In vitro effects of 2-methoxyestradiol, an endogenous estrogen, on MCF-12A and MCF-7 cell cycle progression

2-Methoxyestradiol (2ME) is an endogenous estrogen metabolite with antiproliferative and antiangiogenic properties. 2ME also plays an active role in the induction of apoptosis, especially in cancerous cells. These properties have been confirmed by various in vitro and in vivo studies and render 2ME a potential antitumor agent. The mechanism of action of 2ME, however, is not yet fully elucidated and it is believed that multiple mechanisms are involved that may be dependent on cell type. The aim of this study was to investigate the differential effects of 2ME on cell growth, morphology and spindle formation in the non-tumorigenic MCF-12A breast cell line and the tumorigenic MCF-7 breast cell line. In dose-dependent studies, cell growth was determined spectrophotometrically. Light microscopy was used to investigate the morphological changes induced by 2ME and its effect on spindle formation was investigated by means of indirect immunofluorescence. The estrogen receptor status of the MCF-12A cells was confirmed with immunocytochemistry. In order to investigate the effect of 2ME on the length of the cell cycle, cells were blocked in early S-phase with hydroxyurea, then allowed to continue through the cell cycle and mitotic indices determined at regular time intervals. Checkpoint kinase and Cdc2 kinase assays were used to determine the effect of 2ME on relevant cell cycle kinases. Although 2ME inhibited cell growth in both cell lines, the MCF-7 cells were inhibited from much lower concentrations and growth inhibition was more pronounced than in the MCF-12A cells. Treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent, or not as prominent in the MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells. Although the two cell lines differ in their estrogen receptor status, this could not explain the differential effects, for 2ME has a very low affinity for the estrogen receptor. 2ME had no effect on the length of the cell cycle, but blocked MCF-7 cells in mitosis. There were no significant alterations in the phosphorylation status of Cdc25C after 2ME treatment. However, Cdc2 activity was increased to a greater extend in the MCF-7 cells than in the MCF-12A cells. Therefore, it is suggested that exposure to 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest, which may result in the induction of apoptosis. The tumorigenic MCF-7 cells are especially sensitive to 2ME treatment compared to the normal MCF-12A cells. 2ME shows potential for the treatment of breast cancer. Selecting the concentration of 2ME that has maximum inhibitory effect on tumorigenic, but minimal effect on normal cells is crucial in its possible application as antitumor agent. Furthermore, research concerning the differential action mechanisms of 2ME is essential to create a better understanding regarding the treatment of cancer and may possibly contribute to the development and/or improvement of novel chemotherapeutic agents.Dissertation (MSc (Physiology))--University of Pretoria, 2008.Physiologyunrestricte

Quality and safety of South African hand sanitisers during the COVID-19 pandemic

Health agencies recommend using hand sanitisers as protection against the coronavirus. Thus far, the emphasis on hand sanitiser studies is limited to an analysis of disinfectant content only. This study aims to provide an extended analysis of 60 off-the-shelf alcohol-based hand sanitisers by using gas chromatography to report on alcohol content and the presence of impurities, a recombinant yeast estrogen screen to assess estrogenic activity, and an investigation into labelling compliance with the South African National Standard. Fifty hand sanitisers had an alcohol content of ≥60% v/v alcohol; however, most contained skin irritants and substances that could harm human and environmental health. Estrogenic activity was detected in 29 hand sanitisers and none of the products complied with all the labelling requirements. Since off-the-shelf hand sanitisers in South Africa are not regulated and monitored, evidence-based public awareness programmes on hand sanitiser quality and safety should become a priority.The UNICEF program for Youth Empowerment and Health/Economic Responses to COVID-19 (YEaH) as well as the UNESCO – TWAS (The World Academy of Sciences) for the Advancement of Sciences and the Inter Academy Partnership short-term funding project.http://www.tandfonline.com/loi/cije20hj2024Plant Production and Soil ScienceSDG-03:Good heatlh and well-bein

Removal of pharmaceutical' estrogenic activity of sequencing batch reactor effluents assessed in the T47D-KBluc report

Various water treatment processes may be ineffective to remove pharmaceutical compounds (PhCs) and their by-products, leading to endocrine-disruptive activity that might be detrimental to wildlife and human health. This study investigated the degradation of carbamazepine (CBZ), diclofenac (DCF), ibuprofen (IBP), and their intermediates, as well as estrogenic activity that is not effectively removed by conventional methods. A consortium of isolated South African indigenous fungi A. niger, M. circinelloides, T. polyzona, T. longibrachiatum and R. microsporus, was used in a sequencing batch reactor (SBR) to remove PhCs, their intermediates and strongly reduce their estrogenic activity. The fungal ligninolytic enzymatic activity was determined for laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP) using a spectrophotometric method. The biodegradation of PhCs and their intermediates was monitored by SPE-UPLC/MS. The in vitro estrogenic activity was assessed in the T47D-KBluc reporter gene assay. Lac, MnP and LiP production appeared to be biomass growth dependent. During a lag phase of growth, a constant biomass of about 122.04 mg/100 mL was recorded with average enzymatic activity around 63.62 U/L for Lac, 151.91 U/L for MnP and 42.12 U/L for LiP. The exponential growth phase from day 7 to day 17, was characterised by a biomass increase of 124.46 units, and an increase in enzymatic activity of 9.91 units for Lac, 99.03 units for MnP and 44.24 units for LiP. These enzymes played an important synergistic role in PhCs degradation in the cytochrome P450 system. A decrease of 13.89%, 29.7% and 16.15% in PhC concentrations was observed for CBZ, DCF and IBP, respectively, and their intermediates were identified within 4 h of incubation. The removal efficiency achieved after 24 h in the SBR was about 89.77%, 95.8% and 91.41% for CBZ, DCF and IBP, respectively. The estradiol equivalent (EEq) values of 1.71 ± 0.30 ng/L and 2.69 ± 0.17 ng/L were recorded at the start-up time and after 4 h, respectively. The presence of intermediates was found to induce estrogenic activity. The EEq values after 24 h incubation was found to be below the LoQ and below the LoD of the assay. None of the samples exhibited any anti-estrogenic activity. The fungal consortium inoculum was found to induce toxicity at a 0.4× concentration, as observed under a microscope. This study revealed that the use of the fungal consortium can remove the estrogenic activity of pharmaceutical metabolites, which appeared to be the most significant contributors to the endocrine-disrupting activity of the wastewater treatment plant effluents.The Department of Environment, Water and Earth Sciences of the Tshwane University of Technology and grants from the South African National Research Foundation (NRF) through the South African Research Chairs Initiative in Water Quality and Wastewater Management (SARChI, grant number 87310) at the Faculty of Science, Tshwane University of Technology.http://www.elsevier.com/locate/jenvmanhj2020Urolog

Pesticide residues and estrogenic activity in fruit and vegetables sampled from major fresh produce markets in South Africa

Food is likely to be one of the major pathways through which people are exposed to endocrinedisrupting chemicals. With the exception of residual effects, there are concerns that a number of naturally occurring and synthetic chemicals exert adverse effects upon endocrine systems in wildlife and humans. The current study reports selected pesticide concentrations and the total estrogenic activity of fruit and vegetables using the recombinant yeast oestrogen screen (YES) and T47D-KBluc reporter gene assays. A total of 53 food samples (27 fruit and 26 vegetables) from Johannesburg and Tshwane fresh produce markets (in South Africa) were analysed. Of these, 17 contained one to three different pesticide residues with concentrations ranging between 0.01 and 0.68 mg kg–1, whereas in the rest of the samples no residues were detected. All pesticides detected except in one sample were below the maximum residue level (MRL), but others were unauthorised for use in specified fruit and vegetables. Estrogenic activity was detected in 26.4% (14 samples) of the samples tested, and the estradiol equivalents ranged from 0.007 to 2 pg g–1. Although the estrogenic activity was low, it may contribute to adverse health effects. Continuous monitoring for pesticides in fruit and vegetables is important in view of the unauthorised pesticides detected in produce from South Africa and the endocrine-disrupting chemical activity found.South African Table Grape Industry, the University of Pretoria, Institute of Food Nutrition and Well-Being, Department of Science and Technology and the National Research Foundation administered Centre of Excellence in Food Security (Project No 140701).http://www.tandfonline.com/loi/tfac202017-07-30hb201

Endocrine disrupting chemicals in commercially available cling film brands in South Africa

Cling films comprise of plasticizers which are known endocrine disrupting chemicals (EDCs). Cling films are commonly used for food packaging and EDCs may leach into food, increasing exposure, leading to adverse health outcomes. We determined the levels of common selected EDCs expected in commercial cling film brands sold in South Africa. We tested for para-nonylphenol (p-NP), bisphenol A (BPA), bis(2-ethylhexyl) adipate (DEHA), and phthalates [di-2-ethyhexyl phthalate (DEHP) and dibutylphthalate (DBP)]. We selected eight brands of cling film and used standard chromatography methods to extract compounds. We found p-NP in one brand (2.06 µg/kg) and BPA in two brands (1.48 and 1.61 µg/kg). Five brands contained DEHP (2.1–2.59 mg/kg), DBP (0.62–1.21 mg/kg) and DEHA (165.89–674.07 mg/kg). Levels of DEHA and DEHP in the cling film are associated with potential human health risks. The maximum level of DEHA ranged from 1.6 to 16 times the safe level, with a calculated hazard quotient of 1.4. The two cling film brands made from low density polyethylene (LDPE), both endorsed by the Cancer Association of South Africa (CANSA), had target chemicals below the detection limit.http://www.tandfonline.com/loi/bher202020-05-23hj2019School of Health Systems and Public Health (SHSPH)Urolog

Estrogenic activity, chemical levels and health risk assessment of municipal distribution point water from Pretoria and Cape Town, South Africa

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and have been detected in drinking water from various countries. Although various water treatment processes can remove EDCs, chemicals can also migrate from pipes that transport water and contaminate drinking water. This study investigated the estrogenic activity in drinking water from various distribution points in Pretoria (City of Tshwane) (n = 40) and Cape Town (n = 40), South Africa, using the recombinant yeast estrogen screen (YES) and the T47D-KBluc reporter gene assay. The samples were collected seasonally over four sampling periods. The samples were also analysed for bisphenol A (BPA), nonylphenol (NP), di(2-ethylhexyl) adipate (DEHA), dibutyl phthalate (DBP), di(2-ethylhexyl) phthalate (DEHP), diisononylphthalate (DINP), 17β-estradiol (E2), estrone (E1) and ethynylestradiol (EE2) using ultra-performance liquid chromatography-tandem mass spectrophotometry (UPLC-MS/MS). This was followed by a scenario based health risk assessment to assess the carcinogenic and toxic human health risks associated with the consumption of distribution point water. None of the water extracts from the distribution points were above the detection limit in the YES bioassay, but the EEq values ranged from 0.002 to 0.114 ng/L using the T47D-KBluc bioassay. BPA, DEHA, DBP, DEHP, DINP E1, E2, and EE2 were detected in distribution point water samples. NP was below the detection limit for all the samples. The estrogenic activity and levels of target chemicals were comparable to the levels found in other countries. Overall the health risk assessment revealed acceptable health and carcinogenic risks associated with the consumption of distribution point water.The South African Medical Research Councilhttp://www.elsevier.com/locate/chemosphere2018-11-30hj2017School of Health Systems and Public Health (SHSPH)Urolog

Estrogenic activity, selected plasticizers and potential health risks associated with bottled water in South Africa

Potential endocrine disrupting chemicals (EDCs) are present in bottled water from various countries. In South Africa (SA), increased bottled water consumption and concomitant increases in plastic packaging create important consequences for public health. This study aimed to screen SA bottled water for estrogenic activity, selected target chemicals and assessing potential health risks. Ten bottled water brands were exposed to 20 °C and 40 °C over 10 days. Estrogenic activity was assessed using the recombinant yeast estrogen screen (YES) and the T47D-KBluc reporter gene assay. Solid phase extracts of samples were analyzed for bis(2-ethylhexyl) adipate (DEHA), selected phthalates, bisphenol-A (BPA), 4-nonylphenol (4-NP), 17β-estradiol (E2), estrone (E1), and ethynylestradiol (EE2) using gas chromatography–mass spectrophotometry. Using a scenario-based health risk assessment, human health risks associated with bottled water consumption were evaluated. Estrogenic activity was detected at 20 °C (n = 2) and at 40 °C (n = 8). Estradiol equivalent (EEq) values ranged from 0.001 to 0.003 ng/L. BPA concentrations ranged from 0.9 ng/L to 10.06 ng/L. Although EEqs and BPA concentrations were higher in bottled water stored at 40 °C compared to 20 °C, samples posed an acceptable risk for a lifetime of exposure. Irrespective of temperature, bottled water from SA contained chemicals with acceptable health risks.The Cancer Association of South Africa (CANSA).http://jwh.iwaponline.comhj2019School of Health Systems and Public Health (SHSPH

Endocrine-disrupting activity of the fungicide mancozeb used in the Vhembe District of South Africa

Many chemicals released into the environment are believed to disrupt normal endocrine functions in humans and animals. A major group of endocrine disrupting chemicals (EDCs) that could be responsible for reproductive defects are those that mimic natural oestrogens, known as xenoestrogens. A number of in vivo and in vitro screening strategies are being developed to identify and classify xenoestrogens, in order to determine whether they pose a health risk to humans and animals. Oestrogens and androgens mediate their activity via intracellular receptors, directly in muscular tissue, as well as indirectly via stimulation of growth hormones from the pituitary glands and other growth factors from liver plus several other organs. Mancozeb is a metal ethylenebisdithiocarbamate (EBDC) fungicide used to protect many fruits and vegetables and field crops against pathogenic fungal. The T47D-KBluc, GH3.TRE-Luc and MDA-kb2 reporter gene assays were used to determine the possible endocrine disrupting activity/potential of mancozeb. No activity was detected in any of the assays and no mancozeb was detected in any of the dams either. Oestrogenic activity was detected in Albasini Dam, Nandoni Dam and Xikundu weir, but all values were below 0.7 ng l−1 trigger value for oestrogenic activity in drinking water.The University of Vendahttps://www.tandfonline.com/loi/taas202021-09-30hj2021Urolog

Data on UPLC/MS method validation for the biodegradation of pharmaceuticals and intermediates by a fungal consortium and on T47DK-Bluc reporter gene assay to assess the reduction of their estrogenic activity

In term of pharmaceutical and their intermediate compounds analysis, UPLC/MS method is a valuable equipment to achieve better confirmation on their biodegradation by fungi. The T47DKBluc reporter gene assay is an appropriate tool to investigate to removal of estrogenic and antiestrogenic activities of pharmaceuticals and their metabolites from a synthetic wastewater. A consortium of isolated South African indigenous fungi Aspergillus niger, Mucor circinelloides, Trichoderma longibrachiatum, Trametes polyzona and Rhizopus microspores was found to perform a removal of pharmaceuticals and their metabolites and to reduce their estrogenic activity below the limit of detection in a sequencing batch reactor. Here are presented data regarding the phenolic compounds list and the method validation for UPLC/MS analysis used for selected pharmaceutical compounds namely carbamazepine, diclofenac, ibuprofen and their metabolites, as well as the T47DKBluc bioassay using as positive control, the agonist E2 for estrogenic activity and the antagonist ICI 182,780 for antiestrogenic activity. For better understanding of the data presented in this paper, please see the research paper “Removal of pharmaceutical’ estrogenic activity of sequencing batch reactor effluents assessed in the T47DK-Bluc reporter gene assay” [1].The South African National Research Foundation (NRF) through the grant chair of the Water Quality and Wastewater Management under the SARChI 87310 at the Faculty of Science, Tshwane University of Technology.http://www.elsevier.com/locate/dibam2020Urolog

Pesticide residues and estrogenic activity in fruit and vegetables sampled from major fresh produce markets in South Africa

Food is likely to be one of the major pathways through which people are exposed to endocrinedisrupting chemicals. With the exception of residual effects, there are concerns that a number of naturally occurring and synthetic chemicals exert adverse effects upon endocrine systems in wildlife and humans. The current study reports selected pesticide concentrations and the total estrogenic activity of fruit and vegetables using the recombinant yeast oestrogen screen (YES) and T47D-KBluc reporter gene assays. A total of 53 food samples (27 fruit and 26 vegetables) from Johannesburg and Tshwane fresh produce markets (in South Africa) were analysed. Of these, 17 contained one to three different pesticide residues with concentrations ranging between 0.01 and 0.68 mg kg–1, whereas in the rest of the samples no residues were detected. All pesticides detected except in one sample were below the maximum residue level (MRL), but others were unauthorised for use in specified fruit and vegetables. Estrogenic activity was detected in 26.4% (14 samples) of the samples tested, and the estradiol equivalents ranged from 0.007 to 2 pg g–1. Although the estrogenic activity was low, it may contribute to adverse health effects. Continuous monitoring for pesticides in fruit and vegetables is important in view of the unauthorised pesticides detected in produce from South Africa and the endocrine-disrupting chemical activity found.South African Table Grape Industry, the University of Pretoria, Institute of Food Nutrition and Well-Being, Department of Science and Technology and the National Research Foundation administered Centre of Excellence in Food Security (Project No 140701).http://www.tandfonline.com/loi/tfac202017-07-30hb201