Towards Omics Reporting Standards in Regulatory Toxicology: Introducing the OECD Transcriptomics and Metabolomics Reporting Framework

Presentation to the Environmental Mutagenesis and Genomics Society meeting September 2021Search for CCTE records in EPA’s Science Inventory by typing in the title at this link.https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25</p

Additional file 1: of Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing

Figure S1. Comparison of mutant read proportions for substitutions at each nucleotide position across all samples. Figure S2. Example density graphs (smoothed histograms) for variant calls at two different nucleotide positions. Figure S3. Distribution of read starts and ends. Figure S4. Comparison of the density of false mutation proportions between base substitutions (red) and indels (blue). Figure S5. Relative proportion of control and BaP-induced lacI mutations in liver from reference [29]. Figure S6. Bone marrow mutation spectra of control and BaP treatments. Figure S7. Comparison of spontaneous mutation spectra between our NGS dataset and other published datasets [4, 5, 7, 9-11]. Figure S8. Comparison of BaP-induced mutation spectrum in bone marrow measured using NGS with the mean mutation spectrum measured from four tissues (forestomach, spleen, colon, glandular stomach) using Sanger sequencing [6]. Figure S9. Distribution of non-unique base substitutions across the lacZ transgene for control and BaP samples. Figure S10. Comparison of lacZ mutation hotspots identified using Sanger sequencing [5-10] with mutations identified using NGS at the same nucleotide positions in the present study. Figure S11. Distribution of non-unique indels across the lacZ transgene for control and BaP samples. Figure S12. Results of simulations that use random sampling of BaP mutants to approximate the number of mutants per sample required to achieve a consistent mutation spectrum. Table S1. Thresholds used to detect mutations from each sample library. Table S2. Comparison of expected and observed mutant count when the number of each mutant plaque was controlled for in each NGS library. Table S3. Percent Clonality and Adjusted Mutant Frequencies in Control and BaP-treated samples using the LOD/linear model to adjust mutant counts. Table S4. Comparison of BaP-induced and spontaneous mutation spectra. (DOCX 418 kb

Additional file 1: Table S1. of Meta-analysis of transcriptomic responses as a means to identify pulmonary disease outcomes for engineered nanomaterials

A list of 2334 differentially expressed genes that were consistent to all of the microarray platforms employed in the studies included in the meta-analysis. (PPTX 123 kb

Identification of the target genes of miR-206 that are regulated by TH.

<p>A. Levels of miR-206 in AML 12 cells stably transfected to ectopically express miR-206. AML12 cells were transfected with pEGP-mmu-mir-206 expression vector or pEGP-mir null control vector and selected with puromycin and green florescence protein by microscopy. The expression of miR-206 was examined with the Taqman miRNA Assay (n = 5). B. The expression of genes that are putative targets of miR-206 in the two cell types shown in figure 4A was examined with RT-PCR (n = 3). C. The effects of TH on the expression of miR-206 in the transfected cells. The miR-206 transfected cells were treated with 10 nm T3 for 24 hours. MiR-206 expression was analyzed with Taqman miRNA Assay (n = 3). D. The effect of TH on the expression of the miR-206 target genes in the transfected cells. The miR-206 transfected cells were treated with 10 nm T3 for 24 hours. The expression of target genes was examined with RT-PCR (n = 3). Data are presented as mean ± SE. A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.</p

Serum T4 levels and liver malic enzyme mRNA levels in mouse pups rendered hypothyroid by 3 day exposure of dams and their litters to drinking water containing MMI and perchlorate from PND 12 to 15.

<p>A. Serum T4 data are expressed as mean ± standard error (SE, n = 10) and * indicates significant difference (p<0.001). B. qRT-PCR for malic enzyme mRNA (positively regulated by direct TH action) was performed with RNA derived from male pups. Data are presented as mean ± SE (n = 5). A two-tailed Student's t-test was used to calculate significance and * indicates p = 0.03.</p

Expression of miRs-1, 206, 133a and 133b in other animal models with altered TH levels.

<p>A. Hepatic miRNA expression in livers of PND 15 mouse pups rendered hypothyroid by treatment with drinking water containing 0.04% (wt/vol) of PTU from GD 13 to PND 15. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). B. Hepatic miRNA expression in livers of PND 15 mice whose TH levels were modulated as follows: hyperthyroid pups (hyper) received a <i>s.c.</i> injection of T4+T3 (50 µg+5 µg, respectively per 100g bw) four hours prior to sacrifice and corrected hypothyroid pups received drinking water containing MMI and perchlorate (0.05 and 1% wt/vol, respectively) from PND 12 to 15 and an injection of T4+T3 (20 µg+2 µg, respectively per 100g bw) four hours prior to sacrifice, while control mice received an injection of PBS only. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). Data are presented as mean ± SE (n = 3). A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.</p

Expression of miRs-1, 206, 133a and 133b as well as their precursors in the AML 12 cells.

<p>A. AML 12 cells were treated with 10 nm T3 for 1 hour or 24 hours. The expression of miRs-1, 206, 133a and 133b was examined with the Taqman miRNA Assay. U6 was used as an internal control. Five batches of cultured cells were considered as 5 biological replicates. B. The expression of the precursors of miRs-1, 206, 133a and 133b was examined in AML 12 cell treated with 10 nm T3 for 24 hours with RT-PCR. <i>Hprt</i> was used as internal control. Three batches of cultured cells were used as 3 biological replicates. Data are presented as mean ± SE. A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.</p

Differentially expressed miRNAs in hypothyroid mouse liver (p<0.1, Fold change >4).

<p>*indicates they are in the same chromosome and apart less than 10kb (based on database of TargetScan).</p

Serum T4 levels of male pups in the various animal models (n = 3).

<p>Serum T4 levels of male pups in the various animal models (n = 3).</p

Additional file 1: Table S1. of Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

Particle physicochemical properties and exposure information. (XLSX 10 kb