Physiological Regulation of Drug Metabolism and Transport: Pregnancy, Microbiome, Inflammation, Infection, and Fasting

Abstract

This article is a report on a symposium entitled "Physiological Regulation of Drug Metabolism and Transport" sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2017 meeting in Chicago, IL. The contributions of physiologic and pathophysiological regulation of drug-metabolizing enzymes and transporters to interindividual variability in drug metabolism are increasingly recognized but in many cases are not well understood. The presentations herein discuss the phenomenology, consequences, and mechanism of such regulation. CYP2D6 transgenic mice were used to provide insights into the mechanism of regulation of this enzyme in pregnancy, via hepatocyte nuclear factor 4α, small heterodimer partner, and retinoids. Regulation of intestinal and hepatic drug-processing enzymes by the intestinal microbiota via tryptophan and its metabolites was investigated. The potential impact of parasitic infections on human drug metabolism and clearance was assessed in mice infected with Schistosoma mansoni or Plasmodium chabaudi chabaudi AS, both of which produced widespread and profound effects on murine hepatic drug-metabolizing enzymes. Finally, the induction of Abcc drug efflux transporters by fasting was investigated. This was demonstrated to occur via a cAMP, protein kinase A/nuclear factor-E2-related factor 2/Sirtuin 1 pathway via antioxidant response elements on the Abcc genes.

Fig. 1.

Female hormones up-regulate expression of P450 genes in female human hepatocytes. Primary human hepatocytes (HH) from different donors (n = 3/batch) were treated with (A) estradiol (1 µM), (B) progesterone (10 µM), or vehicle (ethanol) for 72 hours with frequent media changes. The mRNA levels of P450 isoforms were measured by RT-qPCR, and estimated relative to glyceraldehyde 3-phosphate dehydrogenase expression using the ΔΔCt method. Data shown are P450 expression in hormone-treated cells relative to that in vehicle-treated cells (mean ± S.D.). The data are taken from Figs. 2 and 5 of Choi et al. (2013).

Fig. 2.

(A) Relative abundances of bacteria in large intestinal content of CV mice (n = 3 per group). The 16S ribosomal RNA sequencing data were analyzed by QIIME and are expressed as the percentage of operational taxonomic units (OTUs). Examples of differentially regulated intestinal bacteria with a mean composition greater than 0.01% for at least one treatment group are shown at the class level. Letters (a and b) represent treatment subgroups with significantly differentiated means (analysis of variance followed by Duncan’s post hoc test, P < 0.05). (B) RT-qPCR quantification of mRNAs in various sections of intestine of CV mice treated with corn oil (vehicle) (n = 3) or tryptophan (10, 20, or 40 mg/kg; n = 5 per group) (left panel), as well as GF mice treated with phosphate-buffered saline (PBS, vehicle) or IPA (40 mg/kg; n = 3 per group). Data are expressed as percentage of β-actin. Statistical significance between groups was determined by analysis of variance followed by Duncan’s post hoc test (P < 0.05) for the tryptophan study and Student’s t test (P < 0.05) for the IPA study. Data are presented as mean ± S.E.M. *Statistically significant differences between vehicle and treatment groups.

Fig. 3.

(A) RT-qPCR quantification of mRNAs in the liver of CV mice orally gavaged with corn oil (vehicle) (n = 3) or tryptophan (10, 20, or 40 mg/kg; n = 5 per group), as well as GF mice orally gavaged with PBS (vehicle) or IPA (40 mg/kg; n = 3 per group). Data are expressed as percentage of β-actin. (B) Quantification of IPA by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) in liver of CV mice normalized to liver mass. (C) RT-qPCR quantification of mRNAs in HepaRG cells treated with 0.1% dimethyl sulfoxide (DMSO, vehicle), tryptophan (10, 100, or 250 µM), and IPA (100 or 250 µM). Statistical significance between groups was determined by analysis of variance followed by Duncan’s post hoc test (P < 0.05) for the tryptophan study and Student’s t test (P < 0.05) for the IPA study. Data are presented as mean ± S.E.M. *Statistically significant differences between vehicle and treatment groups.

Fig. 4.

Effect of S. mansoni infection on protein expression of hepatic P450s in female Swiss Webster mice. Reproduced from Mimche et al. (2014). Mice were infected with S. mansoni and sacrificed at 30 and 45 dpi, and livers were harvested. P450 protein levels were assessed by Western blotting S9 fractions. Each lane corresponds to a different mouse and contains 32 μg S9 protein, except the 45-days-infected group, which has 128 μg. All the primary antibodies used for Western blotting were polyclonal, from rabbit: Cyp2a, anti-human CYP2A13 (Sc-33214) from SantaCruz Biotechnology (Dallas, TX); Cyp 2b and 3a, anti-rat CYP2B1 and CYP3A1 from Dr. James Halpert; Cyp2d9, anti-mouse Cyp2d9 from Dr. Masahiko Negishi; Cyp4a, anti-human CYP4A11 (Ab-140635) from Abcam (Cambridge, MA); and Cyp2c, anti-rat CYP2C11 prepared in our own laboratory. Additional Western blotting details are in Mimche et al. (2014).

Fig. 5.

Fasting-mediated induction of Abcc2-4 is cAMP-PKA and Nrf2 dependent. Adult, male C57BL/6 mice (n = 6–8/group) were fed ad libitum or food withheld (fasted) for 18–24 hours. (A) Biliary concentration of DBSP was measured spectrophometrically 45 minutes after the fed and fasted mice were injected with DBSP (150 μmol/kg per 5 ml i.p.) or saline. (B) Liver protein expression of Abcc2–4. Crude liver membrane preparations were isolated from fed and fasted mice in a sucrose/Tris-based buffer using differential centrifugation followed by Western blot analysis for expression of Abcc2–4. (C) The mRNA expression of Nqo1 and Nrf2. Total RNA was isolated from livers of fed and fasted mice, and mRNA expression of Nqo1 and Nrf2 was quantified using RT-qPCR. (D) Increased nuclear translocation of Nrf2 primary mouse hepatocytes treated with cAMP-PKA activator (8-Br-cAMP). Cytosolic and nuclear fractions were obtained from control and 8-Br-cAMP–treated hepatocytes and immunoblotted for Nqo1 and Nrf2 expression. All data are expressed as average ± S.E.M. (n = 6–8/ group mouse livers, n = 3–4 replicates for in vitro experiments). P ≤ 0.05 was considered statistically significant. Groups without a common letter are statistically significantly different. Figures reprinted with permission from Kulkarni et al. (2014).

Fig. 6.

The cAMP-PKA pathways activate NRF2 and increase NRF2 and SIRT1 recruitment at ABCC2-ARE: (A) Treatment of hepatic cells with cAMP analog induces dose-dependent activation of antioxidant response element (ARE). Huh7 cells were transiently transfected with an ARE–luciferase pRL–CMV for 24 hours and then treated with 0.1 or 1.0 mM 8-Br-cAMP for 24 hours; luciferase activity was measured using a Dual Luciferase Assay (Promega, Madison, WI). (B) The activation of Nrf2 is diminished when treated with the PKA inhibitor H-89. Primary mouse hepatocytes were obtained from ARE-hPAP reporter mice and treated with 8-Br-cAMP with or without H-89 for 24 hours. Total RNA was isolated, and hPAP expression was quantified using RT-qPCR. (C) NRF2 and SIRT1 enrichment at ABCC2 promoter ARE was determined by chromatin immunoprecipitation assay. Briefly, Huh-7 cells were treated with 8-Br-cAMP (0.1 mM) for 45 minutes. The cells were then crosslinked, sheared, and incubated overnight with ProteinG beads and anti-NRF2 or SIRT1 antibody as well as control preimmune IgG. The chromatins and input DNA were analyzed by PCR for DNA fragments containing NRF2 bound-ARE-like element. All data are expressed as average ± S.E.M. (n = 3–4 replicates for in vitro experiments). P ≤ 0.05 was considered statistically significant. Groups without a common letter are statistically significantly different. Figures reprinted with permission from Kulkarni et al. (2014).