27. Danis, BEG; Marlatt, VL. (2021) Investigating Acute and Subchronic Effects of Neonicotinoids on Northwestern Salamander Larvae.Arch. Environ. Contam. Toxicol. 80: 691-707 Investigating Acute and Subchronic Effects of Neonicotinoids on Northwestern Salamander Larvae
This research investigated the adverse effects of neonicotinoids on the Northwestern salamander (Ambystoma gracile; NWS) after acute and subchronic exposures during early aquatic life stages via whole organism (i.e., growth, development) and molecular (i.e., gene expression) level endpoints. In a 96-h exposure, NWS larvae were exposed to four imidacloprid concentrations (250, 750, 2250, 6750 mu g/L) and a water control treatment, and no effects on survival, body weight, snout-vent length (SVL), and total body length were observed. However, a significant 1.70- and 2.33-fold decrease in thyroid receptor beta (TR beta) mRNA expression levels were detected in the larvae exposed to 750 and 2250 mu g/L imidacloprid, respectively, compared with the larvae in the water control. In subsequent subchronic experiments, NWS larvae were exposed for 35 days to imidacloprid alone and an equal part mixture of neonicotinoids (imidacloprid, clothianidin, and thiamethoxam (ICT)) at three concentrations (10, 100 and 1000 mu g total neonicotinoids/L) and a water control. In these experiments, there were no effects on larval survival, body weight, SVL, and total body length. However, advanced development of larvae in the 100 mu g/L imidacloprid treatment was observed compared with the control after 35-day imidacloprid exposure, providing some evidence of disruption of the thyroid endocrine axis at an environmentally relevant concentration. Ultimately, there is a paucity of studies conducted examining the sensitivity of salamanders to pollutants; thus, this study reports novel findings that will contribute to understanding the sensitivity of a Caudate amphibian model to a common environmental pollutant. DOI PubMed
26. Hegeman, KA; Marlatt, VL. (2021) Reproductive and thyroid endocrine axis cross-talk in rainbow trout (Oncorhynchus mykiss) alevins.Gen. Comp. Endocrinol. 312 Reproductive and thyroid endocrine axis cross-talk in rainbow trout (Oncorhynchus mykiss) alevins
Thyroid hormone; Estrogen; Endocrine cross-talk; Gene expression; Rainbow trout; Early development
The goal of this study was to characterize morphological and molecular effects in rainbow trout alevins after waterborne exposures to 17 beta-estradiol (E2; 0.0008 to 0.5 mu g/L), triiodothyronine (T3; 0.52 to 65 mu g/L), and various co-treatments for 21 to 23 days. Interestingly, there was no consistent evidence that E2 alone influenced growth, development or deformity rates, however, 65 mu g/L T3 alone expedited development, and both 13 mu g/L and 65 mu g/L alone caused a unique opercular deformity not previously reported. In addition, some potentiation between E2 and T3 at lower concentrations suggests some cross-talk between these two hormonal pathways may also contribute to the development of this opercular deformity. Gene expression changes were observed, including induction of vtg in rainbow trout alevins at 0.02 mu g/L concentration of E2, which is the lowest concentration reported to induce vtg in rainbow trout alevins. These data suggest low-level E2 does not negate abnormal growth and development caused by hyperthyroidism, and examining more time points is likely required to demonstrate a stronger response profile for individual hormones and endocrine axes cross-talk. DOI PubMed
25. McCuaig, LM; Martyniuk, CJ; Marlatt, VL. (2020) Morphometric and proteomic responses of early-life stage rainbow trout (Oncorhynchus mykiss) to the aquatic herbicide diquat dibromide.Aquat. Toxicol. 222 Morphometric and proteomic responses of early-life stage rainbow trout (Oncorhynchus mykiss) to the aquatic herbicide diquat dibromide
Reward (R); Proteomics; Diquat dibromide; Rainbow trout; Pulse exposure; Sub-network enrichment analysis
The objective of this study was to examine the acute toxicity and sub-lethal effects of the commercial formulation of diquat dibromide, Reward (R) Landscape and Aquatic Herbicide, on multiple life stages of rainbow trout. The continuous exposure 96 h LC50 derived for juvenile feeding fry aged 85 d post-hatch was 9.8 mg/L. Rainbow trout eyed embryos and juvenile feeding fry were also exposed to concentrations of Reward (R) ranging from 0.12 to 10 mg/L during two 24 h pulse exposures separated by 14 d of rearing in fresh water to mimic the manufacturers instructions for direct applications to water bodies. Decreased survival and body morphometrics were evident at 9.3 mg/L during the embryo/alevin exposures, but not in feeding juveniles, indicating a higher sensitivity of the early life stage fish. Quantitative proteomics and subnetwork enrichment analyses were conducted in the livers for both life stages to evaluate protein profiles after exposure to 0.37 mg/L diquat via Reward (R) exposure. Unique protein profiles were revealed for pre-feeding swim-up fry and for feeding juvenile fish, reflecting differences between the two life stages in sub-cellular responses after diquat dibromide exposure. Hepatic proteome effects were more dramatic in the pre-feeding swim-up fry with 315 proteins differentially expressed between the control and exposed fish while in the later life stage feeding fry, only 84 proteins were different after Reward (R) exposure. Exposure to Reward (R) significantly increased RNA/mRNA processes, induced activation of Atk/mTOR and caspase activity, and altered energy homeostasis. Proteomic alterations are associated with reduced growth observed in embryo/alevin at higher exposure concentrations, offering insight into key events underlying growth impairment within the adverse outcome pathway framework. This study is the first to report the sub-cellular and whole organism level effects of diquat dibromide in a commercial formulation and demonstrates that concentrations based on aquatic application rates alter the hepatic proteome. DOI PubMed
24. Moreton, ML; Lo, BP; Simmons, DBD; Marlatt, VL. (2020) Toxicity of the aquatic herbicide, reward (R), on the fathead minnow with pulsed-exposure proteomic profile.Comp. Biochem. Physiol. D-Genomics Proteomics 33 Toxicity of the aquatic herbicide, reward (R), on the fathead minnow with pulsed-exposure proteomic profile
Toxicology; Aquatic herbicide; Diquat dibromide; Fathead minnow; Proteomics
The objectives of this study were to assess the lethal and sub-lethal effects of the aquatic herbicide commercial formulation, Reward (R) (373 g/L DB), using application scenarios prescribed by the manufacturer. Specifically, a 14 d period between applications of Reward (R) in a water body undergoing treatment is required, yet the effects of these 'pulse' exposure scenarios on aquatic wildlife such as fish are unknown. In the first experiment early life stage FHM were exposed to a continuous DB concentrations from 0.105-12.6 mg/L which yielded a larval 7 d LC50 of 2.04 mg/L as well as a significant decrease in body mass (25.0 +/- 11.6%) at the 1.18 mg/L Reward (R) concentration. In a second experiment, FHM larvae were exposed for 24 h and then reared in clean water for 14 d followed by a second 24 h exposure to Reward (R). The 16 d LC50 value was 4.19 mg/L. In a third experiment, adult FHM were exposed in a pulse/discontinuous manner to Reward (R) with a calculated 21 d LC50 value of 6.71 mg/L. No significant changes in gonadosomatic index or fecundity of the F-1 generation's hatch success were found when eggs from exposed adults were then reared in clean water. Proteome analyses of whole FHM larvae from the discontinuous/pulse exposure showed the primary gene ontology molecular functions of the proteins in fish exposed to 3.78 mg/L DB that resulted in similar to 30% mortality with positive or negative differential abundance (p-value < .2) were: structural molecule activity; identical protein binding; structural constituent of cytoskeleton; ion binding; calcium ion binding; cytoskeletal protein binding; actin binding; and, ATP binding. These findings suggest that concentrations causing adverse effects occur above the maximum concentration predicted by the manufacturer when applied according to the label (i.e. > 0.37 mg/L). DOI PubMed
22.Marlatt, VL; Leung, TYG; Calbick, S; Metcalfe, C; Kennedy, C. (2019) Sub-lethal effects of a neonicotinoid, clothianidin, on wild early life stage sockeye salmon (Oncorhynchus nerka).Aquat. Toxicol. 217 Sub-lethal effects of a neonicotinoid, clothianidin, on wild early life stage sockeye salmon (Oncorhynchus nerka)
Growth; Development; Estradiol; Testosterone; Gene expression; Glucocorticoid receptor
One of the categories of environmental contaminants possibly contributing to declining sockeye salmon (Oncorhynchus nerka) in the Fraser River, British Columbia, Canada is pesticides. In this 4-month study, the effects of environmentally relevant concentrations of a waterborne neonicotinoid, clothianidin (0.15, 1.5, 15 and 150 mu g/L), on embryonic, alevin and early swim-up fry sockeye salmon derived from four unique genetic crosses of the Pitt River, BC stock were investigated. There were no significant effects of clothianidin on survival, hatching, growth or deformities, although genetic variation significantly affected these endpoints. Clothianidin caused a significant 4.7-fold increase in whole body 17 beta-estradiol levels in swim-up fry after exposure to 0.15 mu g/L, but no effects were observed on testosterone levels. In addition, hepatic expression of the gene encoding glucocorticoid receptor 2 was also impacted at the highest concentration of clothianidin tested, and was found to be similar to 4-fold lower compared to the sockeye reared in control water. These results indicate additional examination of clothianidin and its effects on salmonid gonad development and the reproductive and stress endocrine axes in general, is warranted. DOI PubMed
21. Moreton, ML; Marlatt, VL. (2019) Toxicity of the aquatic herbicide, reward (R), to the northwestern salamander.Environ. Sci. Pollut. Res. 26: 31077-31085 Toxicity of the aquatic herbicide, reward (R), to the northwestern salamander
Toxicology; Aquatic herbicide; Ecology; Diquat dibromide; Northwestern salamander; Amphibians
Diquat dibromide (DB) is the active ingredient in several herbicide products used around the world for industrial and recreational control of terrestrial and aquatic pest plants. This study aimed to assess the adverse effects of the commercial formulation of the aquatic herbicide, Reward (R), on the Pacific Northwest amphibian species, the northwestern salamander (Ambystoma gracile). Larvae were exposed to the Reward (R) herbicide in a 96-h acute bioassay (0.37-151.7 mg/L DB) and a continuous 21-day exposure (0.37-94.7 mg/L DB). The 96-h LC50 was 71.5 mg/L and the 21-day LC50 was 1.56 mg/L. Collectively, the results of this study demonstrate that early life stage A. gracile larvae appear largely insensitive to acute Reward (R) exposures compared to early life stage fish. However, A. gracile larvae are considerably more sensitive during sub-chronic exposure (21 days) with lethal and sub-lethal effects on growth occurring in the 1-2 mg/L range, which more closely resembles the larval fish lethal sensitivity to this active ingredient. This is the first study examining the toxicity of the aquatic herbicide formulation Reward (R) on A. gracile under acute and sub-chronic exposure scenarios. DOI PubMed
20. Du Gas, LM; Ross, PS; Walker, J; Marlatt, VL; Kennedy, CJ. (2017) Effects of atrazine and chlorothalonil on the reproductive success, development, and growth of early life stage Sockeye Salmon (Oncorhynchus nerka).Environmental Toxicology and Chemistry 36: 1354-1364 Effects of atrazine and chlorothalonil on the reproductive success, development, and growth of early life stage Sockeye Salmon (Oncorhynchus nerka)
Salmon; Pesticides; Commercial formulations; Emergence; Hatch
The effects of 2 currently used commercial pesticide formulations on Pacific sockeye salmon (Oncorhynchus nerka), from fertilization to emergence, were evaluated in a gravel-bed flume incubator that simulated a natural streambed. Embryos were exposed to atrazine at 25 mg/L (low atrazine) or atrazine at 250 mg/L (high atrazine) active ingredient (a.i.), and chlorothalonil at 0.5 mu g/L (low chlorothalonil) or chlorothalonil at 5 mg/L a. i. (high chlorothalonil) and examined for effects on developmental success and timing, as well as physical and biochemical growth parameters. Survival to hatch was reduced in the high chlorothalonil group (55% compared with 83% in controls), accompanied by a 24% increase in finfold deformity incidence. Reduced alevin condition factor (2.9-5.4%) at emergence and elevated triglyceride levels were seen in chlorothalonil-exposed fish. Atrazine exposure caused premature hatch (average high atrazine time to 50% hatch [H50] = 100 d postfertilization [dpf]), and chlorothalonil exposure caused delayed hatch (high chlorothalonil H50 = 108 dpf; controls H50 = 102 dpf). All treatments caused premature emergence (average time to 50% emergence [E50]: control E50 = 181 dpf, low chlorothalonil E50 = 175 dpf, high chlorothalonil E50 = 174 dpf, high atrazine E50 = 175 dpf, low atrazine E50 = 174 dpf), highlighting the importance of using a gravel-bed incubator to examine this subtle, but critical endpoint. These alterations indicate that atrazine and chlorothalonil could affect survival of early life stages of sockeye salmon in the wild. (C) 2017 SETAC DOI
19.Marlatt, VL; Martyniuk, CJ. (2017) Biological responses to phenylurea herbicides in fish and amphibians: New directions for characterizing mechanisms of toxicity.Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 194: 9-21 Biological responses to phenylurea herbicides in fish and amphibians: New directions for characterizing mechanisms of toxicity
Embryos; Fish; Amphibian; Agriculture; Comparative transcriptomics; Chemical structure-function relationships
Urea-based herbicides are applied in agriculture to control broadleaf and grassy weeds, acting to either inhibit photosynthesis at photosystem II (phenylureas) or to inhibit acetolactate synthase acetohydroxyacid synthase (sulfonylureas). While there are different chemical formulas for urea-based herbicides, the phenylureas are a widely used class in North America and have been detected in aquatic environments due to agricultural runoff. Here, we summarize the current state of the literature, synthesizing data on phenylureas and their biological effects in two non-target animals, fish and amphibians, with a primary focus on diuron and linuron. In fish, although the acutely lethal effects of diuron in early life stages appear to be >1 mg/L, recent studies measuring sub-lethal behavioural and developmental endpoints suggest that diuron causes adverse effects at lower concentrations (i.e. <0.1 mg/L). Considerably less toxicity data exist for amphibians, and this is a knowledge gap in the literature. In terms of sub-lethal effects and mode of action (MOA), linuron is well documented to have anti-androgenic effects in vertebrates, including fish. However, there are other MOAs that are not adequately assessed in toxicology studies. In order to identify additional potential MOAs, we conducted in silico analyses for linuron and diuron that were based upon transcriptome studies and chemical structure-function relationships (i.e.ToxCast (TM), Prediction of Activity Spectra of Substances). Based upon these analyses, we suggest that steroid biosynthesis, cholesterol metabolism and pregnane X receptor activation are common targets, and offer some new endpoints for future investigations of phenylurea herbicides in non-target animals. (C) 2017 Elsevier Inc. All rights reserved. DOI
18. Knight, R; Marlatt, VL; Baker, JA; Lo, BP; Debruyn, AMH; Elphick, JR; Martyniuk, CJ. (2016) Dietary selenium disrupts hepatic triglyceride stores and transcriptional networks associated with growth and Notch signaling in juvenile rainbow trout.Aquatic Toxicology 180: 103-114 Dietary selenium disrupts hepatic triglyceride stores and transcriptional networks associated with growth and Notch signaling in juvenile rainbow trout
Dietary; Growth; Transcriptomics; Metabolites; Lipid peroxidation; Oxidative stress
Dietary Se has been shown to adversely affect adult fish by altering growth rates and metabolism. To determine the underlying mechanisms associated with these observations, we measured biochemical and transcriptomic endpoints in rainbow trout following dietary Se exposures. Treatment groups of juvenile rainbow trout were fed either control Lumbriculus variegatus worms or worms cultured on selenized yeast. Selenized yeast was cultured at four nominal doses of 5, 10, 20 or 40 mg/kg Se dry weight (measured dose in the worms of 7.1, 10.7, 19.5, and 31.8 mg/kg Se dw respectively) and fish were fed for 60 days. At 60 d, hepatic triglycerides, glycogen, total glutathione, 8-isoprostane and the transcriptome response in the liver (n = 8/group) were measured. Fish fed the nominal dose of 20 and 40 mg/kg Se dry weight had lower body weight and a shorter length, as well as lower triglyceride in the liver compared to controls. Evidence was lacking for an oxidative stress response and there was no change in total glutathione, 8-isoprostane levels, nor relative mRNA levels for glutathione peroxidase isoforms among groups. Microarray analysis revealed that molecular networks for long-chain fatty acid transport, lipid transport, and low density lipid oxidation were increased in the liver of fish fed 40 mg/kg, and this is hypothesized to be associated with the lower triglyceride levels in these fish. In addition, up-regulated gene networks in the liver of 40 mg/kg Se treated fish included epidermal growth factor receptor signaling, growth hormone receptor, and insulin growth factor receptor 1 signaling pathways. These molecular changes are hypothesized to be compensatory and related to impaired growth. A gene network related to Notch signaling, which is involved in cell-cell communication and gene transcription regulation, was also increased in the liver following dietary treatments with both 20 and 40 mg/kg Se. Transcriptomic data support the hypothesis that dietary Se increases the expression of networks for growth-related signaling cascades in addition to those related to fatty acid synthesis and metabolism. We propose that the disruption of metabolites related to triglyceride processing and storage, as well as gene networks for epidermal growth factor and Notch signaling in the liver, represent key molecular initiating events for adverse outcomes related to growth and Se toxicity in fish. (C) 2016 Elsevier B.V. All rights reserved. DOI
17.Marlatt, VL; Sherrard, R; Kennedy, CJ; Elphick, JR; Martyniuk, CJ. (2016) Application of molecular endpoints in early life stage salmonid environmental biomonitoring.Aquatic Toxicology 173: 178-191 Application of molecular endpoints in early life stage salmonid environmental biomonitoring
Environmental monitoring; Gene expression; Metals; PAH; Salmonid; Development
Molecular endpoints can enhance existing whole animal bioassays by more fully characterizing the biological impacts of aquatic pollutants. Laboratory and field studies were used to examine the utility of adopting molecular endpoints for a well-developed in situ early life stage (eyed embryo to onset of swim-up fry) salmonid bioassay to improve diagnostic assessments of water quality in the field. Coastal cutthroat trout (Oncorhynchus clarki clarki) were exposed in the laboratory to the model metal (zinc, 40 mu g/L) and the polycyclic aromatic hydrocarbon (pyrene, 100 mu g/L) in water to examine the resulting early life stage salmonid responses. In situ field exposures and bioassays were conducted in parallel to evaluate the water quality of three urban streams in British Columbia (two sites with anthropogenic inputs and one reference site). The endpoints measured in swim-up fry included survival, deformities, growth (weight and length), vitellogenin (vtg) and metallothionein (Mt) protein levels, and hepatic gene expression (e.g., metallothioneins [mta and mtb], endocrine biomarkers [vtg and estrogen receptors, esr] and xenobiotic-metabolizing enzymes [cytochrome P4501A3, cyp1a3 and glutathione transferases, gstk]). No effects were observed in the zinc treatment, however exposure of swim-up fry to pyrene resulted in decreased survival, deformities and increased estrogen receptor alpha (er1) mRNA levels. In the field exposures, xenobiotic-metabolizing enzymes (cyp1a3, gstk) and zinc transporter (zntBigM103) mRNA were significantly increased in swim-up fry deployed at the sites with more anthropogenic inputs compared to the reference site. Cluster analysis revealed that gene expression profiles in individuals from the streams receiving anthropogenic inputs were more similar to each other than to the reference site. Collectively, the results obtained in this study suggest that molecular endpoints may be useful, and potentially more sensitive, indicators of site-specific contamination in real-world, complex exposure scenarios in addition to whole body morphometric and physiological measures. (C) 2016 Elsevier B.V. All rights reserved. DOI
16.Marlatt, VL; Sun, JY; Curran, CA; Bailey, HC; Kennedy, CK; Elphick, JR; Martyniuk, CJ. (2014) Molecular responses to 17 beta-estradiol in early life stage salmonids.General and Comparative Endocrinology 203: 203-214 Molecular responses to 17 beta-estradiol in early life stage salmonids
Rainbow trout; Estrogen receptor; Eyed embryo; Gene expression; 17 beta-estradiol; Vitellogenin
Environmental estrogens (EE) are ubiquitous in many aquatic environments and biological responses to EEs in early developmental stages of salmonids are poorly understood compared to juvenile and adult stages. Using 17 beta-estradiol (E2) as a model estrogen, waterborne exposures were conducted on early life stage rainbow trout (Oncorhynchus mykiss; egg, alevin, swim-up fry) and both molecular and physiological endpoints were measured to quantify the effects of E2. To investigate developmental stage-specific effects, laboratory exposures of 1 mu g/L E2 were initiated pre-hatching as eyed embryos or post-hatching upon entering the alevin stage. High mortality (similar to 90%) was observed when E2 exposures were initiated at the eyed embryo stage compared to the alevin stage (similar to 35% mortality), demonstrating stage-specific sensitivity. Gene expression analyses revealed that vitellogenin was detectable in the liver of swim-up fry, and was highly inducible by 1 mu g/L E2 (>200-fold higher levels compared to control animals). Experiments also confirmed the induction of vitellogenin protein levels in protein extracts isolated from head and tail regions of swim-up fry after E2 exposure. These findings suggest that induction of vitellogenin, a well-characterized biomarker for estrogenic exposure, can be informative measured at this early life stage. Several other genes of the reproductive endocrine axis (e.g. estrogen receptors and androgen receptors) exhibited decreased expression levels compared to control animals. In addition, chronic exposure to E2 during the eyed embryo and alevin stages resulted in suppressive effects on growth related genes (growth hormone receptors, insulin-like growth factor 1) as well as premature hatching, suggesting that the somatotropic axis is a key target for E2-mediated developmental and growth disruptions. Combining molecular biomarkers with morphological and physiological changes in early life stage salmonids holds considerable promise for further defining estrogen action during development, and for assessing the impacts of endocrine disrupting chemicals in vivo in teleosts. (C) 2014 Elsevier Inc. All rights reserved. DOI PubMed
15.Marlatt, VL; Lo, BP; Ornostay, A; Hogan, NS; Kennedy, CJ; Elphick, JR; Martyniuk, CJ. (2013) The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas).Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 157: 24-32 The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas)
Anti-androgen; Linuron; Steroidogenic genes; Vitellogenin; Endocrine disruption
Linuron is a widely used urea-based herbicide that has anti-androgenic activity in both fish and rodents. To further elucidate the potential mode of action (MOA) of linuron on the vertebrate endocrine system, adult male and female fathead minnows were exposed for 21 days to dechlorinated water, a solvent control, 17 beta-estradiol (E2; 0.1 mu g/L), dihydrotestosterone (DHT; 100 mu g/L), linuron (1, 10, 100 mu g/L) and one co-treatment of DHT (100 mu g/L) and linuron (100 mu g/L). There were no effects of linuron on egg hatching, 7 day egg survival, nuptial tubercle formation or gonadal histopathology. Administration of DHT and 1 and 100 mu g/L linuron reduced plasma vitellogenin in females, while male plasma vitellogenin were induced after E2 exposure and co-exposure of DHT and linuron. Ovarian mRNA levels were examined for several genes involved in steroidogenesis (e.g. p450scc, cyp19a, star, tspo, hsd17b and hsd11b) and estrogen-mediated responses (esr1, esr26, esr2a). Only p450scc mRNA was significantly decreased with DHT+linuron co-treatment. Clustering of steroidogenic mRNA transcript expression patterns revealed that patterns for linuron were more similar to E2 compared to DHT. Collectively, this study supports the hypothesis that linuron may not be a pure anti-androgen and may have multiple MOAs that affect vertebrate reproduction. (C) 2012 Elsevier Inc. All rights reserved. DOI
14.Marlatt, VL; Veldhoen, N; Lo, BP; Bakker, D; Rehaume, V; Vallee, K; Haberl, M; Shang, DY; van Aggelen, GC; Skirrow, RC; Elphick, JR; Helbing, CC. (2013) Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA).Aquatic Toxicology 126: 85-94 Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA)
Tree frog tadpole; Postembryonic development; Thyroid hormone receptor; Gelatinase B; Proliferating nuclear antigen; Metamorphosis assay
The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5 mu g/L thyroxine (T-4), 0.3, 3 and 30 mu g/L (nominal) TCS, or combined T-4/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T-4 treatment alone accelerated development concomitant with altered levels of TH receptors alpha and beta, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30 mu g/L) was protective against tadpole mortality, this protection was lost in the presence of T-4. The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent. (C) 2012 Elsevier B.V. All rights reserved. DOI
13. Atkinson, SK; Marlatt, VL; Kimpe, LE; Lean, DRS; Trudeau, VL; Blais, JM. (2012) The occurrence of steroidal estrogens in south-eastern Ontario wastewater treatment plants.Science of the Total Environment 430: 119-125 The occurrence of steroidal estrogens in south-eastern Ontario wastewater treatment plants
Steroidal estrogen; Biodegradation; Wastewater treatment plant; Removal efficiency; Estrogenic potency
We measured steroidal estrogens in wastewater in Ottawa and Cornwall (Ontario, Canada) to determine removal efficiency of these steroids during the treatment process, and whether removal varies during a seasonal cycle. Estrone (E1), 17 beta-estradiol (E2) and 17 alpha-ethinylestradiol (EE2) were found at maximum concentrations in raw sewage (RS), at 104, 66.9 and 5.7 ng L-1, respectively. For the Ottawa wastewater treatment plant (WWTP), there was sufficient data to show that El concentrations in RS correlated with both ambient air temperature and mean daily flow of the WWTP (R-2 = 0.792, p = 0.003 and R-2 = 0.757, p = 0.005). El removal was correlated with the percent difference in cBOD from RS to FE (final effluent) (R-2 = 0.435, p = 0.075). However estrogenic potency, as determined by a sensitive in vitro reporter gene assay, did not decrease during the water treatment process, suggesting that many estrogenic chemicals are conserved in FE. El and EE2 were found in river water, both upstream and downstream of the WWTPs, and at much lower concentrations than in FE Our study demonstrates the persistence of steroidal estrogens and estrogenic potency in Ontario WWTP effluents and surface waters, and has uncovered temporal patterns of release that may be used to help predict risks to aquatic organisms in these environments. (C) 2012 Elsevier B.V. All rights reserved. DOI
12.Marlatt, VL; Gerrie, E; Wiens, S; Jackson, F; Moon, TW; Trudeau, VL. (2012) Estradiol and triiodothyronine differentially modulate reproductive and thyroidal genes in male goldfish.Fish Physiology and Biochemistry 38: 283-296 Estradiol and triiodothyronine differentially modulate reproductive and thyroidal genes in male goldfish
Thyroid hormone; Estrogen; Hormone receptors; Deiodinase; Aromatase B
While the reproductive and thyroidal systems are extensively studied in fish, they are largely studied in isolation from one another, but there is evidence supporting cross-regulation between these two systems. To better understand hormone action and the potential cross-regulation between estrogen and thyroid hormones, we examined gene expression changes in estrogen receptor (ER) and thyroid receptor (TR) subtypes and key enzymes responsible for the local synthesis and availability of estrogen and thyroid hormones (aromatase B and deiodinase, respectively) in sexually regressed, adult, male goldfish in response to 3 days waterborne exposures to 17 beta-estradiol (E2; 1 nM), triiodothyronine (T3; 20 and 100 nM), and co-treatments thereof. Treatments with E2 alone did not effect ER subtype transcripts in the liver, telencephalon, or testis; however, in the testis, 1 nM T3 decreased ER alpha and ER beta 1 and co-treatments of T3 and E2 decreased ER beta 1 levels. TR alpha-1 and TR beta transcripts were not auto-regulated by T3 or cross-regulated by E2. Although deiodinase type I levels were also unaffected, deiodinase type II decreased in response to T3 treatments. Liver deiodinase type III transcripts increased in response to T3 treatments, while E2 exhibited antagonistic effects on this T3-mediated induction. These results provide novel evidence of cross-talk between the reproductive and thyroid endocrine axes in a model teleost. DOI
11. Martyniuk, CJ; Alvarez, S; Lo, BP; Elphick, JR; Marlatt, VL. (2012) Hepatic Protein Expression Networks Associated with Masculinization in the Female Fathead Minnow (Pimephales promelas).Journal of Proteome Research 11: 4147-4161 Hepatic Protein Expression Networks Associated with Masculinization in the Female Fathead Minnow (Pimephales promelas)
iTRAQ; subnetwork enrichment analysis; androgens; vitellogenin; ecotoxicoproteomics
Endocrine disruptors that act via the androgen receptor (AR) are less well studied than environmental estrogens, and there is evidence that treatment with AR agonists can result in masculinization of female fish. In this study, female fathead minnows (FHM) were exposed to the model nonaromatizable androgen S-alpha dihydrotestosterone (DHT) (100 mu g/L), the ureic-based herbicide linuron (UN) (100 mu g/L), and a mixture of DHT and LIN (100 mu g/L each) to better characterize androgen action in females. UN was used because of reports that this chemical has an antiandrogenic mode of action in fish. After 21d, DHT and LIN treatments resulted in a significant depression of plasma vitellogenin (Vtg) and DHT and DHT + LIN increased the prevalence of nuptial tubercles in female FHMs indicating masculinization. Using iTRAQ and an LTQ Orbitrap Velos, similar to 2000 proteins were identified in the FHM liver and the number of proteins quantified after exposures was >1200. Proteins that significantly and consistently changed in abundance across biological replicates included prostaglandin E synthase 3, programmed cell death 4a, glutathione S transferases, canopy, selenoprotein U, and ribosomal proteins. Subnetwork enrichment analysis identified that interferon and epidermal growth factor signaling were regulated by DHT and UN, suggesting that these signaling pathways are correlated to depressed plasma vitellogenin. These data provide novel insight into hepatic protein networks that are associated with the process of masculinization in teleosts. DOI
10. Atkinson, SK; Marlatt, VL; Kimpe, LE; Lean, DRS; Trudeau, VL; Blais, JM. (2011) Environmental Factors Affecting Ultraviolet Photodegradation Rates and Estrogenicity of Estrone and Ethinylestradiol in Natural Waters.Archives of Environmental Contamination and Toxicology 60: 1-7 Environmental Factors Affecting Ultraviolet Photodegradation Rates and Estrogenicity of Estrone and Ethinylestradiol in Natural Waters
The environmental fate and persistence of steroidal estrogens is influenced by their photodegradation. This can potentially occur both in the presence of the ultraviolet (UV) portion of solar radiation and in tertiary wastewater treatment plants that use UV radiation for disinfection purposes. To determine patterns of UV photodegradation for estrone (E1) and 17 alpha-ethinylestradiol (EE2), water samples containing these compounds were exposed to levels of UVB radiation that would simulate exposure to ambient sunlight. E1 degraded with a pseudo-first-order rate law constant that was directly proportional to UVB radiation intensity (R(2) = 0.999, P < 0.001) and inversely proportional to dissolved organic carbon (DOC) concentration (R(2) = 0.812, P = 0.037). DOC acted as a competitive inhibitor to direct photolysis of E1 by UV. In contrast to E1, EE2 was more persistent under similar UVB treatment. A reporter gene assay showed that the estrogenicity of UVB-exposed estrogens did not decrease relative to non-UVB-exposed estrogens, suggesting that some of the photoproducts may also have estrogenic potency. These results show that environmental degradation rates of steroidal estrogens are predictable from the UV intensity reaching surface waters, and the DOC concentrations in these surface waters. DOI
8.Marlatt, VL; Lakoff, J; Crump, K; Martyniuk, CJ; Watt, J; Jewell, L; Atkinson, S; Blais, JM; Sherry, J; Moon, TW; Trudeau, VL. (2010) Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish.Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology 156: 92-101 Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish
Aromatase; Estrogen receptors; Fish; Gene expression; Vitellogenin
This research examined the gene expression profile of three goldfish estrogen receptor (ER) subtypes in multiple tissues in relation to mRNA levels of aromatase B and vitellogenin (VTG) following waterborne estrogen exposures. The protocol consisted of: i) adult male goldfish in late gonadal recrudescence exposed to 1 nM 17 beta-estradiol (E2); ii) adult male and female goldfish in early sexual regression exposed to 1 nM E2 for 3, 6, 12 and 24 h; and, iii) sexually mature, adult male goldfish exposed to 0.3 nM 17 alpha-ethynylestradiol (EE2) for 24 h. Liver produced the most consistent response with up-regulation of ER alpha in sexually regressed, mature and recrudescing males and in sexually regressed females. The dose and length of exposure, reproductive state and sex affected the auto-regulation of ER beta 1 by E2. ER beta 2 was not affected in any experiments suggesting it may not be auto-regulated by E2. Aromatase B and VTG gene expression were affected by E2, but also by other experimental conditions. EE2 induced liver ER alpha and VTG mRNA levels indicating that high environmental EE2 levels induce E2-mediated gene expression in a model teleost. These studies reveal a more complicated action of estrogenic compounds that has important implications on estrogenic endocrine disruptors in teleosts. (C) 2010 Elsevier Inc. All rights reserved. DOI
7. Simmons, DBD; Marlatt, VL; Trudeau, VL; Sherry, JP; Metcalfe, CD. (2010) Interaction of Galaxolide (R) with the human and trout estrogen receptor-alpha.Science of the Total Environment 408: 6158-6164 Interaction of Galaxolide (R) with the human and trout estrogen receptor-alpha
Estrogen receptor; Polycyclic musks; HHCB; DEP; Galaxolide
Synthetic musks have been detected in sewage effluents surface waters and fish tissues where the polycyclic musk compound HHCB (Galaxolide (R)) is the dominant compound in those matrices In the present study the Galaxolide (R) formulation was tested in the yeast estrogenicity screening (YES) assay and also tested in in vitro and in vivo teleost systems to determine whether it interacts with the estrogen receptor as either an agonist or antagonist. In those tests Galaxolide (R) did not act as an estrogen agonist, however there was strong evidence of antagonistic activity as Galaxolide (R) inhibited the estrogenic activity of 17 beta-estradiol (E2) In the YES assay based on a recombinant strain of yeast containing the human estrogen receptor (i e hER alpha), Galaxolide (R) inhibited the effects of E2 in a dose-dependent manner (IC50 =1 63 x 10(-5) M) In a luciferase reporter gene assay based on the rainbow trout estrogen receptor e rtER) transfected into a rainbow trout gonadal (RTG-2) cell line the IC50 for the antagonistic effect of Galaxolide (R) was 2 79 x 10-9 M In an in vivo assay based on modulation of vitellogenin in rainbow trout Galaxolide (R) i p injected into trout at a dose of 3 64 mg/kg caused inhibition of E2-induced vitellogenin production That dose is within the range of concentrations of Galaxolide (R) that have been detected in tissues of fish from contaminated locations (C) 2010 Elsevier B V All rights reserved DOI
6. Zhang, DP; Xiong, HL; Mennigen, JA; Popesku, JT; Marlatt, VL; Martyniuk, CJ; Crump, K; Cossins, AR; Xia, X; Trudeau, VL. (2009) Defining Global Neuroendocrine Gene Expression Patterns Associated with Reproductive Seasonality in Fish.PLOS One 4 Defining Global Neuroendocrine Gene Expression Patterns Associated with Reproductive Seasonality in Fish
Background: Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. Methodology/Principal Findings: In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-day-length photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABA(A) gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays. Conclusions/Significance: Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development. DOI
5.Marlatt, VL; Martyniuk, CJ; Zhang, D; Xiong, H; Watt, J; Xia, X; Moon, T; Trudeau, VL. (2008) Auto-regulation of estrogen receptor subtypes and gene expression profiling of 17 beta-estradiol action in the neuroendocrine axis of male goldfish.Molecular and Cellular Endocrinology 283: 38-48 Auto-regulation of estrogen receptor subtypes and gene expression profiling of 17 beta-estradiol action in the neuroendocrine axis of male goldfish
estrogen receptors; fish; aromatase; gene expression
Auto-regulation of the three goldfish estrogen receptor (ER) subtypes was examined simultaneously in multiple tissues, in relation to mRNA levels of liver vitellogenin (VTG) and brain transcripts. Male goldfish were implanted with a silastic implant containing either no steroid or 17 beta-estradiol (E2) (100 mu g/g body mass) for one and seven days. Liver transcript levels of ER alpha were the most highly up-regulated of the ERs, and a parallel induction of liver VTG was observed. In the testes (7 d) and telencephalon (7 d), E2 induced ER alpha. In the liver (1 d) and hypothalamus (7 d) ER beta 1 was down-regulated, while ER beta 2 remained unchanged under all conditions. Although aromatase B levels increased in the brain, the majority of candidate genes identified by microarray in the hypothalamus (1 d) decreased. These results demonstrate that ER subtypes are differentially regulated by E2, and several brain transcripts decrease upon short-term elevation of circulating E2 levels. (C) 2007 Elsevier Ireland Ltd. All rights reserved. DOI
4. Simmons, DBD; Trudeau, VL; Marlatt, VL; Moon, TW; Sherry, JP; Metcalfe, CD. (2008) Interaction of stilbene compounds with human and rainbow trout estrogen receptors.Environmental Toxicology and Chemistry 27: 442-451 Interaction of stilbene compounds with human and rainbow trout estrogen receptors
estrogen receptor; stilbene; yeast estrogen screen; personal care products; pharmaceuticals
Compounds with stilbene structures are widely used as pharmaceuticals and personal care products (PPCPs) and are present in plants. A suite of stilbene-related compounds, including PPCPs and plant-derived compounds were tested in vitro for interactions with the human and rainbow trout estrogen receptors and in vivo with rainbow trout using vitellogenin levels as a biomarker. Among the compounds with antagonistic activity, the common structural similarity was (in addition to the stilbene backbone) the presence of 4-hydroxy substitution. Stilbene-related compounds found to act as inhibitors at the estrogen receptor included the plant-derived compound resveratrol and two formulations of fluorescent whitening agents used in detergents, 4,4'-bis(2-sulfostyryl)biphenyl and diaminostilbene-1. In the yeast estrogenicity screening assay, the concentrations which caused a 50% inhibition in estrogenic response (IC50s) with the human estrogen receptor ranged from 2.56 x 10(-6) to 2.56 x 10(-6) M. In the rainbow trout estrogen receptor assay, the IC50s ranged from 7.75 X 10(-1) to 1.11 X 10(-5) M. However, in the in vivo rainbow trout vitellogenin assay, tamoxifen was the only stilbene of the compounds tested to have a significant effect as an inhibitor of estrogenicity. DOI
3.Marlatt, VL; Hewitt, LM; Van der Kraak, G. (2006) Utility of in vitro test methods to assess the activity of xenoestrogens in fish.Environmental Toxicology and Chemistry 25: 3204-3212 Utility of in vitro test methods to assess the activity of xenoestrogens in fish
estrogen receptors; vitellogenin induction; fish reproduction; pulp mill effluent; endocrine disruptors
The results of the present study have demonstrated the utility of an estrogen receptor (ER) competitive ligand-binding assay, a hepatocyte vitellogenin (VTG) induction bioassay, and an ER reporter gene bioassay in characterizing the activity of model estrogen agonists (17 beta-estradiol [E(2)], ethynylestradiol, and nonylphenol) and antagonists (tamoxifen and ZM 189154) in rainbow trout (Oncorhynchus mykiss). The in vitro results were validated against in vivo trout waterborne exposures to E(2) and tamoxifen. The results showed that all three in vitro assays were capable of detecting the hormonal activities of the selected model compounds in a dose-dependent manner, with the exception of nonylphenol in the ER reporter gene bioassay. However, the relative potency rankings of the model compounds were not consistent between these assays, which suggests that the relative potencies obtained within assays may have limited predictive value between assays. Discrepancies in potencies most likely can be attributed to the different levels of cellular organization in each assay. In addition to model compounds, we also evaluated the responses of the ER-binding assay and the hepatocyte VTG induction bioassay to complex mixtures associated with endocrine effects in fish, specifically extracts of pulp mill effluent. Of the 14 pulp mill effluent extracts tested, only six showed activity in both assays, whereas the remaining eight samples showed activity in only one of the two assays. The hepatocyte VTG induction bioassay consistently showed that the pulp mill effluent extracts were antiestrogenic, which to our knowledge has not been reported in previous studies. Collectively, these results suggest that a combination of in vitro assays that depend on differing endpoints is required to identify potential xenoestrogens and to characterize their modes of action. DOI
