Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines.

Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cells and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100 × 10(3) and 50 × 10(3) dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10 × 10(3) and 5 × 10(3) dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p<0.05 at the 50 × 10(3) dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, ß-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The proliferative effect of the mixture on CAMA-1 cells in the presence of sex steroids appears mostly due to the antiandrogenic properties of p,p'-DDE, a major constituent of the mixture. Other mixtures of contaminants that include emerging compounds of interest such as brominated flame retardants and perfluoroalkyl compounds should be tested for their capacity to induce breast cancer cell proliferation.

1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) disrupts the estrogen-androgen balance regulating the growth of hormone-dependent breast cancer cells.

INTRODUCTION: Estrogen and androgen signalling pathways exert opposing influences on the proliferation of mammary epithelial and hormone-dependent breast cancer cells. We previously reported that plasma concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), the main metabolite of the insecticide DDT (1,1,1-trichloro-2,2-bis [p-chlorophenyl]ethane) and a potent androgen antagonist, were associated with tumor aggressiveness in women diagnosed with breast cancer. We sought to examine the biological plausibility of this association by testing the effect of p,p'-DDE on the proliferation of CAMA-1 cells, a human breast cancer cell line that expresses the estrogen receptor alpha (ERalpha) and the androgen receptor (AR), in the presence of physiological concentrations of estrogens and androgens in the cell culture medium. METHODS: The proliferation of CAMA-1 cells was determined in 96-well plates following a 9-day treatment with p,p'-DDE alone (0.1 to 10 muM) or in combination with 17beta-estradiol (E2) (100 pM) and dihydrotestosterone (DHT) (100, 500, or 1,000 pM). We also assessed p,p'-DDE-induced modifications in cell cycle entry and the expression of the sex-steroid-dependent genes ESR1, AR, CCND1, and TFF1 (pS2) (mRNA and/or protein). RESULTS: We found that treatment with p,p'-DDE induced a dose-response increase in the proliferation of CAMA-1 cells when cultivated in the presence of physiological concentrations of estrogens and androgens, but not in the absence of sex steroids in the cell culture medium. A similar effect of p,p'-DDE was noted on the proliferation of MCF7-AR1 cells, an estrogen-responsive cell line that was genetically engineered to overexpress the AR. DHT added together with E2 to the cell culture medium decreased the recruitment of CAMA-1 cells in the S phase and the expression of ESR1 and CCND1 by comparison with cells treated with E2 alone. These androgen-mediated effects were blocked with similar efficacy by p,p'-DDE and the potent antiandrogen hydroxyflutamide. CONCLUSION: Our results suggest that p,p'-DDE could increase breast cancer progression by opposing the androgen signalling pathway that inhibits growth in hormone-responsive breast cancer cells. The potential role of environmental antiandrogens in breast carcinogenesis deserves further investigation.

Biomarker measurements in a coastal fish-eating population environmentally exposed to organochlorines.

The Lower North Shore region of the St. Lawrence River is home to a fish-eating population that displays an unusually high body burden of several organochlorines, including polychlorinated biphenyls (PCBs) and dioxin-like compounds (DLCs). We measured biomarkers indicative of liver enzyme induction and investigated the relationship with organochlorine body burden in adult volunteers from this population. We determined plasma concentrations of PCBs and chlorinated pesticides by high-resolution gas chromatography (HRGC) with electron capture detection. DLC concentrations were measured by the dioxin-receptor chemically activated luciferase expression (DR-CALUX) assay and in a subset of participants, by HRGC/high-resolution mass spectrometry. We measured cotinine, d-glucaric acid, and porphyrins in morning urine samples and determined liver CYP1A2 activity in vivo using the caffeine breath test. Neither DLC concentrations as measured by the DR-CALUX nor PCB-153 concentrations, the latter representing total PCB exposure, were correlated with biomarkers of effects. Smoking (morning urinary cotinine concentration) was positively related to CYP1A2 activity as measured by the caffeine breath test (p < 0.01). Liver CYP1A2 activity was in turn negatively correlated with PCB-105:PCB-153 and PCB-118:PCB-153 congener ratios (p < 0.05). Hence, despite the relatively high body burden of PCBs and DLCs in this population, only smoking had a significant correlation with biomarkers of hepatic enzyme induction. Our data are consistent with smoking-induced liver CYP1A2 activity altering heme metabolism and increasing the biotransformation of mono-ortho PCB congeners.

Determinants of AhR-mediated transcriptional activity induced by plasma extracts from Nunavik Inuit adults.

The exposure of Inuit people to polychlorinated biphenyls (PCBs) and chlorinated pesticides has been well characterised but little is known regarding their exposure to dioxin-like compounds, which induce toxic effects through binding to the aryl hydrocarbon receptor (AhR). In order to obtain a global measure of persistent organic pollutants in plasma that interact with this signalling pathway, we used a luciferase reporter gene assay to assess the AhR-mediated transcriptional activity elicited by plasma sample extracts from 874 Inuit adults who were recruited in the course of a prospective epidemiological study conducted in Nunavik (Québec, Canada). Several sociodemographic, anthropometric, dietary and lifestyle variables were considered as possible modulating factors of the AhR-mediated activity in multivariate statistical analyses. The geometric mean AhR-mediated activity expressed as 2,3,7,8-tetachlorodibenzo-p-dioxin equivalents was 8.9 pg g(-1) lipids (range: <5-144 pg g(-1) lipids). PCB-153 concentration measured by high-resolution gas chromatography-mass spectrometry was moderately correlated to AhR-mediated activity (Pearson's r=0.53, p<0.001). Multiple linear regression analyses revealed that age and omega-3 fatty acids in erythrocyte membranes (an index of marine food consumption) were positively associated with plasma AhR-mediated activity (p<0.001), whereas a negative association was noted with body fat mass (p=0.037). These results suggest that AhR-mediated transcriptional activity of Inuit plasma extracts is linked to their organochlorine body burden, most likely that of dioxin-like PCBs, polychlorinated dibenzo-p-dioxins and polychlorodibenzofurans. AhR-mediated transcriptional activity measures may prove useful in investigating possible associations between exposure to AhR agonists and adverse health effects in this indigenous population.

Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo.

BACKGROUND: The genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation. METHODOLOGY/PRINCIPAL FINDINGS: We have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation. SIGNIFICANCE: Our observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.