Carcinoma of the gallbladder has a very unusual geographical distribution with pockets of high incidence seen in Chile, Poland, India, Japan and Israel,1, 2, 3, 4 it occurs rarely in China. However, over recent decades, the reported incidence has risen in China.5 The etiology of gallbladder cancer is incompletely understood, like many other malignancies, this cancer is a disease of multifactorial etiology6, 7, 8; gallstone are strongly associated with gallbladder cancer in both sexes and represent the most important risk factor for gallbladder cancer9, 10, 11, 12; other risk factors are some occupational exposures and chronic bacterial infections of gallbladder.13, 14, 15 Although just how the biological mechanisms underlying them are involved in its development is not fully understood, one of the proposed mechanism involves oxidative DNA damage caused by reactive oxygen species (ROS).16, 17 ROS are formed continuously in the living cells through both endogenous and exogenous processes.18 The reaction of ROS results in numerous forms of both cellular and DNA damage.19 Oxidative damage to DNA is thought to cause mutations, which in turn can activate oncogenes or inactive tumor suppresser genes and finally lead to cancer.20 Thus, organisms have developed a highly efficient DNA repair machinery consisting of several genes and pathways responsible for removing and repairing damaged DNA; interindividual variation in the repair capacity have been implicated as a cancer susceptibility factor.21
Of the oxidative DNA damages, the formation of 8-hydroxyguanine (Oh8Gua) is believed to play an important role in carcinogenesis because it is abundant and highly mutagenic,22 the Oh8Gua lesions are repaired primarily through the base excision repair mechanism, by which the specific DNA glycosylase/apurinic (AP) lyase catalyzes the release of Oh8Gua and the cleavage of DNA at the AP site appears to be a key component.23 Repair of oxidative DNA damage is initiated by hOGG1. Loss of OGG1 activity in yeast is associated with a mutator phenotype that accumulates G:C→to-T:A transversions.24, 25 Many epidemiological studies have investigated the association between the Ser326Cys polymorphism in the hOGG1 gene and the risk for different types of cancers, significant risk increases were observed for the hOGG1326polymorphism in the risk for esophageal,26, 27 orolaryngeal,28 lung cancer,29, 30 colon cancer31 and gastric cancer.32, 33 However, there are few reports about studying the association between gallbladder cancer and Ser326Cys hOGG1 polymorphism. We sought to investigate the association of the polymorphisms in the hOGG1 gene with gallbladder cancer in a relatively large population-based, case control study conducted in China.
Material and methods
Patients with gallbladder cancer and controls recruited for our study were essentially the same as those in our previous case-control study for genomic determination of the CR1 (CD35) density polymorphism on erythrocytes of patients with gallbladder carcinoma.34 In the present study, we have expanded the size of sample of patients and control to 204 and 209, respectively. Briefly, all subjects were residents in Guangzhou, Southern China. The cases undergoing surgery were recruited from January 2001 to January 2005 at 3 local hospitals (First Affiliated Hospital, and Cancer Center Research Institute of Zhongshan University, and Second Affiliated Hospital of Guangzhou Medical College), yielding a 100% response rate. These hospitals were chosen because that they have a history of treating large numbers of patients with gallbladder cancer. All the cases were histologically confirmed as gallbladder adenocarcinoma by the Department of Pathology at Zhongshan University School of Medicine, Zhongshan University. Population-based controls were recruited from the same area among those who visited any of the 3 hospitals for a health check-up, randomly selected cancer-free controls were gender and age matched to the cases. A questionnaire that contained detailed questions on demographics and gallstone was administered to all study subjects. Gallstone was diagnosed by a panel of specialists using real-time ultrasound sonography (TOSHIBA nemio SSA-55A, Japan) to examine the abdominal region after fasting of the subjects for at least 2 hr based on the presence of “movable hyperechoic foci with acoustic shadow.” For smaller subgroups, gallstone size was categorized as 2 cm or greater or less than 2 cm,35 then the observer measured the largest diameter of stones presence. In case of an acoustic shadow, the largest diameter had to match at least the width of this shadow. The display of the measuring instrument (PAV electronic sliding caliper, type classic 6511) showed the distance digitally in one-hundredths of a millimeter corresponding to an actual distance of 1 cm, indicated by the scale printed on the hard copy of B-ultrasound sonography was also measured in one-hundredths of a millimetre. This distance was used to compute the actual stone size. To set up a consistent diagnosis of gallstone among the specialists, the Kappa statistic was used to assess the agreement of inter-observer reliability among study specialists. A pilot study was performed using 90 randomly selected healthy subjects other than the study participants. For inter-observer reliability, the Kappa valve for diagnosis of gallstone between specialists was 0.84 (95% CI:0.74–0.95). The study protocol was approved by the Institutional Review Board of Zhongshan University and Guangzhou Medical College.
Genotyping of hOGG1 Ser326Cys polymorphism
Genomic DNA was obtained from the peripheral blood of gallbladder cancer patients and healthy controls. Buffy coats from 4.9 ml venous blood in EDTA were isolated and DNA was extracted by a standard phenol–chloroform procedure. Polymorphisms at codon 326 in the hOGG1 gene were determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) based method.36 Laboratory personnel were blinded to the case control status of the subjects. A 200 bp fragment of the hOGG1 gene was amplified from healthy control DNA by PCR using forward primer 5′-ACTGTCACTAGTCTCACCAG-3′ and reverse primer 5′-GGAAGGTGCTTGGGGAAT-3′. The final volume of PCR reaction mixture was 25 μl and each reaction tube contained 1XPCR buffer, 0.4 μl of each primer, 3.5 mM of MgCI2, 0.4 mM of each deoxynucleotide triphosphate, 50 ng genomic DNA and 2.5 units of Taq DNA polymerase (Roche Diagnostics). Cycling conditions (Mastercycler Gradient, Eppendorf, Germany) were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles, each of that consisted of incubations at 95°C for 30 sec, 60°C for 30 sec and 72°C for 1 min. The final extension was allowed to occur at 60°C for 10 min. A simple RFLP method was used to identify the Ser326Cys variant,37 because the C-to-G transversion creates a new Fnu4HI restriction site. The PCR product is 200 bp in length and can be digested by the Fnu4HI restriction enzyme into two 100-bp fragments for the 326cys allele and cannot be digested for the 326Ser allele. Fragments were separated on a 2% agarose gel and stained with ethidium bromide. The Cys/Cys homozygote can be cleaved by Fnu4HI and yields a 100 bp band. The Ser/Ser homozygote cannot be cleaved by Fnu4HI and remains the single 200 bp band. The Ser/Cys heterozygote contains both the 200-bp and 100-bp bands. The image of a representative gel is showed in Figure 1.
Gallbladder cancer risk in relation to hOGG1 genotypes was estimated by using unconditional logistic regression to calculate odds ratios and 95% confidence intervals. The ORs were adjusted for several confounding factors such as age, gender and gallstone status. χ2 test was used for analysis of allelic prevalence while a Student's t-test was used to compare gallstone status between the cases and control subjects. The χ2 test for trend was used to examine potential associations between predicted high-risk hOGG1 genotypes and gallbladder cancer risk. All the analyses were carried out with Statistical Analysis System software (Version 6.12, SAS Institute, Cary, NC, USA).
Table I shows the distribution of age, gender and gallstone status among cases and controls. The mean age ±SD (52.8 ± 9.1 years vs. 52.1 ± 9.7 years) and proportion of gender (male 32.1%, vs. male 30.9%) between the cases and controls were not statistically significant. The difference between cases and controls for gallstone status was statistically significant (p < 0.001), with the OR for gallstone-associated gallbladder cancer being 3.8 (95% CI = 2.4–4.3).
Table I. Distribution of Age, Gender, and Gallstone Among Cases and Controls
We ascertained the identity of each study subject hOGG1 alleles by PCR–RELP analysis. Among the control subjects, the prevalence of the hOGG1 326cys allele was 0.401, which is similar to the results reported for Chinese in previous studies.26 The distribution of genotypes in controls as followed the Hardy–Weinberg equilibrium (p = 0.995).
To determine whether the hOGG1 326cys allele contributed to increased association for gallbladder cancer, we examined the prevalence of hOGG1 alleles in gallbladder cancer cases versus controls. The distribution of hOGG1 genotypes among controls (Ser/Ser, 37.3; Ser/Cys, 53.6% and Cys/Cys 9.1%), the frequencies of the 3 genotypes among gallbladder cancer cases were Ser/Ser, 43.1%-; Ser/Cys, 36.3%- and Cys/Cys, 20.6%. The Cys/Cys genotype was more prevalent in the cases than in the controls (p < 0.001). Significantly increased association for gallbladder cancer was observed for subjects with both the hOGG1 326Ser/Cys (OR = 1.8, 95% CI = 0.9–3.6) and hOGG1 326Cys/Cys (OR = 4.4, 95% CI = 1.0–22.0) genotype (Table II). This association was not affected by adjusting for other factors (age, sex and gallstone) via regression analysis (ORadjust =1.9, 95% CI = 1.0–3.7 for the hOGG1 326Ser/Cys genotype; OR = 4.5, 95% CI =1.1–22.4 for the hOGG1 326 Cys/Cys genotype). These results were consistent with the fact that a significant trend towards increased association was observed with predicted less protective hOGG1 genotypes (p < 0.001, χ2 trend test, Table II).
Table II. Prevalence of hOGG1 Genotypes and Gallbladder Cancer Risk by Gallbladder Stone
To determine the relationship between hOGG1 genotype and gallbladder cancer association by exposure to gallstone, we stratified study subjects by hOGG1 genotype and gallstone status (Table II). We observed no statistically significant association between hOGG1 genotype and gallbladder cancer association in gallstone absence. In contrast, a near-significant increase in risk for gallbladder cancer was observed for gallstone presence with the hOGG1 326Ser/Cys genotype (OR = 2.2, CI = 1.4–3.5) whereas a significant increase in association for gallbladder cancer was observed for gallstone presence with the 326Cys/Cys genotype (OR = 6.1, CI = 2.1–27.2). These data corresponded with the fact that a significant trend towards increased association for gallbladder cancer was observed with potentially higher-risk hOGG1 genotypes in gallstone presence (p < 0.001, χ2 trend test) but not in gallstone absence (p = 0.89, χ2 trend test). A significant increase in risk for gallbladder cancer was observed for larger gallstone (those with stone diameters 2 cm or greater) with the hOGG1 326Ser/Cys(OR = 1.9, 95% CI = 1.1–2.9) and hOGG1 326Cys/Cys genotypes (OR = 5.9, 95% CI = 1.6–18.0). These data are consistent with the observation that a significant trend towards increased risk for gallbladder cancer was observed with potentially higher-risk hOGG1 genotypes in gallbladder cancer patients with larger gallstone (p < 0.001, χ2 trend test). However, we observed no statistically significant association between hOGG1 genotype and gallbladder cancer risk in gallbladder cancer patients with smaller gallstone (those with stone diameters 2 cm smaller) (hOGG1 326Ser/Cys:OR = 2.2, 95% CI = 0.8–4.0; hOGG1 326Cys/Cys:OR = 2.9, 95% CI = 0.6–29.4; p=0.06, χ2 tread test).
This study, which to our knowledge is the first one to show the potential association between hOGG1 polymorphism and gallbladder cancer in Chinese population, showed that the hOGG1 Ser326Cys polymorphism was associated with susceptibility to gallbladder cancer. Elevated risk of gallbladder cancer has been linked to poor antioxidant status.38, 39 Oxidative DNA damage induced by ROS is involved in the process of carcinogenesis, which suggests a role of oxidative DNA damages in its etiology. One of the major forms of DNA adducts induced by oxidative damage is 8-OH-dG and increased 8-OH-dG formation in DNA is likely to be involved in mutagenesis and carcinogenesis.40, 41, 42 Previous studies have suggested that hOGG1 is one of the major enzymes involved in the repair of 8-OH-dG adducts in DNA.23 In the current study, individuals who were homozygous for the Cys/Cys genotype were at a markedly increased risk for developing this cancer. Our finding provides evidence for the first time that the hOGG1 Ser326Cys polymorphism might be a genetic basis for the hypothesis that poor antioxidant status or oxidative damage plays a role in the carcinogenesis of the gallbladder.
Gallstone is well known as a strong risk factor for gallbladder cancer.35, 43 The prevalence of gallstones reported in patients with cancer of the gallbladder in western countries ranges from 74 to 92%; the prevalence of gallstones in the general U.S. population is estimated to be ∼10%44; however, according to the data from the Chinese Society of Surgery, the Chinese Medical Association,45 based on a review of a total of 3922 cases of gallbladder cancer from 28 provinces in China from 1986 to 1998, the incidence of coexistence of gallbladder cancer and gallstone in gallbladder was 49.7% (1,951/3,922), the prevalence of gallstones in the general population of China was estimated to be 7.2% (7,023/105,019). In our present study, the incidence of coexistence of gallbladder cancer was as high as 57.8% (118/204), in contrast to the ratio of gallstones in normal individuals (5.6%, 98/209). Nervi et al. reported that the incidence of gallbladder cancer in patients with gallstones was 7 times higher than that in patients without gallstones.46 physical trauma produced by the stone and carcinogenic substance in bile might result in epithelia dysplasia and ultimately progress to carcinoma.47, 48 If the induction by physical trauma hypothesis is correct, it may be that duration for trauma rather stone size itself is the critical factor. It may be that large stones more effectively initiate this process than small stones. Finally, gallstone size may reflect duration of cholelithiasis. Large stones might plausibly be assumed to have been present (and enlarging) for longer periods than have smaller stones.35, 43, 49 The potential mechanism of carcinogenesis in gallbladder tissue may involve tissue damage and regeneration in the presence of highly ROS released from inflammatory cells that can interact with DNA in epithelial cells to produce permanent genomic mutations.39, 40 One of these polymorphisms, a Ser-Cys substitution at codon 326, results in decreased DNA-repair activity, and decreased hOGG1 activity might be expected to increase gallbladder cancer risk, especially among those who are exposed to high levels of ROS generated by gallstone.
However, we are aware of potential limitation of this study. The number of controls with gallstone recruited into this study was small, so we do not know the correlation between hOGG1 genotype and phenotypes in gallstone status and gallstone size in the risk for gallbladder cancer. Although a significant increase in association for gallbladder cancer was observed for presence of gallstone with the hOGG1 326Ser/Cys and hOGG1 326Cys/Cys genotype, a significant increase in association for gallbladder cancer was also observed for larger gallstone (those with stone diameters 2 cm or greater) with the hOGG1 326Ser/Cys and hOGG1 326Cys/Cys genotypes, therefore these results must be confirmed in large-scale studies.
In conclusion, these results suggest that the hOGG1 Ser326Cys allele might play a role in the carcinogenesis of the gallbladder.
The authors thank the medical staff of the Second Affiliated Hospital, Guangzhou Medical College, the First Affiliated Hospital for their generous assistance, and Cancer Center Research Institute of Zhongshan University for data collection. The authors also grateful to the laboratory staff of Department of General Surgery and Transplantation Surgery, University Hospital Duisburg-Essen, Essen, Germany, for their valuable technical assistance with laboratory assays.