DOI: 10.5061/dryad.7wm37pw47
title: Profiling the cytotoxic effects of naled and other pesticides in primary human placental cytotrophoblasts

description: 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&lt;0.01, absolute fold change &gt; 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.
description: # Profiling the cytotoxic effects of naled and other pesticides in primary
 human placental cytotrophoblasts Dataset DOI:
 [10.5061/dryad.7wm37pw47](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 (&gt;0.5 log 2 cpm) in &gt; 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 &lt; 0.01 and an absolute average fold change &gt; 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 &lt; 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 &gt; 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 &gt; 20 (Column I). In cases of BMC/BMCL
 &gt; 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 &lt; 0.005) and specified the number of DE genes (&gt; 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 NA ## Access information NA