Dynamics of growth zone patterning in the milkweed bug Oncopeltus fasciatus.

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.

Latent carcinogenicity of early-life exposure to dichloroacetic acid in mice.

Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH2O, control); dH2O with 0.06% phenobarbital (PB), a mouse liver tumor promoter; or DCA (1.0, 2.0 or 3.5g/l) for 10 weeks followed by dH2O or PB (n = 20-30/group/sex). Pathology and molecular assessments were performed at 98 weeks of age. In the absence of PB, early-life exposure to DCA increased the incidence and number of hepatocellular tumors in male and female mice compared with controls. Significant dose trends were observed in both sexes. At the high dose level, 10 weeks of prior DCA treatment induced comparable effects (≥85% tumor incidence and number) to those seen after continuous lifetime exposure. Prior DCA treatment did not enhance or inhibit the carcinogenic effects of PB, induce persistent liver cytotoxicity or preneoplastic changes on histopathology or alter DNA sequence variant profiles within liver tumors compared with controls. Distinct changes in liver messenger RNA and micro RNA profiles associated with prior DCA treatment were not apparent at 98 weeks. Our findings demonstrate that early-life exposure to DCA may be as carcinogenic as life-long exposures, potentially via epigenetic-mediated effects related to cellular metabolism.

A multi-cell, multi-scale model of vertebrate segmentation and somite formation.

Somitogenesis, the formation of the body's primary segmental structure common to all vertebrate development, requires coordination between biological mechanisms at several scales. Explaining how these mechanisms interact across scales and how events are coordinated in space and time is necessary for a complete understanding of somitogenesis and its evolutionary flexibility. So far, mechanisms of somitogenesis have been studied independently. To test the consistency, integrability and combined explanatory power of current prevailing hypotheses, we built an integrated clock-and-wavefront model including submodels of the intracellular segmentation clock, intercellular segmentation-clock coupling via Delta/Notch signaling, an FGF8 determination front, delayed differentiation, clock-wavefront readout, and differential-cell-cell-adhesion-driven cell sorting. We identify inconsistencies between existing submodels and gaps in the current understanding of somitogenesis mechanisms, and propose novel submodels and extensions of existing submodels where necessary. For reasonable initial conditions, 2D simulations of our model robustly generate spatially and temporally regular somites, realistic dynamic morphologies and spontaneous emergence of anterior-traveling stripes of Lfng. We show that these traveling stripes are pseudo-waves rather than true propagating waves. Our model is flexible enough to generate interspecies-like variation in somite size in response to changes in the PSM growth rate and segmentation-clock period, and in the number and width of Lfng stripes in response to changes in the PSM growth rate, segmentation-clock period and PSM length.

Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples.

Formalin fixation of biological specimens damages nucleic acids and limits their use in genomic analyses. Previously, we showed that RNA isolation with an organocatalyst (2-amino-5-methylphenyl phosphonic acid, used to speed up reversal of formalin-induced adducts) and extended heated incubation (ORGΔ) improved RNA-sequencing data from formalin-fixed paraffin-embedded (FFPE) tissue samples. The primary goal of this study was to evaluate whether ORGΔ treatment improves DNA-sequencing data from clinical FFPE samples. We isolated RNA and DNA ± ORGΔ from paired FFPE and frozen human renal and ovarian carcinoma specimens collected as part of the National Cancer Institute Biospecimen Pre-analytical Variables program. Tumor types were microscopically confirmed from adjacent tissue sections. Following extraction, DNA was fragmented and sequenced and differences were compared between frozen and FFPE sample pairs. Treatment with ORGΔ improved concurrent SNP calls in FFPE DNA compared to non-ORGΔ FFPE samples and enhanced confidence in SNP calls for all FFPE DNA samples, beyond that of matched frozen samples. In general, the concordant SNPs identified in paired frozen and FFPE DNA samples agreed for both genotype and homozygosity vs. heterozygosity of calls regardless of ORGΔ treatment. The increased confidence in ORGΔ FFPE DNA variant calls relative to the matched frozen DNA suggests a novel application of this method. With further optimization, this method may improve quality of DNA-sequencing data in FFPE as well as frozen tissue samples.

Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action.

Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to nucleic acid damage from formalin fixation. Older FFPE samples with highly degraded RNA pose a particularly difficult technical challenge. Probe-based targeted sequencing technologies show promise in addressing this issue but have not been directly compared to standard whole-genome RNA-Sequencing (RNA-Seq) methods. In this study, we evaluated dose-dependent transcriptional changes from paired frozen (FROZ) and FFPE liver samples stored for over 20 years using targeted resequencing (TempO-Seq) and whole-genome RNA-Seq methods. Samples were originally collected from male mice exposed to a reference chemical (dichloroacetic acid, DCA) at 0, 198, 313, and 427 mg/kg-day (n = 6/dose) by drinking water for 6 days. TempO-Seq showed high overlap in differentially expressed genes (DEGs) between matched FFPE and FROZ samples and high concordance in fold-change values across the two highest dose levels of DCA vs. control (R2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R2 ≥ 0.92). In contrast, RNA-Seq FFPE samples showed few overlapping DEGs compared to FROZ RNA-Seq (≤5 for all dose groups). Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples within 1.4-fold of RNA-Seq FROZ samples (93.9 mg/kg-d), whereas RNA-Seq FFPE samples were 3.3-fold higher (310.3 mg/kg-d). This work demonstrates that targeted sequencing may provide a more robust method for quantifying gene expression profiles from aged archival FFPE samples.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

Gene expression of normal human epidermal keratinocytes modulated by trivalent arsenicals.

Chronic exposure to inorganic arsenic (iAs) is associated with the development of benign and malignant human skin lesions including nonmelanoma skin cancers. The precise arsenical form(s) responsible for this carcinogenic effect are unknown, although trivalent inorganic arsenic (iAs(III)) and two of its toxic metabolites, monomethylarsonous acid (MMA(III)) and methylarsinous acid (DMA(III)), are attractive candidates. In an effort to better understand and compare their toxic effects in the skin, we compared the global gene expression profiles of normal human epidermal keratinocytes (NHEKs) exposed to varying noncytotoxic/slightly cytotoxic concentrations of iAs(III), MMA(III), and DMA(III) for 24 h. Exposure to each arsenical treatment group exhibited a dose effect in the number of altered genes and the magnitude of expression change in NHEKs. The most significant gene expression changes associated with iAs(III) and MMA(III) exposure were consistent with several key events believed to be important to As-driven skin carcinogenesis, namely induction of oxidative stress, increased transcript levels of keratinocyte growth factors, and modulation of MAPK and NF-κB pathways. At both comparable arsenical concentrations and comparable NHEK toxicity, greater potential carcinogenic effects were observed in MMA(III)-exposed NHEKs than those exposed to iAs(III), including involvement of more proinflammatory signals and increased transcript levels of more growth factor genes. In contrast, none of these above-mentioned transcriptional trends were among the most significantly altered functions in the DMA(III) treatment group. This study suggests the relative capacity of each of the tested arsenicals to drive suspected key events in As-mediated skin carcinogenesis is MMA(III) > iAs(III) with little contribution from DMA(III).

Direct formalin fixation induces widespread transcriptomic effects in archival tissue samples.

Sequencing technologies now provide unprecedented access to genomic information in archival formalin-fixed paraffin-embedded (FFPE) tissue samples. However, little is known about artifacts induced during formalin fixation, which could bias results. Here we evaluated global changes in RNA-sequencing profiles between matched frozen and FFPE samples. RNA-sequencing was performed on liver samples collected from mice treated with a reference chemical (phenobarbital) or vehicle control for 7 days. Each sample was divided into four parts: (1) fresh-frozen, (2) direct-fixed in formalin for 18 h, (3) frozen then formalin-fixed, and (4) frozen then ethanol-fixed and paraffin-embedded (n = 6/group/condition). Direct fixation resulted in 2,946 differentially expressed genes (DEGs) vs. fresh-frozen, 98% of which were down-regulated. Freezing prior to formalin fixation had ≥ 95% fewer DEGs vs. direct fixation, indicating that most formalin-derived transcriptional effects in the liver occurred during fixation. This finding was supported by retrospective studies of paired frozen and FFPE samples, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription initiation pathways with direct fixation. Notably, direct formalin fixation in the parent study did not significantly impact response profiles resulting from chemical exposure. These results advance our understanding of FFPE samples as a resource for genomic research.

Three conazoles increase hepatic microsomal retinoic acid metabolism and decrease mouse hepatic retinoic acid levels in vivo.

Conazoles are fungicides used in agriculture and as pharmaceuticals. In a previous toxicogenomic study of triazole-containing conazoles we found gene expression changes consistent with the alteration of the metabolism of all trans-retinoic acid (atRA), a vitamin A metabolite with cancer-preventative properties (Ward et al., Toxicol. Pathol. 2006; 34:863-78). The goals of this study were to examine effects of propiconazole, triadimefon, and myclobutanil, three triazole-containing conazoles, on the microsomal metabolism of atRA, the associated hepatic cytochrome P450 (P450) enzyme(s) involved in atRA metabolism, and their effects on hepatic atRA levels in vivo. The in vitro metabolism of atRA was quantitatively measured in liver microsomes from male CD-1 mice following four daily intraperitoneal injections of propiconazole (210 mg/kg/d), triadimefon (257 mg/kg/d) or myclobutanil (270 mg/kg/d). The formation of both 4-hydroxy-atRA and 4-oxo-atRA were significantly increased by all three conazoles. Propiconazole-induced microsomes possessed slightly greater metabolizing activities compared to myclobutanil-induced microsomes. Both propiconazole and triadimefon treatment induced greater formation of 4-hydroxy-atRA compared to myclobutanil treatment. Chemical and immuno-inhibition metabolism studies suggested that Cyp26a1, Cyp2b, and Cyp3a, but not Cyp1a1 proteins were involved in atRA metabolism. Cyp2b10/20 and Cyp3a11 genes were significantly over-expressed in the livers of both triadimefon- and propiconazole-treated mice while Cyp26a1, Cyp2c65 and Cyp1a2 genes were over-expressed in the livers of either triadimefon- or propiconazole-treated mice, and Cyp2b10/20 and Cyp3a13 genes were over-expressed in the livers of myclobutanil-treated mice. Western blot analyses indicated conazole induced-increases in Cyp2b and Cyp3a proteins. All three conazoles decreased hepatic atRA tissue levels ranging from 45-67%. The possible implications of these changes in hepatic atRA levels on cell proliferation in the mouse tumorigenesis process are discussed.

Multicell simulations of development and disease using the CompuCell3D simulation environment.

Mathematical modeling and computer simulation have become crucial to biological fields from genomics to ecology. However, multicell, tissue-level simulations of development and disease have lagged behind other areas because they are mathematically more complex and lack easy-to-use software tools that allow building and running in silico experiments without requiring in-depth knowledge of programming. This tutorial introduces Glazier-Graner-Hogeweg (GGH) multicell simulations and CompuCell3D, a simulation framework that allows users to build, test, and run GGH simulations.

Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples.

Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing. We examined RNA quality metrics conducted prior to RNA-sequencing in paired frozen and FFPE samples with varying levels of quality based on age in block and time in formalin. RNA quality was measured by the RNA integrity number (RIN), a modified RIN called the paraffin-embedded RNA metric, the percentage of RNA fragments >100-300 nucleotides in size (DV100-300), and 2 quantitative PCR-based methods. This information was correlated to sequencing read quality, mapping, and gene detection. Among fragmentation-based methods, DV and PCR-based metrics were more informative than RIN or paraffin-embedded RNA metric in determining sequencing success. Across low- and high-quality FFPE samples, a minimum of 80% of RNA fragments >100 nucleotides (DV100 > 80) provided the best indication of gene diversity and read counts upon sequencing. The PCR-based methods further showed quantitative reductions in amplifiable RNA of target genes related to sample age and time in formalin that inform input quantity of FFPE RNA for sequencing. These results should aid in screening and prioritizing archival FFPE samples for retrospective analyses of gene expression.

Authentic Inquiry through Modeling in Biology (AIM-Bio): An Introductory Laboratory Curriculum That Increases Undergraduates' Scientific Agency and Skills.

Providing opportunities for science, technology, engineering, and mathematics undergraduates to engage in authentic scientific practices is likely to influence their view of science and may impact their decision to persist through graduation. Laboratory courses provide a natural place to introduce students to scientific practices, but existing curricula often miss this opportunity by focusing on confirming science content rather than exploring authentic questions. Integrating authentic science within laboratory courses is particularly challenging at high-enrollment institutions and community colleges, where access to research-active faculty may be limiting. The Authentic Inquiry through Modeling in Biology (AIM-Bio) curriculum presented here engages students in authentic scientific practices through iterative cycles of model generation, testing, and revision. AIM-Bio university and community college students demonstrated their ability to propose diverse models for biological phenomena, formulate and address hypotheses by designing and conducting experiments, and collaborate with classmates to revise models based on experimental data. Assessments demonstrated that AIM-Bio students had an enhanced sense of project ownership and greater identification as scientists compared with students in existing laboratory courses. AIM-Bio students also experienced measurable gains in their nature of science understanding and skills for doing science. Our results suggest AIM-Bio as a potential alternative to more resource-intensive curricula with similar outcomes.

Identification of interspecies concordance of mechanisms of arsenic-induced bladder cancer.

Exposure to arsenic causes cancer by inducing a variety of responses that affect the expression of genes associated with numerous biological pathways leading to altered cell growth and proliferation, signaling, apoptosis and oxidative stress response. Affymetrix GeneChip arrays were used to detect gene expression changes following dimethylarsinic acid (DMA) exposure to human bladder cells (UROtsa) or rat bladder cells (MYP3) and rat bladder epithelium in vivo at comparable doses. Using different experimental models coupled with transcriptional profiling allowed investigation of the correlation of mechanisms of DMA-induced toxicity between in vitro and in vivo treatment and across species. Our observations suggest that DMA-induced gene expression in UROtsa cells is distinct from that observed in the MYP3 cells. Principal component analysis shows a more distinct separation by treatment and dose in MYP3 cells as compared to UROtsa cells. However, at the level of pathways and biological networks, DMA affects both common and unique processes in the bladder transitional cells of human and rats. Twelve pathways were found common between human in vitro, rat in vitro and rat in vivo systems. These included signaling pathways involved in adhesion, cellular growth and differentiation. Fifty-five genes found to be commonly expressed between rat in vivo and rat in vitro systems were involved in diverse functions such as cell cycle regulation, lipid metabolism and protein degradation. Many of the genes, processes and pathways have previously been associated with arsenic-induced toxicity. Our finding reiterates and also identifies new biological processes that might provide more information regarding the mechanisms of DMA-induced toxicity. The results of our analysis further suggest that gene expression profiles can address pertinent issues of relevance to risk assessment, namely interspecies extrapolation of mechanistic information as well as comparison of in vitro to in vivo response.

Editor's Highlight: Genetic Targets of Acute Toluene Inhalation in Drosophila melanogaster.

Interpretation and use of data from high-throughput assays for chemical toxicity require links between effects at molecular targets and adverse outcomes in whole animals. The well-characterized genome of Drosophila melanogaster provides a potential model system by which phenotypic responses to chemicals can be mapped to genes associated with those responses, which may in turn suggest adverse outcome pathways associated with those genes. To determine the utility of this approach, we used the Drosophila Genetics Reference Panel (DGRP), a collection of ∼200 homozygous lines of fruit flies whose genomes have been sequenced. We quantified toluene-induced suppression of motor activity in 123 lines of these flies during exposure to toluene, a volatile organic compound known to induce narcosis in mammals via its effects on neuronal ion channels. We then applied genome-wide association analyses on this effect of toluene using the DGRP web portal (http://dgrp2.gnets.ncsu.edu), which identified polymorphisms in candidate genes associated with the variation in response to toluene exposure. We tested ∼2 million variants and found 82 polymorphisms located in or near 66 candidate genes that were associated with phenotypic variation for sensitivity to toluene at P < 5 × 10-5, and human orthologs for 52 of these candidate Drosophila genes. None of these orthologs are known to be involved in canonical pathways for mammalian neuronal ion channels, including GABA, glutamate, dopamine, glycine, serotonin, and voltage sensitive calcium channels. Thus this analysis did not reveal a genetic signature consistent with processes previously shown to be involved in toluene-induced narcosis in mammals. The list of the human orthologs included Gene Ontology terms associated with signaling, nervous system development and embryonic morphogenesis; these orthologs may provide insight into potential new pathways that could mediate the narcotic effects of toluene.

Metabolic Disruption Early in Life is Associated With Latent Carcinogenic Activity of Dichloroacetic Acid in Mice.

Early-life environmental factors can influence later-life susceptibility to cancer. Recent evidence suggests that metabolic pathways may mediate this type of latency effect. Previously, we reported that short-term exposure to dichloroacetic acid (DCA) increased liver cancer in mice 84 weeks after exposure was stopped. Here, we evaluated time course dynamics for key events related to this effect. This study followed a stop-exposure design in which 28-day-old male B6C3F1 mice were given the following treatments in drinking water for up to 93 weeks: deionized water (dH2O, control); 3.5 g/l DCA continuously; or 3.5 g/l DCA for 4-52 weeks followed by dH2O. Effects were evaluated at eight interim time points. A short-term biomarker study was used to evaluate DCA effects at 6, 15, and 30 days. Liver tumor incidence was higher in all DCA treatment groups, including carcinomas in 82% of mice previously treated with DCA for only 4 weeks. Direct effects of DCA in the short-term study included decreased liver cell proliferation and marked mRNA changes related to mitochondrial dysfunction and altered cell metabolism. However, all observed short-term effects of DCA were ultimately reversible, and prior DCA treatment did not affect liver cell proliferation, apoptosis, necrosis, or DNA sequence variants with age. Key intermediate events resulting from transient DCA exposure do not fit classical cytotoxic, mitogenic, or genotoxic modes of action for carcinogenesis, suggesting a distinct mechanism associated with early-life metabolic disruption.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Quantitation of intracellular NAD(P)H can monitor an imbalance of DNA single strand break repair in base excision repair deficient cells in real time.

DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or during the base excision repair pathways. Here we established a new real-time assay to assess an imbalance of DNA SSB repair by indirectly measuring PARP-1 activation through the depletion of intracellular NAD(P)H. A water-soluble tetrazolium salt is used to monitor the amount of NAD(P)H in living cells through its reduction to a yellow colored water-soluble formazan dye. While this assay is not a direct method, it does not require DNA extraction or alkaline treatment, both of which could potentially cause an artifactual induction of SSBs. In addition, it takes only 4 h and requires less than a half million cells to perform this measurement. Using this assay, we demonstrated that the dose- and time-dependent depletion of NAD(P)H in XRCC1-deficient CHO cells exposed to methyl methanesulfonate. This decrease was almost completely blocked by a PARP inhibitor. Furthermore, methyl methanesulfonate reduced NAD(P)H in PARP-1+/+ cells, whereas PARP-1-/- cells were more resistant to the decrease in NAD(P)H. These results indicate that the analysis of intracellular NAD(P)H level using water-soluble tetrazolium salt can assess an imbalance of SSB repair in living cells in real time.

Editor's Highlight: Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded Samples.

Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here, we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from 2 archival studies in mice, one <2 years old and the other >20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively. Total RNA was ribo-depleted and sequenced using the Illumina HiSeq platform. In the recently archived study, FFPE samples had 35% lower total counts compared to FROZ samples but high concordance in fold-change values of differentially expressed genes (DEGs) (r2 = 0.99), highly enriched pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (<5% difference vs FROZ). In contrast, older FFPE samples had markedly lower total counts (3% of FROZ) and poor concordance in global DEGs and pathways. However, counts from FFPE and FROZ samples still positively correlated (r2 = 0.84 across all transcripts) and showed comparable dose responses for more highly expressed target genes. These findings highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Recently archived FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older lower-quality FFPE samples. This work should help advance the use of archival resources in chemical safety and translational science.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.

Gene expression profiling of responses to dimethylarsinic acid in female F344 rat urothelium.

Gene expression profiling has been shown to be useful for identifying underlying mechanisms of toxicity, determining patterns of biological response, and elucidating candidate markers of exposure and response. Inorganic arsenic (iAs) is a human carcinogen and epidemiologic evidence implicates it in the development of urinary bladder cancer. Dimethylarsinic acid (DMA), the major excreted metabolite of iAs in humans, is a known rat bladder carcinogen. To examine the changes associated with DMA exposure, microarray analysis of the urothelium was performed in female F344 rats exposed to non-toxic and toxic doses of DMA in their drinking water for 28 days. A novel method for isolating predominantly urothelial cells was developed. Gene expression profiling of the urothelium using a custom 2-dye spotted array revealed that DMA treatment modulated the expression of transcripts of genes that regulate apoptosis, cell cycle regulation and the oxidative stress response. Expression of genes mapping to pathways involved in cancer control processes were also altered after DMA exposure. Morphological data suggested a dose dependent increase in cellular toxicity. Significant changes in differential gene expression were present after all treatments event at doses where standard toxicological responses were not detectable. The greatest perturbation in gene expression was present in rats after treatment with 40 ppm DMA. Doses which produced no histologic or ultrastructural evidence of toxicity (non-toxic) could be differentiated from toxic doses based on the expression of a subset of genes, which control cell signaling and the stress response. These reported changes in gene expression show similarities between the mechanisms of action of DMA in vivo and those previously described for iAs in vitro. These data illustrate the utility of transcriptional profiling and its potential in predicting key mechanistic pathways involved in toxicity and as a time efficient tool to inform the mode of action analysis in risk assessment.