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Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.

ABSTRACT: The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing enzymes (DMEs) and transporters. This study evaluated differential detergent fractionation (DDF) of membrane protein from progressively smaller numbers of primary mouse hepatocytes (5 million down to 50,000 cells) and limited liver tissue (25-50 mg) in quantifying select DMEs and transporters by QTAP. Two non-ionic detergents, digitonin and Triton-X-100, were applied in sequence to permeabilize cells and extract membrane proteins. Comparison was made with a membrane protein extraction kit and with homogenization in hypotonic buffer and subsequent differential centrifugation (DC). DDF produced linear membrane protein yields with increasing hepatocyte numbers and better permeabilization evidenced by the higher ratio of cytosolic to membrane protein yields. DDF produced 5-times more membrane protein from liver tissue than DC. The concentration of DMEs and transporters remained consistent in the fractions prepared by DDF from progressively smaller numbers of hepatocytes, but declined in kit fractions. In liver tissue, the concentrations were comparatively higher in DDF versus kit and DC. In conclusion, sequential digitonin and Triton-X-100 fractionation of membrane protein from limited samples is efficient, reproducible and cost-effective for QTAP of DMEs and transporters.

SUBMITTER: Qasem RJ

PROVIDER: S-EPMC7750260 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:INTRODUCTION:Sandwich-cultured human hepatocytes (SCHHs) are the most common in vitro hepatocyte model used for studying hepatic drug disposition and hepatotoxicity. Targeted quantification of key DME and transporter protein expression is useful for in vitro-in vivo extrapolation of drug and xenobiotic clearance and developing corresponding PBPK models. However, established methods for comprehensive quantification of drug metabolizing enzyme (DMEs) and transporter expression in SCHHs are lacking. In this study, a targeted quantitative proteomic isotope dilution nanoLC-MS/MS method developed in our laboratory was adapted to quantify a panel of phase I & II DMEs and transporter proteins in SCHHs under basal and induced conditions. METHODS:SCHHs were treated with known inducers of DMEs (Rifampin: PXR activator, CITCO: CAR activator) and transporters (CDCA: FXR activator) or with vehicle control (DMSO) for 72?h. Membrane protein was isolated from the SCHHs using a membrane extraction kit and 30??g membrane protein was digested with trypsin. The resulting peptides were analyzed by isotope dilution nanoLC-MS/MS to quantify the DMEs and transporters. RESULTS:Using the method, we could quantify fourteen phase I and ten phase II DMEs, and twelve uptake/efflux transporters, under basal and induced conditions in the SCHHs. Analysis showed donor to donor variation in basal protein levels of CYP450s, UGTs and transporters, and that basal protein expression of CYP450s and UGTs was higher than that of transporters. In addition, induction of key proteins in response to rifampin, CITCO and CDCA was observed. DISCUSSION:We have successfully quantified protein abundance of multiple phase I and II DMEs and uptake and efflux transporters in SCHHs using a method previously developed in our laboratory. Our method is sufficiently sensitive to quantify inter-donor differences in protein concentrations at the basal level as well as changes in protein expression in response to endogenous and exogenous stimuli.

Project description: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.

Project description:PurposeChronic exposure to pesticides has been associated with thyroid dysfunction owing to their endocrine disruption ability. Genetic variations in genes encoding phase I and II enzymes and phase III transporters are partly responsible for individual responses to chemical pesticides. This study investigated the association between variations in genes involved in pesticide metabolism and altered thyroid hormone concentrations.MethodsWe assessed thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) in organic agriculture workers (n = 216) and workers who used chemical pesticides (n = 229). A questionnaire was used to collect sociodemographic, pesticide exposure, and health status data. Blood samples were analyzed for TSH, FT3, and FT4. Genomic DNA was extracted and genotyped using the TaqMan real-time PCR genotyping assay and restriction fragment length polymorphism method for 15 metabolically related genes.ResultsSignificant differences in the TSH (1.58 vs 1.12 µIU/mL) and FT3 (0.34 vs 0.31 ng/dL) concentrations between the chemical and organic worker groups were observed. The frequencies of all single nucleotide polymorphisms were in Hardy-Weinberg equilibrium and were mostly consistent with Asian populations. The findings showed the association between SNPs of enzymes and transporters and TSH, FT3, and FT4. The odd ratio and adjusted odd ratio (with sex, age, smoking status, alcohol consumption and exposure parameters) for subclinical thyroid disease by the variant alleles CYP1A1 rs1048943, CYP2B6 rs2279343, CYP2C19 rs4244285, NAT2 rs1799931, and PON1 rs662 in the chemical workers compared with the organic workers were found (P values < 0.05).ConclusionThis is the first study to assess gene-environment interactions in Thai agricultural workers by investigating disruptions of the hypothalamic-pituitary-thyroid axis. The investigated SNP profiles revealed several gene-thyroid hormone associations in which even low levels of pesticide exposure could disturb thyroid homeostasis. These findings provide a foundation for planning future studies investigating associations between complex diseases and occupational pesticide exposure.

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Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.

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