Transcriptomic point of departure determination: a comparison of distribution-based and gene set-based approaches.

A key step in assessing the potential human and environmental health risks of industrial and agricultural chemicals is to determine the toxicity point of departure (POD), which is the highest dose level that causes no adverse effect. Transcriptomic POD (tPOD) values have been suggested to accurately estimate toxicity POD values. One step in the most common approach for tPOD determination involves mapping genes to annotated gene sets, a process that might lead to substantial information loss particularly in species with poor gene annotation. Alternatively, methods that calculate tPOD values directly from the distribution of individual gene POD values omit this mapping step. Using rat transcriptome data for 79 molecules obtained from Open TG-GATEs (Toxicogenomics Project Genomics Assisted Toxicity Evaluation System), the hypothesis was tested that methods based on the distribution of all individual gene POD values will give a similar tPOD value to that obtained via the gene set-based method. Gene set-based tPOD values using four different gene set structures were compared to tPOD values from five different individual gene distribution methods. Results revealed a high tPOD concordance for all methods tested, especially for molecules with at least 300 dose-responsive probesets: for 90% of those molecules, the tPOD values from all methods were within 4-fold of each other. In addition, random gene sets based upon the structure of biological knowledge-derived gene sets produced tPOD values with a median absolute fold change of 1.3-1.4 when compared to the original biological knowledge-derived gene set counterparts, suggesting that little biological information is used in the gene set-based tPOD generation approach. These findings indicate using individual gene distributions to calculate a tPOD is a viable and parsimonious alternative to using gene sets. Importantly, individual gene distribution-based tPOD methods do not require knowledge of biological organization and can be applied to any species including those with poorly annotated gene sets.

From Pipeline to Plant Protection Products: Using New Approach Methodologies (NAMs) in Agrochemical Safety Assessment.

The human population will be approximately 9.7 billion by 2050, and food security has been identified as one of the key issues facing the global population. Agrochemicals are an important tool available to farmers that enable high crop yields and continued access to healthy foods, but the average new agrochemical active ingredient takes more than ten years, 350 million dollars, and 20,000 animals to develop and register. The time, monetary, and animal costs incentivize the use of New Approach Methodologies (NAMs) in early-stage screening to prioritize chemical candidates. This review outlines NAMs that are currently available or can be adapted for use in early-stage screening agrochemical programs. It covers new in vitro screens that are on the horizon in key areas of regulatory concern. Overall, early-stage screening with NAMs enables the prioritization of development for agrochemicals without human and environmental health concerns through a more directed, agile, and iterative development program before animal-based regulatory testing is even considered.

Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Applying genomics in regulatory toxicology: a report of the ECETOC workshop on omics threshold on non-adversity.

In a joint effort involving scientists from academia, industry and regulatory agencies, ECETOC's activities in Omics have led to conceptual proposals for: (1) A framework that assures data quality for reporting and inclusion of Omics data in regulatory assessments; and (2) an approach to robustly quantify these data, prior to interpretation for regulatory use. In continuation of these activities this workshop explored and identified areas of need to facilitate robust interpretation of such data in the context of deriving points of departure (POD) for risk assessment and determining an adverse change from normal variation. ECETOC was amongst the first to systematically explore the application of Omics methods, now incorporated into the group of methods known as New Approach Methodologies (NAMs), to regulatory toxicology. This support has been in the form of both projects (primarily with CEFIC/LRI) and workshops. Outputs have led to projects included in the workplan of the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) group of the Organisation for Economic Co-operation and Development (OECD) and to the drafting of OECD Guidance Documents for Omics data reporting, with potentially more to follow on data transformation and interpretation. The current workshop was the last in a series of technical methods development workshops, with a sub-focus on the derivation of a POD from Omics data. Workshop presentations demonstrated that Omics data developed within robust frameworks for both scientific data generation and analysis can be used to derive a POD. The issue of noise in the data was discussed as an important consideration for identifying robust Omics changes and deriving a POD. Such variability or "noise" can comprise technical or biological variation within a dataset and should clearly be distinguished from homeostatic responses. Adverse outcome pathways (AOPs) were considered a useful framework on which to assemble Omics methods, and a number of case examples were presented in illustration of this point. What is apparent is that high dimension data will always be subject to varying processing pipelines and hence interpretation, depending on the context they are used in. Yet, they can provide valuable input for regulatory toxicology, with the pre-condition being robust methods for the collection and processing of data together with a comprehensive description how the data were interpreted, and conclusions reached.

A Transformative Vision for an Omics-Based Regulatory Chemical Testing Paradigm.

Use of molecular data in human and ecological health risk assessments of industrial chemicals and agrochemicals has been anticipated by the scientific community for many years; however, these data are rarely used for risk assessment. Here, a logic framework is proposed to explore the feasibility and future development of transcriptomic methods to refine and replace the current apical endpoint-based regulatory toxicity testing paradigm. Four foundational principles are outlined and discussed that would need to be accepted by stakeholders prior to this transformative vision being realized. Well-supported by current knowledge, the first principle is that transcriptomics is a reliable tool for detecting alterations in gene expression that result from endogenous or exogenous influences on the test organism. The second principle states that alterations in gene expression are indicators of adverse or adaptive biological responses to stressors in an organism. Principle 3 is that transcriptomics can be employed to establish a benchmark dose-based point of departure (POD) from short-term, in vivo studies at a dose level below which a concerted molecular change (CMC) is not expected. Finally, Principle 4 states that the use of a transcriptomic POD (set at the CMC dose level) will support a human health-protective risk assessment. If all four principles are substantiated, this vision is expected to transform aspects of the industrial chemical and agrochemical risk assessment process that are focused on establishing safe exposure levels for mammals across numerous toxicological contexts resulting in a significant reduction in animal use while providing equal or greater protection of human health. Importantly, these principles and approaches are also generally applicable for ecological safety assessment.

A microRNA or messenger RNA point of departure estimates an apical endpoint point of departure in a rat developmental toxicity model.

Traditional developmental toxicity testing practice examines fetal apical endpoints to identify a point of departure (POD) for risk assessment. A potential new testing paradigm involves deriving a POD from a comprehensive analysis of molecular-level change. Here, the rat ketoconazole endocrine-mediated developmental toxicity model was used to test the hypothesis that maternal epigenomic (miRNA) and transcriptomic (mRNA) PODs are similar to fetal apical endpoint PODs. Sprague-Dawley rats were exposed from gestation day (GD) 6-21 to 0, 0.063, 0.2, 0.63, 2, 6.3, 20, or 40 mg/kg/day ketoconazole. Dam systemic, liver, and placenta PODs, along with GD 21 fetal resorption, body weight, and skeletal apical PODs were derived using BMDS software. GD 21 dam liver and placenta miRNA and mRNA PODs were obtained using three methods: a novel individual molecule POD accumulation method, a first mode method, and a gene set method. Dam apical POD values ranged from 2.0 to 38.6 mg/kg/day; the lowest value was for placenta histopathology. Fetal apical POD values were 10.9-20.3 mg/kg/day; the lowest value was for fetal resorption. Dam liver miRNA and mRNA POD values were 0.34-0.69 mg/kg/day, and placenta miRNA and mRNA POD values were 2.53-6.83 mg/kg/day. Epigenomic and transcriptomic POD values were similar across liver and placenta. Deriving a molecular POD from dam liver or placenta was protective of a fetal apical POD. These data support the conclusion that a molecular POD can be used to estimate, or be protective of, a developmental toxicity apical POD.

A Collaborative Initiative to Establish Genomic Biomarkers for Assessing Tumorigenic Potential to Reduce Reliance on Conventional Rodent Carcinogenicity Studies.

There is growing recognition across broad sectors of the scientific community that use of genomic biomarkers has the potential to reduce the need for conventional rodent carcinogenicity studies of industrial chemicals, agrochemicals, and pharmaceuticals through a weight-of-evidence approach. These biomarkers fall into 2 major categories: (1) sets of gene transcripts that can identify distinct tumorigenic mechanisms of action; and (2) cancer driver gene mutations indicative of rapidly expanding growth-advantaged clonal cell populations. This call-to-action article describes a collaborative approach launched to develop and qualify biomarker gene expression panels that measure widely accepted molecular pathways linked to tumorigenesis and their activation levels to predict tumorigenic doses of chemicals from short-term exposures. Growing evidence suggests that application of such biomarker panels in short-term exposure rodent studies can identify both tumorigenic hazard and tumorigenic activation levels for chemical-induced carcinogenicity. In the future, this approach will be expanded to include methodologies examining mutations in key cancer driver gene mutation hotspots as biomarkers of both genotoxic and nongenotoxic chemical tumor risk. Analytical, technical, and biological validation studies of these complementary genomic tools are being undertaken by multisector and multidisciplinary collaborative teams within the Health and Environmental Sciences Institute. Success from these efforts will facilitate the transition from current heavy reliance on conventional 2-year rodent carcinogenicity studies to more rapid animal- and resource-sparing approaches for mechanism-based carcinogenicity evaluation supporting internal and regulatory decision-making.

Bridging Sex-Specific Differences in the CAR-Mediated Hepatocarcinogenesis of Nitrapyrin Using Molecular and Apical Endpoints.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in B6C3F1 mice. In a 2-year oncogenicity study, increased incidence of mice with hepatocellular tumors was observed following exposure to 125 (females only) or 250 mg/kg/day (males and females) nitrapyrin in the diet. Previous data was generated in male mice to support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor (NR) activation, increased hepatocellular proliferation, and subsequent hepatocellular foci and tumor formation. Uncertainty as to the relevance of this MoA for females remained given the increased sensitivity to tumor formation in female mice. A targeted MoA study was conducted to evaluate CAR activation and hepatic responses in female mice treated with the female carcinogenic dose of nitrapyrin for 4 days. Nitrapyrin induced a treatment-related increase in hepatocellular hypertrophy and hepatocellular proliferation. Nitrapyrin also induced a dose-related increase in the Cyp2b10/CAR-associated transcript and liver weights. Nitrapyrin-induced liver weights and Cyp2b10 gene expression for both males and females were compared to data generated from three other established CAR activators; methyl isobutyl ketone, phenobarbital, and sulfoxaflor. The response observed in female mice following exposure to nitrapyrin was within range of the degree of change observed in mice following exposure to tumorigenic doses of other CAR activators. Consistent with the liver MoA in male mice, these data support a CAR-mediated mode of action for nitrapyrin-induced liver tumors in female mice, with the understanding that a focused approach minimizing animal use can bridge male and female datasets when sex-specific carcinogenic differences are observed.

A rat subchronic study transcriptional point of departure estimates a carcinogenicity study apical point of departure.

Considerations of human relevance and animal use are driving research to identify new approaches to inform risk assessment of chemicals and replace guideline-based rodent carcinogenicity tests. Here, the hypothesis was tested across four agrochemicals that 1) a rat 90-day transcriptome-based BEPOD is protective of a rat carcinogenicity study and 2) a subchronic liver or kidney BEPOD would approximate a cancer bioassay apical POD derived from other organs and a rat subchronic BEPOD would approximate a mouse cancer bioassay apical POD. Using RNA sequencing and BMDExpress software, liver and/or kidney BEPOD values were generated in male rats exposed for 90 days to either Triclopyr Acid, Pronamide, Sulfoxaflor, or Fenpicoxamid. BEPOD values were compared to benchmark dose-derived apical POD values generated from rat 90-day and rodent carcinogenicity studies. Across all four agrochemicals, findings showed that a rat 90-day study BEPOD approximated the most sensitive apical POD (within 10-fold) generated from the 90-day rat study and long-term rodent carcinogenicity studies. This study supports the conclusion that a subchronic transcriptome-based BEPOD could be utilized to estimate an apical POD within a risk-based approach of chronic toxicity and carcinogenicity agrochemical assessment, abrogating the need for time- and resource-intensive rodent carcinogenicity studies and minimizing animal testing.

Identification of early liver toxicity gene biomarkers using comparative supervised machine learning.

Screening agrochemicals and pharmaceuticals for potential liver toxicity is required for regulatory approval and is an expensive and time-consuming process. The identification and utilization of early exposure gene signatures and robust predictive models in regulatory toxicity testing has the potential to reduce time and costs substantially. In this study, comparative supervised machine learning approaches were applied to the rat liver TG-GATEs dataset to develop feature selection and predictive testing. We identified ten gene biomarkers using three different feature selection methods that predicted liver necrosis with high specificity and selectivity in an independent validation dataset from the Microarray Quality Control (MAQC)-II study. Nine of the ten genes that were selected with the supervised methods are involved in metabolism and detoxification (Car3, Crat, Cyp39a1, Dcd, Lbp, Scly, Slc23a1, and Tkfc) and transcriptional regulation (Ablim3). Several of these genes are also implicated in liver carcinogenesis, including Crat, Car3 and Slc23a1. Our biomarker gene signature provides high statistical accuracy and a manageable number of genes to study as indicators to potentially accelerate toxicity testing based on their ability to induce liver necrosis and, eventually, liver cancer.

A Rat Liver Transcriptomic Point of Departure Predicts a Prospective Liver or Non-liver Apical Point of Departure.

Identifying a toxicity point of departure (POD) is a required step in human health risk characterization of crop protection molecules, and this POD has historically been derived from apical endpoints across a battery of animal-based toxicology studies. Using rat transcriptome and apical data for 79 molecules obtained from Open TG-GATES (Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System) (632 datasets), the hypothesis was tested that a short-term exposure, transcriptome-based liver biological effect POD (BEPOD) could estimate a longer-term exposure "systemic" apical endpoint POD. Apical endpoints considered were body weight, clinical observation, kidney weight and histopathology and liver weight and histopathology. A BMDExpress algorithm using Gene Ontology Biological Process gene sets was optimized to derive a liver BEPOD most predictive of a systemic apical POD. Liver BEPODs were stable from 3 h to 29 days of exposure; the median fold difference of the 29-day BEPOD to BEPODs from earlier time points was approximately 1 (range: 0.7-1.1). Strong positive correlation (Pearson R = 0.86) and predictive accuracy (root mean square difference = 0.41) were observed between a concurrent (29 days) liver BEPOD and the systemic apical POD. Similar Pearson R and root mean square difference values were observed for comparisons between a 29-day systemic apical POD and liver BEPODs derived from 3 h to 15 days of exposure. These data across 79 molecules suggest that a longer-term exposure study apical POD from liver and non-liver compartments can be estimated using a liver BEPOD derived from an acute or subacute exposure study.

Short-term toxicogenomics as an alternative approach to chronic in vivo studies for derivation of points of departure: A case study in the rat with a triazole fungicide.

The derivation of an apical endpoint point of departure (POD) from animal-intensive testing programs has been the traditional cornerstone of human health risk assessment. Replacement of in vivo chronic studies with novel approaches, such as toxicogenomics, holds promise for future alternative testing paradigms that significantly reduce animal testing. We hypothesized that a toxicogenomic POD following a 14 day exposure in the rat would approximate the most sensitive apical endpoint POD derived from a battery of chronic, carcinogenicity, reproduction and endocrine guideline toxicity studies. To test this hypothesis, we utilized myclobutanil, a triazole fungicide, as a model compound. In the 14 day study, male rats were administered 0 (vehicle), 30, 150, or 400 mg/kg/day myclobutanil via oral gavage. Endpoints evaluated included traditional apical, hormone, and liver and testis transcriptomic (whole genome RNA sequencing) data. From the transcriptomic data, liver and testis biological effect POD (BEPOD) values were derived. Myclobutanil exposure for 14 days resulted in increased liver weight, altered serum hormones, liver histopathology, and differential gene expression in liver and testis. The liver and testis BEPODs from the short-term study were 22.2 and 25.4 mg/kg/day, respectively. These BEPODs were approximately an order of magnitude higher than the most sensitive apical POD identified from the two year cancer bioassay based on testis atrophy (1.4 mg/kg/day). This study demonstrates the promise of using a short-term study BEPOD to derive a POD for human health risk assessment while substantially reducing animal testing.

Dioxin male rat reproductive toxicity mode of action and relative potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran characterized by fetal pituitary and testis transcriptome profiling.

Fetal rat exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces epididymal sperm number involving altered pituitary-testicular hormonal signaling as the proposed mode-of-action (MOA). To evaluate this MOA and compare TCDD to 2,3,7,8-tetrachlorodibenzofuran (TCDF), an in utero rat exposure and study was conducted. Endpoints included congener tissue levels and transcriptomes of maternal liver and fetal liver, testis, and pituitary. Decreased gonadotropin subunit mRNAs levels (Lhb and Fshb) and enriched signaling pathways including GNRH Signaling and Calcium Signaling were observed in fetal pituitary after TCDD (but not TCDF) exposure. TCDD (but not TCDF) decreased fetal testis cholesterologenic and steroidogenic pathway genes. TCDD tissue concentrations in dam liver, dam adipose, and whole fetus were approximately 3- to 6-fold higher than TCDF. These results support a MOA for dioxin-induced rat male reproductive toxicity involving key events in both the fetal pituitary (e.g., reduced gonadotropin production) and fetal testis (e.g., reduced Leydig cell cholesterologenesis and steroidogenesis).

A Novel Open Access Web Portal for Integrating Mechanistic and Toxicogenomic Study Results.

Applying toxicogenomics to improving the safety profile of drug candidates and crop protection molecules is most useful when it identifies relevant biological and mechanistic information that highlights risks and informs risk mitigation strategies. Pathway-based approaches, such as gene set enrichment analysis, integrate toxicogenomic data with known biological process and pathways. Network methods help define unknown biological processes and offer data reduction advantages. Integrating the 2 approaches would improve interpretation of toxicogenomic information. Barriers to the routine application of these methods in genome-wide transcriptomic studies include a need for "hands-on" computer programming experience, the selection of 1 or more analysis methods (eg pathway analysis methods), the sensitivity of results to algorithm parameters, and challenges in linking differential gene expression to variation in safety outcomes. To facilitate adoption and reproducibility of gene expression analysis in safety studies, we have developed Collaborative Toxicogeomics, an open-access integrated web portal using the Django web framework. The software, developed with the Python programming language, is modular, extensible and implements "best-practice" methods in computational biology. New study results are compared with over 4000 rodent liver experiments from Drug Matrix and open TG-GATEs. A unique feature of the software is the ability to integrate clinical chemistry and histopathology-derived outcomes with results from gene expression studies, leading to relevant mechanistic conclusions. We describe its application by analyzing the effects of several toxicants on liver gene expression and exemplify application to predicting toxicity study outcomes upon chronic treatment from expression changes in acute-duration studies.

Polybrominated diphenyl ether (PBDE) neurotoxicity: a systematic review and meta-analysis of animal evidence.

A recent systematic review (SR) and meta-analysis of human studies found an association between prenatal serum polybrominated diphenyl ethers (PBDE) concentrations and a decrease in the IQ of children. A SR of experimental developmental animal PBDE-mediated neurotoxicity studies was performed in the present study. Outcomes assessed included measures related to learning, memory, and attention, which parallel the intelligence-related outcomes evaluated in the human studies SR. PubMed, Embase, and Toxline were searched for relevant experimental non-human mammalian studies. Evaluation of risk of bias (RoB) and overall body of evidence followed guidance developed by the National Toxicology Program. Animal studies using varying designs and outcomes were available for BDEs 47, 99, 153, 203, 206, and 209 and the technical mixture DE-71. Study reporting of methods and results was often incomplete leading to concerns regarding RoB. A meta-analysis of 6 Morris water maze studies showed evidence of a significant increase in last trial latency (effect size of 25.8 [CI, 20.3 to 31.2]) in PBDE-exposed animals with low heterogeneity. For most endpoints, there were unexplained inconsistencies across studies and no consistent evidence of a dose-response relationship. There is a "moderate" level of evidence that exposure to BDEs 47, 99, and 209 affects learning. For other PBDEs and other endpoints, the level of evidence was "low" or "very low". The meta-analysis led to stronger conclusions than that based upon a qualitative review of the evidence. The SR also identified RoB concerns that might be remedied by better study reporting.

Systematic reviews and meta-analyses of human and animal evidence of prenatal diethylhexyl phthalate exposure and changes in male anogenital distance.

Male reproductive alterations found in animals and humans following in utero phthalate exposure include decreased anogenital distance (AGD) and other reproductive-tract malformations. The aim of this investigation was to conduct systematic reviews of human and animal evidence of the effect of in utero exposure to diethylhexyl phthalate (DEHP) on anogenital distance (AGD) in males. PubMed, Embase, and Toxline were searched for relevant human and experimental animal studies on August 15, 2016. Search results were screened for relevance, and studies that met the inclusion criteria were evaluated for quality and data extracted for analysis. Confidence in the human and animal bodies of evidence was assessed and hazard conclusions reached by integrating evidence streams. The search yielded 6 relevant human studies and 19 animal studies. Meta-analysis of 5 human observational prospective cohort studies showed that increased maternal urinary concentrations of DEHP metabolites were associated with decreased AGD in boys (-4.07 [CI, -6.49 to -1.66] % decrease per log10 rise in DEHP metabolites). Meta-analysis and meta-regression of the 19 experimental animal studies found reduced AGD with DEHP treatment, with a dose-response gradient, and with heterogeneity explained by species and strain. There is a moderate level of evidence from human investigations and a high level of data from animal studies that in utero exposure to DEHP decreases AGD. Based upon the available human and animal evidence, and consideration of mechanistic data, DEHP is presumed to be a reproductive hazard to humans on the basis of effects on AGD.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants.

Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants.

Comparative response of rat and rabbit conceptuses in vitro to inhibitors of histiotrophic nutrition.

Histiotrophic nutrition via the visceral yolk sac is an essential nutritional pathway of the rodent conceptus, and inhibition of this pathway may cause growth retardation, malformations, and death in rodent embryos. Morphologic differences among species during early development indicate that the visceral yolk sac histiotrophic nutrition pathway may be of lesser importance in nonrodent species, including humans. Here, comparative studies were conducted with inhibitors of different steps in the visceral yolk sac histiotrophic nutrition pathway to determine whether the rabbit is similarly responsive to the rat. Early somite stage New Zealand White rabbit and Crl:CD(SD) rat conceptuses (gestation day 9, rabbits; gestation day 10, rats) were exposed for 48 hr to three different histiotrophic nutrition pathway inhibitors using whole embryo culture techniques, after which they were evaluated for growth and malformations. Cubilin antibody, an inhibitor of endocytosis, reduced growth and development and increased malformations in both rat and rabbit embryos, although the rabbit appeared more sensitive. Leupeptin, a lysosomal cysteine protease inhibitor, also impaired growth and development and increased malformations in rat embryos, while in the rabbit it induced malformations and a slight decrease in morphology score but had no effect upon growth. Trypan blue, an inhibitor of endocytosis and endosome maturation, affected all measures in both species to a similar degree at the highest concentration (2500 µg/ml), but rat embryos responded to a greater extent at lower concentrations. Although the specific adverse outcomes appear to be different, these results demonstrate that rabbits, like rats, are sensitive to inhibitors of the histiotrophic nutrition pathway.

Identification of gene expression changes in postnatal rat foreskin after in utero anti-androgen exposure.

In utero human phthalate exposure has been associated with male reproductive disorders in epidemiological studies, but discovering relationships is hindered by the lack of identifying markers. This study identified gene expression changes following in utero dibutyl phthalate (DBP) and flutamide exposures in Sprague-Dawley rat foreskin. Dams were exposed to 100 or 500mg/kg/day dibutyl phthalate or 5mg/kg/day flutamide from gestational days 16-20. Microarray analysis was performed on foreskin tissue from gestational day 20 and postnatal day 5. Expression changes found following DBP exposure were not present following flutamide treatment, indicating that expression changes were specific to DBP exposure and not caused by altered androgen signaling. Genes that were expressed at lower levels in tissue from pups treated with the low dose of DBP were reduced more in pups treated with the high dose of DBP, demonstrating a dose response effect of this compound. Changes in expression of Marcks, Pum1, Nupr1, and Penk caused by in utero phthalate exposure were confirmed by qRT-PCR. Changes in expression of these genes were maintained after birth and consequently their expression could serve as markers of chemical exposure and biological response.