• bladder cancer;
  • XRCC1;
  • XRCC3;
  • XPD;
  • DNA adducts


  1. Top of page
  2. Abstract

Individuals differ widely in their ability to repair DNA damage, and DNA-repair deficiency may be involved in modulating cancer risk. In a case-control study of 124 bladder-cancer patients and 85 hospital controls (urological and non-urological), 3 DNA polymorphisms localized in 3 genes of different repair pathways (XRCC1-Arg399Gln, exon 10; XRCC3-Thr241Met, exon 7; XPD-Lys751Gln, exon 23) have been analyzed. Results were correlated with DNA damage measured as 32P-post-labeling bulky DNA adducts in white blood cells from peripheral blood. Genotyping was performed by PCR-RFLP analysis, and allele frequencies in cases/controls were as follows: XRCC1-399Gln = 0.34/0.39, XRCC3-241Met = 0.48/0.35 and XPD-751Gln = 0.42/0.42. Odds ratios (ORs) were significantly greater than 1 only for the XRCC3 (exon 7) variant, and they were consistent across the 2 control groups. XPD and XRCC1 appear to have no impact on the risk of bladder cancer. Indeed, the effect of XRCC3 was more evident in non-smokers [OR = 4.8, 95% confidence interval (CI) 1.1–21.2]. XRCC3 apparently interacted with the N-acetyltransferase type 2 (NAT-2) genotype. The effect of XRCC3 was limited to the NAT-2 slow genotype (OR = 3.4, 95% CI 1.5–7.9), suggesting that XRCC3 might be involved in a common repair pathway of bulky DNA adducts. In addition, the risk of having DNA adduct levels above the median was higher in NAT-2 slow acetylators, homozygotes for the XRCC3-241Met variant allele (OR = 14.6, 95% CI 1.5–138). However, any discussion of interactions should be considered preliminary because of the small numbers involved. Our results suggest that bladder-cancer risk can be genetically modulated by XRCC3, which may repair DNA cross-link lesions produced by aromatic amines and other environmental chemicals. © 2001 Wiley-Liss, Inc.

A potentially important source of interindividual variability in response to carcinogens is DNA-repair capability. Apart from rare recessive inherited syndromes such as ataxia-telangiectasia, Fanconi's anemia and Bloom's syndrome, all of which are characterized by both chromosomal instability and high risk of cancer, and xeroderma pigmentosum, characterized by extreme susceptibility to UV-induced skin cancer,1 individuals differ widely in the ability to repair DNA damage.2 Polymorphisms in DNA-repair genes have been described.3, 4 Their role in modulating the risk of environmentally induced cancer is potentially important but has been studied only occasionally. In particular, XPD, XRCC1 and XRCC3 have been proposed as polymorphic genes that might be involved in environmental carcinogenesis.5–10

The XRCC1 protein is involved in the base excision-repair pathway,11 acting apparently as a scaffold protein, facilitating the repair reaction by binding DNA ligase III at its carboxy and DNA polymerase β to its amino terminus.12 XRCC3 participates in DNA double-strand breaks/recombinational repair and belongs to an emerging family of Rad51-related proteins that participate in homologous recombination (HR) to maintain chromosomal stability.13, 14 It is likely that removal of interstrand cross-links is strongly dependent on efficient HR repair, as suggested by the extraordinary sensitivity of the ERCC1, XPF/ERCC4, XRCC2 and XRCC3 mutants to cross-linking chemicals.15 XRCC3 interacts directly with HsRad51 and, like Rad55 and Rad57 in yeast, may co-operate with HsRad51 during recombinational repair.16 XPD is involved in the nucleotide excision repair (NER) pathway, which recognizes and repairs a wide range of structurally unrelated lesions such as bulky adducts and thymidine dimers.17, 18 XPD functions as an ATP-dependent 5′–3′ helicase joint to the basal transcription factor IIH complex.19 Two of the polymorphisms analyzed, XRCC3-T241M and XRCC1-R399Q, are non-conservative amino acid changes. The XPD-Lys751Gln polymorphism is a conservative substitution which does not reside in known or hypothesized helicase/ATPase domains.

A high frequency of chromosomal instability or aberrations has been reported in bladder cancer. Aberrations in chromosomes 8 and 11 and allelic losses on chromosomes 4, 8, 9, 11 and 17 have been described.20–24

The risk of bladder cancer can be modulated by genetic metabolic polymorphisms. GSTM1 is polymorphic25 and plays a role in the metabolism of organic epoxides and peroxides; in particular, it conjugates known carcinogens such as epoxides of polycyclic aromatic hydrocarbons (PAHs). Other GSTs potentially relevant to the risk of bladder cancer are T1 and P1. N-Acetyltransferase-2 (NAT-2) differentially acetylates arylamines, well-known bladder carcinogens, to arylamides. Slow acetylators are at higher risk for developing bladder cancer.25

Previously, we studied the association between the risk of bladder cancer and white blood cell (WBC)–DNA adducts, including modulation by consumption of fruit and vegetables.26, 27 In the present study, we have analyzed 3 DNA-repair polymorphisms and their interaction with WBC–DNA adducts in 124 bladder-cancer cases and 85 controls.


  1. Top of page
  2. Abstract


We performed a hospital-based case-control investigation at the urology departments of 2 Turin hospitals (Gradenigo and S. Giovanni Battista), where about half the cases of newly diagnosed bladder cancer in the Turin metropolitan area are treated. The men making up the case group were aged 45 to 74 years, resident in the Turin metropolitan area and treated from 1994 to 1996 for histologically confirmed bladder cancer. All were incident (newly diagnosed) cases. Cases were identified by daily contact between a trained interviewer and the urology departments. Histological confirmation was obtained from the pathology departments. Controls were recruited daily in random fashion (i) from patients treated at the same urology departments for benign diseases, mainly prostatic hyperplasia and cystitis (all newly diagnosed), and (ii) from patients treated at the medical and surgical departments for hernias, vasculopathies, diabetes, heart failure, asthma or other benign diseases (none was represented in >10% of controls). Patients with cancer, liver or renal diseases and smoking-related conditions were excluded. Like the cases, controls were men aged 40 to 74 years and living in the Turin metropolitan area. Before therapy began, a trained interviewer used a standard questionnaire to interview cases and controls on history of tobacco smoking (including brands and tobacco type) and a 24 hr recall interview to collect information about dietary habits, drug use and occupational history. Before therapy and after informed consent, blood was collected from cases and controls.

DNA analysis

WBC DNA was isolated and purified from stored buffy coats by enzymatic digestion of RNA and proteins, followed by phenol-chloroform extraction. WBC-DNA adduct levels were measured using the 32P-DNA post-labeling technique, as described previously.26, 27 DNA-adduct analyses were carried out at the Istituto Nazionale per la Ricerca sul Cancro (Genoa, Italy). Three known slow-acetylator alleles (NAT2*5A/5B, NAT2*6A/6B and NAT2*7A/7B) were identified as described,26, 27 with slight modifications. Rapid-acetylator genotypes are wild-type allele homo-/heterozygotes; slow-acetylator genotypes are those with 2 slow-acetylator alleles.28

PCR followed by enzymatic digestion was also used for genotyping of XRCC1-Arg399Gln, XPD-Lys751Gln and XRCC3-Thr241Met polymorphisms. All PCRs were performed in a total reaction volume of 20 μl containing 10 ng genomic DNA, 0.4 units of Taq polymerase (Perkin-Elmer Applied Biosystems, Foster City, CA) in PCR buffer 1×, 1.5 mM MgCl2, 50 mM dNTPs and 250 nM of each primer. Thermal cycling conditions were as follows: initial denaturation step at 95°C for 3 min; 35 cycles of PCR consisting of 95°C for 20 sec, 20 sec at the appropriate annealing temperature and 72°C for 20 sec; then a final extension step at 72°C for 5 min. The XRCC1-Arg399Gln polymorphism, a transition G[RIGHTWARDS ARROW]A in exon 10 (position 28152), was determined using the primers (sense) 5′-CAAGTACAGCCAGGTCCTAG-3′ and (anti-sense) 5′-ccttccctca tctggagtac-3′ at 55°C annealing temperature for PCR. The 248 bp PCR product was digested with NciI (Promega, Madison, WI): the Arg allele was cut into 89 and 159 bp fragments (Gln allele not digested). The XPD-Lys751Gln polymorphism, a transversion A[RIGHTWARDS ARROW]C in exon 23 (position 35931), was determined using the primers (sense) 5′-CTGCTCAGCCTGGAGCAGCTAGA ATCAGAGGAGACGCTG-3′ and (anti-sense) 5′-AAGACCTTCTAGCACCACCG-3′ at 67°C annealing temperature for PCR. The 161 bp PCR product was digested with PstI (Promega): the Gln allele was cut into 41 and 120 bp fragments (Lys allele not digested). The XRCC3-Thr241Met polymorphism, a transition C[RIGHTWARDS ARROW]T in exon 7 (position 18067), was determined using the primers (sense) 5′-GCCTGGTGGTCATCGACTC-3′ and (anti-sense) 5′-ACAGGGCTCTGGAAGGCACTGCTCAGC TCACGCACC-3′ (underlined base modifies primer sequence introducing a cut site in the presence of the Met allele) at 60°C annealing temperature for PCR. The 136 bp PCR product was digested with NcoI (Promega): the Met allele was cut into 39 and 97 bp fragments (Thr allele not digested).

DNA typing quality control

Methodological validation included a comparison between PCR-RFLP, direct sequencing and denaturing high-performance liquid chromatography (DHPLC).29 Due to the small amount of DNA available, we checked the accuracy of PCR-RFLP genotyping for the 3 polymorphisms using the primer extension technique with DHPLC (Transgenomic, Santa Clara, CA) on a set of 50 individuals from a cardiovascular study, and complete agreement in the typing was obtained. The primer extension technique30 is based on specific incorporation of the complementary dideoxynucleotide (ddNTP) into the base substitution; the rate of right incorporation completely overwhelms ddNTP misincorporations, and heterozygotes are easily detectable. The following primers flanking the base substitution were used: XRCC1-Arg399Gln, 5′-cggcggctgccctccc-3′; XRCC3-Thr241Met, 5′-GGCATCTGCAGTCCCTGGGGGCCA-3′; XPD-Lys751Gln, 5′- ATCTGCTCTATCCTCT-3′. Due to the results described below for XRCC3, we decided to re-analyze the XRCC3-Thr241Met polymorphism by DHPLC in the whole sample (124 cases and 85 controls). All DHPLC typings confirmed the PCR-RFLP results.

Statistical analysis

Odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were computed with the SAS (Cary, NC) package for personal computers. Unconditional logistic regression models were fitted. Age was not associated with the distribution of polymorphisms, and age-adjusted ORs were identical to unadjusted estimates. Since we noticed in a previous analysis that the 2 control groups (urological and non-urological) had different levels of DNA adducts,31 we analyzed them separately. In addition, we performed a “case-only analysis” to avoid bias related to the choice of the control group. Case-only studies have proven to be a valid design to test gene–environment and gene–gene interactions, with accurate and precise estimates of association.32 A case-only approach is justified if the variables considered for interaction are independent in the control series.

The size of the study was sufficient to detect an OR of 2.3 with α = 0.05 and β (type-II error) = 20%, assuming a frequency of the relevant allele of 50% (XRCC3-241 exon 7, Thr/Thr genotype).


  1. Top of page
  2. Abstract

We recruited overall 162 bladder-cancer cases and 104 controls, who provided biological samples. All were interviewed except 2 cases and 2 controls who refused and 3 cases who were too ill to answer the questions. We also interviewed 42 cases who did not provide biological samples. Although they did not differ from the others on clinical or pathological traits or smoking habits,27 they were excluded from the present analysis nonetheless. DNA was of a sufficient amount to perform analyses of DNA-repair gene polymorphisms for 124 cases and 85 controls. The following data refer only to these subjects. Table I gives information about the main characteristics of cases and controls. Smoking habits were analyzed in a previous report.26

Table I. Distribution of Bladder-Cancer Cases and Controls by Relevant Characteristics
 Controls(n = 85)Cases(n = 124)
Age groups (years)
 45–5420 (23%)17 (14%)
 55–6434 (40%)45 (36%)
 65–6920 (23%)36 (29%)
 70–7411 (13%)26 (21%)
 χ2 5.33 (p = 0.15)
Smoking habits
 Never smoked38 (45%)13 (10%)
 Ex-smoker22 (26%)37 (30%)
 Current smoker25 (29%)74 (60%)
 χ2 34 (p = 0.01)
Number of cigarettes smoked (highest amount throughout life)
 038 (45%)13 (10%)
 1–1511 (13%)25 (20%)
 15–2924 (28%)58 (47%)
 30+12 (14%)28 (23%)
 χ2 32 (p = 0.001)
Histological grade
 117 (14%)
 248 (39%)
 329 (23%)
 Unknown30 (24%)

No polymorphism showed deviations from the Hardy-Weinberg equilibrium (HWE) in either cases or controls, except XRCC3 in controls (χmath image = 7.61, p ≤ 0.01), though the XRCC3 allele frequencies in our control group (Met = 0.35 and Thr = 0.65) were comparable with those described by Shen et al.3 (Met = 0.38 and Thr = 0.62). Specifically, the non-urological control group was not in HWE (χmath image = 7.83, p ≤ 0.01), whereas the urological control group was (χmath image = 0.83, p > 0.05). There appears to be no biological or functional explanation for this result. We calculated the heterogeneity between samples as G2 (het.) = 7.30, p ≤ 0.01. Even when the deviation from HWE was taken into account (see Appendix), the difference remained statistically significant.

In the whole control group, the XRCC1-399Gln variant frequency (0.39) was similar to that reported by Lunn et al.7 for the Caucasoid population (0.37) but different from that estimated by Shen et al.3 analyzing DNA sequences in 12 individuals (0.25). The XPD-751Gln frequency was higher (0.42) than that estimated by Shen et al.3 in the United States (0.29) and that estimated by Broughton et al.5 in England (0.30) but similar to that estimated by Duell et al.10 in the United States (0.39). Allele frequencies in the case group were as follows: XRCC1-399Arg/Gln = 0.66/0.34, XRCC3-241Thr/Met = 0.52/0.48 and XPD-751Lys/Gln = 0.58/0.42.

The NAT-2 genetic polymorphism was associated with the risk of bladder cancer, with a statistically significant OR of 1.72 (95% CI 1.03–2.87) for slow acetylators compared with rapid acetylators. This was the only metabolic polymorphism among those analyzed that showed a statistically significant association with bladder cancer.26

Table II shows genotype distributions for the 3 DNA-repair gene polymorphisms analyzed (XRCC1-Arg399Gln, exon 10; XRCC3-Thr241Met, exon 7; XPD-Lys751Gln, exon 23). The results are shown separately for the 2 control groups, urological and non-urological. ORs are significantly greater than 1 only for XRCC3 (exon 7), and they are consistent across the 2 control groups. Smoking habits appear to play a modulating role since the effect of the XRCC3 polymorphism was twice as high in non-smokers than in smokers (Table III) (however, the difference among smoking categories was not statistically significant: χmath image for heterogeneity = 1.046, p = 0.593). No significant difference in bulky DNA-adduct levels was observed for XRCC1-Arg399Gln and XPD-Lys751Gln polymorphisms when stratifying by smoking status (Table III).

Table II. Case-Control Study on Bladder Cancer (124 Cases, 85 Controls): Distribution by Selected DNA-Repair Polymorphisms1
 CasesUrological controlsNon-urological controls
  • 1

    ORs are for Arg/Gln + Gln/Gln vs. Arg/Arg (XRCC1), for Thr/Met + Met/Met vs. Thr/Thr (XRCC3) and for Lys/Gln + Gln/Gln vs. Lys/Lys (XPD), separately for the 2 control groups. p values refer to 3 × 3 contingency table.

XRCC1, exon 10
 Arg/Arg53 (43%)12 (33%)19 (40%)
 Arg/Gln58 (47%)19 (51%)22 (46%)
 Gln/Gln13 (10%)6 (16%)6 (14%)
 Arg/Gln + Gln/Gln72 (57%)25 (67%)28 (60%)
 OR (95% CI)0.60 (0.21–1.71)0.80 (0.28–2.25)
 p = 0.69 (1 missing value)
XRCC3, exon 7
 Thr/Thr33 (26%)19 (50%)23 (49%)
 Thr/Met64 (52%)14 (37%)13 (28%)
 Met/Met27 (22%)5 (13%)11 (23%)
 Thr/Met + Met/Met91 (74%)19 (50%)24 (51%)
 OR (95% CI)2.84 (1.34–6.04)2.72 (1.37–5.43)
 OR (95% CI), controls combined2.77 (1.55–4.93)
 p = 0.007
 Lys/Lys39 (32%)12 (32%)12 (26%)
 Lys/Gln66 (53%)23 (60%)27 (57%)
 Gln/Gln19 (15%)3 (8%)8 (17%)
 Lys/Gln + Gln/Gln85 (68%)26 (68%)35 (74%)
 OR (95% CI)0.94 (0.42–2.10)0.71 (0.32–1.58)
 p = 0.69
Table III. DNA-Repair Genotype Distribution of 124 Bladder Cancer Cases and 85 Controls by Smoking Habits1
 Current smokersEx-smokersNon-smokers
  • 1

    Means ± SE of DNA-adduct levels are reported as relative adduct labeling × 108 for cases/controls. ORs are for subjects with at least a variant allele vs. wild-type homozygotes. ORs are adjusted by age.

XRCC1, exon 10
 Arg/Arg31 (0.55 ± 0.10)7 (0.31 ± 0.18)16 (0.33 ± 0.06)5 (0.15 ± 0.08)6 (0.29 ± 0.10)19 (0.13 ± 0.05)
 Arg/Gln + Gln/Gln43 (0.55 ± 0.08)17 (0.26 ± 0.07)21 (0.30 ± 0.06)17 (0.26 ± 0.13)7 (0.55 ± 0.15)19 (0.17 ± 0.04)
 OR (95% CI)0.43 (0.15–1.25)0.27 (0.07–1.02)1.05 (0.29–3.89)
XRCC3, exon 7
 Thr/Thr18 (0.54 ± 0.11)10 (0.22 ± 0.10)13 (0.31 ± 0.16)13 (0.37 ± 0.08)2 (0.28 ± 0.06)19 (0.11 ± 0.04)
 Thr/Met + Met/Met56 (0.55 ± 0.08)15 (0.29 ± 0.09)24 (0.28 ± 0.05)9 (0.12 ± 0.05)11 (0.45 ± 0.11)19 (0.18 ± 0.05)
 OR (95% CI)1.8 (0.7–4.8)5.2 (1.2–21.5)4.8 (1.1–21.2)
XPD, exon 23
 Lys/Lys24 (0.55 ± 0.11)13 (0.16 ± 0.04)10 (0.28 ± 0.08)4 (0.63 ± 0.53)5 (0.43 ± 0.15)7 (0.13 ± 0.08)
 Lys/Gln + Gln/Gln50 (0.55 ± 0.08)12 (0.37 ± 0.13)27 (0.32 ± 0.05)18 (0.15 ± 0.04)8 (0.43 ± 0.14)31 (0.15 ± 0.04)
 OR (95% CI)2.53 (0.92–6.96)0.60 (0.14–2.68)0.36 (0.09–1.55)

Table IV shows that XRCC3 apparently interacts with the NAT genotype. Although based on small numbers, Table IV shows that the effect of XRCC3 is limited to the NAT-2 slow genotype, suggesting that XRCC3 might be involved in a common repair pathway of bulky DNA adducts. In a logistic regression model including age, XRCC3 (exon 7, dichotomized as Thr/Thr vs. Thr/Met+Met/Met), NAT-2 and an interactive term for XRCC3 and NAT-2, ORs were 1.38 (95% CI 0.60–3.17) for XRCC3, 1.09 (95% CI 0.52–2.29) for NAT-2 and 2.28 (95% CI 0.78–6.66) for the interactive term. The effect of XRCC3 was present also in the case-only analysis, which was not influenced by the choice of controls, and when DNA adducts in WBCs were considered in relation to the NAT-2 and XRCC3 genotypes. In a logistic regression model including a 3-way interactive term (based on NAT-2, the median of DNA adducts and dichotomized XRCC3), ORs were 1.42 (95% CI 0.64–3.13) for XRCC3, 0.95 (95% CI 0.47–1.92) for NAT-2, 3.17 (95% CI 1.77–5.69) for the median of DNA adducts and 1.81 (95% CI 0.92–3.56) for the interactive term. Since intake of fruit and vegetables modified the association between DNA adducts and bladder cancer, we also included this variable in the analyses, but there was no change in the estimates.

Table IV. Distribution of 124 Bladder Cancer Cases and 85 Controls by the XRCC3 (Exon 7) Polymorphism, NAT-2 and DNA-Adduct Levels Above/Below Median1
Case-control analysis: Cases/controls (OR and 95% CI in parentheses)
XRCC3NAT-2 rapidOR (95% CI)NAT-2 slowOR (95% CI)NAT-2 rapid2NAT-2 slow2
ABOR (95% CI)ABOR (95% CI)
Thr/Thr15/171.018/251.08/57/122.7 (0.6–11.7)11/87/173.3 (0.9–11.8)
Thr/Met18/151.4 (0.5–3.6)45/125.2 (2.2–12.2)8/410/112.2 (0.5–9.6)32/513/73.4 (0.9–12.8)
Met/Met6/61.1 (0.3–4.3)21/102.9 (1.1–7.6)3/33/31.0 (0.1–9.6)13/18/914.6 (1.5–138)
Thr/Met + Met/Met24/211.3 (0.5–3.2)66/223.4 (1.5–7.9)11/713/142.2 (0.6–8.5)45/621/165.2 (1.8–15.1)
Case-only analysis
XRCC3NAT-2 rapidNAT-2 slowOR (95% CI)NAT-2 rapid3NAT-2 slow3OR (95% CI)
  • 1

    ORs are adjusted by age. Adducts are expressed as relative adduct labeling × 108; the overall median (0.23) was used as threshold for the case-control analysis and the median in cases (0.33), as the threshold for the case-only analysis. One missing value.

  • 2

    DNA adducts: A, above; B, below median, cases/controls; OR, above vs. below median.

  • 3

    DNA adducts: A, above; B, below median (median in cases 0.33). OR = above vs. below median, reference group rapid acetylators.

Thr/Thr15181.0871081.1 (0.3–4.3)
Thr/Met18452.1 (0.9–5.0)71124211.8 (0.6–5.5)
Met/Met6212.9 (0.95–9.1)2411102.2 (0.3–14.9)
Thr/Met + Met/Met24662.25 (1.0–4.9)91535311.9 (0.7–4.9)


  1. Top of page
  2. Abstract

In a case-control study, we found a statistically significant association between the Thr241Met polymorphism of the XRCC3 gene, involved in DNA repair, and the risk of bladder cancer. The association was particularly apparent among those with the slow-acetylator genotype and among non-smokers. In addition, increased levels of bulky DNA adducts in WBCs were more frequent in bladder-cancer patients with the XRCC3-241Met variant who were slow acetylators.

XRCC3 participates in DNA double-strand break and cross-link repair through homologous recombination and contributes, as other RAD51-related proteins, to the maintenance of chromosomal stability.13, 14, 33 One study reported an association between a rare microsatellite polymorphism in the XRCC3 gene and cancer in patients with varying radiosensitivity.6 The Thr241Met substitution in XRCC3 is a non-conservative change with possible biological implications for the functionality of the enzyme and/or for the interaction with other proteins involved in DNA repair.

The association between bulky DNA-adduct formation and XRCC3 Thr/Met and Met/Met genotypes (particularly in the slow NAT-2 group) may be related to environmental exposure to genotoxic aromatic amines, such as trans-4-dimethylaminostilbene and 4-trans-acetylaminostilbene,34 which are capable of forming DNA adducts to guanine and adenine and of inducing other secondary lesions of equal or greater importance, e.g., cross-links between bases. 4-Aminostilbene has been reported to induce high levels of chromosomal aberrations.35 The association between DNA adducts and the XRCC3 polymorphism may also be due to oxidation reactions, which might cause formation of intrastrand cross-links between adjacent nucleotides, leading to bulky oxidative DNA modification, i.e., dimer formation, detectable by 32P-DNA post-labeling.36

In studies on tobacco use, smoking has been clearly associated with the risk of bladder cancer, yet no relationship between smoking and WBC-DNA adduct levels (p > 0.05) was observed in the present study.26 The level of DNA adducts was strongly associated with case/control status.27 The age-adjusted OR associated with an adduct level above the limit of detection was 3.7 (95% CI 2.2–6.3), and a dose-response relationship with adduct levels was apparent.27

Surprisingly, the association between bladder cancer and the XRCC3 polymorphism was higher in non-smokers and ex-smokers (Table III), which is difficult to explain but consistent with observations made on the association between DNA adducts and DNA-repair polymorphisms in a group of healthy subjects (data not shown). A potential explanation is that smoking induces DNA-repair enzymes so that the difference in adduct levels observed in peripheral leukocytes among genotypes is overcome in current smokers, whereas the difference becomes detectable in non-smokers.

Our results concerning bulky DNA adducts are rather surprising because such adducts are known to undergo NER rather than recombinational repair. This might means that an important fraction of cross-links could be detectable by the 32P-post-labeling36 and/or that XRCC3 can lower DNA-adduct levels by acting on different repair pathways.

If our findings are correct, a high frequency of chromosomal instability or aberrations induced by carcinogens in bladder cancer is to be expected. Indeed, a number of studies have shown aberrations and allelic losses in different chromosomes,20–24 so bladder cancer can be considered one of the cancers prone to chromosomal instability.

Our study has some limitations. Specifically, statistical power was relatively limited and the HWE was not met in 1 of the control groups (surgical controls). The latter problem might be explained by selection bias. However, the association with XRCC3 was statistically significant when using either control group; also, the response rate was very high, both control groups were heterogeneous (with few subjects having the same diagnosis) and there was no evidence of selection bias. In addition, the association with the XRCC3 polymorphism was confirmed in a case-only analysis, i.e., excluding controls. Despite limited power, 95% CIs were reasonably narrow. Multiple comparisons were not a major problem in this study since we looked at 3 polymorphisms, based on a priori choice.

In conclusion, the XRCC3-241Met variant can be associated with human bladder cancer, showing an interaction with the NAT-2 gene. However, any discussion of interactions should be considered preliminary because of the small numbers involved. Our results suggest the need to further investigate possible synergistic effects between DNA repair and metabolic genetic polymorphisms in modulating cancer risk.


  1. Top of page
  2. Abstract

We tested for deviations from the HWE using the G2 statistic:37

  • equation image

This statistic is used to test whether an observed number is significantly different from the number expected on the basis of a specific hypothesis (e.g., HWE), and it is distributed as a χ2 statistic. When phenotypes (p) are compared in a number of samples (n), the heterogeneity between their gene frequencies (g), which takes into account the effect of the deviation from HWE, can be written as follows:

  • equation image

where G2 (T) with (p – 1)(n – 1) degrees of freedoms (df) tests for the difference of phenotypes as the usual (p × n) contingency table; G2 (H.W.) with (p – g) df tests for the deviation from HWE in the total sample formed by pooling n samples; G2 (Shw) with n(p – g) df is the sum of n tests of deviation from HWE calculated in each sample; and G2 (het.) with (g – 1)(n – 1) tests for the heterogeneity of the gene frequencies (g) between n samples.


  1. Top of page
  2. Abstract
  • 1
    Friedberg EC, Walker GC, Siede W. DNA repair and mutagenesis. Washington DC: ASM Press, 1995.
  • 2
    Berwick M, Vineis P. Markers of DNA repair and susceptibility to cancer in humans: an epidemiologic review. J Natl Cancer Inst 2000;92: 87497.
  • 3
    Shen MR, Jones IM, Mohrenweiser H. Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair in healthy humans. Cancer Res 1998;58: 6048.
  • 4
    Kaur TB, Travaline JM, Gaughan JP, Richie JP, Stellman SD, Lazarus P. Role of polymorphisms in codons 143 and 160 of the O6-alkylguanine DNA alkyltransferase gene in lung cancer risk. Cancer Epidemiol Biomarkers Prev 2000;9: 33942.
  • 5
    Broughton BC, Steingrimsdottir H, Lehmann AR. Five polymorphisms in the coding sequence of the xeroderma pigmentosum group D gene. Mutat Res 1996;362: 20911.
  • 6
    Price EA, Bourne SL, Radbourne R, Lawton PA, Lamerdin J, Thompson LH, Arrand JE. Rare microsatellite polymorphisms in the DNA repair genes XRCC1, XRCC3 and XRCC5 associated with cancer in patients of varying radiosensitivity. Somat Cell Mol Genet 1997;23: 23747.
  • 7
    Lunn RM, Helzlsouer KJ, Parshad R, Umbach DM, Harris EL, Sanford KK, Bell DA. XPD polymorphisms: effects on DNA repair proficiency. Carcinogenesis 2000;4: 5515.
  • 8
    Dybdahl M, Vogel U, Frentz G, Wallin H, Nexo BA. Polymorphisms in the DNA repair gene XPD: correlations with risk and age at onset of basal cell carcinoma. Cancer Epidemiol Biomarkers Prev 1999;8: 7781.
  • 9
    Sturgis EM, Castillo EJ, Li L, Zheng R, Eicher SA, Clayman GL, Strom SS, Spitz MR, Wei Q. Polymorphisms of DNA repair gene XRCC1 in squamous cell carcinoma of the head and neck. Carcinogenesis 1999;20: 21259.
  • 10
    Duell EJ, Wiencke JK, Cheng TJ, Varkonyi A, Zuo ZF, Ashok TD, Mark EJ, et al. Polymorphisms in the DNA repair genes XRCC1 and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells. Carcinogenesis 2000;21: 96571.
  • 11
    Thompson LH, Brookman KW, Jone NJ, Allen SA, Carrano AV. Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange. Mol Cell Biol 1990;10: 616071.
  • 12
    Caldecott KW, Aoufouchi S, Johnson P, Shall S. XRCC1 polypeptide interacts with DNA polymerase beta and possibly poly(ADP-ribose) polymerase, and DNA ligase III is a novel molecular “nick-sensor” in vitro. Nucleic Acids Res 1996;24: 438794.
  • 13
    Liu N, Lamerdin JE, Tebbs RS, Schild D, Tucker JD, Shen MR, Brookman KW, et al. XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. Mol Cell 1998;1: 78393.
  • 14
    Tebbs RS, Zhao Y, Tucker JD, Scheerer JB, Siciliano MJ, Hwang M, Liu N, et al. Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene. Proc Natl Acad Sci USA 1995;92: 63548.
  • 15
    Thompson LH, Schild D. The contribution of homologous recombination in preserving genome integrity in mammalian cells. Biochimie 1999;81: 87105.
  • 16
    Bishop D, Ear U, Bhattacharyya A, Calderone C, Beckett M, Weichselbaum RR, Shinohara A. Xrcc3 is required for assembly of Rad51 complexes in vivo. J Biol Chem 1998;273: 214828.
  • 17
    Flejter WL, McDaniel LD, Johns D, Friedberg EC, Schultz RA. Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of the human ERCC2 DNA repair gene. Proc Natl Acad Sci USA 1992;89: 2615l.
  • 18
    Braithwaite E, Wu X, Wang Z. Repair of DNA lesions induced by polycyclic aromatic hydrocarbons in human cell-free extracts: involvement of two excision repair mechanisms in vitro. Carcinogenesis 1998;19: 123946.
  • 19
    de Laat WL, Jaspers NG, Hoeijmakers JH. Molecular mechanism of nucleotide excision repair. Genes Dev 1999;13: 76885.
  • 20
    Czerniak B, Li L, Chaturvedi V, Ro JY, Johnston DA, Hodges S, Benedict WF. Genetic modeling of human urinary bladder carcinogenesis. Genes Chromosomes Cancer 2000;27: 392402.
  • 21
    Awata S, Sakagami H, Tozawa K, Sasaki S, Ueda K, Kohri K. Aberration of chromosomes 8 and 11 in bladder cancer as detected by fluorescence in situ hybridization. Urol Res 2000;28: 18590.
  • 22
    Ohgaki K, Minobe K, Kurose K, Iida A, Habuchi T, Ogawa O, Kubota Y, Akimoto M, Emi M. Two target regions of allelic loss on chromosome 9 in urinary-bladder cancer. Jpn J Cancer Res 1999;90: 95764.
  • 23
    Louhelainen J, Wijkstrom H, Hemminki K. Allelic losses demonstrate monoclonality of multifocal bladder tumors. Int J Cancer 2000;87: 5227.
  • 24
    Hartmann A, Rosner U, Schlake G, Dietmaier W, Zaak D, Hofstaedter F, Knuechel R. Clonality and genetic divergence in multifocal low-grade superficial urothelial carcinoma as determined by chromosome 9 and p53 deletion analysis. Lab Invest 2000;80: 70918.
  • 25
    Vineis P, Caporaso N, Cuzick J, Lang M, Malats N, Boffetta P. Metabolic polymorphsims and susceptibility to cancer. IARC Scientific Publication 148, Lyon: IARC, 1999.
  • 26
    Peluso M, Airoldi L, Armelle M, Martone T, Coda R, Malaveille C, Giacomelli G, et al. White blood cell DNA adducts, smoking, and NAT2 and GSTM1 genotypes in bladder cancer: a case-control study. Cancer Epidemiol Biomarkers Prev 1998;7: 3416.
  • 27
    Peluso M, Airoldi L, Magagnotti C, Fiorini L, Munnia A, Hautefeuille A, Malaveille C, Vineis P. White blood cell DNA adducts and fruit and vegetable consumption in bladder cancer. Carcinogenesis 2000;21: 1837.
  • 28
    Bell DA, Taylor JA, Butler MA, Stephens EA, Wiest J, Brubaker LH, Kadlubar FF, Lucier GW. Genotype/phenotype discordance for human N-acetyltransferase (NAT2) reveals a new slow-acetylator allele common in African-Americans. Carcinogenesis 1993;14: 168992.
  • 29
    Underhill PA, Jin L, Lin AA, Mehdi SQ, Jenkins T, Vollrath D, Davis RW, Cavalli-Sforza LL, Oefner PJ. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography. Genome Res 1997;7: 9961005.
  • 30
    Austin J, Buckland P, Cardno AG, Williams N, Spurlock G, Hoogendoorn B, Zammit S, Jones G, Sanders R, Jones L, McCarthy G, Jones S, Bray NJ, McGuffin P, Owen MJ, O'Donovan MC. Comparative sequencing of the proneurotensin gene and association studies in schizophrenia. Mol Psychiatry 2000;5: 20812.
  • 31
    Sacerdote C, Peluso M, Munnia A, Malaveille C, Vineis P. The choice of controls in a case-control study on WBC-DNA addcuts and metabolic polymorphisms. Biomarkers 2000;5: 30713.
  • 32
    Piegorsch WW, Weinberg CR, Taylor JA. Non-hierarchical logistic models and case-only designs for assessing susceptibility in population-based case-control studies. Stat Med 1994;13: 15362.
  • 33
    Brenneman MA, Weiss AE, Nickoloff JA, Chen DJ. XRCC3 is required for efficient repair of chromosome breaks by homologous recombination. Mutat Res 2000;459: 8997.
  • 34
    Wildschutte M, Franz R, Neumann HG. The tentative identification of DNA-adducts generated by trans-4-dimethylaminostilbene and the 4-trans-acetylaminostilbene in rats. Chem Biol Interact 1990;76: 4762.
  • 35
    Das L, Das SK, Hooberman BH, Chu EH, Sinsheimer JE. Chromosomal aberrations in mouse lymphocytes exposed in vitro and in vivo to benzidine and 5 related aromatic amines. Chem Res Toxicol 1994;320: 6974.
  • 36
    Randerath K, Randerath E, Smith CV, Chang J. Structural origins of bulky oxidative DNA adducts (type II I-compounds) as deduced by oxidation of oligonucleotides of known sequences. Chem Res Toxicol 1996;9: 24754.
  • 37
    Sokal RR, Rohlf FJ. Biometry. San Francisco: Freeman, 1969.