Evaluating the Role of the Steroid and Xenobiotic Receptor (SXR/PXR) in PCB-153 Metabolism and Protection against Associated Adverse Effects during Perinatal and Chronic Exposure in Mice.

BACKGROUND
Polychlorinated biphenyls (PCBs) are environmental toxicants; PCB exposure has been associated with adverse effects on wildlife and humans. However, the mechanisms underlying these adverse effects are not fully understood. The steroid and xenobiotic receptor [SXR; also known as the pregnane X receptor (PXR) and formally known as NR1I2] is a nuclear hormone receptor that regulates inducible metabolism of drugs and xenobiotics and is activated or inhibited by various PCB congeners.


OBJECTIVES
The aim of this study was to investigate the effects of exposure to PCB-153, the most prevalent PCB congener in human tissues, on SXR knockout mice (SXRKO) and to elucidate the role of SXR in PCB-153 metabolism and promotion of its harmful effects.


METHODS
Wild-type (WT) and SXRKO mice were chronically or perinatally exposed to a low dose (54μg/kg/d) of PCB-153. Blood, livers, and spleens were analyzed using transcriptome sequencing (RNA-seq) and molecular techniques to investigate the impacts of exposure on metabolism, oxidative stress, and hematological parameters.


RESULTS
SXRKO mice perinatally exposed to PCB-153 displayed elevated oxidative stress, symptoms of hemolytic anemia, and premature death. Transcriptomal analysis revealed that expression of genes involved in metabolic processes was altered in SXRKO mice. Elevated levels of the PCB-153 metabolite, 3-OH-PCB-153, were found in exposed SXRKO mice compared to exposed WT mice. Blood hemoglobin (HGB) levels were lower throughout the lifespan, and the occurrence of intestinal tumors was larger in SXRKO mice chronically exposed to PCB-153 compared to vehicle and WT controls.


DISCUSSION
Our results suggest that altered metabolism induced by SXR loss of function resulted in the accumulation of hydroxylated metabolites upon exposure to PCB-153, leading to oxidative stress, hemolytic anemia, and tumor development in a mouse model. These results support a major role for SXR/PXR in protection against xenobiotic-induced oxidative stress by maintaining proper metabolism in response to PCB-153 exposure. This role of SXR could be generally applicable to other environmental toxicants as well as pharmaceutical drugs. https://doi.org/10.1289/EHP6262.

. Breeding Results. Table S2. qPCR primer sequences. Table S3. Proportion of deaths at 5 weeks of age in WT, hSXRki, and SXRKO mice exposed to PCB-153 or vehicle in chronic exposure study. Table S4. Wild-type blood parameters over time in chronic exposure study. Table S5. SXRKO blood parameters over time in chronic exposure study.   Figure S3. Gene Ontology (GO) term enrichment of spleen RNA-seq data set comparing SXRKO mice exposed to PCB-153 vs SXRKO mice exposed to vehicle (DMSO) in perinatal exposure study.    Figure S7. qPCR validation of differentially expressed genes (DEGs) related to oxidative stress in the spleen of SXRKO mice exposed to PCB-153 vs vehicle (DMSO). Figure S8. DNA repair genes in the spleen of WT and SXRKO mice exposed to vehicle or PCB-153. Figure S9. Gene Ontology (GO) term enrichment of liver RNA-seq dataset comparing SXRKO vs wild-type mice exposed to vehicle (DMSO). Figure S10. Differential expression of genes related to transmembrane transporter activity between WT and SXRKO mice with DMSO and PCB-153 exposure.

Figure S11. Differential expression of various UGT and SULT genes between livers of WT and SXRKO mice with DMSO and PCB-153 exposure.
Figure S12. Tumor images, body weights, and spleen weights of mice chronically exposed to PCB-153.

RNA-seq script
RNA quality: Bioanalyzer (Agilent Bioanalyzer 2100) results for RNA used for RNA-seq Additional File-Excel Document S1        Figure S1: Blood hemoglobin levels throughout lifespan. Blood hemoglobin levels of wild-type (WT) mice from 6 to 40 weeks of age exposed to either PCB-153 (n= 23) or vehicle/DMSO (n= 14) as measured by the blood analyzer and plotted as mean ± SEM (A). Blood hemoglobin values of vehicle/DMSO exposed WT (n= 14) and SXRKO (n= 18) mice from 6 to 40 weeks of age (B). Data plotted as mean ± SEM. *= p-value <0.05 determined by Student's t-test comparing to WT mice at the same timepoint in panel B. Figure S2: Relative liver and spleen weights of dams and pups from perinatal PCB-153 exposure study. Relative liver weights (A) and spleen weights (B) for DMSO and PCB-153 exposed WT and SXRKO dams (WT DMSO: n=9; WT PCB-153: n=9; SXRKO DMSO: n=5; SXRKO PCB-153: n=7). Relative liver weights (C) and spleen weights (D) for perinatally exposed WT and SXRKO mice at 4 weeks of age (WT DMSO: n= 19; WT PCB-153: n=18; SXRKO DMSO: n=16; SXRKO PCB-153: n=13). Plotted as mean ± SEM. Figure S3: Gene Ontology (GO) term enrichment of spleen RNA-seq data set comparing SXRKO mice exposed to PCB-153 vs SXRKO mice exposed to vehicle (DMSO) in perinatal exposure study. GO term enrichment of select biological processes (BP) of SXRKO PCB vs SXRKO DMSO in the spleen RNA-seq dataset. Number of significant genes within each term is displayed to the right of the bar. Figure S4: qPCR validation of select differentially expressed genes (DEGs) related to erythrocyte development and heme metabolism in the spleen. Relative mRNA expression of Erythropoietin receptor (Epor) (A), Ferrochelatase (Fech) (B), Erythrocyte membrane protein band 4.2 (Epb42) (C), δ-aminolevulinic acid dehydratase (Alad) (D), hemoglobin subunit theta 1B (Hbq1b) (E), and Redox-regulatory protein Fam213a (F) in the spleen (n= 5 per group) determined by qPCR analysis compared to β-actin/Actb housekeeping gene. Plotted as mean ± SEM. a= statistically significant compared to WT DMSO, b= statistically significant compared to WT PCB, c= statistically significant compared to SXRKO DMSO determined by two-way ANOVA and Tukey's multiple comparisons test.

Figure S7: qPCR validation of differentially expressed genes (DEGs) related to oxidative stress in the spleen of SXRKO mice exposed to PCB-153 vs vehicle (DMSO).
Heatmap of DEGs in the GO term of Response to reactive oxygen species (ROS) (A). qPCR validation of the antioxidant gene Gpx1 (B) and glutathione S-transferase genes, Mgst3 (C) and Gstp3 in the spleen (n= 5 per group) compared to Actb housekeeping gene, plotted as mean ± SEM. a= statistically significant compared to WT DMSO, b= statistically significant compared to WT PCB, c= statistically significant compared to SXRKO DMSO determined by two-way ANOVA and Tukey's multiple comparisons test. Figure S8:DNA repair genes in the spleen of WT and SXRKO mice exposed to vehicle or PCB-153. Heatmap of significant differentially expressed genes within the GO term of DNA repair between spleens of SXRKO mice exposed to PCB-153 vs DMSO (A). qPCR validation of Growth Arrest and DNA Damage Inducible Alpha (Gadd45a) (B) and RAD23 Homolog A (Rad23a) (C) in the spleen (n= 5 per group) compared to Actb housekeeping gene, plotted as mean ± SEM. Figure S9: Gene Ontology (GO) term enrichment of liver RNA-seq dataset comparing SXRKO vs wild-type mice exposed to vehicle (DMSO). GO term enrichment of select biological processes (BP) of SXRKO DMSO vs WT DMSO in the liver RNA-seq dataset. Number of significant genes within each term is displayed to the right of the bar. Figure S10: Differential expression of genes related to transmembrane transporter activity between WT and SXRKO mice with DMSO and PCB-153 exposure. Heatmap of DEGs related to transmembrane transporter activity in the livers between SXRKO and WT mice exposed to PCB-153 (A). Normalized counts of solute carrier organic anion transporter family member 1A1 (Sclo1a1/OATP-1) obtained from DESeq2 (B). Lines demonstrate the comparisons that were significant as determined by DESeq2 using the criteria listed in the Methods. Adjusted p-values determined by DESeq2 analysis are displayed above lines for corresponding comparisons.

Figure S11: Differential expression of various UGT and SULT genes between livers of WT and SXRKO mice with DMSO and PCB-153 exposure.
Heatmap of DEGs related to glucuronosyltransferase activity in the livers between SXRKO and WT mice exposed to PCB-153 (A). Normalized counts of Ugt3a1 obtained from DESeq2 (B). Heatmap of DEGs related to sulfotransferase activity, comparing all groups (C). Normalized counts of Sult2a1 obtained from DESeq2 analysis (D). Lines demonstrate the comparisons that were significant as determined by DESeq2 using the criteria listed in the Methods. Adjusted p-values determined by DESeq2 analysis are displayed above lines for corresponding comparisons. Figure S12: Tumor images, body weights, and spleen weights of mice chronically exposed to PCB-153. Representative images of SXRKO mice chronically exposed to PCB-153 displaying tumors in the upper small intestine near the duodenum-jejunum junction (A). Representative histology images of the intestinal tumors at 40X total magnification (B). Spleen weights (C) and body weights (D) of chronically exposed mice (WT DMSO: n= 14; WT PCB-153: n=23; SXRKO DMSO: n=33; SXRKO PCB-153: n=35). Tumor bearing mice are indicated with red shaded symbols in panel C and D. Data plotted as mean ± SEM. b= statistically significant compared to WT PCB, c= statistically significant compared to SXRKO DMSO determined by two-way ANOVA and Tukey's multiple comparisons test.