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Evaluation of 5-Day In Vivo Rat Liver and Kidney with High-Throughput Transcriptomics for Estimating Benchmark Doses of Apical Outcomes

ABSTRACT: The Division of the National Toxicology Program (DNTP) is currently evaluating high-throughput transcriptomics (HTT) as an approach to provide estimates of chemical exposure that may pose minimal risk. HTT was evaluated in a 5-day in vivo rat model (with repeated dosing) with the objective to determine if benchmark doses (BMD) values for transcriptional pathway changes in the liver and kidney could estimate BMD values for traditional toxicological (apical) endpoints. Eighteen chemicals, most having been tested by the NTP in 2-year bioassays, were chosen for this study. Some of these chemicals are known to be potent hepatotoxicants (e.g. DE71, PFOA, furan, and methyl eugenol) in rodents, some exhibit toxicity but have minimal effects on the liver (e.g. acrylamide and α,β-thujone), and some exhibit little overt toxicity (e.g. ginseng and milk thistle extract) based on traditional toxicological evaluations. Male Sprague Dawley rats were exposed daily for 5 consecutive days by oral gavage to 8 to 10 dose levels of each chemical. Liver and kidney were collected 24 hours after the final exposure and assayed using HTT with the rat S1500+ platform. HTT dose-response data were analyzed using BMD Express 2.2 to determine transcriptional BMD values for liver and kidney. BMDS Wizard was used to determine apical BMD values for histopathological effects from historical chronic or sub-chronic toxicity studies. For many of the chemicals, the lowest transcriptional BMDs from the 5-day assays were within a factor of 5 of the lowest histopathological BMDs estimated from the traditional toxicity studies. These data suggest that using HTT in a 5-day in vivo model provides reasonable estimates of BMD values for traditional apical endpoints. This approach may be useful to prioritize chemicals for further testing while providing actionable data in a timely and cost-effective manner.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE147072 | GEO | 2020/05/12

REPOSITORIES: GEO

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Project description:The number of legacy chemicals without toxicity reference values combined with the rate of new chemical development are overwhelming the capacity of the traditional risk assessment paradigm. More efficient approaches are needed to quantitatively estimate chemical risks. In this study, rats were dosed orally with multiple doses of six chemicals for 5 days, 2, 4, and 13 weeks. Target organs were analyzed for traditional histological and organ weight changes and transcriptional changes using microarrays. Histological and organ weight changes in this study and the tumor incidences in the original cancer bioassays were analyzed using benchmark dose (BMD) methods to identify noncancer and cancer points-of-departure. The dose-response changes in gene expression were also analyzed using BMD methods and the responses grouped based on signaling pathways. A comparison of transcriptional BMD values for the most sensitive pathway with BMD values for the noncancer and cancer apical endpoints showed a high degree of correlation at all time points. When the analysis included data from an earlier study with 8 additional chemicals, transcriptional BMD values for the most sensitive pathway were significantly correlated with noncancer (r = 0.827, p = 0.0031) and cancer-related (r = 0.940, p = 0.0002) BMD values at 13 weeks. The average ratio of apical-to-transcriptional BMD values was less than two suggesting that for the current chemicals, transcriptional perturbation did not occur at significantly lower doses than apical responses. Based on our results, we propose a practical framework for application of transcriptomic data to chemical risk assessment. Four to five week-old rats were exposed in dose-response and for multiple durations to 6 chemicals. The chemicals were 1,2,4-tribromobenzene (CAS No. 615-54-3), 2,3,4,6-tetrachlorophenol (CAS No. 58-90-2), bromobenzene (CAS No. 108-86-1), 4,4'-methylenebis(N,N-dimethyl)benzenamine (CAS No. 101-61-1), hydrazobenzene (CAS No. 122-66-7), N-nitrosodiphenylamine (CAS No. 86-30-6). For 1,2,4-tribromobenzene and 2,3,4,6-tetrachlorophenol, male Sprague Dawley (Rat/Crl:CD(SD)) rats were used. For bromobenzene and hydroazobenzene, male F344/DuCrl rats were used. For 4,4'-methylenebis(N,N-dimethyl)benzenamine and N-nitrosodiphenylamine, female F344/DuCrl rats were used. The exposure durations were 5 days, 2 weeks, 4 weeks, and 13 weeks. The dose and route of exposure are provided with the individual sample annotations. With each chemical treatment, a matched vehicle control group was run concurrently with the exposure. Gavage exposures were administered 7 days per week and feed exposures were provided 7 days per week. At the appropriate time point, animals were euthanized with a lethal intraperitoneal injection of sodium pentobarbital. For the liver, slices from each of the lobes were mixed together and used for microarray analysis. For the thyroid, the gland was transversely sectioned and the bottom portion was used for microarray analysis. For the bladder, the organ was longitudinally bisected, a slice was removed for histology and the remainder of the organ was minced and used for microarray analysis. Microarrays were run on five rats per dose per time point.

Project description:Current methods to support chemical hazard assessment face significant challenges given the rapidly growing number of compounds of emerging concern (CEC) requiring assessment. Standard testing strategies rely upon live animal testing, which are time-consuming, expensive, and ethically questionable. These concerns serve as an impetus to develop new approach methodologies (NAMs) that do not rely on live animal tests. This study explored the application of the molecular benchmark dose (BMD) approach in a 7-day embryo-larval fathead minnow (FHM) assay to derive determine transcriptomic points-of-departure (tPODs) which can be useds to predict apical BMDs of fluoxetine (FLX), a highly prescribed and potent selective serotonin reuptake inhibitor frequently detected in surface waters. Fertilized FHM embryos were exposed to graded concentrations of FLX (confirmed at <LOD, 0.19, 0.74, 3.38, 10.2, 47.5 µg/L) for 32 days. Subsets of fish were subjected to omics and locomotor analyses at 7 days post-fertilization (dpf) and to histological and biometric measurements at 32 dpf. Enrichment analyses of transcriptomics and proteomics data revealed significant perturbations in gene sets associated with serotonergic and axonal functions. BMD analysis resulted in tPOD values of 0.56 µg/L (median of the 20 most sensitive gene-level BMDs), 5.0 µg/L (tenth percentile of all gene-level BMDs), 7.51 µg/L (mode of the first peak of all gene-level BMDs) , and 5.66 µg/L (pathway-level BMD). These tPODs are protective of apical locomotor and reduced weight effects (LOEC of 10.2 µg/L) observed in this study and are well within the range of chronic apical BMDs of FLX reported in the literature. Furthermore, the distribution of gene-level BMDs followed a bimodal pattern, revealing disruption of sensitive neurotoxic pathways at low concentrations and metabolic pathway perturbations at higher concentrations. This is one of the first studies to derive tPODs for FLX using a short-term embryo assay at a life stage not considered to be a live animal under current legislations.

Project description:To provide insights into the mode of action for Ni3S2 lung carcinogenicity by examining gene expression changes in target cells after inhalation exposure. Gene expression changes were determined in micro-dissected lung broncho-alveolar cells from Fischer F344 rats following inhalation of Ni3S2 at 0.0, 0.04, 0.08, 0.15, and 0.60 mg/m3 (0.03, 0.06, 0.11, and 0.44 mg Ni/m3) for one and four weeks (6 hours per day, 5 days per 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 mg Ni/m3, for apoptosis/survival signaling. When conducted on the basis of lung Ni concentration the lowest pathway BMD was 0.64 M-BM-5g Ni/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. Gene expression changes were determined in micro-dissected lung broncho-alveolar cells from Fischer F344 rats following inhalation of Ni3S2 at 0.0, 0.04, 0.08, 0.15, and 0.60 mg/m3 (0.03, 0.06, 0.11, and 0.44 mg Ni/m3) for one and four weeks (6 hours per day, 5 days per week).

Project description:Furan is a widely used industrial chemical and a common contaminant in heated foods. Chronic furan exposure has been shown to cause cholangiocarcinoma and hepatocellular tumors at doses of 2 mg/kg bw/day with gender differences in frequency and severity. The hepatic transcriptional alterations induced by low doses of furan (doses below those inducing liver tumors) and the potential mechanisms underlying gender differences are largely unexplored. We used DNA microarrays to examine the global hepatic mRNA and microRNA transcriptional profiles of male and female rats exposed to 0, 0.03, 0.12, 0.5 or 2 mg/kg bw/day furan over 90 days. Marked gender differences in gene expression responses to furan were observed, with many more altered genes in exposed males than females, confirming the increased sensitivity of males even at the low doses. Pathway analysis supported that key events in furan-induced hepatotoxicity in males included gene expression changes related to oxidative stress, apoptosis and inflammatory response, while pathway changes in females were consistent with primarily adaptive responses (regeneration). Pathway benchmark doses (BMDs) were estimated and compared to relevant apical endpoints. Transcriptional BMDs could be examined in males, they ranged from 0.08 – 1.43 mg/kg bw/day and approximated those derived from traditional histopathology. MiR-34a (a target of P53 signalling) was the only microRNA significantly increased at the 2 mg/kg bw/day, providing evidence in support of the importance of apoptosis and cell proliferation in furan hepatotoxicity. Overall, this study demonstrates the use of transcriptional profiling to discern mode of action and mechanisms involved in gender differences. Total RNAs from 16 liver samples (4 males and 4 females from control and 2 mg/kg bw/day dose groups) were processed using the miRNA Complete Labelling and Hybridization kit (Agilent Technologies Inc.). Labelled RNA was hybridized on 8 X 15K Agilent rat miRNA microarray slides. Arrays were scanned using an Agilent G2505B scanner (5 ?M resolution). Feature extraction (version 10.7.3.1, Agilent Tech. Inc.) was used to acquire the fluorescence intensity of each probe.

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Evaluation of 5-Day In Vivo Rat Liver and Kidney with High-Throughput Transcriptomics for Estimating Benchmark Doses of Apical Outcomes

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