TPS 711: The exposome, Exhibition Hall, Ground floor, August 26, 2019, 3:00 PM - 4:30 PM Background: Untargeted metabolomics analysis is a valuable tool in the field of metabolic profiling of biological samples, but it has been less widely applied to characterization of environmental mixtures. The current study explores using untargeted analysis of environmental samples of air pollution for component analysis and source appointment. Methods: Particulate matter (PM) in wood smoke and diesel exhaust were collected in laboratory settings at the University of Washington. Total suspended particles were collected using 37 mm Teflon filtration membranes (PALL Corporation, USA) in open-face cassettes attached to air sampling pumps (AirChek XR5000, SKC Inc., USA). After sample extraction (Methanol), concentration and reconstitution (25μL Methanol and 25μL 0.4% (v/v) acetic acid), PM samples were analyzed by accurate-mass Q-TOF (Agilent 6500) for separation and qualification. We used MetaboAnalyst (version 4.0) to perform principal component analysis (PCA) to visualize the data and t-tests to compare chemical features between wood smoke and diesel exhaust groups. Results: We collected and analyzed six wood smoke PM samples (weight = 65 ± 3 μg) and five diesel exhaust PM samples (average weight = 150 ± 1 μg). Using Q-TOF HPLC/MS, we detected 1513 chemical features from the PM samples. In a volcano plot, a total of 112 chemical features were identified that exhibited differences (fold-change > 2, and p-value < 0.05) between PM from the wood smoke and diesel exhaust. PCA showed that 81.3% of the variance was explained when including the 5 most significant principal components (PC). Both the 2-D scores plot and 3-D score plot between selected PCs totally separated wood smoke and diesel exhaust PM samples. Conclusion: Untargeted metabolomics analysis detected numerous chemical features from wood smoke and diesel exhaust PM samples. The untargeted analysis has the potential to be used in air pollution studies for components analysis and source appointment of PM.

It was previously shown that CYP3A4 is induced in the human intestinal Caco-2 cell model by treatment with 1 alpha ,25-dihydroxy vitamin D-3 (1,25-D-3). We demonstrate the vitamin D analog, 19-nor-1 alpha ,25-dihydroxy vitamin D-2, is also an effective inducer of CYP3A4 in Caco-2 cells, but with half the potency of 1,25-D-3. We report that treatment of LS180 cells, a human intestinal cell line, with 1 to 10 nM 1,25-D-3 dose dependently increased CYP3A4 protein and CYP3A4 mRNA expression. CYP3A4- and CYP3A23-promoter-Luciferase reporter constructs transiently transfected into LS180 cells were transcriptionally activated in a dose-dependent manner by 1,25-D-3, whereas mutation of the nuclear hormone receptor binding motif (ER6) in the CYP3A4 promoter abrogated 1,25-D-3 activation of CYP3A4. Although the CYP3A4 ER6 promoter element has been shown to bind the pregnane X receptor (PXR), this receptor does not mediate 1,25-D-3 induction of CYP3A4 because a) PXR is not expressed in Caco-2 cells; b) PXR mRNA expression is not induced by 1,25-D-3 treatment of LS180 cells; and c) the ligand binding domain of human PXR was not activated by 1,25-D-3. 1,25-D-3 uses the vitamin D receptor to induce CYP3A4 because a) the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimer binds specifically to the CYP3A4 ER6; b) selective mutation of the CYP3A4 ER6 disrupted the binding of VDR-RXR; and c) reporter constructs containing only three copies of the CYP3A4 ER6 linked to a TK-CAT reporter were activated by 1,25-D-3 only in cells cotransfected with a human VDR expression plasmid. These data support the hypothesis that 1,25-D-3 and VDR induce expression of intestinal CYP3A by binding of the activated VDR-RXR heterodimer to the CYP3A PXR response element and promoting gene transcription.

Activity of cytochrome P450 3A4 (CYP3A4), the most abundant human P450 isoform and responsible for metabolizing approximately half of all therapeutic agents, has been speculated to vary during the menstrual cycle. This investigation evaluated CYP3A4 activity during the menstrual cycle, using midazolam clearance as a metabolic probe. Midazolam (1 mg IV) was administered to nonsmoking, nonpregnant female volunteers (N = 11, age 26 ± 5 years) with normal menstrual cycles on three separate occasions during the same cycle: days 2 (menstrual phase), 13 (estradiol peak), and 21 (progesterone peak). Venous plasma midazolam concentrations were determined by gas chromatography—mass spectrometry. Midazolam clearance was determined by noncompartmental and compartmental analysis. Midazolam plasma disposition did not differ between phases of the menstrual cycle. There was no significant difference in any measure of midazolam clearance. Noncompartmental clearances (mean ± SD) were 7.36 ± 2.73, 6.34 ± 3.59, and 6.23 ± 2.04 ml/kg/min, respectively, on days 2, 13, and 21 of the menstrual cycle. These results suggest no difference in hepatic CYP3A4 activity on menstrual cycle days 2, 13, and 21. Consideration of menstrual cycle variability in the metabolism of CYP3A4 substrates does not appear indicated in the dosing or design of clinical trials.

Clinical investigations using isoform‐selective probes to phenotype cytochrome P450 activity and interaction studies using isoform‐selective inhibitors to determine P450 involvement in drug metabolism assume minimal interday variability in P450 activity. CYP3A4 is the most abundant human P450 isoform and metabolizes approximately half of all therapeutic agents. This investigation evaluated interday variability in hepatic CYP3A4 activity in males, using the clearances of midazolam and alfentanil as metabolic probes. Midazolam (1 mg) followed 1 hour later by alfentanil (20 μg/kg) were administered by intravenous bolus to 9 nonsmoking male volunteers (ages 30 8 years). Drug administration was repeated 12 and 20 days later. Venous plasma midazolam and alfentanil concentrations were determined by gas chromatography/mass spectrometry. Drug clearances were determined by noncompartmental and multiexponential analysis. There were no significant interday differences in plasma drug concentrations or clearances (3.9 1.4, 3.9 1.7, and 4.2 1.7 ml/kg/min for alfentanil, respectively, and 6.6 2.0, 7.9 2.4, and 7.9 2.5 ml/kg/min for midazolam, respectively, on days 1, 13, and 21 [mean SD]). Interday variability in clearance was 13% 6% and 19% 12% for alfentanil and midazolam, respectively. Interday variability in the clearance of these probes, and presumably hepatic CYP3A4 activity, was small compared with interindividual variability. Consideration of interday variability in the hepatic metabolism of CYP3A4 substrates does not appear significant in the design of clinical trials.