Dose Response, Dosimetric, and Metabolic Evaluations of Replacement PFAS Perfluoro-(2,5,8-trimethyl-3,6,9-trioxadodecanoic) Acid (HFPO-TeA).

Few studies are available on the environmental and toxicological effects of perfluoroalkyl ether carboxylic acids (PFECAs), such as GenX, which are replacing legacy PFAS in manufacturing processes. To collect initial data on the toxicity and toxicokinetics of a longer-chain PFECA, male and female Sprague Dawley rats were exposed to perfluoro-(2,5,8-trimethyl-3,6,9-trioxadodecanoic) acid (HFPO-TeA) by oral gavage for five days over multiple dose levels (0.3-335.2 mg/kg/day). Clinically, we observed mortality at doses >17 mg/kg/day and body weight changes at doses ≤17 mg/kg/day. For the 17 mg/kg/day dose level, T3 and T4 thyroid hormone concentrations were significantly decreased (p < 0.05) from controls and HFPO-TeA plasma concentrations were significantly different between sexes. Non-targeted analysis of plasma and in vitro hepatocyte assay extractions revealed the presence of another GenX oligomer, perfluoro-(2,5-dimethyl-3,6-dioxanonanoic) acid (HFPO-TA). In vitro to in vivo extrapolation (IVIVE) parameterized with in vitro toxicokinetic data predicted steady-state blood concentrations that were within seven-fold of those observed in the in vivo study, demonstrating reasonable predictivity. The evidence of thyroid hormone dysregulation, sex-based differences in clinical results and dosimetry, and IVIVE predictions presented here suggest that the replacement PFECA HFPO-TeA induces a complex and toxic exposure response in rodents.

Short-Term Transcriptomic Points of Departure Are Consistent with Chronic Points of Departure for Three Organophosphate Pesticides across Mouse and Fathead Minnow.

New approach methods (NAMs) can reduce the need for chronic animal studies. Here, we apply benchmark dose (concentration) (BMD(C))-response modeling to transcriptomic changes in the liver of mice and in fathead minnow larvae after short-term exposures (7 days and 1 day, respectively) to several dose/concentrations of three organophosphate pesticides (OPPs): fenthion, methidathion, and parathion. The mouse liver transcriptional points of departure (TPODs) for fenthion, methidathion, and parathion were 0.009, 0.093, and 0.046 mg/Kg-bw/day, while the fathead minnow larva TPODs were 0.007, 0.115, and 0.046 mg/L, respectively. The TPODs were consistent across both species and reflected the relative potencies from traditional chronic toxicity studies with fenthion identified as the most potent. Moreover, the mouse liver TPODs were more sensitive than or within a 10-fold difference from the chronic apical points of departure (APODs) for mammals, while the fathead minnow larva TPODs were within an 18-fold difference from the chronic APODs for fish species. Short-term exposure to OPPs significantly impacted acetylcholinesterase mRNA abundance (FDR p-value <0.05, |fold change| ≥2) and canonical pathways (IPA, p-value <0.05) associated with organism death and neurological/immune dysfunctions, indicating the conservation of key events related to OPP toxicity. Together, these results build confidence in using short-term, molecular-based assays for the characterization of chemical toxicity and risk, thereby reducing reliance on chronic animal studies.

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.

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.

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.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

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.

Developmental toxicity of Nafion byproduct 2 (NBP2) in the Sprague-Dawley rat with comparisons to hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) and perfluorooctane sulfonate (PFOS).

Nafion byproduct 2 (NBP2) is a polyfluoroalkyl ether sulfonic acid that was recently detected in surface water, drinking water, and human serum samples from monitoring studies in North Carolina, USA. We orally exposed pregnant Sprague-Dawley rats to NBP2 from gestation day (GD) 14-18 (0.1-30 mg/kg/d), GD17-21, and GD8 to postnatal day (PND) 2 (0.3-30 mg/kg/d) to characterize maternal, fetal, and postnatal effects. GD14-18 exposures were also conducted with perfluorooctane sulfonate (PFOS) for comparison to NBP2, as well as data previously published for hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). NBP2 produced stillbirth (30 mg/kg), reduced pup survival shortly after birth (10 mg/kg), and reduced pup body weight (10 mg/kg). Histopathological evaluation identified reduced glycogen stores in newborn pup livers and hepatocyte hypertrophy in maternal livers at ≥ 10 mg/kg. Exposure to NBP2 from GD14-18 reduced maternal serum total T3 and cholesterol concentrations (30 mg/kg). Maternal, fetal, and neonatal liver gene expression was investigated using RT-qPCR pathway arrays, while maternal and fetal livers were also analyzed using TempO-Seq transcriptomic profiling. Overall, there was limited alteration of genes in maternal or F1 livers from NBP2 exposure with significant changes mostly occurring in the top dose group (30 mg/kg) associated with lipid and carbohydrate metabolism. Metabolomic profiling indicated elevated maternal bile acids for NBP2, but not HFPO-DA or PFOS, while all three reduced 3-indolepropionic acid. Maternal and fetal serum and liver NBP2 concentrations were similar to PFOS, but ∼10-30-fold greater than HFPO-DA concentrations at a given maternal oral dose. NBP2 is a developmental toxicant in the rat, producing neonatal mortality, reduced pup body weight, reduced pup liver glycogen, reduced maternal thyroid hormones, and altered maternal and offspring lipid and carbohydrate metabolism similar to other studied PFAS, with oral toxicity for pup loss that is slightly less potent than PFOS but more potent than HFPO-DA.

An international, interlaboratory ring trial confirms the feasibility of an open-source, extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open-access, direct RT-PCR assays are a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

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.

'All In': a pragmatic framework for COVID-19 testing and action on a global scale.

Current demand for SARS-CoV-2 testing is straining material resource and labor capacity around the globe. As a result, the public health and clinical community are hindered in their ability to monitor and contain the spread of COVID-19. Despite broad consensus that more testing is needed, pragmatic guidance toward realizing this objective has been limited. This paper addresses this limitation by proposing a novel and geographically agnostic framework (the 4Ps framework) to guide multidisciplinary, scalable, resource-efficient, and achievable efforts toward enhanced testing capacity. The 4Ps (Prioritize, Propagate, Partition, and Provide) are described in terms of specific opportunities to enhance the volume, diversity, characterization, and implementation of SARS-CoV-2 testing to benefit public health. Coordinated deployment of the strategic and tactical recommendations described in this framework has the potential to rapidly expand available testing capacity, improve public health decision-making in response to the COVID-19 pandemic, and/or to be applied in future emergent disease outbreaks.

Microbiota alter metabolism and mediate neurodevelopmental toxicity of 17ß-estradiol.

Estrogenic chemicals are widespread environmental contaminants associated with diverse health and ecological effects. During early vertebrate development, estrogen receptor signaling is critical for many different physiologic responses, including nervous system function. Recently, host-associated microbiota have been shown to influence neurodevelopment. Here, we hypothesized that microbiota may biotransform exogenous 17-ßestradiol (E2) and modify E2 effects on swimming behavior. Colonized zebrafish were continuously exposed to non-teratogenic E2 concentrations from 1 to 10 days post-fertilization (dpf). Changes in microbial composition and predicted metagenomic function were evaluated. Locomotor activity was assessed in colonized and axenic (microbe-free) zebrafish exposed to E2 using a standard light/dark behavioral assay. Zebrafish tissue was collected for chemistry analyses. While E2 exposure did not alter microbial composition or putative function, colonized E2-exposed larvae showed reduced locomotor activity in the light, in contrast to axenic E2-exposed larvae, which exhibited normal behavior. Measured E2 concentrations were significantly higher in axenic relative to colonized zebrafish. Integrated peak area for putative sulfonated and glucuronidated E2 metabolites showed a similar trend. These data demonstrate that E2 locomotor effects in the light phase are dependent on the presence of microbiota and suggest that microbiota influence chemical E2 toxicokinetics. More broadly, this work supports the concept that microbial colonization status may influence chemical toxicity.

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.

Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples.

Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and transcriptional responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n = 6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 h followed by ethanol (18F); or 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. Additional sections from 3F received the following demodification protocols to mitigate RNA damage: short heated incubation with Tris-Acetate-EDTA buffer; overnight heated incubation with an organocatalyst using 2 different isolation kits; or overnight heated incubation without organocatalyst. Ribo-depleted, stranded, total RNA libraries were built and sequenced using the Illumina HiSeq 2500 platform. Overnight incubation (± organocatalyst) increased RNA yield >3-fold and RNA integrity numbers and fragment analysis values by > 1.5- and >3.0-fold, respectively, versus 3F. Postsequencing metrics also showed reduced bias in gene coverage and deletion rates for overnight incubation groups. All demodification groups had increased overlap for differentially expressed genes (77%-84%) and enriched pathways (91%-97%) with FR, with the highest overlap in the organocatalyst groups. These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.

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.

Effects of the antimicrobial contaminant triclocarban, and co-exposure with the androgen 17ß-trenbolone, on reproductive function and ovarian transcriptome of the fathead minnow (Pimephales promelas).

Triclocarban (TCC) is an antimicrobial agent routinely detected in surface waters that has been hypothesized to interact with the vertebrate endocrine system. The present study examined the effects of TCC alone and in combination with the model endocrine disruptor 17ß-trenbolone (TRB) on fish reproductive function. Adult Pimephales promelas were continuously exposed to either 1 µg TCC/L or 5 µg TCC/L, to 0.5 µg TRB/L, or to a mixture (MIX) of 5 µg TCC/L and 0.5 µg TRB/L for 22 d, and a variety of reproductive and endocrine-related endpoints were examined. Cumulative fecundity was significantly reduced in fathead minnows exposed to TRB, MIX, or 5 µg TCC/L. Exposure to 1 µg TCC/L had no effect on reproduction. In general, both TRB and MIX treatments caused similar physiological effects, evoking significant reductions in female plasma vitellogenin, estradiol, and testosterone, and significant increases in male plasma estradiol. Based on analysis of the ovarian transcriptome, there were potential pathway impacts that were common to both TRB- and TCC-containing treatment groups. In most cases, however, those pathways were more plausibly linked to differences in reproductive status than to androgen-specific functions. Overall, TCC was reproductively toxic to fish at concentrations at or near those that have been measured in surface water. There was little evidence that TCC elicits reproductive toxicity through a specific mode of endocrine or reproductive action, nor that it could augment the androgenic effects of TRB. Nonetheless, the relatively small margin of safety between some measured environmental concentrations and effect concentrations suggests that concern is warranted. Environ Toxicol Chem 2017;36:231-242. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

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.

Developing a Novel Embryo-Larval Zebrafish Xenograft Assay to Prioritize Human Glioblastoma Therapeutics.

Glioblastoma is an aggressive brain cancer requiring improved treatments. Existing methods of drug discovery and development require years before new therapeutics become available to patients. Zebrafish xenograft models hold promise for prioritizing drug development. We have developed an embryo-larval zebrafish xenograft assay in which cancer cells are implanted in a brain microenvironment to discover and prioritize compounds that impact glioblastoma proliferation, migration, and invasion. We illustrate the utility of our assay by evaluating the well-studied, phosphatidylinositide 3-kinase inhibitor LY294002 and zinc oxide nanoparticles (ZnO NPs), which demonstrate selective cancer cytotoxicity in cell culture, but the in vivo effectiveness has not been established. Exposures of 3.125-6.25 µM LY294002 significantly decreased proliferation up to 34% with concentration-dependent trends. Exposure to 6.25 µM LY294002 significantly inhibited migration/invasion by ∼27% within the glioblastoma cell mass (0-80 µm) and by ∼32% in the next distance region (81-160 µm). Unexpectedly, ZnO enhanced glioblastoma proliferation by ∼19% and migration/invasion by ∼35% at the periphery of the cell mass (161+ µm); however, dissolution of these NPs make it difficult to discern whether this was a nano or ionic effect. These results demonstrate that we have a short, relevant, and sensitive zebrafish-based assay to aid glioblastoma therapeutic development.

Phenotype anchoring in zebrafish reveals a potential role for matrix metalloproteinases (MMPs) in tamoxifen's effects on skin epithelium.

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen--a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥ 10 µM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3-7 fold), consistent with AP-1 activity--a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin.

Toward safer multi-walled carbon nanotube design: Establishing a statistical model that relates surface charge and embryonic zebrafish mortality.

Given the increased utility and lack of consensus regarding carbon nanotube (CNT) environmental and human health hazards, there is a growing demand for guidelines that inform safer CNT design. In this study, the zebrafish (Danio rerio) model is utilized as a stable, sensitive biological system to evaluate the bioactivity of systematically modified and comprehensively characterized multi-walled carbon nanotubes (MWNTs). MWNTs were treated with strong acid to introduce oxygen functional groups, which were then systematically thermally reduced and removed using an inert temperature treatment. While 25 phenotypic endpoints were evaluated at 24 and 120 hours post-fertilization (hpf), high mortality at 24 hpf prevented further resolution of the mode of toxicity leading to mortality. Advanced multivariate statistical methods are employed to establish a model that identifies those MWNT physicochemical properties that best estimate the probability of observing an adverse outcome. The physicochemical properties considered in this study include surface charge, percent surface oxygen, dispersed aggregate size and morphology and electrochemical activity. Of the five physicochemical properties, surface charge, quantified as the point of zero charge (PZC), was determined as the best predictor of mortality at 24 hpf. From a design perspective, the identification of this property-hazard relationship establishes a foundation for the development of design guidelines for MWNTs with reduced hazard.