Changes in the gut microbiota of mice orally exposed to methylimidazolium ionic liquids

Ionic liquids are salts used in a variety of industrial processes, and being relatively non-volatile, are proposed as environmentally-friendly replacements for existing volatile liquids. Methylimidazolium ionic liquids resist complete degradation in the environment, likely because the imidazolium moiety does not exist naturally in biological systems. However, there is limited data available regarding their mammalian effects in vivo. This study aimed to examine the effects of exposing mice separately to 2 different methylimidazolium ionic liquids (BMI and M8OI) through their addition to drinking water. Potential effects on key target organs-the liver and kidney-were examined, as well as the gut microbiome. Adult male mice were exposed to drinking water containing ionic liquids at a concentration of 440 mg/L for 18 weeks prior to examination of tissues, serum, urine and the gut microbiome. Histopathology was performed on tissues and clinical chemistry on serum for biomarkers of hepatic and renal injury. Bacterial DNA was isolated from the gut contents and subjected to targeted 16S rRNA sequencing. Mild hepatic and renal effects were limited to glycogen depletion and mild degenerative changes respectively. No hepatic or renal adverse effects were observed. In contrast, ionic liquid exposure altered gut microbial composition but not overall alpha diversity. Proportional abundance of Lachnospiraceae, Clostridia and Coriobacteriaceae spp. were significantly greater in ionic liquid-exposed mice, as were predicted KEGG functional pathways associated with xenobiotic and amino acid metabolism. Exposure to ionic liquids via drinking water therefore resulted in marked changes in the gut microbiome in mice prior to any overt pathological effects in target organs. Ionic liquids may be an emerging risk to health through their potential effects on the gut microbiome, which is implicated in the causes and/or severity of an array of chronic disease in humans

Investigation into the role of Phthalates in Breast Cancer carcinogenesis

Plasticisers are used in the manufacture of polymers and increase the spacing be-tween chains of crystalline polymers to make them more flexible and durable. Phthalates are one of many such plasticisers used in a wide range of consumer goods. Phthalates are not chemically bound to the polymer matrix of plastics and easily leach into the environment and can be absorbed into the human body through the skin, inhalation, or ingestion. Growing evidence suggests that phthalates are environmental toxins and they have been found to be linked to the growth of breast cancer and non-cancerous cell lines in-vitro or involved in inva-sion and metastasis of primary tumours in breast tissue. The mechanism of this is thought to be via phthalates binding to the oestrogen receptor. The work presented in this thesis explores the mechanism(s) through which differ-ent phthalate chemicals promote the progression of breast cancers by exploring phthalates effects on in-vitro breast cancer cells, MCF-7 (oestrogen receptor posi-tive (ER+)) and MDA-MB-231 (oestrogen negative (ER-)) cell line.Presto Blue cell proliferation assays on the effects of phthalates Dibutyl Phthalate (DBP), Diethyl Phthalate (DEP), and phthalate metabolites, Monobutyl Phthalate MBP and Diisocytyl Phthalate (DiCP) on MCF-7 and MDA-MB-231 cells over 72 hours showed subtle and long-term proliferative effects of phthalates on MCF-7 cells and an inhibition of cell proliferation in MDA-MB-231 cells. Clonogenic assays performed over 10 days showed a significant increase in colony number of MCF-7 cells after treatment with 10M DBP and DiCP. Cell proliferation and clonogenic assays suggest phthalates have proliferative effect on ER+ cancer cell line MCF-7. These results suggest phthalates could have clinical implications in the progres-sion of cancer via long term proliferative effects on ER+ cancer cells. The possible mechanisms of phthalates effect on cells was also explored by inves-tigating intracellular signalling pathways of MCF-7 and MDA-MB-231 using western blotting and exploring ER and ER expression in MCF-7 cells using qPCR after treatment with phthalates. The results suggested increased expression of ER after treatment with DBP. Western blotting results showed increased levels of phospho-ERK and decreased levels of phospho-Akt after 72 hours compared to the control treated cells for MCF-7 cells and decreased levels of phospho-ERK in MDA-MB-231 cells after 72 hours of phthalate treatment. Results from confocal microscopy showed a subtle increase in oestrogen receptor expression in MCF-7 cells. These results correlate with the cell proliferation results that showed a proliferative effect of phthalates on ER+ MCF-7 cells and a decrease in cell proliferation compared to the control of MDA-MB-231 cells. Conclusions from this work would suggest the proliferative effects of phthalates are associated with the presence of the ER pres-ence on cells. The latter part of the project included initiating localisation studies of phthalates within cells. As phthalates do not auto-fluoresce the work in this section involved synthesis of a phthalate that was conjugated to a fluorescent chemical. As phthalates do not have a binding site for any of the commercially available fluores-cent tags an amine group was successfully synthesised onto a phthalic acid and could be used in future phthalate localisation studies.The work in this project therefore concludes that phthalates have a moderate long-term effect on breast cancer cell lines and the ability to tag phthalates as demon-strated in this work has the potential to allow more information on the exact site and mechanism of phthalate action in cells to be investigated

Fate and behaviour of drugs in the environment

Sewage treatment works (STWs) are routes through which treated wastewater effluents often containing myriads of chemicals are passed into receiving waters due to incomplete removal processes as have been identified in several studies. The current work aimed to determine the levels of these chemicals in the effluents from Stoke Bardolph STW, Nottingham and to determine the fate and behaviour of compounds by conducting degradation batch studies under different treatment conditions. The selection of representative illicit compounds; cocaine and its metabolite benzoylecgonine, heroin and its metabolites 6-monoacetylmorphine and morphine and a pharmaceutical (diazepam) was based on their presence in the STW effluent. The results obtained using solid phase extraction gas-chromatography technique (SPE-GCMS) showed thirteen compounds detected at concentrations between 1.9 and 3147 ng L-1 in effluents from Stoke Bardolph STW. Procaine, bromacil, codeine, lidocaine, ibruprofen, caffeine, nicotine and diazepam were the most abundant compounds in the final effluent with concentrations of 99.2, 1806.8, 33.5, 71.8, 3147, 213.4, 252.5 and 105.2 ng L-1, respectively. The percentage recoveries ranged from 74.5 – 109.6%, with the instrumental limits of detection (LODs) ranges of 0.2 – 12.7 ng L-1, and relative standard deviation (RSD) values of 0.6 – 4.7% were achieved for all the compounds

Surfaktanti u okolišu

Surfactants are a diverse group of chemicals that are best known for their wide use in detergents and other cleaning products. After use, residual surfactants are discharged into sewage systems or directly into surface waters, and most of them end up dispersed in different environmental compartments such as soil, water or sediment. The toxic effects of surfactants on various aquatic organisms are well known. In general, surfactants are present in the environment at levels below toxicity and in Croatia below the national limit. Most surfactants are readily biodegradable and their amount is greatly reduced with secondary treatment in wastewater treatment plants. The highest concern is the release of untreated wastewater or wastewater that has undergone primary treatment alone. The discharge of wastewater polluted with massive quantities of surfactants could have serious effects on the ecosystem. Future studies of surfactant toxicities and biodegradation are necessary to withdraw highly toxic and non-biodegradable compounds from commercial use and replace them with more environmentally friendly ones.Surfaktanti ili površinski aktivne tvari raznolika su skupina molekula najpoznatijih po uporabi u sastavu deterdženata i ostalih sredstava za pranje i čišćenje. Nakon uporabe u kućanstvu ili industriji, surfaktanti se ispuštaju u kanalizacijski sustav ili izravno u površinske vode te većina surfaktanata završi raspršena u vodi, sedimentu ili tlu. Toksični utjecaj surfaktanata na vodne organizme dobro je istražen i opisan u literaturi. U većini slučajeva surfaktanti su u okolišu prisutni u koncentracijama nižim od toksične te nižim od maksimalne koncentracije dopuštene hrvatskim zakonskim odredbama. Većina surfaktanata klasifi cirana je kao biološki razgradiva i njihova se koncentracija znatno smanjuje biološkom obradom otpadne vode pa je najveći rizik za okoliš ispuštanje prethodno pročišćene ili nepročišćene otpadne vode. Takva otpadna voda opterećena visokim koncentracijama surfaktanata može nepovoljno utjecati na okoliš. Potrebno je proučavati toksičnost i biološku razgradnju surfaktanata u svrhu uklanjanja visoko štetnih i biološki nerazgradljivih surfaktanata iz komercijalne uporabe te njihovu zamjenu tvarima manje štetnim za okoliš

Highlighting the gaps in hazard and risk assessment of unregulated Endocrine Active Substances in surface waters: retinoids as a European case study

Regulatory hazard and risk assessment of endocrine-active substances currently specifies four modes of action: interference with sex hormone (oestrogen, androgen) pathways, steroidogenesis, and thyroid hormone signalling. This does not encompass the full complexity of the endocrine system and its extended interfaces with environmental pollutants that can potentially disrupt the carefully maintained balance. Here we take the retinoid signalling pathway as a European case study for both, under- and unregulated endocrine pathways and outline the different levels of interference, discuss their adversity, and indicate crosstalk to other signalling pathways. Retinoid compounds already exist in drinking water sources, occur naturally in cyanobacterial blooms and/or enter surface waters via wastewater discharge, where they pose a potential hazard to the environment and human health - a situation that can be expected to worsen due to water shortages induced by climate-change and population growth. We briefly review relevant aspects of current endocrine disruptor (ED) testing for regulatory purposes and then expand upon the needs for inclusion of disruption of retinoid signalling in (ED) regulatory safety assessment contributing to adverse health outcomes that include cognitive function and neurological disease. An overview of developmental effects of retinoid signalling disruption across species highlights critical processes and potential crosstalk with other signalling pathways. A focused weight of evidence-based evaluation of the biologically plausible associations between neurological disorders and altered retinoid signalling highlights the evidence gaps. We show that monitoring only a limited number of anthropogenic priority chemicals in water is insufficient to address the environmental risks of retinoid signalling disruption. To comprehensively assess impacts on the endpoints, processes, and pathways of the endocrine system that are most vulnerable to chemical interference we need further investigation of the true mixture composition in environmental matrices. On a weight of evidence-basis this information can then be integrated into a reliable, inclusive, quantitative approach that ultimately accommodates all the critical pathways. By focusing on the retinoid signalling pathway, we intend to improve the scope and relevance of an integrated approach for the risk assessment of endocrine disruptors

Functional Characterization of Nuclear Receptor and Co-activator Binding Loci in the Human Genome

Steroid hormones, such as oestrogen, mediate their effects via activation of oestrogen regulated genes using nuclear receptors. Selective oestrogen receptor modulators (SERMs), such as Tamoxifen, are used to treat oestrogen responsive breast cancers, hctioning to act as oestrogen antagonists, preventing the oestrogen receptor biding DNA and blocking gene expression. However, Tamoxifen has been identified as an oestrogen agonist in other tissues which can often lead to secondary tumors in the years following the treatment. Identification of the genomic regions where SERMs can act as oestrogen agonists can possibly lead to the development of gene targeted therapies or other alternatives to prevent this from occurring. Investigations of a previously constructed ERE reporter library were directed towards the isolation of SERM responsive plasmids. Identified SERM responsive plasmids were compared to oestrogen responsive reporter piasmids on the basis of their ability to mediate trauscription in conjunction with other nuclear -tors and coactivators to establish potential novel interactions. ERa mutation analysis was performed to attempt to identify the mechanism by which these sequences have the ability to be activated with the addition of SERMs. It was found that SRC-3, a member of the SRC family of coactivators, had the ability to corepress SERMs via oestrogen receptors, a response not documented previously. Further to results obtained from the investigations of the SERM responsive reporter plasmids, DamID technology was employed to attempt to construct two libraries of sequences that associated, indirectly, with SRC-1A or SRC-3, via ligand bound oestrogen or progesterone. Gateway technology was used to transfer cloned sequences in to reporter plasmids to enable the luciferase assay to be employed in order to determine the functionality of the isolated sequences. - ~.... - Transactivation assays suggested that the attempt to direct the SRC-1A:Dam fusion protein to bind to DNA via either ligand bound oestrogen or progesterone failed, however following reporter plasmid sequencing bioinformatical studies were performed q d a number of possible transcription factor binding,motifs were identified in the sequences. Further transactivation assays indicated SRC-1A:Dam had, in a subset of the reporter plasmids, bound via the orphan nuclear receptors RORa and COUP TFI. Results suggest that SRC-1A may mediate this association and coactivate gene transcription; the mechanism behind this novel finding remains to be elucidated

Effect-based analyses as tools to assess the impact of differently treated effluents on fish and aquatic ecosystem health

Many surface waters are suffering from anthropogenic pressures, including pollutant emissions. Anthropogenic substances enter the aquatic environment through various pathways with wastewater treatment plants belonging to the most important sources. The technologies that are presently used in conventional wastewater treatment often fail to remove all substances. Consequently, numerous compounds are continuously discharged via effluents into surface waters, where they can be detected on a regular basis. Many studies revealed that traces of chemicals, so-called micropollutants, may pose a considerable threat to aquatic organisms. These findings led to the development of different strategies to reduce trace substances in surface waters, ranging from source control to so-called “end-of-pipe” strategies. One important measure is the expansion of conventional wastewater treatment plants by additional purification steps. Here, removing substances via adsorption to powdered activated carbon is a promising approach for large-scale application. Yet, little is known about the efficiency of this purification process with respect to ecosystem health. In my doctoral thesis, I used effect-based analyses to characterize the effect of differently treated wastewater effluents on fish health. For this purpose, I combined in situ exposure of rainbow trout at two conventionally equipped wastewater treatment plants with subsequent biomarker analyses. In addition, the present thesis includes studies conducted in the context of the research project SchussenAktivplus, which aimed to examine the effect of expanding a wastewater treatment plant by an additional powdered activated carbon stage on the ecosystem of the receiving river Schussen. For this, a comprehensive approach combining different chemical and biological analyses prior and subsequent to the implementation of the additional purification stage was adopted. My studies showed that fish health can be considerably impaired by discharges of conventionally equipped wastewater treatment plants. An additional adsorption stage significantly reduced micropollutant loads in the effluent and thus mitigated the adverse effects in exposed fish. Moreover, this positive effect was also reflected on other levels of biological organisation, thus showing the benefit of wastewater treatment plant upgrading. However, during my studies, adverse effects could sometimes also be detected in fish exposed at the reference site located upstream of the respective wastewater treatment plant. Thus, the degree of adverse reactions in exposed fish was not only linked to the type of wastewater treatment. The general water quality of the receiving river, which also depends on pollution inputs upstream of the wastewater treatment plants under investigation, and the composition of the raw wastewater also seemed to play an important role. Hence, the local conditions should be taken into account when decisions on the necessity and the type of wastewater treatment plant upgrading have to be made

Antiprogestins reduce epigenetic field cancerization in breast tissue of young healthy women

Background: Breast cancer is a leading cause of death in premenopausal women. Progesterone drives expansion of luminal progenitor cells, leading to the development of poor-prognostic breast cancers. However, it is not known if antagonising progesterone can prevent breast cancers in humans. We suggest that targeting progesterone signalling could be a means of reducing features which are known to promote breast cancer formation. Methods: In healthy premenopausal women with and without a BRCA mutation we studied (i) estrogen and progesterone levels in saliva over an entire menstrual cycle (n = 20); (ii) cancer-free normal breast-tissue from a control population who had no family or personal history of breast cancer and equivalently from BRCA1/2 mutation carriers (n = 28); triple negative breast cancer (TNBC) biopsies and healthy breast tissue taken from sites surrounding the TNBC in the same individuals (n = 14); and biopsies of ER+ve/PR+ve stage T1–T2 cancers and healthy breast tissue taken from sites surrounding the cancer in the same individuals (n = 31); and (iii) DNA methylation and DNA mutations in normal breast tissue (before and after treatment) from clinical trials that assessed the potential preventative effects of vitamins and antiprogestins (mifepristone and ulipristal acetate; n = 44). Results: Daily levels of progesterone were higher throughout the menstrual cycle of BRCA1/2 mutation carriers, raising the prospect of targeting progesterone signalling as a means of cancer risk reduction in this population. Furthermore, breast field cancerization DNA methylation signatures reflective of (i) the mitotic age of normal breast epithelium and (ii) the proportion of luminal progenitor cells were increased in breast cancers, indicating that luminal progenitor cells with elevated replicative age are more prone to malignant transformation. The progesterone receptor antagonist mifepristone reduced both the mitotic age and the proportion of luminal progenitor cells in normal breast tissue of all control women and in 64% of BRCA1/2 mutation carriers. These findings were validated by an alternate progesterone receptor antagonist, ulipristal acetate, which yielded similar results. Importantly, mifepristone reduced both the TP53 mutation frequency as well as the number of TP53 mutations in mitotic-age-responders. Conclusions: These data support the potential usage of antiprogestins for primary prevention of poor-prognostic breast cancers