Impact of Microbiome on Hepatic Metabolizing Enzymes and Transporters in Mice during Pregnancy

Abstract

The microbiome and pregnancy are known to alter drug disposition, yet the interplay of the two physiologic factors on the expression and/or activity of drug metabolizing enzymes and transporters (DMETs) is unknown. This study investigated the effects of microbiome on host hepatic DMETs in mice during pregnancy by comparing four groups of conventional (CV) and germ-free (GF) female mice and pregnancy status, namely, CV nonpregnant, GF non-pregnant, CV pregnant, and GF pregnant mice. Transcriptomic and targeted proteomics of hepatic DMETs were profiled by using multiomics. Plasma bile acid and steroid hormone levels were quantified by liquid chromatography tandem mass spectrometry. CYP3A activities were measured by mouse liver microsome incubations. The trend of pregnancy-induced changes in the expression or activity of hepatic DMETs in CV and GF mice was similar; however, the magnitude of change was noticeably different. For certain DMETs, pregnancy status had paradoxical effects on mRNA and protein expression in both CV and GF mice. For instance, the mRNA levels of Cyp3a11, the murine homolog of human CYP3A4, were decreased by 1.7-fold and 3.3-fold by pregnancy in CV and GF mice, respectively; however, the protein levels of CYP3A11 were increased similarly ∼twofold by pregnancy in both CV and GF mice. Microsome incubations revealed a marked induction of CYP3A activity by pregnancy that was 10-fold greater in CV mice than that in GF mice. This is the first study to show that the microbiome can alter the expression and/or activity of hepatic DMETs in pregnancy. SIGNIFICANCE STATEMENT: We demonstrated for the first time that microbiome and pregnancy can interplay to alter the expression and/or activity of hepatic drug metabolizing enzymes and transporters. Though the trend of pregnancy-induced changes in the expression or activity of hepatic drug metabolizing enzymes and transporters in conventional and germ-free mice was similar, the magnitude of change was noticeably different.

Fig. 1.

Comparison of expression of selected genes determined by RNA-seq and qRT-PCR analysis. Log twofold change is relative to the CVNP group for both RNA-seq and qRT-PCR data. Shown are means ± S.D. of gene expression data from five to six different mouse liver tissues.

Fig. 2.

Effect of pregnancy and microbiome on mRNA expression of hepatic phase I enzymes. Shown are boxplots with individual scatters of RNA-seq analysis data of hepatic phase I enzymes from female C57BL/6 mice. Data illustrates individual log2 counts per million for each DMET. All pregnant mice used were on gestation day 15. *FDR < 0.1; **FDR < 0.01; ***FDR < 0.001.

Fig. 3.

Effect of pregnancy and microbiome on mRNA expression of hepatic phase II enzymes. Shown are boxplots with individual scatters of RNA-seq analysis data of hepatic phase II enzymes from female C57BL/6 mice. Data illustrates individual log2 counts per million for each DMET. All pregnant mice used were on gestation day 15. *FDR < 0.1; **FDR < 0.01; ***FDR < 0.001.

Fig. 4.

Effect of pregnancy and microbiome on mRNA expression of hepatic transporters. Shown are boxplots with individual scatters of RNA-seq analysis data of hepatic transporters from female C57BL/6 mice. Data illustrates individual log2 counts per million for each DMET. All pregnant mice used were on gestation day 15. *FDR < 0.1; **FDR < 0.01; ***FDR < 0.001.

Fig. 5.

Effect of pregnancy and microbiome on protein expression of hepatic P450 enzymes. Shown are boxplots with individual data points of relative protein levels of hepatic P450 enzymes in C57BL/6 female mice. Shown are means ± S.E. for selected enzymes or transporters in the liver from four to six mice. Data illustrates ratio-of-ratio estimates for each DMET. All pregnant mice used were on gestation day 15. *P < 0.05; **P < 0.01 by Wilcoxon signed-rank test.

Fig. 6.

Effect of pregnancy and microbiome on protein expression of hepatic transporters. Shown are boxplots with individual data points of relative protein levels of hepatic transporters in C57BL/6 female mice. Shown are means ± S.E. for selected enzymes or transporters in the liver from four to six mice. Data illustrates ratio-of-ratio estimates for each DMET. All pregnant mice used were on gestation day 15. *P < 0.05; **P < 0.01 by Wilcoxon signed-rank test.

Fig. 7.

Effect of pregnancy and microbiome on hepatic Cyp3a activity. Shown are boxplots of individual data points of hepatic Cyp3a activity, which is presented as relative light unit. Circles represent incubations without an inhibitor, and triangles indicate incubations in the presence of the inhibitor ketoconazole at 5 μM. Data shown are means ± S.E. of hepatic Cyp3a activity in liver microsomes isolated from four to six mice from one representative experiment. The experiment was done in triplicate and repeated, and similar results were obtained. All pregnant mice used were on gestation day 15. *P < 0.05; **P < 0.01 by Wilcoxon signed-rank test.

Fig. 8.

Effect of pregnancy and microbiome on plasma concentrations of primary bile acids. Shown are boxplots with individual data points of plasma concentrations of primary bile acids in C57BL/6 female mice. Concentrations were determined using targeted LC-MS/MS quantification and reported as nanograms per milliliter. Shown are means ± S.E. for selected bile acids or steroid hormones in the plasma from five to six mice. All pregnant mice used were on gestation day 15. *P < 0.05; **P < 0.01 by Wilcoxon signed-rank test.

Fig. 9.

Effect of pregnancy and microbiome on plasma concentrations of secondary bile acids. Shown are boxplots with individual data points of plasma concentrations of secondary bile acids in C57BL/6 female mice. Concentrations were determined using targeted LC-MS/MS quantification and reported as nanograms per milliliter. Shown are means ± S.E. for selected bile acids or steroid hormones in the plasma from five to six mice. All pregnant mice used were on gestation day 15. *P < 0.05; **P < 0.01; by Wilcoxon signed-rank test.

Fig. 10.

Effect of pregnancy and microbiome on plasma concentrations of steroid hormones. Shown are boxplots with individual data points of plasma concentrations of steroid hormones in C57BL/6 female mice. Concentrations were determined using targeted LC-MS/MS quantification and reported as nanograms per milliliter. Shown are means ± S.E. for selected bile acids or steroid hormones in the plasma from five to six mice. All pregnant mice used were on gestation day 15. *P < 0.05; **P < 0.01 by Wilcoxon signed-rank test.