The Boundary of the Market for Biosecurity Risk.

Imported goods create value in destination countries but also create biosecurity risk. Although widely used in other domains of the economy, risk markets have not been created to manage losses that occur when exotic pests and diseases are introduced with traded goods. In this article we show that not all biosecurity risks are insurable. Losses arising from effort needed to detect and respond to exotic pests and diseases that breach national borders appear to be insurable because entry of these threats and consequent response costs, can be regarded as random events. As pests and diseases establish and spread, however, loss of access to export markets and productivity losses display systematic risk and appear to be uninsurable. Other insurability criteria support this definition of the boundary of biosecurity risk markets. We use the Australian biosecurity system as an example, although the framework described in this study will be applicable to biosecurity systems worldwide. We argue that biosecurity risk insurance could be incorporated into the current biosecurity system but would require legislation mandating importers to purchase insurance. Advantages of actuarial pricing of biosecurity risk are: (i) an increase in economic efficiency to the extent that importers respond to the price of biosecurity risk; (ii) financial sustainability would improve because actuarial pricing creates a structural link between funds available for biosecurity activities and risk exposure; and (iii) equity issues evident in the current biosecurity system could be addressed because risk creators (importers) would fund response activities through the purchase of insurance.

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.

Workplace Violence Against Nurses: Making It Safe to Care.

This article explores the topic of workplace violence in the health care setting. A definition of workplace violence and those who are most vulnerable is provided. National and state legislation that addresses the topic of workplace violence will be discussed. Other organizations such as the American Nurses Association and The Joint Commission and their position statements will be explored. Lastly, strategies targeting workplace violence prevention and the barriers to implementing identified strategies will be discussed. Workplace violence is a rapidly growing concern for those working in health care. This article provides recommendations for legislative and workplace actions to protect health care workers.

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.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

Relevance of linguistic and cultural training among non-clinical personnel at the University of Mississippi Medical Center: a qualitative study.

BACKGROUND: An academic medical center presents a unique environment where non-clinical staff provide vital services to a cross-section of people. A medical Spanish course was offered to a non-clinical department, campus police, in response to the growing number of Hispanics or Latinos seeking care within the health center. METHOD: In October 2007, a structured group discussion with six course participants was convened at the end of the Occupational Spanish course, using a topic guide to direct the conversation. RESULTS: Content analysis revealed that participation in the course: (1) increased interest and provided a model for other departments, (2) promoted ability to respond, (3) enhanced cultural and linguistic competence, and (4) increased confidence, effectiveness, and value in their work. CONCLUSIONS: Structured cultural and linguistic training within an academic health center can contribute to a safe, secure environment, a more competent, responsive workforce, and enhance services to limited English proficient (LEP) patients.

Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment.

The predominant role of toxicogenomic data in risk assessment, thus far, has been one of augmentation of more traditional in vitro and in vivo toxicology data. This article focuses on the current available examples of instances where toxicogenomic data has been evaluated in human health risk assessment (e.g., acetochlor and arsenicals) which have been limited to the application of toxicogenomic data to inform mechanism of action. This article reviews the regulatory policy backdrop and highlights important efforts to ultimately achieve regulatory acceptance. A number of research efforts on specific chemicals that were designed for risk assessment purposes have employed mechanism or mode of action hypothesis testing and generating strategies. The strides made by large scale efforts to utilize toxicogenomic data in screening, testing, and risk assessment are also discussed. These efforts include both the refinement of methodologies for performing toxicogenomics studies and analysis of the resultant data sets. The current issues limiting the application of toxicogenomics to define mode or mechanism of action in risk assessment are discussed together with interrelated research needs. In summary, as chemical risk assessment moves away from a single mechanism of action approach toward a toxicity pathway-based paradigm, we envision that toxicogenomic data from multiple technologies (e.g., proteomics, metabolomics, transcriptomics, supportive RT-PCR studies) can be used in conjunction with one another to understand the complexities of multiple, and possibly interacting, pathways affected by chemicals which will impact human health risk assessment.

Use of genomic data in risk assessment case study: II. Evaluation of the dibutyl phthalate toxicogenomic data set.

An evaluation of the toxicogenomic data set for dibutyl phthalate (DBP) and male reproductive developmental effects was performed as part of a larger case study to test an approach for incorporating genomic data in risk assessment. The DBP toxicogenomic data set is composed of nine in vivo studies from the published literature that exposed rats to DBP during gestation and evaluated gene expression changes in testes or Wolffian ducts of male fetuses. The exercise focused on qualitative evaluation, based on a lack of available dose-response data, of the DBP toxicogenomic data set to postulate modes and mechanisms of action for the male reproductive developmental outcomes, which occur in the lower dose range. A weight-of-evidence evaluation was performed on the eight DBP toxicogenomic studies of the rat testis at the gene and pathway levels. The results showed relatively strong evidence of DBP-induced downregulation of genes in the steroidogenesis pathway and lipid/sterol/cholesterol transport pathway as well as effects on immediate early gene/growth/differentiation, transcription, peroxisome proliferator-activated receptor signaling and apoptosis pathways in the testis. Since two established modes of action (MOAs), reduced fetal testicular testosterone production and Insl3 gene expression, explain some but not all of the testis effects observed in rats after in utero DBP exposure, other MOAs are likely to be operative. A reanalysis of one DBP microarray study identified additional pathways within cell signaling, metabolism, hormone, disease, and cell adhesion biological processes. These putative new pathways may be associated with DBP effects on the testes that are currently unexplained. This case study on DBP identified data gaps and research needs for the use of toxicogenomic data in risk assessment. Furthermore, this study demonstrated an approach for evaluating toxicogenomic data in human health risk assessment that could be applied to future chemicals.

Cost effectiveness in site selection to protect native plant communities from the weed, bitou bush, in New South Wales, Australia.

Government funding to protect native plant communities is usually limited. For cost effectiveness, priority sites for conservation must therefore be identified and funds allocated to protect these sites according to the quantity of communities conserved per dollar of cost. In 1999, invasion of coastal vegetation in New South Wales (NSW) by bitou bush was listed as a key threatening process under the NSW Threatened Species Conservation Act 1995. In accordance with the Act, a Threat Abatement Plan (TAP) was prepared to reduce the impacts of the weed to threatened biodiversity at priority sites. In the present study, data collected for the TAP were analysed by linear programming to determine the feasibility of achieving cost effectiveness in identifying sites and allocating funds, and to explore the impact of associated economic issues on the quantity of native plant communities that are protected. In addition to the total funds and costs per site, the quantity was influenced by alternative funding policies and different site selection strategies. Allocations that recognise these issues can enhance protection outcomes, and promote the cost effectiveness of weed management.

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.

Integrated Omic Analyses Identify Pathways and Transcriptomic Regulators Associated With Chemical Alterations of In Vitro Neural Network Formation.

Development of in vitro new approach methodologies has been driven by the need for developmental neurotoxicity (DNT) hazard data on thousands of chemicals. The network formation assay characterizes DNT hazard based on changes in network formation but provides no mechanistic information. This study investigated nervous system signaling pathways and upstream physiological regulators underlying chemically induced neural network dysfunction. Rat primary cortical neural networks grown on microelectrode arrays were exposed for 12 days in vitro to cytosine arabinoside, 5-fluorouracil, domoic acid, cypermethrin, deltamethrin, or haloperidol as these exposures altered network formation in previous studies. RNA-seq from cells and gas chromatography/mass spectrometry analysis of media extracts collected on days in vitro 12 provided gene expression and metabolomic identification, respectively. The integration of differentially expressed genes and metabolites for each neurotoxicant was analyzed using ingenuity pathway analysis. All 6 compounds altered gene expression that linked to developmental disorders and neurological diseases. Other enriched canonical pathways overlapped among compounds of the same class; eg, genes and metabolites altered by both cytosine arabinoside and 5-fluorouracil exposures are enriched in axonal guidance pathways. Integrated analysis of upstream regulators was heterogeneous across compounds, but identified several transcriptomic regulators including CREB1, SOX2, NOTCH1, and PRODH. These results demonstrate that changes in network formation are accompanied by transcriptomic and metabolomic changes and that different classes of compounds produce differing responses. This approach can enhance information obtained from new approach methodologies and contribute to the identification and development of adverse outcome pathways associated with DNT.

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.

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.

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.

High-throughput toxicogenomic screening of chemicals in the environment using metabolically competent hepatic cell cultures.

The ToxCast in vitro screening program has provided concentration-response bioactivity data across more than a thousand assay endpoints for thousands of chemicals found in our environment and commerce. However, most ToxCast screening assays have evaluated individual biological targets in cancer cell lines lacking integrated physiological functionality (such as receptor signaling, metabolism). We evaluated differentiated HepaRGTM cells, a human liver-derived cell model understood to effectively model physiologically relevant hepatic signaling. Expression of 93 gene transcripts was measured by quantitative polymerase chain reaction using Fluidigm 96.96 dynamic arrays in response to 1060 chemicals tested in eight-point concentration-response. A Bayesian framework quantitatively modeled chemical-induced changes in gene expression via six transcription factors including: aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, farnesoid X receptor, androgen receptor, and peroxisome proliferator-activated receptor alpha. For these chemicals the network model translates transcriptomic data into Bayesian inferences about molecular targets known to activate toxicological adverse outcome pathways. These data also provide new insights into the molecular signaling network of HepaRGTM cell cultures.

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.

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.