Perinatal Lead (Pb) Exposure Increases Mouse Embryonic Weight and Alters Neuronal Gene Expression

Perinatal Lead (Pb) Exposure Increases Mouse Embryonic Weight and Alters Neuronal Gene Expression | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Perinatal Lead (Pb) Exposure Increases Mouse Embryonic Weight and Alters Neuronal Gene Expression Bambarendage P. U. Perera , Minghua Li , Anagha Tapaswi , Junru Pan , Dongyue Wang , Tejas Goswami , Rachel K. Morgan , View ORCID Profile Kelly M. Bakulski , Jaclyn M. Goodrich , Maureen A. Sartor , Dana C. Dolinoy , Justin A. Colacino doi: https://doi.org/10.1101/2025.09.18.677210 Bambarendage P. U. Perera 1 Department of Environmental Health Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA 2 Division of Environmental Health Sciences, School of Public Health, University of California , Berkeley, Berkeley, CA 94704, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: piniperera{at}berkeley.edu colacino{at}umich.edu Minghua Li 3 Department of Computational Medicine and Bioinformatics, Medical School, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anagha Tapaswi 1 Department of Environmental Health Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Junru Pan 4 Department of Nutritional Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dongyue Wang 1 Department of Environmental Health Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tejas Goswami 5 Department of Chemistry, College of Literature, Science , and the Arts, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rachel K. Morgan 1 Department of Environmental Health Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kelly M. Bakulski 6 Department of Epidemiology, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kelly M. Bakulski Jaclyn M. Goodrich 1 Department of Environmental Health Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maureen A. Sartor 3 Department of Computational Medicine and Bioinformatics, Medical School, University of Michigan , Ann Arbor, MI 48109, USA 7 Department of Biostatistics, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dana C. Dolinoy 1 Department of Environmental Health Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA 4 Department of Nutritional Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Justin A. Colacino 1 Department of Environmental Health Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA 4 Department of Nutritional Sciences, School of Public Health, University of Michigan , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: piniperera{at}berkeley.edu colacino{at}umich.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Acute and chronic exposure to lead (Pb) during pregnancy is linked to adverse health outcomes, including delayed neurodevelopment in offspring. However, the pathways by which Pb exposure influences long-term health remain poorly understood. To address this, we measured the effects of perinatal Pb exposure on gene expression including imprinted genes, X-linked genes, and sexually dimorphic genes. Female mice were given control or Pb acetate dosed (32 ppm) drinking water two weeks prior to timed mating until embryonic day (E)10–12, upon which whole embryos were collected, weighed, and sexed at E13–15. From a subset of embryo heads ( n ≥9 per sex per group), we extracted and sequenced RNA. We used linear regression to assess Pb impacts on embryonic weight and gene expression across all mice and stratified by sex. Among the differentially expressed genes, we identified significantly enriched pathways. Pb-exposed embryos weighed more than controls ( p =0.007), across both sexes. Collectively, we identified 2,920 differentially expressed genes (FDR<0.05), including 31 imprinted genes and 120 X-linked genes upon Pb exposure. Pb exposure altered expression in gene pathways related to neuronal structure and function as well as sexually dimorphic genes (44 for females; 76 for males). These findings highlight perinatal Pb-linked alterations that may drive later-life health outcomes. 1. INTRODUCTION Environmental and occupational lead (Pb) exposure impacts human populations in the US and around the world and has neurotoxic, cardiovascular, and metabolic effects 1 , 2 . Sources of Pb exposure include leaded paint, pipes, electronics, industrial activities, and contaminated soil and water. There is no known ‘safe’ level of Pb exposure. An estimated 500,000 children under the age of 6 in the U.S. are reported to have ≥5 μg/dL concentrations of blood Pb 3 . Children and pregnant women are particularly susceptible to the health effects of Pb, with gestational exposure linked to adverse birth outcomes, delayed neurodevelopment, growth defects, and potential contributions to long-term neurological health issues such as Alzheimer’s disease 4 – 6 . Despite abundant literature on Pb-induced toxicity, the gene pathways driving its health effects, particularly during early stages of neuronal development, remain poorly understood. According to the Developmental Origins of Health and Disease hypothesis, environmental exposure during development predisposes offspring to diseases later in life 7 . Early development is largely driven by the epigenome, which regulates gene expression via DNA methylation (DNAm), histone modifications, and non-coding RNA (ncRNA), and is highly sensitive to environmental exposures. In therian mammals, including placental mammals and marsupials, offspring inherit one maternal allele and one paternal allele, giving rise to biallelic autosomal gene expression. However, a subset of these genes is exclusively expressed from one parental allele, resulting in parent-of-origin specific monoallelic expression through the epigenetically regulated process of genomic imprinting 8 . Dysfunctional genomic imprinting is implicated in several human diseases such as Angelman syndrome and Prader-Willi syndrome 9 – 12 . For instance, loss of function of the maternally-inherited ubiquitin protein ligase E3A ( UBE3A ) contributes to the onset of Angelman syndrome, while the loss of paternally expressed genes including small nuclear ribonucleoprotein polypeptide N ( SNRPN ) contributes to the onset of Prader-Willi syndrome 13 – 15 . Imprinted genes are highly sensitive to the environment during early gestation and are critical for proper embryonic growth and development 16 – 19 . Human cohort studies and perinatal Pb exposure mouse models support that Pb exposure can alter imprinted genes and related mechanisms 20 – 22 . Several reported effects of Pb neurotoxicity are sex-specific, with notable changes in gene expression and epigenetically regulated mechanisms 23 – 26 . Among the latter is the regulation of X-linked genes, which are located on the X chromosome and have differing copy numbers between females and males 27 . Therian mammals undergo X-chromosome inactivation (XCI) as dosage compensation for balancing the X-linked genes in females 28 . Therefore, X-linked genes are critical for proper brain development and neuronal functions, with XCI being established during the early stages of embryogenesis and extensively studied in mouse models 27 . XCI is a complex epigenetic mechanism involving the expression of the X-inactive specific transcript ( XIST ) long ncRNA (lncRNA) among others to facilitate imprinted and random XCI in females 28 . The process of XCI may be modulated by environmental cues, potentially leading to skewed XCI and altered gene expression. However, the impact of Pb exposure on X-linked genes is not fully understood. Developmental exposure to Pb has been linked to sex-dependent effects in human and animal studies 29 , 30 . Sexual dimorphism refers to the species-specific differences between females and males in their physical or biological characteristics 31 . Pb alters DNAm signatures in a sex-specific manner in vivo , impacting autosomal genes linked to neurodevelopment, cardiovascular disease, and immune functions differently in sexes 23 , 30 , 32 . These molecular changes may be modulated by sex hormones, contributing to differential disease susceptibility and phenotypic outcomes in brain and behavior between the sexes following Pb exposure 33 . We hypothesized that Pb exposure impacts gene expression including of imprinted genes, X-linked genes, and sexually dimorphic autosomal genes. The current work evaluated offspring brain-specific transcriptomic changes associated with Pb exposure using a mouse model of prenatal and gestational exposure using embryonic heads at E13–15. The study identified differentially expressed genes, differentially expressed imprinted genes, differentially expressed X-linked genes, sexually dimorphic genes, and enriched pathways among these genes associated with Pb exposure. The current study is the first to report that Pb exposure during preconception and early gestation is associated with increased mid-gestation embryonic weight, and that sexually dimorphic genes are particularly sensitive to Pb exposure in the mouse nervous system. This work will lay the foundation for developing targeted interventions to alleviate long-term health consequences of early life exposure to Pb. 2. MATERIALS AND METHODS 2.1. Animal exposure study design C57BL/6J adult female mice at 10–12 weeks of age were randomly selected and exposed to either control or Pb acetate drinking water (pH 4.5; adjusted using glacial acetic acid) at 32 ppm starting 2 weeks before mating (preconception) and continued until E10–12, and euthanized at E13–15 26 , 34 . All animals were maintained on a phytoestrogen-free modified AIN-93 G diet (Td.95092, 7% corn oil diet, Envigo) while housed in polycarbonate-free cages with 12-hour light and dark cycles 25 , 26 , 34 . Study dams were time-mated by assembling randomly selected 3-month-old males in breeding cages for approximately 48 hours (spanning three days and two nights), two weeks after the exposure initiation. Pair breeding using one male and female per breeding cage was conducted to minimize stress, overcrowding, and other variables. All sires were physically removed from the breeding cages to ensure proper timing of pregnancy. Pb-exposed animals were switched to control water at E10–12 until euthanasia. Study animals were euthanized via CO 2 asphyxiation followed by double pneumothorax. All animal procedures were approved by the University of Michigan Institutional Animal Care and Use Committee (IACUC). Cardiac puncture was used to collect whole blood from control and Pb-exposed females immediately after euthanasia. Maternal blood Pb levels were measured on an inductively coupled plasma mass spectrometer (ICP-MS) through the Michigan Department of Health and Human Services ( n =2–3 per group; limit of detection <1.0 μg/dL). The start of animal exposure, mating, removal of sire, switching to control water bottles, and animal euthanasia occurred during the hours of 09:00 to 14:00. 2.2. Tissue collection, nucleic acid extraction, and sex determination The mouse uterus was dissected and placed in a 100mm x 15mm petri dish (Fisher Scientific, #FB0875712) and submerged in 1X PBS, pH 7.4 (Gibco, #10-010-023) on ice. Visual observations including litter size, embryonic viability (determined based on morphological embryonic growth for the given stage), and the number of viable embryos per litter were recorded 35 . Each embryonic sac and whole embryo were carefully dissected and underwent 1– 2 washes with cold 1X PBS to remove excess blood. The whole embryo tissues were gently blotted on lint-free wipes before recording their weights on a digital weighing scale (Fisher Science Education, #SLF103). All tissues including the embryonic sac and head were collected, labeled according to the embryonic position in the uterus, snap frozen in liquid nitrogen, and placed at –80°C for long-term storage. DNA was extracted from frozen embryonic sac tissues using the QIAamp DNA Blood Mini Kit (Qiagen, #51106) according to the manufacturer’s protocol. DNA isolated from embryonic sacs for each exposure group (total n =147) was used to determine the sex of E13–15 embryos using an established protocol via PCR 36 . A subset of control and Pb-exposed embryonic head samples representing 1 female and 1 male from each litter (using n ≥9 per sex per group) were used for RNA extraction using the AllPrep DNA/RNA Mini Kit (Qiagen, #80204) supplemented with the RNase-free DNase Set (Qiagen, #79254), according to the manufacturer’s protocol. The nucleic acid concentration and quality was assessed using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, #ND-2000). A total of n =38 embryonic heads consisting of n =18 control (9 females and 9 males) and n =20 Pb-exposed (10 females and 10 males) samples were selected for RNA-seq experiments. 2.3. Whole embryo weight analysis Data for a total of n =147 samples were recorded for exposure and sex-specific assessment. The Pearson’s chi-square test was conducted to determine statistical significance between number of viable embryos by exposure group and sex. Whole embryo weight data for n =146 representing a total n =71 for the control group ( n =39 females and n =32 males) and a total n =75 for the Pb-exposed group ( n =36 females and n =40 males) was recorded at animal euthanasia and used for embryo weight assessment by exposure group and sex. Weight data for one control male embryo was excluded from analysis as the data was not recorded during tissue collection. Linear mixed-effects models were used assess the effect of Pb exposure on embryonic weight by accounting for sex as a fixed effect and litter as a random effect. The analysis was performed in R statistical software (v2025.5.1.513) using the lmer function per the lmertest package, where test statistic was used to calculate statistical significance, and which is built on the lme4 package 37 , 38 . Models were also stratified by sex, accounting for litter as a random effect. Trends were visualized using ggplot2 in RStudio. Results of p <0.05 were considered statistically significant. 2.4. RNA quantification, library preparation, and RNA-seq RNA quantification and library preparation were conducted using previously established protocols, and RNA-seq was executed at the University of Michigan (UM) Advanced Genomics Core 24 , 39 . Briefly, the Quant-IT RiboGreen RNA Assay kit (ThermoFisher, #R11490) was used to quantify the RNA concentrations. A plexWell (SeqWell) plate-based approach was used for cDNA preparation, quality control, and library preparation. cDNA was prepared using the seq well cDNA module (SeqWell, #301035), purified using MAGwise Paramagnetic beads (SeqWell, #MG10000), and cDNA concentrations were quantified using Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher Scientific, #P11496). The resulting fluorescence was measured by the SpectraMax M5e using a pre-set protocol for PicoGreen. The plexWell LP384 Library Preparation Kit (SeqWell, #LP684X) was used to barcode cDNA libraries using approximately 10ng of cDNA input per sample. Barcoded samples were pooled, bead purified and quantified on the Agilent Bioanalyzer (High Sensitivity DNA 5000 Kit). Randomly pooled libraries were sequenced on the Illumina NovaSeq 6000 using 151bp paired-end reads at a sequencing depth of >30 million reads per sample. 2.5. Differentially expressed genes based on exposure group To process the RNA-sequencing data, reads (FASTQ files) were demultiplexed back into individual samples based on individual barcodes. The sequence quality was subsequently determined using FastQC (v 0.12.1) 40 . Reads were aligned and counted using Salmon (v1.9.0), relative to the Gencode M35 mouse reference genome 41 . Read count matrices were loaded into R via edgeR (v4.6.3) 42 . All samples had library sizes >10 million mapped reads for downstream analyses. Lowly expressed genes were filtered by using filterByExpr , normalized by using calcNormFactors , and the sample dispersion was estimated by using estimateDisp (default settings were utilized for each function). Differentially expressed genes by exposure group (comparison between Pb-exposed embryonic head relative to control) were identified in all mice and then stratified by sex. Each gene comparison was calculated using the quasi-likelihood F-test (QLFT) negative binomial generalized linear model (GLM). Genes significant at a false discovery rate (FDR)-adjusted p -values (FDR<0.05) were considered significant differentially expressed genes. The data distribution by exposure group ( n =18 for control and n =20 for Pb) and subsequent sex stratification ( n =9 per sex for control and n =10 per sex for Pb) were visualized by multidimensional scaling plots using the plotMDS function 43 . Differentially expressed genes by exposure group for all tested embryonic heads and for sex-specific models were visualized using volcano plots (supplemented by ggrepel). Differentially expressed genes from the combined sex, and sex-specific model comparisons were used to generate a Venn diagram of differentially expressed gene overlap between sexes. The LogFC by exposure group between females and males were calculated using the Pearson’s correlation test for all genes. Data visualization was conducted using ggplot2 . 2.6. Gene set enrichment analysis based on exposure group Gene Set Enrichment Analysis was performed using LRpath on Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in both sex-combined, and sex-stratified models 44 . Enriched GO categories including biological process (GOBP), cellular components (GOCC), and molecular functions (GOMF), as well as KEGG pathways, were identified. A stringent threshold for LRpath analysis (FDR<0.001) was used to select the most relevant pathways associated with Pb exposure. Terms containing more than 1000 genes were excluded. The ten most statistically significant GO terms and KEGG pathways from each of the combined sex and sex-stratified models were selected for visualization. 2.7. Differentially expressed imprinted genes, X linked genes, and associated pathways based on exposure group, stratified by sex The differentially expressed genes by exposure group using the combined sex model and sex-stratified models were further filtered by a list of known 300 gametic and non-canonical imprinted genes including those from the geneimprint database 25 . An X-linked gene list was generated using the UCSC Genome Browser mouse assembly (GRCm39/mm39; Gencode VM35) for the X chromosome (chrX: 1–169,476,592). All differentially expressed imprinted genes and differentially expressed X-linked genes upon Pb exposure were confirmed by using the log 2 counts per million changes calculated using the cpm function of normalized data. The resulting imprinted genes and X-linked genes from the combined sex, F, and male data comparisons were used to generate a Venn diagram of overlapping genes between sexes. A comparison of the LogFC by exposure group between females and males were calculated using the Pearson’s correlation test. A heatmap of differentially expressed imprinted genes within significantly enriched GOBP terms or KEGG pathways was generated using ComplexHeatmap (v2.24.1) 45 . Gene expression was normalized with the cpm function in edgeR (v4.6.3). For genes present in multiple enriched categories, annotation was assigned to the category containing the largest number of differentially expressed imprinted or X-linked genes. 2.8. Differentially expressed genes and associated pathways based on sex, stratified by exposure group to identify sexually dimorphic autosomal genes Sexually dimorphic differentially expressed genes were assessed by sex using a comparison between male embryonic heads relative to F, exclusively for the control group. Data distribution by sex for each exposure group ( n =9 females and 9 males for control; n=10 females and 10 males for Pb) was visualized using the MDS plots, after filtering and normalizing for low read counts using edgeR 42 . Each sex-based comparison was calculated using the QLFT negative binomial GLM. The sexually dimorphic genes were compared against the differentially expressed genes identified based on the exposure for the combined model, stratified by sex to generate the Venn diagrams. Over-representation analyses of sexually dimorphic Pb-sensitive differentially expressed genes were conducted using the enrichGO and enrichKEGG functions in clusterProfiler (v4.16.0) 46 . Enrichment was determined at FDR<0.05. Fisher’s exact tests were applied to evaluate overlaps between sexually dimorphic and Pb-sensitive differentially expressed genes using the fisher.test function in stats (v4.5.1). 3. RESULTS 3.1. Pb exposure during preconception and gestation increases embryonic weight Adult mice were exposed to either control or Pb in drinking water at 32 ppm two weeks before mating until E10–12 and euthanized at E13–15 at mid-gestation. The average maternal blood Pb level of 9.7 μg/dL was detected for the Pb-exposed group, while the control group was below the limit of detection. This is a human-relevant exposure level and was measured after 4 weeks of exposure to experimental water and food provided ad libitum 47 . The control group produced 7.89 viable embryos per litter from 9 successful pregnancies at 89.87% viability, while 7.60 viable embryos per litter were produced from 10 successful pregnancies at 90.48% viability for the Pb group ( Table 1 and Supplementary Figure 1). No significant differences were observed in litter sizes and the number of viable embryos between exposure group ( Supplementary Figure 1). The sex distribution did not significantly differ between the control (39 females and 32 males) and the Pb-exposed groups (36 females and 40 males) (χ² (1, N=147) = 0.56, p =0.45). View this table: View inline View popup Download powerpoint Table 1. Summary of Litter Sizes and Embryo Viability. Whole embryo weights at E13–15 were recorded to evaluate embryonic weight differences by exposure group and sex. Weight records for each embryo by litter, exposure group, and sex were analyzed for n =146. The raw data show a trend toward higher absolute embryonic weights 0.11–0.42g for the Pb-exposed group relative to the control group 0.08–0.29g ( Supplementary Figure S2 ). This trend was observed in both sexes ( Supplementary Figure S3 ) for each exposure group (control F: 0.08–0.26g and M: 0.08–0.29g; Pb-exposed F: 0.11–0.42g and M: 0.12–0.41g).The linear mixed-effects model for exposure group ( Figure 2A ), controlling for litter effects showed that Pb exposure had a significant effect on embryonic weight, relative to control ( p =0.0068). Pb-exposed whole embryos (average weight at 0.24g) had significantly higher total weights compared to control (average weight at 0.16g), independent of sex and litter. A separate linear mixed-effects model was used to analyze the Pb exposure effects on embryonic weights stratified by sex, while controlling for litter effects ( Figure 2B ). The Pb group displayed a significantly higher ( p =0.00793) embryonic weights for females (average weight at 0.25g), relative to the control females (average weight at 0.15g). Similarly, the Pb-exposed male embryonic weights (average weight at 0.24g) were significantly higher ( p =0.00746), relative to control males (average weight at 0.17g). Collectively, these results suggest that the Pb-exposed whole embryos weigh significantly more than controls, independent of sex and litter, upon preconception and gestational exposure to Pb. Download figure Open in new tab Figure 1. Experimental design for the perinatal Pb exposure study. C57BL/6J adult females at 10–12 weeks of age were subjected to either control (yellow) or Pb-spiked water at 32 ppm (dark blue) starting 2 weeks before mating, continued until embryonic day (E)13–15. All animals were time-mated with their male counterparts for two nights at two weeks after the start of exposure. Pb-exposed animals were switched to control water at E10–12 prior to euthanasia. The whole embryo tissues were collected at E13–15 and weighted prior to embryonic head collection. DNA was extracted from the embryonic sac for sex genotyping. A female (F) and male (M) embryo head RNA were extracted from each litter (n=9 for control and n=10 for the Pb-exposed group), to account for litter effects. RNA was converted to cDNA and libraries were prepared for sequencing, which was subsequently used for gene expression data analysis using edgeR and gene set enrichment assessment using LRpath-based methods. DEGs: differentially expressed genes; IG: imprinted gene. Download figure Open in new tab Figure 2. Whole embryo weight assessment by exposure group and sex. The data represent n =146 total embryo weights representing all animals used for the study; n =1 whole embryo weight (control male) was excluded from analysis. Statistical significance is shown as p <0.01 (**). ( A ) Linear mixed effects model for the whole embryo weight by exposure group. The box plot represents the combined sex model indicating control (yellow) and Pb (dark blue) exposure groups that were assessed using the exposure group as a fixed effect and litter as a random effect. ( B ) Linear mixed effects model for the whole embryo weight by exposure group and sex. The box plot represents the model accounting for exposure group and sex, indicating females (F; purple) and males (M; grey) for control and Pb exposure groups that were assessed using exposure group and sex as a fixed effect, and litter as a random effect. 3.2. Perinatal Pb exposure leads to differential neuronal gene expression in the embryonic head Bulk RNA-seq was conducted to determine the overall gene expression changes between control and Pb-exposed groups, using 1 female and 1 male representative embryonic heads per litter and selected at random (control: n =9 females and 9 males; Pb: n =10 females and 10 males). The final library sizes after filtering for lowly expressed genes and normalizing ranged between 14,032,468–39,354,980, with 22,432,193 average reads per sample ( Supplementary Table S1 ), indicating consistent sequencing depth between biological replicates within each exposure and sex group. The MDS plots indicate that sample clustering for the overall transcriptomic data is driven mainly by exposure group rather than by sex ( Supplementary Figure S4 ). A total of 26,937 genes were detected, with 15,372 genes remaining after filtering and normalization ( Table 2 ). The magnitude of gene expression changes by exposure for the combined sex and sex-stratified models are shown with volcano plots in Figures 3 – 5 and Supplementary Figure S5. Download figure Open in new tab Figure 3. Differentially expressed genes based on exposure group for the combined sex model. The combined model shows results related to n =38 RNA-seq samples, including n =18 for control and n =20 for the Pb exposure groups. ( A ) The volcano plot represents upregulated (teal; LogFC>0; FDR<0.05) and downregulated (red; LogFC<0; FDR<0.05) differentially expressed genes by Pb exposure relative to control. The dotted lines indicate 0.5 LogFC, while non-significant (NS) genes are shown in grey. ( B ) The top 5 significantly upregulated differentially expressed genes (based on FDR) by exposure group. Gene expression of Gsdme , Ube2w , Alg11 , Rpgrip1 , and St13 is represented by boxplots, based on control (yellow) and Pb (dark blue) exposure groups. ( C ) The top 5 significantly downregulated differentially expressed genes (based on FDR) by exposure group. Gene expression of Supt16 , Ptpn13 , Krt81 , Gm11703 , and Fam193b is represented by boxplots, based on control (yellow) and Pb (dark blue) exposure groups. DEGs: differentially expressed genes. Download figure Open in new tab Figure 4. Differentially expressed genes based on exposure group for the female model. The female model shows results related to n =19 RNA-seq samples, including n =9 for control and n =10 for the Pb exposure groups. ( A ) The volcano plot represents upregulated (teal; LogFC>0; FDR<0.05) and downregulated (red; LogFC<0; FDR<0.05) differentially expressed genes in Pb-exposed females, relative to control females. The dotted lines indicate 0.5 LogFC, while non-significant (NS) genes are shown in grey. ( B ) The top 5 significantly upregulated differentially expressed genes (based on FDR) by exposure group for females. Gene expression of Gsdme , Cep15 , Ier3ip1 , Smim10l1 , and Scn2b is represented by boxplots, based on control (yellow) and Pb (dark blue) exposure groups. ( C ) The top 5 significantly downregulated differentially expressed genes (based on FDR) by exposure group for females. Gene expression of Lrwd1 , Wnt4 , Trpc2 , Dlec1 , and Evx1 is represented by boxplots, based on control (yellow) and Pb (dark blue) exposure groups. DEGs: differentially expressed genes. Download figure Open in new tab Figure 5. Differentially expressed genes based on exposure group for the male model. The male model shows results related to n =19 RNA-seq samples, including n =9 for control and n =10 for the Pb exposure groups. ( A ) The volcano plot represents upregulated (teal; LogFC>0; FDR<0.05) and downregulated (red; LogFC<0; FDR<0.05) differentially expressed genes in Pb-exposed males, relative to control males. The dotted lines indicate 0.5 LogFC, while non-significant (NS) genes are shown in grey. ( B ) The top 5 significantly upregulated differentially expressed genes (based on FDR) by exposure group for males. Gene expression of Gh , Mobp , Mbp , Plp1 , and Ntsr2 is represented by boxplots, based on control (yellow) and Pb (dark blue) exposure groups. ( C ) The top 5 significantly downregulated differentially expressed genes (based on FDR) by exposure group for males. Gene expression of Krt6b , Krt81 , Gm11703 , Krt83 , and Krt6a is represented by boxplots, based on control (yellow) and Pb (dark blue) exposure groups. DEGs: differentially expressed genes. View this table: View inline View popup Download powerpoint Table 2. Number of Differentially Expressed Genes Based on Exposure and Sex for the Embryonic Head at E13–15. For the combined sex model, 17.3% (2,662) of total sequenced genes were differentially expressed genes upon exposure to Pb, relative to the control group ( Table 2 and Supplementary Table S2 ). Approximately 58.3% (1,551) and 41.7% (1,111) of the combined sex differentially expressed genes were significantly increased and decreased upon Pb exposure, respectively ( Figure 3A ). For Pb exposure in the combined model, the most significantly upregulated differentially expressed genes included Gsdme , Ube2w , Alg11 , Rpgrip1 , and St13 ( Figure 3B ), while the most downregulated differentially expressed genes contained Supt16 , Ptpn13 , Krt81 , Gm11703 , and Fam193b ( Figure 3C ). Sexually dimorphic effects were shown in males for Krt81 and Gm11703 ( Supplementary Figure 5A). The sex-stratified assessment for the female model indicated that 4.4% (668) of total sequenced genes were dysregulated upon Pb exposure ( Table 2 and Supplementary Table S3 ). Approximately 66.8% (446) and 33.2% (222) of the differentially expressed genes for the female model were significantly increased and decreased upon Pb exposure, respectively ( Figure 4A ). The most significantly upregulated differentially expressed genes for the female model included Gsdme , Cep15 , Ier3ip1 , Smim10l1 , and Scn2b ( Figure 4B ), while the most downregulated differentially expressed genes included Lrwd1 , Wnt4 , Trpc2 , Dlec1 , and Evx1 ( Figure 4C ) by Pb exposure, with sex-specific gene expression comparisons to the combined sex model ( Supplementary Figure 5B). For males, 4.61% (709) of total captured genes were dysregulated upon Pb exposure ( Table 2 and Supplementary Table S4 ). Approximately 43.2% (306) and 56.8% (403) of the differentially expressed genes among males were significantly increased and decreased upon Pb exposure, respectively ( Figure 5A ). The most significantly upregulated differentially expressed genes among males included Gh , Mobp , Mbp , Plp1 , and Ntsr2 ( Figure 5B ), while the most downregulated differentially expressed genes included Krt6b , Krt81 , Gm11703 , Krt83 , and Krt6a ( Figure 5C ), showing sexually dimorphic effects of Pb exposure ( Supplementary Figure 5C). Taken together, 3.5% (103) of total differentially expressed genes were found common between all three tested models, with 3.8% (112) and 4.9% (144) found exclusively in the female and male models, respectively ( Supplementary Figure S6 ). A statistically significant positive correlation (r=0.52, p <0.001) was observed between the LogFCs between the two sexes ( Supplementary Figure S7 ). These results indicate that Pb exposure significantly dysregulates gene expression with some effects in common and some sex-specific effects. 3.3. Pb exposure dysregulates neurodevelopmental pathways To assess the biological pathways and disease mechanisms impacted by gene expression changes upon exposure and sex during preconception and gestational Pb exposure, Gene Ontology and KEGG enrichment analyses were conducted using LRpath ( Figures 6 and 7 , Supplementary Tables S5–S10 ). Comparison of the combined sex and sex-stratified models showed that GOBP (total 249), GOCC (total 91), GOMF (total 87), and KEGG (total 74) pathways were significantly impacted by gestational exposure to Pb ( Figure 6 ). For GOBP, 23.7% (59) of common biological pathways were enriched between all three tested models, while 16.5% (41) and 18.9% (47) were enriched exclusively in the female and male models, respectively. The most significantly dysregulated GOBP pathways upon Pb exposure included synaptic signaling, embryonic morphogenesis, epigenetic regulation of gene expression, among others ( Supplementary Tables S5 , S7 , and S9 ). Pb exposure significantly downregulated the GOBP DNA metabolic process in the female model, while DNA damage response, sexual reproduction, and keratinization processes were significantly downregulated in the male model ( Figure 7 ). Download figure Open in new tab Figure 6. Overview of the most significant gene ontology and KEGG pathways disrupted by gestational Pb exposure. The Venn diagrams represent data related to the combined sex model (blue), female model (purple), and male model (grey) for the most significant pathways impacted by Pb exposure (FDR<0.001). ( A ) Gene Ontology Biological processes (GOBP) impacted by exposure and sex. ( B ) Gene Ontology Cellular components (GOCC) impacted by exposure and sex. ( C ) Gene Ontology Molecular functions (GOMF) impacted by exposure and sex. ( D ) KEGG pathways impacted by exposure and sex. Collectively, 249 GOBP, 91 GOCC, 87 GOMF, and 74 KEGG pathways are dysregulated by gestational Pb exposure in the embryonic head, based on the three models assessed. Download figure Open in new tab Figure 7. The most enriched biological processes and KEGG pathway terms associated with gestational Pb exposure. Results are shown for combined sex, female, and male models (top subheadings) with gene ontology biological process (GOBP; diamond) and Kyoto Encyclopedia of Genes and Genomes (KEGG; circle). The model-specific GOBP and KEGG comparisons are listed in subheadings on the right. Pathway enrichment direction is indicated by teal (upregulated) and red (downregulated). Point size reflects the number of genes contributing to enrichment. For GOCC, 31.9% (29) of common cellular components were enriched between all three tested models, while 14.3% (13) and 18.7% (17) were enriched exclusively in the female and male models, respectively. The most significantly dysregulated GOCC upon Pb exposure included chromatin, presynapse, and transmembrane transporter complex, among others ( Supplementary Tables S5 , S7 , and S9 ). For GOMF, 23.0% (20) of common molecular functions were enriched between all three tested models, while 11.5% (10) and 20.7% (18) were enriched exclusively in the female and male models, respectively. The most significantly dysregulated GOMF upon Pb exposure were chromatin binding, cis-regulatory region seq-specific DNA binding, neurotransmitter receptor activity, and modification-dependent protein binding, among others ( Supplementary Tables S5 , S7 , and S9 ). For KEGG, 32.4% (24) of common disease pathways were enriched between all three tested models, while 12.2% (9) and 24.3% (4) were enriched exclusively in the female and male models, respectively. The most significantly dysregulated KEGG pathways upon Pb exposure included oxidative phosphorylation, neuroactive ligand-receptor interaction, Parkinson’s disease, and diabetic cardiomyopathy, among others ( Supplementary Tables S6 , S8 , and S10 ). The KEGG glutamatergic synapse was significantly upregulated upon Pb exposure in the female model ( Figure 7 ). Similarly, the Polycomb repressive complex (PRC) and the tricarboxylic acid (TCA) cycle were significantly down and upregulated, respectively, upon Pb exposure in the male model ( Figure 7 ). Collectively, these findings reveal sex-specific dysregulation of neuronal and metabolic pathways in the developing brain following preconception and gestational exposure to Pb that contribute to disease risk. 3.4. Pb exposure dysregulates imprinted genes in a sex-specific manner Given the role of imprinted genes in fetal growth and regulation, a list of 300 canonical and non-canonical imprinted genes were used to filter differentially expressed genes by exposure for the combined sex and sex-stratified models ( Supplementary Tables S11–S13 ). A total of 161 imprinted genes were captured after filtering ( Table 3 ). The magnitude of imprinted gene expression changes by exposure for the combined sex and sex-stratified models are shown with volcano plots in Supplementary Figures S8–S10 . View this table: View inline View popup Download powerpoint Table 3. Number of Differentially Expressed Imprinted Genes Based on Exposure and Sex for the Embryonic Head at E13–15. For the combined sex model, 23 (14.3%) of total captured imprinted genes were differentially expressed upon Pb exposure relative to the control group ( Table 3 and Supplementary Table S11 ). Approximately 21 (91.3%) and 2 (8.7%) of the combined sex differentially expressed imprinted genes were significantly increased and decreased upon Pb exposure, respectively ( Figure 8 ). The most upregulated differentially expressed imprinted genes for combined sex included Ifitm10 , Rasgrf1 , Qpct , Slc22a2 , and Snrpn , while the most downregulated differentially expressed imprinted genes included Etv6 , and Id1 ( Figure 8B and Supplementary Figure S8 ). Download figure Open in new tab Figure 8. Combined sex, female, and male model-specific differentially expressed imprinted gene comparisons. ( A ) Venn diagram of differentially expressed imprinted gene comparisons between each tested model. The combined model shows 23 differentially expressed imprinted genes related to n =38 RNA-seq samples, including n =18 for control and n =20 for the Pb exposure groups. The female/male models show 9/10 differentially expressed imprinted genes, respectively, related to n =19 RNA-seq samples, including n =9 for control and n =10 for the Pb exposure groups. Collectively, 31 differentially expressed imprinted genes were dysregulated by gestational exposure to Pb in the embryonic head. ( B ) Complete list of differentially expressed imprinted genes is recorded based on directionality of gene expression changes. Upregulated (teal; LogFC>0; FDR<0.05) and downregulated (red; LogFC<0; FDR<0.05) differentially expressed imprinted genes upon Pb exposure relative to control are indicated in respective boxes, comparing the three tested models. IGs: imprinted genes. Among females, 9 (5.6%) of total captured imprinted genes were dysregulated upon exposure to Pb, with 100% of the differentially expressed imprinted genes significantly upregulated upon Pb exposure ( Table 3 and Supplementary Table S12 ). The most significantly differentially expressed imprinted genes by Pb exposure for the female model included Ifitm10 , Htra3, Rasgrf1 , Nap1l5 , and Qpct ( Figure 8B and Supplementary Figure S9 ). Among males, 10 (6.2%) of total captured imprinted genes were dysregulated upon Pb exposure ( Table 3 and Supplementary Table S13 ). Three of these imprinted genes were upregulated and seven were downregulated with Pb exposure ( Figure 8B ). The most significantly upregulated differentially expressed imprinted genes for the male model included Xist and Rasgrf1, while the most downregulated differentially expressed imprinted genes contained Ube3a , Meg3 , and Slc38a1 ( Supplementary Table S13 ), with Ube3a showing pronounced sexually dimorphic effects from Pb exposure ( Supplementary Figure S10 ). In summary, 2 (6.5%) of the total differentially expressed imprinted genes were common between all three tested models, and 8 (25.8%) were found exclusively among males ( Figure 8A ). A statistically significant moderate positive correlation (r=0.40, p <0.001) was observed between the LogFCs of female vs. male models, indicating a tendency for concordance in differentially expressed imprinted gene expression between sexes ( Supplementary Figure S11 ). These results indicate that Pb exposure significantly dysregulates imprinted genes, with some observed sex-specific effects. The overall differentially expressed imprinted genes by exposure group and sex are enriched in GOBP and KEGG pathways ( Figure 9 ) responsible for metabolism and biosynthetic processes (amide and organophosphate metabolic processes), gene regulation (negative regulation of transcription), cell division, transport and localization (import into cell and localization within membrane), phagosome, sexual reproduction, and nervous system/diseases (synaptic signaling and behavior). Download figure Open in new tab Figure 9. Heatmap of differentially expressed imprinted gene expression within enriched GOBP and KEGG pathways in combined sex and sex-stratified models of Pb exposure. Pathways are indicated by the colored left annotation bar, while exposure (Control: yellow; Pb: dark blue) and sex (Female: F; Male: M) are shown in the top annotation bar. Gene expression levels are represented as z-score scaled log counts per million (logCPM), with gene names displayed on the right. 3.5. Pb exposure dysregulates X-linked genes in a sex-specific manner Given the observed sex-stratified impact on Pb toxicity ( Supplementary Figure S5 ), a list of 1,529 X-linked genes was used to filter differentially expressed genes by exposure for the combined sex and sex-stratified models ( Supplementary Tables S14–S16 ). A total of 535 X-linked genes were captured after filtering ( Table 4 ). The magnitude of X-linked gene expression changes by exposure for the combined sex and sex-stratified models are shown using volcano plots ( Supplementary Figures S12–S14 ). View this table: View inline View popup Download powerpoint Table 4. Number of Differentially Expressed X-linked Genes Based on Exposure and Sex for the Embryonic Head at E13–15. For the combined sex model, 107 (20.0%) of total sequenced X-linked genes were differentially expressed upon Pb exposure relative to the control group ( Table 4 and Supplementary Table S14 ). Approximately 79 (73.8%) and 28 (26.1%) of the combined sex differentially expressed X-linked genes were significantly increased and decreased upon Pb exposure, respectively ( Figure 10 ). The most significantly upregulated differentially expressed X-linked genes for combined sex included Tspan7 , Dynlt3 , Gabra3 , Tceal1 , and Nxt2 , while the most downregulated differentially expressed X-linked genes included Med12 , Bcor , Brwd3 , Huwe1 , and Hcfc1 ( Figure 10B and Supplementary Figure S12 ). The sex stratified assessment for females indicated that 27 (5.1%) of total sequenced X-linked genes were dysregulated upon Pb exposure ( Table 4 and Supplementary Table S15 ). Approximately 22 (81.9%) and 5 (18.5%) of the differentially expressed X-linked genes for the female model were significantly increased and decreased upon Pb exposure, respectively ( Figure 10 ). The most upregulated differentially expressed X-linked genes for the female model included Vma21 , Nxt2 , Klhl13 , Slitrk4 , and Slitrk2 , while the most downregulated differentially expressed X-linked genes included Dusp9 , Abcd1 , Suv39h1 , Kdm5c , and Stard8 ( Figure 10B and Supplementary Figure S13 ). The sex-stratified assessment for the male model indicated that 24 (4.49%) of total captured X-linked genes were dysregulated upon Pb exposure ( Table 4 and Supplementary Table S16 ). Download figure Open in new tab Figure 10. Combined sex, Female, and Male model-specific differentially expressed X-linked gene comparisons. ( A ) Venn diagram of differentially expressed X-linked gene comparisons between each tested model. The combined model shows 107 differentially expressed X-linked genes related to n =38 RNA-seq samples, including n =18 for control and n =20 for the Pb exposure groups. The female/male models show 27/24 differentially expressed X-linked genes, respectively, related to n =19 RNA-seq samples, including n =9 for control and n =10 for the Pb exposure groups. Collectively, 120 differentially expressed X-linked genes were dysregulated by gestational exposure to Pb in the embryonic head. ( B ) The top differentially expressed X-linked genes were recorded based on directionality of gene expression changes. Upregulated (teal; LogFC>0; FDR<0.05) and downregulated (red; LogFC<0; FDR<0.05) differentially expressed X-linked genes upon Pb exposure relative to control are indicated in respective boxes, comparing the three tested models. Approximately 10 (41.7%) and 14 (58.3%) of the differentially expressed X-linked genes for the male model were significantly increased and decreased upon Pb exposure, respectively ( Figure 10B ). The most upregulated differentially expressed X-linked genes for the male model included Plp1 , Xist , Dynlt3 , Tspan7 , and Armcx6 , while the most downregulated differentially expressed X-linked genes included Huwe1 , Med12 , Aff2 , Ddx3x , and Atrx ( Figure 10B ), with Plp1 showing pronounced sexually dimorphic effects of exposure ( Supplementary Figure S14 ). Collectively, 2 (1.7%) of total X-linked genes were common between all three tested models, with 5 (4.2%) and 8 (6.7%) were found exclusively in the female and male models, respectively ( Figure 10A ). A modest but statistically significant positive correlation (r=0.43, p <0.001) was observed between the LogFCs of female vs. male models, indicating a tendency for concordance in differentially expressed X-linked gene expression between sexes ( Supplementary Figure S15 ). These results suggest that Pb exposure significantly dysregulate X-linked genes by exposure group and sex. The total differentially expressed X-linked genes by exposure group and sex are enriched in GOBP and KEGG pathways, with the heatmap revealing a clear separation by exposure ( Figure 11 ). The observed enriched pathways are responsible for metabolism and biosynthetic processes (carbohydrate derivative and organophosphate metabolic processes), gene regulation (negative regulation of transcription by RNA polymerase II and RNA processing), transport and localization (import into cell, inorganic ion transmembrane transport, and membrane organization), development and morphogenesis (chordate embryonic and head development), DNA damage response, and nervous system/diseases (Alzheimer’s disease and synapse organization). Download figure Open in new tab Figure 11. Heatmap of differentially expressed X-linked gene expression within enriched GOBP and KEGG pathways in combined sex and sex-stratified models of Pb exposure. Pathways are indicated by the colored left annotation bar, while exposure (Control: yellow; Pb: dark blue) and sex (Female: F; Male: M) are shown in the top annotation bar. Gene expression levels are represented as z-score scaled log counts per million (logCPM), with gene names displayed on the right. Only GOBP and KEGG pathways containing at least two differentially expressed X-linked genes were included. 3.6. Sexually dimorphic autosomal genes are particularly susceptible to gestational Pb exposure in the embryonic head at E13–15 To specifically assess the sexually dimorphic genes within the RNA-seq dataset, differentially expressed genes were identified based on sex exclusively in the control group for n =9 females and 9 males. While the control samples cluster distinctly by sex, this pattern disappeared after Pb exposure, as observed by the MDS plots ( Supplementary Figure S16 ). For the control sex-specific model, 3.3% (498) of total captured genes were differentially expressed genes in control males relative to control females ( Supplementary Table S17 ). Approximately 62.7% (312) and 37.4% (186) of the genes were significantly upregulated and downregulated in control males relative to control females, respectively. The differentially expressed genes identified by sex for the control group indicate sexually dimorphic genes for the embryonic head at E13–15. The sexually dimorphic autosomal genes were significantly enriched and sensitive to gestational Pb exposure for each sex ( p =8.8×10 −6 for females and p <2.2×10 −16 for males; Table 5 ). Our data thus indicates that sexually dimorphic genes are more likely than expected by chance to be transcriptionally responsive to Pb exposure in both sexes. For females, 8.8% (44) of sexually dimorphic genes were significantly dysregulated upon Pb exposure ( Figure 12A ). The most significantly enriched pathways of sexually dimorphic genes dysregulated by Pb exposure for females ( Figure 12B ) include GOCC (CHD-type complex, NuRX complex, and the chromosome, centromeric core domain) and GOMF (GTPase and kinase binding). For males, 15.3% (76) of sexually dimorphic genes were significantly dysregulated upon Pb exposure ( Figure 12C ). The most significantly enriched GOBP pathways of sexually dimorphic genes dysregulated by Pb exposure for males included myelination and axon ensheathment, glial cell development, and cytoskeletal filament organization ( Figure 12D ), while the most significantly enriched KEGG pathways included neurotransmitter and synaptic signaling (amphetamine addiction and cholinergic, dopaminergic, and glutamatergic synapses), Ras signaling pathway, diabetic cardiomyopathy, cellular structure and barrier function (cornfield envelope formation). Download figure Open in new tab Figure 12. Summary of potential sexually dimorphic genes sensitive to gestational Pb exposure. A total of 498 genes is differentially expressed based on sex at E13–15 in the embryo head data analysis of the control group alone ( n =9 per sex). According to data presented in the combined model, 668 and 709 genes are differentially expressed based on gestational exposure to Pb at E13–15 in the embryo head in females and males, respectively ( n =9 per sex for control; n =10 per sex for Pb). Data interpretation is based on sex (yellow) and exposure (blue) comparisons for each sex. ( A ) Differentially expressed gene overlap between sex vs. exposure-specific assessment, focusing on females. ( B ) Summary of biological processes significantly dysregulated by Pb exposure for the 44 sexually dimorphic genes, representing the female data. ( C ) Differentially expressed gene overlap between sex vs. exposure-specific assessment, focusing on males. ( D ) Summary of biological processes and KEGG pathways significantly dysregulated by Pb exposure for the 76 sexually dimorphic genes, representing the male data. View this table: View inline View popup Download powerpoint Table 5. Fisher’s Exact Test for Sexually Dimorphic Differentially Expressed Gene Enrichment for the Embryonic Head at E13–15. Overall, these data support that preconception and gestational Pb exposure alter neuronal structure, function, and biological processes particularly related to sexually dimorphic genes that may contribute to neurodegenerative diseases. 4. DISCUSSION 4.1. Key findings Pb exposure during preconception and gestation significantly increased embryonic weight at E13–15 relative to controls, which was independent of sex and litter. Bulk RNA-sequencing revealed substantial Pb-induced gene expression changes in the mid-gestation embryonic head among all mice and in sex-stratified analyses, including 2,920 differentially expressed genes, 31 affected imprinted genes, and 120 affected X-linked genes that collectively dysregulate neuronal and metabolic pathways and diseases. The current study also reports that sexually dimorphic autosomal genes are particularly sensitive to preconception and gestational Pb exposure, with 44 genes significantly changed in females and 76 changed in males. Pb exposure dysregulated molecular pathways of GOBP (gene expression and RNA metabolism, membrane transport, developmental morphogenesis, and stress responses), GOCC (nuclear structure and gene regulation, synaptic structure and function, and membrane transport complexes), and GOMF (chromatin structure and regulation, neuronal signaling, and protein interactions). The current study also highlights KEGG pathways related to neurotransmission processes, metabolism, cell cycle and cardiometabolic diseases, and neurodegenerative disease risk. These findings suggest that Pb exposure perturbs fetal development by disrupting multiple biological pathways, including those governing imprinting, X-linked genes, and sexually dimorphic autosomal genes. The evidence of such widespread dysregulation underscores the urgent need to elucidate the precise epigenetic regulatory mechanisms involved and to assess the lasting sex-specific impacts on offspring health from birth to adulthood. 4.2. The developmental impact of Pb exposure Our results indicated no significant differences between the number of total embryos, viable embryos, litter sizes, or sex distribution upon Pb exposure at 32 ppm during preconception and gestation. The ratio of males to females were close to an expected Mendelian ratio of 50:50. This observation is consistent with a Swiss mouse model of in utero Pb exposure ( n =6 dams per doses of 14, 28, 56 and 112 mg/kg), which reported that the number of implants, fetuses (alive and dead), and sex ratio were unaffected 48 . However, embryogenesis is sensitive to Pb, as high doses of 1000–2000 ppm Pb in rats significantly reduces litter sizes and fetal weights 49 . Our results demonstrate that Pb exposure increases embryonic weight in both sexes ( Table 1 and Figure 2 ). Although this finding is consistent with previous reports of prenatal Pb exposure altering fetal growth parameters, the effects on weight can be heavily dependent on experimental design. For instance, the Swiss mouse study reports no significant differences in fetal body weights at E18, and some epidemiological studies associate maternal blood Pb levels with small-for-gestational-age, low birth weight, and preterm birth 48 , 50 – 53 . As such, these variations in experimental design include differences in the route of exposure, mouse strain, or endpoints measured, as well as the observational nature and inherent limitations of human cohort analyses. Given that our study used a 48-hour, two-night window for mating, it is possible that the observed embryo weight differences arose from Pb-exposed animals mating consistently on the first night and control animals mating consistently on the second night. The likelihood of this scenario is extremely low, as the disparity in embryo weights was observed across n≥9 separate litters per exposure group ( Supplementary Figure 2). The observed increase in embryonic weight may reflect a compensatory dysregulation in growth or metabolism, aligning with reports that early toxicant exposures trigger adaptive, and potentially maladaptive, responses in the developing embryo 54 . For a more comprehensive understanding of Pb-induced toxicity and its impact during early development, investigation of maternal-fetal crosstalk is warranted, with a particular focus on the dynamic communication between the maternal brain, the placenta, and the developing embryo 55 . 4.3. Pb exposure dysregulates neuronal gene expression and biological pathways Pb exposure induced widespread exposure and sex-specific transcriptomic changes in the embryonic head as detected from RNA-seq analysis of all mice and stratified by sex ( Figures 3 – 5 ), collectively showing 2,920 differentially expressed genes ( Table 2 and Supplementary Figure S6 ). The most significantly dysregulated differentially expressed genes by exposure group ( Figure 3 ) were not sex dependent. Among these, Gsdme is a tumor suppressor gene involved in pyroptosis and inflammatory responses, and its upregulation suggests a generalized stress or cell death pathways activated by Pb in the developing brain 56 . Conversely, Supt16 , a chromatin remodeler, was the most significantly downregulated differentially expressed gene observed, suggesting broad repressive effects on transcriptional regulation and neurodevelopment associated with Pb exposure 57 , 58 . The biological processes associated with Pb exposure ( Figure 7 ) suggest a shift towards neuronal synaptic activity and reduced gene regulatory plasticity during development. For instance, the upregulation of synaptic signaling and vesicle-mediated transport, neurotransmitter transport, and ion transmembrane transport points to an increased demand for efficient synaptic communication, while the downregulation of mRNA metabolic processes, chromatin organization, and embryonic development pathways indicates suppression of neuronal plasticity at a transcriptional and epigenetic level. According to the KEGG results, these transcriptional changes are associated with neurodegenerative and addiction disease processes (including Parkinson’s disease and nicotine addiction). The enriched pathways identified in our study is consistent with known mechanisms of Pb neurotoxicity and disease associations. Pb disrupts presynaptic vesicular release by altering synaptic proteins, inhibits N-methyl-D-aspartate receptor (NMDAR) signaling, and impairs brain-derived neurotrophic factor (BDNF) release, thereby reducing synaptic plasticity and function 59 . There is evidence confirming Pb exposure as a significant risk factor for Parkinson’s disease 60 . Thus, our results linking synaptic hyperactivity and oxidative metabolism upregulation with suppressed gene regulatory systems echo these toxicological insights and highlight a mechanistic basis for Pb-related cognitive decline. In the female model ( Figure 4 ), dysregulated Scn2b (encoding a subunit of the voltage-gated sodium channel), Wnt4 (involved in key developmental signaling pathways and homeostasis in the brain), and Trpc2 (a cation channel linked to sex-specific social behaviors), point to potential impairments in neuronal signaling pathways critical for development and sensory processing 61 – 63 . Collectively, the female-specific gene expression changes are involved in overall downregulation of DNA metabolic processes, contributing to genome instability and cellular dysfunction, while upregulation of glutamatergic synapse-related disease pathways suggest disruption or heightened neuronal excitatory signaling ( Figure 7 ). The most notable trends were observed in the male model ( Figure 5 ), indicating upregulation of myelin-related genes, including Mobp (myelin-associated oligodendrocytic basic protein), Mbp (myelin basic protein), and Plp1 (Proteolipid protein 1; an essential component of myelin). Excessive levels of these proteins underlie several neurodegenerative conditions and psychiatric disorders 64 , 65 . Our findings closely align with a reported single-cell RNA-seq analysis indicating that perinatal Pb exposure is associated with a 12.4% increase in oligodendrocyte abundance, even following a four-month cessation of 32 ppm Pb exposure in mice 66 . Interestingly, these oligodendrocyte markers were also sensitive to gestational nicotine exposure in rats in a sex-specific manner 67 . On the other hand, striking differentially expressed gene patterns of male downregulation were dominated by keratin genes ( Krt6b , Krt81 , Krt83 , Krt6a ), which can serve as stress response proteins, and this downregulation might reduce protection against Pb-induced damage in epithelial tissues 68 . Our pathway analysis for males revealed distinct biological disruptions associated with Pb exposure, reflecting both DNA damage responses and altered metabolic processes ( Figure 7 ). Despite the lack of a sex-specific assessment of Pb-induced DNA damage response, our data is consistent with previous literature showing that Pb impairs DNA repair mechanisms by downregulating key repair genes involved in base excision repair, nucleotide excision repair, and double-strand break repair, thus contributing to mutagenic outcomes 69 . The male-specific KEGG pathway upregulation of the TCA cycle suggests a shift in cellular metabolism, likely due to increased energy demands under Pb-induced oxidative stress, while the downregulation of PRC pathways suggests that Pb-induced epigenetic dysregulation of chromatin remodelers is likely involved in transcriptional repression. Taken together, our results suggest that Pb exposure in females may induce greater disruption in regulatory neuronal signaling and developmental control, while males may undergo more neurostructural and cytoskeletal alterations. Certain gene expression changes (such as upregulated Gh ) could plausibly support enhanced growth and subsequent weight gain in Pb-exposed samples. However, the simultaneous dysregulation of multiple genes essential to development and tissue homeostasis identified in enriched pathways ( Figure 6 ) makes it difficult to pinpoint the basis of Pb neurotoxicity. Future studies integrating longitudinal transcriptional profiles with functional assays of synaptic and oxidative stress will be essential to define causality and identify therapeutic targets for later-life neurological diseases. 4.4. Pb exposure disrupts imprinted genes and X-linked genes Even though imprinted genes and X-linked genes represent a subset of the transcriptome, they play crucial roles for proper embryonic development. Pb-induced exposure and sex-specific differentially expressed genes are associated with 31 imprinted genes ( Table 3 , Figure 8 , and Supplementary Figures S8–S11 ). Imprinted genes cluster at imprinted domains that typically cover 100–1700 kb genomic regions, including at least one lncRNA, differentially methylated regions, and an imprinting control region (ICR) that regulates the entire canonical imprinting domains 8 . Thus, each imprinting cluster is regulated through distinct epigenetic mechanisms such as the lncRNA-mediated silencing (insulin-like growth factor 2 receptor; Airn / Igf2r cluster) and the insulator (H19/ Igf2 cluster) models. ICRs have been shown to be sensitive to Pb exposure during development in human and cell culture models 20 – 22 , 25 . Our results provide strong evidence that imprinted genes are dysregulated following gestational Pb exposure in the developing embryonic brain that include maternally and paternally imprinted genes ( Figure 8 ). The Angelman syndrome and Prader-Willi syndrome imprinted domain cluster is in the same genomic region, where the antisense RNA ( Ube3a -ATS) originates from the paternal Snrpn promoter involved in the imprinting setting of the Ube3a paternal allele in the brain 70 . As indicated by the differential expression of Ube3a (downregulated in males) and Snrpn (upregulated in combined sex) and their associated pathways in sexual reproduction ( Figures 8 and 9 ), Pb-induced transcriptional dysregulation may impact this mechanism, but confirmation of a direct association is needed. Along with de novo DNAm, ncRNA including PIWI-interacting RNA (piRNA) and histone modifications can also contribute to non-canonical imprinting regulation 71 . The upstream genomic regions of the Ras protein-specific guanine nucleotide releasing factor 1 ( Rasgrf1 ) generate piRNA that recruits DNAm at the Rasgrf1 -ICR to maintain its paternal imprinting status 72 . In this study, Rasgrf1 is among the most significantly upregulated imprinted genes following gestational Pb exposure ( Figure 8 ), with its altered expression linked to disrupted synaptic signaling pathways in the embryonic brain ( Figure 9 ). Our previous work demonstrated that piRNA is highly expressed in the hippocampus and that perinatal Pb exposure (32 ppm) alters piRNA expression in the male brain, suggesting that Pb influences piRNA-mediated epigenetic regulation 26 , 73 . Similarly, perinatal Pb exposure at the same dose and time window alters heart-specific piRNA expression in a sex-specific manner, further suggesting that Pb-induced piRNA dysregulation during development may influence cardiovascular disease risk 34 . These findings, together with existing literature, highlight the critical role of Rasgrf1 in hippocampal memory formation and its calcium-dependent activation via NMDAR signaling 74 . The impact of developmental Pb exposure on imprinted genes and the consequences for sex differences in neurotoxicity remain largely unexplored and warrant further investigation. Sex linked genes perform essential roles in sexual differentiation and drive sex-specific brain development even before gonadal formation 75 . In this study, Pb exposure and sex-specific differentially expressed genes were associated with 120 X-linked genes ( Table 4 , Figure 10 , and Supplementary Figures S12–S15 ). Notably, Figure 11 highlights the overwhelming impact of Pb toxicity on these genes and illustrates that the most significantly dysregulated X-linked genes are linked to key biological and disease pathways. The oligodendrocyte marker Plp1 is among the most differentially expressed X-linked gene following Pb exposure, and is associated with inorganic ion transmembrane transport, suggesting its role in neuronal signaling and overall neuronal cellular functions. One of the most enriched KEGG pathways linked in this analysis is Alzheimer’s disease ( Figure 11 ), suggesting that gestational Pb exposure may contribute to early molecular changes associated with neurodegenerative risk and underscoring the long-term implications of X-linked gene dysregulation by Pb. This finding is consistent with some research suggesting that early life Pb exposure is linked to Alzheimer’s disease risk 5 , 76 . X-linked imprinted genes contribute to sex differences in brain function and may increase vulnerability to disease when affected by Pb exposure 23 . Normally, Xist is a prominent X-linked imprinted gene exclusively expressed in females and involved in random XCI; it is often used as a marker for sex genotyping. Our results are consistent with this, showing high Xist expression in females with no significant difference between control and Pb-exposed groups ( Figure 10 and Supplementary Figure S14 ). In contrast, Xist expression in males is nearly absent under control conditions but increases sharply (by a 2.9 log fold change) following Pb exposure ( Supplementary Table S16 ). Further research is needed to determine whether this transcriptional change results from Pb-induced de-repression via altered DNAm or disrupted histone modifications. Given the complex epigenetic mechanisms involved in the process of genomic imprinting, interpretation of our findings on Pb-induced changes in imprinted and X-linked gene expression should be approached with caution and incorporated in future studies, as these results must consider the intricate nature of imprinting clusters, sex-, tissue-, developmental stage-specific regulation, and Pb-induced neurotoxicity. 4.5. Pb exposure elicits sexually dimorphic responses Differential gene expression between the sexes, including sex-linked and autosomal genes, are influenced by the unique content of the sex chromosomes 31 . In the exposure-specific comparison of differentially expressed genes (control vs. Pb-exposed groups), we observed a sex-stratified impact on Pb toxicity ( Supplementary Figure S5 ), suggesting that sexually dimorphic autosomal genes are particularly sensitive to Pb exposure. This observation was further supported by the sex-specific comparison of differentially expressed genes using the same dataset (control females vs. male groups; Supplementary Figure S16 ). In the control group, samples cluster by sex, indicating underlying biological or molecular distinctions between males and females. The suppression of this trend by Pb exposure suggests that it either masks, reduces, or overrides these inherent sex-based differences. The current study reports that sexually dimorphic genes are particularly sensitive to preconception and gestational Pb exposure, males appear to be more susceptible to Pb-induced changes in sexually dimorphic gene expression, with 76 genes significantly altered in males compared to 44 in females ( Figure 12 ). Collectively, our findings reveal that Pb-induced sexually dimorphic gene expression changes are linked to distinct molecular pathways including chromatin remodeling processing in females; changes in males were associated with neural structure and functional processes, as well as those associated with nervous system and cardiovascular diseases ( Figure 12 ). The sexually dimorphic gene responses observed in our study echo reports demonstrating that males and females differ in their transcriptomic and phenotypic susceptibility to Pb 22 , 23 , 30 . Animal studies often indicate greater or unique neurobehavioral, metabolic, or epigenetic impacts in one sex following gestational Pb exposure 29 , 30 , 77 . For example, one study found that gestational Pb exposure is associated to male-specific late-onset obesity and decreased spontaneous motor activity, implicating cholinergic and glutamatergic neurons (cell populations highlighted in male-specific assessment in our study) as being affected by Pb exposure 29 . Another study demonstrates that molecular and neurobehavioral effects of gestational Pb exposure is parent and sex-dependent, further suggesting that Pb-induced sexually dimorphic effects are likely regulated via epigenetic mechanisms 77 . There is extensive research indicating Pb-induced sexually dimorphic effects in cardiovascular disease following gestational exposure 22 , 30 , 78 . Taken together, these insights emphasize the critical need to consider sex as a biological variable in assessing the neurodevelopmental and metabolic risks of early life Pb exposure, as targeted interventions may be necessary to protect the most vulnerable populations. 5. STRENGTHS AND LIMITATIONS The current study is among the first to identify significant association between perinatal Pb exposure effects, including increased embryonic weight at E13–15 and expression changes in sexually dimorphic genes in the embryonic head, specifically at a human exposure-relevant dose. By carefully cataloging the whole embryonic weight phenotypes using linear regression models and subsequent RNA-seq assessment, this study explored genome-wide gene expression differences upon Pb exposure (in the combined sex model) and sex (male and female models) during a critical exposure time window at murine mid-gestation. Findings from this study fill a critical knowledge gap in understanding the association between Pb toxicity and gestational neurodevelopment as it relates to genomic imprinting, sex-specific effects on gene expression in the brain and the associated biological and disease pathways. There are several limitations to the current study. First, while this study was designed using the embryonic head as a proxy to capture gene expression changes in the developing brain, it may also contain other tissues such as the connective tissues, craniofacial skeleton, and mesenchyme and connective tissues among others, that could contribute to the obtained results. Second, although bulk-RNA seq captured genome-wide gene expression, the method is unable to detect cell type-specific changes linked to Pb exposure. Thus, future work will address how these gene expression alterations perturb temporal neurodevelopmental functions. Third, the current study is conducted using a murine model from the C57BL/6J wildtype background, which does not differentiate between the allele-specific gene expression changes. As such, future confirmation is needed for the allelic contribution of gene expression changes linked to Pb toxicity, especially for imprinted gene assessment. Fourth and finally, as the study design draws conclusions of Pb-induced increased embryonic weight and its association to sexually dimorphic gene expression changes and pathways, the establishment of causality is limited. As such, the associations should be interpreted cautiously. Future research is encouraged to confirm the overall epigenetic pathways of Pb-induced toxicity, including the evaluation of DNAm, histone modifications, and ncRNA in the maternal-fetal crosstalk. Similarly, repeated measurements of Pb-associated gene expression changes in the brain using mouse models of disease relevance at multiple timepoints in development would provide a robust causal inference to the disease pathways highlighted in this study. 6. CONCLUSION This study demonstrates that Pb exposure during preconception and gestation, corresponding to a human-relevant maternal blood Pb level average of 9.7 μg/dL, disrupts key developmental processes by broadly changing embryonic gene expression, including imprinted genes, X-linked genes, and neuronal and metabolic pathways. Taken together, these molecular alterations and pathways are linked to the Pb-induced increased embryonic weight phenotype. Furthermore, these molecular disturbances implicate Pb as a potent developmental toxicant, affecting pathways fundamental to brain function, metabolism, and overall embryonic development. The pronounced sensitivity of sexually dimorphic genes to gestational Pb exposure highlights potential sex-specific vulnerabilities and associated long-term health impacts of early life exposure. As such, a deeper investigation into Pb toxicity and its effects on epigenetic programming will lay the foundation for developing targeted interventions to alleviate long-term health consequences of early life Pb exposure. Future research will assess epigenetic mechanisms altered by Pb exposure to inform strategies for alleviating its associated adverse health outcomes instigated during development. Authorship contribution statement BPUP, DCD, and JAC conceived and designed the experiments. BPUP, AT, JP, MAS, DCD, JAC planned the experimental setup, while BPUP, AT, JP, DW, TG, RKM, and JAC carried out various aspects of the experiments. BPUP, ML, AT, JP, DW, and JAC contributed to experimental data collection and curation. BPUP, ML, TG, JMG, and JAC analyzed and interpreted the results. BPUP, KMB, JMG, MAS, DCD, and JAC supervised the experimental work. BPUP, ML, AT, and JAC drafted the original manuscript, with JP, DW, TG, RKM, KMB, JMG, MAS, and DCD contributing to writing and revision of the manuscript. All authors have read and approved the final version of the manuscript draft. Funding This work was supported by the National Institute of Environmental Health Sciences (NIEHS) Transition to Independent Environmental Health Research Career Award to BPUP K01 ES035064, Michigan Lifestage Environmental Exposures and Disease (M-LEEaD) NIEHS Core Center P30 ES017885, Michigan Nutrition Obesity research Center (MNORC) through the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) P30 DK089503, National Institute of Aging (NIA) Awarded to JAC and KMB R01 AG072396 and U01 AG088407, and NIEHS Environmental Epigenomics and Precision Environmental Health Award to DCD R35 ES031686. Declaration of competing interests DCD serves as an expert legal consultant for toxicology and epigenetics lawsuits. The current study (funding, design, experimental setup, experimentation, data collection, analysis and interpretation of results, supervision of experimental work, decision to publish, and preparation of manuscript) was not impacted by this service. The remaining authors have no conflicts of interest to disclose. Data availability The data submission for GEO and Github are in progress. Acknowledgements The authors would like to thank Dr. Sundeep Kalantry and Dr. Vasantha Padmanabhan for their thoughtful discussion of results interpretation, the University of Michigan Advance Genomics Core for their assistance with RNA sequencing, and the University of Michigan Unit for Laboratory Animal Medicine for ensuring proper animal care. We would like to also thank Dr. Mathia Colwell and Jennifer Smith for their assistance during animal dissections. Funder Information Declared National Institute of Environmental Health Sciences , ES035064 , ES017885 , ES031686 National Institute of Diabetes and Digestive and Kidney Diseases, https://ror.org/00adh9b73 , DK089503 National Institute of Aging , AG072396 , AG088407 REFERENCES 1. ↵ Bhasin T , Lamture Y , Kumar M , Dhamecha R . Unveiling the Health Ramifications of Lead Poisoning: A Narrative Review . Cureus . Oct 2023 ; 15 ( 10 ): e46727 . doi: 10.7759/cureus.46727 OpenUrl CrossRef 2. ↵ Chowdhury R , Ramond A , O’Keeffe LM , et al. Environmental toxic metal contaminants and risk of cardiovascular disease: systematic review and meta-analysis . BMJ. Aug 29 2018 ; 362 : k3310 . doi: 10.1136/bmj.k3310 OpenUrl Abstract / FREE Full Text 3. ↵ Ruckart PZ , Jones RL , Courtney JG , et al. Update of the Blood Lead Reference Value – United States, 2021 . MMWR Morb Mortal Wkly Rep . 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