High-Throughput Screening Data Interpretation in the Context of In Vivo Transcriptomic Responses to Oral Cr(VI) Exposure

The toxicity of hexavalent chromium [Cr(VI)] in drinking water has been studied extensively, and available in vivo and in vitro studies provide a robust dataset for application of advanced toxicological tools to inform the mode of action (MOA). This study aimed to contribute to the understanding of Cr(VI) MOA by evaluating high-throughput screening (HTS) data and other in vitro data relevant to Cr(VI), and comparing these findings to robust in vivo data, including transcriptomic profiles in target tissues. Evaluation of Tox21 HTS data for Cr(VI) identified 11 active assay endpoints relevant to the Ten Key Characteristics of Carcinogens (TKCCs) that have been proposed by other investigators. Four of these endpoints were related to TP53 (tumor protein 53) activation mapping to genotoxicity (KCC#2), and four were related to cell death/proliferation (KCC#10). HTS results were consistent with other in vitro data from the Comparative Toxicogenomics Database. In vitro responses were compared to in vivo transcriptomic responses in the most sensitive target tissue, the duodenum, of mice exposed to ≤ 180 ppm Cr(VI) for 7 and 90 days. Pathways that were altered both in vitro and in vivo included those relevant to cell death/proliferation. In contrast, pathways relevant to p53/DNA damage were identified in vitro but not in vivo. Benchmark dose modeling and phenotypic anchoring of in vivo transcriptomic responses strengthened the finding that Cr(VI) causes cell stress/injury followed by proliferation in the mouse duodenum at high doses. These findings contribute to the body of evidence supporting a non-mutagenic MOA for Cr(VI)-induced intestinal cancer

Reduction of hexavalent chromium by fasted and fed human gastric fluid. II. Ex vivo gastric reduction modeling

AbstractTo extend previous models of hexavalent chromium [Cr(VI)] reduction by gastric fluid (GF), ex vivo experiments were conducted to address data gaps and limitations identified with respect to (1) GF dilution in the model; (2) reduction of Cr(VI) in fed human GF samples; (3) the number of Cr(VI) reduction pools present in human GF under fed, fasted, and proton pump inhibitor (PPI)-use conditions; and (4) an appropriate form for the pH-dependence of Cr(VI) reduction rate constants. Rates and capacities of Cr(VI) reduction were characterized in gastric contents from fed and fasted volunteers, and from fasted pre-operative patients treated with PPIs. Reduction capacities were first estimated over a 4-h reduction period. Once reduction capacity was established, a dual-spike approach was used in speciated isotope dilution mass spectrometry analyses to characterize the concentration-dependence of the 2nd order reduction rate constants. These data, when combined with previously collected data, were well described by a three-pool model (pool 1 = fast reaction with low capacity; pool 2 = slow reaction with higher capacity; pool 3 = very slow reaction with higher capacity) using pH-dependent rate constants characterized by a piecewise, log-linear relationship. These data indicate that human gastric samples, like those collected from rats and mice, contain multiple pools of reducing agents, and low concentrations of Cr(VI) (<0.7 mg/L) are reduced more rapidly than high concentrations. The data and revised modeling results herein provide improved characterization of Cr(VI) gastric reduction kinetics, critical for Cr(VI) pharmacokinetic modeling and human health risk assessment

Comparison of the Effects of Hexavalent Chromium in the Alimentary Canal of F344 Rats and B6C3F1 Mice Following Exposure in Drinking Water: Implications for Carcinogenic Modes of Action

Exposure to high concentrations of hexavalent chromium (Cr[VI]) in drinking water is reported to induce oral mucosa tumors in F344 rats and intestinal tumors in B6C3F1 mice. To investigate the modes of action underlying these tumors, 90-day drinking water studies (with interim necropsy at day 8) were conducted with concentrations of 0.1–182 mg/l Cr(VI), administered as 0.3–520 mg/l sodium dichromate dihydrate. Blood and tissue samples were analyzed for chromium content, oxidative stress, iron levels, and gross and microscopic lesions. Results for the F344 rats are described herein and compared with results from B6C3F1 mice published previously. After 90 days of exposure, total chromium concentrations in the rat and mouse oral mucosae were comparable, yet significant dose-dependent decreases in the reduced-to-oxidized glutathione ratio (GSH/GSSG) were observed only in rats. In the duodenum, changes in GSH/GSSG were only observed in mice. Levels of 8-hydroxydeoxyguanosine were not increased in the oral or duodenal mucosae of either species. Glutathione levels were increased in the duodenum but decreased in the jejunum of both species, indicating potential differential responses in the intestinal segments. Histiocytic infiltration was observed in the duodenum of both species, yet duodenal cytokines were repressed in mice but increased in rats. Serum and bone marrow iron levels were more decreased in rats than mice. Collectively, these data suggest that Cr(VI)-induced carcinogenesis in the rodent alimentary canal involves oxidative stress; however, differences in histopathology, cytokines, and iron status suggest potential contributions from other factors as well

Application of the U.S. EPA Mode of Action Framework for Purposes of Guiding Future Research: A Case Study Involving the Oral Carcinogenicity of Hexavalent Chromium

Mode of action (MOA) analysis provides a systematic description of key events leading to adverse health effects in animal bioassays for the purpose of informing human health risk assessment. Uncertainties and data gaps identified in the MOA analysis may also be used to guide future research to improve understanding of the MOAs underlying a specific toxic response and foster development of toxicokinetic and toxicodynamic models. An MOA analysis, consistent with approaches outlined in the MOA Framework as described in the Guidelines for Carcinogen Risk Assessment, was conducted to evaluate small intestinal tumors observed in mice chronically exposed to relatively high concentrations of hexavalent chromium (Cr(VI)) in drinking water. Based on review of the literature, key events in the MOA are hypothesized to include saturation of the reductive capacity of the upper gastrointestinal tract, absorption of Cr(VI) into the intestinal epithelium, oxidative stress and inflammation, cell proliferation, direct and/or indirect DNA modification, and mutagenesis. Although available data generally support the plausibility of these key events, several unresolved questions and data gaps were identified, highlighting the need for obtaining critical toxicokinetic and toxicodynamic data in the target tissue and in the low-dose range. Experimental assays that can address these data gaps are discussed along with strategies for comparisons between responsive and nonresponsive tissues and species. This analysis provides a practical application of MOA Framework guidance and is instructive for the design of studies to improve upon the information available for quantitative risk assessment

sj-tif-2-tpx-10.1177_01926233231159078 – Supplemental material for Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death

Supplemental material, sj-tif-2-tpx-10.1177_01926233231159078 for Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death by Chad M. Thompson, Melissa M. Heintz, Jeffrey C. Wolf, Roza Cheru, Laurie C. Haws and John M. Cullen in Toxicologic Patholog

Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate (HFPO-DA) is a short chain member of per- and polyfluoroalkyl substances (PFAS). To better understand the relevance of histopathological effects seen in livers of mice exposed to HFPO-DA for human health risk assessment, histopathological effects were summarized from hematoxylin and eosin (H&E)-stained sections in several repeat-dose toxicity studies in mice. Findings across studies revealed histopathological changes consistent with peroxisomal proliferation, whereas two reports of steatosis could not be confirmed in the published figures. In addition, mechanisms of hepatocellular death were assessed in H&E sections as well as with the apoptotic marker cleaved caspase-3 (CCasp3) in newly cut sections from archived liver blocks from select studies. A comparison of serially CCasp3 immunolabeled and H&E-stained sections revealed that mechanisms of hepatocellular death cannot be clearly discerned in H&E-stained liver sections alone as several examples of putatively necrotic cells were positive for CCasp3. Published whole genome transcriptomic data were also reevaluated for enrichment of various forms of hepatocellular death in response to HFPO-DA, which revealed enrichment of apoptosis and autophagy, but not ferroptosis, pyroptosis, or necroptosis. These morphological and molecular findings are consistent with transcriptomic evidence for peroxisome proliferator-activated receptor alpha (PPARα) signaling in HFPO-DA exposed mice

sj-tif-1-tpx-10.1177_01926233231159078 – Supplemental material for Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death

Supplemental material, sj-tif-1-tpx-10.1177_01926233231159078 for Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death by Chad M. Thompson, Melissa M. Heintz, Jeffrey C. Wolf, Roza Cheru, Laurie C. Haws and John M. Cullen in Toxicologic Patholog

Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate (HFPO-DA) is a short chain member of per- and polyfluoroalkyl substances (PFAS). To better understand the relevance of histopathological effects seen in livers of mice exposed to HFPO-DA for human health risk assessment, histopathological effects were summarized from hematoxylin and eosin (H&E)-stained sections in several repeat-dose toxicity studies in mice. Findings across studies revealed histopathological changes consistent with peroxisomal proliferation, whereas two reports of steatosis could not be confirmed in the published figures. In addition, mechanisms of hepatocellular death were assessed in H&E sections as well as with the apoptotic marker cleaved caspase-3 (CCasp3) in newly cut sections from archived liver blocks from select studies. A comparison of serially CCasp3 immunolabeled and H&E-stained sections revealed that mechanisms of hepatocellular death cannot be clearly discerned in H&E-stained liver sections alone as several examples of putatively necrotic cells were positive for CCasp3. Published whole genome transcriptomic data were also reevaluated for enrichment of various forms of hepatocellular death in response to HFPO-DA, which revealed enrichment of apoptosis and autophagy, but not ferroptosis, pyroptosis, or necroptosis. These morphological and molecular findings are consistent with transcriptomic evidence for peroxisome proliferator-activated receptor alpha (PPARα) signaling in HFPO-DA exposed mice

sj-tif-3-tpx-10.1177_01926233231159078 – Supplemental material for Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death

Supplemental material, sj-tif-3-tpx-10.1177_01926233231159078 for Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death by Chad M. Thompson, Melissa M. Heintz, Jeffrey C. Wolf, Roza Cheru, Laurie C. Haws and John M. Cullen in Toxicologic Patholog