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Human Transcriptomic Integrated Approach for Testing and Assessment Oral Reference Dose for Steatosis

ABSTRACT: Integrated Approaches to Testing and Assessment (IATA) are emerging as a means to strategically assess and test chemicals for toxicity, using existing information, and helping to identify potential data gaps. Adverse Outcome Pathways (AOPs) have recently been shown to be a good starting point for IATA development. Our interest in IATAs and AOP Networks (AOPNs) focuses on developing efficient testing and risk assessment strategies for new materials, new chemical entities, and setting environmental cleanup goals. We combine the maths logic of causality, specifically the notion of sufficiency to infer an adverse outcome, with AOPNs to develop Minimally Sufficient Sets of Key Events (MinSSKEs). These MinSSKEs represent a set of key events (can just be one key event) that taken together allow assessors to predict if an adverse outcome is likely or not. For our case study we use the military explosive trinitrotoluene (TNT). TNT is a relatively data rich chemical where the US Environmental Protection Agency has set a reference dose of 0.0005mg/kg-day, based on a point of departure for liver toxicity in dogs of 0.5mg/kg-day (a lowest observed adverse effect level; LOAEL) with uncertainty of 1,000X. Our IATA measures gene expression for genes in involved in our AOPN for energy metabolism, steatosis and oxidative stress in human induced pluripotent stem cells differentiated to hepatocytes exposed to TNT. We used a Bayesian statistical approach to identify differentially expressed genes, and a nonlinear data-driven dose-response modeling approach (Good Risk Assessment Values for Environmental Exposures; GRAVEE) to estimate points of departure for reference dose estimation. We obtained an RfD of 0.12mg/kg-day, with a 90% credible interval spanning from 0.02-0.19mg/kg-day using our IATA approach. Our RfD is based on a POD of 1.22mg/kg-day with a 90% credible interval spanning from 0.24-1.94mg/kg-day, and uncertainty of 10X after applying the IRIS uncertainty factors. Our PODs are highly comparable, with a difference of only 0.72mg/kg-day (or 2.44x). Our RfDs differ by 240x, owing to the fact that we have only a 10x uncertainty factor for human variability, whereas the IRIS assessment uses 1000x uncertainty for 10x animal-to-human extrapolation, 10x LOAEL-to-NOAEL extrapolation, and 10x for subchronic-to-chronic extrapolation. In this case study, our IATA resulted in a highly similar POD compared to the IRIS assessment, and an overall decrease in the uncertainty factors that needed to be applied. Our IATA approach worked well in this instance, and demonstrated several advantages by focusing on using a human-relevant in vitro model, data-driven POD modeling, and a net decrease in the overall risk assessment uncertainty factors, while maintaining the uncertainty associated with the data.

ORGANISM(S): Homo sapiens

PROVIDER: GSE135117 | GEO | 2022/07/02

REPOSITORIES: GEO

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Project description:Motivation: Monitoring, assessment and prediction of environmental risks that chemicals pose demand rapid and accurate diagnostic assays. A variety of toxicological effects have been associated with explosive compounds TNT and RDX. One important goal of microarray experiments is to discover novel biomarkers for toxicity evaluation. We have developed an earthworm microarray containing 15,208 unique oligo probes. Our objective was to identify biomarker genes that can separate earthworm samples into three groups: control, TNT-treated, and RDX-treated. Results: We developed a discriminant analysis and cluster (DAC) pipeline to analyze a 248-array dataset. First, a total of 869 significantly changed genes in response to TNT or RDX exposure were inferred by class comparison statistical algorithms. Then, nine decision tree-based algorithms were applied to generate classification rules and a set of 286 classifier genes. These classifier genes were ranked by their overall weight of significance, and were used to build support vector machines (SVMs). An SVM containing all 286 classifier genes had the highest classification accuracy (91.5%). An unsupervised clustering method was used to cluster the worm samples and results show that the use of the top 100 classifier genes can assign the largest number of worm samples into the three reference clusters obtained by using all the 14,188 filtered genes, suggesting that these top-ranked genes may be potential candidates for biomarkers. This study demonstrates that the DAC pipeline can be used to identify a small set of biomarker genes from high dimensional datasets and generate a reliable SVM classification model for multiple classes. Adult earthworms (E. fetida) were exposed in soil spiked with TNT (0, 6, 12, 24, 48, or 96 mg/kg) or RDX (8, 16, 32, 64, or 128 mg/kg) for 0, 4 or 14 days. The 4-day treatment was repeated with RDX concentration being 2, 4, 8, 16 or 32 mg/kg soil. Each treatment originally had 10 replicate worms with 8-10 survivors at the end of exposure. Total RNA was isolated from the surviving worms. A total of 248 worm RNA samples were hybridized to a custom-designed oligo array using Agilentâs one-color Low RNA Input Linear Amplification Kit. The array contains 15,208 non-redundant 60-mer probes, each targeting a unique E. fetida transcript (Gong et al. 2009). After hybridization and scanning, gene expression data were acquired using Agilentâs Feature Extraction Software (v.9.1.3). The 248-array dataset consists of three worm groups: 32 untreated controls, 96 TNT-treated, and 120 RDX-treated.

Project description:Individuals with Urea Cycle Disorders (UCD) cannot remove ammonia, a waste product, from the blood. At present there is little information on the use of sodium phenylbutyrate (Buphenyl™) in children with argininosuccinic aciduria (ASA). It is hoped that this drug may help lower ammonia and argininosuccinic acid levels in patients with ASA. Through this study we hope to learn whether the use of sodium phenylbutyrate (Buphenyl™) in patients with ASA, in addition to diet and arginine therapy, will decrease liver damage and the number of periods during which ammonia levels are high. The primary study objective is to determine if the treatment of ASA patients with sodium phenylbutyrate (Buphenyl™) in conjunction with lowered doses of arginine improves liver function as measured by short-term assessment of synthetic activity and the use of stable isotope tracers to assess ureagenesis and nitric oxide production. Twelve patients with ASA will be studied using a randomized, cross-over design. Medical records will be reviewed to confirm the diagnosis prior to enrollment. Patients will be on the each arm of the study for one week each preceded by a three day washout period. Patients will come to the Baylor College of Medicine General Clinical Research Center in Texas Children's Hospital for an inpatient hospitalization. This hospitalization is for diet control, clinical evaluation, and stable isotope infusions for measurement of in vivo rates of ureagenesis and nitric oxide production. During the washout periods Buphenyl™ or other alternative route medications, e.g., benzoate, will be discontinued and arginine will be administered at the standard therapeutic dose of 500 mg/kg/day or 10 grams/m2. Following randomization, subjects will be maintained on either high-dose arginine (500 mg/kg/day or 10 grams/m2) alone, or the alternative of low-dose arginine (100 mg/kg/d or 2 grams/m2) along with sodium phenylbutyrate (500 mg/kg/day or 10 grams/m2). For the second week they will be crossed over to the other arm of the study preceded by another three day washout. Previous medications and dietary protein intake (0.6 g/kg/day or current metabolic diet) will be continued during both arms of the study. The two arms may be performed as separate ten-day admissions. The decision to design the study with two arms, high-dose arginine and low-dose arginine/Buphenyl™, was reached after weighing several considerations. First, the current accepted practice for treatment of ASA is high-dose arginine. Based on metabolic flux studies, we have quantified the effects of this dose of arginine as compared to standard doses of Buphenyl™/phenylacetate on ureagenesis. Since the study is not designed or intended to address the independent effects of Buphenyl™ as compared to arginine alone, Buphenyl™ was added for safety reasons to accommodate loss of clearance of nitrogen by decreasing the arginine dose. Moreover, based on our experience with recruiting for previous versions of this study, most ASA patients with liver dysfunction are on an alternative route ammonia scavenger medication in addition to arginine. We found that this has in fact become an accepted practice. Hence, when considering study design from both a safety and recruitment perspective, it was most reasonable to design the study to investigate specifically the hypothesis that the production and presence of argininosuccinic acid leads to liver dysfunction. Since both low-dose arginine with Buphenyl™ and high-dose arginine alone lead to a decreased production of argininosuccinic acid, and both are currently accepted therapies in ASA and UCDs in general, the need to address their effects distinctly is not being investigated at this time. Sodium phenylbutyrate dosage will be 500 mg/kg/day in patients weighing less than 20kg and 10 g/m2/day in larger patients. Daily dosage will be given in equally divided doses 4 times per day. In order to ensure that the study remains double-blind to investigators, study staff and subjects, the Investigational Pharmacy Service at Texas Children's Hospital will dispense both the Buphenyl™ and arginine in powder form which is compounded into capsules or will be mixed with syrpalta (up to 5 grams will dissolve in 10cc liquid) and administered orally or through nasogastric or gastrostomy tube. No nasogastric tube should be placed or gastrostomy performed solely for the purpose of inclusion in this study. Because the reported adverse effects of sodium phenylbutyrate include gastritis, participants will be started on a standard dose of ranitidine (Zantac™), or comparable drug, during both arms of the study.

Project description:Munitions constituents (MCs) including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and TNT derivatives are recognized to elicit aberrant neuromuscular responses in many species. The onset of seizures resulting in death was observed in the avian model Northern bobwhite after oral dosing with RDX beginning at 8 mg/kg/day in subacute (14 days) exposures, whereas affective doses of the TNT derivative, 2,6-dinitrotoluene (2,6-DNT), caused gastrointestinal impacts, lethargy, and emaciation in subacute and subchronic (60 days) exposures. To assess and contrast the potential neurotoxicogenomic effects of these MCs, a Northern bobwhite microarray was developed consisting of 4119 complementary DNA (cDNA) features enriched for differentially-expressed brain transcripts from exposures to RDX and 2,6-DNT. RDX affected hundreds of genes in brain tissue, whereas 2,6-DNT affected few (M-bM-^IM-$ 17), indicating that 2,6-DNT exposure had relatively little impact on the brain in comparison to RDX. Birds exhibiting RDX-induced seizures accumulated over 20M-CM-^W more RDX in brain tissues in comparison to non-seizing birds even within a common dose. In parallel, expression patterns were unrelated among seizing and non-seizing birds exposed to equivalent RDX doses. In birds experiencing seizures, genes related to neuronal electrophysiology and signal transduction were significantly affected. Comparative toxicology revealed strong similarity in acute exposure effects between RDX and the organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) regarding both molecular mechanisms and putative mode of action. In a manner similar to DDT, we hypothesize that RDX elicits seizures by inhibition of neuronal cell repolarization postaction potential leading to heightened neuronal excitability and seizures facilitated by multiple molecular mechanisms. Female Northern Bobwhite 14 Day 2,6-DNT Exposure, Brain Tissue Investigation (Low Laser Intensity Scan): Juvenile female Northern bobwhite (12 weeks of age) were dosed with 2,6-DNT by daily gavage (0, 50, 100, 190, or 350 mg/kg/day). Each treatment group included seven birds (at least three of each sex per treatment). Microarray experiments were conducted using an interwoven-loop design using Cyanine-3 (Cy3) and A647. This experiment investigated the effects of the 14-day 2,6-DNT exposure in females including controls, 60, and 100 mg/kg/day treatments, each including 3 biological replicates. To broaden signal detection, each microarray was scanned at high and low laser power to resolve low-intensity spots and reduce signal saturation, respectively (Skibbe et al., 2006). This dataset represents the Low Intensity Scan.

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Human Transcriptomic Integrated Approach for Testing and Assessment Oral Reference Dose for Steatosis

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