RESUMO
It is postulated that below a transcriptomic-based point of departure, adverse effects are unlikely to occur, thereby providing a chemical concentration to use in screening level hazard assessment. The present study extends previous work describing a high-throughput fathead minnow assay that can provide full transcriptomic data after exposure to a test chemical. One-day post-hatch fathead minnows were exposed to ten concentrations of three representatives of four chemical modes of action: organophosphates, ecdysone receptor agonists, plant photosystem II inhibitors, and estrogen receptor agonists for 24 h. Concentration response modeling was performed on whole body gene expression data from each exposure, using measured chemical concentrations when available. Transcriptomic points of departure in larval fathead minnow were lower than apical effect concentrations across fish species but not always lower than toxic effect concentrations in other aquatic taxa like crustaceans and insects. The point of departure was highly dependent on measured chemical concentration which were often lower than the nominal concentration. Differentially expressed genes between chemicals within modes of action were compared and often showed statistically significant overlap. In addition, reproducibility between identical exposures using a positive control chemical (CuSO4) and variability associated with the transcriptomic point of departure using in silico sampling were considered. Results extend a transcriptomic-compatible fathead minnow high-throughput assay for possible use in ecological hazard screening.
Assuntos
Cyprinidae , Larva , Transcriptoma , Poluentes Químicos da Água , Animais , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Larva/efeitos dos fármacosRESUMO
The objective of this study was to characterize vitellogenin (VTG) protein in male fathead minnow (Pimephales promelas) mucus compared with more conventional measures in plasma and mRNA isolated from liver. To assess the intensity and duration of changes in mucus VTG concentrations, male fathead minnows were exposed to 17α-ethinylestradiol (EE2) for 7 days with a subsequent depuration period of 14 days. The experiment was conducted in a flow-through system to maintain a consistent concentration of EE2 at a nominal EC50 concentration of 2.5 ng/L and high concentration of 10 ng/L as a positive control. Mucus, plasma and liver were sampled at regular intervals throughout the study. Relative abundance of vtg mRNA increased after 2 days of exposure and returned to control levels after 4 days of depuration. VTG protein concentration displayed similar induction kinetics in both mucus and plasma, however, it was found to be significantly increased after 2 days of exposure using the mucus-based assays and 7 days with the plasma-based assay. Significantly elevated levels of VTG were detected by both assays throughout the 14-day depuration period. The elimination of the laborious plasma collection step in the mucus-based workflow allowed sampling of smaller organisms where blood volume is limiting. It also resulted in significant gains in workflow efficiency, decreasing sampling time without loss of performance.
Assuntos
Cyprinidae , Vitelogeninas , Animais , Cyprinidae/metabolismo , Fígado/metabolismo , Masculino , Muco/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vitelogeninas/metabolismoRESUMO
BACKGROUND: A very large and rapidly growing collection of transcriptomic profiles in public repositories is potentially of great value to developing data-driven bioinformatics applications for toxicology/ecotoxicology. Modeled on human connectivity mapping (Cmap) in biomedical research, this study was undertaken to investigate the utility of an analogous Cmap approach in ecotoxicology. Over 3500 zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) transcriptomic profiles, each associated with one of several dozen chemical treatment conditions, were compiled into three distinct collections of rank-ordered gene lists (ROGLs) by species and microarray platforms. Individual query signatures, each consisting of multiple gene probes differentially expressed in a chemical condition, were used to interrogate the reference ROGLs. RESULTS: Informative connections were established at high success rates within species when, as defined by their mechanisms of action (MOAs), both query signatures and ROGLs were associated with the same or similar chemicals. Thus, a simple query signature functioned effectively as an exposure biomarker without need for a time-consuming process of development and validation. More importantly, a large reference database of ROGLs also enabled a query signature to cross-interrogate other chemical conditions with overlapping MOAs, leading to novel groupings and subgroupings of seemingly unrelated chemicals at a finer resolution. This approach confirmed the identities of several estrogenic chemicals, as well as a polycyclic aromatic hydrocarbon and a neuro-toxin, in the largely uncharacterized water samples near several waste water treatment plants, and thus demonstrates its future potential utility in real world applications. CONCLUSIONS: The power of Cmap should grow as chemical coverages of ROGLs increase, making it a framework easily scalable in the future. The feasibility of toxicity extrapolation across fish species using Cmap needs more study, however, as more gene expression profiles linked to chemical conditions common to multiple fish species are needed.
Assuntos
Transcriptoma/genética , Animais , Cyprinidae/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/fisiologia , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genéticaRESUMO
Aromatase, a member of the cytochrome P450 superfamily, is a key enzyme in estradiol synthesis that catalyzes the aromatization of androgens into estrogens in ovaries. Here, we used an integrated approach to assess the mechanistic basis of the direct effects of aromatase inhibition, as well as adaptation and recovery processes in fish. We exposed female fathead minnows (Pimephales promelas) via the water to 30 µg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phases. Transcriptomics, metabolomics, and network inference were used to understand changes and infer connections at the transcript and metabolite level in the ovary. Apical endpoints directly indicative of endocrine function, such as plasma estradiol, testosterone, and vitellogenin levels were also measured. An integrated analysis of the data revealed changes in gene expression consistent with increased testosterone in fadrozole-exposed ovaries. Metabolites such as glycogen and taurine were strongly correlated with increased testosterone levels. Comparison of in vivo and ex vivo steroidogenesis data suggested the accumulation of steroidogenic enzymes, including aromatase, as a mechanism to compensate for aromatase inhibition.
Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Cyprinidae/genética , Cyprinidae/metabolismo , Fadrozol/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Androgênios/sangue , Animais , Estradiol/sangue , Feminino , Glicogênio/sangue , Humanos , Masculino , Metabolômica , Taurina/sangue , Testosterona/sangue , Transcriptoma/efeitos dos fármacos , Vitelogeninas/sangueRESUMO
Propranolol is a heavily prescribed, nonspecific beta-adrenoceptor (bAR) antagonist frequently found in wastewater effluents, prompting concern over its potential to adversely affect exposed organisms. In the present study, the transcriptional responses of 4, 5, and 6 days postfertilization (dpf) ±1 h fathead minnow, exposed for 6, 24, or 48 h to 0.66 or 3.3 mg/L (nominal) propranolol were characterized using RNA sequencing. The number of differentially expressed genes (DEGs) was used as an estimate of sensitivity. A trend toward increased sensitivity with age was observed; fish >7 dpf at the end of exposure were particularly sensitive to propranolol. The DEGs largely overlapped among treatment groups, suggesting a highly consistent response that was independent of age. Cluster analysis was performed using normalized count data for unexposed and propranolol-exposed fish. Control fish clustered tightly by age, with fish ≥7 dpf clustering away from younger fish, reflecting developmental differences. When clustering was conducted using exposed fish, in cases where propranolol induced a minimal or no transcriptional response, the results mirrored those of the control fish and did not appreciably cluster by treatment. In treatment groups that displayed a more robust transcriptional response, the effects of propranolol were evident; however, fish <7 dpf clustered away from older fish, despite having similar numbers of DEGs. Increased sensitivity at 7 dpf coincided with developmental milestones with the potential to alter propranolol pharmacokinetics or pharmacodynamics, such as the onset of exogenous feeding and gill functionality as well as increased systemic expression of bAR. These results may have broader implications because toxicity testing often utilizes fish <4 dpf, prior to the onset of these potentially important developmental milestones, which may result in an underestimation of risk for some chemicals. Environ Toxicol Chem 2024;43:807-820. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.
Assuntos
Cyprinidae , Poluentes Químicos da Água , Animais , Propranolol/toxicidade , Propranolol/metabolismo , Cyprinidae/fisiologia , Poluentes Químicos da Água/análiseRESUMO
Aquatic organisms are exposed to many toxic chemicals and interpreting the cause and effect relationships between occurrence and impairment is difficult. Toxicity Identification Evaluation (TIE) provides a systematic approach for identifying responsible toxicants. TIE relies on relatively uninformative and potentially insensitive toxicological end points. Gene expression analysis may provide needed sensitivity and specificity aiding in the identification of primary toxicants. The current work aims to determine the added benefit of integrating gene expression end points into the TIE process. A cDNA library and a custom microarray were constructed for the marine amphipod Ampelisca abdita. Phase 1 TIEs were conducted using 10% and 40% dilutions of acutely toxic sediment. Gene expression was monitored in survivors and controls. An expression-based classifier was developed and evaluated against control organisms, organisms exposed to low or medium toxicity diluted sediment, and chemically selective manipulations of highly toxic sediment. The expression-based classifier correctly identified organisms exposed to toxic sediment even when little mortality was observed, suggesting enhanced sensitivity of the TIE process. The ability of the expression-based end point to correctly identify toxic sediment was lost concomitantly with acute toxicity when organic contaminants were removed. Taken together, this suggests that gene expression enhances the performance of the TIE process.
Assuntos
Anfípodes/genética , Organismos Aquáticos/genética , Determinação de Ponto Final , Genoma/genética , Testes de Toxicidade , Poluentes Químicos da Água/toxicidade , Anfípodes/efeitos dos fármacos , Animais , Organismos Aquáticos/efeitos dos fármacos , Biomarcadores/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sedimentos Geológicos/química , Rhode Island , Rios/químicaRESUMO
Concentrations at which global gene expression profiles in cells or animals exposed to a test substance start to differ significantly from those of controls have been proposed as an alternative point of departure for use in screening level hazard assessment. The present study describes pilot testing of a high throughput compatible transcriptomics assay with larval fathead minnows. One day post hatch fathead minnows were exposed to eleven different concentrations of three metals, three selective serotonin reuptake inhibitors, and four neonicotinoid-like compounds for 24 h and concentration response modeling was applied to whole body gene expression data. Transcriptomics-based points of departure (tPODs) were consistently lower than effect concentrations reported in apical endpoint studies in fish. However, larval fathead minnow-based tPODs were not always lower than concentrations reported to elicit apical toxicity in other aquatic organisms like crustaceans or insects. Random in silico subsampling of data from the pilot assays was used to evaluate various assay design and acceptance considerations such as transcriptome coverage, number of replicate individuals to sequence per treatment, and minimum number of differentially expressed genes to produce a reliable tPOD estimate. Results showed a strong association between the total number of genes for which a concentration response relationship could be derived and the overall variability in the resulting tPOD estimates. We conclude that, for our current assay design and analysis pipeline, tPODs based on fewer than 15 differentially expressed genes are likely to be unreliable for screening and that interindividual variability in gene expression profiles appears to be a more significant driver of tPOD variability than sample size alone. Results represent initial steps toward developing high throughput transcriptomics assays for use in ecological hazard screening.
RESUMO
The fathead minnow is a widely used model organism in environmental toxicology. The lack of a high-quality fathead minnow reference genome, however, has severely hampered its uses in toxicogenomics. We present the de novo assembly and annotation of the fathead minnow genome using long PacBio reads, Bionano and Hi-C scaffolding data, and large RNA-sequencing data sets from different tissues and life stages. The new annotated fathead minnow reference genome has a scaffold N50 of 12.0 Mbp and a complete benchmarking universal single-copy orthologs score of 95.1%. The completeness of annotation for the new reference genome is comparable to that of the zebrafish GRCz11 reference genome. The fathead minnow genome, revealed to be highly repetitive and sharing extensive syntenic regions with the zebrafish genome, has a much more compact gene structure than the zebrafish genome. Particularly, comparative genomic analysis with zebrafish, mouse, and human showed that fathead minnow homologous genes are relatively conserved in exon regions but had strikingly shorter intron regions. The new fathead minnow reference genome and annotation data, publicly available from the National Center for Biotechnology Information and the University of California Santa Cruz genome browser, provides an essential resource for aquatic toxicogenomic studies in ecotoxicology and public health. Environ Toxicol Chem 2022;41:448-461. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.
Assuntos
Cyprinidae , Peixe-Zebra , Animais , Cyprinidae/genética , Ecotoxicologia , Genoma , Camundongos , Software , Peixe-Zebra/genéticaRESUMO
The number of chemicals requiring risk evaluation exceeds our capacity to provide the underlying data using traditional methodology. This has led to an increased focus on the development of novel approach methodologies. This work aimed to expand the panel of gene expression-based biomarkers to include responses to estrogens, to identify training strategies that maximize the range of applicable concentrations, and to evaluate the potential for two classes of small non-coding RNAs (sncRNAs), microRNA (miRNA) and piwi-interacting RNA (piRNA), as biomarkers. To this end larval Pimephales promelas (96 hpf +/- 1h) were exposed to 5 concentrations of 17α- ethinylestradiol (0.12, 1.25, 2.5, 5.0, 10.0 ng/L) for 48 h. For mRNA-based biomarker development, RNA-seq was conducted across all concentrations. For sncRNA biomarkers, small RNA libraries were prepared only for the control and 10.0 ng/L EE2 treatment. In order to develop an mRNA classifier that remained accurate over the range of exposure concentrations, three different training strategies were employed that focused on 10 ng/L, 2.5 ng/L or a combination of both. Classifiers were tested against an independent test set of individuals exposed to the same concentrations used in training and subsequently against concentrations not included in model training. Both random forest (RF) and logistic regression with elastic net regularizations (glmnet) models trained on 10 ng/L EE2 performed poorly when applied to lower concentrations. RF models trained with either the 2.5 ng/L or combination (2.5 + 10 ng/L) treatments were able to accurately discriminate exposed vs. non-exposed across all but the lowest concentrations. glmnet models were unable to accurately classify below 5 ng/L. With the exception of the 10 ng/L treatment, few mRNA differentially expressed genes (DEG) were observed, however, there was marked overlap of DEGs across treatments. Overlapping DEGs have well established linkages to estrogen and several of the 81 DEGs identified in the 10 ng/L treatment have been previously utilized as estrogenic biomarkers (vitellogenin, estrogen receptor-ß). Following multiple test correction, no sncRNAs were found to be differentially expressed, however, both miRNA and piRNA classifiers were able to accurately discriminate control and 10 ng/L exposed organisms with AUCs of 0.83 and 1.0 respectively. We have developed a highly discriminative estrogenic mRNA biomarker that is accurate over a range of concentrations likely to be found in real-world exposures. We have demonstrated that both miRNA and piRNA are responsive to estrogenic exposure, suggesting the need to further investigate their regulatory roles in the estrogenic response.
Assuntos
Estrogênios/toxicidade , MicroRNAs , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Cyprinidae/fisiologia , Etinilestradiol , Expressão Gênica , RNA Mensageiro , RNA Interferente Pequeno , Vitelogeninas/genéticaRESUMO
The canonical mode of action (MOA) of microcystins (MC) is the inhibition of protein phosphatases, but complete characterization of toxicity pathways is lacking. The existence of over 200 MC congeners complicates risk estimates worldwide. This work employed RNA-seq to provide an unbiased and comprehensive characterization of cellular targets and impacted cellular processes of hepatocytes exposed to either MC-LR or MC-RR congeners. The human hepatocyte cell line, HepaRG, was treated with three concentrations of MC-LR or -RR for 2 h. Significant reduction in cell survival was observed in LR1000 and LR100 treatments whereas no acute toxicity was observed in any MR-RR treatment. RNA-seq was performed on all treatments of MC-LR and -RR. Differentially expressed genes and pathways associated with oxidative and endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) were highly enriched by both congeners as were inflammatory pathways. Genes associated with both apoptotic and inflammatory pathways were enriched in LR1000. We present a model of MC toxicity that immediately causes oxidative stress and leads to ER stress and the activation of the UPR. Differential activation of the three arms of the UPR and the kinetics of JNK activation ultimately determine whether cell survival or apoptosis is favored. Extracellular exosomes were enrichment of by both congeners, suggesting a previously unidentified mechanism for MC-dependent extracellular signaling. The complement system was enriched only in MC-RR treatments, suggesting congener-specific differences in cellular effects. This study provided an unbiased snapshot of the early systemic hepatocyte response to MC-LR and MC-RR congeners and may explain differences in toxicity among MC congeners.
RESUMO
As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.
Assuntos
Inibidores da Aromatase/toxicidade , Encéfalo/metabolismo , Fadrozol/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Ovário/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise Serial de Proteínas , Peixe-ZebraRESUMO
Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. Two different concentration of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite or observed for development and survival through larval stage. Global transcript profiling using RNA-seq was conducted (nâ¯=â¯16 per treatment) to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. A transcriptional fingerprint using Random Forests was developed that was able to perfectly discriminate exposed vs. non-exposed individuals in test sets. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals and that the early life stage Fundulus system may provide a convenient test system for exposure characterization. More broadly, this study advances the usefulness of a biological testing and analysis system utilizing non-targeted transcriptomics profiling and early developmental endpoints that complements current screening methods to characterize chemicals of ecological and human health concern.
Assuntos
Retardadores de Chama/toxicidade , Fundulidae/embriologia , Ácidos Ftálicos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Retardadores de Chama/análise , Fundulidae/metabolismo , Fundulidae/fisiologia , Perfilação da Expressão Gênica , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo , Poluentes Químicos da Água/análiseRESUMO
We describe initial development of microarray-based assays for detecting 4 pyrethroid pesticides (bifenthrin, cypermethrin, esfenvalerate, and permethrin) in water. To facilitate comparison of transcriptional responses with gross apical responses, we estimated concentration-mortality curves for these pyrethroids using flow-through exposures of newly hatched Daphnia magna, Pimephales promelas adults, and 24 h posthatch P. promelas. Median lethal concentration (LC50) estimates were below most reported values, perhaps attributable to the use of flow-through exposures or of measured rather than nominal concentrations. Microarray analysis of whole P. promelas larvae and brains from exposed P. promelas adults showed that assays using either tissue type can detect these pyrethroids at concentrations below LC50 values reported for between 72 and 96% of aquatic species, depending on the pesticide. These estimates are conservative because they correspond to the lowest concentrations tested. This suggests that the simpler and less expensive whole-larval assay provides adequate sensitivity for screening contexts where acute aquatic lethality is observed, but the responsible agent is not known. Gene set analysis (GSA) highlighted several Gene Ontology (GO) terms consistent with known pyrethroid action, but the implications of other GO terms are less clear. Exploration of the sensitivity of results to changes in data processing suggests robustness of the detection assay results, but GSA results were sensitive to methodological variations. Environ Toxicol Chem 2019;38:2436-2446. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America.
Assuntos
Biomarcadores/metabolismo , Cyprinidae/genética , Daphnia/genética , Exposição Ambiental/análise , Piretrinas/toxicidade , Animais , Cyprinidae/crescimento & desenvolvimento , Daphnia/efeitos dos fármacos , Ontologia Genética , Larva/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidadeRESUMO
The measurement of vitellogenin (vtg) gene transcription has been shown to be a reliable indicator of exposure to estrogenic compounds. Unfortunately, the relatively poor molecular characterization of North American fish species has hindered its application to a larger number of ecologically important species. The current research aimed to demonstrate specific amplification of vtg gene transcripts in three model (zebrafish, rainbow trout, and medaka) and six nonmodel (emerald shiner, pearl dace, smallmouth bass, creek chub, white sucker, and golden redhorse) fish species. Quantitative polymerase chain reaction (QPCR) primers for model species were designed from publicly available vtg sequences. Successful amplification of vtg was demonstrated in fish exposed to 17alpha-ethinylestradiol (EE(2)) for all model species. Vitellogenin primers for selected nonmodel species were designed from published sequences of closely related species. Multiple primers were developed targeting different regions of the vtg gene. The successful amplification of vtg was confirmed through size and sequence analysis for all nonmodel species with the exception of the white sucker, in which amplifications failed. Furthermore, QPCR primers and conditions were quantitative over five orders of magnitude in at least one species (pearl dace) exposed to 5 ng/L of EE(2) for 24 h. The selected species are found in a wide array of ecological habitats that span the United States. Inclusion of vtg transcriptional analysis for wild, ecologically relevant fish in monitoring studies may aid in understanding the extent of estrogenic exposure in aquatic ecosystems across the United States.
Assuntos
Peixes/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vitelogeninas/genética , Animais , Feminino , Regulação da Expressão Gênica/genética , Masculino , Modelos Animais , RNA Mensageiro/biossíntese , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Ecological risk assessors have a growing need for sensitive and rapid indicators of environmental exposures in aquatic ecosystems resulting from natural and synthetic estrogen-like compounds. Investigators developing subcellular exposure markers in traditional sentinel organisms must be vigilant about inherent variability of analyses, especially regarding regulatory and policy statements. Here, we report a quantitative real-time polymerase chain reaction (QPCR) assay for the detection of vitellogenin transcripts environmentally triggered in fathead minnows (Pimephales promelas). We demonstrate that our QPCR assay exhibits little inter- or intra-assay variability (21.7 and 11.9%, respectively). This method appears to be robust in terms of variability stemming from extrinsic sources, indicating that it may be readily transferable to laboratories having the requisite equipment. Our primary focus in development of this method derived from the observation that transcriptional responses of the vitellogenin gene (vtg) in fathead minnows demonstrated high biological variability between identically treated individuals, even under controlled laboratory conditions (coefficient of variation, > 100%). This variability was not seen in other genes from the same RNA preparations that we examined, suggesting that it is specific to the vitellogenin response. Our data and those of others suggest that variability in vtg expression is common to a number of aquatic vertebrates, which is indicative of genetic causation. Despite a relatively high degree of variability in vtg transcription, this method is sensitive enough to detect exposures of 5.0 ng 17alpha-ethinylestradiol (EE2)/L within 24 h of exposure, and it has the ability to discriminate 10.0 and 5.0 ng EE2/L within 48 h. The vitellogenin QPCR assay is a highly sensitive, comparatively rapid, and inexpensive method for the detection and characterization of exposure to environmental estrogens and estrogen mimics.
Assuntos
Cyprinidae/genética , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Vitelogeninas/genética , Animais , Exposição Ambiental , Masculino , Poluentes Químicos da Água/toxicidadeRESUMO
Fundamental questions remain about the application of omics in environmental risk assessments, such as the consistency of data across laboratories. The objective of the present study was to determine the congruence of transcript data across 6 independent laboratories. Male fathead minnows were exposed to a measured concentration of 15.8 ng/L 17α-ethinylestradiol (EE2) for 96 h. Livers were divided equally and sent to the participating laboratories for transcriptomic analysis using the same fathead minnow microarray. Each laboratory was free to apply bioinformatics pipelines of its choice. There were 12 491 transcripts that were identified by one or more of the laboratories as responsive to EE2. Of these, 587 transcripts (4.7%) were detected by all laboratories. Mean overlap for differentially expressed genes among laboratories was approximately 50%, which improved to approximately 59.0% using a standardized analysis pipeline. The dynamic range of fold change estimates was variable between laboratories, but ranking transcripts by their relative fold difference resulted in a positive relationship for comparisons between any 2 laboratories (mean R2 > 0.9, p < 0.001). Ten estrogen-responsive genes encompassing a fold change range from dramatic (>20-fold; e.g., vitellogenin) to subtle (â¼2-fold; i.e., block of proliferation 1) were identified as differentially expressed, suggesting that laboratories can consistently identify transcripts that are known a priori to be perturbed by a chemical stressor. Thus, attention should turn toward identifying core transcriptional networks using focused arrays for specific chemicals. In addition, agreed-on bioinformatics pipelines and the ranking of genes based on fold change (as opposed to p value) should be considered in environmental risk assessment. These recommendations are expected to improve comparisons across laboratories and advance the use of omics in regulations. Environ Toxicol Chem 2017;36:2593-2601. © 2017 SETAC.
Assuntos
Cyprinidae/genética , Disruptores Endócrinos/toxicidade , Etinilestradiol/toxicidade , Laboratórios/normas , Fígado/metabolismo , Transcriptoma/efeitos dos fármacos , Animais , Cyprinidae/metabolismo , Ensaio de Imunoadsorção Enzimática , Fígado/efeitos dos fármacos , Masculino , Modelos Químicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/isolamento & purificação , RNA/metabolismo , Vitelogeninas/sangueRESUMO
17α-ethinylestradiol (EE2) is a synthetic estrogen that is an active ingredient in oral contraception and hormone replacement therapy. Surveys of wastewater treatment plant effluents and surface waters throughout the world have reported EE2 concentrations in the ng/L range, and these low levels can cause significant reproductive effects in fish. This study tested the effects of three environmentally relevant EE2 concentrations: 0.47, 1.54 and 3.92 ng/L using a 21 d short-term reproductive assay to investigate the effects of EE2 on fathead minnow (Pimephales promelas) reproduction. The two highest EE2 concentrations tested in this study caused significant liver gene expression and induction of vitellogenin plasma protein in male fathead minnows. Exposure to 3.92 ng EE2/L increased the production of plasma vitellogenin in the females. Plasma estradiol concentrations were significantly reduced in females exposed to 1.54 and 3.92 ng EE2/L. All three tested concentrations significantly reduced fathead minnow egg production after a 21 d exposure to EE2. The results of this study indicate that the previously reported no observed adverse effect concentration (NOAEC) for EE2 on fathead minnow egg production (1.0 ng/L) may be too high. Because all three treatments resulted in significantly reduced egg production, the lowest observed adverse effect concentration (LOAEC) for EE2 on fathead minnow egg production is 0.47 ng EE2/L. This research estimates a NOAEC for fathead minnow reproduction at 0.24 ng EE2/L following a 21 d exposure. Additionally, induction of vitellogenin is a sensitive indicator of estrogen exposure but does not appear to be predictive of fathead minnow egg production.
Assuntos
Cyprinidae/fisiologia , Estrogênios/toxicidade , Etinilestradiol/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Cyprinidae/sangue , Cyprinidae/genética , Estradiol/sangue , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Nível de Efeito Adverso não Observado , Reprodução/efeitos dos fármacos , Vitelogeninas/sangueRESUMO
Omics technologies have long since promised to address a number of long standing issues related to environmental regulation. Despite considerable resource investment, there are few examples where these tools have been adopted by the regulatory community, which is in part due to a focus of most studies on discovery rather than assay development. The current work describes the initial development of an omics based assay using 48h Pimephales promelas (FHM) larvae for identifying aquatic exposures to pyrethroid pesticides. Larval FHM were exposed to seven concentrations of each of four pyrethroids (permethrin, cypermethrin, esfenvalerate and bifenthrin) in order to establish dose response curves. Then, in three separate identical experiments, FHM were exposed to a single equitoxic concentration of each pyrethroid, corresponding to 33% of the calculated LC50. All exposures were separated by weeks and all materials were either cleaned or replaced between runs in an attempt to maintain independence among exposure experiments. Gene expression classifiers were developed using the random forest algorithm for each exposure and evaluated first by cross-validation using hold out organisms from the same exposure experiment and then against test sets of each pyrethroid from separate exposure experiments. Bifenthrin exposed organisms generated the highest quality classifier, demonstrating an empirical Area Under the Curve (eAUC) of 0.97 when tested against bifenthrin exposed organisms from other exposure experiments and 0.91 against organisms exposed to any of the pyrethroids. An eAUC of 1.0 represents perfect classification with no false positives or negatives. Additionally, the bifenthrin classifier was able to successfully classify organisms from all other pyrethroid exposures at multiple concentrations, suggesting a potential utility for detecting cumulative exposures. Considerable run-to-run variability was observed both in exposure concentrations and molecular responses of exposed fish across exposure experiments. The application of a calibration step in analysis successfully corrected this, resulting in a significantly improved classifier. Classifier evaluation suggested the importance of considering a number of aspects of experimental design when developing an expression based tool for general use in ecological monitoring and risk assessment, such as the inclusion of multiple experimental runs and high replicate numbers.
Assuntos
Biomarcadores/metabolismo , Expressão Gênica/efeitos dos fármacos , Praguicidas/toxicidade , Piretrinas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Área Sob a Curva , Cyprinidae/crescimento & desenvolvimento , Cyprinidae/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Larva/efeitos dos fármacos , Larva/metabolismo , Praguicidas/análise , Piretrinas/análise , RNA/isolamento & purificação , Curva ROC , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/químicaRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant with well-known adverse effects in fish. In this study, we initially exploited suppression subtractive hybridization (SSH) as a screening tool to assess qualitative gene expression changes in whole brain, liver, and testis of adult male Japanese medaka (Oryzias latipes) exposed for 48 h to a single intraperitoneal-injected dose of TCDD (10 microg TCDD/kg body weight). Across these three organs, SSH identified a total of 335 unique genes. Each set of forward- and reverse-subtracted organ cDNA libraries consisted of a distinct gene list and corresponding distribution of biological processes, suggesting that transcript profiles of these libraries were highly organ-specific. Based on sequence match significance and frequencies within each set of organ libraries, genes hypothesized to be strongly responsive (42 total) within male medaka brain, liver, or testis were semi-quantitatively screened with replicate cDNA nylon membrane arrays. In addition, TCDD-treated male medaka were surveyed for gross histological analysis of brain, liver, and testis. In general, adverse histopathological changes were not observed in the brain, and glycogen depletion was observed only in the liver. However, significant histological changes occurred in the testis, and included disorganization of spermatogenesis at the testis periphery, disruption of the interstitium, Leydig cell swelling, and Sertoli cell vacuolation. Of the 42 genes screened by cDNA array analysis, cytochrome P450 1A (CYP1A) mRNA was the only transcript significantly higher in TCDD-exposed brain, whereas 12 transcripts (including CYP1A) were significantly higher in TCDD-exposed liver, and 34 transcripts were significantly lower in TCDD-exposed testis. Therefore, the degree of TCDD-induced alterations observed in each organ at a gross histological level corresponded well with the number and ontology of gene transcripts affected on the array. Based on real-time reverse transcription polymerase chain reaction (RT-PCR), relative CYP1A (but not AHR1) transcript levels were confirmed to be significantly higher in TCDD-treated brain and liver. However, CYP1A was not significantly induced in TCDD-exposed testis, suggesting that gene expression and histopathological responses observed in the testis at 48 h may be CYP1A-independent. Based on these data, unique liver-specific and testis-specific mRNA-level targets in male medaka were identified as promising biomarkers of acute TCDD-induced toxicity.
Assuntos
Poluentes Ambientais/toxicidade , Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Oryzias/genética , Dibenzodioxinas Policloradas/toxicidade , Testículo/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Perfilação da Expressão Gênica , Injeções Intraperitoneais , Fígado/metabolismo , Fígado/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/metabolismo , Testículo/patologiaRESUMO
In this age of modern biology, aquatic toxicological research has pursued mechanisms of action of toxicants. This has provided potential tools for ecotoxicologic investigations. However, problems of biocomplexity and issues at higher levels of biological organization remain a challenge. In the 1980s and 1990s and continuing to a lesser extent today, organisms residing in highly contaminated field sites or exposed in the laboratory to calibrated concentrations of individual compounds were carefully analyzed for their responses to priority pollutants. Correlation of biochemical and structural analyses in cultured cells and tissues, as well as the in vivo exposures led to the production and application of biomarkers of exposure and effect and to our awareness of genotoxicity and its chronic manifestations, such as neoplasms, in wild fishes. To gain acceptance of these findings in the greater environmental toxicology community, "validation of the model" versus other, better-established often rodent models, was necessary and became a major focus. Resultant biomarkers were applied to heavily contaminated and reference field sites as part of effects assessment and with investigations following large-scale disasters such as oil spills or industrial accidents. Over the past 15 years, in the laboratory, small aquarium fish models such as medaka (Oryzias latipes), zebrafish (Danio rerio), platyfish (Xiphophorus species), fathead minnow (Pimephales promelas), and sheepshead minnow (Cyprinodon variegatus) were increasingly used establishing mechanisms of toxicants. Today, the same organisms provide reliable information at higher levels of biological organization relevant to ecotoxicology. We review studies resolving mechanisms of toxicity and discuss ways to address biocomplexity, mixtures of contaminants, and the need to relate individual level responses to populations and communities.