Neonatal Exposure to BPA, BDE-99, and PCB Produces Persistent Changes in Hepatic Transcriptome Associated With Gut Dysbiosis in Adult Mouse Livers

Abstract

Recent evidence suggests that complex diseases can result from early life exposure to environmental toxicants. Polybrominated diphenyl ethers (PBDEs), and polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) and remain a continuing risk to human health despite being banned from production. Developmental BPA exposure mediated-adult onset of liver cancer via epigenetic reprogramming mechanisms has been identified. Here, we investigated whether the gut microbiome and liver can be persistently reprogrammed following neonatal exposure to POPs, and the associations between microbial biomarkers and disease-prone changes in the hepatic transcriptome in adulthood, compared with BPA. C57BL/6 male and female mouse pups were orally administered vehicle, BPA, BDE-99 (a breast milk-enriched PBDE congener), or the Fox River PCB mixture (PCBs), once daily for three consecutive days (postnatal days [PND] 2-4). Tissues were collected at PND5 and PND60. Among the three chemicals investigated, early life exposure to BDE-99 produced the most prominent developmental reprogramming of the gut-liver axis, including hepatic inflammatory and cancer-prone signatures. In adulthood, neonatal BDE-99 exposure resulted in a persistent increase in Akkermansia muciniphila throughout the intestine, accompanied by increased hepatic levels of acetate and succinate, the known products of A. muciniphila. In males, this was positively associated with permissive epigenetic marks H3K4me1 and H3K27, which were enriched in loci near liver cancer-related genes that were dysregulated following neonatal exposure to BDE-99. Our findings provide novel insights that early life exposure to POPs can have a life-long impact on disease risk, which may partly be regulated by the gut microbiome.

Keywords: adverse health outcomes; bioinformatics; environmental chemicals; epigenetic; microbiome; toxicogenomics.

Figure 1.

Persistent dysregulation of the liver transcriptome following early-life exposure to environmental toxicants. A, Corn oil (vehicle control), BPA, BDE-99, and the Fox River PCB mixture were used to orally expose neonatal mice on PND2, PND3, and PND4. Twenty-four hours after the final dose, RNA-seq in the liver and 16s rDNA-seq in the small and large intestines were performed to characterize the acute effects from the exposure. When the exposed mice reached young adulthood at PND60, RNA-seq, metabolomics, ChIP-seq, and 16s rDNA-seq in the small and large intestines were conducted to investigate the persistent effects from the early life exposure. B, Number of dysregulated protein-coding genes (PCGs) at PND5 (black) and PND60 (red) for males (top) and females (bottom). C, Venn diagram showing intersections of dysregulated genes in neonates. D, Venn diagram showing intersections of dysregulated genes in young adulthood. E, Summary of gene ontology enrichment results by age, sex, and exposure groups. Up-regulated (left panel, red) and down-regulated (right panel, blue) biological terms related to liver function are included. The color gradient describes the average significance (p < .01) in each category per group. The top enriched proportion (size of circles) indicates the proportion of GO terms in each category relative to the top 50 terms (p < .01), i.e., larger circles indicate a higher proportion relative to the top 50 GO terms. Please refer to the article in the online version for colored figures.

Figure 2.

Specific categories of persistently dysregulated genes following early-life exposure to environmental toxicants in males. Two-way hierarchical clustering of differentially regulated genes involved in xenobiotic biotransformation (A) and oxidative stress and inflammation (B) in livers of PND60 adult mice following neonatal chemical exposure. C, Disease enrichment of persistently dysregulated genes following early-life exposure to BDE-99. The color gradient in the legend represents the adjusted p value in −log₁₀ scale. D, Gene expression values of PCG and long noncoding RNAs (lncRNAs) in the Dlk1-Dio3 imprinted cluster. Expression values are shown in the same y-axis scale for comparison. E, Two-way hierarchical clustering of differentially regulated microRNAs (miRNAs) in the Dlk1-Dio3 imprinted cluster. F, An example of differentially regulated lncRNAs associated with liver tumors. All individual bar graphs with gene symbols show the average transcripts per million (TPM) ± standard error (SE) of the genes in the cluster. Asterisks represent statistically significant differences compared with vehicle-exposed groups. Asterisks (“*” p value < .05) were placed for lncRNAs that were differentially expressed but are not visible due to low expression values within the same y-axis scale. Colored bars in heatmaps represent genes that are differentially regulated in a particular exposure group, i.e., BPA—red, BDE-99—blue, PCB mixuture—green. Please refer to the article in the online version for colored figures.

Figure 3.

Persistent dysbiosis of the gut microbiome and upregulation of key metabolites involved in liver reprogramming and inflammation in the liver from early life exposure to environmental toxicants. A, Diagram of the duodenum (duo), jejunum (jej), ileum (ile), colon (col), and feces (fec) in physical order from left to right. Note: the intestinal contents were kept in each of the tissue sections. B, Top 10 most abundant taxa in each compartment at the family level in adult age. Asterisks represent statistically significant differences compared with the vehicle control. C, Persistently increased abundance of Akkermansia municiphila in all bio-compartments from neonatal exposure to BDE-99 in males. D, Box plots of persistently increased metabolites from early life exposure to BDE-99 in males. E, Persistently dysregulated colon microbiota significantly correlated with the persistently upregulated lactate and succinate in livers of males neonatally exposed to BDE-99. F, Significant linkage (r > 0.8, FDR < 0.1) between dysregulated colon microbiota and upregulated liver metabolites involved in epigenetic reprogramming and inflammatory signaling. Please refer to the article in the online version for colored figures.

Figure 4.

Developmentally reprogrammed liver epigenome following early-life exposure to BDE-99. Top 20 gene ontology enrichment terms from overlay results (left panel), examples of peak alterations (middle panel), and average peak and gene expression values comparing vehicle control and BDE-99 for up (A) and downregulated (B) genes. Colored bars show histone marks with altered peaks (> 30% in A and < 30% in B) compared with the control for genes that comprise particular GO terms, i.e., purple—H3K4me1, orange—H3K4me3, blue—H3K27ac. Pound signs represent absolute peak fold change > 30%, and asterisks show differential regulation. Please refer to the article in the online version for colored figures.

Figure 5.

Summary figure of results. Neonatal oral exposure to human health-relevant environmental toxicants BPA, BDE-99, or PCBs (Fox River Mix) was sufficient to acutely and persistently alter the liver transcriptome and changing the ontogenetic trajectory of many key signaling pathways, including cell cycle, epigenetic remodeling, drug metabolism, lipid metabolism, and immune response. In general, the effect of neonatal chemical exposure is markedly amplified in adult age: at the given dose, BDE-99 had the most prominent hepatic response, followed by BPA, then the PCB mixture, for which males were more susceptible than females. For BDE-99, the persistent transcriptomic changes revealed downregulated signatures of endogenous liver functions, i.e., xenobiotic and lipid metabolism, and upregulated cellular proliferation signatures. These changes are possible due to developmental reprogramming through upregulated liver metabolites involved in the epigenetic modification and inflammatory signaling, i.e., acetate, lactate, succinate. Interestingly, neonatal exposure to BDE-99 persistently upregulated gut microbiota capable of producing these metabolites, suggesting a persistently dysregulated cross-talk between the gut microbiome. The persistent effect due to neonatal exposure may be a point source for altered hepatic biotransformation leading to increased risk for chronic disease. Please refer to the article in the online version for colored figures.