Transcriptomic points of departure for 6PPD-Quinone derived from human Caco-2 and HepG2 cells
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PROVIDER: PRJNA1291191 | ENA |
REPOSITORIES: ENA
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Project description:N-(1,3-Dimethyl butyl)-N′-phenyl-phenylenediamine-quinone (6ppd-quinone) is of emerging concern due to its widespread presence and toxicity to aquatic species. The chemical has been detected in human biofluids though little is known about its effects on human tissues. The objective of this study was to increase understanding of 6ppd-quinone’s potential human health effects by deriving transcriptomic points of departure (tPOD) values in two human cell lines using the TPD-seq workflow. An EC20 for cytotoxicity was calculated for Caco-2 (104µg/L) but not for HepG2 cells. Even in the absence of cytotoxicity, tPOD values (20th gene, 10th percentile, mode) were calculated in Caco-2 (6.5-25µg/L) and HepG2 (0.36-35µg/L) cells. These ranges capture values from 16 statistical and bioinformatic tests that examined mapping methods (CLC and Deplexer), algorithms (Limma and DESeq2), and filters (log2FC and BMR). The most common and sensitive genes with calculable benchmark doses (BMDs) in Caco-2 (DPF2, CD44, PGAP1, GDF15, H4C16) and HepG2 (SLC5A3, DKK1, ARG2, PHLDA1, TM4SF1) cells are listed. Pathway BMDs were also calculated for Caco-2 (systemic lupus erythematosus, 9.7-18µg/L; alcoholism, 9.7-20µg/L; viral carcinogenesis, 9.3-18.1µg/L), and HepG2 (metabolic pathways, 50-60µg/L) cells. These findings highlight TPD-seq as an efficient workflow to yield quantitative and mechanistic data relevant for human health risk assessment.
2025-08-15 | GSE302559 | GEO
Project description:N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone; 6PPD-Q), a transformation product of an antiozonant chemical widely used in automobile tires (6PPD; N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine) was identified as the causative agent for pre-spawn mortality in coho salmon resulting from exposure to stormwater run-off. This study employed a 24 h, 96-well plate based, exposure to larval fathead minnows (5 days post-fertilization) to examine the effects of 6PPD and 6PPD-Q exposure on global gene expression. Concentration-response modeling of the resulting gene expression changes identified a transcriptomic point of departure of 1.3 uM for 6PPD and 2.5 uM for 6PPD-Q, although they are effectively equivalent considering the range of uncertainty. Examination of the functional roles of differentially expressed genes provided little insight into the mechanism of idiosyncratic toxicity observed in coho salmon and certain other salmonid species. Overall, larval fathead minnows do not appear sensitive to the same mechanisms.
2025-07-30 | GSE241136 | GEO
Project description:Short-term transcriptomic points-of-departure are consistent with chronic points-of departure for three organophosphate pesticides across mouse and fathead minnow.
| PRJNA1005574 | ENA
Project description:Evaluating the bioactivity of the ubiquitous tire preservative 6PPD and degradant, 6PPD-quinone
| PRJNA1006627 | ENA
Project description:There is growing scientific and regulatory interest in transcriptomic points of departure (tPOD) values from high-throughput in vitro experiments. To further help democratize tPOD research, here we outline ‘tPOD-seq’ which links microplate-based exposure methods involving cell lines for human (Caco-2, Hep G2) and environmental (rainbow trout RTgill-W1) health, with a commercially available RNA-seq kit, with a cloud-based bioinformatics tool (ExpressAnalyst.ca). To demonstrate tPOD-seq’s utility, we applied it to derive tPODs for solvents (dimethyl sulfoxide, DMSO; methanol) and positive controls (3,4-dichloroaniline, DCA; hydrogen peroxide, H2O2) commonly used in toxicity testing. The majority of reads mapped to protein coding genes (~10.5k for fish cells; ~7k for human cells), and about 50% of differentially expressed genes were curve-fitted from which 90% yielded gene benchmark doses. The most robust transcriptomic responses were caused by DMSO exposure, and tPOD values were 31-136.6 mM across the cell lines. OECD test guideline 249 (RTgill-W1 cells) recommends the use of DCA and here we calculated a tPOD of ~0.005 to ~0.076 mMmg/L. Finally, exposure of the two human cell lines to H2O2 resulted in similar tPOD values that ranged from 0.68-1.1 in Caco-2 cells and 0.0048-0.03 in Hep G2 cells. The methods outlined here are designed to be performed in laboratories with basic molecular and cell culture facilities, and the performance and scalability of the tPOD-seq approach can be determined with additional case studies.
2024-12-31 | GSE284070 | GEO
Project description:New approach methods (NAMs) can reduce the need for chronic, animal-based toxicity and carcinogenicity bioassays. Here, we apply benchmark dose (BMD) response modeling (BMDExpress 2.3) to transcriptomic data after short-term exposures to three organophosphate pesticides (OPPs) to estimate chronic adverse effect levels in mouse. Specifically, mouse-liver were collected for RNA sequencing after 7-days of exposure, respectively, to several dose-levels of well-known OPPs: fenthion, methidathion and parathion. Transcriptional points-of-departure (TPODs) were compared to chronic, apical points-of-departures (APOD) for each OPP to inform more efficient means of estimating chemical potency and risk. The mouse-liver TPODs for fenthion, methidathion, and parathion were 0.009, 0.093, and 0.046 mg/Kg-day, respectively. These short-term TPODs reflected the relative potencies of the most sensitive, apical effects from the chronic studies with fenthion identified as the most potent OPP. Moreover, the TPODs were 2.3 to 17-fold more sensitive than the chronic APODs for mouse, suggesting utility in using short-term gene response for estimating long-term chemical risk. Further investigation into OPPs’ modes of action identified significant impacts on acetylcholinesterase mRNA abundance (FDR p-value <0.05, |FC|>2) as well as enrichment of canonical pathways (IPA, z-score>|2|) associated with organism death and neurological and immune dysfunctions, which have been known to be affected by OPPs. Despite bioassay-related differences, our results indicate the conservation of key events potentially important to OPPs biological activity and potency across species. While additional research is needed, these results build confidence in using short-term, molecular-based assays for the characterization of toxicity and risk, thereby reducing reliance on chronic, rodent-based studies.
2023-11-13 | GSE240853 | GEO
Project description:It is postulated that below a transcriptomic-based point of departure, adverse effects are unlikely to occur, thereby providing a chemical concentration to use in screening level hazard assessment. The present study extends previous work describing a high throughput fathead minnow assay that can provide full transcriptomic data after exposure to a test chemical. One day post-hatch fathead minnows were exposed to ten concentrations of three representatives of four chemical modes of action: organophosphates, ecdysone receptor agonists, plant photosystem II inhibitors, and estrogen receptor agonists for 24 hours. Concentration response modeling was performed on whole body gene expression data from each exposure, using measured chemical concentrations when available. Transcriptomic points of departure in larval fathead minnow were lower than apical effect concentrations across fish species but not always lower than toxic effect concentrations in other aquatic taxa like crustaceans and insects. The point of departure was highly dependent on measured chemical concentration which were often lower than the nominal concentration. Differentially expressed genes between chemicals within modes of action were compared and often showed statistically significant overlap. In addition, reproducibility between identical exposures using a positive control chemical (CuSO4) and variability associated with the transcriptomic point of departure using in-silico sampling were considered. Results extend a transcriptomic-compatible fathead minnow high throughput assay for possible use in ecological hazard screening.
2024-10-18 | GSE252223 | GEO
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.
2013-04-22 | GSE45892 | GEO
Project description:There is an urgent demand for more efficient and ethical approaches in chemical risk assessment. Using 17α-ethinylestradiol (EE2) as a model compound, this study established a live-animal alternative embryo benchmark-dose (BMD) assay for rainbow trout (RBT; Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs). Embryos were exposed to graded concentrations of EE2 (0, 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects, while transcriptomics revealed responses that were in agreement with apical effects including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L; the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the tenth percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive overrepresented pathway, respectively) were within the same order of magnitude as empirically derived apical PODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving protective tPODs for EE2.
2021-08-17 | PXD025251 | Pride