| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Epidemiology and Prevention |
Departments of Gastrointestinal Oncology and Digestive Diseases [D. L., P. F. F.], Epidemiology [L-E. W., C. H. B., M. R. S., Q. W.], and Thoracic and Head and Neck Medical Oncology [W. K. H.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Interindividual variation in susceptibility to chemical carcinogenesis depends on carcinogen metabolism and DNA repair. For example, the level of in vivo tobacco smoke-induced DNA adducts is determined by an equilibrium between the metabolism of tobacco carcinogens, such as BP,3 and the rate of adduct removal by DNA repair enzymes. Both processes are modulated by genetic polymorphisms and epigenetic factors (3 , 4) . Biomarkers reflecting such susceptibility may therefore be useful for identifying high-risk individuals. Genotypic biomarkers for carcinogen activation, detoxification, and DNA repair are being used extensively to characterize genetic susceptibility to carcinogenesis (5 , 6) . However, the functional relevance of some of these polymorphisms has not been determined. DNA adducts induced by ultimate carcinogens such as BPDE are phenotypic markers that reflect cellular response to the exposure, including detoxification and DNA repair. Therefore, individuals who are sensitive to BPDE-induced DNA damage may have suboptimal DNA repair capacity. To measure such DNA repair capacity, we developed and reported an in vitro-induced DNA-adduct assay using cultured peripheral lymphocytes that are exposed in vitro to BPDE (7) . To further investigate the utility of this assay as a biomarker for genetic susceptibility to lung cancer, we used the assay for a large epidemiological study of 221 lung cancer cases and 229 healthy controls. Here we report an association between the level of in vitro BPDE-induced DNA adducts and risk of lung cancer.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Study Population.
The cases were patients with newly diagnosed, histopathologically
confirmed lung cancer seen at The University of Texas M. D. Anderson
Cancer Center between 1996 and 1999 with a participation rate of 77.4%
upon contact. Most nonparticipants were too ill to participate. Healthy
control subjects were recruited from a large managed-care organization
in the Houston metropolitan area (Kelsey Seybold Clinic) during the
same time period (8)
. These control subjects were first
surveyed with a short questionnaire for willingness to participate in
research studies and to provide data on smoking behavior and
demographics. A computer database of more than 50,000 potential control
subjects was used to identify eligible control subjects who were
individually selected to match the case patients by age (±5 years),
sex, ethnicity, and smoking status (8)
. Of those
identified and contacted, 73.3% participated. Each eligible subject
was then scheduled for an interview to collect information on
demographic data and risk factors such as smoking history. A 30-ml
blood sample was obtained from each participant for the laboratory
assays. The exclusion criteria were prior chemotherapy or radiotherapy
for the cases, prior cancer for the controls, and recent (within the
last 6 months) blood transfusion for any participant. The study
protocol was approved by the M. D. Anderson and Kelsey Seybold Clinic
Institutional Review Boards, and informed consent was obtained from all
study participants.
Histology and Clinical Stage of Cancer.
Histopathology and clinical stage of cancer were obtained from the
clinical records. NSCC was staged in accordance with the 1997 revision
of the International System for Staging Lung Cancer (9)
.
SCC was divided into "limited" and "extensive" disease, based
on the absence or presence, respectively, of distant metastases. For
this analysis, stages I and II NSCC were defined as "limited
disease," stage III NSCC and limited SCCs as "regional spread"
(because most of the SCCs had mediastinal lymph node involvement at the
time of diagnosis), and stage IV NSCC and extensive SCC as "extensive
spread."
Blood Sample Collection, Cell Culture, and BPDE Treatment.
Within 8 h (the same day) after the blood samples were drawn, they
were processed for cell culture as described previously
(7)
. Briefly, 1 ml of whole blood was inoculated into each
of two T-25 flasks (containing 9 ml of standard RPMI 1640 supplemented
with 15% fetal bovine serum and 112.5 µg/ml PHA) and incubated at
37°C for 72 h. One flask was used as the in vivo
background adduct control, and one was used for BPDE treatment later.
Because unstimulated lymphocytes have little nucleotide excision repair
of BPDE adducts (10
, 11)
, PHA stimulation was
necessary. No statistical difference was observed in the blastogenic
rates in response to PHA stimulation between cases and controls
(71.1% ± 18.3 and 73.4% ± 23.5, respectively;
P = 0.238). To measure differences in the
levels of in vitro BPDE-induced DNA adducts in the host
cells, BPDE (a final concentration of 4 µM; see
Fig. 1
for the dose optimization) was added after 67 h of PHA
stimulation, and the incubation was continued for 5 h, which was
sufficient for adduct formation and removal because: (a) the
level of induced adducts peaks in the host cells within 15 min of BPDE
exposure; (b) nearly half of the damage is removed by the
host cells within 5 h; and (c) the level of adducts
remains virtually unchanged thereafter for 24 h (12)
.
This short-term treatment was optimal and also necessary to minimize
reduction in induced DNA adducts because of DNA replication during cell
division. After 72 h of PHA stimulation, the cells were
harvested. The cell pellets were washed three times with
methanol:acetic acid (3:1, v/v) to remove free DNA released by dead
cells and to fix viable cells. The cleaned cell pellets were then
stored in a -20°C freezer until used for DNA extraction.
|
The nuclease P1-enhanced version of the 32P-postlabeling assay (13) was used for the DNA adduct analysis, and 2.5 µg of DNA from each sample were used. All cells and DNA samples were coded during laboratory analysis; therefore, case-control status was not known by the laboratory personnel. The codes were broken at the time of data analysis. An internal standard DNA from mouse skin treated with dibenzo[a,j]acridine was included in each DNA sample to monitor the quality of enzymatic digestion, radioactive labeling, and chromatography. As demonstrated previously (7) , because DNA replication (at least twice) occurred as the cells transmitted the cell cycles, the levels of background in vivo DNA adducts in untreated cells were negligible (100-fold lower) compared with the levels of in vitro BPDE-induced DNA adducts in treated cells. Therefore, only BPDE-treated cells were analyzed. The adduct levels determined by this assay in DNA sample duplicates were highly reproducible, with a coefficient of variance of <15%. The average of the duplicates was used for comparison.
Assay Validation.
The dose-response of four blood samples to 0–8 µM BPDE
is shown in Fig. 1
. In general, the BPDE-adduct level increased as the
dose increased, although for one blood sample there was a fluctuation
for subject 4 that suggested a possible toxic effect at the highest
dose of 8 µM. Therefore, 4 µM was chosen
for the all experiments in this report.
To assure the high quality of the data, 
13% of the samples
(n = 58) were assayed twice. As demonstrated
in Fig. 2
, the repeated measurements were highly correlated
(r = 0.931; P = 0.0001). These results justify measuring each sample only once for this
study. In addition, the background (untreated) level of adducts in
cultured cells from 16 controls had a mean of 2.29 (±SD, 2.29) per
107 nucleotides with a range of 0.15–9.28.
Compared with a mean of >63.7/107 nucleotides
for the treated 229 controls, the background levels were negligible.
These results indicate that use of the adduct levels from BPDE-treated
samples alone did not alter the statistical validity of the study (but
did greatly reduce the assay cost).
|
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The cases and controls were well matched on age (51 and 50% >60
years, respectively), sex (52 and 58% males, respectively), ethnicity
(86 and 85% non-Hispanic whites, respectively), and smoking status (90
and 91% ever-smokers, respectively). Although more cases reported to
have smoked in the last 24 h (26%) and a family history of cancer
in a first-degree relative (67%) compared with controls (20 and 63%,
respectively), these differences were not statistically significant.
However, significantly more cases (45%) had experienced weight loss in
the previous year before diagnosis compared with the controls (21%).
|
). The average pack-years smoked were significantly higher
(51.3 ± 36.9) among the cases than among the controls
(43.6 ± 32.6; P = 0.020;
Table 2
). The cases also had a significantly lower BMI (25.8 ± 4.5) and experienced significant loss of weight
(-0.02 ± 0.13 kg) in the last year than did the
controls (27.2 ± 4.6 and 0.02 ± 0.13
kg, respectively; P = 0.002 and
P = 0.008, respectively; Table 2
).
Furthermore, the levels of the induced DNA adducts were higher in cases
(93.2 ± 89.3/107 nucleotides)
than in controls (63.7 ± 61.1/107
nucleotides), and this difference was statistically significant
(P < 0.001; Table 2
). After logarithmic
transformation of these levels, the log-adduct levels were
1.76 ± 0.50 for cases and 1.57 ± 0.56
for controls, and the difference remained statistically significant
(P < 0.001; Table 2
).
|
. The nonsignificant differences observed between cases and controls in
other subgroups (Hispanics, Blacks, and never-smokers) were probably
attributable to the small numbers of subjects (<25 for each subgroup).
There was no evidence of interactions between these variables and the
level of induced adducts. When subgroup differences in the levels of
DNA adducts within cases and within controls were compared,
respectively, the only statistically significant finding was for age
within the control group (P < 0.001) but not
within the case group (P = 0.213) because of
larger variation (Table 3)
.
|
, and the crude OR associated with a high adduct level was 1.82 (95%
CI, 1.25–2.65). Logistic regression analysis revealed that having a
high level of induced DNA adducts was an independent risk factor for
lung cancer after adjustment for age, sex, smoking status, smoking in
the last 24 h, BMI, weight loss, and history of first-degree
relatives with cancer (OR, 2.23; 95% CI, 1.45–3.44). Furthermore,
when the quartiles of DNA adduct levels in the controls were used as
cutoff points to calculate the ORs, a significant dose-response
relationship between increasing levels of induced DNA adducts and risk
of lung cancer was evident (ORs of 1.11, 1.62, and 3.23; trend test,
P < 0.001) after adjustment for age, sex,
smoking status, smoking in the last 24 h, BMI, weight loss, and
history of first-degree relatives with cancer (Table 4)
.
|
. There were 36 limited stages I-II, 67 regional spread, 55 extensive
spread, and 63 unspecified. Although the cases in each subgroup (except
those with <25 subjects) had significantly higher induced DNA adduct
levels than did the controls, there were no significant differences in
the levels of DNA adducts between different histopathological types and
clinical stages (data not shown). For example, patients with the large
cell carcinoma had the highest level of adducts (mean ± SD, 114.3 ± 98.0/107
nucleotides), whereas those with the SCCs had the lowest level of
adducts (80.3 ± 73.4/107
nucleotides), but the difference was not statistically significant
(P = 0.161). These findings suggest that
in vitro induced DNA adducts are a constitutional rather
than a tumor-specific marker.
|
. It is clear that the level of induced DNA adducts remained an
independent predictor (P < 0.001) of lung
cancer risk in the presence of other variables, although pack-years of
smoking, BMI, and weight loss were also independent predictors of risk
in the same model.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Because BP is an abundant carcinogen present in cigarette smoke, sensitivity to BPDE may have a significant implication in risk assessment of tobacco-related cancers. Because BPDE, the active form of BP, is used in this assay, the main determinants of the level of induced adducts are carcinogen detoxification and DNA repair capacities of the test cells. Although the initial level of BPDE-induced DNA adducts was not measured in this study (because of technical difficulty when a large number of samples are tested), DNA binding of BPDE usually peaks within 15 min of exposure (12) . Because we previously reported a significant correlation between reduced DNA repair capacity and higher level of BPDE-induced DNA adducts (21) , we believe that variation in cellular DNA repair capacity may be the major underlying mechanism for the difference in in vitro induced DNA-adduct levels between cases and controls.
Early age of onset of disease is a hallmark of genetic predisposition, which can result in high sensitivity to low levels of exposure (22) . Indeed, our results showed that younger cases tended to have higher levels of BPDE-induced DNA adducts than older cases and controls, although there was no clear evidence of interaction between age and level of the adducts. The significant high level of BPDE-induced DNA adducts observed in younger cases suggests a reduced DNA repair capacity in young cancer patients, as reported previously (23) . It is documented that women have higher risks of developing smoking-related cancer than men at similar levels of exposure (24) . However, we did not find a significant sex difference in the sensitivities to BPDE-induced DNA damage.
In a pilot study of lung cancer, we found a tendency for higher levels of BPDE-induced adducts in nonsmoking cases than smoking cases (7) . This trend was observed in the present study as well. Although the difference was not statistically significant, possibly because of the small number of nonsmokers studied, this trend suggests the importance of genetic background in cancer risk among individuals with no or low levels of carcinogen exposure. An early study showed that younger lung cancer patients with a family history of cancer had a significantly higher level of DNA adducts in their monocytes exposed in vitro to BP than did patients with no family history (22) . This difference suggests a carcinogen sensitivity-related genetic predisposition to lung cancer. However, our study did not reveal any significant difference in the levels of BPDE-induced DNA adducts between individuals with or without family history of cancer. This observation suggests that genetic differences in biological functions other than nucleotide excision repair are also involved in inherited predisposition to lung cancer.
Although this in vitro BPDE-induced adduct assay may be useful in risk assessment of lung cancer, it has some limitations. One is the use of lymphocytes, the repair of which may not reflect that of lung epithelial cells. However, it has been demonstrated that the levels of smoking-induced DNA adducts in lymphocytes and lung are significantly correlated (25) . Therefore, lymphocytes are a relevant and very accessible surrogate tissue for the lung. In this study, we assumed that if this assay measures DNA repair capacity that is genetically determined (as seen in xeroderma pigmentosum) and even tissue specificity exists, the individuals’ levels of in vitro BPDE-induced adducts can be compared in the same type of tissue such as lymphocytes. Nevertheless, these assumptions could not be validated in this study. Although the BPDE-induced adduct level is probably determined by the host cells’ DNA repair capacity, the fact that we did not measure the initial level of BPDE-induced adducts excludes the possibility of precisely measuring the repair rate. Furthermore, the large variation in the measurements suggests that this assay should be used for research purposes rather than diagnosis.
Another limitation of this study is that it was a retrospective study; therefore, we cannot rule out that there was a systemic effect on the level of BPDE-induced adducts in lung cancer patients. A tested case-control study would validate the findings in this study. Although it is possible that smoking and overall sickness of the lung cancer patients lowered their DNA repair levels, our detailed analyses of the effect of smoking and weight loss as well as disease stage did not support this possibility. The uniform concentration and time of in vitro BPDE exposure, uniform assay conditions, and the high level of BPDE-induced adducts in the treated cells measured by the sensitive 32P-postlabeling method also minimize the possibility of artifacts in the measurements. Our consistent findings of statistically significant differences between relatively large numbers of cases and controls and the dose-response relationship between the levels of BPDE-induced adducts and risk of lung cancer are probably not attributable to chance. Furthermore, the findings in this study are consistent with those from another plasmid-based transfection (host-cell reactivation) assay we reported recently, in which consistently poorer repair of BPDE-induced adducts in the plasmids was evident in lung cancer patients compared with controls (26) . These two assays should be complementary in defining DNA repair capacity in that the in vitro BPDE-induced adduct assay measures the repair of direct damage to genomic DNA, and the host-cell reactivation assay measures the repair of fixed damage to foreign (plasmids) DNA that have been transfected into the cells (27) . The consistent results of these two assays in lung cancer patients further strengthen biological plausibility of our preliminary findings.
There is a growing body of evidence that reduced DNA repair is associated with increased cancer risk. Individuals with defective DNA repair have a >1000-fold increased risk for developing cancer as seen in xeroderma pigmentosum patients with skin cancer who have defective nucleotide excision repair genes (28) ; patients with hereditary nonpolyposis colon cancer who have defective mismatch repair genes (29) ; and patients with breast cancer who have defective BRCA1 and BRCA2 (30) . This etiological association between deficient DNA repair and increased cancer risk is also supported by evidence from studies of sporadic cancers of the skin (23) , lung (26) , colon (31) , and breast (32) . This study presented the analysis of the main effect of the level of in vitro BPDE-induced DNA adducts as an independent risk factor. Because increased cancer risk is well documented in patients with genetically inherited defective DNA repair, our findings are biologically plausible. However, this assay alone may not provide a risk profile as complete as is needed in risk assessment. Further analyses of this biomarker in combination with other genetic polymorphisms such as GSTM1, GSTT1, and GSTP1 and DNA repair genes that may modulate the level of induced adducts will provide further information on the interaction between genes and BPDE exposure. Using a battery of complementary assays for genetic susceptibility to cancer may increase our ability to identify individuals at high risk and further our understanding of the underlying mechanisms of smoking-related carcinogenesis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This investigation was supported by NIH Grants
CA 74851 and CA 70334 (to Q. W.), CA 55769 (to M. R. S.), and CA
68437 (to W. K. H.). 
2 To whom requests for reprints should be
addressed, at Department of Epidemiology, Box 189, The University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,
TX 77030. Phone: (713) 792-3020; Fax: (713) 792-0807; E-mail: qwei{at}mdanderson.org
3 The abbreviations used are: BP,
benzo(a)pyrene; BPDE, BP diol epoxide; NSCC, non-small
cell carcinoma; SCC, small cell carcinoma; PHA, phytohemagglutinin; OR,
odds ratio; CI, confidence interval; BMI, body mass index; GST,
glutathione S-transferase.
Received 4/27/00. Accepted 12/13/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  L. Zhou, Y. Jiang, A. Tan, A. R. Greenlee, Y. Shen, L. Liu, and Q. Yang Silencing of N-Ras Gene Expression Using shRNA Decreases Transformation Efficiency and Tumor Growth in Transformed Cells Induced by Anti-BPDE Toxicol. Sci., October 1, 2008; 105(2): 286 - 294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhao, L.-E Wang, D. Li, R. M. Chamberlain, E. M. Sturgis, and Q. Wei Genotypes and haplotypes of ERCC1 and ERCC2/XPD genes predict levels of benzo[a]pyrene diol epoxide-induced DNA adducts in cultured primary lymphocytes from healthy individuals: a genotype-phenotype correlation analysis Carcinogenesis, August 1, 2008; 29(8): 1560 - 1566. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Orlow, B. J. Park, U. Mujumdar, H. Patel, P. Siu-Lau, B. A. Clas, R. Downey, R. Flores, M. Bains, N. Rizk, et al. DNA Damage and Repair Capacity in Patients With Lung Cancer: Prediction of Multiple Primary Tumors J. Clin. Oncol., July 20, 2008; 26(21): 3560 - 3566. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-E Wang, P. Xiong, H. Zhao, M. R. Spitz, E. M. Sturgis, and Q. Wei Chromosome Instability and Risk of Squamous Cell Carcinomas of Head and Neck Cancer Res., June 1, 2008; 68(11): 4479 - 4485. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Marin, M. F. Lopez-Cima, L. Garcia-Castro, T. Pascual, M. G. Marron, and A. Tardon Poly (AT) Polymorphism in Intron 11 of the XPC DNA Repair Gene Enhances the Risk of Lung Cancer Cancer Epidemiol. Biomarkers Prev., November 1, 2004; 13(11): 1788 - 1793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ito, K. Matsuo, N. Hamajima, T. Mitsudomi, T. Sugiura, T. Saito, T. Yasue, K.-M. Lee, D. Kang, K.-Y. Yoo, et al. Gene-environment interactions between the smoking habit and polymorphisms in the DNA repair genes, APE1 Asp148Glu and XRCC1 Arg399Gln, in Japanese lung cancer risk Carcinogenesis, August 1, 2004; 25(8): 1395 - 1401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Feng, W. Hu, and M.-s. Tang Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: A possible mechanism for lipid peroxidation-induced carcinogenesis PNAS, June 8, 2004; 101(23): 8598 - 8602. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Caporaso The Molecular Epidemiology of Oxidative Damage to DNA and Cancer J Natl Cancer Inst, September 3, 2003; 95(17): 1263 - 1265. [Full Text] [PDF]
|
|
|
|
|
|
S. Qiuling, Z. Yuxin, Z. Suhua, X. Cheng, L. Shuguang, and H. Fengsheng Cyclin D1 gene polymorphism and susceptibility to lung cancer in a Chinese population Carcinogenesis, September 1, 2003; 24(9): 1499 - 1503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G E Goodman Lung cancer * 1: Prevention of lung cancer Thorax, November 1, 2002; 57(11): 994 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Li, F. L. Walcott, P. Chang, W. Zhang, J. Zhu, E. Petrulis, S. E. Singletary, A. A. Sahin, and M. L. Bondy Genetic and Environmental Determinants on Tissue Response to in Vitro Carcinogen Exposure and Risk of Breast Cancer Cancer Res., August 15, 2002; 62(16): 4566 - 4570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Xiong, M. L. Bondy, D. Li, H. Shen, L.-E Wang, S. E. Singletary, M. R. Spitz, and Q. Wei Sensitivity to Benzo(a)pyrene Diol-Epoxide Associated with Risk of Breast Cancer in Young Women and Modulation by Glutathione S-Transferase Polymorphisms: A Case-Control Study Cancer Res., December 1, 2001; 61(23): 8465 - 8469. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
