A BRCA1 Mutation Is Not Associated with Increased Indicators of Oxidative Stress

original contribution A BRCA1 Mutation Is Not Associated with Increased Indicators of Oxidative Stress Joanne Kotsopoulos,1,2 HongLei Shen,2 A. Venketeshwer Rao,2 Aletta Poll,1 Peter Ainsworth,3 Neil Fleshner,4 Steven A. Narod1 Abstract Background: Several functions have been attributed to the BRCA1 protein. A recent study suggests that BRCA1 is involved in the cellular antioxidant response by inducing the expression of genes involved in the antioxidant defense system and thus conferring resistance to oxidative stress. It is possible that individuals with a BRCA1 mutation might be susceptible to the effects of oxidative stress. The aim of this study was to evaluate whether women with a BRCA1 mutation exhibit increased indicators of oxidative stress. Patients and Methods: We measured 3 markers of oxidative stress in vivo, the amounts of serum malondialdehyde and protein thiols, and 8-oxo-2'-deoxyguanosine (8-oxodG) levels in 25 unaffected BRCA1 mutation carriers and 25 noncarrier control subjects. Results: There was no significant difference in serum malondialdehyde levels (P = .41), serum thiol levels (P = .85), or the number of 8-oxodG lesions (P = .49) in BRCA1 mutation carriers versus noncarriers. Conclusion: The results of this study suggest that the presence of a heterozygous BRCA1 mutation is not associated with increased levels of indicators of oxidative stress in serum or lymphocytes. Future studies are warranted to evaluate whether strategies aimed at minimizing oxidative stress might aid in the prevention of hereditary breast cancer.

Clinical Breast Cancer, Vol. 8, No. 6, 506-510, 2008; DOI: 10.3816/CBC.2008.n.061 Keywords: DNA damage, Lipid peroxidation, Malondialdehyde, Protein oxidation

Introduction The inheritance of a deleterious mutation in the breast cancer susceptibility gene BRCA1 is associated with a high lifetime risk of breast cancer.1 The protein product of the BRCA1 gene plays roles in DNA transcriptional regulation, cell-cycle checkpoint control, DNA damage repair, protein ubiquitylation, regulation of apoptosis, and chromatin remodeling.2-4 It is believed that some effects of BRCA1 protein deficiency can be observed in the nonmalignant cells of the BRCA1 carrier. In these cells, the BRCA1 protein is derived from the single normal copy of the gene. (In cancer cells, no wild-type protein is produced as a consequence of the loss of expression of the wild-type allele.) Baldeyron et al have shown that steady-state levels of wild-type BRCA1 protein are lower among mutation carriers than noncarriers.5 Other studies have described 1Women’s

College Research Institute, Women’s College Hospital, Toronto of Nutritional Sciences, Faculty of Medicine, University of Toronto, 3London Health Sciences Center, Molecular Diagnostic Laboratory 4Division of Urology, University Health Network, Department of Surgery, University of Toronto Ontario, Canada 2Department

Submitted: Jan 18, 2008; Revised: Aug 11, 2008; Accepted: Aug 25, 2008 Address for correspondence: Steven A. Narod, MD, FRCP, Women’s College Research Institute, 790 Bay St, Rm 750, Toronto, Ontario M5G 1N8, Canada Fax: 416-351-3767; e-mail: [email protected]

enhanced DNA sensitivity to mutagens or radiation in cells from women with a BRCA1 mutation compared with noncarrier control subjects.6-9 These studies suggest that deficient DNA repair might be a phenotype displayed by cells that are functionally heterozygous for a BRCA1 mutation. BRCA1 plays a role in maintaining genomic stability because of its role in the repair of DNA double-strand breaks by means of homologous recombination.10 BRCA1 also upregulates the expression of multiple genes involved in the antioxidant response, including glutathione-S-transferases (GSTs), oxidoreductases, and other antioxidant genes.11 This suggests that an impaired inherent antioxidant defense system among BRCA1 mutation carriers might be associated with an accumulation of oxidative damage and increased susceptibility to cancer development. Furthermore, other repair functions of BRCA1 include transcription-coupled repair of oxidative DNA damage.12 Based on these roles, it is possible that oxidative stress might be an important risk factor in women with an inherited deficiency in functional BRCA1 protein for the development of breast cancer. The aim of this study was to evaluate whether heterozygosity for a BRCA1 mutation affects levels of oxidative stress. Using a panel of markers that reflect oxidative stress in vivo, our overall goal of this study was to quantify and compare the levels of oxidized lipids, proteins, and DNA molecules in sera from populations of BRCA1

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mutation carriers and noncarrier control subjects. If so, then ≥ 1 indices of oxidative status could lead to the identification of a biomarker of risk for use in future intervention trials to evaluate the preventive role of lifestyle or dietary factors, especially those with antioxidant properties. If an association between BRCA1 mutation carrier status and markers of oxidative stress could be shown, then dietary supplementation with antioxidants might alleviate the burden of oxidative stress caused by the inherited mutation and possibly translate to a reduced risk of breast cancer.

Patients and Methods Subjects and Study Design Eligible subjects were healthy women with no previous history of breast cancer or of another cancer and were between the ages of 20 and 60 years. We included 25 healthy BRCA1 mutation carriers (BRCA1+/−) and 25 healthy mutation-negative women as control subjects (BRCA1WT). The control subjects were drawn from the first- and second-degree relatives of women with a BRCA1 mutation but who had negative test results for the family mutation. Patients and control subjects were participants in previous and ongoing clinical research protocols from the Centre for Research in Women’s Health, Toronto, Ontario, Canada, and the London Health Sciences Centre, London, Ontario, Canada. All study subjects received counseling and provided their written informed consent for genetic testing. The study was approved by the institutional review boards of the host institutions. In most cases, testing was initially offered to women who had been affected with breast or ovarian cancer. When a BRCA1 (or BRCA2) mutation was identified in a proband or her relative, genetic testing was offered to other at-risk women in the family. Mutation detection was performed by using a range of techniques, but all nucleotide sequences were confirmed by means of direct sequencing of DNA. A woman was eligible for the current study when the molecular analysis established that she was a carrier of a pathogenic mutation. Most (> 95%) of the mutations identified in the study subjects were nonsense mutations, deletions, insertions, or small frame shifts. Women were invited to participate in the study by letter. Women were excluded if they were pregnant or had a serious illness. The majority of women who were approached agreed to participate. The reasons given for declining our invitation included (1) travel time to our clinic in downtown Toronto (many individuals live outside the greater Toronto area), (2) dealing with other family matters, (3) lack of interest, or (4) loss to follow-up.

Data Collection Participants completed 3 questionnaires before their visit to the clinic. These were all self-administered and included the Diet History Questionnaire (a food frequency questionnaire that was developed by staff at the Risk Factor Monitoring and Methods Branch at the National Cancer Institute and reflects Canadian food availability and food fortification practices)13; a “Follow-up Questionnaire for a Study of Breast and Ovarian Cancer in High-risk Families” with questions directed at reproductive histories, prophylactic surgery, use of exogenous estrogens and other lifestyle factors; and a shorter “Research Questionnaire for a Study of Genetic and Non-genetic Factors Associated with Breast Cancer Risk in High-risk Women” that asked questions regarding use of dietary supplements and physical activity.

The follow-up questionnaire has been used in ≥ 25 studies of BRCA mutation carriers14,15 and, more importantly, is currently being used in an ongoing international collaborative study of BRCA mutation carriers that includes > 55 participating centers in 11 countries. The physical activity portion of the research questionnaire was adapted from the Harvard Alumni Questionnaire that has been used in the ongoing Harvard Alumni Health Study16 and has previously been tested for validity and reliability.17-19 The supplementary questions were extracted from an ongoing clinical study of dietary factors and prostate cancer. Studies have shown that the Diet History Questionnaire provides reasonable nutrient estimates, and 2 studies have been conducted to assess its validity.20,21 At an onsite visit, blood was collected from female heterozygous BRCA1 mutation carriers and noncarrier control subjects by means of venipuncture (one 5-mL sample in a heparin-containing tube and one 5-mL sample in a red-top tube with no additives). After blood collection, the heparin-containing tubes were gently inverted several times and immediately placed on ice for no longer than 3 hours until further processing. The samples with no additives were allowed to stand at room temperature to coagulate for no more than 1 hour (on ice after 1 hour) and then processed for serum collection (see below). Genomic DNA was extracted from the peripheral blood lymphocytes isolated from the blood sample of one of the ethylenediamine tetraacetic acid (EDTA)–containing tubes by using a Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN) and stored at 4°C for subsequent DNA-oxidation analysis. The tubes with no additives were centrifuged for 10 minutes at 3000 rpm and 4°C. The sera were then collected and stored at −70°C in multiple 500-μL Eppendorf tubes for lipid peroxidation and protein oxidation analysis. Standardized procedures were used to obtain various anthropometric measurements (weight, waist and hip circumference, and height).

Measurement of Oxidative Stress One week before testing, serum and lymphocyte DNA samples were transferred on ice to the laboratory of A. V. Rao, PhD, at the Department of Nutritional Sciences, University of Toronto, for analysis of oxidative stress and total antioxidant potential. These samples were stored for an average of 12 months from the date of sample collection to the date of lipid and serum oxidation analysis. The mean difference between sample collection and testing for DNA oxidation was 16 months. Lipid Peroxidation. Malondialdehyde (MDA) was measured by using the thiobarbituric acid (TBA)–MDA assay and reported as TBA-reactive substances.22,23 Serum samples were incubated with TBA and orthophosphoric acid in the presence of butylated hydroxytoluene for 45 minutes at 95°C, cooled to room temperature, and extracted with n-butanol. The absorbance of the butanol extract was read at 535 nm with a spectrophotometer. Results were calculated by using ε535 1.56 × 105 mol/L−1 and reported as serum MDA per micromole per liter of serum. Protein Oxidation. Protein oxidation was estimated by measuring the loss of reduced thiol (SH) groups with the 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) assay.24 Serum samples were diluted with 0.25 mol/L tris-EDTA buffer, pH 8.2, and incubated

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BRCA1 Mutation and Oxidative Stress Table 1 Principal Characteristics of the Study Participants by Mutation Status BRCA1 Mutation Carriers (BRCA1+/−) n = 25

Noncarriers (BRCA1WT) n = 25

P Value*

Mean Age at Interview, Years (SD)

43.56 (9.81)

44.62 (11.19)

.74

Mean Age at Menarche, Years (SD)

12.31 (1.40)

12.36 (1.70)

.92

(SD)†

Variable

Mean Age at First Birth, Years

26.50 (5.00)

26.79 (4.80)

.87

Mean Height, Inches (SD)

64.38 (2.52)

64.02 (2.15)

.59

Mean Weight, Pounds (SD)

147.84 (30.46)

154.04 (34.77)

.51

25.02 (4.70)

26.36 (5.63)

.37

Premenopausal

10 (40)

17 (68)

Postmenopausal

15 (60)

8 (32)

Nonuser

21 (84)

25 (100)

User

4 (16)

0

23 (92)

20 (80)

2 (8)

5 (20)

Mean BMI, kg/m2 (SD) Menopausal Status, n (%)

.05

Hormone Replacement Therapy Use,‡ n (%) .04

Oral Contraceptive Use,‡ n (%) Nonuser User

.22

Smoking Status,‡ n (%) Never Past Current Mean Total Alcoholic Drinks Per Day (SD) Mean Energy Intake, kcal/Day (SD) Mean Total Hours of Physical Activity Per Week (SD)

18 (72)

8 (32)

7 (28)

14 (56)

.01

0

3 (12)

0.73 (1.57)

0.70 (0.69)

.94

1730.09 (540.23)

1718.02 (443.41)

.93

19.03 (7.25)

22.60 (5.17)

.07

P values are univariate and were derived by using the Student t test for continuous variables and the C2 test for categorical variables. parous women. ‡Current use. Abbreviations: BMI = body mass index; SD = standard deviation *All

†Among

with 100 μmol/L DTNB (final concentration) and methanol for 15 minutes at room temperature. Samples were centrifuged, and the absorbance of the supernatant was measured at 412 nm against a blank. Thiols were calculated by using the extinction coefficient of 13.6 mmol/L−1 and reported as serum thiols per micromole per liter of serum. DNA Oxidation. DNA oxidation in the lymphocyte DNA was measured by means of 8-oxo-2'-deoxyguanosine (8-oxodG) analysis by using high-performance liquid chromatography with an electrochemical detector. DNA was then hydrolyzed at 37°C with nuclease P1 (20 μg) for 1 hour, pH 4.8, and alkaline phosphatase (2 U) for 1 hour after the pH was adjusted to 7.4. The separation of 8-oxodG in the DNA hydrolysate was made on a 3-μm Supelcosil LC-18-DB (15 cm × 4.6 mm) analytic column with 7.5% methanol in 50-mmol/L phosphate buffer, pH 5.5, as mobile phase at a flow rate of 0.8 mL/min and an ESA electrochemical detector (Coulochem II) equipped with an analytic cell (model 5010; ESA Biosciences, Inc; Chelmsford, MA) operating at 300 mV. Peaks were identified and quantified by using authentic 8-oxodG as a standard.25 Results were reported as femtomoles of 8-oxodG per microgram of DNA.

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Statistical Analysis The objective of this study was to examine whether BRCA1 mutation status was associated with any of several markers of oxidative stress. The Student t test was used to compare normally distributed continuous variables between BRCA1 mutation carriers and the noncarrier control subjects. The χ2 test was used to test for significance of differences in categorical variables. Unadjusted and adjusted values of serum MDA and SH, as well as 8-oxodG from peripheral blood lymphocyte DNA, were compared between the 2 groups of women. Multivariate linear regression analysis was performed on the 3 markers of oxidative stress to adjust for the potential confounding effect or effects of age (years), body mass index (kg/m2), current smoking (yes/no), physical activity (total hours per week), total daily caloric intake (kilocalories), and multivitamin use (yes/no). All statistical tests were 2-sided. A P value of .05 was taken to be significant. All analyses were performed with the SPSS statistical package, version 12.0.1 for Windows (SPSS, Inc, Chicago, IL).

Results Fifty women were enrolled in the current study, including 25 BRCA1 mutation carriers and 25 noncarrier control subjects. BRCA1 mutation carriers and control subjects were similar with respect

Joanne Kotsopoulos et al Table 2 Crude and Adjusted Means of 3 Markers of Oxidative Stress Stratified by BRCA1 Mutation Carriers and Noncarriers BRCA1+/−

BRCA1WT

No.

No.

BRCA1+/−

BRCA1WT

Serum SH, μmol/L

24

23

387.19 (7.75)

Serum MDA, μmol/L

24

23

8-oxodG, fmol/μg DNA

25

24

Oxidative Biomarker

P Value*

BRCA1+/−

BRCA1WT

No.

No.

BRCA1+/−

BRCA1WT

391.06 (8.49)

.74

19

21

392.72 (5.04)

394.12 (5.39)

.85

9.88 (0.42)

10.50 (0.47)

.33

19

21

10.32 (0.14)

10.11 (0.21)

.41

1.55 (0.16)

1.24 (0.14)

.18

20

22

1.51 (0.13)

1.41 (0.09)

.49

Crude Mean (± SEM)

Adjusted Mean‡ (± SEM)

P Value*

Student t test was used to test for differences in the crude and adjusted means of the oxidative biomarkers between BRCA1 mutation carriers and noncarriers. linear regression included terms for age (years), body mass index (kilograms per square meter), current smoking (yes/no), physical activity (total hours per week), total caloric intake (kilocalories), and multivitamin use (yes/no). Abbreviations: MDA = malondialdehyde; SEM = standard error of the mean; SH = thiols

*The

†Multivariate

to current age, age at menarche, age at first birth, body mass index, and oral contraceptive use (Table 1). The number of postmenopausal women was slightly higher among carrier women (15 vs. 8; P = .05), and a higher proportion of carriers were current users of hormone replacement therapy (HRT; 16% vs. 0 in carriers and noncarriers, respectively; P = .04). Smoking status, energy intake per day, alcohol consumption, and the total hours of physical activity per week were similar for carriers and noncarriers (Table 1). Serum MDA, protein SH, and 8-oxodG values of lymphocyte DNA were determined to provide a measure of lipid peroxidation, protein oxidation, and DNA oxidation, respectively. The crude and adjusted mean values of the 3 biomarkers of oxidative stress are shown in Table 2. There was no significant difference in lipid peroxidation (measured as serum MDA) or protein oxidation (measured as serum SH) in BRCA1 mutation carriers and noncarriers (Table 2). Similarly, the number of 8-oxodG lesions in lymphocytes (measure of DNA oxidation) did not differ. Adjustment for known potential confounders did not significantly affect the final results.

Discussion Oxidative stress occurs when there is an imbalance between the rate of oxidant production and the antioxidant defense mechanisms. Oxidative stress has been implicated in the cause of numerous diseases, including heart disease, cancer, inflammatory disease, and aging.26-28 Although numerous assays exist, the assessment of oxidative stress is evaluated mainly through the measurement of oxidatively modified biomolecules, predominantly DNA, lipids, and protein.29 Using in vitro models, Bae et al found that overexpression of BRCA1 was associated with the upregulation of genes involved in natural antioxidant defense mechanisms.11 Genes that were upregulated in BRCA1-overexpressing cells included microsomal GSTs (MGST1 and MGST2), cytoplasmic GSTs (GSTT1 and GSTZ1), a glutathione peroxidase (GPX3), and various oxidoreductases (NQO1, alcohol dehydrogenase 5, and malic enzyme). Based on this report, we compared the oxidative status of DNA, proteins, and lipids from the sera of women with and without a BRCA1 mutation. We were unable to show that inheriting a deleterious BRCA1 mutation influences in vivo oxidation of these biomarkers in this study population. When comparing women with and without a BRCA1 mutation, we found no significant difference in levels of MDA (P = .41), SH (P = .85), or 8-oxodG (P = .49). Our null findings might reflect the limitations of the study, including a small sample size. The sample used in the final analysis was even smaller because of missing data on covariates or specific measures of oxidative stress.

A limited number of studies have shown higher levels of DNA damage in the sera of women given diagnoses of breast cancer,30,31 in breast cancer tissue compared with that seen in unaffected control subjects,32 and in the urine of patients with breast cancer.33 These studies did not evaluate the BRCA1 status of the patients. Increased levels of MDA have been observed in the sera of patients with breast cancer34,35 and in the urine of women with mammographic dysplasia, a risk factor for breast cancer.36-38 In the present study of women without breast cancer, the presence of a (heterozygous) BRCA1 mutation did not influence oxidative stress. We measured lipid and protein peroxidation in the sera of the study subjects. DNA oxidation was quantified in the genetic material derived from peripheral blood lymphocytes. These samples might not be representative of the breast tissue; oxidative stress could be higher in the breast tissue than in the serum because of increased levels of estrogen.30,39 Quantifying oxidative status directly in the breast tissue (through biopsy) or in nipple aspirate fluid (ie, potentially sampling more directly from tumor tissue) might clarify a role of oxidative stress in the cause of both hereditary and sporadic breast cancer. Also, polymorphisms in antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase, might affect the endogenous antioxidant defense mechanisms, resulting in increased levels of oxidative stress. Genetic, reproductive, and environmental factors have all been suggested to influence breast cancer risk in BRCA1 mutation carriers.40 The variability in risk, even among women with the same mutation, suggests that the penetrance of a BRCA1 mutation is highly variable. Levels of oxidative damage might also be increased among mutation carriers with breast cancer compared with those who remain cancer free. We compared unaffected BRCA1 mutation carriers and control subjects and did not address the hypothesis that among BRCA1 carriers oxidative stress might be related to cancer penetrance. Menopause has been associated with an increase in the levels of several indicators of oxidative stress in human subjects41 and in rodents.42 This has been attributed to a decrease in estrogen synthesis43,44 as well as a possible decrease in endogenous antioxidant activity.45 However, adjusting for HRT in our final analysis did not affect the results (data not shown). There is an increase in oxidant activity that occurs during the isolation, handling, and storage of the serum and DNA samples (ie, the time between sample collection and actual testing). This time lapse might have also affected our results; however, there was no significant difference in the median times from sample collection to testing for patients and control subjects. We tried to minimize the potential for bias by having a single technician who was blinded to the mutation status

Clinical Breast Cancer December 2008 • 509

BRCA1 Mutation and Oxidative Stress of the samples analyze the samples in a single run. Also, the carriers and noncarriers were tested in a single batch.

Conclusion The results of this study suggest the presence of a heterozygous BRCA1 mutation is not associated with increased levels of indicators of oxidative stress. In view of these findings, it is possible that 1 unaffected copy of the allele is sufficient to carry out normal antioxidant functions. The tissue specificity of BRCA1-related cancers suggests that estrogen might be important in the carcinogenic process in mutation carriers, especially during periods of active cellular proliferation and differentiation in the breast (ie, puberty and pregnancy), when both BRCA1 expression and ovarian hormone production are normally increased. Women with reduced expression of the BRCA1 protein because of the inheritance of a BRCA1 mutation (1 functional allele and subsequent decreased expression of BRCA1) might be particularly susceptible to the carcinogenic effects of hormonal exposure.46-48 Because loss of heterozygosity might be limited to the breast tissue (and/or ovaries), the BRCA1-null cells are probably not represented in the lymphocytes from BRCA1+/− individuals. Based on this, we cannot entirely exclude the possibility that estrogen-induced oxidative stress is not an important phenomenon occurring only in the breast tissue (and other hormonally regulated tissues) of mutation carriers. If so, then strategies aimed at reducing oxidative stress in the breast epithelium, particularly during critical periods of cellular change, might help in the prevention of hereditary breast cancer.

Acknowledgements Joanne Kotsopoulos is supported by a fellowship from the Canadian Breast Cancer Foundation, Ontario Chapter. We thank Nancy Scanlan for help in the recruitment of patients from the London Health Sciences Centre, London, Ontario, Canada; Tania Correa for her help with the blood collection; and Dr Ping Sun for his statistical support.

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