Ultrafast functional profiling of RNA-seq data for nonmodel organisms.

Computational time and cost remain a major bottleneck for RNA-seq data analysis of nonmodel organisms without reference genomes. To address this challenge, we have developed Seq2Fun, a novel, all-in-one, ultrafast tool to directly perform functional quantification of RNA-seq reads without transcriptome de novo assembly. The pipeline starts with raw read quality control: sequencing error correction, removing poly(A) tails, and joining overlapped paired-end reads. It then conducts a DNA-to-protein search by translating each read into all possible amino acid fragments and subsequently identifies possible homologous sequences in a well-curated protein database. Finally, the pipeline generates several informative outputs including gene abundance tables, pathway and species hit tables, an HTML report to visualize the results, and an output of clean reads annotated with mapped genes ready for downstream analysis. Seq2Fun does not have any intermediate steps of file writing and loading, making I/O very efficient. Seq2Fun is written in C++ and can run on a personal computer with a limited number of CPUs and memory. It can process >2,000,000 reads/min and is >120 times faster than conventional workflows based on de novo assembly, while maintaining high accuracy in our various test data sets.

Characterizing Variability and Uncertainty Associated with Transcriptomic Dose-Response Modeling.

Transcriptomics dose-response analysis (TDRA) has emerged as a promising approach for integrating toxicogenomics data into a risk assessment context; however, variability and uncertainty associated with experimental design are not well understood. Here, we evaluated n = 55 RNA-seq profiles derived from Japanese quail liver tissue following exposure to chlorpyrifos (0, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, and 40 µg/g; n = 5 replicates per group) via egg injection. The full dataset was subsampled 637 times to generate smaller datasets with different dose ranges and spacing (designs A-E) and number of replicates (n = 2-5). TDRA of the 637 datasets revealed substantial variability in the gene and pathway benchmark doses, but relative stability in overall transcriptomic point-of-departure (tPOD) values when tPODs were calculated with the "pathway" and "mode" methods. Further, we found that tPOD values were more dependent on the dose range and spacing than on the number of replicates, suggesting that optimal experimental designs should use fewer replicates (n = 2 or 3) and more dose groups to reduce uncertainty in the results. Finally, tPOD values ranged by over ten times for all surveyed experimental designs and tPOD types, suggesting that tPODs should be interpreted as order-of-magnitude estimates.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Parental Whole Life Cycle Exposure to Dietary Methylmercury in Zebrafish (Danio rerio) Affects the Behavior of Offspring.

Methylmercury (MeHg) is an established neurotoxicant of concern to fish-eating organisms. While most studies have focused on the fish consumers, much less is known about the effects of MeHg on the fish themselves, especially following exposures to chronic and environmentally relevant scenarios. Here we evaluated the behavioral effects of developmental MeHg insult by exposing parental generations of zebrafish to an environmentally realistic MeHg dietary concentration (1 ppm) and two higher concentrations (3 and 10 ppm) throughout their whole life span. Upon reaching adulthood, their offspring were analyzed through a series of behavioral tests, including the visual-motor response (VMR) assay, analysis of spontaneous swimming and evaluation of foraging efficiency. The VMR assay identified decreased locomotor output in the 6 day postfertilization (dpf) offspring of fish exposed to 3 and 10 ppm MeHg. However, in a second test 7 dpf fish revealed an increase in locomotor activity in all MeHg exposures tested. Increases in locomotion continued to be observed until 16 dpf, which coincided with increased foraging efficiency. These results suggest an association between MeHg and hyperactivity, and imply that fish chronically exposed to MeHg in the wild may be vulnerable to predation.

Potency of polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity in hepatocyte cultures from chicken, Pekin duck, and greater scaup.

The potency of tetrachlorodibenzo-p-dioxin (TCDD) and 18 polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity was assessed in primary hepatocyte cultures prepared from chicken (Gallus domesticus), Pekin duck (Anas platyrhynchos domesticus), and greater scaup (Aythya marila). TCDD and 8 of the PAHs induced EROD activity in a concentration-dependent manner. Seven of these were previously shown to be acutely toxic to avian embryos, while the 10 congeners that did not produce an EROD response caused limited mortality. The rank order potency of the EROD-active congeners in all three species was as follows: TCDD>dibenz[ah]anthracene>benzo[k]fluoranthene>indeno[1,2,3-cd]pyrene>benzo[a]pyrene>chrysene≈benz[a]anthracene≈benz[ghi]perylene>benzo[b]naphtho[2,3-d]thiophene. Chicken hepatoctyes were more sensitive than duck hepatocytes to EROD induction by all test compounds, but the gap in species sensitivity was 100-fold for TCDD, and generally ≤10-fold for PAHs. This study is the first to use in vitro methods to rank the AHR-mediated potency of PAHs in birds. These data may be useful for assessing risks associated with exposure to PAHs in the environment.

Hepatic Transcriptomic Responses to Ethinylestradiol in Embryonic Japanese Quail and Double-Crested Cormorant.

Understanding species differences in sensitivity to toxicants is a critical issue in ecotoxicology. We recently established that double-crested cormorant (DCCO) embryos are more sensitive than Japanese quail (JQ) to the developmental effects of ethinylestradiol (EE2). We explored how this difference in sensitivity between species is reflected at a transcriptomic level. The EE2 was dissolved in dimethyl sulfoxide and injected into the air cell of eggs prior to incubation at nominal concentrations of 0, 3.33, and 33.3 µg/g egg weight. At midincubation (JQ 9 days; DCCO 16 days), livers were collected from five embryos/treatment group for RNA sequencing. Data were processed and analyzed using EcoOmicsAnalyst and ExpressAnalyst. The EE2 exposure dysregulated 238 and 1,987 genes in JQ and DCCO, respectively, with 78 genes in common between the two species. These included classic biomarkers of estrogen exposure such as vitellogenin and apovitellenin. We also report DCCO-specific dysregulation of Phase I/II enzyme-coding genes and species-specific transcriptional ontogeny of vitellogenin-2. Twelve Kyoto Encyclopedia of Genes and Genomes pathways and two EcoToxModules were dysregulated in common in both species including the peroxisome proliferator-activated receptor (PPAR) signaling pathway and fatty acid metabolism. Similar to previously reported differences at the organismal level, DCCO were more responsive to EE2 exposure than JQ at the gene expression level. Our description of differences in transcriptional responses to EE2 in early life stage birds may contribute to a better understanding of the molecular basis for species differences. Environ Toxicol Chem 2024;43:772-783. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cytotoxicity of 19 Pesticides in Rainbow Trout Gill, Liver, and Intestinal Cell Lines.

The rainbow trout gill cell line (RTgill-W1), via test guideline 249 of the Organisation for Economic Co-operation and Development, has been established as a promising New Approach Methodology, although to advance confidence in the method more case studies are needed that: 1) expand our understanding of applicability domains (chemicals with diverse properties); 2) increase methodological throughput (96-well format); and 3) demonstrate biological relevance (in vitro to in vivo comparisons; gill vs. other cells). Accordingly, the objective of our study was to characterize the cytotoxicity of 19 pesticides against RTgill-W1 cells, and also liver (RTL-W1) and gut epithelial (RTgutGC) cell lines, and then to compare the in vitro and in vivo data. Of the 19 pesticides tested, 11, 9, and 8 were cytotoxic to the RTgill-W1, RTL-W1, and RTgutGC cells, respectively. Six pesticides (carbaryl, chlorothalonil, chlorpyrifos, dimethenamid-P, metolachlor, and S-metolachlor) were cytotoxic to all three cell lines. Aminomethylphosphonic acid, chlorantraniliprole, dicamba, diquat, imazethapyr, and permethrin exhibited cell-line-specific toxicity. No cytotoxic responses were observed for three herbicides (atrazine, glyphosate, and metribuzin) and four insecticides (clothianidin, diazinon, imidacloprid, and thiamethoxam). When cytotoxicity was measured, there was a strong correlation (rs = 0.9, p < 0.0001) between in vitro median effect concentration (EC50) values (based on predicted concentrations using the In Vitro Mass Balance Model Equilibrium Partitioning (IV-MBM EQP) Ver. 2.1) derived from RTgill-W1 and RTL-W1 cells with in vivo median lethal concentration (LC50) values from 96-h acute toxicity studies with trout. In all 28 cases, the in vitro EC50 was within 18-fold of the in vivo LC50. These data help increase our understanding of the ecotoxicological domains of applicability for in vitro studies using cultured rainbow trout cells, while also demonstrating that these assays performed well in a 96-well format and have promise to yield data of biological relevance. Environ Toxicol Chem 2024;00:1-13. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Comparing Transcriptomic Responses to Chemicals Across Six Species Using the EcoToxChip RNASeq Database.

The EcoToxChip project includes RNA-sequencing data from experiments involving model (Japanese quail, fathead minnow, African clawed frog) and ecological (double-crested cormorant, rainbow trout, northern leopard frog) species at multiple life stages (whole embryo and adult) exposed to eight chemicals of environmental concern known to perturb a wide range of biological systems (ethinyl estradiol, hexabromocyclododecane, lead, selenomethionine, 17ß trenbolone, chlorpyrifos, fluoxetine, and benzo[a]pyrene). The objectives of this short communication were to (1) present and make available this RNA-sequencing database (i.e., 724 samples from 49 experiments) under the FAIR principles (FAIR data are data which meet principles of findability, accessibility, interoperability, and reusability), while also summarizing key meta-data attributes and (2) use ExpressAnalyst (including the Seq2Fun algorithm and EcoOmicsDB) to perform a comparative transcriptomics analysis of this database focusing on baseline and differential transcriptomic changes across species-life stage-chemical combinations. The database is available in NCBI GEO under accession number GSE239776. Across all species, the number of raw reads per sample ranged between 13 and 58 million, with 30% to 79% of clean reads mapped to the "vertebrate" subgroup database in EcoOmicsDB. Principal component analyses of the reads illustrated separation across the three taxonomic groups as well as some between tissue types. The most common differentially expressed gene was CYP1A1 followed by CTSE, FAM20CL, MYC, ST1S3, RIPK4, VTG1, and VIT2. The most common enriched pathways were metabolic pathways, biosynthesis of cofactors and biosynthesis of secondary metabolites, and chemical carcinogenesis, drug metabolism, and metabolism of xenobiotics by cytochrome P450. The RNA-sequencing database in the present study may be used by the research community for multiple purposes, including, for example, cross-species investigations, in-depth analyses of a particular test compound, and transcriptomic meta-analyses. Environ Toxicol Chem 2024;00:1-6. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

ExpressAnalyst: A unified platform for RNA-sequencing analysis in non-model species.

The increasing application of RNA sequencing to study non-model species demands easy-to-use and efficient bioinformatics tools to help researchers quickly uncover biological and functional insights. We developed ExpressAnalyst ( www.expressanalyst.ca ), a web-based platform for processing, analyzing, and interpreting RNA-sequencing data from any eukaryotic species. ExpressAnalyst contains a series of modules that cover from processing and annotation of FASTQ files to statistical and functional analysis of count tables or gene lists. All modules are integrated with EcoOmicsDB, an ortholog database that enables comprehensive analysis for species without a reference transcriptome. By coupling ultra-fast read mapping algorithms with high-resolution ortholog databases through a user-friendly web interface, ExpressAnalyst allows researchers to obtain global expression profiles and gene-level insights from raw RNA-sequencing reads within 24 h. Here, we present ExpressAnalyst and demonstrate its utility with a case study of RNA-sequencing data from multiple non-model salamander species, including two that do not have a reference transcriptome.

Development and Initial Testing of EcoToxChip, a Novel Toxicogenomics Tool for Environmental Management and Chemical Risk Assessment.

New approach methods (NAMs) are increasingly important to help accelerate the pace of ecological risk assessment and offer more ethical, affordable, and efficient alternatives to traditional toxicity tests. In the present study, we describe the development, technical characterization, and initial testing of a toxicogenomics tool, EcoToxChip (384-well quantitative polymerase chain reaction [qPCR] array), to support chemical management and environmental monitoring for three laboratory model species-fathead minnow (Pimephales promelas), African clawed frog (Xenopus laevis), and Japanese quail (Coturnix japonica). Chip design, including gene selection, was informed by a diverse end-user group and quality control metrics (e.g., primer assay, reverse transcription, and PCR efficiency) performed well based on a priori established criteria. Correlation with RNA sequencing (seq) data provided additional confidence in this novel toxicogenomics tool. Although the present study represents an initial testing of only 24 EcoToxChips for each of the model species, the results provide increased confidence in the robustness/reproducibility of EcoToxChips for evaluating perturbations in gene expression associated with chemical exposure and thus, this NAM, combined with early-life stage toxicity testing, could augment current efforts for chemical prioritization and environmental management. Environ Toxicol Chem 2023;42:1763-1771. © 2023 SETAC.

Absence of fractionation of mercury isotopes during trophic transfer of methylmercury to freshwater fish in captivity.

We performed two controlled experiments to determine the amount of mass-dependent and mass-independent fractionation (MDF and MIF) of methylmercury (MeHg) during trophic transfer into fish. In experiment 1, juvenile yellow perch (Perca flavescens) were raised in captivity on commercial food pellets and then their diet was either maintained on unamended food pellets (0.1 µg/g MeHg) or was switched to food pellets with 1.0 µg/g or 4.0 µg/g of added MeHg, for a period of 2 months. The difference in δ(202)Hg (MDF) and Δ(199)Hg (MIF) between fish tissues and food pellets with added MeHg was within the analytical uncertainty (δ(202)Hg, 0.07 ‰; Δ(199)Hg, 0.06 ‰), indicating no isotope fractionation. In experiment 2, lake trout (Salvelinus namaycush) were raised in captivity on food pellets and then shifted to a diet of bloater (Coregonus hoyi) for 6 months. The δ(202)Hg and Δ(199)Hg of the lake trout equaled the isotopic composition of the bloater after 6 months, reflecting reequilibration of the Hg isotopic composition of the fish to new food sources and a lack of isotope fractionation during trophic transfer. We suggest that the stable Hg isotope ratios in fish can be used to trace environmental sources of Hg in aquatic ecosystems.

Consideration of metabolomics and transcriptomics data in the context of using avian embryos for toxicity testing.

Early-life stage (ELS) avian toxicity tests have been proposed as a more ethical alternative to traditional standardized tests with adult birds. At the same time, 'omics approaches are gaining traction in the field of avian toxicology, but little has been done to characterize the metabolome and transcriptome at different life stages. The present study uses 'omics data from toxicity tests of 8 environmental chemicals in ELS and adult Japanese quail (Coturnix japonica) to address this data gap. Previous analyses of these data focused on responses to each of the individual chemicals. Here, we consider data from all studies to describe variation in the metabolome and transcriptome between life stages and across independent experiments, irrespective of chemical treatment. Of the 230 metabolites detected in liver, 163 were shared between the two life stages. However, many of the targeted bile acids that were present in the adult liver were absent from ELS samples. For the transcriptome, >90% of the 18,364 detected transcripts were common to both life stages. Based on the 213 genes solely detected in ELS liver, the neuroactive ligand-receptor interaction pathway was significantly enriched. Multivariate and hierarchical clustering analyses revealed that variability among independent experiments was higher for the adult than the ELS studies at both the metabolomic and transcriptomic levels. Our results indicate concordance of the two approaches, with less variation between independent experiments in the ELS metabolome and transcriptome than in adults, lending support for the use of ELS as an alternative toxicity testing strategy.

Hepatic Transcriptomic Responses to Ethinylestradiol in Two Life Stages of Japanese Quail.

Chemical risk assessment for avian species typically depends on information from toxicity tests performed in adult birds. Early-life stage (ELS) toxicity tests have been proposed as an alternative, but incorporation of these data into existing frameworks will require knowledge about the similarities/differences between ELS and adult responses. The present study uses transcriptomics to assess hepatic gene expression in ELS and adult Japanese quail following exposure to ethinylestradiol (EE2). Prior to incubation, ELS quail were dosed with measured EE2 concentrations of 0.54, 6.3, and 54.2 µg/g egg weight via air cell injection. Adult quail were fed a single dose of EE2 at nominal concentrations of 0, 0.5, and 5 mg/kg body weight by gavage. Liver tissue was collected from five to six individuals per dose group at mid-incubation for ELS quail and 4 days after dosing for adults. A total of 283 and 111 differentially expressed genes (DEGs) were detected in ELS and adult quail, respectively, 16 of which were shared across life stages. Shared DEGs included estrogenic biomarkers such as vitellogenin genes and apovitellenin-1. For the dose groups that resulted in the highest number of DEGs (ELS, 6.3 µg/g; adult, 5 mg/kg), 21 and 35 Kyoto Encyclopedia of Genes and Genomes pathways were enriched, respectively. Ten of these pathways were shared between life stages, including pathways involved with signaling molecules and interaction and the endocrine system. Taken together, our results suggest conserved mechanisms of action following estrogenic exposure across two life stages, with evidence from differential expression of key biomarker genes and enriched pathways. The present study contributes to the development and evaluation of ELS tests and toxicogenomic approaches and highlights their combined potential for screening estrogenic chemicals. Environ Toxicol Chem 2022;41:2769-2781. © 2022 SETAC.

Retrospective analysis of mercury content in feathers of birds collected from the state of Michigan (1895-2007).

Museum specimens were used to analyze temporal trends in feather mercury (Hg) concentrations in birds collected from the state of Michigan between the years 1895 and 2007. Hg was measured in flank and secondary feathers from three species of birds that breed in the Great Lakes region; common terns (n = 32), great blue herons (n = 35), and herring gulls (n = 35). More than 90% of the Hg in feathers should be organic, but some of the heron and gull feathers collected prior to 1936 showed evidence of contamination with inorganic Hg, likely from museum preservatives. The data presented here therefore consist of organic Hg in pre-1936 samples and total Hg in post-1936 samples. Insufficient tissue was available from terns to assess organic Hg content. Mean Hg concentrations ranged from 2.9 ± 2.5 µg/g Hg in tern flank feathers to 12.4 ± 10.6 µg/g Hg in gull flank feathers. No linear trend of Hg contamination over time was detected in herons and gulls. Though a significant decrease was noted for terns, these data are presented with caution given the strong likelihood that earlier samples were preserved with inorganic mercury. When data were separated into 30-year intervals, Hg content in heron and gull feathers collected from birds sampled between 1920 and 1949 were consistently highest but not to a level of statistical significance. For example, Hg concentrations in gull secondary feathers collected in the second time interval (1920-1949) were 11.5 ± 7.8. This value was 67% higher than the first time interval (1890-1919), 44% higher than the third interval (1950-1979), and 187% higher than the fourth interval (1980-2009). Studies on Great Lakes sediments also showed greatest Hg accumulations in the mid-twentieth century. Through the use of museum specimens, these results present a unique snapshot of Hg concentrations in Great Lakes biota in the early part of the twentieth century.

Effects of acute stressors experienced by five strains of layer breeders on measures of stress and fear in their offspring.

Stressors experienced by layer breeders during egg production can lead to changes in the egg hormone content, potentially impacting their offspring, the commercial layers. Genetic differences might also affect the offspring's susceptibility to maternal experiences. In this study, we tested if maternal stress affects measures of stress and fear in five strains of layer breeders: commercial brown 1 & 2, commercial white 1 & 2 and a pure line White Leghorn. Each strain was equally separated into two groups: "Maternal Stress" (MS), where hens were subjected to a series of 8 consecutive days of acute psychological stressors, and "Control," which received routine husbandry. Additional eggs from Control were injected either with corticosterone diluted in a vehicle solution ("CORT") or just "Vehicle." Stress- and fear-responses of the offspring were measured in a plasma corticosterone test and a combined human approach and novel object test. While the stress treatments did not  affect the measured endpoints in the offspring, significant strain differences were found. The offspring of the white strains showed a higher physiological response compared to brown strains and the White 2 offspring was the least fearful strain in the human approach test. Our study found that neither the acute psychological stressors experienced by layer breeders nor the egg injections of corticosterone affected the parameters tested in their offspring. Post hoc power analyses suggest that the lack of treatment effects might be due to a small sample size (type II error). Although studies on larger flocks of layers are still needed, our results provide an initial understanding of an important subject, as in poultry production, layer breeders are often subjected to short-term stressors. In addition, our results suggest the dissociation between the physiological and behavioural parameters of stress response in laying hens, showing that increased concentrations of plasma corticosterone in response to stress might not be directly associated with high levels of fear.

Targeted Metabolomics to Assess Exposure to Environmental Chemicals of Concern in Japanese Quail at Two Life Stages.

This proof-of-concept study characterizes the Japanese quail (Coturnix japonica) hepatic metabolome following exposure to benzo[a]pyrene, chlorpyrifos, ethinylestradiol, fluoxetine hydrochloride, hexabromocyclododecane, lead(II)nitrate, seleno-L-methionine, and trenbolone in embryos and adults. The analysis revealed effects on lipid metabolism following exposure to several chemicals at both life stages. The most pronounced effects were observed in embryos exposed to 41.1 µg/g chlorpyrifos. This work highlighted challenges and the need for further avian metabolomics studies.

Effects on Apical Outcomes of Regulatory Relevance of Early-Life Stage Exposure of Double-Crested Cormorant Embryos to 4 Environmental Chemicals.

Environmental risk assessment is often challenged by a lack of toxicity data for ecological species. The overall goal of the present study was to employ an avian early-life stage toxicity test to determine the effects of 4 chemicals (benzo[a]pyrene [BaP], chlorpyrifos, fluoxetine hydrochloride [FLX], and ethinyl estradiol [EE2]) on an ecologically relevant avian species, the double-crested cormorant (Phalacrocorax auritus), and to compare our results with those we previously reported for a laboratory model species, Japanese quail. Chemicals were dissolved in dimethyl sulfoxide and administered via air cell injection to fertilized, unincubated double-crested cormorant eggs at 3 nominal concentrations, the highest selected to approximate the 20% lethal dose. Of the 4 chemicals, only chlorpyrifos and FLX were detected in liver tissue of embryos at midincubation (day 14) and termination (day 26; 1-2 d prior to hatch); EE2 and BaP were not detectable, suggesting embryonic clearance/metabolism. No apical effects were observed in double-crested cormorant embryos up to the highest concentrations of chlorpyrifos (no-observed-effect level [NOEL] = 25 µg/g) or FLX (NOEL = 18 µg/g). Exposure to EE2 reduced embryonic viability and increased deformities at a concentration of 2.3 µg/g (NOEL = 0.18 µg/g), and BaP decreased embryonic viability (median lethal dose = 0.015 µg/g; NOEL = 0.0027 µg/g). Compared with Japanese quail, double-crested cormorant were more sensitive with regard to embryolethality and deformities for EE2 and embryolethality for BaP, whereas they were less sensitive to embryonic deformities associated with chlorpyrifos exposure. These data reinforce the idea that standardized toxicity tests using a laboratory model species may not always be protective of wild birds, and thus they stress the importance of developing such alternative testing strategies (e.g., the EcoToxChip Project) for ecologically relevant species to augment risk assessment efforts. Environ Toxicol Chem 2021;40:390-401. © 2020 SETAC.

Using Transcriptomics and Metabolomics to Understand Species Differences in Sensitivity to Chlorpyrifos in Japanese Quail and Double-Crested Cormorant Embryos.

Modern 21st-century toxicity testing makes use of omics technologies to address critical questions in toxicology and chemical management. Of interest are questions relating to chemical mechanisms of toxicity, differences in species sensitivity, and translation of molecular effects to observable apical endpoints. Our study addressed these questions by comparing apical outcomes and multiple omics responses in early-life stage exposure studies with Japanese quail (Coturnix japonica) and double-crested cormorant (Phalacrocorax auritus), representing a model and ecological species, respectively. Specifically, we investigated the dose-dependent response of apical outcomes as well as transcriptomics and metabolomics in the liver of each species exposed to chlorpyrifos, a widely used organophosphate pesticide. Our results revealed a clear pattern of dose-dependent disruption of gene expression and metabolic profiles in Japanese quail but not double-crested cormorant at similar chlorpyrifos exposure concentrations. The difference in sensitivity between species was likely due to higher metabolic transformation of chlorpyrifos in Japanese quail compared to double-crested cormorant. The most impacted biological pathways after chlorpyrifos exposure in Japanese quail included hepatic metabolism, oxidative stress, endocrine disruption (steroid and nonsteroid hormones), and metabolic disease (lipid and fatty acid metabolism). Importantly, we show consistent responses across biological scales, suggesting that significant disruption at the level of gene expression and metabolite profiles leads to observable apical responses at the organism level. Our study demonstrates the utility of evaluating effects at multiple biological levels of organization to understand how modern toxicity testing relates to outcomes of regulatory relevance, while also highlighting important, yet poorly understood, species differences in sensitivity to chemical exposure. Environ Toxicol Chem 2021;40:3019-3033. © 2021 SETAC.

Correlation between an in vitro and an in vivo measure of dioxin sensitivity in birds.

We describe a statistically significant correlation between two well-characterized responses to dioxin-like compounds in birds; induction of 7-ethoxyresorufin-O-deethylase (EROD) activity in cultured hepatocytes, and embryo mortality. Data were obtained from a review of the literature. EROD EC50 values for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 6 polychlorinated biphenyls (PCBs) were strongly correlated with LD50 values in chicken embryos (r(2) = 0.93, P < 0.005). Similarly, EROD EC50 values for TCDD and a potent dioxin-like compound, PCB 126, were correlated with embryonic LD50 values in different species of birds (chicken, ring-necked pheasant, turkey, double-crested cormorant, and common tern) (r(2) = 0.92, P < 0.005). Our findings contribute to a developing understanding of the molecular basis for differential dioxin sensitivity in birds, and validate the EROD bioassay as a useful predictive tool for ecological risk assessment.

An Early-Life Stage Alternative Testing Strategy for Assessing the Impacts of Environmental Chemicals in Birds.

Early-life stage (ELS) toxicity tests are recognized as an advancement over current testing methodologies in terms of cost, animal use, and biological relevance. However, standardized ELS tests are not presently available for some vertebrate taxa, including birds. The present study describes a Japanese quail (Coturnix japonica) ELS test that is a promising candidate for standardization and applies it to test 8 environmental chemicals (ethinylestradiol, benzo[a]pyrene, chlorpyrifos, fluoxetine, lead(II)nitrate, trenbolone, seleno-L-methionine, hexabromocyclododecane). Individual chemicals were injected into the air cell of unincubated Japanese quail eggs at 3 concentrations, all predicted to cause ≤20% mortality. Survival to embryonic day 16 was consistently high (>90%) among the vehicle-injected controls. All chemicals, except ethinylestradiol, were detected in liver tissue, most at concentrations suggestive of embryonic clearance. Adverse effects were observed for 5 of the 8 chemicals; chlorpyrifos (41.1 µg/g) significantly increased developmental abnormalities and decreased embryo and gallbladder mass. Ethinylestradiol (54.2 µg/g) and hexabromocyclododecane (0.02 µg/g) decreased embryo mass and tarsus length, respectively. Benzo[a]pyrene (0.83 µg/g) and fluoxetine hydrochloride (32.7 µg/g) exceeded the 20% mortality cutoff. No effects were observed following lead(II)nitrate, seleno-L-methionine, or trenbolone exposure up to 10.7, 0.07, and 4.4 µg/g, respectively. Overall, our ELS approach was time- and cost-effective, caused minimal mortality in controls, effectively delivered diverse chemicals to the embryo, and permitted identification of apical outcomes, all of which provide support toward standardization. Environ Toxicol Chem 2019;39:141-154. © 2019 SETAC.