Preface to the special issue of Food and Chemical Toxicology on "New approach methodologies and machine learning in food safety and chemical risk assessment: Development of reproducible, open-source, and user-friendly tools for exposure, toxicokinetic, and toxicity assessments in the 21st century".

This Special Issue contains articles on applications of various new approach methodologies (NAMs) in the field of toxicology and risk assessment. These NAMs include in vitro high-throughput screening, quantitative structure-activity relationship (QSAR) modeling, physiologically based pharmacokinetic (PBPK) modeling, network toxicology analysis, molecular docking simulation, omics, machine learning, deep learning, and "template-and-anchor" multiscale computational modeling. These in vitro and in silico approaches complement each other and can be integrated together to support different applications of toxicology, including food safety assessment, dietary exposure assessment, chemical toxicity potency screening and ranking, chemical toxicity prediction, chemical toxicokinetic simulation, and to investigate the potential mechanisms of toxicities, as introduced further in selected articles in this Special Issue.

Searching for LINCS to Stress: Using Text Mining to Automate Reference Chemical Curation.

Adaptive stress response pathways (SRPs) restore cellular homeostasis following perturbation but may activate terminal outcomes like apoptosis, autophagy, or cellular senescence if disruption exceeds critical thresholds. Because SRPs hold the key to vital cellular tipping points, they are targeted for therapeutic interventions and assessed as biomarkers of toxicity. Hence, we are developing a public database of chemicals that perturb SRPs to enable new data-driven tools to improve public health. Here, we report on the automated text-mining pipeline we used to build and curate the first version of this database. We started with 100 reference SRP chemicals gathered from published biomarker studies to bootstrap the database. Second, we used information retrieval to find co-occurrences of reference chemicals with SRP terms in PubMed abstracts and determined pairwise mutual information thresholds to filter biologically relevant relationships. Third, we applied these thresholds to find 1206 putative SRP perturbagens within thousands of substances in the Library of Integrated Network-Based Cellular Signatures (LINCS). To assign SRP activity to LINCS chemicals, domain experts had to manually review at least three publications for each of 1206 chemicals out of 181,805 total abstracts. To accomplish this efficiently, we implemented a machine learning approach to predict SRP classifications from texts to prioritize abstracts. In 5-fold cross-validation testing with a corpus derived from the 100 reference chemicals, artificial neural networks performed the best (F1-macro = 0.678) and prioritized 2479/181,805 abstracts for expert review, which resulted in 457 chemicals annotated with SRP activities. An independent analysis of enriched mechanisms of action and chemical use class supported the text-mined chemical associations (p < 0.05): heat shock inducers were linked with HSP90 and DNA damage inducers to topoisomerase inhibition. This database will enable novel applications of LINCS data to evaluate SRP activities and to further develop tools for biomedical information extraction from the literature.

Evaluation of Growth Performance and Environmental Impact of Hermetia illucens Larvae Reared on Coffee Silverskins Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens is a promising insect due to its ability to convert low-value substrates as food chain by-products into highly nutritious feed. Its feeding and nutrition are important issues. The aim of this work was to investigate the effect of different substrates consisting of coffee silverskin, a by-product of the roasting process, enriched with different inclusions of microalgae (5%, 10%, 20%, and 25%), Schizochytrium limacinum, and Isochrysis galbana, combined with the assessment of environmental sustainability by LCA. In general, the addition of microalgae led to an increase in larval growth performance due to the higher content of protein and lipids, although S. limacinum showed the best results with respect to larvae fed with coffee silverskin enriched with I. galbana. A higher prepupal weight was observed in larvae fed with 10%, 20%, and 25% S. limacinum; shorter development times in larvae fed with 25% of both S. limacinum and I. galbana; and a higher growth rate in larvae fed with 25% S. limacinum. The 10% S. limacinum inclusion was only slightly different from the higher inclusions. Furthermore, 10% of S. limacinum achieved the best waste reduction index. The greater the inclusion of microalgae, the greater the environmental impact of larval production. Therefore, the addition of 10% S. limacinum appears to be the best compromise for larval rearing, especially considering that a higher inclusion of microalgae did not yield additional benefits in terms of the nutritional value of H. illucens prepupae.

Exploring the effects of experimental parameters and data modeling approaches on in vitro transcriptomic point-of-departure estimates.

Multiple new approach methods (NAMs) are being developed to rapidly screen large numbers of chemicals to aid in hazard evaluation and risk assessments. High-throughput transcriptomics (HTTr) in human cell lines has been proposed as a first-tier screening approach for determining the types of bioactivity a chemical can cause (activation of specific targets vs. generalized cell stress) and for calculating transcriptional points of departure (tPODs) based on changes in gene expression. In the present study, we examine a range of computational methods to calculate tPODs from HTTr data, using six data sets in which MCF7 cells cultured in two different media formulations were treated with a panel of 44 chemicals for 3 different exposure durations (6, 12, 24 hr). The tPOD calculation methods use data at the level of individual genes and gene set signatures, and compare data processed using the ToxCast Pipeline 2 (tcplfit2), BMDExpress and PLIER (Pathway Level Information ExtractoR). Methods were evaluated by comparing to in vitro PODs from a validated set of high-throughput screening (HTS) assays for a set of estrogenic compounds. Key findings include: (1) for a given chemical and set of experimental conditions, tPODs calculated by different methods can vary by several orders of magnitude; (2) tPODs are at least as sensitive to computational methods as to experimental conditions; (3) in comparison to an external reference set of PODs, some methods give generally higher values, principally PLIER and BMDExpress; and (4) the tPODs from HTTr in this one cell type are mostly higher than the overall PODs from a broad battery of targeted in vitro ToxCast assays, reflecting the need to test chemicals in multiple cell types and readout technologies for in vitro hazard screening.

The probiotic SLAB51 as agent to counteract BPA toxicity on zebrafish gut microbiota -liver-brain axis.

A plethora of studies have so far described the toxic effects of bisphenol A (BPA) on organism health, highlighting the urgent need to find new strategies not only to reduce the presence of this toxicant but also to counteract its adverse effects. In this context, probiotics emerged as a potential tool since they promote organism welfare. Using a multidisciplinary approach, this study explores the effects of SLAB51 dietary administration to counteract BPA toxicity using zebrafish as a model. Adult males and females were maintained under standard conditions (control group; C), exposed for 28 days via the water to an environmental relevant dose of BPA (10 µg/L; BPA), dietary treated with SLAB51 (109 CFU/g of body weight; P) and co-treated with BPA plus SLAB51 (BPA + P). In the gut, exposure to BPA resulted in altered architecture in both males and females, with females also experiencing an increase of pathogenic bacterial species. Co-administration of BPA + P led to the restoration of normal gut architecture, favored beneficial bacteria colonization, and decreased the abundance of pathogenic species. In the liver, male BPA exposure led to steatosis and glycogen depletion, which was partially mitigated by SLAB51 co-administration. In contrast, in females exposed to BPA, the lack of steatosis along with the greater glycogen depletion, suggested an increase in energy demand as supported by the metabolomic phenotype. The analysis of liver metabolites in BPA + P males revealed increased levels of anserine and reduced levels of glutamine, which could lie behind the counteraction of the brain histopathological damage caused by BPA. In BPA + P females, a reduction of retinoic acid was found in the liver, suggesting an increase in retinoids responsible for BPA detoxification. Overall, these results demonstrate that SLAB51 exerts its beneficial effects on the gut microbiota-brain-liver axis through distinct molecular pathways, effectively mitigating the pleiotropic toxicity of BPA.

Genes-to-Pathways Species Conservation Analysis: Enabling the Exploration of Conservation of Biological Pathways and Processes Across Species.

The last two decades have witnessed a strong momentum toward integration of cell-based and computational approaches in safety assessments. This is fueling a global regulatory paradigm shift toward reduction and replacement of the use of animals in toxicity tests while promoting the use of new approach methodologies. The understanding of conservation of molecular targets and pathways provides an opportunity to extrapolate effects across species and ultimately to determine the taxonomic applicability domain of assays and biological effects. Despite the wealth of genome-linked data available, there is a compelling need for improved accessibility, while ensuring that it reflects the underpinning biology. We present the novel pipeline Genes-to-Pathways Species Conservation Analysis (G2P-SCAN) to further support understanding on cross-species extrapolation of biological processes. This R package extracts, synthetizes, and structures the data available from different databases, that is, gene orthologs, protein families, entities, and reactions, linked to human genes and respective pathways across six relevant model species. The use of G2P-SCAN enables the overall analysis of orthology and functional families to substantiate the identification of conservation and susceptibility at the pathway level. In the present study we discuss five case studies, demonstrating the validity of the developed pipeline and its potential use as species extrapolation support. We foresee this pipeline will provide valuable biological insights and create space for the use of mechanistically based data to inform potential species susceptibility for research and safety decision purposes. Environ Toxicol Chem 2023;42:1152-1166. © 2023 UNILEVER GLOBAL IP LTD. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Data-driven learning of narcosis mode of action identifies a CNS transcriptional signature shared between whole organism Caenorhabditis elegans and a fish gill cell line.

With the large numbers of man-made chemicals produced and released in the environment, there is a need to provide assessments on their potential effects on environmental safety and human health. Current regulatory frameworks rely on a mix of both hazard and risk-based approaches to make safety decisions, but the large number of chemicals in commerce combined with an increased need to conduct assessments in the absence of animal testing makes this increasingly challenging. This challenge is catalysing the use of more mechanistic knowledge in safety assessment from both in silico and in vitro approaches in the hope that this will increase confidence in being able to identify modes of action (MoA) for the chemicals in question. Here we approach this challenge by testing whether a functional genomics approach in C. elegans and in a fish cell line can identify molecular mechanisms underlying the effects of narcotics, and the effects of more specific acting toxicants. We show that narcosis affects the expression of neuronal genes associated with CNS function in C. elegans and in a fish cell line. Overall, we believe that our study provides an important step in developing mechanistically relevant biomarkers which can be used to screen for hazards, and which prevent the need for repeated animal or cross-species comparisons for each new chemical.

Latent Variables Capture Pathway-Level Points of Departure in High-Throughput Toxicogenomic Data.

Estimation of points of departure (PoDs) from high-throughput transcriptomic data (HTTr) represents a key step in the development of next-generation risk assessment (NGRA). Current approaches mainly rely on single key gene targets, which are constrained by the information currently available in the knowledge base and make interpretation challenging as scientists need to interpret PoDs for thousands of genes or hundreds of pathways. In this work, we aimed to address these issues by developing a computational workflow to investigate the pathway concentration-response relationships in a way that is not fully constrained by known biology and also facilitates interpretation. We employed the Pathway-Level Information ExtractoR (PLIER) to identify latent variables (LVs) describing biological activity and then investigated in vitro LVs' concentration-response relationships using the ToxCast pipeline. We applied this methodology to a published transcriptomic concentration-response data set for 44 chemicals in MCF-7 cells and showed that our workflow can capture known biological activity and discriminate between estrogenic and antiestrogenic compounds as well as activity not aligning with the existing knowledge base, which may be relevant in a risk assessment scenario. Moreover, we were able to identify the known estrogen activity in compounds that are not well-established ER agonists/antagonists supporting the use of the workflow in read-across. Next, we transferred its application to chemical compounds tested in HepG2, HepaRG, and MCF-7 cells and showed that PoD estimates are in strong agreement with those estimated using a recently developed Bayesian approach (cor = 0.89) and in weak agreement with those estimated using a well-established approach such as BMDExpress2 (cor = 0.57). These results demonstrate the effectiveness of using PLIER in a concentration-response scenario to investigate pathway activity in a way that is not fully constrained by the knowledge base and to ease the biological interpretation and support the development of an NGRA framework with the ability to improve current risk assessment strategies for chemicals using new approach methodologies.

A Comparison of Reproductive Performances in Young and Old Females: A Case Study on the Atlantic Bluefin Tuna in the Mediterranean Sea.

In the Mediterranean Sea, a demographic substructure of the Atlantic bluefin tuna Thunnus thynnus has emerged over the last decade, with old and young individuals exhibiting different horizontal movements and spatial-temporal patterns of gonad maturation. In the present study, histology and molecular reproductive markers were integrated with the gonad-specific mir-202 gene expression and ovarian localization to provide a comprehensive picture of the reproductive performances in young and old females and investigate the role played by the mir-202 during gonadal maturation. During the reproductive period, old females (>100 kg; 194.6 ± 33.9 cm straight fork length; 11.3 ± 2.7 years old) were found to have greater reproductive performances than younger females (<80 kg; 139.3 ± 18.8 cm straight fork length; 8.4 ± 1.1 years old) according to gene expression results, suggesting a prolonged spawning season, earlier arrival on spawning grounds and/or better condition in older females. The mir-202-5p showed no global changes; it was abundantly expressed in granulosa cells and faintly present in the ooplasm. On the other hand, the mir-202-3p expression profile reflected levels of oocyte maturation molecular markers (star, lhr) and both histological and molecular (casp3) levels of follicular atresia. Overall, old females exhibited greater reproductive performances than younger females, likely reflecting different reproductive dynamics linked to the physical condition, habitat usage and migratory behaviour. These results highlight the importance of preserving large and old females in the context of fishery management. Finally, the mir-202 appears to be a good candidate to regulate the reproductive output of this species in an autocrine/paracrine manner through either stage- or age-dependent processes.

The probiotic Lactobacillus rhamnosus mimics the dark-driven regulation of appetite markers and melatonin receptors' expression in zebrafish (Danio rerio) larvae: Understanding the role of the gut microbiome.

The use of probiotics has been recently considered a novel therapeutic strategy to prevent pathologies such as obesity; however, the specific mechanisms of action by which probiotics exert their beneficial effects on metabolic health remain unclear. The aim of the present study was to investigate the short-term effects of a probiotic Lactobacillus rhamnosus supplementation (PROB) on appetite regulation, growth-related markers, and microbiota diversity in zebrafish (Danio rerio) larvae, compared to a group subjected to a constant darkness photoperiod (DARK), as well as to evaluate the effects of both treatments on melatonin receptors' expression. After a 24 h treatment, both PROB and DARK conditions caused a significant increase in leptin a expression. Moreover, mRNA abundances of leptin b and proopiomelanocortin a were elevated in the PROB group, and DARK showed a similar tendency, supporting a negative regulation of appetite markers by the treatments. Moreover, both PROB and DARK also enhanced the abundances of melatonin receptors transcript (melatonin receptor 1 ba and bb) and protein (melatonin receptor 1) suggesting a potential involvement of melatonin in mediating these effects. Nevertheless, treatments did not exhibit a significant effect on the expression of most of the growth hormone/insulin-like growth factor axis genes evaluated. Finally, only the DARK condition significantly modulated gut microbiota diversity at such short time, altogether highlighting the rapid effects of this probiotic on modulating appetite regulatory and melatonin receptors' expression, without a concomitant variation of gut microbiota.

Opsins and gonadal circadian rhythm in the swordfish (Xiphias gladius) ovary: Their potential roles in puberty and reproductive seasonality.

Circadian rhythm is well known to play a pivotal role in reproduction but the presence of a gonadal circadian rhythm is opening a lot of questions about a local regulation of reproduction. In the present study, we first set to identify the key genes driving circadian rhythmicity from the gonadal transcriptome of the swordfish, a commercially relevant species undergoing overfishing, and we then investigated whether their transcriptional activity was influenced by the maturation stage. Finally, we explored whether seasonality had the ability to modulate the expression of these genes. Interestingly, we identified a wide number of circadian rhythm related genes in the transcriptome of the swordfish gonad including, among the others, clock, bmal1, cry2 and per2, which have been found to be differentially expressed between sexually immature and mature individuals sampled during the breeding season. This differential modulation was also found for melatonin biosynthesis genes (mel1b, asmt and tph1) as well as opsin genes (opsin4, tmt opsin, parapinopsin, VA opsin, rho and sws), known to be the primary receptors of light stimuli. These differences were not observed between mature and immature individuals when considering the non-breeding season suggesting that, despite the molecular machinery of mature gonads is able to respond to signals driving ovarian maturation, these signals are not present hence highlighting the potential role of seasonality in modulating the gonadal circadian rhythm. These results confirm the presence of a gonadal circadian rhythm in the swordfish and open new interesting questions about its role in driving puberty onset.

New Associations between Drug-Induced Adverse Events in Animal Models and Humans Reveal Novel Candidate Safety Targets.

To improve our ability to extrapolate preclinical toxicity to humans, there is a need to understand and quantify the concordance of adverse events (AEs) between animal models and clinical studies. In the present work, we discovered 3011 statistically significant associations between preclinical and clinical AEs caused by drugs reported in the PharmaPendium database of which 2952 were new associations between toxicities encoded by different Medical Dictionary for Regulatory Activities terms across species. To find plausible and testable candidate off-target drug activities for the derived associations, we investigated the genetic overlap between the genes linked to both a preclinical and a clinical AE and the protein targets found to interact with one or more drugs causing both AEs. We discuss three associations from the analysis in more detail for which novel candidate off-target drug activities could be identified, namely, the association of preclinical mutagenicity readouts with clinical teratospermia and ovarian failure, the association of preclinical reflexes abnormal with clinical poor-quality sleep, and the association of preclinical psychomotor hyperactivity with clinical drug withdrawal syndrome. Our analysis successfully identified a total of 77% of known safety targets currently tested in in vitro screening panels plus an additional 431 genes which were proposed for investigation as future safety targets for different clinical toxicities. This work provides new translational toxicity relationships beyond AE term-matching, the results of which can be used for risk profiling of future new chemical entities for clinical studies and for the development of future in vitro safety panels.

De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage.

In the present work, we assembled and characterized a de novo larval transcriptome of the Atlantic bluefin tuna Thunnus thynnus by taking advantage of publicly available databases with the goal of better understanding its larval development. The assembled transcriptome comprised 37,117 protein-coding transcripts, of which 13,633 full-length (>80% coverage), with an Ex90N50 of 3061 bp and 76% of complete and single-copy core vertebrate genes orthologues. Of these transcripts, 34,980 had a hit against the EggNOG database and 14,983 with the KEGG database. Codon usage bias was identified in processes such as translation and muscle development. By comparing our data with a set of representative fish species, 87.1% of tuna transcripts were included in orthogroups with other species and 5.1% in assembly-specific orthogroups, which were enriched in terms related to muscle and bone development, visual system and ion transport. Following this comparative approach, protein families related to myosin, extracellular matrix and immune system resulted significantly expanded in the Atlantic bluefin tuna. Altogether, these results provide a glimpse of how the Atlantic bluefin tuna might have achieved early physical advantages over competing species in the pelagic environment. The information generated lays the foundation for future research on the more detailed exploration of physiological responses at the molecular level in different larval stages and paves the way to evolutionary studies on the Atlantic bluefin tuna.

Photoperiod Manipulation Affects Transcriptional Profile of Genes Related to Lipid Metabolism and Apoptosis in Zebrafish (Danio rerio) Larvae: Potential Roles of Gut Microbiota.

Gut microbiota plays a fundamental role in maintaining host's health by controlling a wide range of physiological processes. Administration of probiotics and manipulation of photoperiod have been suggested as modulators of microbial composition and are currently undergoing an extensive research in aquaculture as a way to improve health and quality of harvested fish. However, our understanding regarding their effects on physiological processes is still limited. In the present study we investigated whether manipulation of photoperiod and/or probiotic administration was able to alter microbial composition in zebrafish larvae at hatching stage. Our findings show that probiotic does not elicit effects while photoperiod manipulation has a significant impact on microbiota composition. Moreover, we successfully predicted lipid biosynthesis and apoptosis to be modulated by microbial communities undergoing continuous darkness. Interestingly, expression levels of caspase 3 gene (casp3) and lipid-related genes (hnf4a, npc1l1, pparγ, srebf1, agpat4 and fitm2) were found to be significantly overexpressed in dark-exposed larvae, suggesting an increase in the occurrence of apoptotic processes and a lipid metabolism impairment, respectively (p < 0.05). Our results provide the evidence that microbial communities in zebrafish at early life stages are not modulated by a short administration of probiotics and highlight the significant effect that dark photoperiod elicits on zebrafish microbiota and potentially on health.

Nitrogen and chlorophyll status determination in durum wheat as influenced by fertilization and soil management: Preliminary results.

Handheld chlorophyll meters as Soil Plant Analysis Development (SPAD) have proven to be useful tools for rapid, no-destructive assessment of chlorophyll and nitrogen status in various crops. This method is used to diagnose the need of nitrogen fertilization to improve the efficiency of the agricultural system and to minimize nitrogen losses and deficiency. The objective of this study is to evaluate the effect of repeated conservative agriculture practices on the SPAD readings, leaves chlorophyll concentration and Nitrogen Nutrition Index (NNI) relationships in durum wheat under Mediterranean conditions. The experimental site is a part of a long-term-experiment established in 1994 and is still on-going where three tillage managements and three nitrogen fertilizer treatments were repeated in the same plots every year. We observed a linear relationship between the SPAD readings performed in the central and distal portion of the leaf (R2 = 0.96). In fertilized durum wheat, we found all positive exponential relationships between SPAD readings, chlorophyll leaves concentration (R2 = 0.85) and NNI (R2 = 0.89). In the unfertilized treatment, the SPAD has a good attitude to estimate leaves chlorophyll concentration (R2 = 0.74) and NNI (R2 = 0.77) only in crop grow a soil with relative high content of soil organic matter and nitrogen availability, as observed in the no tilled plots. The results show that the SPAD can be used for a correct assessment of chlorophyll and nitrogen status in durum wheat but also to evaluate indirectly the content of soil organic matter and nitrogen availability during different growth stages of the crop cycle.

A de novo transcriptome assembly approach elucidates the dynamics of ovarian maturation in the swordfish (Xiphias gladius).

The Mediterranean swordfish (Xiphias gladius) has been recently classified as overfished and in 2016, the International Commission for the Conservation of the Atlantic Tunas (ICCAT) established a multi-annual management plan to recover this stock. To successfully achieve this goal, knowledge about swordfish biology is needed. To date, few studies on swordfish have been performed and none of them has provided useful insights into the reproductive biology at molecular level. Here we set to characterise the molecular dynamics underlying ovarian maturation by employing a de novo transcriptome assembly approach. Differential gene expression analysis in mature and immature ovaries identified a number of differentially expressed genes associated with biological processes driving ovarian maturation. Focusing on ovarian steroidogenesis and vitellogenin uptake, we depict the molecular dynamics characterizing these processes while a phylogenetic analysis let us identify a candidate vitellogenin receptor. This is the first swordfish transcriptome assembly and these findings provide in-depth understanding of molecular processes describing ovarian maturation. Moreover, the establishment of a publicly available database containing information on the swordfish transcriptome aims to boost research on this species with the long-term of developing more comprehensive and successful stock management plans.

Endocrine-disrupting chemicals in aquatic environment: what are the risks for fish gametes?

Over the past 25 years, extensive research in vertebrate species has identified several genomic pathways altered by exposures to anthropogenic chemicals with hormone-like activity mediated by their interaction with nuclear receptors. In addition, many pollutants have been shown to interfere with non-genomic (non-classical) pathways, but this mechanism of endocrine disruption is still poorly understood. Recently, the number of publications describing the effects of Endocrine disrupting chemicals (EDCs) on fish reproduction, focusing on the deregulation of the hypothalamus-pituitary-gonadal axis as well as on gamete quality, significantly increased. Depending on their ability to mimic endogenous hormones, the may differently affect male or female reproductive physiology. Inhibition of gametogenesis, development of intersex gonads, alteration of the gonadosomatic index, and decreased fertility rate have been largely documented. In males, alterations of sperm density, motility, and fertility have been observed in several wild species. Similar detrimental effects were described in females, including negative outcomes on oocyte growth and maturation plus the occurrence of apoptotic/autophagic processes. These pathways may affect gamete viability considered as one of the major indicators of reproductive endocrine disruption. Pollutants act also at DNA level producing DNA mutations and changes in epigenetic pathways inducing specific mechanisms of toxicity and/or aberrant cellular responses that may affect subsequent generation(s) through the germline. In conclusion, this review summarizes the effects caused by EDC exposure on fish reproduction, focusing on gametogenesis, giving a general overview of the different aspects dealing with this issue, from morphological alteration, deregulation of steroidogenesis, hormonal synthesis, and occurrence of epigenetic process.

In Silico Computational Transcriptomics Reveals Novel Endocrine Disruptors in Largemouth Bass ( Micropterus salmoides).

In recent years, decreases in fish populations have been attributed, in part, to the effect of environmental chemicals on ovarian development. To understand the underlying molecular events we developed a dynamic model of ovary development linking gene transcription to key physiological end points, such as gonadosomatic index (GSI), plasma levels of estradiol (E2) and vitellogenin (VTG), in largemouth bass ( Micropterus salmoides). We were able to identify specific clusters of genes, which are affected at different stages of ovarian development. A subnetwork was identified that closely linked gene expression and physiological end points and by interrogating the Comparative Toxicogenomic Database (CTD), quercetin and tretinoin (ATRA) were identified as two potential candidates that may perturb this system. Predictions were validated by investigation of reproductive associated transcripts using qPCR in ovary and in the liver of both male and female largemouth bass treated after a single injection of quercetin and tretinoin (10 and 100 µg/kg). Both compounds were found to significantly alter the expression of some of these genes. Our findings support the use of omics and online repositories for identification of novel, yet untested, compounds. This is the first study of a dynamic model that links gene expression patterns across stages of ovarian development.