A PBPK model describing the pharmacokinetics of γ-HBCD exposure in mice.

The brominated flame retardant, hexabromocyclododecane (HBCD), is added-but not bound-to consumer products and is eventually found in the environment and human tissues. Commercial-grade HBCD mixtures contain three major stereoisomers, alpha (α), beta (ß), and gamma (γ), that are typically at a ratio of 12%:6%:82%, respectively. Although HBCD is widely used, the toxicological effects from its exposure in humans are not clearly understood. Using a physiologically based pharmacokinetic (PBPK) model could help improve our understanding of the toxicity of HBCD. The aim of this work was to develop a PBPK model, consisting of five permeability limited compartments (i.e., brain, liver, adipose tissue, blood, and rest of the body), to evaluate the pharmacokinetics of γ-HBCD in C57BL/6 mice. Physiological parameters related to body size, organ weights, and blood flow were taken from the literature. All partition coefficients were calculated based on the log Kow. The elimination in urine and feces was optimized to reflect the percent dose eliminated, as published in the literature. Compared with data from the literature for brain, liver, blood, and adipose tissue, the model simulations accurately described the mouse data set within 1.5-fold of the data points. Also, two examples showing the utility of the PBPK model supplement the information regarding the internal dose that caused the health effects observed during these studies. Although this version of the PBPK model expressly describes γ-HBCD, more efforts are needed to clarify and improve the model to discriminate between the α, ß, and γ stereoisomers.

KRAS-retroviral fusion transcripts and gene amplification in arsenic-transformed, human prostate CAsE-PE cancer cells.

CAsE-PE cells are an arsenic-transformed, human prostate epithelial line containing oncogenic mutations in KRAS compared to immortalized, normal KRAS parent cells, RWPE-1. We previously reported increased copy number of mutated KRAS in CAsE-PE cells, suggesting gene amplification. Here, KRAS flanking genomic and transcriptomic regions were sequenced in CAsE-PE cells for insight into KRAS amplification. Comparison of DNA-Seq and RNA-Seq showed increased reads from background aligning to all KRAS exons in CAsE-PE cells, while a uniform DNA-Seq read distribution occurred in RWPE-1 cells with normal transcript expression. We searched for KRAS fusions in DNA and RNA sequencing data finding a portion of reads aligning to KRAS and viral sequence. After generation of cDNA from total RNA, short and long KRAS probes were generated to hybridize cDNA and KRAS enriched fragments were PacBio sequenced. More KRAS reads were captured from CAsE-PE cDNA versus RWPE-1 by each probe set. Only CAsE-PE cDNA showed KRAS viral fusion transcripts, primarily mapping to LTR and endogenous retrovirus sequences on either 5'- or 3'-ends of KRAS. Most KRAS viral fusion transcripts contained 4 to 6 exons but some PacBio sequences were in unusual orientations, suggesting viral insertions within the gene body. Additionally, conditioned media was extracted for potential retroviral particles. RNA-Seq of culture media isolates identified KRAS retroviral fusion transcripts in CAsE-PE media only. Truncated KRAS transcripts suggested multiple retroviral integration sites occurred within the KRAS gene producing KRAS retroviral fusions of various lengths. Findings suggest activation of endogenous retroviruses in arsenic carcinogenesis should be explored.

Toxicokinetics of perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) in male and female Hsd:Sprague dawley SD rats following intravenous or gavage administration.

Poly- and perfluorinated alkyl substances (PFAS) are environmentally persistent chemicals associated with many adverse health outcomes. The National Toxicology Program evaluated the toxicokinetics (TK) of several PFAS to provide context for toxicologic findings.Plasma TK parameters and tissue (liver, kidney, brain) concentrations are reported for perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA) or perfluorodecanoic acid (PFDA) after single-dose administration in male and female Hsd:Sprague-Dawley® (SD) rats.Generally, longer Tmax and elimination half-lives, and slower clearance f, were correlated with longer chain length. Male rats administered PFOA had a prolonged half-life compared to females (215 h vs. 2.75), while females had faster clearance and smaller plasma area under the curve (AUC). Females administered PFHxA had a shorter half-life (2 h vs. 9) than males and faster clearance with a smaller plasma AUC, although this was less pronounced than PFOA. There was no sex difference in PFDA half-life. Female rats administered PFDA had a higher plasma AUC/dose than males, and a slower clearance. PFDA had the highest levels in the liver of the PFAS evaluated.Profiling the toxicokinetics of these PFAS allows for comparison among subclasses, and more direct translation of rodent toxicity to human populations.

An Intuitive Approach for Predicting Potential Human Health Risk with the Tox21 10k Library.

In vitro-in vivo extrapolation (IVIVE) analyses translating high-throughput screening (HTS) data to human relevance have been limited. This study represents the first report applying IVIVE approaches and exposure comparisons using the entirety of the Tox21 federal collaboration chemical screening data, incorporating assay response efficacy and quality of concentration-response fits, and providing quantitative anchoring to first address the likelihood of human in vivo interactions with Tox21 compounds. This likelihood was assessed using a maximum blood concentration to in vitro response ratio approach (Cmax/AC50), analogous to decision-making methods for clinical drug-drug interactions. Fraction unbound in plasma (fup) and intrinsic hepatic clearance (CLint) parameters were estimated in silico and incorporated in a three-compartment toxicokinetic (TK) model to first predict Cmax for in vivo corroboration using therapeutic scenarios. Toward lower exposure scenarios, 36 compounds of 3925 unique chemicals with curated activity in the HTS data using high-quality dose-response model fits and ≥40% efficacy gave "possible" human in vivo interaction likelihoods lower than median human exposures predicted in the United States Environmental Protection Agency's ExpoCast program. A publicly available web application has been designed to provide all Tox21-ToxCast dose-likelihood predictions. Overall, this approach provides an intuitive framework to relate in vitro toxicology data rapidly and quantitatively to exposures using either in vitro or in silico derived TK parameters and can be thought of as an important step toward estimating plausible biological interactions in a high-throughput risk-assessment framework.

Tissue time course and bioavailability of the pyrethroid insecticide bifenthrin in the Long-Evans rat.

1. Pyrethroids are neurotoxic and parent pyrethroid appears to be toxic entity. This study evaluated the oral disposition and bioavailability of bifenthrin in the adult male Long-Evans rat. 2. In the disposition study, rats were administered bifenthrin (0.3 or 3 mg/kg) by oral gavage and serially sacrificed (0.25 h to 21 days). Blood, liver, brain and adipose tissue were removed. In the bioavailability study, blood was collected serially from jugular vein cannulated rats (0.25 to 24 h) following oral (0.3 or 3 mg/kg) or intravenous (0.3 mg/kg) administration of bifenthrin. Tissues were extracted and analyzed for bifenthrin by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). 3. Bifenthrin concentration in blood and liver peaked 1-2-h postoral administration and were approximately 90 ng/ml (or g) and 1000 ng/ml (or g) for both tissues at 0.3 and 3 mg/kg, respectively. Bifenthrin was rapidly cleared from both blood and liver. Brain concentrations peaked at 4-6 h and were lower than in blood at both doses (12 and 143 ng/g). Bifenthrin in adipose tissue peaked at the collected time points of 8 (157 ng/g) and 24 (1145 ng/g) h for the 0.3 and 3 mg/kg doses, respectively and was retained 21 days postoral administration. Following intravenous administration, the blood bifenthrin concentration decreased bi-exponentially, with a distribution half-life of 0.2 h and an elimination half-life of 8 h. Bifenthrin bioavailability was approximately 30%. These disposition and kinetic bifenthrin data may decrease uncertainties in the risk assessment for this pyrethroid insecticide.

In vitro metabolism of thyroxine by rat and human hepatocytes.

1. The liver metabolizes thyroxine (T(4)) through two major pathways: deiodination and conjugation. Following exposure to xenobiotics, T(4) conjugation increases through the induction of hepatic uridine diphosphate glucuronosyltransferase (UGT) in rodents; however, it is uncertain to what degree different species employ deiodination and conjugation in the metabolism of T(4). The objective of this study was to compare the metabolism of T4 in untreated and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-treated primary sandwich-cultured hepatocytes from rat (SCRH) and human (SCHH). 2. Basal metabolite concentrations were 13 times higher in the medium of SCRH compared to SCHH. Metabolite distribution in the medium of SCRH versus SCHH was as follows: T(4)G, (91.6 versus 5.3%); T4S, (3.6 versus 4.4%) and T(3) + rT(3), (4.9 versus 90.3%). PCB 153 induced T(4)G in the medium of SCRH and SCHH; however, T(4)S and T(3) + rT(3) were changed but to a much lesser degree. 3. The results indicate that baseline T(4) glucuronidation is greater in SCRH compared to SCHH. These data also suggest that glucuronidation may be a more important pathway for T(4) metabolism in rats and deiodination may be a favored pathway in humans; however, with PCB 153 treatment these data support glucuronidation as a primary route of T(4) metabolism in both rat and humans.

Exploration of xenobiotic metabolism within cell lines used for Tox21 chemical screening.

The Tox21 Program has investigated thousands of chemicals with high-throughput screening assays using cell-based assays to link thousands of chemicals to individual molecular targets/pathways. However, these systems have been widely criticized for their suspected lack of 'metabolic competence' to bioactivate or detoxify chemical exposures. In this study, 9 cell line backgrounds used in Tox21 assays (i.e., HepG2, HEK293, Hela, HCT116, ME180, CHO-K1, GH3.TRE-Luc, C3H10T1/2 and MCF7) were evaluated via metabolite formation rates, along with metabolic clearance and metabolite profiling for HepG2, HEK293, and MCF-7aroERE, in comparison to pooled donor (50) suspensions of primary human hepatocytes (PHHs). Using prototype clinical drug substrates for CYP1A2, CYP2B6, and CYP3A4/5, extremely low-to-undetectable CYP450 metabolism was observed (24 h), and consistent with their purported 'lack' of metabolic competence. However, for Phase II metabolizing enzymes and metabolic clearance, surprisingly proficient metabolism was observed for bisphenol AF, bisphenol S, and 7-hydroxycoumarin. Here, comparatively low glucuronidation relative to sulfation was observed in contrast to equivalent levels in PHHs. Overall, while a lack of CYP450 metabolism was confirmed in this benchmarking effort, Tox21 cell lines were not 'incompetent' for xenobiotic metabolism, and displayed surprisingly high proficiency for sulfation that rivaled PHHs. These findings have implications for the interpretation of Tox21 assay data, and establish a framework for evaluating of 'metabolic competence' with in vitro models.

Benchmark Concentrations for Untargeted Metabolomics Versus Transcriptomics for Liver Injury Compounds in In Vitro Liver Models.

Interpretation of untargeted metabolomics data from both in vivo and physiologically relevant in vitro model systems continues to be a significant challenge for toxicology research. Potency-based modeling of toxicological responses has served as a pillar of interpretive context and translation of testing data. In this study, we leverage the resolving power of concentration-response modeling through benchmark concentration (BMC) analysis to interpret untargeted metabolomics data from differentiated cultures of HepaRG cells exposed to a panel of reference compounds and integrate data in a potency-aligned framework with matched transcriptomic data. For this work, we characterized biological responses to classical human liver injury compounds and comparator compounds, known to not cause liver injury in humans, at 10 exposure concentrations in spent culture media by untargeted liquid chromatography-mass spectrometry analysis. The analyte features observed (with limited metabolites identified) were analyzed using BMC modeling to derive compound-induced points of departure. The results revealed liver injury compounds produced concentration-related increases in metabolomic response compared to those rarely associated with liver injury (ie, sucrose, potassium chloride). Moreover, the distributions of altered metabolomic features were largely comparable with those observed using high throughput transcriptomics, which were further extended to investigate the potential for in vitro observed biological responses to be observed in humans with exposures at therapeutic doses. These results demonstrate the utility of BMC modeling of untargeted metabolomics data as a sensitive and quantitative indicator of human liver injury potential.

Development of a quantitative model of pregnane X receptor (PXR) mediated xenobiotic metabolizing enzyme induction.

The pregnane X receptor plays an integral role in the regulation of hepatic metabolism. It has been shown to regulate CYP3A4, which is the most abundant cytochrome P450 in the human liver. With its large and flexible ligand-binding domain, PXR can be activated by an enormous range of relatively small, hydrophobic, exogenous compounds. Upon activation, PXR partners with the retinoid X receptor (RXR) to form a heterodimer. The newly formed heterodimer binds to an appropriate DNA response element, causing increased transcription. This leads to an induction in the level of CYP3A4. These mechanistic steps are included into a biologically-based mathematical model. The quantitative model predicts fold level inductions of CYP3A4 mRNA and protein in response to PXR activation. Model parameter values have been taken from literature when appropriate. Unknown parameter values are estimated by optimizing the model results to published in vivo and in vitro data sets. A sensitivity analysis is performed to evaluate the model structure and identify future data needs which would be critical to revising the model.

Elevated Arsenic and Lead Concentrations in Natural Healing Clay Applied Topically as a Treatment for Ulcerative Dermatitis in Mice.

Ulcerative dermatitis in laboratory mice remains an ongoing clinical problem and animal welfare issue. Many products have been used to treat dermatitis in mice, with varying success. Recently, the topical administration of healing clays, such as bentonite and green clays, has been explored as a viable, natural treatment. We found high concentrations of arsenic and lead in experimental samples of therapeutic clay. Given the known toxic effects of these environmental heavy metals, we sought to determine whether the topical administration of a clay product containing bioavailable arsenic and lead exerted a biologic effect in mice that potentially could introduce unwanted research variability. Two cohorts of 20 singly housed, shaved, dermatitis free, adult male CD1 mice were dosed daily for 2 wk by topical application of saline or green clay paste. Samples of liver, kidney and whole blood were collected and analyzed for total arsenic and lead concentrations. Hepatic and renal concentrations of arsenic were not different between treated and control mice in either cohort; however, hepatic and renal concentrations of lead were elevated in clay treated mice compared to controls in both cohorts. In addition, in both cohorts, the activity of δ-aminolevulinate acid dehydratase, an enzyme involved with heme biosynthesis and a marker of lead toxicity, did not differ significantly between the clay-treated mice and controls. We have demonstrated that these clay products contain high concentrations of arsenic and lead and that topical application can result in the accumulation of lead in the liver and kidneys; however, these concentrations did not result in measurable biologic effects. These products should be used with caution, especially in studies of lead toxicity, heme biosynthesis, and renal α2 microglobulin function.

Evaluation of 5-day In Vivo Rat Liver and Kidney With High-throughput Transcriptomics for Estimating Benchmark Doses of Apical Outcomes.

A 5-day in vivo rat model was evaluated as an approach to estimate chemical exposures that may pose minimal risk by comparing benchmark dose (BMD) values for transcriptional changes in the liver and kidney to BMD values for toxicological endpoints from traditional toxicity studies. Eighteen chemicals, most having been tested by the National Toxicology Program in 2-year bioassays, were evaluated. Some of these chemicals are potent hepatotoxicants (eg, DE71, PFOA, and furan) in rodents, some exhibit toxicity but have minimal hepatic effects (eg, acrylamide and α,ß-thujone), and some exhibit little overt toxicity (eg, ginseng and milk thistle extract) based on traditional toxicological evaluations. Male Sprague Dawley rats were exposed once daily for 5 consecutive days by oral gavage to 8-10 dose levels for each chemical. Liver and kidney were collected 24 h after the final exposure and total RNA was assayed using high-throughput transcriptomics (HTT) with the rat S1500+ platform. HTT data were analyzed using BMD Express 2 to determine transcriptional gene set BMD values. BMDS was used to determine BMD values for histopathological effects from chronic or subchronic toxicity studies. For many of the chemicals, the lowest transcriptional BMDs from the 5-day assays were within a factor of 5 of the lowest histopathological BMDs from the toxicity studies. These data suggest that using HTT in a 5-day in vivo rat model provides reasonable estimates of BMD values for traditional apical endpoints. This approach may be useful to prioritize chemicals for further testing while providing actionable data in a timely and cost-effective manner.

Mixed organic and inorganic tapwater exposures and potential effects in greater Chicago area, USA.

Safe drinking water at the point of use (tapwater, TW) is a public-health priority. TW exposures and potential human-health concerns of 540 organics and 35 inorganics were assessed in 45 Chicago-area United States (US) homes in 2017. No US Environmental Protection Agency (EPA) enforceable Maximum Contaminant Level(s) (MCL) were exceeded in any residential or water treatment plant (WTP) pre-distribution TW sample. Ninety percent (90%) of organic analytes were not detected in treated TW, emphasizing the high quality of the Lake Michigan drinking-water source and the efficacy of the drinking-water treatment and monitoring. Sixteen (16) organics were detected in >25% of TW samples, with about 50 detected at least once. Low-level TW exposures to unregulated disinfection byproducts (DBP) of emerging concern, per/polyfluoroalkyl substances (PFAS), and three pesticides were ubiquitous. Common exceedances of non-enforceable EPA MCL Goal(s) (MCLG) of zero for arsenic [As], lead [Pb], uranium [U], bromodichloromethane, and tribromomethane suggest potential human-health concerns and emphasize the continuing need for improved understanding of cumulative effects of low-concentration mixtures on vulnerable sub-populations. Because DBP dominated TW organics, residential-TW concentrations are potentially predictable with expanded pre-distribution DBP monitoring. However, several TW chemicals, notably Pb and several infrequently detected organic compounds, were not readily explained by pre-distribution samples, illustrating the need for continued broad inorganic/organic TW characterization to support consumer assessment of acceptable risk and point-of-use treatment options.

In vitro metabolism of pyrethroid pesticides by rat and human hepatic microsomes and cytochrome p450 isoforms.

Species differences in the intrinsic clearance (CL(int)) and the enzymes involved in the metabolism of pyrethroid pesticides were examined in rat and human hepatic microsomes. The pyrethroids bifenthrin, S-bioallethrin, bioresmethrin, beta-cyfluthrin, cypermethrin, cis-permethrin, and trans-permethrin were incubated in rat and human hepatic microsomes in the presence or absence of NADPH. Metabolism was measured using a parent depletion approach. The CL(int) of the pyrethroids was 5- to 15-fold greater in rat relative to human microsomes except for trans-permethrin, which was approximately 45% greater in human microsomes. The metabolism of bifenthrin, S-bioallethrin, and cis-permethrin in rat and human hepatic microsomes was solely the result of oxidative processes. The metabolism of bioresmethrin and cypermethrin in human hepatic microsomes was solely the result of hydrolytic processes. Bioresmethrin and cypermethrin in rat hepatic microsomes and beta-cyfluthrin and trans-permethrin in microsomes from both species were metabolized by both oxidative and hydrolytic pathways. The metabolism of trans-permethrin was reduced when incubated with its diastereomer, cis-permethrin, in both rat and human hepatic microsomes. Rat cytochrome P450 (P450) isoforms that showed activity toward several pyrethroids included CYP1A1, CYP1A2, CYP2C6, CYP2C11, CYP3A1, and CYP3A2. Human P450 isoforms that showed activity toward multiple pyrethroids were CYP2C8, CYP2C9, CYP2C19, and CYP3A4. Species-specific differences in metabolism may result in variable detoxification of pyrethroids, which may in turn result in divergent neurotoxic outcomes. These species differences and isomer interactions in metabolism of pyrethroids should be considered when assessing the potential adverse health effects of pyrethroid pesticides.

Using Tox21 High-Throughput Screening Assays for the Evaluation of Botanical and Dietary Supplements.

Introduction: Recent nationwide surveys found that natural products, including botanical dietary supplements, are used by ∼18% of adults. In many cases, there is a paucity of toxicological data available for these substances to allow for confident evaluations of product safety. The National Toxicology Program (NTP) has received numerous nominations from the public and federal agencies to study the toxicological effects of botanical dietary supplements. The NTP sought to evaluate the utility of in vitro quantitative high-throughput screening (qHTS) assays for toxicological assessment of botanical and dietary supplements. Materials and Methods: In brief, concentration-response assessments of 90 test substances, including 13 distinct botanical species, and individual purported active constituents were evaluated using a subset of the Tox21 qHTS testing panel. The screen included 20 different endpoints that covered a broad range of biologically relevant signaling pathways to detect test article effects upon endocrine activity, nuclear receptor signaling, stress response signaling, genotoxicity, and cell death signaling. Results and Discussion: Botanical dietary supplement extracts induced measurable and diverse activity. Elevated biological activity profiles were observed following treatments with individual chemical constituents relative to their associated botanical extract. The overall distribution of activity was comparable to activities exhibited by compounds present in the Tox21 10K chemical library. Conclusion: Botanical supplements did not exhibit minimal or idiosyncratic activities that would preclude the use of qHTS platforms as a feasible method to screen this class of compounds. However, there are still many considerations and further development required when attempting to use in vitro qHTS methods to characterize the safety profile of botanical/dietary supplements.

Evaluating Sufficient Similarity of Botanical Dietary Supplements: Combining Chemical and In Vitro Biological Data.

Botanical dietary supplements are complex mixtures with numerous potential sources of variation along the supply chain from raw plant material to the market. Approaches for determining sufficient similarity (ie, complex mixture read-across) may be required to extrapolate efficacy or safety data from a tested sample to other products containing the botanical ingredient(s) of interest. In this work, screening-level approaches for generating both chemical and biological-response profiles were used to evaluate the similarity of black cohosh (Actaea racemosa) and Echinacea purpurea samples to well-characterized National Toxicology Program (NTP) test articles. Data from nontargeted chemical analyses and gene expression of toxicologically important hepatic receptor pathways (aryl hydrocarbon receptor [AhR], constitutive androstane receptor [CAR], pregnane X receptor [PXR], farnesoid X receptor [FXR], and peroxisome proliferator-activated receptor alpha [PPARα]) in primary human hepatocyte cultures were used to determine similarity through hierarchical clustering. Although there were differences in chemical profiles across black cohosh samples, these differences were not reflected in the biological-response profiles. These findings highlight the complexity of biological-response dynamics that may not be reflected in chemical composition profiles. Thus, biological-response data could be used as the primary basis for determining similarity among black cohosh samples. Samples of E. purpurea displayed better correlation in similarity across chemical and biological-response measures. The general approaches described herein can be applied to complex mixtures with unidentified active constituents to determine when data from a tested mixture (eg, NTP test article) can be used for hazard identification of sufficiently similar mixtures, with the knowledge of toxicological targets informing assay selection when possible.

The Power of Resolution: Contextualized Understanding of Biological Responses to Liver Injury Chemicals Using High-throughput Transcriptomics and Benchmark Concentration Modeling.

Prediction of human response to chemical exposures is a major challenge in both pharmaceutical and toxicological research. Transcriptomics has been a powerful tool to explore chemical-biological interactions, however, limited throughput, high-costs, and complexity of transcriptomic interpretations have yielded numerous studies lacking sufficient experimental context for predictive application. To address these challenges, we have utilized a novel high-throughput transcriptomics (HTT) platform, TempO-Seq, to apply the interpretive power of concentration-response modeling with exposures to 24 reference compounds in both differentiated and non-differentiated human HepaRG cell cultures. Our goals were to (1) explore transcriptomic characteristics distinguishing liver injury compounds, (2) assess impacts of differentiation state of HepaRG cells on baseline and compound-induced responses (eg, metabolically-activated), and (3) identify and resolve reference biological-response pathways through benchmark concentration (BMC) modeling. Study data revealed the predictive utility of this approach to identify human liver injury compounds by their respective BMCs in relation to human internal exposure plasma concentrations, and effectively distinguished drug analogs with varied associations of human liver injury (eg, withdrawn therapeutics trovafloxacin and troglitazone). Impacts of cellular differentiation state (proliferated vs differentiated) were revealed on baseline drug metabolizing enzyme expression, hepatic receptor signaling, and responsiveness to metabolically-activated toxicants (eg, cyclophosphamide, benzo(a)pyrene, and aflatoxin B1). Finally, concentration-response modeling enabled efficient identification and resolution of plausibly-relevant biological-response pathways through their respective pathway-level BMCs. Taken together, these findings revealed HTT paired with differentiated in vitro liver models as an effective tool to model, explore, and interpret toxicological and pharmacological interactions.

Arsenite malignantly transforms human prostate epithelial cells in vitro by gene amplification of mutated KRAS.

Inorganic arsenic is an environmental human carcinogen of several organs including the urinary tract. RWPE-1 cells are immortalized, non-tumorigenic, human prostate epithelia that become malignantly transformed into the CAsE-PE line after continuous in vitro exposure to 5µM arsenite over a period of months. For insight into in vitro arsenite transformation, we performed RNA-seq for differential gene expression and targeted sequencing of KRAS. We report >7,000 differentially expressed transcripts in CAsE-PE cells compared to RWPE-1 cells at >2-fold change, q<0.05 by RNA-seq. Notably, KRAS expression was highly elevated in CAsE-PE cells, with pathway analysis supporting increased cell proliferation, cell motility, survival and cancer pathways. Targeted DNA sequencing of KRAS revealed a mutant specific allelic imbalance, 'MASI', frequently found in primary clinical tumors. We found high expression of a mutated KRAS transcript carrying oncogenic mutations at codons 12 and 59 and many silent mutations, accompanied by lower expression of a wild-type allele. Parallel cultures of RWPE-1 cells retained a wild-type KRAS genotype. Copy number analysis and sequencing showed amplification of the mutant KRAS allele. KRAS is expressed as two splice variants, KRAS4a and KRAS4b, where variant 4b is more prevalent in normal cells compared to greater levels of variant 4a seen in tumor cells. 454 Roche sequencing measured KRAS variants in each cell type. We found KRAS4a as the predominant transcript variant in CAsE-PE cells compared to KRAS4b, the variant expressed primarily in RWPE-1 cells and in normal prostate, early passage, primary epithelial cells. Overall, gene expression data were consistent with KRAS-driven proliferation pathways found in spontaneous tumors and malignantly transformed cell lines. Arsenite is recognized as an important environmental carcinogen, but it is not a direct mutagen. Further investigations into this in vitro transformation model will focus on genomic events that cause arsenite-mediated mutation and overexpression of KRAS in CAsE-PE cells.

A Chemical Category-Based Prioritization Approach for Selecting 75 Per- and Polyfluoroalkyl Substances (PFAS) for Tiered Toxicity and Toxicokinetic Testing.

Per- and polyfluoroalkyl substances (PFASs) are a group of fluorinated substances of interest to researchers, regulators, and the public due to their widespread presence in the environment. A few PFASs have comparatively extensive amounts of human epidemiological, exposure, and experimental animal toxicity data (e.g., perfluorooctanoic acid), whereas little toxicity and exposure information exists for much of the broader set of PFASs. Given that traditional approaches to generate toxicity information are resource intensive, new approach methods, including in vitro high-throughput toxicity (HTT) testing, are being employed to inform PFAS hazard characterization and further (in vivo) testing. The U.S. Environmental Protection Agency (EPA) and the National Toxicology Program (NTP) are collaborating to develop a risk-based approach for conducting PFAS toxicity testing to facilitate PFAS human health assessments. This article describes the construction of a PFAS screening library and the process by which a targeted subset of 75 PFASs were selected. Multiple factors were considered, including interest to the U.S. EPA, compounds within targeted categories, structural diversity, exposure considerations, procurability and testability, and availability of existing toxicity data. Generating targeted HTT data for PFASs represents a new frontier for informing priority setting. https://doi.org/10.1289/EHP4555.

The Influence of Obesity on the Pharmacokinetics of Dioxin in Mice: An Assessment Using Classical and PBPK Modeling.

The effects of body fat mass on the elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in mice. When male C57BL/6J mice are fed a high-fat, simple carbohydrate diet (HFD) for 13 weeks, they develop an obese phenotype. In contrast, A/J mice fed an HFD do not become obese. After 13 weeks on a normal diet (ND) or HFD, male C57BL/6J and A/J mice received a single dose by gavage of 0.1 or 5.0 µg of 2,3,7,8-tetrachloro[1,6-3H] dibenzo-p-dioxin per kg body weight. Using classical pharmacokinetics, the blood elimination half-life of TCDD was approximately 10 and 2 times longer in the C57BL/6J on the HFD compared with the mice on the ND at 0.1 and 5.0 µg/kg doses, respectively. The diet did not increase the blood half-life of TCDD in the A/J mice, which did not get obese. Using a physiologically based pharmacokinetic model for TCDD that incorporated experimentally derived percent body fat mass and tissue partition coefficients, as well as data on hepatic sequestration, did not provide accurate predictions to the data and could not explain the increase in half-life of TCDD in the HFD groups. This work demonstrates that obesity influences the half-life of TCDD, but other undetermined factors are involved in its elimination because the increase in body fat mass, decreases in cytochrome P4501A2, and altered partition coefficients could not completely explain the prolonged half-life.

Evaluation and Optimization of Pharmacokinetic Models for in Vitro to in Vivo Extrapolation of Estrogenic Activity for Environmental Chemicals.

BACKGROUND: To effectively incorporate in vitro data into regulatory use, confidence must be established in the quantitative extrapolation of in vitro activity to relevant end points in animals or humans. OBJECTIVE: Our goal was to evaluate and optimize in vitro to in vivo extrapolation (IVIVE) approaches using in vitro estrogen receptor (ER) activity to predict estrogenic effects measured in rodent uterotrophic studies. METHODS: We evaluated three pharmacokinetic (PK) models with varying complexities to extrapolate in vitro to in vivo dosimetry for a group of 29 ER agonists, using data from validated in vitro [U.S. Environmental Protection Agency (U.S. EPA) ToxCast™ ER model] and in vivo (uterotrophic) methods. In vitro activity values were adjusted using mass-balance equations to estimate intracellular exposure via an enrichment factor (EF), and steady-state model calculations were adjusted using fraction of unbound chemical in the plasma ([Formula: see text]) to approximate bioavailability. Accuracy of each model-adjustment combination was assessed by comparing model predictions with lowest effect levels (LELs) from guideline uterotrophic studies. RESULTS: We found little difference in model predictive performance based on complexity or route-specific modifications. Simple adjustments, applied to account for in vitro intracellular exposure (EF) or chemical bioavailability ([Formula: see text]), resulted in significant improvements in the predictive performance of all models. CONCLUSION: Computational IVIVE approaches accurately estimate chemical exposure levels that elicit positive responses in the rodent uterotrophic bioassay. The simplest model had the best overall performance for predicting both oral (PPK_EF) and injection (PPK_[Formula: see text]) LELs from guideline uterotrophic studies, is freely available, and can be parameterized entirely using freely available in silico tools. https://doi.org/10.1289/EHP1655.