Cancer Research 67, 11123, December 1, 2007. doi: 10.1158/0008-5472.CAN-07-3061
© 2007 American Association for Cancer Research
Plasma Cysteinylglycine Levels and Breast Cancer Risk in Women
Jennifer Lin1,
JoAnn E. Manson1,2,4,
Jacob Selhub5,
Julie E. Buring1,3,4 and
Shumin M. Zhang1,4
1 Division of Preventive Medicine, 2 Channing Laboratory, Department of Medicine, and 3 Department of Ambulatory Care and Prevention, Brigham and Women's Hospital and Harvard Medical School; 4 Department of Epidemiology, Harvard School of Public Health; and 5 Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
Requests for reprints: Jennifer Lin, Division of Preventive Medicine, Brigham and Women's Hospital, Harvard Medical School, 900 Commonwealth Avenue East, Boston, MA 02215. Phone: 617-278-0894; Fax: 617-232-3541; E-mail: jhlin{at}rics.bwh.harvard.edu.
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Abstract
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Cysteinylglycine, a prooxidant generated during the catabolism of glutathione, has been suggested to induce oxidative stress and lipid peroxidation, leading to the development of human cancers. Observational data relating cysteinylglycine status to breast cancer risk are lacking. We prospectively evaluated plasma cysteinylglycine levels and invasive breast cancer risk among 812 case-control pairs nested in the Women's Health Study, a completed randomized trial evaluating low-dose aspirin and vitamin E in middle-aged and older women. We additionally evaluated the effect modification by risk factors for oxidative stress, such as vitamin E assignment, alcohol consumption, obesity, and postmenopausal hormone use. Logistic regression controlling for matching factors, as well as other risk factors for breast cancer, was used to estimate relative risks (RR) and 95% confidence intervals (95% CI). All statistical tests were two sided. We observed no overall association between plasma cysteinylglycine and invasive breast cancer risk. However, higher cysteinylglycine levels were marginally associated with an increased risk of breast cancer in the high oxidative stress groups. Women in the highest quintile group of cysteinylglycine relative to the lowest group had multivariate RRs (95% CIs) of 1.64 (1.01–2.66; Ptrend = 0.04) in the vitamin E placebo group, 2.51 (1.01–6.24; Ptrend = 0.07) in the high alcohol intake group (
9 g/day), and 1.66 (0.97–2.84; Ptrend = 0.03) in the overweight and obese group. Our findings suggest that women who are susceptible to experiencing oxidative stress may be at a greater risk for developing breast cancer. [Cancer Res 2007;67(23):11123–7]
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Introduction
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Oxidative stress, known to play a role in the promotion and progression of human cancers, including breast cancer (1–3), is a disturbance in the balance between reactive oxygen species production and antioxidant defenses, favoring the overproduction of reactive oxygen species (4). Cysteinylglycine, a sulfhydryldipeptide, is generated from the extracellular glutathione, a nonprotein thiol, through the catalytic activity of 
-glutamyltransferase (5). Cysteinylglycine is a highly reactive metabolite which has been suggested to cause the reduction of ferric iron Fe3+ to ferrous iron Fe2+, resulting in an iron redox cycling process liable to the production of reactive oxygen species and stimulation of oxidative reactions (6). The prooxidant, cysteinylglycine, has been shown to cause lipid peroxidation in human plasma LDL lipoproteins (7), as well as oxidative damage on DNA bases (8).
A small case-control study reported a significantly positive association between serum cysteinylglycine levels and risk for ischemic heart disease (9). However, there are no data evaluating cysteinylglycine status in relation to risk for developing breast cancer. We prospectively evaluated the association between plasma levels of cysteinylglycine and invasive breast cancer risk in a case-control study nested in a large female cohort.
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Materials and Methods
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Participants in this study were drawn from the Women's Health Study, a completed randomized trial evaluating low-dose aspirin and vitamin E for the primary prevention of cancer and cardiovascular disease among 39,876 women who were 45 years or older and free of cancer and cardiovascular disease at the time of enrollment beginning in 1992 (10, 11). Upon enrollment into the study, participants completed a baseline questionnaire about their medical history and life-style factors. Participants also filled out a 131-item food frequency questionnaire, which has been validated in a large female cohort (12). After an average of 10 years of treatment, the Women's Health Study trial observed no overall benefit of 600 IU of vitamin E or aspirin at a dose of 100 mg given every other day in the prevention of breast cancer development; the observed relative risks (RR) were 1.00 [95% confidence intervals (95% CI), 0.90–1.12] for randomized vitamin E treatment and 0.98 (95% CI, 0.89–1.08) for randomized aspirin treatment (10, 11).
Blood samples were collected from 28,345 women (71% among the total) at baseline. Baseline characteristics of women who gave blood samples were largely similar to those who did not (13). Briefly, both groups were not different in mean age in years (54.7 and 54.4), proportions of women with body mass index (BMI) of 25kg/m2 (48.4% versus 51.1%), alcohol intake (1.1 versus 0.9 g/day), physical activity (median expenditure per week, 599 versus526 kcal/week), and current use of multivitamin supplements (29.4% versus 28.6%).
In the present study, we included 812 cases who had a confirmed diagnosis of invasive breast cancer incidence during an average of 10 years of follow-up between 1992 and 2004. Each breast cancer case was individually matched to one control with no diagnosis of cancer on age (up to 5 years of difference), ethnicity, menopausal status (premenopausal, postmenopausal, or uncertain/unknown), fasting status (8 h or fewer), month and year of blood return (4-month interval), postmenopausal hormone use (never, past, or current), and trial randomization date (12-month difference). Plasma cysteinylglycine levels were determined using high-performance liquid chromatography with fluorescence detection (14). Blood samples were handled identically and together in the same batch. The mean coefficient of variation for quality control samples (78 repeated samples) was 14.7%.
We first loge-transformed plasma cysteinylglycine and then categorized plasma cysteinylglycine into quintiles on the basis of the distribution in the controls. Differences between case-control pairs in mean levels of plasma nutrients and other continuous covariates were tested using a paired t test. The McNemar test was used to compare the difference between case-control pairs in proportions of covariates as categorical variables. We used conditional logistic regression to estimate RRs and 95% CIs for invasive breast cancer with adjustment for age and randomized treatment assignment, matching factors, and, additionally, potential risk factors for breast cancer assessed at baseline.
Several risk factors, including lower antioxidant intakes, smoking, high alcohol consumption, overweight/obesity, and elevated estrogen levels have been suggested to contribute to lipid peroxidation and oxidative damage to DNA, leading to breast cancer development (2, 15, 16). Accordingly, we also evaluated the effect modification by randomized vitamin E treatment assignment (treatment, placebo group), alcohol consumption (<9, 9 g/day, 75th percentile among drinkers), smoking (current, past, never), obesity (normal, overweight/obese), menopause (premenopause, postmenopause), and postmenopausal hormone therapy use (never, current) with unconditional logistic regression controlling for matching factors, as well as risk factors for breast cancer. Tests for trend were performed by fitting the median value of each category as a continuous variable in the models. Tests for multiplicative interaction between cysteinylglycine levels (continuous variable with the median value of each category) and other modifiers in relation to risk of breast cancer were performed by entering the product term of cysteinylglycine levels and a modifier into the multivariate model with a Wald statistic. All P values were two sided.
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Results
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As shown in Table 1
, plasma cysteinylglycine was assayed with a wide range of concentrations. Breast cancer patients were more likely, than control subjects, to have previous history of benign breast disease and to report being current smokers, having fewer childbirths, being older at first child birth, and at onset of menopause. Baseline dietary intakes of antioxidants, such as β-carotene, selenium, vitamin A, and vitamin C, and number of participants receiving 600 IU of randomized vitamin E treatment on alternate days, were not different between cancer patients and control subjects. However, plasma cysteinylglycine levels in cancer patients were moderately higher than those in control subjects.
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Table 1. Baseline characteristics (mean ± SD or %) among invasive breast cancer cases and their matched controls in the Women's Health Study
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Plasma cysteinylglycine levels were nonsignificantly positively associated with overall risk of invasive breast cancer in the analysis controlling for matching factors and age and random treatment assignment or additionally controlling for risk factors for breast cancer (Table 2
). Further adjustment for dietary intakes of β-carotene, selenium, vitamin A, and vitamin C did not materially change the results (data not shown). Similarly, the results were not substantially changed when we additionally adjusted for plasma levels of folate, vitamin B6, and vitamin B12. However, we observed a marginally positive association among women assigned to vitamin E placebo; women in the highest quintile group of cysteinylglycine relative to those in the lowest group had a multivariate RR of 1.64 (95% CI, 1.01–2.66; Ptrend = 0.04). No association was observed among women assigned to vitamin E treatment group (Pinteraction = 0.34). The effect modification by aspirin treatment on the association between cysteinylglycine levels and breast cancer was not significant (Pinteraction = 0.90).
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Table 2. RRs and 95% CIs of invasive breast cancer according to quintiles of plasma levels of cysteinylglycine in the Women's Health Study
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Higher plasma cysteinylglycine levels were marginally associated with an increased risk for invasive breast cancer among women who consumed alcohol of 9 g or more daily; women in the highest quintile group relative to the lowest group had RR of 2.51 (95% CI, 1.01–6.24; Ptrend = 0.07). In addition, cysteinylglycine levels were positively associated with an increased risk for breast cancer among overweight or obese women (BMI, 25 kg/m2); the multivariate RR (95% CI) in the highest quintile group relative to the lowest group was 1.66 (0.97–2.84; Ptrend = 0.03). In contrast, no association was observed among women with alcohol consumption of 9 g or less daily and among normal-weight women (Pinteraction, 0.11 for BMI and 0.12 for alcohol consumption). Higher plasma cysteinylglycine levels were also marginally associated with an increased risk for developing postmenopausal breast cancer (Table 2). The positive association was mostly attributable to women who had never received hormone therapy, whereas no association was observed among current users of hormone therapy (Table 2; Pinteraction, 0.36 for menopause status and 0.32 for hormone therapy use). Finally, the association was not significant in the stratified analysis according to smoking status and mammogram screening test (yes, no; data not shown).
To evaluate what factors best predict cysteinylglycine concentrations in the study population, we first conducted partial correlation analysis among controls with adjustment for matching factors between cysteinylglycine levels and several oxidative variables, including BMI, alcohol consumption, current cigarette smoking, current or never use of hormone therapy, vitamin E treatment, and aspirin treatment. BMI (partial r = 0.10, P value = 0.03) was shown significantly associated with circulating cysteinylglycine levels. We also performed a stepwise regression among controls with adjustment for age and BMI on all energy-adjusted nutrients derived from the baseline food frequency questionnaire, using the criteria of P value of <0.10 for variable entry into the model and P value of <0.05 for staying in the model. Four nutrients selected as best predictors for cysteinylglycine levels were vitamin E from diet (partial r = -0.09, P = 0.01), vitamin D from diet (partial r = 0.09, P = 0.01), vitamin B2 from diet (partial r = –0.09, P = 0.01), and total sodium (partial r = –0.07, P = 0.04).
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Discussion
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In this first prospective cohort study of plasma cysteinylglycine and breast cancer risk, we observed no overall statistically significant association. However, a marginally positive association between plasma cysteinylglycine levels and breast cancer risk was seen among women who did not receive vitamin E treatment, had higher alcohol consumption, or among those who were overweight or obese. It is possible that women in these groups are more susceptible to the attack of lipid peroxidation and oxidative stress, giving rise to oxidative damage of the mammary membrane. Alcohol has been shown to alter the absorption and metabolism of protective antioxidant nutrients and increase oxidative stress in cell membranes (17). Similarly, excess adiposity due to obesity or lower intakes of antioxidants, such as vitamin E, may increase the formation of reactive oxygen species in cells (18).
We, however, observed a marginally positive association between cysteinylglycine levels and breast cancer risk among postmenopausal women, especially among those who had never used hormone therapy. It remains unclear whether estrogens inhibit or increase lipid peroxidation in mammary tumorigenesis. In contrast to the findings that estrogens may increase risk for oxidative stress in mammary tissue (16), estrogens have also been shown to inhibit lipid peroxidation, resulting in the overexpression of Bcl-2 and antiapoptotic effects on the mammary cells (19). More studies are necessary to clarify the relationship between estrogens and oxidative stress to the development of mammary tumorigenesis.
Given the significant associations with oxidative factors, plasma cysteinylglycine may potentially serve as a marker of enhanced oxidative stress. Because we only had cysteinylglycine concentrations in the study, we were unable to determine whether cysteinylglycine may be a better measure than other oxidative measures in relation to breast cancer risk. A recent case-control study of endogenous levels of F2-isoprostanes and 8-hydroxydeoxyguanosine and breast cancer risk observed a modest positive association between F2-isoprostanes, but not 8-hydroxydeoxyguanosine, levels and breast cancer risk (3). Notably, the study also observed the effect modification by current cigarette smoking in relation to breast cancer risk. In our study, however, the significant modifiers of the association between cysteinylglycine levels and breast cancer risk did not include cigarette smoking. It is possible that cysteinylglycine, F2-isoprostanes, and 8-hydroxydeoxyguanosine may be linked to different oxidative pathways in the development of breast cancer. For instance, the cysteinylglycine-associated oxidation of the cells may be more sensitive than F2-isoprostanes and 8-hydroxydeoxyguanosine to metabolic syndrome (20).
Several limitations present in the study merit further consideration. First, we had only a single baseline measurement for cysteinylglycine levels, which may not reflect the true cysteinylglycine status over time. It remains unclear whether circulating cysteinylglycine levels sufficiently represent oxidative stress exposure in the development of mammary tumorigenesis and whether cysteinylglycine may be a better measure than other oxidative biomarkers, such as F2-isoprostanes or 8-hydroxydeoxyguanosine. We also cannot exclude the possibility of residual confounding by uncontrolled variables.
In conclusion, our findings suggest that higher cysteinylglycine levels, a prooxidant produced by the catabolism of glutathione, may predict an increased risk for developing breast cancer among women exposed to peroxidation-prone environments, such as high alcohol consumption, low antioxidant intakes, or excess weight. Additional studies are warranted to confirm our present findings.
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Acknowledgments
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Grant support: National Cancer Institute grants CA47988, CA104871, and CA112529, and National Heart, Lung, and Blood Institute grant HL43851.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the entire staff of the Women's Health Study under the leadership of David Gordon, as well as Mary Breen, Susan Burt, Marilyn Chown, Georgina Friedenberg, Inge Judge, Jean Mac-Fadyean, Geneva McNair, David Potter, Claire Ridge, and Harriet Samuelson, the Endpoints Committee of the Women's Health Study (Dr. Wendy Y. Chen and Jim Taylor), Eduardo Pereira, and Natalya Gometskaya for technical assistance with the manuscript.
Received 8/10/07.
Revised 10/ 4/07.
Accepted 10/12/07.
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Abstract
Introduction
