolute values, thoughrelativelythese values were highly correlated. Comparison of the absolute values obtained by the two assays should be done with caution, because the assays are based on different principles. The 32P-post-labeling assay detects nuclease PI-resistant aromatic DNA adducts that still can be labeled with 32P at the 5' position by a kinase reaction. The immunoassay detects DNA adducts that react with an anti-BPDE-DNA antiserum and measures BPDE-DNA adducts but also DNA adducts of other PAHs with similar structures (33). The affinity of the antiserum with unknown adducts, as a result of exposure to complex PAH mixtures, is unknown and these unknown adducts may be responsible for the higher values found by ELJSA. The present 32 P-post-labeling procedure is still not sufficiently accurate for absolute quantification and can be expected to result in an underestimation of the actual DNA adduct levels. For instance, nuclease PI treatment may attack unknown adducts and they may not be labeled (35). Furthermore, the labeling efficiency of different adducts of carcinogens and nucleotide monophosphates appears to be different and depends on the class of adduct; labeling efficiencies might be as low as 10% (36). Nevertheless, adducts derived from BPDE arerelativelyresistantand recoveries of 80-100% were achieved when a [3H]BPDE-DNA standard with a modification level of 1 adduct/107 nucleotides was assayed. The total aromatic DNA adduct levels as found by 32P-postlabeling of WBC DNA from the lung cancer patients ranged up to 407 adducts in 108 nucleotides. These levels are high 991
FJ.van Scbooten et at.
compared to other WBC studies in which levels were found up to 200 adducts/108 nucleotides in coke oven workers (22) and up to 68 adducts/108 nucleotides in healthy smokers (37). The same holds true for our ELJSA results, which ranged up to > 150 adducts in 108 nucleotides, whereas in a previous study on coke oven workers we found in WBC DNA levels up to 20.4 adducts in 108 nucleotides (19). Perera et al. (24) have also observed increased DNA adduct levels in WBC of current smokers who had developed lung cancer compared to healthy smokers. These enhanced PAH—DNA adduct levels in the WBC for lung cancer patients may be the result of the genetic background of this group of persons. It is known that a genetic predisposition plays a relevant role in susceptibility of smokers to lung cancer (38). Nowak et al. (39) observed a genetically determined enhancement of B[a]P—DNA adduct formation in vitro in monocytes of lung cancer patients compared to healthy persons. The same authors recently showed that the in vitro formation of B[a]P-DNA adducts in peripheral blood monocytes from lung cancer patients with at least one first-degree relative with lung cancer was slightly but significantly higher when compared with healthy controls (40). In the present study a large interindividual variation was observed. Moreover, samples taken from the same individual at different time points also show considerable variation. As shown in Figure 1, these changes in adduct patterns were qualitative as well as quantitative. The high intraindividual variation is not restricted to lung cancer patients but has also been observed in studies on foundry workers (17,18), coke oven workers (19) and aluminium workers (FJ.van Schooten et al., unpublished data). The reason for this phenomenon may be the relatively short lifetime ( < 2 4 h) of the majority of WBC in the peripheral blood. Only 15—30% of peripheral WBC, monocytes and lymphocytes are long lived (weeks to years). Other factors contributing to the large variation may be the efficiency of metabolic activation of genotoxic compounds and the capacity to repair DNA adducts in the various subsets of WBC. Preliminary results in our laboratory (41) showed that mononuclear cells contain at least 10 times higher B[a]P—DNA adduct levels than polymorphonuclear cells after incubation with B[a]P in vitro, indicating that the composition of WBC at the moment of sampling is very important. The PAH—DNA adduct levels in non-tumorous lung tissue of the patients studied in the present study range between 2 and 134 adducts in 108 nucleotides for ELISA and between 1.9 and 34 adducts in 108 nucleotides for 32P-post-labeling. We observed that the use of filter cigarettes resulted in lower adduct levels in the lung (28). In contrast, levels of PAH—DNA adducts in WBC do not seem to be influenced appreciably by smoking habits. This observation is consistent with other reports (23,25,42) and suggests that PAH—DNA adducts in WBC are derived from other sources. Associations have, however, been reported between cigarette smoking and the level of adducts in placenta (29,30), oral mucosa (43), cervix (44) and lung (26,27). Food is a significant source of human exposure to PAH (45). A recent study on Dutch total diet showed that the major daily PAH intake came from sugar and sweets, cereals, oils, fats and nuts (46). In addition, PAH have been identified in broiled, barbequed and smoked meat or fish (47). In a recent study, Rothman et al. (48) reported that DNA adducts in an individual's WBC may result from ingestive exposure; these authors found high adduct levels that decreased rapidly in 1 —4 days after consumption of charcoal broiled meat. Such rapid loss of adducts is also in line with the
992
large fluctuations in WBC DNA adduct levels within one person observed in our present study. In the present study, we did not find a significant correlation between the adduct levels in lung DNA and in total WBC DNA. Several factors may be involved in this difference. DNA adduct formation in human tissues is complex because of irregular exposure and large differences in the rates by which DNA adducts will be formed and removed in the respective cell types. Adduct levels will be dependent on pharmacodynamic and pharmacokinetic parameters as well as DNA repair rates, cell turnover and adduct stability. The observed discrepancy between WBC and lung DNA adduct levels is in agreement with a recent study of Cuzick et al. (49) on autopsy material from smokers and nonsmokers which showed no correlation between an individual's adduct levels in different organs flung, bladder, pancreas, breast and cervix). Furthermore, smoking-related adducts in placental DNA were not found in WBC DNA from the same subjects (29). The lack of an observable effect of smoking on adduct levels in WBC DNA led Phillips et al. (42) to question the use of WBC DNA as a non-target source of DNA for determining exposure in susceptible tissues. The absence of a relationship between PAH-DNA adduct levels in WBC and lung from lung cancer patients in our study again stresses the limitations of using total WBC for DNA adduct determinations. Perhaps better correlations would have been obtained if a specific cell type, e.g. lymphocytes, had been used. Recent reports support this assumption. Savela and Hemminki (37) demonstrated a difference between smokers and non-smokers when mononuclear cells were assayed. Schoket et al. (51) also detected smoking exposure when utilizing lymphocytes. Acknowledgements We thank Dr J.T.Lutgerink for valuable advice on the 32P-post-labeling analysis, Dr D.H.Phillips (Haddow Laboratories, Sutton, Surrey, UK) for analyzing a number of our samples, and M.Camps for performing the statistical analyses. This study was supported by the Program Committee for Toxicological Research (grant PCT99-2050).
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Publications no. 74, IARC, Lyon, pp. 11-12. 2. Doll.R. and Hill,A.B. (1952) The study of the aetiology of the carcinoma of the lung. Br. J. Mai., U, 271-286. 3. Doll.R. and Peto.R.J. (1987) Cigarette smoking and bronchial carcinoma: dose and time relationships among regular smokers and lifelong non-smokers. J. Epidemiol. Commun. Health, 32, 303-313. 4. Wynder,E.L. and Hoffmann.D. (1982) Tobacco. In Schottenfeld,D. and FraumeniJ.F. Jr, (eds), Cancer Epidemiology and Prevention. W.B.Saunders, Philadelphia, pp. 277-292. 5. IARC (1986) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 38: Tobacco Smoking. IARC, Lyon. 6. IARC (1983) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 32: Polynuclear Aromatic Compounds, Part I, Chemical, Environmental and Experimental Data. IARC, Lyon. 7. Lindsted.G. and SollenbergJ. (1982) Porycyclic aromatic hydrocarbons in the occupational work environment. Scand. J. Work Environ. Health, 8, 1 - 1 9 . 8. Pelkonen.O. and Nebert.D.W. (1982) Metabolism of polycyclic aromatic hydrocarbons: ctiologic role in carcinogencsis. Pharmacol. Rev., 34, 189-222. 9. Harvey,R.G. (1986) Activated metabolites of carcinogenic hydrocarbons. Accts. Chem. Res., 14, 218-226. 10. Gupta.R.C, Earley,K. and Sharma.S. (1988) Use of human lymphocytes to measure DNA binding capacity of chemical carcinogens. Proc. Natl. Acad. Set. USA. 85, 3513-3517. 11. Wogan.G.N. and Gorclick.N.J. (1985) Chemical and biochemical dosimctry of exposure to genotoxic chemicals. Environ. Health Perspect., 62, 5 - 1 8 . 12. Phillips.D.H., Grover,P.L. and Sims.P.A. (1979) Quantitative determination
PAH-DNA adducts in lung cancer cells of the covalent binding of a scries of polycyclic aromatic hydrocarbons to DNA in mouse skin. Int. J. Cancer, 23, 201-208. 13. Stowers.S.J. and Anderson,M.W. (1985) Formation and persistence of benzo(a)pyrene metabolite - DNA adducts. Environ. Health Perspect., 62, 31-39. 14. Shamsuddin.A.K., Sinopoli.N.T., Hemminki,K., Boesch.R.R. and Harris.C.C. (1985) Detection of benzo(a)pyrene-DNA adducts in human white Mood cells. Cancer Res.. 45, 6 6 - 6 8 . 15. Hams.C.C, Vahakangas.K., Newman.NJ., Trivcrs.G.E., Shamsuddin.A., Sinopoli.N., Mann.D.L. and Wright.W. (1985) Detection of benzo(a)py rene diol epoxide-DNA adducts in peripheral Mood lymphocytes and antibodies to the adducts of serum from coke oven workers. Proc. Nail. Acad. Set. USA, 82, 6672-6676. 16. Haugen.A., Becher.G., Benestad.C, Vahakangas.K., Trivers.G.E., Newman,M.J. and Harris.C.C. (1986) Determination of polycyclic aromatic hyrocarbons in the urine, benzo(a)pyrene diol expoxide-DNA adducts in lymphocyte DNA, and antibodies to the adducts in sera from coke oven workers to measured amounts of polycyclic aromatic hydrocarbons in the work atmosphere. Cancer Res., 46, 4178-4183. 17. Perera.F.P., Hemminki.K., Young,T.-L., Brenner.D., Kelly.G. and Santella.R.M. (1988) Detection of polycyclic aromatic hydrocarbon - DNA adducts in white Mood cells of foundry workers. Cancer Res., 48, 2288-2291. 18. Phillips.D.H., Hemminki.K., Alhonen.A., Hewer.A. and Grover.P.L. (1988) Monitoring occupational exposure to carcinogens: detection by Ppostlabeling of aromatic DNA adducts in white blood cells from iron foundry workers. Mural. Res., 204, 531-541. 19. Van Schooten.F.J., Van Leeuwen.F.E., Hillebrand.M.J.X., De Rijke.M.E. Hart.A.A.M., Van Vcen.H.G., Oosterink.S. and Kriek.E. (1990) Determination of benzo(a)pyrene diol epoxide DNA adducts in white blood cell DNA from coke oven workers with polyclonal antibodies. The impact of smoking. J. Nail. Cancer Int., 82, 927-933. 20. Herbert.R., Marcus.M., Wolff.M.S., Perera,F.P., Andrews.L. GodbokU.H., Rivera.M., Stefarodis.M., Lu,X.-Q., Landrigan.PJ. and Santella.R.M. (1990) Detection of adducts of deoxyribonucleic acid in white blood cells of roofers by 32P-postlabeling. Relationship of adduct levels to measures of polycyclic aromatic hydrocarbons. Scand. J. Work Environ. Health, 16, 135-143. 21.Hemminki,K., Randerath.K., Reddy.M.V., Putman.K.L., Santella.R.M., Perera.F.P., Young,T.-L., Phillips.D.H., Hewer.A. and Savela.K. (1990) Postlabeling and immunoassay of polycyclic aromatic hydrocarbons-adducts of deoxyribonucleic acid in white blood cells of foundry workers. Scand. J. Work Environ. Health, 16, 158-162. 22. Hemminki.K., Grzybowsky,E., Chorazy.M., Twardowska-Saucha.K., Sroczynski.J.W., Putman.K.L., Randerath.K., Phillips.D.H., Hewer.A., Santella.R.M., Young,T.L. and Perera.F.P. (1990) DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland. Carcinogenesis, 11, 1229-1231. 23. Perera.F.P., Santella.R.M., Brenner.D., Poirier.M.C, Munshi.A.A., Fischman.H.K. and Van Ryzin J. (1987) DNA adducts, protein adducts and sister chromatid exchange in cigarette smokers and nonsmokers. J. Nail. Cancer Inst., 79, 449-456. 24. Perera.F.P., MayerJ., Jaretzki.A., Hearne,S. Brenner.D., Young.T.L., Fishman.H.K., Grimcs.M., Grantham.S., Tang.M.X., Tsai,W.-Y. and Santella.R.M. (1989) Comparison of DNA adducts and sister chromatid exchange in lung cancer cases and controls. Cancer Res., 49, 4446-4451. 25. Jahnke.G.D., Thompson.C.L., Walker.M.P., GallagherJ.E., Lucier.G.W. and DLAugustine.R.P. (1990) Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis. Carcinogenesis, 11, 205 - 211. 26. Phillips.D.H., Hewer.A., Martin.C.N., Gamer.R.C. and King.M.M. (1988) Correlation of DNA adduct levels in human lung with cigarette smoking. Nature, 336, 790-792. 27.Randerath,E., Miller.R.H., Mittal.D., Avitts.T.A., Dunsford.H.A. and Randerath.K. (1989) Covalent DNA damage in tissues of cigarette smokers as determined by 32P-postlabeling assay. J. Natl. Cancer Inst., 81, 341 -347. 28. VanSchooten.F.J., Hillebrand.M.J.X., Van Leeuwen.F.E., LutgerinkJ.T., Van Zandwijk.N., Jansen.H.M. and Kriek.E. (1990) Polycyclic aromatic hydrocarbon — DNA adducts in lung tissue from lung cancer patients. Carcinogenesis, 11, 1677—1681. 29. Everson.R.B., Randerath.E., Avitts.T.A., Schut.H.A.J. and Randerath.K. (1987) Preliminary investigations of tissue specificity, species specificity, and strategies for identifying chemicals causing DNA adducts in human placenta. Prog. Exp. Tumor Res., 31, 86-103. 30. Everon.R.B., Randerath.E., Santella.R.M., Avitts.T.A., Weinstein.I.B. and Randerath.K. (1988) Quantitative association between DNA damage in human placenta and maternal smoking and birth weight. J. Natl. Cancer Inst., 80, 567-575.
31. Stowers.S.J. and Anderson,M.W. (1984) Ubiquitous binding of benzo(a)py rene metabolites to DNA and protein in tissues of mouse and rabbits. Chem.-Biol. Interactions, 51, 151-166. 32.Ross,J., Nelson.G., Ligerman.A., Erexson.G., Bryant,M.t Earley.K., Gupta.R. and Nesnow.S. (1990) Formation and persistence of novel benzo(a)pvrene adducts in rat lung, liver, and peripheral blood lymphocyte DNA. Cancer Res., 50, 5088-5094. 33. Van Schooten.F.J., Kriek.E., Steenwinkel.M.-J.S.T., Notebom.H.PJ.M., Hillebrand.M.J.X. and Van Leeuwen.F.E. (1987) The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo(a)pyrene diol expoxide is dependent on the level of modification. Implications for quantitation of benzo(a)pyrene — DNA adducts in vivo. Carcinogenesis, 8, 1263-1269. 34. Reddy.M.V. and Randerath.K. (1986) Nuclease Pl-mediated enhancement of sensitivity of 32P-postlabeling test for structural diverse DNA adducts. Carcinogenesis, 7, 1543-1551. 35. GaUagrwJ.E., Jackson.M.A., George.M.H., LewtasJ. and Robertson.I.G.C. (1989) Differences in the detection of DNA adducts in ^P-postlabeling assay after either 1-butanol extraction or nuclease PI treatment. Cancer Lett., 45, 7-12. 36. Shields.P.G., Harris.C.C., Petruzelli.S., Bowman.E.D. and Weston,A. (1991) Standardization of the 32P-postlabeling assay for polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Proc. Am. Assoc. Cancer Res., 32, 1323. 37. Savela.K. and Hemminki.K. (1991) DNA adducts in lymphocytes and granulocytes of smokers and nonsmokers detected by the ^P-postlabeling assay. Carcinogenesis, 12, 503—508. 38.SametJ.M., Humble.C.G. and Pathak.D.C. (1988) Personal and familial history of respiratory disease and lung cancer risk. Am. Rev. Respir. Dis., 134, 466-470. 39. Nowak.D., Schmidt-Preuss.U., Jorres.R., Liebke.F. and ROdiger.H.W. (1988) Formation of adducts and water-soluble metabolites of benzo(a)pyrene in human monocytes is genetically controlled. Int. J. Cancer, 41, 169-173. 40. Nowak.D., Meyer.A., Schmidt-Preuss.U., Gatzemeier.U., Magnussen.H. and Rudiger.H.W. (1992) Formation of benzo[a]rjyrene - DNA adducts in blood monocytes from lung cancer patients with a familial history of lung cancer. J. Cancer Res. Clin. Oncol., 118, 6 7 - 7 1 . 41. Van Schooten.F.J., Hillebrand.M.J.X., Van Engelen.J.G.M., Van Leeuwen.F.E., Den Engelse.L. and Kriek.E. (1991) DNA adduct levels in mouse tissues and human white blood cells after exposure to polycyclic aromatic hydrocarbons. Proc. Am. Assoc. Cancer Res., 32, 545. 42. Phillips.D.H., Schoket.B., Hewer.A., Bailey.E., Kostic.S. and Vincze.I. (1990) Influence of cigarette smoking on the levels of DNA adducts in human bronchial epithelium and white blood cells. Int. J. Cancer, 46, 569-575. 43. Foiles.P.G , Miglieta.L.M., Quart.A.M., Quart.E., Kabat.G.C. and Hecht.S.S. (1989) Evaluation of 32P-postlabeling analysis of DNA from exfoliated cells as means of monitoring exposure of the oral cavity to genotoxk agents. Carcinogenesis, 10, 1429-1434. 44. Phillips.D.H., Hewer.A., Malcolm,A.D.B., Ward.P. and Coleman.D.V. (1990) Smoking and DNA damage to cervical cells. Lancet, 335, 417. 45. Lioy.P.L., WaldmanJ.M., Greenberg.A. Harkov.R. and Pietarinen.C. (1988) The total human environmental exposure study (THEES) to benzo(a)pyrene: comparison of the inhalation and food pathways. Arch. Environ. Health, 43, 304-312. 46. De Vos.R.H., Van Dokkum.W., Schouten,A. and De Jong-Berkhout.P. (1990) Polycyclic aromatic hydrocarbons in Dutch total diet samples (1984-1986). Food Chem. Toxicoi., 28, 263-268. 47. Fazio.T. and HowardJ.W. (1983) Polycyclic aromatic hydrocarbons in foods. In Bjorseth,A. (ed.), Handbook of'Polycyclic Aromatic Hydrocarbons. Marcel
Dekker, New York, Vol. 1, pp. 461-505. 48. Rothman.N., Poirier.M.C., Baser.M.E., Hansen.J.A., Gentile.C, Bowman.E.D. and Strickland,P.T. (1990) Formation of polycyclic aromatic hydrocarbon-DNA adducts in peripheral white blood cells during consumption of charcoal-broiled beef. Carcinogenesis, 11, 1241 — 1243. 49. CuacU., Rouoedge.M.N., Jenkins.D. and Gamer.R.C. (1990) DNA adducts in different tissues of smokers and non-smokers. Int. J. Cancer, 45, 673—678. 50. Weston,A. and Bowman.E.D. (1991) Fluorescence detection of benzo{a]pyrene-DNA adducts in human lung. Carcinogenesis, 12, 1445-1449. 51. Schoket.B., Phillips.D.H., Hewer.A. and Vincze.I. (1991) ^P-postlabeling detection of aromatic DNA adducts in peripheral Mood lymphocytes from aluminium production plant workers. Mutat. Res., 260, 89—98. Received on September 12, 1991; revisedon March II, 1992; accepted on March 16, 1992
993
Polycyclic aromatic hydrocarbon—DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung
F.J.van Schooten1, M.J.X.Hillebrand, F.E.van Leeuwen2, N.van Zandwijk3, H.M.Jansen4, L.den Engelse and E.Kriek5 Division of Molecular Carcinogenesis, 2Division of Psychosocial Research and Epidemiology and 'Division of Clinical Oncology, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), 121 Plesmanlaan, 1066 CX Amsterdam and 4Department of Pulmonology, University Hospital of Amsterdam, Academic Medical Center, 15 Meibergdreef, 1105 AZ Amsterdam, The Netherlands 'Present address: Department of Health Risk Analysis, University of Limburg, Becldsnijdersdreef 101, 6200 MD Maastricht, The Netherlands 'To whom correspondence should be addressed
Smokers of cigarettes are exposed to a number of carcinogens, including polycylic aromatic hydrocarbons (PAHs), and are at a high risk for lung cancer. PAHs exert their carcinogenic activity after metabolic activation to reactive intermediates that can damage DNA through adduct formation. Measuring DNA adducts in peripheral white blood cells (WBC) could serve as a means of monitoring human exposure to genotoxic agents and subsequently risk assessment. In this study, DNA from WBC obtained from 39 lung cancer patients was examined for PAH-DNA adducts both in an ELISA using a polyclonal antibody against benzo[a]pyrene 7,8-diol-9,10epoxide (BPDE)-DNA and the ^P-post-labeling technique. The ELISA results showed BPDE-DNA antigenidty in WBC DNA from 12/38 (32%) patients and adduct levels ranged from 1.5 to > 150 adducts in 108 nucleotides. The autoradiographs of chromatograms of ^P-post-labeled digests of WBC DNA from the 38 patients showed a variety of adduct spots; relative adduct labeling (RAL) values ranged from 0.3 to 407 adducts in 108 nucleotides. In 18 of the 38 (47%) persons an adduct spot was detected that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG); RAL values ranged from 0.03 to 382 adducts in lO8 nucleotides. Correlations were not significant between the adduct levels in WBC and smoking habits, age or sex. From 20 patients of the same group lung tissue was collected at surgery and examined for PAH-DNA adducts by ELISA and %-post-labeling assay. No significant correlation was found between DNA adduct levels in blood and lung. This finding stresses the limitations of the use of WBC as a surrogate for adduct levels in the target organ.
Introduction Tobacco smoking is the most important and well-documented cause of lung cancer currently known (1). It has been well established that the risk of lung cancer increases with the number •Abbreviations: PAH, polycyclic aromatic hydrocarbon; B[a]P, benzo(a)pyTene; WBC, white blood cells; BPDE, (±)/ra/is-7,8-dihydroxy-150
ND ND 382.3
2.0 ± 0.7 3.9 ± 1.3 407.4 ± 19.7
34.3 ± 10.9 8.2 ± 2.7 21.1 ± 10.8
10.8 ± 2.7 >150 4.8 ± 0.9 >150 ND >150 ND ND ND 1.5 ± 0.2 ND ND ND 1.5 ± 0.2 ND ND ND ND ND >150 ND ND ND ND ND ND ND 150 ± 50.7 ND >150 ND >150 ND >150 ND 1.5 ± 0.3 ND ND ND ND ND
1.2 ± 79.8 ± 3.2 ± 57.8 ± ND 123.4 ± 0.2 ± ND ND 0.15 ± ND ND ND 0.6 ± ND 0.1 0.3 ± ND ND 40.2 ± ND ND ND ND ND ND ND 9.8 ± ND 45.9 ± 0.2 ± 70.6 ± 0.03 ± 127.8 ± 0.15 ± 0.33 ± ND ND ND ND ND
ELISAC
Last year"
AC ACr ACh
51 68 59
M M F
0 0 10
* 5 10
no no no
AC
42
M
0
23
yes
SC1
58
M
18
18
no
SC AC SC SC AC LC
75 53 54 69 56 43
M F M M M M
10 0 0 1 75 18
10 4 0 41 55
yes yes no yes no no
LC SC SC SC SC * SC SC SC SC AC SC *
73 72 64 77 68 53 69 73 54 78 83 49 58
M M M M M M M M F M M M F
0 0 20 0 8 0 0 0 0 * 0 10 0
*
LC * SC AC Sc Sc SC SC * * LC * Sc *
79 40 54 57 71 65 71 62 32 54 39 57 63 63
M M M F M M M M M F M M M M
0 * 0 0 0 0 0 7 * 11 0 0 0 0
•
0 20 0 8 25 0 0 13 * 0 25 25 0 •
26 0 0 0 21 4 * 11 0 0 8 8
yes yes no no no no no no yes * no no yes no * no no no no no no * yes no * no no
32
± 4.2 0.1 9.2 1.3 9.9 34 0.1
0.1
0.2
0.1
9.3
2.0 23 0.06 2.2 0.03 52 0.03 0.03
6.2 89.1 16.6 74.7 3.2 134.8 2.7 1.7 1.3 0.2 38.2 65.7 1.4 0.6 3.5 4.5 0.3 2.2 1.7 52.3 1.9 0.6 12.3 0.3 2.2 6.7 6.2 12.4 1.3 48.1 3.8 5.3 1.1 135.0 2.4 3.6 0.8 1.6 1.4 1.6 1.0
± 0.5 ± 7.7 ± 7.5 ± 11.7 ± 0.8 ± 36.6 ± 0.1 ± 0.3 ± 0.6 ± 0.03 ± 5.6 ± 11.7 ± 0.9 ± 0.2 ± 1.6 ± 1.5 ± 0.03 ± 1.0 ± 0.2 ± 9.6 ± 0.5 ± 0.2 ± 3.2 ± 0.1 ± 1.0 ± 2.0 ± 0.5 ± 4.5 ± 0.3 ± 25.5 ± 1.35 ± 1.5 ± 0.5 ± 55.9 ± 0.7 ± 0.8 ± 0.1 ± 0.9 0.6 ± 0.2 ± 0.2
2.3 ±
0.7
19.9 ±
4.7
6.7 5.7 19.6 4.3 11.0 20.0
± ± ± ± ± ±
0.7 1.9 5.4 1.9 0.1 1.5
4.9 3.7 11.9 20.7 5.2 12.8 1.9 2.5 9.3
± ± ± ± ± ± ± ± ±
0.2 0.4 4.2 3.1 0.8 4.1 0.7 0.4 2.4
AC, adenocarcinoma. SC, squamous cell carcinoma. LC, large cell carcinoma; *unknown. ND, non detectable. "Average number smoked during last month. b Average number smoked during last year. c Expressed as equivalents of BPDE-DNA adducts/108 nucleotides, mean ± SD, n = 3. d Expressed as adducts/108 nucleotides calculated from RAL values, mean ± SD, n = 2 - 3 . c Data from Van Schooten el al. (28). f Primary rectum carcinoma. 'Sampled 1 week before surgery. h Primary mammacarcinoma. 'Sampled on the day of surgery. JSampled 3 weeks before surgery. 'Sampled 2 days after surgery. •"Sampled 1 day before surgery. "Sampled 2 weeks after surgery. "Sampled 12 days after sample 1.
described elsewhere (28) and will be summarized here only. The ELISA results showed BPDE-DNA antigenicity in lung DNA from 6/20 (30%) patients and adduct levels ranged beween 2 and
134 adducts in 108 nucleotides. For all 20 patients the autoradiographs of chromatograms of 32P-post-labeling digests of DNA from non-tumorous lung tissue showed a strong diagonal 989
FJ.van Schooten et at.
Fig. 1. Representative autoradiograms of chromatograms of ^P-post-labeled DNA digests from WBC from lung cancer patients: (a) patient no. 5; (b) patient no. 5, sampled 1 week later; (c) patient no. 4; (d) patient no. 4, sampled 3 weeks and 2 days later; (e) patient no. 3; (f) patient no. 3, sampled 1 week later; (g) patient no. 11; (h) patient no. 11, sampled 2 weeks and 1 day later. Spot 1 co-chromatographcd with the major BPDE-DNA adduct (BPDE-dG). The origin is located at the bottom left-hand corner of each chromatogram and excised before autoradiography. The autoradiography was carried out at -80°C for 18 h.
radioactive zone (DRZ). The level of DRZ adducts ranged from 1.9 to 34 adducts in 108 nucleotides. This DRZ was generally absent in tumorous tissue. DNA samples that were positive in the ELJSA contained a dominant spot within the DRZ that cochromatographed with BPDE-dG (see also Figure 2). The quantities of the BPDE—dG spots ranged from 0.2 to 4 adducts in 108 nucleotides. Adduct levels obtained by 32P-post-labeling were 5 — 10 times lower than, but correlated well with, those found in the ELJSA (Kendall W = 1; P < 0.01). Correlation between blood and lung adduct levels The relation between adduct levels in lung and WBC from the 20 patients examined is illustrated in Figure 3. Although a positive correlation was found for eight current smokers (r = 0.515, P = 0.19) and a negative correlation for 12 ex-smokers (r = —0.388, P = 0.21), these correlations were not significant. Possible reasons may be the small number of persons examined and the use of WBC for adduct determination (see Discussion). A significant correlation was seen for the presence of the prominent spot that co-chromatographed with BPDE—dG in blood and lung samples (Kendall W = 0.64; P = 0.003), but no such relationship was seen for the ELISA results (Kendall W = 0.02; P = 0.48). Analysis of variance and multiple regression models showed that putative BPDE-DNA adduct levels in WBC (irrespective of the analysis method) were not significantly related to smoking habits, i.e. the number of cigarettes per day in the year or month before diagnosis, use of filters, time elapsed since the patient stopped smoking, age or sex. A slightly better relationship was found when smoking habits were compared with total PAH—DNA adduct levels by analysis of covariance and multiple regression models. Variation was best explained in a simple model where last year's and last month's smoking habits were entered as categorial variables. Smoking in the year before diagnosis (yes/no), smoking in the month before diagnosis 990
(yes/no), number of years smoked and age, together explain 26% of variation in PAH—DNA adduct levels (Table II). Inclusion of the number of cigarettes smoked did not improve the fit of the model, nor did inclusion of PAH, job or leisure time exposure, alcohol, sex or urban/rural living. The results in Table II, though statistically non-significant, indicate some effect of recent smoking and increasing age on PAH—DNA adduct levels in WBC. Correlation between smoking and DNA adduct levels in lung was poor (28). We observed significantly lower DNA adduct levels when filter cigarettes were used. Other parameters like age, diet, medication, alcohol consumption, occupational exposure to PAH and habitation (urban or rural) were not related with adduct levels in the lung. The high adduct levels we found in WBC DNA from a number of patients prompted us to have these samples examined in another laboratory. The results are compared to Table III. Generally, there is reasonably good agreement between our data and those found by Dr Phillips, with the exception of one sample. At present we have no explanation for this discrepancy. Discussion We have examined the formation of PAH-DNA adducts in WBC from 38 lung cancer patients. The adduct profiles obtained by 32P-post-labeling of WBC DNA digests suggest the presence of a variety of adducts with rather similar chromatographic behavior. We could identify a spot in WBC of 18/38 (47%) patients which co-chromatographed with the major B[a]P—DNA adduct, BPDE—dG. Although physico-chemical evidence (e.g. LC -MS) is still needed to establish whether substantial amounts of BPDE—dG are present, our evidence for die presence of BPDE-DG in human lung tissue reported previously (28) was confirmed by Weston and Bowman (50), who described fluorescence detection and identification of BPDE—DNA adducts
PAH-DNA adducts in lung cancer ceUs
a 'BPDE-DNA
107
1
b"
1
• C
P5
Table D. Multiple regression model for variables predictive of PAH-DNA adduct levels in WBC of lung cancer patients (n = 38)
'Sir'
A ' e P5
WBC
Regression coefficient*
SEb
P value
Intercept Smoking last month (yes/no)c Smoking last year (yes/no)d No. of years smoked Age (years)
-1.760 0.865 0.702 -0.028 0.748
— 0.451 0.436 0.014 0.289
— 0.07 0.07 0.06 0.02
I? = 0.26, P = 0.13. •Regression coefficient indicates log change in DNA adducts (frnol/jig DNA) per unit change in variable. °SE, standard error. c Average number of cigarettes smoked during last month. d Average number of cigarettes smoked during last year.
d
LUNG
Variable
TaWe m . Total PAH-DNA adduct levels in WBC analyzed in two laboratories by the nuclcase PI enhanced 32P-post-labeling assay from nine lung cancer patients Patient no.
Mean level of adducts/108 nucleotides ( ± SD) Van Schooten et al.* Phillips1"
3/1 6 33/2 32 19 28
407.4 (19.7) 134.8 (36.6) 486.7 (76.4) 135.0 (55.9) 52.3 ( 5.3) 48.1 (25.5) 65.7 (11.7) 0.6 ( 0.2) 0.8 ( 0.1)
f
• •
< *
I*
\m
21 37
Fig. 2. Comparison of autoradiograms of chromatograms of ^P-postlabeling DNA digests of: (a, b) BPDE-modified DNA; (c,d) lung tissue from patient no. 5; (e,f) WBC from patient no. 5. For (b), (d) and (f) the chromatography in the D3 development was performed with 0.72 M sodium phosphate, 0.45 M Tris-HCl, 7.6 M urea, pH 8.2. Filter paper wicks were used in D3 and D4 to maneuver spots further from the origin. Autoradiography was carried out at -80°C for 20 h. O
1OO
Adducts/10* nucleot. (Lung) Fig. 3. Relation between the total PAH-DNA adduct levels as determined by 32P-postlabeling in lung and WBC from 20 lung cancer patients: ( • ) current smokers, (A) ex-smokers. The WBC data used were from blood samples collected 2.7 ± 4.1 days before thoracic surgery.
in human lung. According to the ELJSA, 12/38 (32%) patients had BPDE-dG antigenicity in their WBC DNA. The lower percentage of BPDE—dG-positive samples in the ELJSA compared with the post-labeling (32 versus 47%) is thought to be related to the higher sensitivity of the ^P-post-labeling assay.
119.9 95.2 467.2 107.6 34.7 40.4 4.4 0.8 2.8
(36.4) ( 3.0) (57.5 (15.6) ( 5.3) ( 8.2) ( 0.8) ( 0.1) ( 0.4)
"Data from this study (Table I). •"Data obtained by DT D.H.Phillips (Haddow Laboratories, Sutton, UK).
Quantification of the putative BPDE-DNA adducts by ELJSA and by 32P-post-labeling showed a difference in absolute values, thoughrelativelythese values were highly correlated. Comparison of the absolute values obtained by the two assays should be done with caution, because the assays are based on different principles. The 32P-post-labeling assay detects nuclease PI-resistant aromatic DNA adducts that still can be labeled with 32P at the 5' position by a kinase reaction. The immunoassay detects DNA adducts that react with an anti-BPDE-DNA antiserum and measures BPDE-DNA adducts but also DNA adducts of other PAHs with similar structures (33). The affinity of the antiserum with unknown adducts, as a result of exposure to complex PAH mixtures, is unknown and these unknown adducts may be responsible for the higher values found by ELJSA. The present 32 P-post-labeling procedure is still not sufficiently accurate for absolute quantification and can be expected to result in an underestimation of the actual DNA adduct levels. For instance, nuclease PI treatment may attack unknown adducts and they may not be labeled (35). Furthermore, the labeling efficiency of different adducts of carcinogens and nucleotide monophosphates appears to be different and depends on the class of adduct; labeling efficiencies might be as low as 10% (36). Nevertheless, adducts derived from BPDE arerelativelyresistantand recoveries of 80-100% were achieved when a [3H]BPDE-DNA standard with a modification level of 1 adduct/107 nucleotides was assayed. The total aromatic DNA adduct levels as found by 32P-postlabeling of WBC DNA from the lung cancer patients ranged up to 407 adducts in 108 nucleotides. These levels are high 991
FJ.van Scbooten et at.
compared to other WBC studies in which levels were found up to 200 adducts/108 nucleotides in coke oven workers (22) and up to 68 adducts/108 nucleotides in healthy smokers (37). The same holds true for our ELJSA results, which ranged up to > 150 adducts in 108 nucleotides, whereas in a previous study on coke oven workers we found in WBC DNA levels up to 20.4 adducts in 108 nucleotides (19). Perera et al. (24) have also observed increased DNA adduct levels in WBC of current smokers who had developed lung cancer compared to healthy smokers. These enhanced PAH—DNA adduct levels in the WBC for lung cancer patients may be the result of the genetic background of this group of persons. It is known that a genetic predisposition plays a relevant role in susceptibility of smokers to lung cancer (38). Nowak et al. (39) observed a genetically determined enhancement of B[a]P—DNA adduct formation in vitro in monocytes of lung cancer patients compared to healthy persons. The same authors recently showed that the in vitro formation of B[a]P-DNA adducts in peripheral blood monocytes from lung cancer patients with at least one first-degree relative with lung cancer was slightly but significantly higher when compared with healthy controls (40). In the present study a large interindividual variation was observed. Moreover, samples taken from the same individual at different time points also show considerable variation. As shown in Figure 1, these changes in adduct patterns were qualitative as well as quantitative. The high intraindividual variation is not restricted to lung cancer patients but has also been observed in studies on foundry workers (17,18), coke oven workers (19) and aluminium workers (FJ.van Schooten et al., unpublished data). The reason for this phenomenon may be the relatively short lifetime ( < 2 4 h) of the majority of WBC in the peripheral blood. Only 15—30% of peripheral WBC, monocytes and lymphocytes are long lived (weeks to years). Other factors contributing to the large variation may be the efficiency of metabolic activation of genotoxic compounds and the capacity to repair DNA adducts in the various subsets of WBC. Preliminary results in our laboratory (41) showed that mononuclear cells contain at least 10 times higher B[a]P—DNA adduct levels than polymorphonuclear cells after incubation with B[a]P in vitro, indicating that the composition of WBC at the moment of sampling is very important. The PAH—DNA adduct levels in non-tumorous lung tissue of the patients studied in the present study range between 2 and 134 adducts in 108 nucleotides for ELISA and between 1.9 and 34 adducts in 108 nucleotides for 32P-post-labeling. We observed that the use of filter cigarettes resulted in lower adduct levels in the lung (28). In contrast, levels of PAH—DNA adducts in WBC do not seem to be influenced appreciably by smoking habits. This observation is consistent with other reports (23,25,42) and suggests that PAH—DNA adducts in WBC are derived from other sources. Associations have, however, been reported between cigarette smoking and the level of adducts in placenta (29,30), oral mucosa (43), cervix (44) and lung (26,27). Food is a significant source of human exposure to PAH (45). A recent study on Dutch total diet showed that the major daily PAH intake came from sugar and sweets, cereals, oils, fats and nuts (46). In addition, PAH have been identified in broiled, barbequed and smoked meat or fish (47). In a recent study, Rothman et al. (48) reported that DNA adducts in an individual's WBC may result from ingestive exposure; these authors found high adduct levels that decreased rapidly in 1 —4 days after consumption of charcoal broiled meat. Such rapid loss of adducts is also in line with the
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large fluctuations in WBC DNA adduct levels within one person observed in our present study. In the present study, we did not find a significant correlation between the adduct levels in lung DNA and in total WBC DNA. Several factors may be involved in this difference. DNA adduct formation in human tissues is complex because of irregular exposure and large differences in the rates by which DNA adducts will be formed and removed in the respective cell types. Adduct levels will be dependent on pharmacodynamic and pharmacokinetic parameters as well as DNA repair rates, cell turnover and adduct stability. The observed discrepancy between WBC and lung DNA adduct levels is in agreement with a recent study of Cuzick et al. (49) on autopsy material from smokers and nonsmokers which showed no correlation between an individual's adduct levels in different organs flung, bladder, pancreas, breast and cervix). Furthermore, smoking-related adducts in placental DNA were not found in WBC DNA from the same subjects (29). The lack of an observable effect of smoking on adduct levels in WBC DNA led Phillips et al. (42) to question the use of WBC DNA as a non-target source of DNA for determining exposure in susceptible tissues. The absence of a relationship between PAH-DNA adduct levels in WBC and lung from lung cancer patients in our study again stresses the limitations of using total WBC for DNA adduct determinations. Perhaps better correlations would have been obtained if a specific cell type, e.g. lymphocytes, had been used. Recent reports support this assumption. Savela and Hemminki (37) demonstrated a difference between smokers and non-smokers when mononuclear cells were assayed. Schoket et al. (51) also detected smoking exposure when utilizing lymphocytes. Acknowledgements We thank Dr J.T.Lutgerink for valuable advice on the 32P-post-labeling analysis, Dr D.H.Phillips (Haddow Laboratories, Sutton, Surrey, UK) for analyzing a number of our samples, and M.Camps for performing the statistical analyses. This study was supported by the Program Committee for Toxicological Research (grant PCT99-2050).
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