DDT (dicophane) and postmenopausal breast cancer in Europe: case-control study

Introduction

Environmental oestrogenic compounds, such as the organochlorines DDT (dicophane (2,2-bis (p -chlorophenyl)-1,1,1-trichloroethane)), polychlorinated biphenyls, and dioxins, have been linked to altered sexual development in various species, to a decrease in semen quality, and to an increased risk of breast cancer in women.1 2 3 Their weak oestrogenic effects may result from altered metabolism and competition for binding to cytosolic and nuclear receptors of steroid hormones.4 5

Because of their lipophilicity and long half lives, organochlorines accumulate in the food chain.6 Typically, consumption of fish, meat, and milk is held responsible for the age related increase in concentrations of DDT and its even more persistent metabolite DDE (1,1-dichloro-2,2-bis (p -chlorophenyl)ethylene) in adipose tissue.7 Since the ban on DDT in Western countries 20-25 years ago, DDE concentrations in adipose tissue and mothers' milk dropped in successive cohorts but less so in the generations exposed before the ban.6 8 9

Apart from some early reports,10 11 12 one nested case-control study in the United States showed a fourfold increased risk of breast cancer at high plasma DDE concentrations,13 which was not confirmed by a larger prospective study, also from the United States.14 We determined the association between DDE and breast cancer among 606 women from five European countries on the basis of DDE measured in adipose tissue aspirated from the buttocks.

Subjects and methods

This investigation is an extension of the European study on antioxidants, myocardial infarction, and cancer of the breast (EURAMIC). This multicentre case-control study included 347 women with breast cancer from Germany, the Netherlands, Northern Ireland, Switzerland, and Spain as well as 374 population and hospital controls.15 16 In short, apparently healthy postmenopausal women, aged 50 to 74 years, with a stable dietary pattern and no history of breast cancer were eligible. To achieve efficiency in study conduct and data analysis we aimed at a similar number of cases and controls by study centre and age using group matching. Incident cases with histologically confirmed ductal breast cancer, primary tumour <5 cm, axillary lymph nodes stage <N3, and no clinical indication of distant metastases at discharge were included. Response rates among women with cancer were 75% (Germany), 76% (Northern Ireland), 92% (Switzerland), and 97% (Netherlands, Spain). Controls were obtained from the hospital catchment area by using registries of local municipalities (Switzerland and Germany with response rates of 22% and 45%) and general practitioners as the sampling frame (Northern Ireland, Netherlands, and Spain with response rates of 46%, 50%, and 91%). Information on factors relevant to breast cancer was obtained by similarly formatted centre specific questionnaires, with a priori specified variables for central data analysis. Procedures for sampling and data collection were approved by local ethics committees in each participant's country. Informed consent was obtained from eligible subjects.

Needle aspirates were taken from the subcutaneous fat of the buttocks in 317 women with cancer and 367 controls.15 16 17 For the cancer patients the fat aspirates were taken within seven days after hospital admission. After the exclusion of five subjects for whom relevant background data were missing, fat aspirates of 265 cases and 341 controls were available for laboratory analysis—that is, DDE was analysed in 84% and 93% of the aspirates obtained. This percentage is lower in the cases because for one of the centres the aspirates had to be collected in several hospitals by different staff, less skilled in fat aspiration. To facilitate the standardising and instructing of the skills for fat aspiration, a videotape was prepared and distributed among all centres before the start of the project. In the four other centres this apparently led to good results as sufficient amounts of fat could be aspirated from 91% of the cases and 93% of the controls in these centres. After local storage at -70°C and shipment on dry ice, samples were analysed centrally for vitamins and fatty acids.15 16

Laboratory analyses of DDE were conducted at the National Institute of Public Health and Environmental Protection (Bilthoven, Netherlands), with sample vials randomised before laboratory analyses and staff blinded to disease status. The initial iso-octane extracts contained the methylated fatty acids and heptadecanoic acid (C17:0) as an internal standard. This solution was subjected to hyphenated chromatography, in which liquid chromatography was coupled on line to capillary chromatography. This analysis was carried out on a commercially available liquid chromatography-gas chromatography system (Dualchrom 2000; Carlo Erba Instrument, Milan, Italy) equipped with a Ni63 electron capture detector. The liquid chromatography injection volume was 50 μl. The on column interface was used to transfer a volume of 200 μl into the column. The chromatographic columns were a liquid chromatography column of 50 mm × 1 mm internal diameter packed with 3 μm hypersil silica and a DB5MS gas chromatography column (30 m × 0.32 mm internal diameter, film thickness 0.5 μm). The C17:0 content was determined by gas chromatographic analysis. When the concentration of DDE was below the detection limit (n=19) half the lowest concentration in the remaining samples was entered. The coefficient of variation within each batch varied from 1% to 5% for C17:0 and from 1% to 8% for DDE and between batches from 7% to 8% for C17:0 and from 8% to 11% for DDE. Concentrations of DDE are expressed in micrograms of DDE per gram of fatty acids in the aspirate.

Data analyses were performed with sas statistical software.18 Although skewed distributions of DDE required log transformations in data analysis, results presented are geometric means. Crude means as well as means adjusted for age and centre were calculated for DDE and major risk factors for breast cancer. To identify potential confounders, mean levels of risk factors for breast cancer were compared among fourths of DDE concentration in controls. Odds ratios (and 95% confidence intervals) for breast cancer were obtained from multivariate logistic regression analysis by modelling DDE, firstly in fourths and then as a continuous variable, and contrasting the risk for subjects on the 75th and 25th centiles. In tests for trend, the median DDE concentration in each fourth was used. Interaction terms between DDE and body mass index, waist : hip ratio, fat mass, parity, hormone replacement therapy, oestrogen receptor status, time since menopause, previous benign breast disease, family history of breast cancer, and current alcohol use were tested, but none was significant.

Results

Table 1 shows risk factors among cases and controls. Age was similar because of the matched design. Women with breast cancer had significantly higher body mass indices, waist : hip ratio, age at first birth, and family history of breast cancer. No other significant differences were found.

Among controls, body mass index (kg/m²) was positively associated with DDE concentration and increased from 24.9 (SD 4.0) in the lowest fourth to 28.2 (4.7) in the highest fourth (P<0.01). Waist : hip ratio increased from 0.83 (0.08) to 0.86 (0.06) (P=0.03). Reported history of benign breast disease differed between fourths of DDE (P=0.05), ranging from 13.1% (10.2%) in the second to 3.6% (10.8%) in the fourth. Current alcohol use was more prevalent in the lowest than in the highest fourth (57.4 (7.9%) v 40.2 (8.5%); P<0.01).

Mean DDE concentration was 1.35 μg/g and 1.51 μg/g among cases and controls, respectively, a difference of -10.5%, which changed little after adjustment for age and centre (-9.2%; P=0.36; table 2). Concentrations were lower among cases than among controls in all but one centre (the Netherlands). DDE concentrations ranged from 2.5-fold to 3-fold between countries, being lowest in Northern Ireland and highest in Spain. Within controls, the median exposure in the lowest (0.40 μg/g) versus the highest fourth (5.07 μg) differed 12.5-fold (table 3). The odds ratio for breast cancer, adjusted for age and centre, for the highest versus the lowest fourth of DDE was 0.73, but the test for trend was not significant (P=0.16). Adjustment for body mass index, age at first birth, and current alcohol consumption strengthened the inverse association (P for trend=0.02), with the odds ratio in the highest fourth showing a significant 52% risk reduction. The largest change in the odds ratio occurred when body mass index was entered into the model, which made further adjustment for waist : hip ratio superfluous. When DDE was modelled as a continuous variable, the odds ratio for a subject at the 75th versus the 25th centile (3.46 v 0.86 μg/g) was 0.84 (95% confidence interval 0.73 to 0.97). Further adjustment for waist : hip ratio, history of previous benign breast disease, family history of breast cancer, and years since menopause did not materially affect the risk estimates.

Discussion

Our study did not confirm the hypothesis that DDE concentrations in adipose tissue are higher in women with breast cancer. In contrast, we observed an overall inverse association between DDE and breast cancer, which was consistent in most of the study centres.