Profiling the cytotoxic effects of naled and other pesticides in primary human placental cytotrophoblasts
Data files
Apr 16, 2025 version files 2.14 MB
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README.md
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Supplemental_Figure_1.png
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Supplemental_Figure_2.png
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Supplemental_Table_1.csv
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Supplemental_Table_2_Subclass.csv
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Supplemental_Table_3_Cytotoxicity.csv
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Supplemental_Table_4_All_genes.csv
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Supplemental_Table_5_DE_genes.csv
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Supplemental_Table_6_GO_Analysis_and_BMC.csv
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Supplemental_Table_7._Benchmark.csv
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Supplemental_Table_8_Taqman_probes.csv
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Supplemental_Table_9_Overrepresented_TFBS.csv
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Abstract
Placental cytotrophoblasts (CTBs) play critical roles in placentation, including implantation, barrier-function, uterine invasion and maternal endovascular remodeling. Impairment of CTB function is linked with common pregnancy complications. In this context, environmental chemicals can contribute to CTB dysfunction. Evidence suggests that prenatal exposures to pesticides affect the placenta and contribute to pregnancy complications and adverse developmental outcomes. Despite being restricted in the European Union, dimethyl 1,2-dibromo-2,2 dichloroethyl phosphate (naled), a common organophosphate pesticide, is widely used in vector control and agriculture in the United States and abroad. In this study, we investigated the placentotoxic activity of naled in 2nd trimester primary human CTBs. We assessed the cytotoxicity of naled and 67 pesticides using the neutral red lysosomal cellular uptake assay and the lactate dehydrogenase release assay. Naled was one of the most toxic compounds (~15th percentile), impairing viability and inducing cell death at levels similar to federally restricted pesticides (methoxychlor and triclosan) and at lower concentrations than other commonly used compounds in the organophosphate class (e.g., chlorpyrifos, dichlorvos, and malathion). Naled significantly altered expression of 297 genes (unadjusted p<0.01, absolute fold change > 1.5 with 10 µM or 30 µM), including molecules important in regulating the environmental stress response and developmental processes. Using a benchmark modeling approach, we identified specific genes and related pathways that may serve as early indicators of naled-response in CTBs at physiologically-relevant exposure levels. Thus, our data suggests that naled may alter critical human CTB functions in vivo.
Dataset DOI: 10.5061/dryad.7wm37pw47
Description of the data and file structure
Supplemental Table 1. Gestational age of placentas used in this study.
Supplemental Table 2. Screening panel of test pesticides.
Supplemental Table 3. Pesticide induced cytotoxicity in primary human placental cytotrophoblasts (CTBs; 24h hours).
Supplemental Table 4. Transcriptomes of naled and DMSO exposed primary human placental cytotrophoblasts (24 hours).
Supplemental Table 5. Genes altered due to naled exposure in primary human placental cytotrophoblasts.
Supplemental Table 6. Enriched Gene Ontology (GO) Biological Processes associated with DE genes due to naled exposure of primary human placental cytotrophoblasts.
Supplemental Table 7. Benchmark concentration analysis of genes identified as differentially expressed due to naled exposure of cytotrophoblasts.
Supplemental Table 8. Taqman probes used for qRT-PCR validation analyses.
Supplemental Table 9. Overrepresented transcription factor binding sites in DE genes due to naled exposure of cytotrophoblasts (24 hours).
Supplemental Figure 1. Raw (A) and VOOM normalized (B) counts by CTB biological replicates.
Supplemental Figure 2. Cytotoxicity as determined by Toxcast assays of pesticides in non-placental cell models
Files and variables
File: Supplemental_Table_1.csv
Description: Gestational age of placentas used in this study.
Variables
- Placenta no.: Placenta number
- Gestational week: Gestational week of placenta collected
- Analysis: Cytotoxicity, RNA sequencing or qRT-PCR
- Sex: Sex was determined via XIST expression
File: Supplemental_Table_2_Subclass.csv
Description: Screening panel of test pesticides. Classifications related to biological functions and chemical groups were obtained via the US EPA Comptox dashboard, the British Crop Production Council (BCPC) or Pubchem databases. US production volumes in 2019 are based on estimates in the Chemical Data Reporting (CDR) database. Estimated usage (pounds/year, 2016-2021) in California was from the Annual Pesticide Use Report (PUR) provided by the California Department of Pesticide Regulation (CDPR). Expocast estimates (pg/kg body weight (BW)/day) were from the USEPA Dashboard that provide a general estimate of human exposure.
Variables
- Preferred Name (USEPA): Chemical name
- CASRN: CAS Number
- Biological Function: US EPA Comptox dashboard
- Chemical Group: US EPA Comptox dashboard
- EXPOCAST_MEDIAN_EXPOSURE_PREDICTION_MG.KG.BW.DAY: US EPA Expocast Dashboard
- Production Volume in US 2019 (lbs): Chemical Data Reporting (CDR) database
- Median Usage 2016-21 in CA(lbs): Annual Pesticide Use Report (PUR)
File: Supplemental_Table_3_Cytotoxicity.csv
Description: Pesticide induced cytotoxicity in primary human placental cytotrophoblasts (CTBs; 24h hours). Cytotoxicity was assessed using neutral red (NR) uptake and lactate dehydrogenase (LDH) activity assays. Absorbance values were normalized to the media-only control (=100%). Average relative values and the standard error to the mean (SEM) were calculated across 3 biological replicates. BMDExpress 2 was used to calculate benchmark concentrations (BMCs) reflective of a 10% relative change (BMC10) in decreased viability or increased cell death (LDH activity) vs. the vehicle control.
Variables
- DTXSID: DSSTox Substance Identifier
- PREFERRED_NAME: Chemical name
- CASRN: CAS Number
- NR_CTB_1_30uM: Neutral red uptake assay at 30uM
- NR_CTB_2_30uM: Neutral red uptake assay at 30uM
- NR_CTB_3_30uM: Neutral red uptake assay at 30uM
- NR_CTB_1_100uM: Neutral red uptake assay at 100uM
- NR_CTB_2_100uM: Neutral red uptake assay at 100uM
- NR_CTB_3_100uM: Neutral red uptake assay at 100uM
- LDH_CTB_1_30uM: Lactate dehydrogenase activity assay at 30uM
- LDH_CTB_2_30uM: Lactate dehydrogenase activity assay at 30uM
- LDH_CTB_3_30uM: Lactate dehydrogenase activity assay at 30uM
- LDH_CTB_1_100uM: Lactate dehydrogenase activity assay at 100uM
- LDH_CTB_2_100uM: Lactate dehydrogenase activity assay at 100uM
- LDH_CTB_3_100uM: Lactate dehydrogenase activity assay at 100uM
File: Supplemental_Table_4_All_genes.csv
Description: Transcriptomes of naled and DMSO exposed primary human placental cytotrophoblasts (24 hours). Gene expression levels (log2 counts per million; CPM) in naled or DMSO exposed cells. Genes with abundances above the lower level of detection (>0.5 log 2 cpm) in > 50% of samples were analyzed.
Variables
- Exposure: naled or DMSO
- DMSO_21.2: The placenta of gestational age 21.2 weeks exposed to DMSO
- naled_10M_21.2: The placenta of gestational age 21.2 weeks exposed to 10M naled
- naled_30M_21.2: The placenta of gestational age 21.2 weeks exposed to 30M naled
- DMSO_21.3: The placenta of gestational age 21.3 weeks exposed to DMSO
- naled_10M_21.3: The placenta of gestational age 21.3 weeks exposed to 10M naled
- naled_30M_21.3: The placenta of gestational age 21.3 weeks exposed to 30M naled
- DMSO_23: The placenta of gestational age 23 weeks exposed to DMSO
- naled_10M_23: The placenta of gestational age 23 weeks exposed to 10M naled
- naled_30M_23: The placenta of gestational age 23 weeks exposed to 30M naled
File: Supplemental_Table_5_DE_genes.csv
Description: Genes altered due to naled exposure in primary human placental cytotrophoblasts. We identified differentially expressed (DE) genes due to naled exposure (Column B) using a generalized linear model that adjusted for biological differences (Column C). We used a cutoff of (uncorrected) p < 0.01 and an absolute average fold change > 1.5 for the naled concentrations vs. vehicle control. Log 2 transformed counts (CPM) are listed in Columns D-L. Sample names include exposure and age (gestational week; GW).
Variables
- Gene: Gene name
- p value_Exposure: p value due to naled exposure
- p value_Placenta: adjusted p value for biological differences
- 21.2GW_DMSO: The placenta of gestational age 21.2 weeks exposed to DMSO
- 21.2GW_Naled 10M: The placenta of gestational age 21.2 weeks exposed to 10M naled
- 21.2GW_Naled 30M: The placenta of gestational age 21.2 weeks exposed to 30M naled
- 21.3GW_DMSO: The placenta of gestational age 21.3 weeks exposed to DMSO
- 21.3GW_Naled 10M: The placenta of gestational age 21.3 weeks exposed to 10M naled
- 21.3GW_Naled 30M: The placenta of gestational age 21.3 weeks exposed to 30M naled
- 23GW_DMSO: The placenta of gestational age 23 weeks exposed to DMSO
- 23GW_Naled 10M: The placenta of gestational age 23 weeks exposed to 10M naled
- 23GW_Naled 30M: The placenta of gestational age 23 weeks exposed to 30M naled
File: Supplemental_Table_6_GO_Analysis_and_BMC.csv
Description: Enriched Gene Ontology (GO) Biological Processes associated with DE genes due to naled exposure of primary human placental cytotrophoblasts. We performed GO analyses to identify enriched biological processes associated of with the DE genes as well as the up- or down-regulated subsets using Database for Annotation, Visualization and Integrated Discovery (DAVID). Overrepresented terms associated with “Level 3” Biological Processes that contained ≥ 10 DE genes were identified using a significance cutoff (p < 0.001). Estimated mean and median benchmark concentrations (i.e., BMC10s) and associated lower bound estimates (i.e., BMCL10s) for each gene subset are shown. In addition, BMCs corresponding to the lowest 5th percentile for each term are listed. GO terms linked with genes that had significantly lower BMC values vs. BMC values of all DE genes are shaded in blue (p > 0.05; t-test).
Variables
- GO ID: GO Term ID
- Term: biological processes
- Category: biological category
- all DE genes: Numebr of all differentially expressed genes
- upregulated DE genes: Numebr of upregulated genes
- downregulated DE genes: Numebr of downregulated genes
- Mean BMC10: Estimated mean benchmark concentration
- Mean BMCL10: Mean lower bound estimates
- Median BMC10: Estimated median benchmark concentrations
- Median BMCL10: Median lower bound estimates
File: Supplemental_Table_7._Benchmark.csv
Description: Benchmark concentration analysis of genes identified as differentially expressed due to naled exposure of cytotrophoblasts. Benchmark concentrations (BMCs) were determined by: 1) performing power, linear or polynomial 2 model regression; 2) selecting the “best-fit model” based on Akaike information criterion (AIC; Column G); and 3) calculating BMC10s using default settings [benchmark response (BMR) = 1.3, 0.95 confidence level, constant variance = 1, and maximum iterations = 250]. A BMR of 1.3 is equivalent to a 10% increase or decrease over the assumed background rate of response. For each gene, we report the BMC10 (Column C) as well as the BMCL10 (Column D; lower bound estimate of the 95th percentile confidence limit) and BMDU (Column E, upper bound estimate of the 95th percentile confidence limit). Statistical values associated with model fit: LogLikelihood, (Column F) and Akaike information criterion (Column G). Column H denotes whether the best-fit model predicted up (1) or down (-1) regulation of each DE gene (Column H). To reduce model overfitting, we removed “best-fit model” estimates with BMC/BMCL > 20 (Column I). In cases of BMC/BMCL > 20, we used secondary estimates identified via AIC (n=2 genes). All curves were analyzed to assure proper fitting.
Variables
- Probe ID: Gene name
- Best BMC10: benchmark concentration
- Best BMDL10: lower bound estimate of the 95th percentile confidence limit
- Best BMDU10: upper bound estimate of the 95th percentile confidence limit
- Best AIC: Akaike information criterion
File: Supplemental_Table_8_Taqman_probes.csv
Description: Taqman probes used for qRT-PCR validation analyses.
Variables
- Gene: Gene symbol
- Name: Gene full name
File: Supplemental_Table_9_Overrepresented_TFBS.csv
Description: Overrepresented transcription factor binding sites in DE genes due to naled exposure of cytotrophoblasts (24 hours). We performed enrichment analysis of transcription factor binding sites (TFBS) associated with the DE genes using EnrichR and the TRRUST Transcription Factor 2019 database. To identify enriched TFBS, we applied a significance cutoff (p < 0.005) and specified the number of DE genes (> 3).
Variables
- TF: transcription factor
- All DE Genes: Numebr of all differentially expressed genes
- UP DE Genes: Numebr of upregulated genes
- Dn DE Genes: Numebr of downregulated genes
- TFBS Genes: transcription factor binding sites associated with the DE genes
- UP_genes: list of upregulated genes
- Dn_genes: list of downregulated genes
File: Supplemental_Figure_1.png
Description: Raw (A) and VOOM normalized (B) counts by CTB biological replicates. CPM, counts per million. GW, gestational week.
File: Supplemental_Figure_2.png
Description: Cytotoxicity as determined by Toxcast assays of pesticides in non-placental cell models. Number of assays that were inactive (grey) or active (red). Compounds that were active in our study are labeled in red.
Code/software
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