Vitamin D receptor antagonist activity in wastewater effluents-potential for endocrine disruption

Background Endocrine disrupting chemicals have been identified for a number of human endocrine systems, but there are no reports on vitamin D-antagonistic activities in environmental samples.Objectives We have investigated if there are compounds present in the environment that can act as Vitamin D receptor (VDR) antagonists.Methods Water samples were collected of the influent and effluent water from five Swedish wastewater treatment facilities and concentrated with solid phase extraction. VDR antagonistic properties of the samples were tested with a cell-based in vitro assay responsive to vitamin D signaling. Cytotoxicity was monitored by three different assays.Results We observed a dose-dependent decrease in the VDR signaling in most studied samples, although the effect was overlapping with cytotoxicity for the influent samples. For effluent samples, we observed clear VDR antagonistic effects also in non-cytotoxic concentrations. The observed effects could not be explained by presence of natural organic matter or cadmium in the water.Discussion The vitamin D endocrine system regulates a broad range of physiological processes, and disruption of this system could be associated with serious health consequences. In this study, we report environmental presence of compounds with VDR antagonistic properties, compounds which constitute a new group of potential endocrine disruptors. The VDR antagonism was observed in wastewater treatment facility effluent waters, which are discharged into water systems used as raw water for drinking water production. The findings reported in this study may indicate a potential hazard to human health and aquatic life. Future research is needed to investigate the presence of VDR antagonists in the environment, identification of the causative compounds, and studies of exposure of humans and aquatic organisms to these compounds

Antiandrogenic activity and bioavailability of magnolol analogs – A potential for prostate cancer therapeutics

Background: Prostate cancer is the second most common form of cancer in men worldwide and there is a great need for novel treatment strategies, especially for castrate-resistant prostate cancers where the proliferation of the cancer cells is stimulated by androgens produced in the adrenal cortex and the cancer cells. Purpose: In this study, we have investigated the antiandrogenic properties of magnolol and ten synthetic analogs in vitro. Study design and methods: The compounds were evaluated for cytotoxicity, antiandrogenic receptor activity, binding to the androgen receptor, effects on the production of Prostate-specific antigen (PSA), and potential to pass over a tight layer of Caco-2 cells mimicking gastrointestinal absorption. Results: We found that almost all investigated compounds were antiandrogenic in an androgen receptor reporter gene assay, with IC50 values ranging from 7 to 86 µM. Magnolol itself had the highest antiandrogenic potency. Five of the compounds were then evaluated for their binding to the androgen receptor and three of these compounds were found to bind to the receptor. These five compounds were also evaluated for their effect on the PSA production and four were found to decrease PSA production at non-cytotoxic concentrations. The antiandrogenic activity after passage through a layer of Caco-2 cells, mimicking gastrointestinal absorption, was also evaluated for three of the compounds. All three compounds were found to have the capacity to be transported from the apical to the basolateral side of the Caco-2 cell layer and exert antiandrogenic effects after the transport. Conclusion: In conclusion, this study shows that magnolol and analogs have antiandrogenic effects in vitro and that selected analogs can pass over a tight layer of Caco-2 cells, indicating a potential for good bioavailability after oral administration. These magnolol analogs thereby constitute an interesting group of compounds worthy of further evaluation as potential anti-prostate cancer therapeutics

Impact of sample acidification and extract storage on hormone receptor-mediated and oxidative stress activities in wastewater

An underemphasized aspect of sampling strategies in effect-based in vitro testing is to determine suitable collection and preparation techniques. In the current study, the impact of sample acidification on bioactivities was assessed using in vitro bioassays for hormone receptor-mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and the oxidative stress response (Nrf2 activity). Sampling was conducted at a recently upgraded Swedish wastewater treatment plant. Future plans for the treated wastewater include reuse for irrigation or as a drinking water source. In the AR and Nrf2 assays, acidification decreased bioactivities in the wastewater influent sample extracts, whereas acidification increased bioactivities following further treatment (disc filtration). In the ER assay, acidification had no impact on the observed bioactivities in the sample extracts. A secondary objective of the study was to assess the stability of the sample extracts over time. Lower activities were detected in the ER and AR assays in all extracts after storage for approximately 1 year. Nrf2 activities did not decrease over time, but rather increased in some of the acidified sample extracts. Overall, the findings suggest that sampling strategies involving acidification may need to be tailored depending on the selected bioassay(s) and the type of wastewater treatments being assessed

Evaluation of in vitro bioassays as a screening tool to monitor chemical hazards in cow’s milk

Studies on cow’s milk have mainly focused on analyzing specific chemical groups and natural components. Therefore, in this study, we evaluated if effect-based in vitro methods could be used as a screening tool to monitor chemical hazards in milk. In total, 32 milk samples were collected from a Swedish dairy company throughout one year. These samples included conventional and organic semi-skimmed as well as raw milk. The milk samples were tested in five in vitro methods covering eight endpoints. These endpoints included cytotoxicity, endocrine disruption (estrogen/androgen induction/inhibition), aryl hydrocarbon receptor activity, oxidative stress and DNA damage. Estrogen and androgen receptor inhibition, in addition to aryl hydrocarbon receptor activity, were the most responsive endpoints, where 10 to 13 out of the 32 milk samples were bioactive. Organic and conventional milk showed no major differences. Overall, no or only low activities were observed in milk samples in the remaining in vitro assays, which is a promising result with regard to applying effect-based methods as a screening tool. Concerning the most responsive assays, more research is needed to understand the normal background variations before they can be used as a screening tool for chemical hazards in milk

An in vitro-based hazard assessment of liquid smoke food flavourings

Liquid smoke products are widely used as a food additive to create a desired smoke flavour. These products may contain hazardous chemicals generated during the wood-burning process. However, the toxic effects of these types of hazardous chemicals constituting in the commercially available products are largely unknown. Therefore, a test battery of cell-based in vitro methods, covering different modes of actions of high relevance to human health, was applied to study liquid smoke products. Ten liquid smoke flavourings were tested as non-extracted and extracted. To assess the potential drivers of toxicity, we used two different solvents. The battery of in vitro methods covered estrogenicity, androgenicity, oxidative stress, aryl hydrocarbon receptor activity and genotoxicity. The non-extracted samples were tested at concentrations 0.002 to 1 mu L liquid smoke flavouring/mL culture medium, while extracted samples were tested from 0.003 to 200 mu L/mL. Genotoxicity was observed for nearly all non-extracted and all hexane-extracted samples, in which the former had higher potency. No genotoxicity was observed for ethyl acetate-extracted samples. Oxidative stress was activated by almost all extracted and non-extracted samples, while approximately half of the samples had aryl hydrocarbon receptor and estrogen receptor activities. This study used effect-based methods to evaluate the complex mixtures of liquid smoke flavourings. The increased bioactivities seen upon extractions indicate that non-polar chemicals are driving the genotoxicity, while polar substances are increasing oxidative stress and cytotoxic responses. The differences in responses indicate that non-extracted products contain chemicals that are able to antagonize toxic effects, and upon extraction, the protective substances are lost

In vitro bioanalytical assessment of toxicity of wetland samples from Spanish Mediterranean coastline

Background Fresh water bodies represent less than 1% of overall amount of water on earth and ensuring their quality and sustainability is pivotal. Although several campaigns have been performed to monitor the occurrence of micropollutants by means of chemical analysis, this might not cover the whole set of chemicals present in the sample nor the potential toxic effects of mixtures of natural and anthropogenic chemicals. In this sense, by selecting relevant toxicity endpoints when performing in vitro bioanalysis, effect-based methodologies can be of help to perform a comprehensive assessment of water quality and reveal biological activities relevant to adverse health effects. However, no prior bioanalytical study was performed in wetland water samples from the Spanish Mediterranean coastline. Methods Eleven samples from relevant water bodies from the Spanish Mediterranean coastline were collected to monitor water quality on 8 toxicity endpoints. Aryl hydrocarbon receptor (AhR), androgenicity (AR+ and AR-), estrogenicity (ER+ and ER-), oxidative stress response (Nrf2) and vitamin D receptor (VDR+ and VDR-) reporter gene assays were evaluated. Results AhR was the reporter gene assay showing a more frequent response over the set of samples (activated by 9 out of 11 samples), with TCDD-eq in the range 7.7-22.2 pM. For AR, ER and VDR assays sporadic activations were observed. Moreover, no activity was observed on the Nrf2 reporter gene assay. Wastewater and street runaway streams from Valencia could be responsible for enhanced activities in one of the water inputs in the Natural Park 'L'Albufera'. Conclusions Water quality of relevant wetlands from the Spanish Mediterranean coastline has been evaluated. The utilization of a panel of 5 different bioassays to cover for different toxicity endpoints has demonstrated to be a good tool to assess water quality

Assessment of source and treated water quality in seven drinking water treatment plants by in vitro bioassays – Oxidative stress and antiandrogenic effects after artificial infiltration.

Drinking water quality and treatment efficacy was investigated in seven drinking water treatment plants (DWTPs), using water from the river Göta Älv, which also is a recipient of treated sewage water. A panel of cell-based bioassays was used, including measurements of receptor activity of aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), peroxisome proliferator-activated receptor alpha (PPARα) as well as induction of oxidative stress (Nrf2) and micronuclei formation. Grab water samples were concentrated by solid phase extraction (SPE) and water samples were analyzed at a relative enrichment factor of 50. High activities of AhR, ER and AR antagonism were present in WWTP outlets along the river. Inlet water from the river exhibited AhR and AR antagonistic activities. AhR activity was removed by DWTPs using granulated activated carbon (GAC) and artificial infiltration. AR antagonistic activity was removed by the treatment plants, except the artificial infiltration plant, which actually increased the activity. Furthermore, treated drinking water from the DWTP using artificial infiltration exhibited high Nrf2 activity, which was not found in any of the other water samples. Nrf2 activity was found in water from eight of the 13 abstraction wells, collecting water from the artificial infiltration. No genotoxic activity was detected at non-cytotoxic concentrations. No Nrf2 or AR antagonistic activities were detected in the inlet or outlet water after the DWTP had been replaced by a new plant, using membrane ultrafiltration and GAC. Neither target chemical analysis, nor chemical analysis according to the drinking water regulation, detected any presence of chemicals, which could be responsible of the prominent effects on oxidative stress and AR antagonistic activity in the drinking water samples. Thus, bioanalysis is a useful tool for detection of unknown hazards in drinking water and for assessment of drinking water treatments

Effect-based monitoring of chemical hazards in drinking water from source to tap: seasonal trends over 2 years of sampling

Drinking water producers have a far-reaching responsibility to provide safe, clean and wholesome drinking water, using water resources possibly effected by the thousands of chemicals used in societies' daily life. This study has monitored chemical hazards in drinking water from source to tap, using effect-based methods. The study was conducted at a Swedish drinking water treatment plant sourcing lake water and aimed to investigate potential seasonal variations in both the raw water and water after different treatment steps. Furthermore, a granular activated carbon (GAC) pilot facility was evaluated. Samples were analyzed for estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity, oxidative stress (Nrf2) response and genotoxicity (micronucleus formation). We observed seasonal differences in oxidative stress and genotoxic effects in both raw and drinking water with higher activities during the late fall of each year. The removal efficiency for both oxidative stress and genotoxicity was limited in the full-scale treatment process and occasionally the genotoxicity was also detected in outgoing drinking water from the treatment plant and in samples collected at consumers tap on the distribution network. AhR activity was present in all raw water samples and the removal was limited. Estrogenic activities were observed in most of the raw water samples but in contrast to the other parameters estrogenicity was effectively reduced by the full-scale conventional treatment. The GAC pilot treatment was generally more efficient than the full-scale conventional treatment in removing all observed bioactivities and could be a viable complement to the current treatment to assure drinking water free from genotoxic compounds. Genotoxic activities in drinking water was observed while all currently regulated chemical parameters were fulfilled. This highlights the need for effect-based monitoring in efforts to ensure the chemical safety of drinking water, as target chemical analysis of single compounds will overlook both unknown hazardous compounds as well as potential mixture effects

Vitamin D receptor antagonist activity in wastewater effluents—potential for endocrine disruption

Abstract Background Endocrine disrupting chemicals have been identified for a number of human endocrine systems, but there are no reports on vitamin D-antagonistic activities in environmental samples. Objectives We have investigated if there are compounds present in the environment that can act as Vitamin D receptor (VDR) antagonists. Methods Water samples were collected of the influent and effluent water from five Swedish wastewater treatment facilities and concentrated with solid phase extraction. VDR antagonistic properties of the samples were tested with a cell-based in vitro assay responsive to vitamin D signaling. Cytotoxicity was monitored by three different assays. Results We observed a dose-dependent decrease in the VDR signaling in most studied samples, although the effect was overlapping with cytotoxicity for the influent samples. For effluent samples, we observed clear VDR antagonistic effects also in non-cytotoxic concentrations. The observed effects could not be explained by presence of natural organic matter or cadmium in the water. Discussion The vitamin D endocrine system regulates a broad range of physiological processes, and disruption of this system could be associated with serious health consequences. In this study, we report environmental presence of compounds with VDR antagonistic properties, compounds which constitute a new group of potential endocrine disruptors. The VDR antagonism was observed in wastewater treatment facility effluent waters, which are discharged into water systems used as raw water for drinking water production. The findings reported in this study may indicate a potential hazard to human health and aquatic life. Future research is needed to investigate the presence of VDR antagonists in the environment, identification of the causative compounds, and studies of exposure of humans and aquatic organisms to these compounds

Antiandrogenic activity and bioavailability of magnolol analogs – A potential for prostate cancer therapeutics

Background: Prostate cancer is the second most common form of cancer in men worldwide and there is a great need for novel treatment strategies, especially for castrate-resistant prostate cancers where the proliferation of the cancer cells is stimulated by androgens produced in the adrenal cortex and the cancer cells. Purpose: In this study, we have investigated the antiandrogenic properties of magnolol and ten synthetic analogs in vitro. Study design and methods: The compounds were evaluated for cytotoxicity, antiandrogenic receptor activity, binding to the androgen receptor, effects on the production of Prostate-specific antigen (PSA), and potential to pass over a tight layer of Caco-2 cells mimicking gastrointestinal absorption. Results: We found that almost all investigated compounds were antiandrogenic in an androgen receptor reporter gene assay, with IC50 values ranging from 7 to 86 µM. Magnolol itself had the highest antiandrogenic potency. Five of the compounds were then evaluated for their binding to the androgen receptor and three of these compounds were found to bind to the receptor. These five compounds were also evaluated for their effect on the PSA production and four were found to decrease PSA production at non-cytotoxic concentrations. The antiandrogenic activity after passage through a layer of Caco-2 cells, mimicking gastrointestinal absorption, was also evaluated for three of the compounds. All three compounds were found to have the capacity to be transported from the apical to the basolateral side of the Caco-2 cell layer and exert antiandrogenic effects after the transport. Conclusion: In conclusion, this study shows that magnolol and analogs have antiandrogenic effects in vitro and that selected analogs can pass over a tight layer of Caco-2 cells, indicating a potential for good bioavailability after oral administration. These magnolol analogs thereby constitute an interesting group of compounds worthy of further evaluation as potential anti-prostate cancer therapeutics