reactive oxygen species ( ros ) are generated during normal cellular metabolism , as a result of the influence of various environmental factors , as well as during pathological processes . ros are responsible for dna , lipid and protein damage and play an important role in the development and progression of many human diseases , including cancer . protein oxidation may result in structural changes and loss of function through peptide bond cleavage due to oxidation of the protein backbone , formation of protein the oxidation of protein side chains occurs mainly on proline , arginine , lysine and threonine residues resulting in carbonyl formation . carbonyl groups formed during the oxidation are chemically stable which is useful for both in terms of detection and stability during sample storage . protein carbonyl levels are the most frequently used biomarker of protein oxidation and their accumulation has been observed with aging and in several human diseases , including cancer ( reviewed in ;) . several methods of protein carbonyl groups detection are available , including a colorimetric assay based on derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine ( dnph ) . a stable 2,4-nitrophenylhydrazone ( dnp ) product results that can be detected spectrophotometrically . because this method is relatively time - consuming and requires higher amount of biological samples , buss et al . the assay is reproducible and correlates well with the classical colorimetric assay . in the present study , we investigated the association of oxidative stress , measured as the levels of protein carbonyl groups in plasma , with breast cancer risk among women who participated in the long island breast cancer study project ( libcsp ) . among controls , we also determined the correlation of plasma protein carbonyl levels with age , average lifetime physical activity , fruit and vegetable intake , alcohol consumption , cigarette smoking , body mass index ( bmi ) and menopausal status , factors that may affect oxidative stress . the libcsp is a population - based study investigating the associations between environmental factors and breast cancer . briefly , eligible cases were women residing in nassau or suffolk counties , who spoke english , and were newly diagnosed with a first primary in situ or invasive breast cancer between august 1 , 1996 , and july 31 , 1997 . population - based controls were identified using random digit dialling ( < 65 years of age ) and health care financing administration rosters ( 65 + years of age ) , and frequency matched to the expected age distribution of the cases by 5-year age group . the main questionnaire was administered by a trained interviewer in the participant 's home and lasted approximately 2 hrs . information was assessed on known and suspected risk factors for breast cancer , including passive and active cigarette smoking , lifetime alcohol use , menstrual and reproductive histories , hormone use , body size by decade of adult life , lifetime participation in recreational activities , prior medical history and family history of breast cancer ( http://epi.grants.cancer.gov/libcsp/projects/questionnaire.html ) . usual dietary intake in the year prior to the interview was assessed using a modified block self - administered food frequency questionnaire , as previously described . we also collected information on cancer treatment undergone prior to blood collection , including whether a participant had radiation and/or chemotherapy . of the eligible participants who completed the main questionnaire ( 1508 cases and 1556 controls ) , 73% of cases and 73% of controls donated a non - fasting blood samples at the time of the interview . the samples were donated on average within 3 months after breast cancer diagnosis , prior to the onset of chemotherapy for most case participants , but after surgery . for controls , blood samples , collected in ethylenediaminetetraacetic acid ( edta)-treated tubes , were shipped at room temperature for overnight delivery to columbia university for processing and storage . the samples were aliquoted and frozen at 80c within 24 hrs of collection . in the present study the levels of plasma protein carbonyl groups were assessed using a non - competitive elisa , essentially as described in , with some modifications . briefly , the oxidized protein standards were prepared by incubation of bovine serum albumin ( bsa ) ( 50 mg / ml ) with 0.73 m h2o2 and 0.42 mm fe for 1 hr at 37c . it was then diluted with native ( unoxidized ) bsa and phosphate - buffered saline ( pbs ) to give a final carbonyl content of 2.0 nmol / mg protein and protein concentration of 4 mg / ml . total protein concentration in the plasma samples was measured using bicinchoninic acid kit ( sigma ) and the samples were diluted with pbs to a final protein concentration of 4 mg / ml . after the derivatiza - tion with dnph , the plate was coated with 200 l of sample ( 1 g ) and incubated overnight at 4c in the dark . the plate was washed with pbs / tween ( 0.05% tween 20 in pbs ) and blocked with 0.1% bsa in pbs for 1.5 hr . after another washing step , biotinylated primary anti - dnp antibody ( molecular probes ; diluted 1:1500 with 0.1% bsa , 0.1% tween 20 in pbs ) was added and the plate was incubated at 37c for 1 hr . another washing was followed by adding the streptavidin - biotynylated horseradish peroxidase conjugate ( amersham ; diluted 1:4000 in 0.1% bsa , 0.1% tween 20 in pbs ) and incubation at room temperature for 1 hr . colour was developed by adding the tetram - ethyl benzidine ( tmb ) liquid substrate system ( sigma ) and the reaction was stopped with h2so4 after 1525 min incubation in the dark . each sample was analysed in duplicate ; the interassay coefficient of variability for the repeat analysis ( n = 53 ) of a quality control sample was 17% . as a quality control , a pooled plasma sample was repeatedly frozen and thawed before analysis ; there was no significant difference in protein carbonyl levels with up to four freeze - thaw cycles . we first compared the differences between 1050 cases and 1107 controls with regard to the plasma protein carbonyl levels ( nmol carbonyl / ml plasma ) . the analysis of variance test   was used for comparing case - control differences as continuous variables and the chi - square test   was used for comparing case - control differences . we also examined case - control differences using unconditional logistic regression   adjusting for reference age ( date of diagnosis for cases and date of identification for controls ) . other factors that were considered as possible confounders include those that have been associated with breast cancer and may also influence oxidative stress pathways , including average physical activity level , fruit and vegetable intake , average lifetime alcohol intake , active cigarette smoking status , bmi , menopausal status , age at menarche , age at first birth ( afb ) , parity , oral contraceptive ( oc ) use and hormone replacement therapy ( hrt ) use ( using definitions that have been previously published [ 10,1517 ] ) . only those that changed the effect estimates by more than 10% were included in the final models . we stratified the association between plasma protein carbonyl levels and breast cancer by the same factors to examine effect modification on a multiplicative scale . we also examined the associations with the cases restricted to those women for whom chemotherapy ( n = 827 ) or radiation therapy ( n = 831 ) had not yet been initiated by the time of the blood collection . we then assessed whether plasma protein carbonyl levels differed by age , average physical activity level , fruit and vegetable intake , average lifetime alcohol intake , active smoking status , bmi , menopausal status , age at menarche , afb , parity , oc use and hrt use among 1107 controls first by using the analysis of variance test and second by using linear regression methods . we used log transformation for the plasma protein carbonyl levels in the linear regression model to meet the assumption of normality . we estimated a saturated model with all factors suspected to influence plasma protein carbonyl levels and a parsimonious model with other those variables which were statistically significantly related to the outcome . the libcsp is a population - based study investigating the associations between environmental factors and breast cancer . briefly , eligible cases were women residing in nassau or suffolk counties , who spoke english , and were newly diagnosed with a first primary in situ or invasive breast cancer between august 1 , 1996 , and july 31 , 1997 . population - based controls were identified using random digit dialling ( < 65 years of age ) and health care financing administration rosters ( 65 + years of age ) , and frequency matched to the expected age distribution of the cases by 5-year age group . the main questionnaire was administered by a trained interviewer in the participant 's home and lasted approximately 2 hrs . information was assessed on known and suspected risk factors for breast cancer , including passive and active cigarette smoking , lifetime alcohol use , menstrual and reproductive histories , hormone use , body size by decade of adult life , lifetime participation in recreational activities , prior medical history and family history of breast cancer ( http://epi.grants.cancer.gov/libcsp/projects/questionnaire.html ) . usual dietary intake in the year prior to the interview was assessed using a modified block self - administered food frequency questionnaire , as previously described . we also collected information on cancer treatment undergone prior to blood collection , including whether a participant had radiation and/or chemotherapy . of the eligible participants who completed the main questionnaire ( 1508 cases and 1556 controls ) , 73% of cases and 73% of controls donated a non - fasting blood samples at the time of the interview . the samples were donated on average within 3 months after breast cancer diagnosis , prior to the onset of chemotherapy for most case participants , but after surgery . for controls , blood samples , collected in ethylenediaminetetraacetic acid ( edta)-treated tubes , were shipped at room temperature for overnight delivery to columbia university for processing and storage . the samples were aliquoted and frozen at 80c within 24 hrs of collection . in the present study the levels of plasma protein carbonyl groups were assessed using a non - competitive elisa , essentially as described in , with some modifications . briefly , the oxidized protein standards were prepared by incubation of bovine serum albumin ( bsa ) ( 50 mg / ml ) with 0.73 m h2o2 and 0.42 mm fe for 1 hr at 37c . it was then diluted with native ( unoxidized ) bsa and phosphate - buffered saline ( pbs ) to give a final carbonyl content of 2.0 nmol / mg protein and protein concentration of 4 mg / ml . total protein concentration in the plasma samples was measured using bicinchoninic acid kit ( sigma ) and the samples were diluted with pbs to a final protein concentration of 4 mg / ml . after the derivatiza - tion with dnph , the plate was coated with 200 l of sample ( 1 g ) and incubated overnight at 4c in the dark . the plate was washed with pbs / tween ( 0.05% tween 20 in pbs ) and blocked with 0.1% bsa in pbs for 1.5 hr . after another washing step , biotinylated primary anti - dnp antibody ( molecular probes ; diluted 1:1500 with 0.1% bsa , 0.1% tween 20 in pbs ) was added and the plate was incubated at 37c for 1 hr . another washing was followed by adding the streptavidin - biotynylated horseradish peroxidase conjugate ( amersham ; diluted 1:4000 in 0.1% bsa , 0.1% tween 20 in pbs ) and incubation at room temperature for 1 hr . colour was developed by adding the tetram - ethyl benzidine ( tmb ) liquid substrate system ( sigma ) and the reaction was stopped with h2so4 after 1525 min incubation in the dark . each sample was analysed in duplicate ; the interassay coefficient of variability for the repeat analysis ( n = 53 ) of a quality control sample was 17% . as a quality control , a pooled plasma sample was repeatedly frozen and thawed before analysis ; there was no significant difference in protein carbonyl levels with up to four freeze - thaw cycles . we first compared the differences between 1050 cases and 1107 controls with regard to the plasma protein carbonyl levels ( nmol carbonyl / ml plasma ) . the analysis of variance test   was used for comparing case - control differences as continuous variables and the chi - square test   was used for comparing case - control differences . we also examined case - control differences using unconditional logistic regression   adjusting for reference age ( date of diagnosis for cases and date of identification for controls ) . other factors that were considered as possible confounders include those that have been associated with breast cancer and may also influence oxidative stress pathways , including average physical activity level , fruit and vegetable intake , average lifetime alcohol intake , active cigarette smoking status , bmi , menopausal status , age at menarche , age at first birth ( afb ) , parity , oral contraceptive ( oc ) use and hormone replacement therapy ( hrt ) use ( using definitions that have been previously published [ 10,1517 ] ) . only those that changed the effect estimates by more than 10% we stratified the association between plasma protein carbonyl levels and breast cancer by the same factors to examine effect modification on a multiplicative scale . we also examined the associations with the cases restricted to those women for whom chemotherapy ( n = 827 ) or radiation therapy ( n = 831 ) had not yet been initiated by the time of the blood collection . we then assessed whether plasma protein carbonyl levels differed by age , average physical activity level , fruit and vegetable intake , average lifetime alcohol intake , active smoking status , bmi , menopausal status , age at menarche , afb , parity , oc use and hrt use among 1107 controls first by using the analysis of variance test and second by using linear regression methods . we used log transformation for the plasma protein carbonyl levels in the linear regression model to meet the assumption of normality . we estimated a saturated model with all factors suspected to influence plasma protein carbonyl levels and a parsimonious model with other those variables which were statistically significantly related to the outcome . we observed a statistically significant trend in breast cancer risk in relation to increasing quartiles of plasma protein carbonyl levels ( multivariate - adjusted or = 1.2 , 95% ci = 0.91.5 ; or = 1.5 , 95% ci = 1.22.0 ; or = 1.6 , 95% ci = 1.22.1 for the 2 , 3 and 4 quartile relative to the lowest quartile , respectively , p for trend = 0.0001 ) ( table 1 ) . estimates differed little between models with and without adjustment for other breast cancer risk factors . among cases , there were no differences in plasma protein carbonyl level between those with or without chemotherapy or radiation therapy prior to blood collection . considering plasma protein carbonyl levels categorized into other quartile groupings ( tertiles , quintiles , deciles ) yielded similar effect estimates ( data not shown ) . thus , after removal of the 214 women who had received chemotherapy and the 209 who had received radiation therapy , results did not materially change ( or = 1.1 , 95% ci = 0.81.5 ; or = 1.7 , 95% ci = 1.22.3 ; or = 1.7 , 95% ci 1.22.3 for the 2 , 3 and 4 quartile relative to the lowest quartile , respectively , p for trend = 0.0001 ) . we also determined whether removal of women currently on hrt impacted results but again they did not materially change ( or = 1.0 , 95% ci = 0.71.4 ; or = 1.4 , 95% ci = 1.02.0;or = 1.3 , 95% ci 0.91.8 for the 2nd , 3rd and 4th quartile relative to the lowest quartile , p for trend = 0.02 ) . removing those who received chemotherapy and radiation therapy in addition to those on hrt resulted in ors of 1.0 ( 95% ci 0.71.4 ) , 1.6 ( 95%ci 1.12.3 and 1.4 ( 95%ci 0.92.0 ) for the 2 , 3 and 4 quartile relative to the lowest quartile ( p for trend = 0.02 ) . adjusted odds ratios and 95% confidence intervals for breast cancer associated with plasma protein carbonyl level * adjusted for age , average physical activity level , fruits and vegetables intake , average alcohol intake , active smoking status , menopausal status , bmi , ever use hrt , ever use oc , age at menarche , parity and age at first birth . the increase in breast cancer risk by quartile of plasma protein carbonyl level was similar for younger ( < 50 years ) and older women or by fruits and vegetables consumption ( table 2 ) . the association between plasma protein carbonyl level and breast cancer risk was more pronounced among women with higher physical activity levels ( 0.7 hrs / week for 4 quartile versus lowest quartile or = 2.0 , 95% ci = 1.43.0 ) , with higher average lifetime alcohol consumption ( 15 grams / day for 4 quartile versus lowest quartile or = 2.3 , 95% ci = 1.14.9 ) and who used hrt ( or = 2.6 , 95%ci = 1.64.4 for 4 quartile versus lowest quartile ) . but , the multiplicative interaction terms were statistically significant only for physical activity ( p= 0.02 ) and use of hrt ( p= 0.04 ) . breast cancer risk associated with plasma protein carbonyl level stratified by age , average physical activity level , fruits and vegetable intake , alcohol intake , cigarette smoking status , bmi , menopausal status , hrt and oc use , age of menarche and first birth and parity adjusted for age , average physical activity level , fruits and vegetables intake , average alcohol intake , active smoking status , menopausal status , bmi , ever use hrt , ever use oc , age at menarche , parity and age at first birth . to assess whether breast cancer risk is related to increased levels of markers of protein oxidation and lipid peroxidation , we compared plasma protein carbonyl levels and urinary 15f2t - isoprostane levels ( analysed in a subset of 400 cases and 401 controls in our previous study ) . women with higher levels of plasma protein carbonyl ( median ) and higher levels of 15f2t - isoprostane ( median ) had a 1.8-fold increase in breast cancer risk ( or = 1.8 , 95% ci = 1.22.6 ) compared to women with levels below the median for both markers of oxidative stress ( table 3 ) . breast cancer risk associated with the 15-f2t - isoprostane and plasma protein carbonyl levels in univariate analyses , among controls , plasma protein carbonyl levels significantly increased with age ( p= 0.02 , table 4 ) , and were significantly higher in post - menopausal compared to pre - menopausal women ( p= 0.01 ) and in those with age of menarche < 13 yrs ( p < 0.01 ) . contrary to our expectations , levels were also higher with increased fruits and vegetables intake ( p= 0.04 ) . no significant associations were found between plasma protein carbonyl levels and average physical activity level , average lifetime alcohol intake , cigarette smoking status , bmi and other reproductive variables . mean ( and sd ) of plasma protein carbonyl levels ( continuous ) within strata of age , average physical activity level , fruits and vegetables intake , average lifetime alcohol intake , cigarette smoking status , body mass index ( bmi ) and menopausal status , ever use hrt , ever use oc , age at menarche , parity and age at first birth among controls comparing differences between mean values by category . table 5 reports the findings from multivariate linear regression models examining the association between selected breast cancer risk factors and plasma protein carbonyl levels among controls . higher average physical activity , higher consumption of alcohol and later age of menarche were associated with decreased plasma protein carbonyl levels . in the best - fitting model , higher fruit and vegetable intake and post - menopausal status were associated with increased plasma protein car - bonyl levels and high alcohol consumption and later age of menarche with decreased levels . multivariate estimates of the association among age , average physical activity level , fruit and vegetable intake , average lifetime alcohol intake , cigarette smoking status , body mass index ( bmi ) , menopausal status , ever used hrt , ever used oc , age at menarche , parity , and age at first birth and the log of plasma protein carbonyl levels among controls * adjusted for age , average physical activity level , fruits and vegetables intake , average lifetime alcohol intake , active smoking status , body mass index , menopausal status , ever use hrt , ever use oc , age at menarche , parity and age at first birth . * * best - fitting model . protein oxidation , measured as plasma levels of protein carbonyl groups , was significantly associated with breast cancer risk in our study . participants with the highest quartile of plasma protein carbonyl levels had a 60% increased risk of developing breast cancer compared to participants with plasma protein carbonyl levels in the lowest quartile . several previous studies have provided evidence that protein oxidation might be associated with cancer risk . increased levels of plasma protein carbonyl groups were observed in patients with hodgkin 's lymphoma , with bladder cancer   and in children with various malignancies , but not in a small study of lung cancer . to the best of our knowledge , our study is the first analysis of the relationship between protein oxidation and breast cancer risk . also , our study completes the investigation of a panel of oxidative stress markers among the participants of libcsp . according to a recent report , it is critical to simultaneously analyse several markers of oxidative stress to better understand the processes leading to the particular pathology being studied . the process of aging is accompanied by increasing levels of oxidative damage , including protein oxidation ( reviewed in ) . increased levels of protein carbonyls have been observed in elderly participants as compared with younger participants in some ,   but not all   studies . we also found increased plasma protein carbonyl levels in older women ( table 4 ) . in the multivariate analysis , higher physical activity was associated with decreased plasma protein carbonyl levels ( table 5 ) . in general , physical exercise can produce an imbalance between ros and antioxidants and thus lead to oxidative stress , but the results of different studies are conflicting . decreased plasma protein carbonyl levels were found in participants after strenuous physical exercise   and in sedentary participants as a result of physical exercise , but levels increased in participants in a super - marathon race . thus , the biological meaning of our observation is not clear;how - ever , it is in agreement with other studies . fruits and vegetables contain both antioxidant and pro - oxidant factors so their effect on oxidative stress markers may be variable . decreased levels of various oxidative stress markers have been observed after administration of fruits and vegetables - rich food in several studies [ 3133 ] . analysis of data from the libcsp showed that higher fruits and vegetables consumption was associated with significantly decreased breast cancer risk among postmenopausal women ( or 0.72 , 95% ci 0.530.99 for the highest quintile compared with the lowest ) with weaker associations among pre - menopausal . contrary to this data , we observed significantly increased plasma protein carbonyl levels among participants with higher fruits and vegetables consumption ( table 4 & 5 ) . where participants receiving a diet rich in fruits and vegetables , or a pill containing vitamins and minerals corresponding to those in the fruits and vegetables diet , had higher protein carbonyl formation at lysine residues than those on placebo . they hypothesized that the pro - oxidant activity of vitamin c might be responsible for their findings . three pathways of ethanol metabolism have been described and each results in free radical production leading to oxidative damage . several studies have observed increased protein carbonyl levels among patients with chronic alcoholism [ 3638 ] suggesting that repeated exposure to high alcohol levels may be necessary to affect protein carbonyl levels . lower levels of oxidative stress markers were reported among moderate alcohol drinkers [ 39 , 40 ] , but these studies concentrated on white and red wine drinkers . it is probable that the presence of antioxidants in wine diminishes the negative effect of ethanol on oxidative stress markers . in the present study , this may reflect the type of alcoholic beverages consumed by the participants in our study ( prevalence of wines rich in antioxidants ) , as well as relatively low levels of alcohol exposure that were not sufficient to increase oxidative stress even in the group of women consuming > 30 g alcohol / day . significantly increased levels of oxidized proteins in smokers than in non - smokers were observed   with similar , but non - significant results in another study . we found a significant increase in plasma protein carbonyl levels among former smokers compared with non - smokers ( table 5 ) . shorter life - time exposure to estrogen as a result of late menarche may decrease oxidative stress induced by estrogen metabolism . in our previous study , we observed an association between urinary 15-f2t - isoproprostane , a marker of lipid peroxidation , and breast cancer risk in a subset of women analysed in the present study . the combination of elevated ( median ) plasma protein carbonyl and 15-f2t - isoproprostane levels resulted in a 1.8-fold increase in breast cancer risk compared to women with levels below the median for both markers of oxidative stress . analysis of a combination of several oxidative stress markers may provide more relevant information about risk than any one measure . a limitation of our study is that the blood samples were shipped at room temperature after collection and haemolysis may have contributed to additional oxidative stress . however , all samples were handled the same way and thus , time between collection of samples and their processing can not account for the differences between cases and controls . although extensive efforts were undertaken to obtain the samples prior to the onset of chemotherapy and radiation therapy for most case participants , all breast cancer cases had undergone surgery before blood draw . it has been shown that surgery results in significant decrease of ros production and levels of oxidative stress markers , including protein carbonyl levels in colon   and laryngeal cancer patients . moreover , total plasma antioxidant capacity increases after a tumour removal . in our study , we observed no effect of non - surgical treatment on carbonyl level and when we restricted our analyses to case participants who had not received chemotherapy and radiation therapy prior to blood collection , our results did not change substantially . a prospective study using samples collected before diagnosis may be more useful for determining if elevated levels of protein oxidation predict breast cancer risk . a major strength of our study is the large number of participants , which helped to ensure that the study had sufficient power . in addition , the population - based study design ensured that cases and controls arose from the same source population . in summary , we found positive associations between plasma protein carbonyl levels and breast cancer risk in this population - based , case - control study . among controls , oxidized protein levels significantly increased with cigarette smoking and , unexpectedly , with higher fruit and vegetable consumption but decreased with alcohol consumption > 30 grams per day . elevation in two oxidative stress biomarkers , urinary 15f2t - isoprostanes and oxidized protein was associated with increased breast cancer risk . our results add additional support to the hypothesis that oxidative stress may play a role in breast cancer etiology .