An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expressio... more An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ngmL(-1) 17beta-estradiol and 17alpha-ethynylestradiol, 10 and 50 ngmL(-1) mestranol, and 100 ngmL(-1) testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17beta-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ngmL(-1). Sample extracts and standards were coded and tested blindly. A decision limit (CCalpha) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ngmL(-1) testosterone or progesterone, were all below the determined CCalpha and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17beta-estradiol, 17alpha-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCalpha. Determined EC(50) values calculated from the 17beta-estradiol dose-response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM.
Targeting the estrogen pathway has been proven effective in the treatment for estrogen receptor p... more Targeting the estrogen pathway has been proven effective in the treatment for estrogen receptor positive breast cancer. There are currently two common groups of anti-estrogenic compounds used in the clinic; Selective Estrogen Receptor Modulators (SERMs, e.g. tamoxifen) and Selective Estrogen Enzyme Modulators (SEEMs e.g. letrozole). Among various naturally occurring, biologically active compounds, resveratrol and melatonin have been suggested to act as aromatase inhibitors, which make them potential candidates in hormonal treatment of breast cancer. Here we used a co-culture model in which we previously demonstrated that primary human breast adipose fibroblasts (BAFs) can convert testosterone to estradiol, which subsequently results in estrogen receptor-mediated breast cancer T47D cell proliferation. In the presence of testosterone in this model, we examined the effect of letrozole, resveratrol and melatonin on cell proliferation, estradiol (E2) production and gene expression of CYP19A1, pS2 and Ki-67. Both melatonin and resveratrol were found to be aromatase inhibitors in this co-culture system, albeit at different concentrations. Our co-culture model did not provide any indications that melatonin is also a selective estrogen receptor modulator. In the T47D-BAF co-culture, a melatonin concentration of 20 nM and resveratrol concentration of 20 μM have an aromatase inhibitory effect as potent as 20 nM letrozole, which is a clinically used anti-aromatase drug in breast cancer treatment. The SEEM mechanism of action of especially melatonin clearly offers potential advantages for breast cancer treatment.
For a better understanding of species-specific relative effect potencies (REPs), responses of dio... more For a better understanding of species-specific relative effect potencies (REPs), responses of dioxin-like compounds (DLCs) were assessed. REPs were calculated using chemical-activated luciferase gene expression assays (CALUX) derived from guinea pig, rat, and mouse cell lines. Almost all 20 congeners tested in the rodent cell lines were partial agonists and less efficacious than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For this reason, REPs were calculated for each congener using concentrations at which 20% of the maximal TCDD response was reached (REP20TCDD). REP20TCDD values obtained for PCDD/Fs were comparable with their toxic equivalency factors assigned by the World Health Organization (WHO-TEF), while those for PCBs were in general lower than the WHO-TEF values. Moreover, the guinea pig cell line was the most sensitive as indicated by the 20% effect concentrations of TCDD of 1.5, 5.6, and 11.0 pM for guinea pig, rat, and mouse cells, respectively. A similar response pattern was observed using multivariate statistical analysis between the three CALUX assays and the WHO-TEFs. The mouse assay showed minor deviation due to higher relative induction potential for 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,6,7,8-hexachlorodibenzofuran and lower for 1,2,3,4,6,7,8-heptachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl (PCB126). 2,3,7,8-Tetrachlorodibenzofuran was more than two times more potent in the mouse assay as compared with that of rat and guinea pig cells, while measured REP20TCDD for PCB126 was lower in mouse cells (0.05) as compared with that of the guinea pig (0.2) and rat (0.07). In order to provide REP20TCDD values for all WHO-TEF assigned compounds, quantitative structure-activity relationship (QSAR) models were developed. The QSAR models showed that specific electronic properties and molecular surface characteristics play important roles in the AhR-mediated response. In silico derived REP20TCDD values were generally consistent with the WHO-TEFs with a few exceptions. The QSAR models indicated that, e.g., 1,2,3,7,8-pentachlorodibenzofuran and 1,2,3,7,8,9-hexachlorodibenzofuran were more potent than given by their assigned WHO-TEF values, and the non-ortho PCB 81 was predicted, based on the guinea-pig model, to be 1 order of magnitude above its WHO-TEF value. By combining in vitro and in silico approaches, REPs were established for all WHO-TEF assigned compounds (except OCDD), which will provide future guidance in testing AhR-mediated responses of DLCs and to increase our understanding of species variation in AhR-mediated effects.
Breast cancer treatment by the aromatase inhibitor Letrozole (LET) or Selective Estrogen Receptor... more Breast cancer treatment by the aromatase inhibitor Letrozole (LET) or Selective Estrogen Receptor Modulator Tamoxifen (TAM) can result in the onset of menopausal symptoms. Women often try to relieve these symptoms by taking menopausal supplements containing high levels of phytoestrogens. However, little is known about the potential interaction between these supplements and breast cancer treatment, especially aromatase inhibitors. In this study, interaction of phytoestrogens with the estrogen receptor alpha and TAM action was determined in an ER-reporter gene assay (BG1Luc4E2 cells) and human breast epithelial tumor cells (MCF-7). Potential interactions with aromatase activity and LET were determined in human adrenocorticocarcinoma H295R cells. We also used the previously described H295R/MCF-7 co-culture model to study interactions with steroidogenesis and tumor cell proliferation. In this model, genistein (GEN), 8-prenylnaringenin (8PN) and four commercially available menopausal supplements all induced ER-dependent tumor cell proliferation, which could not be prevented by physiologically relevant LET and 4OH-TAM concentrations. Differences in relative effect potencies between the H295R/MCF-7 co-culture model and ER-activation in BG1Luc4E2 cells, were due to the effects of the phytoestrogens on steroidogenesis. All tested supplements and GEN induced aromatase activity, while 8PN was a strong aromatase inhibitor. Steroidogenic profiles upon GEN and 8PN exposure indicated a strong inhibitory effect on steroidogenesis in H295R cells and H295R/MCF-7 co-cultures. Based on our in vitro data we suggest that menopausal supplement intake during breast cancer treatment should better be avoided, at least until more certainty regarding the safety of supplemental use in breast cancer patients can be provided.
An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expressio... more An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ngmL(-1) 17beta-estradiol and 17alpha-ethynylestradiol, 10 and 50 ngmL(-1) mestranol, and 100 ngmL(-1) testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17beta-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ngmL(-1). Sample extracts and standards were coded and tested blindly. A decision limit (CCalpha) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ngmL(-1) testosterone or progesterone, were all below the determined CCalpha and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17beta-estradiol, 17alpha-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCalpha. Determined EC(50) values calculated from the 17beta-estradiol dose-response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM.
An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expressio... more An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ngmL(-1) 17beta-estradiol and 17alpha-ethynylestradiol, 10 and 50 ngmL(-1) mestranol, and 100 ngmL(-1) testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17beta-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ngmL(-1). Sample extracts and standards were coded and tested blindly. A decision limit (CCalpha) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ngmL(-1) testosterone or progesterone, were all below the determined CCalpha and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17beta-estradiol, 17alpha-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCalpha. Determined EC(50) values calculated from the 17beta-estradiol dose-response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM.
Targeting the estrogen pathway has been proven effective in the treatment for estrogen receptor p... more Targeting the estrogen pathway has been proven effective in the treatment for estrogen receptor positive breast cancer. There are currently two common groups of anti-estrogenic compounds used in the clinic; Selective Estrogen Receptor Modulators (SERMs, e.g. tamoxifen) and Selective Estrogen Enzyme Modulators (SEEMs e.g. letrozole). Among various naturally occurring, biologically active compounds, resveratrol and melatonin have been suggested to act as aromatase inhibitors, which make them potential candidates in hormonal treatment of breast cancer. Here we used a co-culture model in which we previously demonstrated that primary human breast adipose fibroblasts (BAFs) can convert testosterone to estradiol, which subsequently results in estrogen receptor-mediated breast cancer T47D cell proliferation. In the presence of testosterone in this model, we examined the effect of letrozole, resveratrol and melatonin on cell proliferation, estradiol (E2) production and gene expression of CYP19A1, pS2 and Ki-67. Both melatonin and resveratrol were found to be aromatase inhibitors in this co-culture system, albeit at different concentrations. Our co-culture model did not provide any indications that melatonin is also a selective estrogen receptor modulator. In the T47D-BAF co-culture, a melatonin concentration of 20 nM and resveratrol concentration of 20 μM have an aromatase inhibitory effect as potent as 20 nM letrozole, which is a clinically used anti-aromatase drug in breast cancer treatment. The SEEM mechanism of action of especially melatonin clearly offers potential advantages for breast cancer treatment.
For a better understanding of species-specific relative effect potencies (REPs), responses of dio... more For a better understanding of species-specific relative effect potencies (REPs), responses of dioxin-like compounds (DLCs) were assessed. REPs were calculated using chemical-activated luciferase gene expression assays (CALUX) derived from guinea pig, rat, and mouse cell lines. Almost all 20 congeners tested in the rodent cell lines were partial agonists and less efficacious than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For this reason, REPs were calculated for each congener using concentrations at which 20% of the maximal TCDD response was reached (REP20TCDD). REP20TCDD values obtained for PCDD/Fs were comparable with their toxic equivalency factors assigned by the World Health Organization (WHO-TEF), while those for PCBs were in general lower than the WHO-TEF values. Moreover, the guinea pig cell line was the most sensitive as indicated by the 20% effect concentrations of TCDD of 1.5, 5.6, and 11.0 pM for guinea pig, rat, and mouse cells, respectively. A similar response pattern was observed using multivariate statistical analysis between the three CALUX assays and the WHO-TEFs. The mouse assay showed minor deviation due to higher relative induction potential for 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,6,7,8-hexachlorodibenzofuran and lower for 1,2,3,4,6,7,8-heptachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl (PCB126). 2,3,7,8-Tetrachlorodibenzofuran was more than two times more potent in the mouse assay as compared with that of rat and guinea pig cells, while measured REP20TCDD for PCB126 was lower in mouse cells (0.05) as compared with that of the guinea pig (0.2) and rat (0.07). In order to provide REP20TCDD values for all WHO-TEF assigned compounds, quantitative structure-activity relationship (QSAR) models were developed. The QSAR models showed that specific electronic properties and molecular surface characteristics play important roles in the AhR-mediated response. In silico derived REP20TCDD values were generally consistent with the WHO-TEFs with a few exceptions. The QSAR models indicated that, e.g., 1,2,3,7,8-pentachlorodibenzofuran and 1,2,3,7,8,9-hexachlorodibenzofuran were more potent than given by their assigned WHO-TEF values, and the non-ortho PCB 81 was predicted, based on the guinea-pig model, to be 1 order of magnitude above its WHO-TEF value. By combining in vitro and in silico approaches, REPs were established for all WHO-TEF assigned compounds (except OCDD), which will provide future guidance in testing AhR-mediated responses of DLCs and to increase our understanding of species variation in AhR-mediated effects.
Breast cancer treatment by the aromatase inhibitor Letrozole (LET) or Selective Estrogen Receptor... more Breast cancer treatment by the aromatase inhibitor Letrozole (LET) or Selective Estrogen Receptor Modulator Tamoxifen (TAM) can result in the onset of menopausal symptoms. Women often try to relieve these symptoms by taking menopausal supplements containing high levels of phytoestrogens. However, little is known about the potential interaction between these supplements and breast cancer treatment, especially aromatase inhibitors. In this study, interaction of phytoestrogens with the estrogen receptor alpha and TAM action was determined in an ER-reporter gene assay (BG1Luc4E2 cells) and human breast epithelial tumor cells (MCF-7). Potential interactions with aromatase activity and LET were determined in human adrenocorticocarcinoma H295R cells. We also used the previously described H295R/MCF-7 co-culture model to study interactions with steroidogenesis and tumor cell proliferation. In this model, genistein (GEN), 8-prenylnaringenin (8PN) and four commercially available menopausal supplements all induced ER-dependent tumor cell proliferation, which could not be prevented by physiologically relevant LET and 4OH-TAM concentrations. Differences in relative effect potencies between the H295R/MCF-7 co-culture model and ER-activation in BG1Luc4E2 cells, were due to the effects of the phytoestrogens on steroidogenesis. All tested supplements and GEN induced aromatase activity, while 8PN was a strong aromatase inhibitor. Steroidogenic profiles upon GEN and 8PN exposure indicated a strong inhibitory effect on steroidogenesis in H295R cells and H295R/MCF-7 co-cultures. Based on our in vitro data we suggest that menopausal supplement intake during breast cancer treatment should better be avoided, at least until more certainty regarding the safety of supplemental use in breast cancer patients can be provided.
An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expressio... more An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ngmL(-1) 17beta-estradiol and 17alpha-ethynylestradiol, 10 and 50 ngmL(-1) mestranol, and 100 ngmL(-1) testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17beta-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ngmL(-1). Sample extracts and standards were coded and tested blindly. A decision limit (CCalpha) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ngmL(-1) testosterone or progesterone, were all below the determined CCalpha and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17beta-estradiol, 17alpha-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCalpha. Determined EC(50) values calculated from the 17beta-estradiol dose-response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM.
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Papers by Majorie Van Duursen