Estrogenic contamination of waterways is of world-wide concern due to the adverse effects observed in aquatic biota. Recently, wastes from agricultural activities have been identified as likely sources of steroid estrogens released into the environment. Wastes from dairying activities are of particular concern in New Zealand. This project included development of analytical methods to measure free and conjugated estrogens, measurement of estrogens from the source to receiving environments and an investigation of effluent treatment technologies. The analytical method developed in this study was based on GC-MS measurement of free estrogens (17α-estradiol (17α-E2), 17β-estradiol (17β-E2) and estrone (E1)) and LC-IT-MS measurement of their sulfate-conjugates (17α-E2-3S, 17β-3S, E1-3S) in raw and treated farm dairy shed effluents (DSE). Effluents from farms in the Canterbury and Waikato Regions, two regions where dairy farming is the dominant land-use, were collected and analysed. All effluents demonstrated high concentrations of steroid estrogens, particularly 17α-E2 (median 760 ng/L). Estrogenic activity was also elevated, at up to 500 ng/L 17β-E2 equivalents using the E-Screen, an in vitro cell proliferation bioassay. Comparison to the chemical data indicated that for most samples, the highest proportion of estrogenic activity was derived from steroid estrogens naturally excreted by dairy cows. Conjugated estrogens were measured in several raw effluent samples, at similar concentrations to those of free estrogens, particularly E1. Dairy effluent treatment systems reduced free estrogen concentrations by 63-99% and reduced estrogenic activity by up to 89%. In spite of high removal efficiencies, estrogens remained elevated in the treated effluents that are discharged into waterways. Steroid estrogens and estrogenic activity were detected in streams and groundwater in areas impacted by dairy farming. Although concentrations were generally low, in two streams the concentrations were above levels regarded as safe for aquatic biota (<1 ng/L). The results demonstrate that dairy effluents are indeed a major source of estrogens to the environment and to waterways.

Endocrine disrupting chemicals in wild animals, including fish, can disrupt reproduction by causing intersexuality. Organic pollutants, specifically polychlorinated biphenyls (PCBs), have been shown to be estrogenic or anti-androgenic likely contributing to intersexuality in males. Organic pollutants persist in the environment despite being restricted for use in the United States. Bluegills, Lepomis macrochirus, play a crucial role in lake food webs as forage and sport fish and may be affected by PCBs. In the current study, male bluegills collected in 2010 from selected Illinois lakes were analyzed to quantify PCB body residues and the incidence of intersexuality (n=200). Experiments were then conducted in 2011 and 2012 to assess the reproductive output of mature males with varying PCB body residues paired with mature females containing no detectable PCB body residues held in uncontaminated 0.05-hectare research ponds (Southern Illinois University Touch of Nature Pond Facility). Lake-specific male bluegill whole-body tissue PCB body residues ranged from below reporting limit to 2 mg/kg dry weight (non-lipid normalized basis) and varied significantly among lakes (F7,32= 9.59, p<0.0001). No male intersex bluegills were found. In both reproductive experiments, abundance of young declined as male PCB body residues increased (F 4,18 = 13.28; P=0.002). Adult survival was unrelated to reproductive output. Taken together, these results suggest PCBs, which are ubiquitous in aquatic and terrestrial habitats, have the ability to alter male reproductive performance, and possibly population dynamics and community structure through reduced recruitment, in the absence of overt intersexuality.

Polycyclic aromatic compounds (PACs) are a group of chemicals encompassing thousands of different aromatic, alkyl aromatic and heterocyclic hydrocarbons (i.e.- containing N, S, or O- atoms); 16 of which have been designated as priority pollutants due to their toxicity and prevalence. Several studies have highlighted increases in the concentrations of C1-C4 alkylated PACs and heterocyclic aromatic hydrocarbons such as dibenzothiophenes in the atmosphere, water, soil and sediments, plants, wildlife and fish in the Athabasca Oil Sands Region (AOSR). Although there has been considerable research attention related to the toxic, carcinogenic and mutagenic properties of PACs, there is an increasing awareness that these chemicals may also have profound endocrine disrupting properties in wildlife. North American river otter (Lontra canadensis) are good indicators of ecosystem health due to their ecology and sensitivity to environmental pollutants. In this thesis, we first demonstrated the utility of adopting paleotoxicological frameworks in defining environmental baseline levels of PACs and likely biological effects from exposure to these complex environmental mixtures. These methods allowed us to reconstruct historical PAC deposition patterns to impacted areas while simultaneously determining likely biological effects such as endocrine disruption. Next, we showed how PACs exhibited trophic dilution in a Boreal food chain dominated by river otters. Snails, prey and predator fish, as well as river otters were collected from four main study areas in the AOSR in northeastern Alberta, Canada. Bioaccumulation factors such as biota-sediment accumulation factors (BSAF) and trophic magnification factors (TMF) were used to evaluate the partitioning behavior of PACs in the environment and subsequent risks to biota. Our results revealed localized enrichment of certain PACs and subsequent metabolism in higher order vertebrates. Finally, we successfully combined ecotoxicological and physiological analyses paired with population genetic estimates to investigate endocrine disruption and population-level responses to exposure to PACs. River otters are known for their habitual use of latrine sites. Latrine sites represent a unique opportunity for biomonitoring programs to study river otters using indirect sampling methods. In this thesis, PACs were characterized and evaluated in sediment, lower and higher trophic biota with demonstrated impacts on endocrine processes and river otter population health. Effects-based assessments such as the ones presented in this thesis are more powerful for environmental monitoring programs than stressor-based assessment methods (such as describing presence/absence or levels of contaminants) as they provide greater biological context to monitoring data. In turn, these are helpful in selecting triggers for environmental effects monitoring or adaptive management programs.

Thyroid hormone is essential for normal brain development and recognition of this has led to universal screening of newborns for thyroid function to ensure that circulating levels of thyroid hormone are within a range known to be supportive of normal growth and mental development. Environmental chemicals that interfere with thyroid function are known to inhibit normal growth and mental development. Work from our lab and from labs internationally demonstrates in animal systems that some industrial chemicals such as PCBs, PBDEs, and others may interact with the thyroid hormone receptor(s) in ways that are not predicted by changes in serum thyroid hormone levels. Our work demonstrates that the enzyme CYP1A1 must metabolize some individual PCB congeners before they can interact with the thyroid receptor. In animals, this requirement appears to be manifested in part by a strong correlation between CYP1A1 and TH target gene expression. Here we present that this pattern extends to humans by demonstrating a correlation between increased CYP1A1 mRNA and an abundance of thyroid hormone responsive gene mRNA.

No description available.

Pharmaceuticals have routinely been detected in the environment resulting in a growing concern about whether these drugs could elicit effects on aquatic organisms. The concerns are centered on the highly conserved nature of mammalian therapeutic targets in fish. These pharmaceuticals are found at very low levels in the environment, which can result in sub-lethal effects in aquatic organisms. Therefore, 28 d early-life stage studies were conducted on six pharmaceuticals to assess their impacts on survival and growth fathead minnow larvae. Two pharmaceuticals tested, carbamazepine and fenofibrate, resulted in no alterations to survival and growth. However, amiodarone, clozapine, dexamethasone, and levonorgestrel (LNG) reduced survival at concentrations tested with LNG being the most potent at 462 ng/L. Survival was increased with amiodarone and clozapine; however LNG significantly decreased growth at 86 ng/L. Therefore, the most potent pharmaceutical tested was the synthetic progestin LNG with survival and growth impacts at concentrations less than 1 μg/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 d ELS exposure to LNG. Transcripts of 3β-HSD, 20β-HSD, and FSH were significantly down-regulated following 28 d exposure to both 16.3 and 86.9 ng/L LNG. Also, CYP19a expression was significantly down-regulated at 86.9 and 2392 ng/L LNG. Subsequently, a second study examined time periods that may be most sensitive (e.g., windows of sensitivity) for FHM larvae exposed to LNG. Larvae were exposed to a single concentration of LNG (i.e. LOECgrowth of 86.2 ng/L as determined in the 28 d ELS study) for different time periods starting with fertilized egg through 28 dph. Growth and mRNA expression of the four differentially expressed transcripts from the first study were measured. Regardless of the duration of exposure, LNG significantly decreased growth in fathead minnow larvae at day 28. For both 20β-HSD and CYP19a, mRNA expression was decreased following exposure to LNG; however, these transcripts returned to baseline levels after removal of LNG. 3β-HSD and FSH showed similar trends after exposure to LNG with 7-14 d and 14-28 d exposures exhibiting a decrease in expression; however, FSH expression returned to baseline once removed for LNG exposure. Based on these data, 3β-HSD was the only transcript to remain down regulated after LNG exposure. Together these data suggest LNG can negatively impact FHM larval survival and growth, with significant alterations in endocrine related responses. However, these changes in endocrine related responses may not directly correlate to the changes in growth demonstrated with LNG exposure to fathead minnows. Therefore, additional research is warranted to ascertain additional mechanisms, either endocrine related or non-endocrine functions, related to changes in growth of larval fathead minnows.

The anuran Xenopus tropicalis tadpole is an attractive model animal in toxicological evaluation of suspected thyroid disrupting xenobiotics. Due to its reliance of a functioning hypothalamic-pituitary-thyroid (HPT) axis for normal metamorphosis, effects on the HPT axis produces apical endpoints, which are easy to measure. More sensitive endpoints of mRNA expression and histological evaluation of the thyroid gland itself provide strong indications of in vivo thyroid endocrine disruption. X. tropicalis is traditionally exposed in groups of 20 animals in four replicates for each treatment group. However, exposing tadpoles individually can provide stronger statistics and a reduction of total animal sacrifice. In this study we have developed and optimized an individual exposure system by a method development study. This method was then applied in an exposure experiment of a suspected thyroid endocrine disruptor, propylparaben (PrP). Prometamorphotic (NF stage 51) X. tropicalis tadpoles were distributed in three treatment groups (0.05, 0.5 and 5.0 mg PrP/L water) and maintained semi-statically for 14 days. Afterwards, apical measurements (body weight (BW), total body length (BL), snout to vent length (SVL) and hind limb length (HLL)) and reached developmental stage were assessed. In addition, mRNA expression of HPT axis relevant genes encoding deiodinase 2 (D2, hepatic tissue), deiodinase 3 (D3, hepatic and tail tissue) and transthyretin (Ttr, hepatic tissue) were measured by quantitative reverse transcription PCR (qRT-PCR). The PrP exposure did not affect general growth and development, but it did cause a downregulation of dio3 and ttr. The downregulation of dio3 could possibly be associated with a reduced serum content of thyroid hormone, while ttr might be connected to a previously described xenoestrogenic effect of PrP in vitro and in fish.

<p>Following the phase out of polybrominated diphenyl ethers (PBDEs), manufacturers turned to several alternative flame retardants (FRs) to meet flammability standards. Organophosphate FRs (OPFRs), and in particular tris (1,3-dichloropropyl) phosphate (TDCPP), have been increasingly detected in textiles and foam padding used in a variety of consumer products including camping equipment, upholstered furniture, and baby products. Like PBDEs, OPFRs are additive, meaning that they are not chemically bound to the treated material and can more readily leach out into the surrounding environment. Indeed, OPFRs have been detected in numerous environmental and biological matrices, often at concentrations similar to or exceeding that of PBDEs.</p><p>Although OPFRs have been in use for several decades, relatively little is known regarding their potential for adverse human and environmental health consequences. However, based on their structural similarity to OP pesticides, they may have analogous mechanisms of toxicity. OP pesticide toxicity is classically associated with cholinesterase inhibition, resulting in cholinergic intoxication syndrome. OPFRs have been shown to be ineffective cholinesterase inhibitors, however chlorpyrifos (CPF) and other OP pesticides have been shown to elicit adverse effects on developing organisms through other mechanisms.</p><p>The main objective of this research project was to evaluate the toxicity of four structurally similar OPFRs (TDCPP; tris (2,3-dibromopropyl) phosphate, (TDBPP); tris (1-chloropropyl) phosphate (TCPP) and tris (2-chloroethyl) phosphate (TCEP)) in comparison to chlorpyrifos (CPF), a well-studied OP pesticide. A combination of in vitro and in vivo models was used to elucidate potential mechanisms as well as functional consequences of exposure in developing organisms. </p><p>In the first research aim, a series of in vitro experiments with neurotypic PC12 cells was used to evaluate the effects of four structurally similar OPFRs (TDCPP, TDBPP, TCEP, or TCPP) and CPF on neurodevelopment. The effects of TDCPP were also compared to that of BDE-47, a major component of the commercial PentaBDE mixture. In general, TDCPP elicited similar or greater effects when compared to an equimolar concentration of CPF. All OPFRs tested produced similar decrements in cell number and altered phenotypic differentiation, while BDE-47 had no effect on cell number, cell growth, or neurite growth. </p><p>For the second research aim, zebrafish (Danio rerio) were used to evaluate the effects of the same suite of chemicals on early development. TDCPP, TDBPP, and CPF elicited overt toxicity (e.g., malformations or death) within the concentration range tested (0.033-100 µM). TDBPP was the most potent with 100% mortality by 6 days post fertilization (dpf) at &#8805;3.3 µM. CPF and TDCPP showed equivalent toxicity with malformations observed in at 10 µM and significant mortality (&#8805;75%) at &#8805;33 µM. There was no overt toxicity among TCEP- and TCPP-exposed fish. All test chemicals affected larval swimming behavior on 6 dpf at concentrations below the overt toxicity threshold. Parent chemical was detected in all in embryonic (1 dpf) and larval (5 dpf) tissues. TDCPP and TDBPP showed rapid and extensive metabolism.</p><p>Finally, for the third aim, juvenile (45-55 dpf) zebrafish were exposed to CPF (1 µg/g food) or TDCPP (Low TDCPP = 1 µg/g food; High TDCPP = 40 µg/g food) via diet for 28 days followed by a 7 day depuration period where all treatments received clean food. A dietary exposure was chosen to more closely recapitulate exposure in humans. Samples were collected at seven time points throughout the experiment: days 0, 7, 14, 21, 28, 30, 35. Whole tissues were collected for tissue accumulation and histopathology endpoints. Viscera and brain were dissected and flash frozen separately for DNA damage analyses. </p><p>Tissue measurements of CPF, TDCPP, and the metabolite bis (1,3-dichloropropyl) phosphate (BDCPP) were often below the method detection limit, however when present there was a trend towards increased accumulation with treatment and time. On Day 7 Low TDCPP caused a dramatic but transient increase in DNA damage in both viscera and brain that returned to control levels by Day 14. Similar results have been seen previously with other genotoxicants and may be due to CPF and High TDCPP inducing an adaptive response prior to the 7 day sampling point. All treatments shifted the neurohypophysis to adenohypophysis ratio (NH/AH; Day 7 only) and significantly increased thyroid follicle activation (Day 14). Finally High TDCPP affected gonad maturation, causing a significant increase in ovary follicle development (Day 14) and a transient but marked decrease in testes maturity (Day 7). Taken together these data suggest that dietary exposure to TDCPP and CPF elicits DNA damage in brain and viscera and alters endocrine function in juvenile zebrafish. Importantly, analyses were restricted to the first three time points (Days 0, 7, and 14) due to the emergence a disease among the experimental colony. Although these samples were collected prior to the disease becoming apparent, it remains a potential confounder of the current results.</p> / Dissertation

A desregulação endócrina induzida por contaminação ambiental está entre os principais problemas criados pela sociedade moderna de consumo, responsável pela inserção no ambiente de uma série de substâncias interferentes nos sistemas hormonais dos mais diversos organismos, incluindo o próprio homem. A ação destes compostos acarreta, entre outros efeitos, disfunções reprodutivas e estudos apontam que também podem ser indutores de cânceres. A legislação brasileira através do Conselho Nacional do Meio Ambiente (CONAMA) determina os padrões de qualidade das águas, porém muitas substâncias com potencial de desregulação endócrina não tem suas concentrações e emissões especificadas. O objetivo deste trabalho foi executar um levantamento da presença e possíveis conseqüências dos hormônios estrogênicos, uma das classes mais potentes de desreguladores endócrinos (ED), nos compartimentos água e sedimento do rio do Monjolinho. Este rio cruza parte da malha urbana da cidade de São Carlos - SP e recebe lançamentos localizados e difusos de esgotos domésticos e industriais. Portanto, amostras de água e sedimentos foram analisadas através de cromatografia líquida, e exemplares de peixes capturados no rio investigados quanto à presença da proteína vitelogenina (VTG) um biomarcador de exposição. Também ensaios ecotoxicológicos foram desenvolvidos em laboratório com diferentes abordagens para verificação de efeitos diversos. Em uma tentativa de abordar os dados gerados através de uma perspectiva ampla, foi delineada uma avaliação de risco ambiental discutindo as possíveis ameaças a biota e a população humana, já que concentrações de hormônios, principalmente o sintético etinilestradiol (concentração máxima de 30,1 ± 3,41 ng/L), a indução da VTG e efeitos em ensaios ecotoxicológicos foram confirmados. / The environmental endocrine disruption is among the main problems arrived with the modern society way of life. The hormonal systems of several organisms are injured by a number of chemicals disposal on hydric bodies in erroneous way. These compounds causes reproductive disturbs, and studies pointed it be cancer inductors. The Brazilian National Environmental Council (CONAMA) do not regulated standards for discharges and concentrations of these substances. This work aims to investigate the probable presence and effects of sexual estrogens hormones, one of the most powerful groups of endocrine disruptors (EDCs), at the Monjolinho river. This small urban river is placed in São Carlos; a town located in the São Paulo state, southwest Brazil, and receives concentrated and diffuse sewage effluents as industrials as domestics. Samples of water and sediments were analyzed by liquid chromatography, and male fishes captured were investigated to survey the vitellogenin protein (Vtg), a biomarker of exposition. To complement the study, ecotoxicological tests with different approaches were considered. Moreover an environmental risk analyze delineation was made because hormones concentrations, mainly the synthetic ethynilestradiol (EE2), VTG induction, and positive effects in ecotoxicity tests were found.

O glifosato-Roundup é um herbicida amplamente utilizado em diversas culturas agrícolas. Sua toxicidade reprodutiva está relacionada com a inibição da proteína StAR e da enzima aromatase, causando in vitro redução significativa da produção de testosterona e estradiol. Este trabalho teve como objetivo verificar esse efeito in vivo, utilizando-se ratos Wistar machos pré-púberes como modelo experimental. Utilizou-se 68 machos tratados dos 23 aos 53 dias de idade com as doses de 0, 5, 50 e 250 mg/kg de peso vivo por gavagem uma vez ao dia. Foram avaliados a progressão da puberdade, o desenvolvimento corporal, a produção hormonal de testosterona, estradiol e corticosterona, morfologia testicular e da glândula adrenal, função renal e hepática e histopatologia renal e hepática. As análises estatísticas utilizadas foram a análise de variâncias de uma via ANOVA, de duas vias MANOVA ou Kruskall-Walis e pós-testes de Tukey-Kramer, Fisher ou de Dun. O herbicida glyphosate-Roundup alterou de forma significativa a progressão da puberdade de forma dose dependente, bem como se observou a redução na produção de testosterona e alterações na morfologia dos túbulos seminíferos. A morfologia da glândula adrenal e a produção de corticosterona não foram afetadas pelas doses utilizadas nesse estudo. Observou-se comprometimento da função renal e alterações patológicas nesse órgão. Não foram evidenciadas alterações hepáticas. O crescimento corporal dos animais não foi influenciado pelo tratamento. Pode-se concluir que esse herbicida é um potente disruptor endócrino in vivo, causando distúrbios no desenvolvimento reprodutivo e na produção hormonal dos animais. / The glyphosate-Roundup is a widely pesticide used in several culturas agrícolas. Its reproductive toxicity is associated to inhibition of StAR protein and aromatase enzime that cause in vitro significantly reduction in testosterone and estradiol production. The objective of this study was to evaluate the in vivo effects of inhibition of StAR protein and aromatase enzyme, using prepubertal male Wistar rats like experimental model. 68 animals were exposed once a day by gavage to glyphosate-Roundup in following doses: 0, 5, 50 or 250 mg/kg of body weight. The end points were puberty progression, body development, testosterone, estradiol and corticosterona productions and testicular and adrenal morphology, renal and liver function and histopatology. The statistical analysis used were one-way ANOVA, multi-way ANOVA or Kruskall-Walis and posthoc tests of Tukey-Kramer, Fisher or Dun. The herbicide glyphosate-Roundup changed significantly the puberty progression in dose-dependent manner, as well reduction in testosterone production and alterations in testicular morphology. There were not observed alterations in adrenal morphology or corticosterone production in the doses used in this study. The renal function and histopatology were altered in treated groups while liver function wasn\'t. The body development was not influenced by the exposure. In conclusion, glyphosate-Roundup is a potent endocrine disruptor in vivo that cause problems in reproductive development and hormonal synthesis in exposure animals.