Toxicogenomics of Five Cytostatics in Fathead Minnow (Pimephales promelas) Larvae.

In this study, the toxicogenomic effects of five cytostatics (tamoxifen, methotrexate, capecitabine, cyclophosphamide, and ifosfamide) on fathead minnow (Pimephales promelas) larvae were evaluated. Post-fertilization eggs were exposed to increasing concentrations of the drugs for six days. The expression levels of two genetic biomarkers for toxicity and four thyroid hormone-related gene pathways were measured. Interestingly, the results showed that all concentrations of the five cytostatics affect the transcription levels of both toxicity biomarker genes. Additionally, the thyroid hormone-related genes had different expression levels than the control, with the most significant changes observed in those larvae exposed to cyclophosphamide and ifosfamide. While a previous study found no effects on fish morphology, this study suggests that the five cytostatics modify subtle molecular responses of P. promelas, highlighting the importance of assessing multibiological level endpoints throughout the lifecycle of animals to understand the full portrait of potential effects of cytostatics and other contaminants.

An exploratory spatial contaminant assessment for polar bear (Ursus maritimus) liver, fat, and muscle from northern Canada.

Since the industrial era, chemicals have been ubiquitous in worldwide ecosystems. Despite the discontinued release of highly toxic persistent organic pollutants (POPs) in the environment, the levels of some POPs are still being measured in the Canadian Arctic. These contaminants are of great concern due to their persistence, toxicity, and levels of bioaccumulation in food chains. Animals occupying top trophic positions in the Canadian Arctic, particularly polar bears, are exposed to these contaminants mainly through their diet. Our study investigated the levels of 30 metals (including total and methyl mercury) alkaline and alkaline earth metals, 15 polycyclic aromatic compounds and their alkyl congeners (PACs), 6 chlordanes (CHLs), and 20 polychlorinated biphenyls (PCBs), in 49 polar bears from the Canadian Arctic. Contaminant burden was measured in liver, muscle, and fat in bears of different sex, age, and locations. A principal component analysis did not distinguish differences between age and sex profiles for most contaminants. However, the concentrations measured and their distribution in the tissues confirm findings observed in past studies. This study highlights the importance of continual monitoring of polar bear health (e.g., newly detected PACs were measured within this study) and evaluating those impacts for the next generations of polar bears.

Innovation in regulatory approaches for endocrine disrupting chemicals: The journey to risk assessment modernization in Canada.

Globally, regulatory authorities grapple with the challenge of assessing the hazards and risks to human and ecosystem health that may result from exposure to chemicals that disrupt the normal functioning of endocrine systems. Rapidly increasing number of chemicals in commerce, coupled with the reliance on traditional, costly animal experiments for hazard characterization - often with limited sensitivity to many important mechanisms of endocrine disruption -, presents ongoing challenges for chemical regulation. The consequence is a limited number of chemicals for which there is sufficient data to assess if there is endocrine toxicity and hence few chemicals with thorough hazard characterization. To address this challenge, regulatory assessment of endocrine disrupting chemicals (EDCs) is benefiting from a revolution in toxicology that focuses on New Approach Methodologies (NAMs) to more rapidly identify, prioritize, and assess the potential risks from exposure to chemicals using novel, more efficient, and more mechanistically driven methodologies and tools. Incorporated into Integrated Approaches to Testing and Assessment (IATA) and guided by conceptual frameworks such as Adverse Outcome Pathways (AOPs), emerging approaches focus initially on molecular interactions between the test chemical and potentially vulnerable biological systems instead of the need for animal toxicity data. These new toxicity testing methods can be complemented with in silico and computational toxicology approaches, including those that predict chemical kinetics. Coupled with exposure data, these will inform risk-based decision-making approaches. Canada is part of a global network collaborating on building confidence in the use of NAMs for regulatory assessment of EDCs. Herein, we review the current approaches to EDC regulation globally (mainly from the perspective of human health), and provide a perspective on how the advances for regulatory testing and assessment can be applied and discuss the promises and challenges faced in adopting these novel approaches to minimize risks due to EDC exposure in Canada, and our world.

Impacts of endocrine disrupting chemicals on reproduction in wildlife and humans.

Endocrine disrupting chemicals (EDCs) are ubiquitous in aquatic and terrestrial environments. The main objective of this review was to summarize the current knowledge of the impacts of EDCs on reproductive success in wildlife and humans. The examples selected often include a retrospective assessment of the knowledge of reproductive impacts over time to discern how the effects of EDCs have changed over the last several decades. Collectively, the evidence summarized here within reinforce the concept that reproduction in wildlife and humans is negatively impacted by anthropogenic chemicals, with several altering endocrine system function. These observations of chemicals interfering with different aspects of the reproductive endocrine axis are particularly pronounced for aquatic species and are often corroborated by laboratory-based experiments (i.e. fish, amphibians, birds). Noteworthy, many of these same indicators are also observed in epidemiological studies in mammalian wildlife and humans. Given the vast array of reproductive strategies used by animals, it is perhaps not surprising that no single disrupted target is predictive of reproductive effects. Nevertheless, there are some general features of the endocrine control of reproduction, and in particular, the critical role that steroid hormones play in these processes that confer a high degree of susceptibility to environmental chemicals. New research is needed on the implications of chemical exposures during development and the potential for long-term reproductive effects. Future emphasis on field-based observations that can form the basis of more deliberate, extensive, and long-term population level studies to monitor contaminant effects, including adverse effects on the endocrine system, are key to addressing these knowledge gaps.

Embryotoxicity of Five Cytostatics in Fathead Minnow (Pimephales promelas) Larvae.

Cytostatics are compounds used in chemotherapy, known to be genotoxic, mutagenic, and teratogenic at low concentrations. The amount of cytostatic drugs prescribed increases every year as does their release into the aquatic ecosystems, which possibly is a major concern for the health of aquatic organisms. This study aimed to evaluate the putative toxicity of five cytostatics to fathead minnow (Pimephales promelas) larvae: tamoxifen, capecitabine, methotrexate, cyclophosphamide, and ifosfamide. Eggs collected post-fertilization were exposed for 6 days to a range of concentrations, including one above environmental level. At all environmental concentrations, no significant difference in mortality, hatching time, length, heart rate, and presence of malformations were found. Altogether, these cytostatics do not seem embryotoxic to fish. Although, an increased proportion of complete swim bladder were found after ifosfamide's exposure, suggesting an interaction with the thyroid axis, involved in swim bladder development. Complementary work should address other endpoints, such as behavioral changes, reproductive success, and transgenerational effects.

Polycyclic aromatic compounds (PACs) in the Canadian environment: The challenges of ecological risk assessments.

Ecological risk assessments (ERAs) of polycyclic aromatic compounds (PACs), as single congeners or in mixtures, present technical challenges that raise concerns about their accuracy and validity for Canadian environments. Of more than 100,000 possible PAC structures, the toxicity of fewer than 1% have been tested as individual compounds, limiting the assessment of complex mixtures. Because of the diversity in modes of PAC action, the additivity of mixtures cannot be assumed, and mixture compositions change rapidly with weathering. In vertebrates, PACs are rapidly oxygenated by cytochrome P450 enzymes, often to metabolites that are more toxic than the parent compound. The ability to predict the ecological fate, distribution and effects of PACs is limited by toxicity data derived from tests of a few responses with a limited array of test species, under optimal laboratory conditions. Although several models are available to predict PAC toxicity and rank species sensitivity, they were developed with data biased by test methods, and the reported toxicities of many PACs exceed their solubility limits. As a result, Canadian Environmental Quality Guidelines for a few individual PACs provide little support for ERAs of complex mixtures in emissions and at contaminated sites. These issues are illustrated by reviews of three case studies of PAC-contaminated sites relevant to Canadian ecosystems. Interactions among ecosystem characteristics, the behaviour, fate and distribution of PACs, and non-chemical stresses on PAC-exposed species prevented clear associations between cause and effect. The uncertainties of ERAs can only be reduced by estimating the toxicity of a wider array of PACs to species typical of Canada's diverse geography and environmental conditions. Improvements are needed to models that predict toxicity, and more field studies of contaminated sites in Canada are needed to understand the ecological effects of PAC mixtures.

Polycyclic aromatic compounds (PACs) in the Canadian environment: Exposure and effects on wildlife.

Polycyclic aromatic compounds (PACs) are ubiquitous in the environment. Wildlife (including fish) are chronically exposed to PACs through air, water, sediment, soil, and/or dietary routes. Exposures are highest near industrial or urban sites, such as aluminum smelters and oil sands mines, or near natural sources such as forest fires. This review assesses the exposure and toxicity of PACs to wildlife, with a focus on the Canadian environment. Most published field studies measured PAC concentrations in tissues of invertebrates, fish, and birds, with fewer studies of amphibians and mammals. In general, PAC concentrations measured in Canadian wildlife tissues were under the benzo[a]pyrene (BaP) guideline for human consumption. Health effects of PAC exposure include embryotoxicity, deformities, cardiotoxicity, DNA damage, changes to DNA methylation, oxidative stress, endocrine disruption, and impaired reproduction. Much of the toxicity of PACs can be attributed to their bioavailability, and the extent to which certain PACs are transformed into more toxic metabolites by cytochrome P450 enzymes. As most mechanistic studies are limited to individual polycyclic aromatic hydrocarbons (PAHs), particularly BaP, research on other PACs and PAC-containing complex mixtures is required to understand the environmental significance of PAC exposure and toxicity. Additional work on responses to PACs in amphibians, reptiles, and semi-aquatic mammals, and development of molecular markers for early detection of biological responses to PACs would provide a stronger biological and ecological justification for regulating PAC emissions to protect Canadian wildlife.

Sub-lethal effects of calcium dinonylnaphthalenesulfonate on Western clawed frog embryos.

Naphthalene sulfonic acids (NSAs) are used as additives in lubricants, dyes, and greases and commonly act as surfactants in many industrial processes. The calcium salt of dinonyl NSA (calcium dinonylnaphthalenesulfonate; CaDNS) is listed among thousands of chemicals identified as priorities for assessment by the Government of Canada's Chemical Management Plan due to the limited toxicity data. The purpose of this study was two-fold: 1) to establish the toxicity of CaDNS to Western clawed frog (Silurana tropicalis) embryos and 2) to assess the sub-lethal effects and mechanisms of toxicity of CaDNS in amphibians through targeted gene expression and metabolite analyses. Frog embryos were exposed to water overlying sand spiked with a range of concentrations of CaDNS (17-1393 µg/g) over a 72-h period. Results indicated significantly higher mortality and presence of malformations in frog larvae exposed to over 672 µg/g CaDNS in the sand (14 ng/mL CaDNS in the water) compared to control treatments. An overall decrease in the glutathione redox cycle was observed, including decreases in relative mRNA levels of enzymes (glutathione S-transferase (gst), glutathione reductase (gsr), glutathione peroxidase (gpx)) and decreases in the glutathione (GSH) and glutathione disulfide (GSSG) metabolite concentrations. In addition, transcript levels of genes involved in antioxidant capacity and essential amino acid metabolites decreased significantly in embryos exposed to low levels of CaDNS. This is the first study to assess the toxicity of NSAs in amphibians, contributing important data to aid in the assessment of NSAs.

Morphological and molecular effects of two diluted bitumens on developing fathead minnow (Pimephales promelas).

Canada has experienced a significant increase in the transport of diluted bitumen (dilbit), a predominant oil sands product that combines bitumen with diluents derived from oil-gas condensates and other proprietary compounds. The toxicity of dilbit to fish embryos, which are immobile and thus at a high risk of exposure to oil in the event of a spill, remains largely unknown for most species. This study assessed the toxicity of water accommodated fractions (WAF) and chemically enhanced water accommodated fractions (CEWAF) of two winter dilbit blends, Access Western Blend (AWB) and Cold Lake Blend (CLB), to fathead minnow (Pimephales promelas) embryos. The TPH-F EC50s for malformations were 834 and 1058 µg/L for AWB WAF and CEWAF, respectively, and 500 and 715 µg/L for CLB WAF and CEWAF, respectively. Levels of cyp1a mRNA increased up to 46- and 69-fold, respectively, reflecting increasing exposure to polycyclic aromatic compounds (PACs) in AWB and CLB. Similarly, levels of gst mRNA were elevated up to 3.8-fold and 2.7-fold with increasing total concentrations of PACs in AWB and CLB, respectively. However, there were no significant changes in mRNA levels of p53, sod, cat, and gsr. These results suggest that the expression of cyp1a and gst may serve as biomarkers for dilbit exposure in fathead minnow, furthering our understanding of dilbit-responsive indicators of toxicity in fish species native to North America. This study is important as it utilizes the same exposure methodology to examine the toxicity of two commonly used Canadian dilbits, facilitating comparison of dilbit toxicity.

Thyroid hormones and androgens differentially regulate gene expression in testes and ovaries of sexually mature Silurana tropicalis.

A series of ex vivo exposures using testicular and ovarian tissues of sexually mature Western clawed frogs (Silurana tropicalis) were designed to examine molecular mechanisms of thyroid hormone (TH) and androgen crosstalk sans hypophyseal feedback as well as investigate potential sex-specific differences. Tissues were exposed ex vivo to either triiodothyronine (T3), iopanoic acid (IOP), one co-treatment of IOP + 5α-dihydrotestosterone (5α-DHT), 5α-DHT, 5ß-dihydrotestosterone (5ß-DHT), or testosterone (T). Direct exposure to different androgens led to androgen specific increases in thyroid receptor and deiodinase transcripts in testes (trß and dio1) but a decrease in expression in ovaries (trß and dio3), suggesting that male and female frogs can be differently affected by androgenic compounds. Moreover, exposure to select androgens differentially increased estrogen-related transcription (estrogen receptor alpha (erα) and aromatase (cyp19)) and production (estradiol) in ovaries and testes indicating the activation of alternate metabolic pathways yielding estrogenic metabolites. Sex-steroid-related transcription (i.e., steroid 5α-reductase type 2 (srd5α2) and erα) and production (i.e., 5α-DHT) were also differentially regulated by THs. The presence and frequency of transcription factor binding sites in the putative promoter regions of TH- and sex steroid-related genes were also examined in S. tropicalis, rodent, and fish models using in silico analysis. In summary, this study provides an improved mechanistic understanding of TH- and androgen-mediated actions and reveals differential transcriptional effects as a function of sex in frogs.

Airborne polycyclic aromatic compounds contribute to the induction of the tumour-suppressing P53 pathway in wild double-crested cormorants.

Polycyclic aromatic compounds (PACs), including polycyclic aromatic hydrocarbons (PAHs) and PAH-like compounds are known or probable environmental carcinogens released into the environment as a by-product of incomplete combustion of fossil fuels and other organic materials. Studies have shown that exposure to PACs in the environment can induce both genotoxicity and epigenetic toxicity, but few studies have related PAC exposure to molecular changes in free ranging wildlife. Previous work has suggested that double-crested cormorants (Phalacrocorax auritus; DCCO) exhibited a higher incidence of genetic mutations when their breeding sites were located in heavily industrialized areas (e.g., Hamilton Harbour, Hamilton, ON, Canada) as compared to sites located in more pristine environments, such as in Lake Erie. The aim of this study was to determine if airborne PACs from Hamilton Harbour alter the tumour-suppressing P53 pathway and/or global DNA methylation in DCCOs. Airborne PACs were measured using passive air samplers in the Hamilton Harbour area and low-resolution mass spectrometry analysis detected PACs in livers of DCCOs living in Hamilton Harbour. Further hepatic and lung transcriptional analysis demonstrated that the expression of the genes involved in the DNA repair and cellular apoptosis pathway were up-regulated in both tissues of DCCOs exposed to PACs, while genes involved in p53 regulation were down-regulated. However, global methylation levels did not differ between reference- and PAC-exposed DCCOs. Altogether, data suggest that PACs activate the P53 pathway in free-ranging DCCOs living nearby PAC-contaminated areas.

Diluted bitumen causes deformities and molecular responses indicative of oxidative stress in Japanese medaka embryos.

This study characterized the toxicity and physiological effects of unweathered diluted bitumen (Access Western Blend dilbit; AWB) to fish. Embryos of Japanese medaka (Oryzias latipes) were exposed for 17 days to dilutions of physically-dispersed (water accommodated fraction; WAF) and chemically-dispersed (chemically-enhanced WAF; CEWAF) dilbit. AWB dilbit exposure was not lethal to medaka, but resulted in a high prevalence of blue sac disease (BSD), impaired development, and abnormal or un-inflated swim bladders at hatch. Physiological effects were indicated by the relative mRNA levels of key genes associated with, among others, cell cycling and the response to mutations (p53), xenobiotic metabolism (ahr, arnt2), phase I (cyp1a) and II processes associated with oxidative stress (cat, g6pdh, hsp70, gst, gpx, gsr, nfe2, and sod). AWB dilbit treatment increased p53 and cyp1a transcript levels (1.5-fold and >15-fold, respectively), with significant, but less pronounced changes in indicators of oxidative stress and metabolism. The exposure-related changes in embryotoxicity and mRNA synthesis were consistent with metabolism of polycyclic aromatic hydrocarbons (PAHs) to reactive and toxic metabolites. Medaka embryos responded similarly to WAF and CEWAF treatments, but CEWAF was about 100 times more efficient in delivering toxic concentrations of PAHs. The toxicity of chemically-dispersed nujol, a non-toxic mineral oil used as an experimental control, suggested that a portion of the observed effects of AWB could be attributed to excess dispersant in solution. This first study of the physiological effects of dilbit toxicity to fish embryos provides a baseline to compare toxicity between dilbit and conventional crude oils, and the groundwork for the development of molecular biomarkers of the sensitivity and level of risk of native Canadian fish species to dilbit exposure.

Expression profiles of reproduction- and thyroid hormone-related transcripts in the brains of chemically-induced intersex frogs.

Endocrine disrupting chemicals can induce intersex animals in amphibians and fish. Our previous study in frogs demonstrated that chemically-induced intersex animals can display different hepatic profiles of transcript levels than normal animals. In this study, we extend the observations to the developing frog brain. We investigated the effects of finasteride and fadrozole known to induce female- and male-biased sexual development on Silurana tropicalis brain mRNA levels. Real-time RT-PCR analysis of transcript levels of sex steroid- and thyroid hormone-related genes in the brain demonstrated that in finasteride-induced intersex animals, the mRNA levels of aromatase, estrogen receptor α, thyroid hormone receptor ß and deiodinase type 3 were higher compared to both control males and females. Furthermore, finasteride-induced intersex animals expressed higher mRNA levels of both androgen receptor and estrogen receptor ß compared to control females and to control males, respectively. Furthermore, fadrozole did not affect any of the genes analyzed in the brain but was effective at reducing aromatase activity. Intersex animals display different profiles of transcript levels in the brain whether the intersex condition was induced by an anti-androgen or anti-estrogen treatment. Finally, we conclude that a complex relationship exists between thyroid hormone-responsive genes and androgen status in frogs.

The aromatase inhibitor fadrozole and the 5-reductase inhibitor finasteride affect gonadal differentiation and gene expression in the frog Silurana tropicalis.

Aromatase (cyp19) and the 5alpha- and 5beta-reductases (srd5alpha and srd5beta) are important enzymes for vertebrate sexual development. We investigated the effects of inhibition of cyp19 by fadrozole (FAD), and srd5alpha and srd5beta by finasteride (FIN) during anuran larval development. Chronic exposures of Silurana (Xenopus) tropicalis from Nieuwkoop-Faber stage 12 until stage 60 were performed using either 2 microM FAD or 25 microM FIN. Histological analysis of exposed metamorphic frogs revealed that both treatments induced intersex individuals (presence of testicular oocytes). FAD treatment resulted in 55% male, 30% female and 15% intersex, while FIN treatment produced 27% male, 53% female and 20% intersex. Real-time RT-PCR analysis of hepatic sex steroid- and thyroid hormone-related gene expression demonstrated that FAD-induced intersex animals had higher srd5alpha1, srd5alpha2 and eralpha mRNA levels than control and FAD males. In contrast, FIN-induced intersex had low srd5alpha1, srd5alpha2, srd5beta and dio3 and high dio2 mRNA levels. FIN-treated males exhibited high trbeta, dio2 and a lower dio3 mRNA levels. We conclude that chemically induced intersex animals display different gene expression profiles than non-exposed animals and that, although morphologically similar, intersex animals produced by different chemicals have different endocrine pathophysiologies.

Investigation of pathogenic Escherichia coli and microbial pathogens in pulp and paper mill biosolids.

Biosolids produced from pulp and paper mill wastewater treatment have excellent properties as soil conditioners, but often contain high levels of Escherichia coli. E. coli are commonly used as indicators of fecal contamination and health hazard; therefore, their presence in biosolids causes concern and has lead to restrictions in land-spreading. The objectives of this study were to determine the following: (1) if E. coli from the biosolids of a wastewater-free pulp and paper mill were enteric pathogens, and (2) if other waterborne microbial pathogens were present. E. coli were screened for heat-labile and heat-stable enterotoxin and verocytotoxin virulence genes using a polymerase chain reaction. Ten isolates were also screened for invasion-associated locus and invasion plasmid antigen H genes. None of the 120 isolates carried these genes. Tests for seven other microbial pathogens were negative. Effluents and biosolids from this mill do not contain common microbial pathogens and are unlikely to pose a health hazard.