Molecular and biochemical studies have shown that ROS stimulate epigenetic changes and interact with cellular macromolecules (DNA, proteins, and lipids), both of which can result in shifts in homeostasis that are important in cellular transformation (19
). The most common DNA lesion produced by ROS, 8-oxo-2-deoxyguanosine (8-OxodG), promotes G to T transversion mutations (20
). Further, lipid peroxidation products such as malondialdehyde can also react with DNA to form a number of pro-mutagenic lesions (21
). Metabolism of estrogen, a well established risk factor for breast cancer, generates ROS, indicating that oxidative stress may be an important mediator of estrogen dependent breast carcinogenesis (22
). There is a growing body of epidemiological evidence that oxidative stress biomarkers such as urinary 15-f2t-isoprostanes, malondialdehyde, and urinary and tissue 8-oxodG, are elevated in breast cancer cases compared with controls in population-based studies (1
We selected sisters discordant for breast cancer who are participating in the New York Site of the BCFR to determine whether oxidative damage is also associated with breast cancer in high risk women. Breast cancer in a first degree relative is associated with a 2-fold or more increased risk of breast cancer (10
). Since there is limited information about potentially modifiable factors that could reduce breast cancer risk in those at increased risk due to their family history, we evaluated whether oxidative damage could explain part of this risk.
Carbonyl groups formed by ROS on amino acid side chains are chemically stable moieties making them useful in detecting oxidative damage (24
). Carbonyl modifications are not repaired and the extent of carbonylation depends upon factors that influence oxidant status. Thus, the plasma protein carbonyl biomarker captures the net of pro-oxidant exposures and antioxidant status. The most commonly oxidized amino acids are Arg, Lys, Pro, Thr, and the plasma protein, fibrinogen, is highly susceptible to free radical attack (24
). Intracellular oxidized proteins are rapidly degraded by the 20S proteosome (26
). However, it is unclear whether extracellular oxidized proteins are actively degraded or simply eliminated by normal turnover. Plasma protein carbonyl levels have been shown to increase with fat overload and exercise, and decrease with weight loss, and vitamin treatment (27
In our analysis of factors associated with plasma protein carbonyl levels in unaffected sisters, we found that early age of menarche (<13 yrs) was significantly associated with an increase in plasma protein carbonyl levels. Others have reported differences in girls with early menarche (< 12 yrs) such as higher serum estradiol levels, and earlier onset of regular ovulatory cycles (31
). Further, a combination of early age at menarche and increased waist circumference was associated with higher 17-β estradiol levels in adult women indicating that ones’ age at menarche can contribute to lasting impacts on hormone levels (32
Contrary to our expectation, HRT use was associated with a decrease in plasma protein carbonyls. Others have also reported a decrease in plasma protein carbonyl levels as well as other biomarkers of ROS exposure such as isoprostanes, plasma total thiol levels, and lipid peroxides, and an increase in total antioxidant status and reduced sulfhydryl groups among women supplemented with HRT (33
). Although the mechanisms through which HRT could reduce ROS are not entirely clear, it has been suggested that upregulation of downstream antioxidant systems, such as an increase in glutathione levels, leads to a net reduction in ROS (36
Finally, Hispanic race was associated with a decrease in plasma protein carbonyl level. Race-associated differences in pro-oxidant or antioxidant exposures may have contributed to the differences that we observed in plasma protein carbonyls. For example, in our study 53% of Caucasian versus 33% of Hispanic unaffected sisters reported ever smoking. In addition, racial or ethnic differences in diet have previously been shown to impact on serum antioxidant levels. An analysis of NHANES data found that Mexican-Americans had significantly higher levels of some nutritional biomarkers including β-cryptoxanthin and lutein compared with Caucasians (37
We did not find an association between alcohol consumption and plasma protein carbonyl level. Although ethanol metabolism has been shown to generate free radicals, components of some alcoholic beverages, such as resveratrol in red wine, have antioxidant properties (38
). Since our questionnaire did not collect information on the various types of alcohol consumed, we cannot draw firm conclusions about the relationship between plasma protein carbonyls and alcohol consumption.
Cigarette smoke contains free radicals, which have been shown experimentally to increase protein carbonyl content of plasma in a dose-dependent manner (39
). In humans, smoking has also been associated with a slight but non-significant increase in oxidized plasma proteins in current versus former or never smokers, however, this increase was not found to be dose-dependent (2
). We did not detect an association between smoking and plasma protein carbonyls based on never, former, and current smoking status. The lack of association could have resulted from misclassification due to under-reporting of current smoking or exposure to environmental tobacco smoke.
Age-related increases in a variety of biomarkers of ROS exposure were observed in human lens, brain tissue, fibroblasts, and skeletal muscles (41
). We found a decrease in mean plasma protein carbonyl levels as age increased; however when HRT users were excluded from the analysis mean plasma protein carbonyl levels were similar for cases and unaffected sisters from 18–59 yrs, and increased in those ≥ 60 yrs (data not shown).
When we stratified the plasma protein carbonyl prediction model by menopausal status we found that race, HRT use, parity, BMI, and age at menarche and were predictive of plasma protein carbonyls but the direction and strength of the effects were not the same in pre-and postmenopausal women. The effects of race and HRT use in the stratified model were consistent with the main model. Parity was negatively associated with plasma protein carbonyl levels in postmenopausal women. Parous women were previously reported to have lower circulating estrogen and prolactin levels and higher sex-hormone-binding globulin when compared with nulliparous women (42
). High BMI decreased plasma protein carbonyls in premenopausal women and increased plasma protein carbonyls in postmenopausal women. Though this relationship could have been observed by chance, it is interesting that high BMI in premenopausal women is protective against breast cancer while high BMI in postmenopausal women increases risk (43
). Perhaps part of this risk is mediated through ROS.
There is evidence that menarche, pregnancy, lactation, and menopause are associated with significant and long-term changes in estrogen and other hormone levels. Estrogen, and its metabolites have been shown to generate ROS, and to be strongly associated with another ROS biomarker, F2a
-isoprostane, in premenopausal and/or postmenopausal women depending upon the metabolite (44
). While we did not find a large mean difference in plasma protein carbonyls between premenopausal and postmenopausal women, there are other changes that occur during menopause that could influence oxidant status. Changes in ferritin levels, antioxidant enzyme expression, and body fat amount/distribution may either increase or decrease ROS production (17
). Therefore, it is possible that other factors not included in our analysis impacted on plasma protein carbonyl levels in postmenopausal women. In fact, our stratified model demonstrates that the predictors of plasma protein carbonyls are different depending on menopausal status.
Another aim was to investigate whether oxidative damage was associated with breast cancer in those with a family history. Plasma protein carbonyl levels were also associated with an increase in breast cancer risk, an increase that was significant only in the 3rd
quartile. Individuals with the highest level of plasma protein carbonyls had a slightly attenuated risk of breast cancer. This result may reflect a saturation effect or could be due to other attributes of the unaffected sisters that promote health despite having high levels of ROS. Our finding is in agreement with a recent publication from our laboratory that describes an increase in breast cancer risk associated with plasma protein carbonyl levels in a population-based case-control study of breast cancer in women on Long Island, NY (2
). The strength of the association was higher in the present study, likely because we used a family design, the conditional analysis of which would provide better control for unmeasured genetic or environmental variables. Elevated plasma protein carbonyls have also been observed in bladder, and lung cancer (45
There were several limitations to the current study. First, our breast cancer cases were prevalent and biospecimens were collected months to years after diagnosis. Thus, the plasma protein carbonyl biomarker could have been impacted upon by factors associated with the disease or treatment. However, this limitation is not exclusive to our study, as most of the population-based studies that analyze oxidative stress markers are also case-control designs with cases diagnosed within 6–12 months of study enrollment. Our analysis indicated that there was no impact of treatment or disease on the plasma protein carbonyl biomarker. However, replication in a prospective study is necessary. Finally, we were primarily concerned with reproductive factors and did not include other potentially important variables such as exercise and diet-related variables in the analysis.
Our study also had several strengths. First, this study focused on the association of a potentially modifiable risk factor among women with a family history of breast cancer. Since relatively little is known about familial aggregation of breast cancer this data fills an important niche. Second, our study was well designed to investigate factors that influence oxidative stress levels since our questionnaire data was collected concurrently with biospecimen donation, thereby providing accurate and timely exposure information.
In sum, we found that plasma protein carbonyls can be modified by several factors including BMI, parity, and HRT use. Although we expected to detect associations between plasma protein carbonyls and other factors known to induce oxidative stress such as cigarette smoking and alcohol consumption, exposure misclassification may have hindered detection of these relationships. Finally, our data suggest that oxidative damage is a risk factor for breast cancer even among women with a family history.