Tissue distribution and detection of PCB 11 and its metabolites in maternal dams

PCB 11 is metabolized by P450 enzymes into OH-PCB 11 (Kaminsky et al. 1981), which undergoes further biotransformation, including oxidation, sulfation, glucuronidation, and methylation (Zhang et al. 2020). To assess the distribution of PCB 11 and its metabolites in mouse dams, levels of PCB 11 and OH-PCB 11 metabolites were measured in the brain, liver, and serum of female dams at PD21 using GC–MS/MS and LC-HRMS (Table 1). In brain tissue, the parent congener PCB 11 and its hydroxylated metabolites were below detection limits, with exception of 5-OH-PCB 11, which was detected in 33% of samples from the high-dose group (0.88 ± 0.03 ng/g wet weight [ww]). In the liver, PCB 11 was detected in a few samples in both exposure groups (33% of low-dose and 17% of high-dose samples). 4-OH-PCB 11 (119 ng/g ww) and 5-OH-PCB 11 (2.3 ng/g ww) were each observed with a detection frequency of 17% in low-dose samples. In the serum, PCB 11 was detected in 17% of low-dose samples, and several hydroxylated metabolites were detected. 4-OH-PCB 11 was the most frequently detected PCB 11 metabolite in serum, with a detection frequency of 33% in low-dose samples and 17% in high-dose samples. Additionally, 5-OH-PCB 11 and 6-OH-PCB 11 were detected in 17% and 67% of high-dose samples, respectively.

Table 1 Levels (ng/g wet weight) and detection frequency (%) of PCB 11 and its metabolites determined by GC–MS/MS and LC-HRMS across matrices for the low and high PCB 11 exposure groups.a

LC-HRMS analysis revealed three OH-PCB 11 peaks, X1, X2, and X3 ([M-H]−, m/z 236.98767; Fig. 1, A1 and A2) in serum at retention times of 8.11, 8.21, and 8.34 min, respectively. The detection frequencies of X1 and X2 OH-PCB 11 peaks were 33% and 83% in both the low-dose and high-dose groups, respectively. The X3 metabolite was only detected in serum from the high-dose group, with a detection frequency of 33%. The detection frequencies of the OH-PCB 11 metabolites in the LC-HRMS analysis differ from those observed in the GC–MS/MS analysis, most likely due to differences in the limits of detection between the two analytical methods or a limitation in the liquid–liquid extraction protocol used in the GC–MS/MS analysis workflow. Additionally, two peaks corresponding to PCB 11 sulfates, Y1 and Y2 ([M-H-SO3]−, m/z 236.98800, [M-H]−, m/z 316.94434; Fig. 1, B1 and B2), were observed at 6.66 and 6.78 min, respectively. The PCB 11 sulfate Y1 peak was detected in 50% of low-dose samples, whereas both Y1 and Y2 metabolites were observed in 100% of the high-dose group. Two OH-PCB 11 sulfate peaks, Z1 and Z2 ([M-H-SO3]−, m/z 252.98254; [M-H]−, m/z 332.93951; Fig. 1, C1 and C2), were detected at 6.68 and 6.89 min, respectively. Both OH-PCB 11 sulfate peaks were detected in 50% and 100% of low- and high-dose exposure samples, respectively. These findings suggest that PCB 11 and its metabolites do not accumulate at detectable levels in the dams at the time point investigated, likely due to the rapid elimination of PCB 11 in laboratory animals (Hu et al. 2013; Zhang et al. 2021).

Fig. 1

Identification of putative PCB 11 metabolites in serum of exposed postpartum dams. Representative ion chromatograms and mass spectrometric data support the presence of (A) OH-PCB 11 and (B) PCB 11 sulfate in a high-dose sample and (C) OH-PCB 11 sulfate metabolite in a low-dose sample. (A1) OH-PCB 11 metabolites detected at retention times 8.11 min, 8.21 min, and 8.34 min (m/z 236.98767); (A2) accurate masses of isotope ions corresponding to OH-PCB 11 eluting at 8.21 min; (B1) PCB 11 sulfate detected at 6.66 min and 6.78 min (m/z 316.94434); (B2) accurate masses of PCB 11 sulfate isotope ions and a fragment ion ([M-H-SO3]−, m/z 236.98800) eluting at 6.66 min; (C1) OH-PCB 11 sulfate detected at 6.68 min and 6.89 min (m/z 332.93951); (C2) accurate masses of isotope ions and a fragment ([M-H-SO3]−, m/z 252.98254) of OH-PCB 11 sulfate eluting at 6.89 min. (D) Proposed metabolic pathway of PCB 11 in dam serum samples. CYP: cytochrome; SULT: sulfotransferase

Fig. 2

Hepatic proteome alterations induced by low and high doses of PCB 11 in postpartum day 21 dams. Volcano plots show differentially expressed hepatic proteins following (A) low-dose (1.0 mg/kg; n = 5) and (B) high-dose (6.0 mg/kg; n = 5) PCB 11 exposure, with significance defined by FDR ≤ 0.05. The top ten KEGG pathway enrichments identified via STRING analysis are presented for (C) low-dose and (D) high-dose exposure groups

Impact of PCB 11 exposure during pregnancy on the maternal hepatic proteome

While HC-PCBs are known to alter the hepatic proteome (Rignall et al. 2013), the effects of LC-PCBs, such as PCB 11, on the hepatic proteome remain poorly investigated. Furthermore, the impact of exposure to PCBs during pregnancy and lactation on the maternal liver proteome has not been characterized. To address these knowledge gaps, we first performed global proteomic profiling of liver tissue, followed by an analysis of drug-metabolizing enzymes in dams exposed to PCB 11 throughout pregnancy and lactation, to determine whether PCB 11 exposure elicits proteomic changes similar to those induced by other PCB congeners.

Global alterations of the liver proteome following PCB 11 exposure during pregnancy and lactation

Principal component analysis (PCA) revealed distinct separation between vehicle control and both PCB 11 exposure groups (Online Resource Figure S1). Relative to the vehicle controls, 123 hepatic proteins were significantly altered (FDR ≤ 0.05; Fig. 2A and Online Resource Table S9) in the low-dose group, with 3 increased and 120 decreased. The most decreased proteins included dihydrolipoamide S-acetyltransferase (DLAT), ubiquitin carboxyl-terminal hydrolase 26 (USP26), malic enzyme 1 (ME1), cytochrome P450 2D10 (CYP2D10), and SEC23 homolog A, COPII coat complex component (SEC23A). The most increased proteins were pre-mRNA processing factor 38A (PRPF38A), solute carrier family 6 member 13 (SLC6A13), and elastin microfibril interfacer 1 (EMILIN1).

In the high-dose group, 234 proteins were significantly altered (FDR ≤ 0.05; Fig. 2B, Online Resource Table S12), with 56 increased and 178 decreased. The top five decreased proteins were serine protease 1 (PRSS1) and 2 (PRSS2), chymotrypsin-like (CTRL), Ras-related C3 botulinum toxin substrate 1 (RAC1), and DLAT. The top five increased proteins were solute carrier family 6 member 13 (SLC6A13), CCR4-NOT transcription complex subunit 4 (CNOT4), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE), asparaginyl-tRNA synthetase 1 (NARS1), and chromodomain helicase DNA binding protein 1 (CHD1).

KEGG pathway analysis revealed enrichment of differentially abundant proteins (FDR ≤ 0.05) in the low- (Fig. 2C; Online Resource Table S10) and high-dose (Fig. 2D; Online Resource Table S13) exposure groups across several hepatic metabolic pathways. Key pathways included “metabolic pathways”, “fatty acid degradation”, “carbon metabolism”, “tryptophan metabolism”, “beta-alanine metabolism”, “valine/leucine/isoleucine degradation”, “histidine metabolism”, “biosynthesis of amino acids”, “pyruvate metabolism”. “Fatty acid metabolism” was enriched only in the low-dose group, while the “TCA cycle” was specific to the high-dose group. Protein–protein network analyses for low- and high-group exposure are depicted in the Online Resource Figures S2 and S3, respectively. Comparison of maternal hepatic proteins between exposure doses (Online Resource Figure S4, Table S15) identified 119 common proteins (FDR ≤ 0.05) enriched in pathways related to amino acid, energy, and xenobiotic metabolism, including drug-processing enzymes such as acyl-CoA’s, epoxide hydrolases, carboxylesterases, P450s, and flavin-containing monooxygenases.

Disruption of hepatic phase I biotransformation in the maternal proteomeEffect of PCB 11 exposure on liver P450 enzymes

PCBs are known to induce hepatic P450 enzymes (Robertson et al. 1984), but the effects of PCB 11 during pregnancy and lactation remain poorly understood. Using global proteomics, we identified eleven hepatic P450 enzymes altered by maternal PCB 11 exposure, irrespective of the dose (Fig. 3). Compared to vehicle controls, both low- and high-dose PCB 11 exposure significantly reduced (FDR ≤ 0.05) the protein abundance of CYP1A2, CYP2C23, CYP2C29, CYP2C40, CYP2C70, CYP2D10, CYP2E1, and CYP3A41, enzymes critical for phase I biotransformation and steroid biosynthesis, suggesting a broad suppression of hepatic biotransformation capacity by PCB 11 in mouse dams exposed via the diet.

Fig. 3

Protein abundance of cytochrome P450 enzymes in female adult wildtype mice exposed to PCB 11. Total protein abundance of P450 enzymes was measured in vehicle (sterile peanut butter; 0 mg/kg; n = 5; green), low (1.0 mg/kg; n = 5; yellow) and high (6.0 mg/kg; n = 5; red) exposure groups. P450 enzyme families detected include CYP1A (A), CYP2C subtypes (B-F), CYP2D (G), CYP2E (H), CYP3A subtypes (I-K). Outliers were identified with Grubbs’ test, resulting in the removal of one sample from CYP2C23 protein analyses. Data were analyzed using ordinary one-way ANOVA followed by Dunnett’s multiple comparison test with GraphPad Prism v10.1

We did not detect CYP2B enzymes in the liver, irrespective of the experimental group. Given that many NDL-PCBs induce CYP2B enzymes (Uwimana et al. 2019), CYP2B10 expression was specifically examined but was absent in the proteomics analysis. PCB 11 exposure did not significantly affect (P > 0.05) CYP2B10 mRNA expression, assessed by RT-PCR (Online Resource Figure S5), or protein levels, assessed by western blotting (Online Resource Figure S6).

STRING and KEGG pathway analysis of drug-metabolizing enzymes

To further examine PCB 11 effects on hepatic drug metabolism, STRING analysis was performed at a confidence threshold score ≥ 0.900. Eight enzymes were significantly altered in the low-dose group (CYP1A2, CYP2C23, CYP2C29, CYP2C40, CYP2C70, CYP2E1, EPHX1, EPHX2; FDR ≤ 0.05; Fig. 4A), and nine in the high-dose group, including the eight drug metabolizing enzymes plus CES1D (FDR ≤ 0.05; Fig. 4C).

Fig. 4

Characterization of cytochrome P450 and steroid hormone biosynthesis pathway in female adult mice exposed to PCB 11. Protein–protein interaction (PPI) network and KEGG pathway enrichment analyses were used to assess hepatic drug metabolism pathways following PCB 11 exposure. Panels (A, B) depict the PPI network (interaction score ≥ 0.900) and significantly enriched KEGG pathways (FDR ≤ 0.05) in the low-dose group (1.0 mg/kg; n = 5; yellow), while panels (C, D) show high-dose exposure (6.0 mg/kg; n = 5; red). Panel (E) highlights the steroid biosynthesis pathway and enzymes identified between both exposures

KEGG pathway enrichment of these proteins subsequently identified overlapping pathways in both PCB 11 exposure groups, including “steroid hormone biosynthesis,” “chemical carcinogenesis,” “arachidonic acid metabolism,” “retinol metabolism,” “serotonergic synapse,” “metabolic pathways,” “inflammatory mediator regulation of TRP channels,” “metabolism of xenobiotics by P450s,” and “drug metabolism – cytochrome P450” (FDR ≤ 0.05;  Fig. 4B and D; Online Resource Tables S11 and S14).

“Steroid hormone biosynthesis” was enriched in both groups, suggesting potential endocrine-disrupting effects of PCB 11. While PCBs are established endocrine disruptors (Streifer et al. 2024), evidence for PCB 11 and/or its metabolites remains limited. In the low-dose group, seven hepatic enzymes (CYP1A2, CYP2C23, CYP2C29, CYP2C40, CYP2C70, CYP2D10, and CYP2E1; Fig. 4E) are involved in converting dehydroepiandrosterone (DHEA) to 16α-hydroxydehydroepiandrosterone (16α-OH-DHEA). In the high-dose group, these enzymes plus hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1; Fig. 4E) were altered, supporting disruption of steroid hormone metabolism following PCB 11 exposure during pregnancy and lactation.