Alina Efremenko

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 p53s 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 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.

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.

Transcriptional responses in the rat nasal epithelium following subchronic inhalation of naphthalene vapor

Male and female Fischer 344 rats were exposed to naphthalene vapors at 0 (controls), 0.1, 1, 10, and 30ppm for 6h/d, 5 d/wk, over a 90-day period. Following exposure, the respiratory epithelium and olfactory epithelium from the nasal cavity were dissected separately, RNA was isolated, and gene expression microarray analysis was conducted. Only a few significant gene expression changes were observed in the olfactory or respiratory epithelium of either gender at the lowest concentration (0.1ppm). At the 1.0ppm concentration there was limited evidence of an oxidative stress response in the respiratory epithelium, but not in the olfactory epithelium. In contrast, a large number of significantly enriched cellular pathway responses were observed in both tissues at the two highest concentrations (10 and 30ppm, which correspond to tumorigenic concentrations in the NTP bioassay). The nature of these responses supports a mode of action involving oxidative stress, inflammation and proliferation. These results are consistent with a dose-dependent transition in the mode of action for naphthalene toxicity/carcinogenicity between 1.0 and 10ppm in the rat. In the female olfactory epithelium (the gender/site with the highest incidences of neuroblastomas in the NTP bioassay), the lowest concentration at which any signaling pathway was significantly affected, as characterized by the median pathway benchmark dose (BMD) or its 95% lower bound (BMDL) was 6.0 or 3.7ppm, respectively, while the lowest female olfactory BMD values for pathways related to glutathione homeostasis, inflammation, and proliferation were 16.1, 11.1, and 8.4ppm, respectively. In the male respiratory epithelium (the gender/site with the highest incidences of adenomas in the NTP bioassay), the lowest pathway BMD and BMDL were 0.4 and 0.3ppm, respectively, and the lowest male respiratory BMD values for pathways related to glutathione homeostasis, inflammation, and proliferation were 0.5, 0.7, and 0.9ppm, respectively. Using a published physiologically based pharmacokinetic (PBPK) model to estimate target tissue dose relevant to the proposed mode of action (total naphthalene metabolism per gram nasal tissue), the lowest transcriptional BMDLs from this analysis equate to human continuous naphthalene exposure at approximately 0.3ppm. It is unlikely that significant effects of naphthalene or its metabolites will occur at exposures below this concentration.

Time- and concentration-dependent genomic responses of the rat airway to inhaled nickel subsulfide

OBJECTIVE To provide insights into the mode of action for Ni3S2 lung carcinogenicity by examining gene expression changes in target cells after inhalation exposure. METHODS Gene expression changes were determined in micro-dissected lung broncho-alveolar cells from Fischer 344 rats following inhalation of Ni3S2 at 0.0, 0.04, 0.08, 0.15, and 0.60 mg/m(3) (0.03, 0.06, 0.11, and 0.44 mgNi/m(3)) for one and four weeks (6h/day, 5 days/week). RESULTS Broncho-alveolar lavage fluid evaluation and lung histopathology provided evidence of inflammation only at the two highest concentrations, which were similar to those tested in the 2-year bioassay. The number of statistically significant up- and down-regulated genes decreased markedly from one to four weeks of exposure, suggesting adaptation. Cell signal pathway enrichment at both time-points primarily reflected responses to toxicity, including inflammatory and proliferative signaling. While proliferative signaling was up-regulated at both time points, some inflammatory signaling reversed from down-regulation at 1 week to up-regulation at 4 weeks. CONCLUSIONS These results support a mode of action for Ni3S2 carcinogenicity driven by chronic toxicity, inflammation and proliferation, leading to mis-replication, rather than by direct genotoxicity. Benchmark dose (BMD) analysis identified the lowest pathway transcriptional BMD exposure concentration as 0.026 mgNi/m(3), for apoptosis/survival signaling. When conducted on the basis of lung Ni concentration the lowest pathway BMD was 0.64 μgNi/g lung, for immune/inflammatory signaling. IMPLICATIONS These highly conservative BMDs could be used to derive a point of departure in a nonlinear risk assessment for Ni3S2 toxicity and carcinogenicity.

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 (As2O3) 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.

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

ABSTRACT 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. HIGHLIGHTSA biological pathway approach to testing chemicals for GR‐dependent adipogenesisToxCast GR assays were used to prioritize chemicals for phenotypic in vitro testing.Fit‐for‐purpose assays can be used for comparing in vitro potency to human exposure.Testing prioritized chemicals in vitro can help provide context for human risk.

Time- and concentration-dependent genomic responses of the rat airway to inhaled nickel sulfate

While insoluble nickel subsulfide (Ni3S2) was carcinogenic in the lung in a 2‐year rat bioassay, soluble nickel sulfate hexahydrate (NiSO4*6H2O) was not. To investigate whether differences in the cellular responses to these two nickel compounds could underlie their differential activities, we conducted parallel studies to determine the gene expression changes in micro‐dissected lung distal airway cells from Fischer 344 rats following inhalation of the two compounds for one and four weeks (6 hr per day, 5 days per week). The results of the Ni3S2 study have been reported previously; this paper reports the results for NiSO4 and provides a comparative analysis. The cellular responses to NiSO4 were highly similar to those previously reported for Ni3S2, and a set of genes was identified whose expression could be used as biomarkers for comparing cellular nickel effects from in vitro or in vivo studies with soluble NiSO4 and particulate Ni3S2. Evaluation of the genomic concentration‐responses for the two compounds suggests that the highest inhaled concentration in the tumor bioassay for NiSO4, which was limited by toxicity, may not have achieved the Ni concentrations at which tumors were observed in the Ni3S2 bioassay. However, several key differences in the immune responses to NiSO4 and Ni3S2 were identified that may result from the differential intracellular disposition of Ni from NiSO4 entering the cell as an ion rather than as a slowly soluble Ni3S2 particle. These differences may also contribute to the observation of tumors in the bioassay for Ni3S2 but not NiSO4. Environ. Mol. Mutagen. 58:607–618, 2017.

Abeta systems pharmacology: An initial model for wild type mouse, monkey, and human

P4-296 ABETA SYSTEMS PHARMACOLOGY: AN INITIAL MODEL FORWILD TYPE MOUSE, MONKEY, AND HUMAN Hugh Barton, Ellen Wang, Yasong Lu, Alina Efremenko, Nikki Etxgoien, Harvey Clewell, Melvin Andersen, Jerry Campbell, Timothy Nicholas, Pfizer, Groton, Connecticut, United States; Pfizer, New York, New York, United States; 3 The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina, United States.