Integrating nonclinical in vitro, in silico, and in vivo datasets holistically can improve hazard characterization and risk assessment. In pharmaceutical development, cardiovascular liabilities are a leading cause of compound attrition. Prior to clinical studies, functional cardiovascular data are generated in single-dose safety pharmacology telemetry studies, with structural pathology data obtained from repeat-dose toxicology studies with limited concurrent functional endpoints, eg, electrocardiogram via jacketed telemetry. Relationships between datasets remain largely undetermined. To address this gap, a cross-pharma collaboration collated functional and structural data from 135 compounds. Retrospective functional data were collected from good laboratory practice conscious dog safety pharmacology studies: effects defined as hemodynamic blood pressure or heart rate changes. Morphologic pathology findings (mainly degeneration, vacuolation, inflammation) from related toxicology studies in the dog (3-91 days repeat-dosing) were reviewed, harmonized, and location categorized: cardiac muscle (myocardium, epicardium, endocardium, unspecified), atrioventricular/aortic valves, blood vessels. The prevalence of cardiovascular histopathology changes was 11.1% of compounds, with 53% recording a functional blood pressure or heart rate change. Correlations were assessed using the Mantel-Haenszel Chi-square trend test, identifying statistically significant associations between cardiac muscle pathology and (1) decreased blood pressure, (2) increased heart rate, and between cardiovascular vessel pathology and increased heart rate. Negative predictive values were high, suggesting few compounds cause repeat-dose cardiovascular structural change in the absence of functional effects in single-dose safety pharmacology studies. Therefore, observed functional changes could prompt moving (sub)chronic toxicology studies forward, to identify cardiovascular liabilities earlier in development, and reduce late-stage attrition.

Per- and polyfluoroalkyl substances (PFAS) are used as industrial surfactants and chemical coatings for household goods such as Teflon. Despite regulatory efforts to phase out legacy PFAS, they remain detectable in drinking water throughout the United States. This is due to the stability of legacy PFAS and the continued use of replacement compounds. In humans, PFAS have been detected in placenta and cord blood and are associated with low birth weight and preeclampsia risk. Preeclampsia is a leading cause of maternal mortality and is driven by insufficient endometrial trophoblast invasion, resulting in poor placental blood flow. PFAS alter invasion of other cell types, but their impact on trophoblasts is not understood. We therefore assessed the effects of PFAS on trophoblast migration, invasion, and gene expression in vitro. Trophoblast migration and invasion were assessed using a modified scratch assay in the absence or presence of Matrigel, respectively. Treatment with perfluorooctanoic sulfate (PFOS), perfluorooctanoic acid (PFOA), and GenX (1000 ng/ml) each decreased trophoblast migration over 24 h. However, only GenX (1000 ng/ml) significantly inhibited trophoblast invasion. Treatment with PFOS, PFOA, and GenX also decreased trophoblast expression of chemokines (eg, CCL2), chemokine receptors (eg, CCR4), and inflammatory enzymes (eg, ALOX15) involved in migration. Inhibition of chemokine receptors with pertussis toxin (10 ng/ml), a G-protein inhibitor, inhibited trophoblast migration similar to the PFAS. Taken together, PFAS decrease trophoblast migration, invasion, and inflammatory signaling. By understanding the mechanisms involved, it may be possible to identify the biological and exposure factors that contribute to preeclampsia.

In 2014, it was estimated that more than 1.9 billion adults were overweight with over 600 million classifiable as obese. Approximately two-thirds of U.S. adults over 20 years of age are currently overweight with about 35% classified as obese, a figure thought likely to reach 42% by 2030 in those over 18 years of age. Adipose cells from stored body fat secrete estrogen and a very large number (> 500) of biologically active substances termed adipokines, in addition to inducing, by other cell-driven effects, pathological alterations in insulin pathways. The U.S. National Cancer Institute reports that exposure to the hormone disrupting and proinflammatory effects of excess adipose tissue are associated with an increased risk for 11 different cancers. Obesity is also associated with a number of serious non-neoplastic conditions including metabolic syndrome and type 2 diabetes; menstrual cycle irregularities and lowered fertility (men and women); and abnormal bone morphology in a subset of female patients. In men hypogonadism, low testosterone levels, benign prostatic hyperplasia, and lowered sperm counts have been reported. In developed countries, the endogenous adverse health burden associated with obesity is only matched, quantitatively and qualitatively, by the exogenous toxicity of cigarette smoking. The investigation of possible hormonal and/or proinflammatory effects of chemicals should include an assessment of the profound endocrine alterations associated with obesity.

Zileuton is an orally active inhibitor of leukotriene synthesis for maintenance treatment of asthma, for which clinical usage has been associated with idiosyncratic liver injury. Mechanistic understanding of zileuton toxicity is hampered by the rarity of the cases and lack of an animal model. A promising model for mechanistic study of rare liver injury is the Diversity Outbred (J:DO) mouse population, with genetic variation similar to that found in humans. In this study, female DO mice were administered zileuton or vehicle daily for 7 days (i.g.). Serum liver enzymes were elevated in the zileuton group, with marked interindividual variability in response. Zileuton exposure-induced findings in susceptible DO mice included microvesicular fatty change, hepatocellular mitosis, and hepatocellular necrosis. Inducible nitric oxide synthase and nitrotyrosine abundance were increased in livers of animals with necrosis and those with fatty change, implicating nitrosative stress as a possible injury mechanism. Conversely, DO mice lacking adverse liver pathology following zileuton exposure experienced decreased hepatic concentrations of resistin and increased concentrations of insulin and leptin, providing potential clues into mechanisms of toxicity resistance. Transcriptome pathway analysis highlighted mitochondrial dysfunction and altered fatty acid oxidation as key molecular perturbations associated with zileuton exposure, and suggested that interindividual differences in cytochrome P450 metabolism, glutathione-mediated detoxification, and farnesoid X receptor signaling may contribute to zileuton-induced liver injury (ZILI). Taken together, DO mice provided a platform for investigating mechanisms of toxicity and resistance in context of ZILI which may lead to targeted therapeutic interventions.

Pancreatic cancer is a highly fatal malignancy for which surgery is currently considered to be the only curative treatment. However, less than a quarter of patients have disease amenable to definitive surgical resection. Local treatment with radiation therapy is a promising alternative to surgery for those patients with unresectable disease. However, conventional radiation techniques with computed tomography (CT)-guided therapy have yielded disappointing results due to the inability to deliver ablative doses of ionizing radiation, while sparing the radiosensitive adjacent organs at risk. Magnetic resonance-guided radiotherapy (MRgRT) has emerged as an alternative to CT-guided radiation treatment which allows for the delivery of higher doses of radiation with low toxicity to surrounding structures. Further study into the use of MRgRT and dose escalation for locally advanced unresectable pancreatic cancer is needed.

Determining the in vitro bioavailable concentration is a critical, yet unmet need to refine in vitro-to-in vivo extrapolation for unknown or variable composition, complex reaction product or biological material (UVCB) substances. UVCBs such as petroleum substances are commonly subjected to dimethyl sulfoxide (DMSO) extraction in order to retrieve the bioactive polycyclic aromatic compound (PAC) portion for in vitro testing. In addition to DMSO extraction, protein binding in cell culture media and dilution can all influence in vitro bioavailable concentrations of aliphatic and aromatic compounds in petroleum substances. However, these in vitro factors have not been fully characterized. In this study, we aimed to fill in these data gaps by characterizing the effects of these processes using both a defined mixture of analytical standards containing aliphatic and aromatic hydrocarbons, as well as 4 refined petroleum products as prototypical examples of UVCBs. Each substance was extracted with DMSO, and the protein binding in cell culture media was measured by using solid-phase microextraction. Semiquantitative analysis for aliphatic and aromatic compounds was achieved via gas chromatography-mass spectrometry. Our results showed that DMSO selectively extracted PACs from test substances, and that chemical profiles of PACs across molecular classes remained consistent after extraction. With respect to protein binding, chemical profiles were retained at a lower dilution (higher concentration), but a greater dilution factor (ie, lower concentration) resulted in higher protein binding in cell medium, which in turn altered the ultimate chemical profile of bioavailable PACs. Overall, this case study demonstrates that extraction procedures, protein binding in cell culture media, and dilution factors prior to in vitro testing can all contribute to determining the final bioavailable concentrations of bioactive constituents of UVCBs in vitro. Thus, in vitro-to-in vivo extrapolation for UVCBs may require greater attention to the concentration-dependent and compound-specific differences in recovery and bioavailability.

Mixtures risk assessment needs an efficient integration of in vivo, in vitro, and in silico data with epidemiology and human studies data. This involves several approaches, some in current use and others under development. This work extends the Agency for Toxic Substances and Disease Registry physiologically based pharmacokinetic (PBPK) toolkit, available for risk assessors, to include a mixture PBPK model of benzene, toluene, ethylbenzene, and xylenes. The recoded model was evaluated and applied to exposure scenarios to evaluate the validity of dose additivity for mixtures. In the second part of this work, we studied toluene, ethylbenzene, and xylene (TEX)-gene-disease associations using Comparative Toxicogenomics Database, pathway analysis and published microarray data from human gene expression changes in blood samples after short- and long-term exposures. Collectively, this information was used to establish hypotheses on potential linkages between TEX exposures and human health. The results show that 236 genes expressed were common between the short- and long-term exposures. These genes could be central for the interconnecting biological pathways potentially stimulated by TEX exposure, likely related to respiratory and neuro diseases. Using publicly available data we propose a conceptual framework to study pathway perturbations leading to toxicity of chemical mixtures. This proposed methodology lends mechanistic insights of the toxicity of mixtures and when experimentally validated will allow data gaps filling for mixtures' toxicity assessment. This work proposes an approach using current knowledge, available multiple stream data and applying computational methods to advance mixtures risk assessment.

1,3-Dichloropropene (1,3-D) showed a statistically increased incidence of bronchioloalveolar adenomas in male B6C3F1 mice at 60 ppm air concentration during previous chronic inhalation testing. No tumors were observed in female mice, nor in either sex of F344 rats up to 60 ppm, the highest dose tested. Therefore, to understand if lung tumors observed in high dose male mice are due to saturation of metabolic clearance, the linearity of 1,3-D concentrations in mouse blood was investigated on day 15 of repeated nose-only inhalation exposure to 0, 10, 20, 40, 60, 90, and 120 ppm (6 h/d, 7 d/week). Additional groups were included at 20, 60, and 120 ppm for blood collection at 1.5 and 3 h of exposure and up to 25 or 40 min post-exposure to determine area-under-the-curve. The data provide multiple lines of evidence that systemic exposures to 1,3-D in the mouse become nonlinear at inhalation exposure levels of 30 ppm or above. A reduction in minute volume occurred at the highest exposure concentration. The glutathione (GSH)-dependent metabolism of 1,3-D results in significant depletion of GSH at repeated exposure levels of 30 ppm and above. This loss of GSH results in decreased metabolic clearance of this test material, with a concomitant increase of the 1,3-D isomers in circulating blood at exposure concentrations ≥30 ppm. Shifts in the ratio of cis- and trans-1,3-D also support nonlinear toxicokinetics well below 60 ppm. Based on this data, a kinetically derived maximum dose for 1,3-D in mice for repeated exposures should be at or below 30 ppm. These results support non-relevance of 1,3-D-induced benign pulmonary tumorigenicity in mice for human health risk assessment.

Thyroperoxidase (TPO) is an enzyme essential for thyroid hormone (TH) synthesis and a target site for a number of xenobiotics that disrupt TH homeostasis. An in vitro high-throughput screening assay for TPO inhibition, the Amplex UltraRed-TPO (AUR-TPO), has been used to screen the ToxCast chemical libraries for this action. Output from this assay would be most useful if it could be readily translated into an in vivo response, namely a reduction of TH in serum. To this end, the relationship between TPO inhibition in vitro and serum TH decreases was examined in rats exposed to 2 classic TPO inhibitors, propylthiouracil (PTU) and methimazole (MMI). Serum and gland PTU, MMI, and TH levels were quantified using tandem liquid chromatography mass spectrometry. Thyroperoxidase activity was determined in thyroid gland microsomes treated with PTU or MMI in vitro and ex vivo from thyroid gland microsomes prepared from exposed animals. A quantitative model was constructed by contrasting in vitro and ex vivo AUR-TPO results and the in vivo time-course and dose-response analysis. In vitro:ex vivo correlations of AUR-TPO outputs indicated that less than 30% inhibition of TPO in vitro was sufficient to reduce serum T4 by 20%, a degree of regulatory significance. Although further testing of model estimates using other TPO inhibitors is essential for verification of these initial findings, the results of this study provide a means to translate in vitro screening assay results into predictions of in vivo serum T4 changes to inform risk assessment.

Breast cancer (BC) is the most common cancer among women. Fortunately, BC survival rates have increased because the implementation of adjuvant chemotherapy leading to a growing population of survivors. However, chemotherapy-induced cognitive impairments (CICIs) affect up to 75% of BC survivors and may be driven by inflammation and oxidative stress. Chemotherapy-induced cognitive impairments can persist 20 years and hinder survivors' quality of life. To identify early effects of CMF administration in mice, we chose to evaluate adult female mice at 2-week postchemotherapy. Mice received weekly IP administration of CMF (or saline) for 4 weeks, completed behavioral testing, and were sacrificed 2 weeks following their final CMF injection. Behavioral results indicated long-term memory (LTM) impairments postchemotherapy, but did not reveal short-term memory deficits. Dendritic morphology and spine data found increases in overall spine density within CA1 basal and CA3 basal dendrites, but no changes in DG, CA1 apical, or CA3 apical dendrites. Further analysis revealed decreases in arborization across the hippocampus (DG, CA1 apical and basal, CA3 apical and basal). These physiological changes within the hippocampus correlate with our behavioral data indicating LTM impairments following CMF administration in female mice 2-week postchemotherapy. Hippocampal cytokine analysis identified decreases in IL-1α, IL-1β, IL-3, IL-10, and TNF-α levels.