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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 (CuSO 4 ) 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., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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 EC 50 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., (Published by Elsevier Inc.)

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., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

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-β). 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., (Copyright © 2021. Published by Elsevier B.V.)

The gene expression response thought to underlie the negative apical effects resulting from estrogen exposure have been thoroughly described in fish. Although epigenetics are believed to play a critical role translating environmental exposures into the development of adverse apical effects, they remain poorly characterized in fish species. This study investigated alterations of DNA methylation of estrogen receptor alpha (esr1) in brain and liver tissues from 8 to 10 month old male fathead minnows (Pimephales promelas) after a 2d exposure to either 2.5 ng/L or 10 ng/L 17α-ethynylestradiol (EE2). Changes in the patterns of methylation were evaluated using targeted deep sequencing of bisulfite treated DNA in the 5' region of esr1. Methylation and gene expression were assessed at 2d of exposure and after a 7 and 14d depuration period. After 2d EE2 exposure, males exhibited significant demethylation in the 5' upstream region of esr1 in liver tissue, which was inversely correlated to gene expression. This methylation pattern reflected what was seen in females. No gene body methylation (GBM) was observed for liver of exposed males. Differential methylation was observed for a single upstream CpG site in the liver after the 14d depuration. A less pronounced methylation response was observed in the upstream region in brain tissue, however, several CpGs were necessarily excluded from the analysis. In contrast to the liver, a significant GBM response was observed across the entire gene body, which was sustained until at least 7d post-exposure. No differential expression was observed in the brain, limiting functional interpretation of methylation changes. The identification of EE2-dependent changes in methylation levels strongly suggests the importance of epigenetic mechanisms as a mediator of the organismal response to environmental exposures and the need for further characterization of the epigenome. Further, differential methylation following depuration indicates estrogenic effects persist well after the active exposure, which has implications for the risk posed by repeated exposures.., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Published by Elsevier Ltd.)

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., (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.)

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 (n = 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 R 2  > 0.9, p < 0.001). Ten estrogen-responsive genes encompassing a fold change range from dramatic (>20-fold; e.g., vitellogenin) to subtle (∼2-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., (© 2017 SETAC.)

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., (Published by Elsevier B.V.)

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., (Published by Elsevier Ltd.)

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.

Fathead minnow and zebrafish are among the most intensively studied fish species in environmental toxicogenomics. To aid the assessment and interpretation of subtle transcriptomic effects from treatment conditions of interest, better characterization and understanding are needed for natural variation in gene expression among fish individuals from lab cultures. Leveraging the transcriptomics data from a number of our toxicogenomics studies conducted over the years, we conducted a meta-analysis of nearly 600 microarrays generated from the ovary tissue of untreated, reproductively mature fathead minnow and zebrafish samples. As expected, there was considerable batch-to-batch transcriptomic variation; this "batch-effect" appeared to differentially impact subsets of fish transcriptomes in a nonsystematic way. Temporally more closely spaced batches tended to share a greater transcriptomic similarity among one another. The overall level of within-batch variation was quite low in fish ovary tissue, making it a suitable system for studying chemical stressors with subtle biological effects. The observed differences in the within-batch variability of gene expression, at the levels of both individual genes and pathways, were probably both technical and biological. This suggests that biological interpretation and prioritization of genes and pathways targeted by experimental conditions should take into account both their intrinsic variability and the size of induced transcriptional changes. There was significant conservation of both the genomes and transcriptomes between fathead minnow and zebrafish. The high degree of conservation offers promising opportunities in not only studying fish molecular responses to environmental stressors by a comparative biology approach, but also effective sharing of a large amount of existing public transcriptomics data for developing toxicogenomics applications.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Vitellogenin is frequently used as a biomarker of exposure to environmental estrogens due to its specificity and sensitivity. Appropriate incorporation of this biomarker into environmental monitoring and assessment necessitates evaluation of its critical performance parameters. In this study, we characterize the sensitivity of both vitellogenin gene (vtg) mRNA transcripts in liver and protein (VTG) in plasma over a range of concentrations and exposure durations. Male fathead minnows were exposed to 17α-ethynylestradiol (EE2) in a flow-through system for 2, 4 and 7 days at multiple EE2 concentrations in order to provide information regarding the sensitivity of each of these biomarkers to diagnose exposure to this representative estrogen. Measurements of the expression of the vitellogenin gene and protein both reliably detected exposures to EE2 at concentrations of 5ng/l and higher at all time points. Vtg mRNA and plasma VTG appear to have similar sensitivities, though the lower variability in VTG in control fish may make it more sensitive to small changes in expression compared to vtg. For lower concentrations, sensitivity may be improved by increasing exposure duration. A sample size of ∼12 fish was sufficient in many cases to produce a statistically significant increase in vitellogenin. Larger sample sizes may provide more sensitivity at low concentrations, but detecting exposure to estrogens in the lower range of environmentally relevant concentrations may need larger sample sizes. These data will assist in designing experiments that have sufficient statistical power necessary to determine if fish have been exposed to estrogens., (Published by Elsevier B.V.)

Gamma-aminobutyric acid (GABA) and GABA receptors play an important role in neuroendocrine regulation in fish. Disruption of the GABAergic system by environmental contaminants could interfere with normal regulation of the hypothalamic-pituitary-gonadal axis, leading to impaired fish reproduction. The present study used a 21-d fathead minnow (Pimephales promelas) reproduction assay to investigate the reproductive toxicity of fipronil (FIP), a broad-spectrum phenylpyrazole insecticide that acts as a noncompetitive blocker of GABA receptor-gated chloride channels. Continuous exposure up to 5 µg FIP/L had no significant effect on most of the endpoints measured, including fecundity, secondary sexual characteristics, plasma steroid and vitellogenin concentrations, ex vivo steroid production, and targeted gene expression in gonads or brain. The gonad mass, gonadosomatic index, and histological stage of the gonad were all significantly different in females exposed to 0.5 µg FIP/L compared with those exposed to 5.0 µg FIP/L; however, there were no other significant effects on these measurements in the controls or any of the other treatments in either males and females. Overall, the results do not support a hypothesized adverse outcome pathway linking FIP antagonism of the GABA receptor(s) to reproductive impairment in fish., (Copyright © 2013 SETAC.)

Endocrine-disrupting chemicals can affect reproduction and development in humans and wildlife. We developed a computational model of the hypothalamic-pituitary-gonadal (HPG) axis in female fathead minnows to predict dose-response and time-course (DRTC) behaviors for endocrine effects of the aromatase inhibitor, fadrozole (FAD). The model describes adaptive responses to endocrine stress involving regulated secretion of a generic gonadotropin (LH/FSH) from the hypothalamic-pituitary complex. For model development, we used plasma 17β-estradiol (E2) concentrations and ovarian cytochrome P450 (CYP) 19A aromatase mRNA data from two time-course experiments, each of which included both an exposure and a depuration phase, and plasma E2 data from a third 4-day study. Model parameters were estimated using E2 concentrations for 0, 0.5, and 3 µg/l FAD exposure concentrations, and good fits to these data were obtained. The model accurately predicted CYP19A mRNA fold changes for controls and three FAD doses (0, 0.5, and 3 µg/l) and plasma E2 dose response from the 4-day study. Comparing the model-predicted DRTC with experimental data provided insight into how the feedback control mechanisms in the HPG axis mediate these changes: specifically, adaptive changes in plasma E2 levels occurring during exposure and "overshoot" occurring postexposure. This study demonstrates the value of mechanistic modeling to examine and predict dynamic behaviors in perturbed systems. As this work progresses, we will obtain a refined understanding of how adaptive responses within the vertebrate HPG axis affect DRTC behaviors for aromatase inhibitors and other types of endocrine-active chemicals and apply that knowledge in support of risk assessments.

Adaptive or compensatory responses to chemical exposure can significantly influence in vivo concentration-duration-response relationships. This study provided data to support development of a computational dynamic model of the hypothalamic-pituitary-gonadal axis of a model vertebrate and its response to aromatase inhibitors as a class of endocrine active chemicals. Fathead minnows (Pimephales promelas) were either exposed to the aromatase inhibitor fadrozole (0.5 or 30 μg/l) continuously for 1, 8, 12, 16, 20, 24, or 28 days or exposed for 8 days and then held in control water (no fadrozole) for an additional 4, 8, 12, 16, or 20 days. The time course of effects on ovarian steroid production, circulating 17β-estradiol (E2) and vitellogenin (VTG) concentrations, and expression of steroidogenesis-related genes in the ovary was measured. Exposure to 30 μg fadrozole/l significantly reduced plasma E2 and VTG concentrations after just 1 day and those effects persisted throughout 28 days of exposure. In contrast, ex vivo E2 production was similar to that of controls on day 8-28 of exposure, whereas transcripts coding for aromatase and follicle-stimulating hormone receptor were elevated, suggesting a compensatory response. Following cessation of fadrozole exposure, ex vivo E2 and plasma E2 concentrations exceeded and then recovered to control levels, but plasma VTG concentrations did not, even after 20 days of depuration. Collectively these data provide several new insights into the nature and time course of adaptive responses to an aromatase inhibitor that support development of a computational model (see companion article).

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.

The hypothalamus-pituitary-gonadal (HPG) axis plays a central role in the maintenance of homeostasis and disruptions in its function can have important implications for reproduction and other critical biological processes. A number of compounds found in aquatic environments are known to affect the HPG axis. In the present study, we used two-dimensional electrophoresis to investigate the proteome of female and male zebrafish brain after 96 h exposure to the fungicide prochloraz. Prochloraz has known effects on a number of key HPG molecules, including antagonism of Cyp17 and Cyp19 (aromatase). Twenty-eight proteins were shown to be differentially expressed in the brains of females and 22 in males. Proteins were identified using LC-MS/MS and identities were examined relative to brain function in the context of changing steroid hormone levels. There was little overlap between sexes in proteins exhibiting differential expression. Proteins with known roles in metabolism, learning, neuroprotection, and calcium regulation were determined to be differentially regulated. Relationships between identified proteins were also examined using Ingenuity Pathway Analysis, and females were shown to exhibit enrichment of several metabolic pathways. We used differentially expressed proteins to establish a putative classifier consisting of three proteins that was able to discriminate prochloraz-exposed from control females. Putatively impacted brain functions and specific protein changes that were observed have the potential to be generalized to other that similarly impact steroid hormone levels., (Published by Elsevier B.V.)

The studies presented in this manuscript focus on characterization of transcriptomic responses to anti-androgens in zebrafish (Danio rerio). Research on the effects of anti-androgens in fish has been characterized by a heavy reliance on apical endpoints, and molecular mechanisms of action (MOA) of anti-androgens remain poorly elucidated. In the present study, we examined effects of a short term exposure (24-96h) to the androgen receptor antagonists flutamide (FLU) and vinclozolin (VZ) on gene expression in gonads of sexually mature zebrafish, using commercially available zebrafish oligonucleotide microarrays (4×44K platform). We found that VZ and FLU potentially impact reproductive processes via multiple pathways related to steroidogenesis, spermatogenesis, and fertilization. Observed changes in gene expression often were shared by VZ and FLU, as demonstrated by overlap in differentially-expressed genes and enrichment of several common key pathways including: (1) integrin and actin signaling, (2) nuclear receptor 5A1 signaling, (3) fibroblast growth factor receptor signaling, (4) polyamine synthesis, and (5) androgen synthesis. This information should prove useful to elucidating specific mechanisms of reproductive effects of anti-androgens in fish, as well as developing biomarkers for this important class of endocrine-active chemicals., (2010 Elsevier B.V. All rights reserved.)

Pesticides are nearly ubiquitous in surface waters of the United States, where they often are found as mixtures. The molecular mechanisms underlying the toxic effects of sub-lethal exposure to pesticides as both individual and mixtures are unclear. The current work aims to identify and compare differentially expressed proteins in brains of male fathead minnows (Pimephales promelas) exposed for 72 h to permethrin (7.5 μg/L), terbufos (57.5 μg/L) and a binary mixture of both. Twenty-four proteins were found to be differentially expressed among all three treatments relative to the control using an ANOVA followed by a Dunnett's post hoc test (p ≤0.05). One protein was found to be differentially expressed among all treatment groups and one protein was in common between the terbufos and the mixture group. Fifteen spots were successfully sequenced using LC-MS/MS sequencing. Proteins associated with the ubiquitin-proteasome system, glycolysis, the cytoskeleton and hypoxia were enriched. As a second objective, we attempted to establish protein expression signatures (PES) for individual permethrin and terbufos exposures. We were unable to generate a useable PES for terbufos; however, the permethrin PES was able to distinguish between control and permethrin-exposed individuals in an independent experiment with an accuracy of 87.5%. This PES also accurately classified permethrin exposed individuals when the exposure occurred as part of a mixture. The identification of proteins differentially expressed as a result of pesticide exposure represent a step forward in the understanding of mechanisms of toxicity of permethrin and terbufos. They also allow a comparison of molecular responses of the binary mixture to single exposures. The permethrin PES is the first step in establishing a method to determine exposures in real-world scenarios., (Published by Elsevier B.V.)

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP) 19 aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17beta-estradiol (E2) in female fish. At a prochloraz water concentration of 30 microg/l, inhibitory effects on E2 production were transitory and did not persist during the 8-day exposure phase. At 300 microg/l prochloraz, inhibition of E2 production was evident throughout the 8-day exposure but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by upregulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage), and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes upregulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals.

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.

Knowledge of possible toxic mechanisms (or modes) of action (MOA) of chemicals can provide valuable insights as to appropriate methods for assessing exposure and effects, thereby reducing uncertainties related to extrapolation across species, endpoints and chemical structure. However, MOA-based testing seldom has been used for assessing the ecological risk of chemicals. This is in part because past regulatory mandates have focused more on adverse effects of chemicals (reductions in survival, growth or reproduction) than the pathways through which these effects are elicited. A recent departure from this involves endocrine-disrupting chemicals (EDCs), where there is a need to understand both MOA and adverse outcomes. To achieve this understanding, advances in predictive approaches are required whereby mechanistic changes caused by chemicals at the molecular level can be translated into apical responses meaningful to ecological risk assessment. In this paper we provide an overview and illustrative results from a large, integrated project that assesses the effects of EDCs on two small fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). For this work a systems-based approach is being used to delineate toxicity pathways for 12 model EDCs with different known or hypothesized toxic MOA. The studies employ a combination of state-of-the-art genomic (transcriptomic, proteomic, metabolomic), bioinformatic and modeling approaches, in conjunction with whole animal testing, to develop response linkages across biological levels of organization. This understanding forms the basis for predictive approaches for species, endpoint and chemical extrapolation. Although our project is focused specifically on EDCs in fish, we believe that the basic conceptual approach has utility for systematically assessing exposure and effects of chemicals with other MOA across a variety of biological systems.

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.

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.

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.

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.

The dynamics of energy production and utilization in fish eggs before and shortly after fertilization may be critical for embryo survival. Therefore, the current study examined the turnover of adenosine triphosphate (ATP) as well as examined the possible role and localization of ATP in unfertilized steelhead (Oncorhynchus mykiss) eggs and early embryos. The mean ATP level in unfertilized steelhead eggs was 1.92+/-0.10 (mean+/-S.E.M., n=17) nmol ATP per egg. Exposure of the unfertilized egg to 10 degrees C water (water activation) and fertilization resulted in comparable and substantial decreases (approx. 20-50%) in egg ATP levels within 3 min. This suggests that the energy expended at fertilization is used in response to water activation rather than fertilization per se. Unfertilized eggs maintained in ovarian fluid for 9 days at 10 degrees C under air showed a progressive decline of fertility that reached zero after 6 days. In contrast, no significant changes were seen in ATP levels throughout this 9 days period. Thus, fertility does not positively correlate with egg ATP levels in stored eggs. In the unfertilized egg, the ATP stored in the yolk accounted for approximately 1.5% of the total egg ATP. After fertilization, the concentration of ATP in the yolk increased approximately seven-fold, with the yolk and blastoderm each now accounting for approximately 20% of the total remaining ATP. Finally, to estimate the changes in oxidative metabolism following fertilization, the cyanide (KCN)-sensitive decline in total ATP was determined for unfertilized eggs and 1 day embryos. In the presence of KCN, ATP levels declined to approximately 50% within 24 h in both unfertilized eggs as well as embryos; the rates of ATP decline were not different. Therefore, there was not a discernible increase in ATP generation by oxidative phosphorylation at the time of fertilization.

Medaka fish are an established non-mammalian research model for the study of liver carcinogenesis and exposure to environmental pollutants. Studies have emphasized the development of hepatic neoplasms in medaka following exposure to model carcinogens. To date however, little information is known regarding the mechanisms underlying initiation of hepatic tumors in this species. The aim of this study was to relate our understanding of diethylnitrosamine (DEN)-induced tumor formation to ras gene activation in hepatic neoplasms of exposed medaka. Initial studies were conducted to identify medaka ras exons 1 and 2 by reverse transcriptase polymerase chain reaction (RT-PCR). Amplification of ras exons 1 and 2 from untreated medaka liver resulted in the identification of three polymorphic ras sequence variants exhibiting a high degree of homology to other teleost and mammalian ras genes. Exposure of medaka to 159 ppm of DEN resulted in a wide range of hepatic neoplasms including: hepatocellular adenomas, hepatocellular carcinomas, cholangiomas, and mixed hepatocholangiocellular carcinomas. Individual liver tumors were examined for oncogenically activating ras mutations by probing genomic DNA with probes specific for activating point mutations or by direct cloning and sequencing of ras transcripts using RT-PCR. Using allele-specific oligonucleotide (ASO) analysis, a single point mutation was detected in codon 12 position two in 8/25 (32%) tumors examined. Mutated ras alleles were additionally detected in 12 of 39 (30%) medaka liver tumors by sequence analysis. Ten of the 12 mutations identified contained a single point mutation at codon 12 resulting in a Gly to Asp amino acid substitution. Two unique mutations were identified at codon 16 resulting in either Lys to Asn or Lys to Thr amino acid substitutions. Our results show that ras mutations are induced by DEN and are present in over 30% of the fish that developed tumors. A ras mutation incidence of 30% is similar to that reported in mammalian species exposed to DEN. While mutations at codon 12 have previously been reported, the present study is the first in vivo report of ras point mutations at codon 16.

NASP is an H1 histone-binding protein that is cell cycle-regulated and occurs in two major forms: tNASP, found in gametes, embryonic cells, and transformed cells; and sNASP, found in all rapidly dividing somatic cells (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386). When full-length tNASP fused to green fluorescent protein (GFP) is transiently transfected into HeLa cells, it is efficiently transported into the nucleus within 2 h after translation in the cytoplasm, whereas the NASP nuclear localization signal (NLS) deletion mutant (NASP-DeltaNLS-GFP) is retained in the cytoplasm. In HeLa cells synchronized by a double thymidine block and transiently transfected to overexpress full-length tNASP or NASP-DeltaNLS, progression through the G(1)/S border is delayed. Cells transiently transfected to overexpress the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS) or sNASP were not delayed in progression through the G(1)/S border. By using a DNA supercoiling assay, in vitro binding data demonstrate that H1 histone-tNASP complexes can transfer H1 histones to DNA, whereas NASP-DeltaHBS cannot. Measurement of NASP mobility in the nucleus by fluorescence recovery after photobleaching indicates that NASP mobility is virtually identical to that reported for H1 histones. These data suggest that NASP-H1 complexes exist in the nucleus and that tNASP can influence cell cycle progression through the G(1)/S border through mediation of DNA-H1 histone binding.

We previously cloned and sequenced cDNAs encoding mouse NASP (mNASP), a cell cycle regulated histone H1 binding protein. Here we report the genomic sequence and organization for mNASP along with its 5' regulatory region and compare these with human NASP (hNASP). The mNASP gene contains 16 exons interrupted by 15 introns. The sequence encoding testis mNASP uses all 16 exons while the somatic form uses 13 exons by differential splicing. All the exons conform to the AG/GT splicing rule. Putative TATA box-containing transcription initiation sites are present for somatic NASP in human and mouse and for testis hNASP. Comparison of the promoter regions of mNASP and hNASP approximately 1 kb upstream of the transcription start sites for the two splice variants revealed a number of possible transcription factor binding sites relevant to specific patterns of NASP tissue expression. The presence of single bands on Southern blots of mouse genomic DNA suggests that mNASP is a single copy gene although pseudogenes exist in both the mouse and human genomes. Chromosome fluorescence by in situ hybridization revealed that mNASP is present on chromosome 4, in an area that corresponds to band 4D1, a region syntenic to the locus of hNASP on chromosome 1. Additionally, we report that human somatic and testis NASP mRNAs are expressed at varying levels in all the transformed cell lines and human tumors tested, further supporting NASP's role in the cell cycle of dividing cells.

Successful short-term storage of salmonid milt depends on numerous factors, including temperature, fluid volume, and gaseous environment, with storage at low temperatures under an atmosphere of 100% O2 being the most common method. Salmonid sperm maintained in a storage environment with elevated carbon dioxide (CO2) levels, such as the approximately 4% CO2 in exhaled air, are not motile when activated. While these modest levels of CO2 inhibit sperm motility, the effect is reversible within hours after exposure to a CO2-free oxygenated environment. Therefore, the effect of CO2 (as a component gas in the storage environment) on chinook salmon (Oncorhynchus tshawytscha) sperm motility and viability was examined. The hypothesis of the current investigation was that CO2-exposure with subsequent CO2 removal would be beneficial during short-term chinook salmon milt storage. Milt samples were collected from mature (adult) and precocious (jack) male chinook salmon and stored under various CO2 and O2 levels at 3 to 4 degrees C for up to 14 days. Milt samples were then removed from the incubation environments and maintained under CO2-free humidified air with continuous mixing for 4 h at 10 degrees C before analysis of motility. The resultant motility of samples incubated under 3.5% or less CO2 was not different than controls during the 14 d incubation period; motility of samples stored under higher CO2 tensions were significantly lower. The motility of samples incubated under 3.5% CO2 reached the maximum recovered motility after 2 h exposure to CO2-free humidified air, while the motility of sperm incubated under 13.4% CO2 levels recovered no motility even after 6 h exposure to CO2-free humidified air. The motility of samples incubated under normoxia was significantly greater than that of samples incubated under hyperoxia (approximately 90% O2) at both 7 and 14 d, regardless of the CO2 level. Sperm viability was relatively unaltered by any of the incubation conditions examined. The results of this investigation suggest that there is no apparent advantage to storage of chinook salmon sperm in the presence of CO2 and that storage under hyperoxia negatively affects sperm function compared to storage under normoxia.

Ecto-ATPase activity was measured for red blood cells, white blood cells, and whole blood from a variety of vertebrates. A large range of red blood cell ecto-ATPase activity was observed; for example, at 10 degrees C, red blood cells from a catastomid fish (Catostomus macrocheilus) and a newt (Taricha rivularis) had activities of 56 +/- 9 and 25,000,000 +/- 14,000,000 pmol ATP per 10(6) red blood cells per hour, respectively (mean +/- SD). Several control experiments verified that the measured ATPase activity was not the result of intracellular ATPases released due to cell damage or lysis nor due to the release of intracellular nucleoside triphosphate or uptake of extracellular ATP. Red blood cell ecto-ATPase activity was relatively low within the teleosts, was high within the reptiles, and had the greatest range and single highest value within the amphibians. Within the endotherms, avian red blood cell ecto-ATPase activities were greater than mammalian red blood cell ecto-ATPase activities, which were the lowest for all vertebrates examined. The lowest ecto-ATPase activities measured were for human and skunk red blood cells, which had activities of 13 +/- 1 and 11 +/- 2 pmol ATP per 10(6) red blood cells per hour, respectively, at 35 degrees C. Ecto-ATPase activity was measured in white blood cells of several vertebrate species and appeared generally high and less variable than red blood cell ecto-ATPase activity. Measured whole blood ecto-ATPase activity showed a range of three orders of magnitude and correlated positively with red blood cell ecto-ATPase activities. Ecto-ATPase activity was also determined for red blood cells from fetal, 1-3 d old neonatal, and pregnant garter snakes (Thamnophis elegans); these activities were not significantly different from the activity of red blood cells from nonpregnant adult females. Overall, the data from the present study demonstrate a wide range of red blood cell and whole blood ecto-ATPase activities among vertebrates and include some of the highest ecto-ATPase activities reported to date.