Biological system considerations for application of toxicogenomics in next-generation risk assessment and predictive toxicology.

There is increasing interest in using modern 'omics technologies, such as whole transcriptome sequencing, to inform decisions about human health safety and chemical toxicity hazard. High throughput methodologies using in vitro assays offer a path forward in reducing or eliminating animal testing. However, many aspects of these technologies need assessment before they will gain the trust of regulators and the public as viable alternative test methods for human health and safety. We used a high throughput whole transcriptome sequence assay (TempO-Seq) to assess the use of three widely used cancer cell lines (HepG2, MCF7, and Ishikawa cells) as in vitro systems for determination of cellular modes of action for two well studied compounds with canonical liver responses: ketoconazole and phenobarbital. We evaluated transcriptomic data to infer points of departure for use in risk analyses of compounds. Both compounds displayed shortcomings in evidence for canonical liver-related responses in any cell line, despite a strong dose response in all three. This raises questions about the competence of simple, mono-cultured cancer cell lines as appropriate surrogates for some adverse effects or toxic endpoints. Points of departure derived from benchmark doses were highly consistent across all three cell lines however, indicating the use of transcriptomic BMD analyses for such purposes would be a reliable and consistent approach.

Time-dependent genomic response in primary human uroepithelial cells exposed to arsenite for up to 60 days.

Evidence from both in vivo and in vitro studies suggests that gene expression changes from long-term exposure to arsenite evolve markedly over time, including reversals in the direction of expression change in key regulatory genes. In this study, human uroepithelial cells from the ureter segments of 4 kidney-donors were continuously treated in culture with arsenite at concentrations of 0.1 or 1 µM for 60 days. Gene expression at 10, 20, 30, 40, and 60 days was determined using Affymetrix human genome microarrays and signal pathway analysis was performed using GeneGo Metacore. Arsenic treated cells continued to proliferate for the full 60-day period, whereas untreated cells ceased proliferating after approximately 30 days. A peak in the number of gene changes in the treated cells compared to untreated controls was observed between 30 and 40 days of exposure, with substantially fewer changes at 10 and 60 days, suggesting remodeling of the cells over time. Consistent with this possibility, the direction of expression change for a number of key genes was reversed between 20 and 30 days, including CFOS and MDM2. While the progression of gene changes was different for each subject, a common pattern was observed in arsenic treated cells over time, with early upregulation of oxidative stress responses (HMOX1, NQ01, TXN, TXNRD1) and down-regulation of immune/inflammatory responses (IKKα). At around 30 days, there was a transition to increased inflammatory and proliferative signaling (AKT, CFOS), evidence of epithelial-to-mesenchymal transition (EMT), and alterations in DNA damage responses (MDM2, ATM). A common element in the changing response of cells to arsenite over time appears to involve up-regulation of MDM2 by inflammatory signaling (through AP-1 and NF-κB), leading to inhibition of P53 function.

New framework for a non-animal approach adequately assures the safety of cosmetic ingredients - A case study on caffeine.

This case study on the model substance caffeine demonstrates the viability of a 10-step read-across (RAX) framework in practice. New approach methodologies (NAM), including RAX and physiologically-based kinetic (PBK) modelling were used to assess the consumer safety of caffeine. Appropriate animal systemic toxicity data were used from the most relevant RAX analogue while assuming that no suitable animal toxicity data were available for caffeine. Based on structural similarities, three primary metabolites of the target chemical caffeine (theophylline, theobromine and paraxanthine) were selected as its most relevant analogues, to estimate a point of departure in order to support a next generation risk assessment (NGRA). On the basis of the pivotal mode of action (MOA) of caffeine and other methylxanthines, theophylline appeared to be the most potent and suitable analogue. A worst-case aggregate exposure assessment determined consumer exposure to caffeine from different sources, such as cosmetics and food/drinks. Using a PBK model to estimate human blood concentrations following exposure to caffeine, an acceptable Margin of Internal Exposure (MOIE) of 27-fold was derived on the basis of a RAX using theophylline animal data, which suggests that the NGRA approach for caffeine is sufficiently conservative to protect human health.

Physiologically Based Pharmacokinetic Modeling in Risk Assessment: Case Study With Pyrethroids.

The assessment of potentially sensitive populations is an important application of risk assessment. To address the concern for age-related sensitivity to pyrethroid insecticides, life-stage physiologically based pharmacokinetic (PBPK) modeling supported by in vitro to in vivo extrapolation was conducted to predict age-dependent changes in target tissue exposure to 8 pyrethroids. The purpose of this age-dependent dosimetry was to calculate a Data-derived Extrapolation Factor (DDEF) to address age-related pharmacokinetic differences for pyrethroids in humans. We developed a generic human PBPK model for pyrethroids based on our previously published rat model that was developed with in vivo rat data. The results demonstrated that the age-related differences in internal exposure to pyrethroids in the brain are largely determined by the differences in metabolic capacity and in physiology for pyrethroids between children and adults. The most important conclusion from our research is that, given an identical external exposure, the internal (target tissue) concentration is equal or lower in children than in adults in response to the same level of exposure to a pyrethroid. Our results show that, based on the use of the life-stage PBPK models with 8 pyrethroids, DDEF values are essentially close to 1, resulting in a DDEF for age-related pharmacokinetic differences of 1. For risk assessment purposes, this indicates that no additional adjustment factor is necessary to account for age-related pharmacokinetic differences for these pyrethroids.

Application of a combined aggregate exposure pathway and adverse outcome pathway (AEP-AOP) approach to inform a cumulative risk assessment: A case study with phthalates.

Advancements in measurement and modeling capabilities are providing unprecedented access to estimates of chemical exposure and bioactivity. With this influx of new data, there is a need for frameworks that help organize and disseminate information on chemical hazard and exposure in a manner that is accessible and transparent. A case study approach was used to demonstrate integration of the Adverse Outcome Pathway (AOP) and Aggregate Exposure Pathway (AEP) frameworks to support cumulative risk assessment of co-exposure to two phthalate esters that are ubiquitous in the environment and that are associated with disruption of male sexual development in the rat: di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP). A putative AOP was developed to guide selection of an in vitro assay for derivation of bioactivity values for DEHP and DnBP and their metabolites. AEPs for DEHP and DnBP were used to extract key exposure data as inputs for a physiologically based pharmacokinetic (PBPK) model to predict internal metabolite concentrations. These metabolite concentrations were then combined using in vitro-based relative potency factors for comparison with an internal dose metric, resulting in an estimated margin of safety of ~13,000. This case study provides an adaptable workflow for integrating exposure and toxicity data by coupling AEP and AOP frameworks and using in vitro and in silico methodologies for cumulative risk assessment.

Internal Threshold of Toxicological Concern (iTTC): Where We Are Today and What Is Possible in the Near Future.

The Threshold of Toxicological Concern (TTC) is a risk assessment tool for evaluating low-level exposure to chemicals with limited toxicological data. A next step in the ongoing development of TTC is to extend this concept further so that it can be applied to internal exposures. This refinement of TTC based on plasma concentrations, referred to as internal TTC (iTTC), attempts to convert the chemical-specific external NOAELs (in mg/kg/day) in the TTC database to an estimated internal exposure. A multi-stakeholder collaboration formed, with the aim of establishing an iTTC suitable for human safety risk assessment. Here, we discuss the advances and future directions for the iTTC project, including: (1) results from the systematic literature search for metabolism and pharmacokinetic data for the 1,251 chemicals in the iTTC database; (2) selection of ~350 chemicals that will be included in the final iTTC; (3) an overview of the in vitro caco-2 and in vitro hepatic metabolism studies currently being generated for the iTTC chemicals; (4) demonstrate how PBPK modeling is being utilized to convert a chemical-specific external NOAEL to an internal exposure; (5) perspective on the next steps in the iTTC project.

Development and Application of a Life-Stage Physiologically Based Pharmacokinetic (PBPK) Model to the Assessment of Internal Dose of Pyrethroids in Humans.

To address concerns around age-related sensitivity to pyrethroids, a life-stage physiologically based pharmacokinetic (PBPK) model, supported by in vitro to in vivo extrapolation (IVIVE) was developed. The model was used to predict age-dependent changes in target tissue exposure of 8 pyrethroids; deltamethrin (DLM), cis-permethrin (CPM), trans-permethrin, esfenvalerate, cyphenothrin, cyhalothrin, cyfluthrin, and bifenthrin. A single model structure was used based on previous work in the rat. Intrinsic clearance (CLint) of each individual cytochrome P450 or carboxylesterase (CES) enzyme that are active for a given pyrethroid were measured in vitro, then biologically scaled to obtain in vivo age-specific total hepatic CLint. These IVIVE results indicate that, except for bifenthrin, CES enzymes are largely responsible for human hepatic metabolism (>50% contribution). Given the high efficiency and rapid maturation of CESs, clearance of the pyrethroids is very efficient across ages, leading to a blood flow-limited metabolism. Together with age-specific physiological parameters, in particular liver blood flow, the efficient metabolic clearance of pyrethroids across ages results in comparable to or even lower internal exposure in the target tissue (brain) in children than that in adults in response to the same level of exposure to a given pyrethroid (Cmax ratio in brain between 1- and 25-year old = 0.69, 0.93, and 0.94 for DLM, bifenthrin, and CPM, respectively). Our study demonstrated that a life-stage PBPK modeling approach, coupled with IVIVE, provides a robust framework for evaluating age-related differences in pharmacokinetics and internal target tissue exposure in humans for the pyrethroid class of chemicals.

Updating physiologically based pharmacokinetic models for manganese by incorporating rapid association/dissociation processes in tissues.

Previously, we developed a series of physiologically based pharmacokinetic (PBPK) models for manganese (Mn) in which saturable tissue binding and dose-dependent increases in biliary excretion captured key aspects of Mn homeostasis biology. These models reproduced the non-linear behavior of Mn kinetics in different tissues, accounting for dose-dependent changes in Mn kinetics. The original model construct had relatively slow association and dissociation rate constants for Mn binding in tissues. In this updated model, both rates of entry into tissue and the interaction of Mn with binding sites are rapid, and the step limiting Mn accumulation is the saturation of tissue binding sites. This binding reflects general cellular requirements for Mn with high affinity but rapid exchange between bound and free forms, which we captured using a dissociation constant (KD) of ~ 0.5 µM across tissues while maintaining different maximum binding capacities in each tissue. Variability in the binding capacities accounted for different background levels of Mn in particular tissues. This alternative structure successfully described Mn kinetics in tissues in adult rats exposed to Mn either in their diet or by inhalation, indicating that both the original and the present models capture the dose-dependent and tissue-specific kinetic behavior of Mn in adult rats. Although the published models that emphasize the role of smaller tissue binding rate constants in non-linear behaviors capture all relevant dose-dependent kinetic behaviors of this metal, increasing biological relevance of the model structure and parameters should provide greater confidence in applying the Mn PBPK models to risk assessment.

Evaluation of Age-Related Pyrethroid Pharmacokinetic Differences in Rats: Physiologically-Based Pharmacokinetic Model Development Using In Vitro Data and In Vitro to In Vivo Extrapolation.

An in vitro to in vivo (IVIVE) extrapolation based-physiologically based pharmacokinetic (PBPK) modeling approach was demonstrated to understand age-related differences in kinetics and how they potentially affect age-related differences in acute neurotoxic effects of pyrethroids. To describe the age-dependent changes in pyrethroid kinetics, it was critical to incorporate age-dependent changes in metabolism into the model. As such, in vitro metabolism data were collected for 3 selected pyrethroids, deltamethrin (DLM), cis-permethrin, and trans-permethrin, using liver microsomes and cytosol, and plasma prepared from immature and adult rats. Resulting metabolism parameters, maximum rate of metabolism (Vmax) and Michaelis-Menten constant (Km), were biologically scaled to respective in vivo parameters for use in the age-specific PBPK model. Then, age-dependent changes in target tissue exposure, i.e., brain Cmax, to a given pyrethroid were simulated across ages using the model. The PBPK model recapitulated in vivo time-course plasma and brain concentrations of the 3 pyrethroids in immature and adult rats following oral administration of both low and high doses of these compounds. A single model structure developed for DLM was able to describe the kinetics of the other 2 pyrethroids when used with compound- and age-specific metabolism parameters, suggesting that one generic model for pyrethroids as a group can be used for early age-sensitivity evaluation if appropriate metabolic parameters are used. This study demonstrated the validity of applying IVIVE-based PBPK modeling to development of age-specific PBPK models for pyrethroids in support of pyrethroid risk assessment of potentially sensitive early age populations in humans.

An in vitro approach for prioritization and evaluation of chemical effects on glucocorticoid receptor mediated adipogenesis.

Rising obesity rates worldwide have socio-economic ramifications. While genetics, diet, and lack of exercise are major contributors to obesity, environmental factors may enhance susceptibility through disruption of hormone homeostasis and metabolic processes. The obesogen hypothesis contends that chemical exposure early in development may enhance adipocyte differentiation, thereby increasing the number of adipocytes and predisposing for obesity and metabolic disease. We previously developed a primary human adipose stem cell (hASC) assay to evaluate the effect of environmental chemicals on PPARG-dependent adipogenesis. Here, the assay was modified to determine the effects of chemicals on the glucocorticoid receptor (GR) pathway. In differentiation cocktail lacking the glucocorticoid agonist dexamethasone (DEX), hASCs do not differentiate into adipocytes. In the presence of GR agonists, adipocyte maturation was observed using phenotypic makers for lipid accumulation, adipokine secretion, and expression of key genes. To evaluate the role of environmental compounds on adipocyte differentiation, progenitor cells were treated with 19 prioritized compounds previously identified by ToxPi as having GR-dependent bioactivity, and multiplexed assays were used to confirm a GR-dependent mode of action. Five chemicals were found to be strong agonists. The assay was also modified to evaluate GR-antagonists, and 8/10 of the hypothesized antagonists inhibited adipogenesis. The in vitro bioactivity data was put into context with extrapolated human steady state concentrations (Css) and clinical exposure data (Cmax). These data support using a human adipose-derived stem cell differentiation assay to test the potential of chemicals to alter human GR-dependent adipogenesis.

Evaluation of gene expression changes in human primary lung epithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites and to arsenic trioxide.

The concentration response for altered gene expression in primary lung epithelial cells was determined following two treatments with arsenicals: (1) a mixture of trivalent arsenic compounds representative of urinary arsenic concentrations in exposed human populations, and (2) arsenite (As2 O3 ) a common form of inhaled arsenic dust that is frequently used in both in vivo and in vitro experimental exposures. Biochemical assays did not detect any evidence of cytotoxicity at the concentrations used, apart from a concentration-related increase in cellular heme oxygenase that was also indicated by the genomic analysis. Cell signal pathway enrichment analysis indicated similar responses to both treatments, with concentration-related responses in pathways related to cell adhesion, cytoskeleton remodeling, development (morphogenesis), cell cycle control, and to a lesser extent inflammatory responses. These cellular responses to arsenic were consistent with those observed in a previous study with primary uroepithelial cells. Benchmark dose analysis also demonstrated similar potency of the two treatments as well as comparable sensitivity of the two cell types. A number of genes showing similar concentration-dependent expression across individuals in both bladder and lung cells were identified, including heme oxygenase 1, thioredoxin reductase, DNA damage binding protein 2, and thrombomodulin. The data on human primary lung cells from this study, together with the data from human primary uroepithelial cells, support a conclusion that biological responses to arsenic by human cells under study conditions are unlikely to occur at concentrations below 0.1 µM. Environ. Mol. Mutagen. 56:477-490, 2015. © 2015 Wiley Periodicals, Inc.

Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116). At chemical concentrations causing similar increases in p53 protein expression, p53-mediated protein expression and cellular processes showed substantial chemical-specific differences. These chemical-specific differences in the p53 transcriptional response appear to be determined by augmentation of the p53 response by co-regulators. More importantly, dose-response data for each of the chemicals indicate that the p53 transcriptional response does not prevent micronuclei induction at low concentrations. In fact, the no observed effect levels and benchmark doses for micronuclei induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53's post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. This effort demonstrates the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype.

Evaluation of simple in vitro to in vivo extrapolation approaches for environmental compounds.

As part of an effort to support in silico/in vitro based risk assessment, we evaluated the accuracy associated with conducting simple in vitro to in vivo extrapolation (IVIVE) for environmental compounds using available in vitro human metabolism data. The IVIVE approach was applied to a number of compounds with a wide range of properties spanning the diversity of characteristics of environmental compounds, and where possible the resulting estimates of the in vivo steady-state blood concentration were compared with estimates derived on the basis of human in vivo kinetic data. There appears to be a systematic bias in the estimation of intrinsic clearance (Clint) from in vitro versus in vivo data, with in vitro based estimates underestimating in vivo clearance for small values of Clint but with the opposite relationship at large values of Clint. Nevertheless, the resulting estimates of Css were in good agreement. The chief drawback of the simple approach used in this study, which performs the IVIVE prediction for the parent compound only, is that it is not applicable for toxicity associated with a metabolite.

Evaluation of gene expression changes in human primary uroepithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites.

Gene expression changes in primary human uroepithelial cells exposed to arsenite and its methylated metabolites were evaluated to identify cell signaling pathway perturbations potentially associated with bladder carcinogenicity. Cells were treated with mixtures of inorganic arsenic and its pentavalent or trivalent metabolites for 24 hr at total arsenic concentrations ranging from 0.06 µM to 18 µM. One series (five samples) was conducted with arsenite and pentavalent metabolites and a second (10 samples) with arsenite and trivalent metabolites. Similar gene expression responses were obtained for pentavalent or trivalent metabolites. A suite of eight gene changes was consistently identified across individuals that reflect effects on key signaling pathways: oxidative stress, protein folding, growth regulation, metallothionine regulation, DNA damage sensing, thioredoxin regulation, and immune response. No statistical significance of trend (NOSTASOT) analysis of these common genes identified lowest observed effect levels (LOELs) from 0.6 to 6.0 µM total arsenic and no observed effect levels (NOELs) from 0.18 to 1.8 µM total arsenic. For the trivalent arsenical mixture, benchmark doses (BMDs) ranged from 0.13 to 0.92 µM total arsenic; benchmark dose lower 95% confidence limits (BMDLs) ranged from 0.09 to 0.58 µM total arsenic. BMDs ranged from 0.53 to 2.7 µM and BMDLs from 0.35 to 1.7 µM for the pentavalent arsenical mixture. Both endpoints varied by a factor of 3 across individuals. Thisstudy is the first to examine gene expression response in primary uroepithelial cells from multiple individuals and to identify no effect levels for arsenical-induced cell signaling perturbations in normal human cells exposed to a biologically plausible concentration range.

Characterizing the molecular order of phosphonic acid self-assembled monolayers on indium tin oxide surfaces.

Self-assembled monolayers (SAMs) of alkanephosphonic acids with chain lengths between 8 and 18 carbon units were formed on thin films of indium tin oxide (ITO) sputter-deposited on silicon substrates with 400 nm thermally grown SiO(2). The silicon substrates, while not intended for use in near-IR or visible optics applications, do provide smooth surfaces that permit systematic engineering of grain size and surface roughness as a function of the sputter pressure. Argon sputter pressures from 4 to 20 mTorr show systematic changes in surface morphology ranging from smooth, micrometer-sized grain structures to <50 nm grains with 3× higher surface roughness. Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy experiments are conducted for alkanephosphonic acids deposited on these wide range of ITO surfaces to evaluate the effects of these morphological features on monolayer ordering. Results indicate that long-chain SAMs are more highly ordered, and have a smaller tilt angle, than short-chain SAMs. Surprisingly, the 1-octadecyl phosphonic acids maintain their order as the lateral grain dimensions of the ITO surface shrink to ∼50 nm. It is only when the ITO surface roughness becomes greater than the SAM chain length (∼15 Å) that SAMs are observed to become relatively disordered.