Effects of Dicyclohexyl Phthalate Exposure on PXR Activation and Lipid Homeostasis in Mice

Abstract

Background: Exposure to plastic-associated endocrine disrupting chemicals (EDCs) has been associated with an increased risk of cardiovascular disease (CVD) in humans. However, the underlying mechanisms for this association are unclear. Many EDCs have been shown to function as ligands of the nuclear receptor pregnane X receptor (PXR), which functions as xenobiotic sensor but also has pro-atherogenic effects in vivo.

Objective: We sought to investigate the contribution of PXR to the adverse effects dicyclohexyl phthalate (DCHP), a widely used phthalate plasticizer, on lipid homeostasis and CVD risk factors.

Methods: Cell-based assays, primary organoid cultures, and PXR conditional knockout and PXR-humanized mouse models were used to investigate the impact of DCHP exposure on PXR activation and lipid homeostasis in vitro and in vivo. Targeted lipidomics were performed to measure circulating ceramides, novel predictors for CVD.

Results: DCHP was identified as a potent PXR-selective agonist that led to higher plasma cholesterol levels in wild-type mice. DCHP was then demonstrated to activate intestinal PXR to elicit hyperlipidemia by using tissue-specific PXR-deficient mice. Interestingly, DCHP exposure also led to higher circulating ceramides in a PXR-dependent manner. DCHP-mediated PXR activation stimulated the expression of intestinal genes mediating lipogenesis and ceramide synthesis. Given that PXR exhibits considerable species-specific differences in receptor pharmacology, PXR-humanized mice were also used to replicate these findings.

Discussion: Although the adverse health effects of several well-known phthalates have attracted considerable attention, little is known about the potential impact of DCHP on human health. Our studies demonstrate that DCHP activated PXR to induce hypercholesterolemia and ceramide production in mice. These results indicate a potentially important role of PXR in contributing to the deleterious effects of plastic-associated EDCs on cardiovascular health in humans. Testing PXR activation should be considered for risk assessment of phthalates and other EDCs. https://doi.org/10.1289/EHP9262.

Figure 1.

The effects of DCHP on PXR activity in cell-based transfection assays. (A) Human LS180 intestinal cells were transfected with a full-length hPXR plasmid, hPXR reporter (CYP3A4-luciferase), and $\text{CMX-}\mathrm{\beta }\text{-galactosidase}$ ($\mathrm{\beta }\text{-gal}$) control plasmid. Cells were treated with various phthalates at the indicated concentrations for 24 h ($n=3$). (B,C) LS180 cells were transfected with (B) hPXR and CYP3A4-luciferase reporter together with $\text{CMX-}\mathrm{\beta }\text{-galactosidase}$ plasmid or (C) mPXR and ${(\text{CYP}3\mathrm{A}2)}_3\text{-luciferase}$ reporter together with $\text{CMX-}\mathrm{\beta }\text{-galactosidase}$ plasmid. After transfection, LS180 cells were then treated with DCHP at the indicated concentrations for 24 h ($n=3$). (D) LS180 cells were transfected with GAL4 plasmids in which the GAL4 DNA-binding domain is linked to the indicated nuclear receptor ligand-binding domain and a GAL4 reporter. After transfection, LS180 cells were treated with DMSO control or $10\mathrm{\mu }\mathrm{M}$ DCHP for 24 h ($n=3$, two-sample, two-tailed Student’s $t$-test, ***, $p<0.001$ compared with the control group). Reporter gene activity was normalized to the $\mathrm{\beta }\text{-gal}$ transfection controls, and the results were normalized to relative light units per ${\text{OD}}_{595}$ $\mathrm{\beta }\text{-gal}$ per minute to facilitate comparisons between plates. Fold activation was calculated relative to vehicle DMSO controls. Mean fold activation is shown. Error bars $\text{represent}\pm \text{SEM}$. The numerical data corresponding to this figure are shown in Tables S2–S5. Note: $\mathrm{\beta }\text{-gal}$, beta galactosidase; CARα, constitutive androstane receptor; DAP, diallyl phthalate; DCHP, dicyclohexyl phthalate; DEHP, di(2-ethylhexyl) phthalate; DEP, diethyl phthalate; DHP, dihexyl phthalate; DiBP, diisobutyl phthalate; DiDP, diisodecyl phthalate; DiNP, diisononyl phthalate; DMP, dimethyl phthalate; DMSO, dimethyl sulfoxide; DnBP, di-$n$-butyl phthalate; DnOP, di-$n$-octyl phthalate; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; FXR, farnesyl X Receptor; hPXR, human pregnane X receptor; LXR, liver X receptor; mPXR, mouse pregnane X receptor; OD, optical density; PPARα, peroxisome proliferator-activated receptor alpha; PXR, pregnane X receptor; RARα, retinoic acid receptor alpha; rPXR, rat pregnane X receptor; RXR, retinoid X receptor; SEM, standard error of the mean; VDR, vitamin D receptor.

Figure 2.

Binding of DCHP to PXR ligand-binding domain (LBD) and the impact of DCHP on PXR and co-regulator interactions. (A) Competitive ligand binding assays were performed to evaluate the inhibition of FRET between fluorescein-labeled PXR ligand and recombinant GST-PXR LBD by DCHP and DEHP. DEHP was included as a positive control. Results are presented as mean TR-FRET emission ratios based on the signal from the fluorescein emission divided by the terbium signal ($n=3$). Error bars $\text{represent}\pm \text{SEM}$. (B,C) Human LS180 cells were transfected with a GAL4 reporter, VP16-hPXR vector, and expression vector of GAL4 DNA-binding domain (DBD) or GAL4 DBD linked to the receptor interaction domains of (B) nuclear receptor co-activators (GAL4-SRC1 or GAL4-PBP) or (C) nuclear receptor co-repressors (GAL4-SMRT or GAL4-NCoR). After transfections, LS180 cells were treated with DMSO vehicle control or DCHP at the indicated concentrations for 24 h. Reporter gene activity was normalized to the $\mathrm{\beta }\text{-gal}$ transfection controls, and the results were normalized to relative light units per ${\text{OD}}_{595}$ $\mathrm{\beta }\text{-gal}$ per minute to facilitate comparisons between plates ($n=3$). Mean fold activation was calculated relative to vehicle DMSO controls. Error bars $\text{represent}\pm \text{SEM}$. The numerical data corresponding to this figure are shown in Tables S6–S8. Note: DCHP, dicyclohexyl phthalate; DEHP, di(2-ethylhexyl) phthalate; DMSO, dimethyl sulfoxide; FRET, fluorescence resonance energy transfer; GAL4, galactose-responsive transcription factor; GST, glutathione S-transferase; NCoR, nuclear receptor co-repressor; OD, optical density; PBP, peroxisome proliferator-activated receptor binding protein; PXR, pregnane X receptor; SEM, standard error of the mean; SMRT, silencing mediator of retinoid and thyroid hormone receptors; SRC-1, Steroid receptor co-activator-1; TR-FRET, time-resolved measurement of fluorescence resonance energy transfer.

Figure 3.

The effects of DCHP exposure on plasma lipid levels in wild-type (WT) mice. Eight-wk-old male WT mice were treated with vehicle control or $10\mathrm{mg}/\mathrm{kg}$ BW per day of DCHP daily by oral gavage for 7 d. Fasting plasma (A) total cholesterol and (B) triglyceride levels were measured by enzymatically colorimetric methods. (C) Lipoprotein fractions (VLDL-C, LDL-C, and HDL-C) were isolated from the plasma and the cholesterol levels of each fraction were then measured. ($n=5–8$, two-sample, two-tailed Student’s $t$-test, **, $p<0.01$, and ***, $p<0.001$). Results represent mean values. Error bars $\text{represent}\pm \text{SEM}$. The numerical data corresponding to this figure are shown in Table S9. Note: BW, body weight; DCHP, dicyclohexyl phthalate; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; SEM, standard error of the mean; VLDL-C, very-low-density lipoprotein-cholesterol.

Figure 4.

The impact of DCHP exposure on intestinal PXR target gene expression and plasma lipid levels in ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ mice. Eight-wk-old male ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ littermates were administered vehicle control or $10\mathrm{mg}/\mathrm{kg}$ BW per day of DCHP daily by oral gavage for 7 d. (A) Intestinal expression of PXR target genes was analyzed by qPCR ($n=5$, two-sample, two-tailed Student’s $t$-test, *, $p<0.05$). Fasting plasma (B) total cholesterol and (C) triglyceride levels were analyzed enzymatically by colorimetric methods. (D) Lipoprotein fractions (VLDL-C, LDL-C, and HDL-C) were isolated from the plasma, and the cholesterol levels of each fraction were then measured enzymatically. ($n=5–6$, two-way ANOVA, Bonferroni multiple comparisons test for multiple comparisons, *, $p<0.05$). The numerical data corresponding to this figure are shown in Tables S10–S11. Results represent mean values. Error bars $\text{represent}\pm \text{SEM}$. Note: ANOVA, analysis of variance; BW, body weight; CYP3A11, cytochrome P450, family 3, subfamily A, polypeptide 11; DCHP, dicyclohexyl phthalate; $\text{GST}\mathrm{\alpha }1$, glutathione $S$-transferase; MDR1a, multidrug resistance protein 1a; PXR, pregnane X receptor; ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$, intestinal epithelial cell-specific pregnane X receptor deficient; ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$, pregnane X receptor flox; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

Figure 5.

The effects of DCHP exposure on intestinal NPC1L1 and MTP expression and lipid absorption in ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ mice. Eight-wk-old male ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ littermates were administered vehicle control or $10\mathrm{mg}/\mathrm{kg}$ BW per day of DCHP daily by oral gavage for 7 d. (A) Intestinal expression of NPC1L1 and MTP was measured by qPCR ($n=5$, two-sample, two-tailed Student’s $t$-test, *, $p<0.05$ and **, $p<0.01$). (B) The recruitment of PXR onto the NPC1L1 and MTP promoter in the intestine was evaluated by chromatin immunoprecipitation (ChIP) assays. The PCR products of the ChIP assays were visualized by loading onto 2% agarose gel ($n=3$). (C) Control or DCHP-treated ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ mice were given an oral challenge of oil containing ${[}^3\mathrm{H}]\text{-cholesterol}$, and the distribution of radioactivity in intestinal segments of the mice was evaluated after 2 h ($n=5–6$, two-way ANOVA, Bonferroni multiple comparisons test for multiple comparisons, *, $p<0.05$, **, $p<0.01$ and ***, $p<0.001$). (D) Control or DCHP-treated ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ mice were injected with lipase inhibitor poloxamer-407 and then given an oral challenge of oil containing ${[}^3\mathrm{H}]\text{-cholesterol}$. Plasma samples were collected from those mice at several time points within 6 h, and the presence of ${[}^3\mathrm{H}]\text{-cholesterol}$ was measured ($n=5$, two-way ANOVA, Bonferroni multiple comparisons test for multiple comparisons. *, $p<0.05$, compared with ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ mice treated with control; ^††, $p<0.01$, compared with ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ mice treated with DCHP). The numerical data corresponding to this figure are shown in Tables S12–S14. Results represent mean values. Error bars $\text{represent}\pm \text{SEM}$. Note: ANOVA, analysis of variance; BW, body weight; DCHP, dicyclohexyl phthalate; MTP, microsomal triglyceride transfer protein; NPCIL1, Niemann-Pick C1-Like 1; ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$, intestinal epithelial cell-specific pregnane X receptor deficient; ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$, pregnane X receptor flox; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

Figure 6.

The impact of DCHP exposure on circulating ceramide levels and ceramide-synthesis–related gene expression in ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ mice. Eight-wk-old male ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ littermates were treated with vehicle control or $10\mathrm{mg}/\mathrm{kg}$ BW per day of DCHP daily by oral gavage for 7 d. Plasma ceramide levels of (A) ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$ and (B) ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$ mice were measured by LC-MS/MS ($n=6$, two-sample, two-tailed Student’s $t$-test, *, $p<0.05$). (C) Intestinal expression of ceramide-synthesis–related genes were measured by qPCR ($n=5$, two-sample, two-tailed Student’s $t$-test, *, $p<0.05$). The numerical data corresponding to this figure are shown in Tables S15–S16. Results represent mean values. Error bars $\text{represent}\pm \text{SEM}$. Note: BW, body weight; CERS4, ceramide synthase 4; DCHP, dicyclohexyl phthalate; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; GBA1, glucosylceramidase beta 1; LC-MS/MS, liquid chromatography–tandem mass spectrometry; NEU3, neuraminidase 3; ${\text{PXR}}^{\mathrm{\Delta }\text{IEC}}$, intestinal epithelial cell-specific pregnane X receptor deficient; ${\text{PXR}}^{\mathrm{F}/\mathrm{F}}$, pregnane X receptor flox; SMPD1, sphingomyelin Phosphodiesterase 1; SMPD3, sphingomyelin Phosphodiesterase 3; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

Figure 7.

The effects of DCHP exposure on PXR target gene expression and plasma lipid levels in PXR-humanized (huPXR) mice. Eight-wk-old male huPXR and ${\text{PXR}}^{–/–}$ littermates were treated with $10\mathrm{mg}/\mathrm{kg}$ BW per day of DCHP or DMSO control daily by oral gavage for 7 d. (A) The intestinal mRNA expression of PXR target genes and lipogenic genes were measured by qPCR analysis ($n=5$, two-sample, two-tailed Student’s $t$-test, *, $p<0.05$ and ***, $p<0.001$). Fasting plasma (B) total cholesterol and (C) triglyceride levels were analyzed by enzymatically colorimetric methods. (D) Lipoprotein fractions VLDL, LDL, and HDL were isolated from the plasma, and the cholesterol levels of VLDL, LDL, and HDL were examined by colorimetric methods. ($n=5–6$, two-way ANOVA, Bonferroni multiple comparisons test for multiple comparisons, *, $p<0.05$). Error bars $\text{represent}\pm \text{SEM}$. The numerical data corresponding to this figure are shown in Tables S17–S18. Note: ANOVA, analysis of variance; CYP3A11, cytochrome P450, family 3, subfamily A, polypeptide 11; DCHP, dicyclohexyl phthalate; DMSO, dimethyl sulfoxide; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; MDR1a, multidrug resistance protein 1a; MTP, microsomal triglyceride transfer protein; NPC1L1, Niemann-Pick C 1-Like 1; PXR, pregnane X receptor; ${\text{PXR}}^{–/–}$, pregnane X receptor deficient; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; VLDL, very-low-density lipoprotein-cholesterol.

Figure 8.

The impact of DCHP exposure circulating ceramide levels and ceramide-synthesis–related gene expression in PXR-humanized (huPXR) mice. Eight-wk-old male huPXR and ${\text{PXR}}^{–/–}$ littermates were treated with $10\mathrm{mg}/\mathrm{kg}$ BW per day of DCHP or vehicle control daily by oral gavage for 7 d. Plasma ceramide levels of (A) huPXR and (B) ${\text{PXR}}^{–/–}$ mice were measured by LC-MS/MS. ($n=5–6$, two-sample, two-tailed Student’s $t$-test, *, $p<0.05$). (C) Intestinal expression of ceramide-synthesis–related genes were measured by qPCR ($n=4–5$, two-sample, two-tailed Student’s $t$-test, *, $p<0.05$). (D) Immunoblotting of GBA1, MTP, and Actin proteins in the intestine of huPXR and ${\text{PXR}}^{–/–}$ mice. The densitometry analyses of the protein bands were shown below the immunoblotting panel ($n=3$, two-way ANOVA, Bonferroni multiple comparisons test for multiple comparisons, *, $p<0.05$, **, $p<0.01$, and ***, $p<0.001$). The numerical data corresponding to this figure are shown in Tables S19–S21. Results represent mean values. Error bars $\text{represent}\pm \text{SEM}$. Note: ANOVA, analysis of variance; DCHP, dicyclohexyl phthalate; GBA1, glucosylceramidase beta 1; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MTP, microsomal triglyceride transfer protein; ${\text{PXR}}^{–/–}$, pregnane X receptor deficient; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

Figure 9.

Schematic representation of the potential role of PXR in mediating the adverse effects of DCHP exposure on plasma lipid and ceramide levels. DCHP, a widely used phthalate, is a potent agonist of PXR. Exposure to DCHP leads to higher plasma cholesterol and ceramide levels in mice in an intestinal PXR-dependent manner. DCHP-mediated PXR activation regulates the expression of key lipogenic genes (e.g., NPC1L1 and MTP) (A) and ceramide-synthesis–related genes (e.g., GBA1) (B) in the intestine. Intestinal PXR signaling may contribute to adverse effects of DCHP on cardiovascular health through both hyperlipidemia-dependent and -independent mechanisms. This figure was created using BioRender.com. Note: DCHP, dicyclohexyl phthalate; GBA1, glucosylceramidase beta 1; MTP, microsomal triglyceride transfer protein; NPCIL1, Niemann-Pick C1-Like 1; PXR, pregnane X receptor.