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Manganese is an essential trace element that is required for multiple enzymes in the human body. The general population is mainly exposed to manganese via food intake, in particular plant foods. In areas with elevated concentrations of manganese in groundwater, drinking water can also be an important source of exposure. The gastrointestinal absorption of manganese is below 10%, and it appears to be influenced by the amount of manganese in the diet and by the nutritional status of the individual, especially the iron status. In blood, most of the manganese is found in the cellular fractions. Manganese is primarily eliminated via the bile followed by excretion via faeces. To date, no specific biomarkers of manganese intake have been identified. The dietary intake of manganese in the Nordic countries has been reported to be within the range that has been reported for other European countries (2-6 mg/day). Since manganese is found in nutritionally adequate amounts in food, deficiency is not of public health concern. On the other hand, there is emerging epidemiological evidence that various suggested manganese biomarkers may be negatively associated with children's neurodevelopment. However, the limited number of prospective studies, the lack of appropriate exposure biomarkers, and validated neurodevelopmental outcomes render data uncertain and inconclusive. In 2013, the European Food Safety Authority considered the evidence to be insufficient to derive an average requirement or a population reference intake, and instead an adequate intake for adults was set at 3.0 mg/day.

Molybdenum is an essential element in the form of the molybdenum cofactor (Moco). In humans, Moco is required for four enzymes: xanthine oxidase (XO), aldehyde oxidase, sulfite oxidase (SO), and mitochondrial amidoxime-reducing component (mARC). The enzymes are involved in the oxidation of purines to uric acid, metabolism of aromatic aldehydes and heterocyclic compounds, and in the catabolism of sulfur amino acids. Molybdenum cofactor deficiency is a rare autosomal recessive syndrome due to a defective synthesis of Moco, resulting in a deficiency of all the molybdoenzymes. There are no reports on clinical signs of dietary molybdenum deficiency in otherwise healthy humans. Water-soluble molybdate is efficiently absorbed from the digestive tract. The body retention is regulated by urinary excretion. Plasma molybdenum reflects long-term intake and 24-h urinary excretion is related to recent intake. There are no biochemical markers of molybdenum status. Cereal products are the main contributors to molybdenum dietary intake, estimated to 100-170 μg/day in Nordic studies. Little data are available on molybdenum toxicity in humans. A tolerable upper intake level of molybdenum has been based on reproductive toxicity in rats, but the effects have not been reproduced in more recent studies. The U.S. Institute of Medicine (IOM, present National Academy of Sciences, Engineering, and Medicine; NASEM) established a Recommended Dietary Allowance of 45 μg/day in adult men and women in 2001, based on a small study reporting urinary excretion in balance with intake at 22 μg/day. The European Food Safety Authority (EFSA) considered in 2013 the evidence to be insufficient to derive an Average Requirement and a Population Reference Intake, but proposed an Adequate Intake of 65 μg/day for adults.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular defense against oxidative stress and correlated with classical toxicological endpoints. In vitro methods using fish cell lines for the assessment of aquatic toxicity are needed for mechanistic studies and as an alternative to in vivo . We describe an in vitro assay to study oxidative stress using zebrafish cell lines. Transfection efficiency of twelve commercially available transfection reagents were tested in the zebrafish cell lines ZFL, ZF4, and Pac2. The most efficient reagent for each cell line was selected for further experiments. Cells were transiently transfected with an Nrf2-responsive luciferase plasmid. The assay was tested using the oxidative stress inducing chemicals tert butylhydroquinone, hydrogen peroxide, and sulforaphane. Of the transfected cell lines, ZF4 and ZFL showed higher sensitivity. The latter were used to study potential oxidative stress induced by pesticides (diazinon, deltamethrin, atrazine, metazachlor, terbutylazine, diuron). Besides known inducers, Nrf2 activity was also significantly induced by diazinon, deltametrin, diuron, and metazachlor. Activation of Nrf2 by metazachlor is a novel finding. The described assay could be a valuable tool for research in toxicology to study the stress response of both pure chemicals and environmental water samples.

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 potential 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.

BackgroundEndocrine 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.ObjectivesWe have investigated if there are compounds present in the environment that can act as Vitamin D receptor (VDR) antagonists.MethodsWater 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.ResultsWe 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.DiscussionThe 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.

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. Graphical Abstract

BackgroundContamination of drinking water by hazardous chemicals can be associated with human health risks. Recent studies using effect-based in vitro methods have demonstrated that a large part of the observed toxic effects are caused by unknown chemicals. In this study, we have used a panel of effect-based methods to study the presence of chemical contaminants in a unique material; glass-bottled Swedish tap water collected during the 1990s. These water samples were compared to drinking water from the same source waters and drinking water facilities, yet collected about 25 years later, in 2020.ResultsSamples were concentrated by solid phase extraction and evaluated for the following activities; estrogen receptor activity, androgen receptor activity, antiandrogenic activity, aryl hydrocarbon receptor activity, and oxidative stress response. We observed aryl hydrocarbon receptor activities in almost all studied samples and estrogen receptor activity in three out of ten studied samples. No activities were observed for androgen receptor activity, antiandrogenic activity or oxidative stress response. In general, observed activities were more frequent and higher in the water samples collected during the 1990s as compared to the corresponding samples collected in 2020.ConclusionsThis study demonstrates that it is possible to conduct an effect-based evaluation of the presence of hazardous chemicals in drinking water, with as small starting volume as 330 mL, by using miniaturized bioassays. Further, by comparing the glass-bottled water samples with newly collected water samples from the same drinking water treatment facilities, our results indicate that the presence of aryl hydrocarbon receptor and estrogen receptor activating compounds in the drinking water has decreased over the approximately quarter of a century that is separating the two sampling occasions. This difference could be due to improved raw water quality and/or improved treatment efficiency in the treatment plants.

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 μL liquid smoke flavouring/mL culture medium, while extracted samples were tested from 0.003 to 200 μL/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.

Inflammation in the mammary gland (mastitis) is the most common disease in dairy herds worldwide, often caused by the pathogens Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Little is known about the effects of mastitis on drug transporters and the impact on transporter-mediated excretion of drugs into milk. We used murine mammary epithelial HC11 cells, after lactogenic differentiation into a secreting phenotype, and studied gene expressions of ABC- and SLC- transporters after treatment of cells with S. aureus and lipopolysaccharide, an endotoxin secreted by E. coli. The studied transporters were Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1 and Oct1. In addition, Csn2, the gene encoding β-casein, was analyzed. As biomarkers of the inflammatory response, gene expressions of the cytokines Il6 and Tnfα and the chemokine Cxcl2 were determined. Our results show that S. aureus and LPS treatment of cells, at non-cytotoxic concentrations, induced an up-regulation of Mdr1 and of the inflammatory biomarkers, except that Tnfα was not affected by lipopolysaccharide. By simple regression analysis we could demonstrate statistically significant positive correlations between each of the transporters with each of the inflammatory biomarkers in cells treated with S. aureus. The coefficients of determination (R2) were 0.7-0.9 for all but one correlation. After treatment of cells with lipopolysaccharide, statistically significant correlations were only found between Mdr1 and the two parameters Cxcl2 and Il6. The expression of Csn2 was up-regulated in cells treated with S. aureus, indicating that the secretory function of the cells was not impaired. The strong correlation in gene expressions between transporters and inflammatory biomarkers may suggest a co-regulation and that the transporters have a role in the transport of cytokines and chemokines. Our results demonstrate that transporters in mammary cells can be affected by infection, which may have an impact on transport of essential compounds and contaminants into milk.

BackgroundBioanalytical tools have been shown to be useful in drinking water quality assessments. Here, we applied a panel of in vitro bioassays to assess the treatment efficiency of two pilot-scale treatments: ozonation and granular activated carbon (GAC) filtration at a drinking water treatment plant (DWTP). The pilot-scale systems were studied alongside a full-scale treatment process consisting of biological activated carbon (BAC) filtration, UV disinfection, and monochloramine dosing. Both systems were fed the same raw water treated with coagulation/flocculation/sedimentation and sand filtration. The endpoints studied were oxidative stress (Nrf2 activity), genotoxicity (micronuclei formations), aryl hydrocarbon receptor (AhR) activation, as well as estrogen receptor (ER) and androgen receptor (AR) activity.ResultsNrf2, AhR, and ER activities and genotoxic effects were detected in the incoming raw water and variability was observed between the sampling events. Compared to most of the samples taken from the full-scale treatment system, lower Nrf2, AhR, and ER bioactivities as well as genotoxicity were observed in all samples from the pilot-scale systems across all sampling events. The most pronounced treatment effect was a 12-fold reduction in Nrf2 activity and a sixfold decrease in micronuclei formations following ozonation alone. GAC filtration alone resulted in sevenfold and fivefold reductions in Nrf2 activity and genotoxicity, respectively, in the same sampling event. Higher bioactivities were detected in most samples from the full-scale system suggesting a lack of treatment effect. No androgenic nor anti-androgenic activities were observed in any sample across all sampling events.ConclusionsUsing effect-based methods, we have shown the presence of bioactive chemicals in the raw water used for drinking water production, including oxidative stress, AhR and ER activities as well as genotoxicity. The currently used treatment technologies were unable to fully remove the observed bioactivities. Ozonation and GAC filtration showed a high treatment efficiency and were able to consistently remove the bioactivities observed in the incoming water. This is important knowledge for the optimization of existing drinking water treatment designs and the utilization of alternative treatment technologies.