Introduction

Breast cancer is one of the most common types of cancer in women in China and the second only to lung cancer as a cause of cancer death. The incidence of breast cancer has increased steadily in China over the past few decades. Risk factors for the development of breast cancer can be grouped into categories including familial/genetic factors (family history, known and suspected BRCA1/2, TP53, PTEN, or other gene mutation associated with breast cancer risk), factors related to demographics (age, race), reproductive history (age at menarche, age at first live birth, age at menopause), environmental factors, etc. [1–6]. Women who are at high risk of breast cancer can be offered more intensive surveillance or prophylactic measures to lower the incidence of breast cancer. It is imperative to find novel candidate biomarker for breast cancer risk assessment.

DNA repair plays an essential role in the maintenance of DNA integrity. Deficient DNA repair capacity has been suggested as a predisposing factor in familial and sporadic breast cancer. The mutagen sensitivity assay (MSA) provides a phenotypic marker of DNA repair capacity and genomic stress response, which has been reported as a heritable trait that affects both familial and sporadic breast cancer risk [7–12]. This assay measures the number of chromosomal breaks in cultured lymphocytes following exposure to DNA-damaging agents which bleomycin was used in it. Using this assay, DNA from women in high-risk families and sporadic breast cancer cases exhibits a nearly twofold increase in the mean number of breaks per cell compared with DNA from women without cancer from low-risk families [6, 7, 11]. Epidemiologic studies have also suggested that mutagen sensitivity is a predisposing factor for other cancers [13–16]. Thus, mutagen sensitivity may specifically reflect differences in an individual’s ability to repair DNA through the pathway of interest and homologous repair, and it may be a biomarker for cancer risk.

It is widely acknowledged that tobacco smoke is a human carcinogen [17]. In mainland China, 2010 data show that 26 % of the urban adult population and 30 % of the rural adult population are current smokers, with about 70 % of the adult population exposed to passive smoking in a typical week [18]. Accumulating evidence has implicated active smoking as a contributor to women’s risk of breast cancer [19]. The evidence for a relationship between passive smoking and breast cancer, however, remains tenuous. To shed light on the potential roles of bleomycin-induced mutagen sensitivity in breast cancer susceptibility, we conducted a case–control study focused on the association between mutagen sensitivity, environment tobacco exposure, and breast cancer risk in Chinese women.

Materials and methods

Study population

Eligible cases were women with primary breast carcinoma who underwent mastectomy or breast-conserving surgery in two hospitals in Wuhan city, including Zhongnan Hospital and Renmin Hospital of Wuhan University during the period from January 2009 to February 2011. All of the cases were histopathologically confirmed, and the blood was drawn before any treatment. Controls were matched to cases from the same hospitals with no history of cancer, gynecological disease or endocrine disease, same residing area, and within ±3 years of age. Each participant donated 10 ml of blood after signing written informed consent. Of 219 breast cancer cases identified, in-person interviews were completed for 203 (93.1 %), three cases (1.4 %) did not provide a blood sample, and four blood cultures failed (1.8 %). In-person interviews were completed for 221 (91.7 %) of the 241 eligible controls. Four controls (1.8 %) did not provide a blood sample and six blood cultures failed (2.7 %). Therefore, data analysis for bleomycin sensitivity was performed on 196 cases and 211 controls.

Questionnaire

A thorough, structured questionnaire was completed by all subjects. Three experienced nurses (one interviewer, two collectors) were assigned to obtain information through face-to-face interviews conducted in hospital wards or in the subject’s home. The baseline questionnaire focused on demographic and anthropometric parameters, dietary habits, menstrual and reproductive factors, hormone use, lifestyle, medical history, and family history. Postmenopausal women were defined as those having bilateral oophorectomy or having no menstrual cycle in the 12 months prior to blood sample collection. Passive smoking history was collected for two level of duration. First, the subject was asked whether her husband had ever smoked at home and/or she was exposed to the smoke of others in her workplace, then the subject was asked the average number of hours per day she was exposed to the smoke, and the total number of years she had been exposed to the smoke at home and/or workplace. The passive smoking index was calculated as the total number of years multiplies by hours per day (hour-year).

Mutagen sensitivity assays

The assay was described in detail previously [20]. Briefly, 1 ml of fresh whole blood was added to 9 ml of RPMI-1640 medium supplemented with 15 % bovine serum, 1.5 % of phytohemagglutinin (Wuhan Boster Bio-engineering Limited Co.), 2 mM l-glutamine, and 100 U/ml each of penicillin and streptomycin. After the cells were cultured for 72–90 h at 37 °C, they were incubated for 5 h with 0.03 U/ml bleomycin (Hisun pharmaceutical Co.). To arrest the cells at metaphase, 0.2 μg/ml colcemid was added to the culture 1 h before the harvest. The cells were treated in hypotonic solution (0.06 M KCl) and fixed in fixative (three parts of methanol with one part of acetic acid). The cells were dropped onto clean microscopic slides, air dried and stained with 4 % Gurr’s Giemsa solution (Wuhan Boster Bio-engineering Limited Co). Fifty well-spread metaphase cells per subject were examined to visually score the chromatid breaks. Only frank chromatid breaks or chromatid exchanges were scored. Criteria for a frank chromatid break were a discontinuity of a single chromatid in which the distance of discontinuity region was wider than the diameter of the chromatid, or there was a clear misalignment of one of the chromatids. A chromatid exchange is the result of two or more chromatid breaks and the subsequent rearrangement of chromatid material. Exchanges may be between chromatids of different chromosomes (interchanges), or between or within chromatids of one chromosome (intrachanges). The total number of breaks was divided by the number of the cells examined, and the mean number of breaks per cell was recorded for statistical analysis. Cells with more than 12 breaks were excluded from the calculation of mean breaks per cell to reduce the bias of the results by a very few severely damaged cells. In our study, the frequency of the cells with more than 12 breaks was rare. In the vast majority of the subjects, fifty cells were analyzed from one slide without seeing one cell with more than 12 breaks. The slides were coded and scored without the knowledge of case–control status. Twenty blinded quality control samples were included to assess variability, and each sample was run in triplicate. The coefficient of variation for repeats was 5.1 %.

Statistical analyses

The distribution of demographic information between cases and controls was examined using Fisher’s exact test for categorical variables and Wilcoxon rank-sum test for the means of continuous variables. The number of bleomycin-induced chromatid breaks was analyzed as both continuous and categorical variables. An individual was considered to have high bleomycin sensitivity if the MSA score was equal to or greater than the 50th percentile value in controls (0.75 breaks per cell). Bleomycin sensitivity was also categorized according to the quartiles in control subjects. Passive smoking status was stratified into two categories of tobacco smoke exposure (never: no exposure and ever: some exposure); and four categories (never, low: less than 10 h-year, medium: 10–20 h-year, and high: more than 20 h-year). Family history of female cancers was defined as having breast or ovarian cancer in first- or second-degree biological relatives. Postmenopausal women were defined as those having bilateral oophorectomy or having no menstrual cycle in the 12 months prior to blood sample collection. Alcohol consumption was defined as intake of at least 100 g alcoholic beverage per time per week. Physical activity was defined as any physical activity on a regular basis (at least once a week on average) for at least 20 min at a time. Multivariate logistic regression was used to obtain the odds ratio (OR) and 95 % confidence intervals (CIs) for the strength of the association between breast cancer and mutagen sensitive phenotype, while controlling for known breast cancer risk factors and other potential confounders: age, physical activity, menopausal status, passive smoking, alcohol, body mass index (BMI), family history of female cancer (if inclusion of a factor altered the odds ratio (OR) estimation by 10 %, that factor was retained in the final model). Tests for a linear trend were done using quartile levels as continuous variables based on MSA scores and passive tobacco smoking exposure. We also explored the combined effect of passive smoking and mutagen sensitivity by their categories and the possibility of interactions of the two variables by logistic regression analysis. All p values were two-sided, and p < 0.05 was used as the threshold for statistical significance. All analyses were conducted using STATA/IC 11.2 (StataCorp, College Station, TX, USA).

Results

Characteristics of the study population

The characteristics of cases and controls are summarized in Table 1. The average age was 46.7 years for the cases (range 25–75 years) and 48.6 years for the controls (range 27–75 years). There were no significant differences in the distribution of alcohol use, oral contraceptive pill use, hormone replacement treat (HRT), and family history of female cancers (breast and ovarian) between cases and controls. There were significant case–control differences in passive smoke exposure, age at menopause, age at menarche, age at first full birth, total times of lactation, body mass index (BMI), and education.

Table 1 Characteristics of cases and controls
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Bleomycin sensitivity and breast cancer risk

Table 2 shows case–control comparisons of the mean number of bleomycin-induced breaks per cell. Overall, the mean breaks per cell were significantly higher in cases (mean 0.81) than that in controls (mean 0.73, p = 0.016). When the case–control comparison was stratified by cancer risk, we observed significant differences between subjects over 50 years old, those exposed to passive smoke, those who consumed alcohol, had a BMI <23, had family history of female cancer and those pre-menopause. The relationship between bleomycin sensitivity and breast cancer risk was estimated by calculating ORs and 95 % CIs using unconditional logistic regression analysis, with adjustment for age, physical activity, menopausal status, alcohol, body mass index (BMI), family history of female cancer. Defining bleomycin sensitive as ≥0.75 break/cell (median level in population controls), 59.2 % of the cases were bleomycin sensitive compared with 49.8 % of the controls with an adjusted OR of 1.23. Subjects were categorized into four groups (by quartiles) according to the bleomycin-induced breaks per cell in controls. A dose–response relationship was observed with the highest versus lowest quartile with an adjusted OR of 1.82, p ≤ 0.01 (Table 3). We also stratified the bleomycin-induced chromatid breaks by passive smoking exposure and found that the breast cancer risk was associated with high bleomycin sensitivity in women who had been exposed to passive smoke (adjusted OR = 1.94). When the data were categorized by quartile distribution in the control group, the results showed that there is a significant correlation between bleomycin sensitivity and breast cancer risk in a dose-dependent manner for both women who had never been exposed to smoke, and those who had. The adjusted OR for the fourth quartile compared with the first quartile was 2.06 and 2.75, respectively (Table 5).

Table 2 Case–control comparison of mean bleomycin-induced breaks per cell
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Table 3 Association of bleomycin sensitivity with breast cancer risk
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Joint effect of bleomycin sensitivity and passive smoking on breast cancer risk

Table 4 shows the association between passive smoking and breast cancer risk. Overall, passive smoking had a positive relationship with breast cancer risk in our study population and had a significant dose–response relationship, adjusted OR 2.13, p trend = 0.01. However, there was no significant interaction between passive smoking and bleomycin sensitivity when the interaction was formally tested in the logistic model (p = 0.471). We further examined the combined effect of passive smoking and bleomycin sensitivity on breast cancer risk, using women who were low sensitivity and had never been exposed to passive smoke as the reference group. The combined effect of bleomycin sensitivity and passive smoking on the risk of breast cancer was significantly different from the single effect of either bleomycin sensitivity or passive smoking. Women who had high bleomycin sensitivity phenotype and passive smoking were at 2.77-fold increased risk of breast cancer compared with women who had a low bleomycin sensitivity phenotype and never passive smoking (Tables 5, 6).

Table 4 Association of passive smoking with breast cancer risk
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Table 5 Association of bleomycin sensitivity with breast cancer risk
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Table 6 Combined effect of mutagen sensitivity and passive smoking on breast cancer risk
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Discussion

The failure to maintain genome integrity is central to the problem of carcinogenesis [21]. DNA repair capacity is a cellular defense system designed to protect genomic integrity. There is considerable interindividual variation in DNA repair capacity. Numerous epidemiologic studies have consistently yielded significant associations between reduced DNA repair capacity and increased cancer occurrence [22, 23], suggesting that DNA repair capacity is a cancer susceptibility factor. Mutagen sensitivity, measured by quantifying the chromatid breaks induced in short-term cultures of peripheral blood lymphocytes, has been used as an indirect measure of DNA repair capacity. Mutagens act on cells through different molecular mechanisms and may activate other repair mechanisms. In our study, we chose bleomycin as the mutagen for the MSA. The main reason is that bleomycin is a clastogenic agent that mimics the effects of radiation after formation of a complex with DNA, ferrous ions (Fe2+), and oxygen, which releases oxygen radical [24]. The free oxygen radicals are capable of producing single- and double-strand breaks in DNA. Bleomycin is also relevant to tobacco use because it is similar to numerous compounds in tobacco smoke known to cause oxidative damage which can be repaired by the base excision and recombination repair systems [25, 26].

In this case–control study, we demonstrated that, after adjusting for known breast cancer risk factors, bleomycin sensitivity is positively related to the risk of breast cancer among Chinese women. For nearly two decades, mutagen sensitivity has been a commonly used phenotypic assay in cancer epidemiology. The epidemiologic evidence supporting its association with cancer risk is strong and consistent. Jyothish et al. [27] reported that bleomycin-induced chromosomal breaks were significantly higher in both familial and sporadic breast cancer patients compared with unrelated female controls. Xiong et al. [28] investigated benzo[a]pyrene diol-epoxide sensitivity and breast cancer risk in a case–control study of predominantly white women and reported that it was associated with a threefold increased risk of breast cancer in premenopausal women. A study examined the MSA using gamma radiation as the mutagen and breast cancer risk in a case–control study of African-American women, and gamma radiation sensitivity was found to be associated with an increased breast cancer risk (OR = 4.5) [8]. More recently, Wang et al. [29] reported that high radiosensitivity was related to risk of breast cancer in a case–control study of 515 young women with newly diagnosed sporadic breast cancer and 402 cancer-free controls. Our results are in agreement with these previous reports and provide further evidence (adjusted OR = 1.82).

Our results also suggest that accumulative exposure to tobacco smoke may increase risk for breast cancer among Chinese women, particularly in those who had high bleomycin sensitive phenotypes.

We chose to investigate the effects of tobacco smoke exposure as a risk for female breast cancer because the vast majority of Chinese women have never smoked cigarettes, while the prevalence of smoking is high among adult men and most of them smoke at home. Smoking has not been restricted in public places, including work settings. This exposure pattern provides a unique opportunity to vigorously investigate the hypothesis related to passive smoking. The body of literature on this topic is still relatively small and findings to date have not been consistent. There have been ten prospective cohort studies and 17 case–control studies conducted to examine the relationship between passive smoking and breast cancer risk. Results have been mixed, with four of the ten cohort studies yielding positive results and 11 of the 17 case–control studies reporting positive findings [30]. The largest of these is the prospective Million Women Study from United Kingdom [31]. The authors reported an overall null association (OR = 0.98, 95 % CI = 0.93–1.05) for passive smoking and breast cancer, and the point estimate for risk in premenopausal women actually suggested an inverse association 0.54 (95 % CI = 0.33–0.99). However, our study shows a positive relationship between passive smoking and breast cancer risk and dose–response association with hours-years of exposure. We further investigate the combined effect of passive smoking and bleomycin sensitivity on breast cancer risk and suggest that women who had high bleomycin sensitivity phenotypes and longer passive smoking exposure have greater risk of breast cancer (adjusted OR = 2.77). In our study, stratified analysis suggests that bleomycin sensitivity is a stronger risk factor in women exposed to tobacco smoke than in those not exposed. Among high bleomycin sensitive women, having longer exposure to smoke is associated with a 2.77-fold increased risk of breast cancer. We suggest that while longer exposure to tobacco smoke may be associated with mutagen sensitivity, this needs further investigation.

Our study has several limitations and the results need to be interpreted with caution. We had limited sample size and thus limited power to detect an association. Another limitation of this study is the use of lymphocytes, the repair of which may not reflect that of breast epithelial cells. We recruited the controls in the hospital setting; they might not represent the general population, and the selection bias may influence the results.

In summary, our results showed that bleomycin sensitivity is associated with breast cancer risk and is a promising biomarker for breast cancer risk assessment for Chinese women. Our observation that passive smoking increases breast cancer risk especially for women with high sensitivity to bleomycin is intriguing and worthy of further investigation in large studies.