https://doi.org/10.5061/dryad.pc866t1wp

To elucidate whether HFPO-DA-mediated transcriptomic responses in mouse liver are PPARa-dependent, transcriptomic analyses were conducted on samples from primary PPARa knockout (KO) and wild-type (WT) mouse hepatocytes exposed for 12, 24 or 72 hours with various concentrations of HFPO-DA, or established agonists of PPARa (GW7647) and PPARg (rosiglitazone), or cytotoxic agents (acetaminophen or d-galactosamine).

List of files

A list of each file included in this dataset:

·       Supplementary Files S1 & S2_Sequencing Quality & Cytotoxicity Results.xlsx – Summary of sequencing quality data and observations of cytotoxicity.

·       Supplementary File S3a_DEPs_Mouse_B6129SF2J.xlsx – Differential probe expression data for WT mouse.

·       Supplementary File S3b_DEPs_Mouse_PPARa-KO.xlsx – Differential probe expression data for PPARa-KO mouse.

·       Supplementary File S4_Hypergeometric Gene Set Enrichment.csv – Gene set enrichment results for both mouse strains.

·       Supplementary File S5_Concentration-Responsive Genes.csv – Gene dose-response analysis results for both mouse strains.

·       Supplementary File S6_Concentration-Responsive Pathways.csv – Pathway dose-response analysis results (i.e., enriched gene sets) for both mouse strains.

Description of the data and file structure

Each file (Excel spreadsheet) contains supplementary data related to sequencing quality results, cytotoxicity results, or processed RNA sequencing data including differentially expressed genes/probes, gene set enrichment, concentration-responsive genes, and enriched gene sets among concentration-responsive genes.

Below is a list of each supplementary file along with a description of its contents:

Supplementary Files S1 & S2_Sequencing Quality & Cytotoxicity Results.xlsx

Supplementary File S1 contains two sub-tables: Table 1 lists the mean sequencing quality criteria across mouse strains (WT and PPARa-KO). Table 2 lists the individual samples that failed sequencing quality criteria. The following variables are included in both Tables 1 (means) and 2 (by individual samples):

·      Sequencing depth = total number of reads sequenced and aligned across all probes

·      Number of probes = total number of probes sequenced

Supplementary File S2: Provides hepatocyte cytotoxicity results across strains, chemical doses, and timepoints.

Supplementary File S3a_DEPs_Mouse_B6129SF2J.xlsx

Supplementary File 3a provides differential gene expression analysis results of WT (B6129SF2/J) mouse hepatocytes treated with HFPO-DA, GW7647, rosiglitazone, acetaminophen, or d-galactosamine, as calculated using DESeq2. Each tab provides differential gene expression results for one of the five chemicals tested, with the following variables listed as column headers in each of the five spreadsheet tabs (as defined by Love et al. 2014):

• Dose_Timepoint = concentration and treatment duration of the chemical tested
• gene_source= gene name and TempO-Seq probe ID number
• baseMean = the average of the normalized gene count values, divided by size factors, taken over all samples
• log2FoldChange = the effect size estimate. This value indicates how much the gene's expression has changed between the treatment and control groups. This value is reported on a logarithmic scale to base 2. If the baseMean value = 0 (i.e., no counts for that gene) the log2 fold change will be equal to "NA", meaning a log2 fold change could not be estimated.
• lfcSE = the standard error estimate for the log2 fold change estimate. If the log2 fold change estimate = NA, the lfcSE estimate will also be equal to "NA", meaning that the standard error estimate of the log2 fold change could not be estimated.
• stat = the value of the Wald test statistic for the gene. Wald test statistic is the log2FoldChange divided by lfcSE, which is compared to a standard Normal distribution to generate a two-tailed P-value. If the log2 fold change and standard error estimate are equal to NA, the Wald test statistic with also be equal to "NA", meaning that the Wald test statistic could not be estimated.
• pvalue = P-value of the Wald test statistic for the gene. If the P-value = NA, either the Wald test statistic could not be estimated or the gene contained an outlier count as identified using Cook's distance.
• padj = adjusted P-value for multiple testing for the gene. If the adjusted P-value = NA, either the P-value was also equal to "NA" or the gene did not pass the independent filtering threshold for the selection of genes for multiple test correction.

Supplementary File S3b_DEPs_Mouse_PPARa-KO.xls

Supplementary File 3b provides differential gene expression analysis results of PPARa-KO mouse hepatocytes treated with HFPO-DA, GW7647, rosiglitazone, acetaminophen, or d-galactosamine, as calculated using DESeq2. Each tab provides differential gene expression results for one of the five chemicals tested, with the same variables listed as column headers in each of the five spreadsheet tabs as were listed in Supplementary File 3a (Supplementary File S3a_DEPs_Mouse_B6129SF2J.xlsx).

Supplementary File S4_Hypergeometric Gene Set Enrichment.csv

Supplementary File S4 provides hypergeometric test results for gene set enrichment for each mouse strain and chemical treatment group. The following variables are listed as column headers in this spreadsheet (as defined by Varemo et al. 2013):

·      Species_Strain = species and mouse strain

·      Chemical = chemical treatment

·      Concentration = treatment concentration

·      Timepoint = exposure duration

·      Pathways = canonical pathway/gene set

·      p-value - non-dir = non-directional (direction of differential expression omitted) p-value

·      Adjusted p-value - non-dir = corrected non-directional p-value

·      Significant (in gene set) - non-dir = number of differentially expressed genes in gene set

·      Non-significant (in gene set) - non-dir = number of non-significant genes in gene set

·      Significant (not in gene set) - non-dir = number of differentially expressed genes not in gene set

·      Non-significant (not in gene set) - non-dir = number of non-significant genes not in gene set

·      p-value – down = p-value for downregulation of gene set

·      Adjusted p-value – down = corrected downregulation p-value

·      Significant (in gene set) – down = number of differentially expressed downregulated genes in gene set

·      Non-significant (in gene set) – down = number of non-differentially expressed genes in gene set (downregulation)

·      Significant (not in gene set) – down = number of differentially expressed downregulated genes not in gene set

·      Non-significant (not in gene set) – down = number of non-differentially expressed genes not in gene set (downregulation)

·      p-value - up = p-value for upregulation of gene set

·      Adjusted p-value - up = corrected upregulation p-value

·      Significant (in gene set) - up = number of differentially expressed upregulated genes in gene set

·      Non-significant (in gene set) - up = number of non-differentially expressed genes in gene set (upregulation)

·      Significant (not in gene set) - up = number of differentially expressed upregulated genes not in gene set

·      Non-significant (not in gene set) - up = number of non-differentially expressed genes not in gene set (upregulation)

·      GeneSymbols_all = identify of differentially expressed genes (non-directional)

·      GeneSymbols_up = identify of differentially expressed upregulated genes

·      GeneSymbols_down = identify of differentially expressed downregulated genes

Supplementary File S5_Concentration-Responsive Genes.csv

Supplementary File S5 provides concentration-responsive gene results for each mouse strain and chemical treatment group. BMDExpress evaluates fit across the following models: Hill, Power, Linear, Polynomial 2, Polynomial 3, Exponential 2, Exponential 3, Exponential 4, and Exponential 5. The following variables are listed as column headers in this spreadsheet, noting that column headers regarding statistical modeling results identical across models and therefore only listed once (as defined by Philips et al. 2019; BMDExpress, Undated; EPA, 2022):

·      Species_Strain = species and mouse strain

·      Chemical = chemical treatment

·      Timepoint = exposure duration

·      Probe ID = gene name and Tempo-Seq probe ID number

·      Entrez Gene IDs = universal gene identifier

·      Genes Symbols = gene symbols that are included in the probe  

·      [Model] BMD = benchmark dose; estimated dose level associated with specified benchmark dose response

·      [Model] BMDL = benchmark dose lower bound of 95% confidence interval

·      [Model] BMDU = benchmark dose upper bound of 95% confidence interval

·      [Model] fitPValue = P-value for global goodness of fit of model to data

·      [Model] fitLogLikelihood = measure of model fit to compare between models

·      [Model] AIC = Akaike Information Criterion; estimates quality of model relative to the other models

·      [Model] adverseDirection = direction of the response (up or down)

·      [Model] BMD/BMDL = ratio of model’s BMD to BMDL for gene

·      Flagged [Model] = indicates model flagged for gene based on settings from BMD analysis (1 = flagged; 0 = not flagged)   

·      [Model] Parameters Intercept, alpha, rho, control, slope, power, beta 0-18, v, n, k = various parameters for each different model

·      [Model] Execution Complete = indicates whether model calculation could be completed 

·      Best Model = most appropriate model selected by program for dataset. Parameters listed in column headers following “Best Model” indicate the value of the parameter from the best model.

·      wAUC = weighted area under the curve

·      Prefilter P-Value = prefilter P-value from analysis of variance (ANOVA)

·      Prefilter Adjusted P-Value = prefilter P-value after Benjamini-Hochberg correction

·      Max Fold Change = maximum fold change in response across all dose levels

·      Max Fold Change Absolute Value = absolute value of the maximum fold change in response across all dose levels

·      NOTEL = no-observable transcriptional effect level

·      LOTEL = lowest observable transcriptional effect level

·      FC Dose Level n: fold change at dose n

Blank cells indicate parameters that could not be calculated for a model. “#NAME?” indicate calculations that could not be completed due to missing parameters that could not be calculated for a model.

Supplementary File S6_Concentration-Responsive Pathways

Supplementary File S6 provides enriched gene sets across concentration-responsive genes for each mouse strain and chemical treatment group. The following variables (or sets of variables) are listed as column headers in this spreadsheet that provide information on the gene set, enrichment and modeling results for each Reactome gene set evaluated in BMDExpress (as defined by Philips et al. 2019; BMDExpress, Undated):

·      Species_Strain = species and mouse strain

·      Chemical = chemical treatment

·      Timepoint = exposure duration

·      GO/Pathway/Gene Set/Gene ID = category identifier

·      GO/Pathway/Gene Set/Gene Name = GO category, reactome pathway, or user-defined gene set names

·      All Genes (Expression Data) = total number of genes loaded that were assigned to the pathway/category

·      All Genes (Platform) = total number of genes assigned to the pathway/category from the platform utilized (i.e., Reactome)

·      Input Genes = total number of genes assigned to the pathway/category that passed ANOVA pre-filtering and BMD analyses

·      Genes with BMD <= Highest Dose = number of genes with BMD values less than the highest dose

·      Genes with BMD p-Value >= = number of genes with fit P-values

·      Genes with BMD/BMDL <= = number of genes with BMD to BMDL ratios less than 20

·      Genes with BMDU/BMD <= = number of genes with BMDU to BMD ratios less than 20

·      Genes with BMDU/BMDL <= = number of genes with BMDU to BMDL ratios less than 40

·      Genes with BMD <= N-Fold Lowest Positive Dose = number of genes with BMD values greater than 10-fold below the lowest positive dose

·      Genes with Prefilter P-Value <= = total number of genes that passed P-value pre-filter (0.05)

·      Genes with Prefilter Adjusted P-Value <= = number of genes that passed adjusted P-value filter (0.5)

·      Genes That Passed All Filters = total number of genes that passed all prefilters

·      Fisher's A Parameter = number of genes related to category term with a BMD

·      Fisher’s B Parameter = number of genes not related to category term with a BMD

·      Fisher’s C Parameter = number of genes related to category term without a BMD

·      Fisher’s D Parameter = number of genes not related to category term without a BMD

·      Fisher's Exact Left P-Value = P-value of enrichment analysis of gene sets that indicates significant underrepresentation of gene set’s responsive genes

·      Fisher's Exact Right P-Value = P-value of enrichment analysis of gene sets that indicates significant overrepresentation of gene set’s responsive genes

·      Fisher's Exact Two Tail = P-value of enrichment analysis of gene sets that from two-tailed test that can indicate either significant over- or underrepresentation of gene sets

·      Percentage = percent (0-1) of total number of genes on array input into BMD analysis that had reportable BMDs based on filter criteria in Functional Classification setup

·      Entrez Gene IDs = universal gene identifiers from probes/probe sets used as input in BMD analysis

·      Gene Symbols = unique gene symbols corresponding to Gene IDs input into BMD analysis

·      Probe IDs = probe identifiers input into BMD analysis

·      Genes with Conflicting Probesets = Entrez Gene IDs with conflicting probe sets or probes (with correlation coefficient)

·      BMD/BMDL/BMDU Mean = mean BMD/BMDL/BMDU for genes in category that had a calculated BMD/BMDL/BMDU

·      BMD/BMDL/BMDU Median = median BMD/BMDL/BMDU for genes in category that had a calculated BMD/BMDL/BMDU

·      BMD/BMDL/BMDU Minimum = minimum BMD/BMDL/BMDU for genes in category that had a calculated BMD/BMDL/BMDU BMD/BMDL/BMDU

·      BMD/BMDL/BMDU Standard Deviation = standard deviation of BMD/BMDL/BMDU for genes in category that had a calculated BMD/BMDL/BMDU

·      BMD/BMDL/BMDU wMean = weighted mean BMD/BMDL/BMDU for genes in category that had a calculated BMD/BMDL/BMDU

·      BMD/BMDL/BMDU wSD = weighted standard deviation BMD/BMDL/BMDU for genes in category that had a calculated BMD/BMDL/BMDU

·      5th/10th Percentile Index = Nth gene number representing 5th/10th percentile for all genes in category

·      BMD at 5th/10th Percentile of Total Genes = BMD at 5th/10th percentile for all genes in Reactome category, including genes without significant dose-response

·      BMD/BMDL/BMDU List = list of BMD/BMDL/BMDU values for genes in this set

·      Probes with Adverse Direction Up = number of probes/probe sets in GO category with adverse direction up (increased)

·      Probes with Adverse Direction Down= number of probes/probe sets in GO category with adverse direction down (decreased)

·      Genes with Adverse Direction Up = number of genes in set with adverse direction up

·      Genes Up List = Entrez Gene IDs with adverse direction up

·      Genes Up Probes List = Probes/probe sets with adverse direction up

·      Genes Up BMD/BMDL/BMDU Mean = mean of BMD/BMDL/BMDU with adverse direction up

·      Genes Up BMD/BMDL/BMDU Median = median of BMD/BMDL/BMDU with adverse direction up

·      Genes Up BMD/BMDL/BMDU SD = standard deviation of BMD/BMDL/BMDU with adverse direction up

·      BMD/BMDL/BMDU list (up) = list of BMD/BMDL/BMDU values for genes with adverse direction up

·      Genes with Adverse Direction Down = number of genes in set with adverse direction down

·      Genes Down List = Entrez Gene IDs with adverse direction down

·      Genes Down Probes List = Probes/probe sets with adverse direction down

·      Genes Down BMD/BMDL/BMDU Mean = mean of BMD/BMDL/BMDU with adverse direction down

·      Genes Down BMD/BMDL/BMDU Median = median of BMD/BMDL/BMDU with adverse direction down

·      Genes Down BMD/BMDL/BMDU SD = standard deviation of BMD/BMDL/BMDU with adverse direction down = list of BMD/BMDL/BMDU values for genes with adverse direction down

·      BMD/BMDL/BMDU list (down) = list of BMD/BMDL/BMDU values for genes with adverse direction down

·      Genes with Adverse Conflict Count = number of genes with probes in category that have conflicting adverse directions

·      Genes Conflict List = Entrez Gene IDs with probes not correlated above designated threshold in Functional Classification setup

·      Genes Conflict Probes List = probes mapping to same gene not correlated above designated threshold in Functional Classification setup

·      BMD/BMDL/BMDU list (Conflict) = BMD/BMDL/BMDU values from conflicted probes

·      Model Counts = model counts denoting model, total number of genes in that mode, and percentage of total model fits the model accounts for

·      Mean/Total/Min/Max/Standard Deviation/Median Fold Change = mean, summed, minimum, maximum, standard deviation, and median of absolute value of fold change values for genes in category

·      Lower bound of the 95% confidence interval -  BMD/BMDL/BMDU = lower bound of 95% confidence interval of BMD/BMDL/BMDU values (assumes normal distribution)

·      Upper bound of the 95% confidence interval -  BMD/BMDL/BMDU = upper bound of 95% confidence interval of BMD/BMDL/BMDU values (assumes normal distribution)

·      Overall Direction = considered “Up” if >60% of genes in category were up-regulated; considered “Down” if >60% of genes in category were down-regulated; considered “Conflict” if neither criteria were met

·      Percent Genes With Overall Direction Up = fraction of genes up-regulated based on adverse direction up of genes

·      Percent Genes With Overall Direction Down = fraction of genes down-regulated based on adverse direction down of genes

·      Percent Genes With Overall Direction Conflict = fraction of genes with probes that are both up and down regulated

Blank cells indicate parameters that were not applicable or could not be calculated.

Sharing/Access information

RNA sequencing data are publicly available at NCBI’s Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) (GEO series accession number GSE248251).

References

BMDExpress. (Undated). BMDExpress-2 Documentation. Available from https://bmdexpress-2.readthedocs.io/en/feature-readthedocs/. Accessed 2AUG24.

EPA. (2022). Benchmarck Dose Software. Version 3.3. User Guide. EPA/600/R-21/245. July 2022. 

Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology, 15, 1-21.

Phillips, J. R., Svoboda, D. L., Tandon, A., Patel, S., Sedykh, A., Mav, D., ... & Auerbach, S. S. (2019). BMDExpress 2: enhanced transcriptomic dose-response analysis workflow. Bioinformatics, 35(10), 1780-1782.

Väremo, L., Nielsen, J., & Nookaew, I. (2013). Enriching the gene set analysis of genome-wide data by incorporating directionality of gene expression and combining statistical hypotheses and methods. Nucleic acids research, 41(8), 4378-4391.