The discovery of gut microbial metabolites as modulators of host susceptibility to acetaminophen-induced hepatotoxicity.

The mammalian gut microbiota plays diverse and essential roles in modulating host physiology. Key mediators determining the outcome of the microbiota-host interactions are the small molecule metabolites produced by the gut microbiota. The liver is the organ massively exposed to gut microbial metabolites, and it serves as the nexus, maintaining healthy interactions between the gut microbiota and host. At the same time, the liver is the primary target of harmful gut microbial metabolites. This review provides an up-to-date list of gut microbial metabolites identified to increase or decrease host susceptibility to APAP-induced liver injury. Signaling pathways and molecular factors involved in the progression of APAP-induced hepatotoxicity are well-established, and we propose that the mouse model of APAP-induced hepatotoxicity serves as an excellent system for uncovering gut microbial metabolites of previously unknown function. Moreover, we envision that gut microbial metabolites identified to alter APAP-induced hepatotoxicity likely have broader implications in other liver diseases. Significance Statement This review provides an overview of recent discoveries from investigating whether and how the gut microbiota modulates the host susceptibility to APAP-induced liver injury. It focuses on the roles of gut bacterial small molecule metabolites as mediators of the interaction between the gut microbiota and the liver. It also illustrates the utility of APAP-induced liver injury as a model to identify gut microbial metabolites with biological function.

Phenylpropionic acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity.

The gut microbiota affects hepatic drug metabolism. However, gut microbial factors modulating hepatic drug metabolism are largely unknown. In this study, using a mouse model of acetaminophen (APAP)-induced hepatotoxicity, we identified a gut bacterial metabolite that controls the hepatic expression of CYP2E1 that catalyzes the conversion of APAP to a reactive, toxic metabolite. By comparing C57BL/6 substrain mice from two different vendors, Jackson (6J) and Taconic (6N), which are genetically similar but harbor different gut microbiotas, we established that the differences in the gut microbiotas result in differential susceptibility to APAP-induced hepatotoxicity. 6J mice exhibited lower susceptibility to APAP-induced hepatotoxicity than 6N mice, and such phenotypic difference was recapitulated in germ-free mice by microbiota transplantation. Comparative untargeted metabolomic analysis of portal vein sera and liver tissues between conventional and conventionalized 6J and 6N mice led to the identification of phenylpropionic acid (PPA), the levels of which were higher in 6J mice. PPA supplementation alleviated APAP-induced hepatotoxicity in 6N mice by lowering hepatic CYP2E1 levels. Moreover, PPA supplementation also reduced carbon tetrachloride-induced liver injury mediated by CYP2E1. Our data showed that previously known PPA biosynthetic pathway is responsible for PPA production. Surprisingly, while PPA in 6N mouse cecum contents is almost undetectable, 6N cecal microbiota produces PPA as well as 6J cecal microbiota in vitro, suggesting that PPA production in the 6N gut microbiota is suppressed in vivo. However, previously known gut bacteria harboring the PPA biosynthetic pathway were not detected in either 6J or 6N microbiota, suggesting the presence of as-yet-unidentified PPA-producing gut microbes. Collectively, our study reveals a novel biological function of the gut bacterial metabolite PPA in the gut-liver axis and presents a critical basis for investigating PPA as a modulator of CYP2E1-mediated liver injury and metabolic diseases.

Temporal changes in the systemic concentrations of retinoids in pregnant and postpartum women.

Retinoids and vitamin A are essential for multiple biological functions, including vision and immune responses, as well as the development of an embryo during pregnancy. Despite its importance, alterations in retinoid homeostasis during normal human pregnancy are incompletely understood. We aimed to characterize the temporal changes in the systemic retinoid concentrations across pregnancy and postpartum period. Monthly blood samples were collected from twenty healthy pregnant women, and plasma concentrations of retinol, all-trans-retinoic acid (atRA), 13-cis-retinoic acid (13cisRA), and 4-oxo-retinoic acids were measured using liquid chromatography-tandem mass spectrometry. Significant decreases in 13cisRA concentrations over the pregnancy were observed, with rebound increases in retinol and 13cisRA levels after delivery. Of note, atRA concentrations exhibited a unique temporal pattern with levels peaking at mid-pregnancy. While the 4-oxo-atRA concentration was below the limit of quantification, 4-oxo-13cisRA was readily detectable, and its temporal change mimicked that of 13cisRA. The time profiles of atRA and 13cisRA remained similar after correction by albumin levels for plasma volume expansion adjustment. Together, the comprehensive profiling of systemic retinoid concentrations over the course of pregnancy provides insights into pregnancy-mediated changes in retinoid disposition to maintain its homeostasis.

Size optimization of carfilzomib nanocrystals for systemic delivery to solid tumors.

Carfilzomib (CFZ) is a second-generation proteasome inhibitor effective in blood cancer therapy. However, CFZ has shown limited efficacy in solid tumor therapy due to the short half-life and poor tumor distribution. Albumin-coated nanocrystal (NC) formulation was shown to improve the circulation stability of CFZ, but its antitumor efficacy remained suboptimal. We hypothesize that NC size reduction is critical to the formulation safety and efficacy as the small size would decrease the distribution in the reticuloendothelial system (RES) and selectively increase the uptake by tumor cells. We controlled the size of CFZ-NCs by varying the production parameters in the crystallization-in-medium method and compared the size-reduced CFZ-NCs (z-average of 168 nm, NC168) with a larger counterpart (z-average of 325 nm, NC325) as well as the commercial CFZ formulation (CFZ-CD). Both CFZ-NCs showed similar or higher cytotoxicity than CFZ-CD against breast cancer cells. NC168 showed greater uptake by cancer cells, less uptake by macrophages and lower immune cell toxicity than NC325 or CFZ-CD. NC168, but not NC325, showed a similar safety profile to CFZ-CD in vivo. The biodistribution and antitumor efficacy of CFZ-NCs in mice were also size-dependent. NC168 showed greater antitumor efficacy and tumor accumulation but lower RES accumulation than NC325 in 4T1 breast cancer model. These results support that NC formulation with an optimal particle size can improve the therapeutic efficacy of CFZ in solid tumors.

Syntheses and studies of deuterated Imdiazo[1,2-a]pyridine-3-carboxamides with potent anti-tuberculosis activity and improved metabolic properties.

The imidazo[1,2-a]pyridine-3-carboxyamides (IAPs) are a unique class of compounds endowed with impressive nanomolar in vitro potency against Mycobacterium tuberculosis (Mtb) as exemplified by clinical candidate Telacebec (Q203). These compounds target mycobacterial respiration through inhibition of the QcrB subunit of cytochrome bc1:aa3 super complex resulting in bacteriostatic efficacy in vivo. Our labs have had a long-standing interest in the design and development of IAPs. However, some of these compounds suffer from short in vivo half-lives, requiring multiple daily dosing or the addition of a cytochrome P450 inhibitor for murine efficacy evaluations. Deuteration has been shown to decrease metabolism as the C-D bond is stronger than the CH bond. Herein we describe our efforts on design and synthesis of potent deuterated IAPs and the effect that deuteration has upon metabolism through microsomal stability studies.

Anti-contamination SMART (Spectrum Monitoring Apparatus with Roll-to-roll Transparent film) window for optical diagnostics of plasma systems.

Optical emission spectroscopy is widely used in semiconductor and display manufacturing for plasma process monitoring. However, because of the contamination of the viewport, quantitative analysis is extremely difficult; therefore, qualitative analysis is used to detect species in the process. To extend plasma monitoring in advanced precise processes, the contamination problem of the viewport must be solved. We propose a new spectrum monitoring apparatus with a roll-to-roll transparent film window for optical diagnostics of a plasma system. By moving a transparent film in front of the viewport, contamination in the emission light path becomes negligible. However, the speed of the film should be optimized to reduce the maintenance period and to minimize measurement errors. We calculated the maximum thickness of SiO2, Si3N4, ITO, and the Ar/CHF3 plasma contaminant to suppress the electron temperature error measured by the line-intensity-ratio within 2% at 2 eV. The thickness of the Si3N4, ITO, and Ar/CHF3 plasma contaminant should be thinner than 12.5 nm, 7.5 nm, and 100 nm, respectively.

Gut microbiota in reductive drug metabolism.

Gut bacteria are predominant microorganisms in the gut microbiota and have been recognized to mediate a variety of biotransformations of xenobiotic compounds in the gut. This review is focused on one of the gut bacterial xenobiotic metabolisms, reduction. Xenobiotics undergo different types of reductive metabolisms depending on chemically distinct groups: azo (-NN-), nitro (-NO2), alkene (-CC-), ketone (-CO), N-oxide (-NO), and sulfoxide (-SO). In this review, we have provided select examples of drugs in six chemically distinct groups that are known or suspected to be subjected to the reduction by gut bacteria. For some drugs, responsible enzymes in specific gut bacteria have been identified and characterized, but for many drugs, only circumstantial evidence is available that indicates gut bacteria-mediated reductive metabolism. The physiological roles of even known gut bacterial enzymes have not been well defined.

Methods to study mechanisms underlying altered hepatic drug elimination during pregnancy.

Hepatic drug metabolism is a major route of drug elimination, mediated by multiple drug-metabolizing enzymes. Any changes in the rate and extent of hepatic drug metabolism can lead to altered drug efficacy or toxicity. Accumulating clinical evidence indicates that pregnancy is accompanied by changes in hepatic drug metabolism. In this article, we discuss in vitro and in vivo tools used to study the mechanisms underlying the altered drug metabolism during pregnancy, focusing on primary hepatocyte culture, transgenic animal models, and use of probe drugs to assess change in enzymatic activity. The information obtained from these studies has enabled prediction of clinical PK changes for a given drug in pregnant women.

Characterization of Vitamin A Metabolome in Human Livers With and Without Nonalcoholic Fatty Liver Disease.

Retinoids are essential endogenous compounds involved in regulation of critical biologic processes, including maintenance of metabolic homeostasis in the liver. Much of the knowledge of altered retinoid homeostasis in human disease states is derived from changes in indirect markers such as mRNA expression of retinoid-related genes and circulating concentrations of retinol or its binding protein RBP4. We hypothesized that in the human liver, concentrations of the active retinoid all-trans-retinoic acid (atRA) correlate with the concentrations of retinyl palmitate (RP), the storage form of atRA, retinol, the inactive vitamin A, and the mRNA expression of retinoid-related genes. On the basis of existing knowledge of altered vitamin A homeostasis in metabolic syndrome, we also predicted that in human livers with nonalcoholic fatty liver disease (NAFLD) retinoid concentrations would be decreased. Using novel liquid chromatography-tandem mass spectrometry methods, the hepatic vitamin A metabolome was quantified in normal human livers (n = 50) and 22 livers from donors with NAFLD. The hepatic concentrations of RP, atRA, 13-cisRA, and 4-oxo-atRA were significantly decreased in NAFLD samples in comparison with normal liver samples, whereas retinol levels remained unchanged. The concentrations of atRA were positively correlated with RP and 13-cisRA but not with retinol or the relative mRNA expression of LRAT, ALDH1A1, CYP26A1, RARα, and RARß An active metabolite of atRA, 4-oxo-atRA was, for the first time, detected in human tissues at comparable concentration with RA isomers, suggesting this retinoid may contribute to retinoid signaling in humans. SIGNIFICANCE STATEMENT: This study shows that in NAFLD liver vitamin A homeostasis is disrupted potentially contributing to disease progression. The results show that interpretation of retinoid homeostasis on the basis of indirect markers such as retinol concentrations or mRNA data is probably misleading when evaluating human disease processes, and analysis of the broader retinoid metabolome is needed to characterize disease effects on retinoid signaling.

Determinants of Cytochrome P450 2D6 mRNA Levels in Healthy Human Liver Tissue.

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme that exhibits large interindividual variability. Recent studies suggest that differential transcriptional regulation of CYP2D6 in part may be responsible for the variability. In this study, we characterized potential determinants of CYP 2D6  transcript levels in healthy human liver tissue samples (n = 115), including genetic polymorphisms in CYP2D6 and the genes encoding transcription regulators for CYP2D6 expression; mRNA expression of the transcription factors and their known target genes; and hepatic levels of bile acids and retinoids, agents that modulate the expression/activity of the transcription factors. Their associations with CYP2D6 mRNA levels in the tissues were examined. Results from multivariable linear regression analysis revealed CYP8B1 mRNA level and rs3892097, the single- nucleotide polymorphism defining the nonfunctional CYP2D6*4 allele, as the two most significant predictors of CYP2D6 mRNA levels in the liver tissue samples, explaining 30% of the variability.

Repression of hepatocyte nuclear factor 4 alpha by AP-1 underlies dyslipidemia associated with retinoic acid.

All-trans retinoic acid (atRA) is used to treat certain cancers and dermatologic diseases. A common adverse effect of atRA is hypercholesterolemia; cytochrome P450 (CYP) 7A repression is suggested as a driver. However, the underlying molecular mechanisms remain unclear. We investigated CYP7A1 expression in the presence of atRA in human hepatocytes and hepatic cell lines. In HepaRG cells, atRA increased cholesterol levels dose-dependently alongside dramatic decreases in CYP7A1 expression. Lentiviral-mediated CYP7A1 overexpression reversed atRA-induced cholesterol accumulation, suggesting that CYP7A1 repression mediated cholesterol accumulation. In CYP7A1 promoter reporter assays and gene-knockdown studies, altered binding of hepatocyte nuclear factor 4 α (HNF4α) to the proximal promoter was essential for atRA-mediated CYP7A1 repression. Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) and ERK pathways attenuated atRA-mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP-1 (c-Jun/c-Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression. In DNA pull-down and chromatin immunoprecipitation assays, AP-1 exhibited sequence-specific binding to the proximal CYP7A1 promoter region overlapping the HNF4α binding site, and atRA increased AP-1 but decreased HNF4α recruitment to the promoter. Collectively, these results indicate that atRA activates JNK and ERK pathways and the downstream target AP-1 represses HNF4α transactivation of the CYP7A1 promoter, potentially responsible for hypercholesterolemia.

Commensal Gut Bacteria Convert the Immunosuppressant Tacrolimus to Less Potent Metabolites.

Tacrolimus exhibits low and variable drug exposure after oral dosing, but the contributing factors remain unclear. Based on our recent report showing a positive correlation between fecal abundance of Faecalibacterium prausnitzii and oral tacrolimus dose in kidney transplant patients, we tested whether F. prausnitzii and other gut abundant bacteria are capable of metabolizing tacrolimus. Incubation of F. prausnitzii with tacrolimus led to production of two compounds (the major one named M1), which was not observed upon tacrolimus incubation with hepatic microsomes. Isolation, purification, and structure elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy indicated that M1 is a C-9 keto-reduction product of tacrolimus. Pharmacological activity testing using human peripheral blood mononuclear cells demonstrated that M1 is 15-fold less potent than tacrolimus as an immunosuppressant. Screening of 22 gut bacteria species revealed that most Clostridiales bacteria are extensive tacrolimus metabolizers. Tacrolimus conversion to M1 was verified in fresh stool samples from two healthy adults. M1 was also detected in the stool samples from kidney transplant recipients who had been taking tacrolimus orally. Together, this study presents gut bacteria metabolism as a previously unrecognized elimination route of tacrolimus, potentially contributing to the low and variable tacrolimus exposure after oral dosing.

A Family of Small Intrinsically Disordered Proteins Involved in Flagellum-Dependent Motility in Salmonella enterica.

By screening a collection of Salmonella mutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF, STM14_1829, and yciG) required for wild-type flagellum-dependent swimming and swarming motility. The ymdF, STM14_1829, and yciG genes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829 mutant due to the downregulation of the flhDC operon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829 strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829 mutant, suggesting the indirect repression of the flhDC operon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, including Escherichia coli and Pseudomonas aeruginosa We showed that the inactivation of STM14_1829 homologs in E. coli and P. aeruginosa also alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility.IMPORTANCE This study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility in Salmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.

Inhibitory and Inductive Effects of Opuntia ficus indica Extract and Its Flavonoid Constituents on Cytochrome P450s and UDP-Glucuronosyltransferases.

Opuntia ficus indica (OFI) is grown abundantly in arid areas and its fruits are regarded as an important food and nutrient source owing to the presence of flavonoids, minerals, and proteins. The previous report that OFI exerts phytoestrogenic activity makes it plausible for OFI-containing supplements to be used as alternative estrogen replacement therapy. In the case of polypharmacy with the consumption of OFI-containing botanicals in post- or peri-menopausal women, it is critical to determine the potential drug-OFI interaction due to the modulation of drug metabolism. In the present study, the modulating effects on the hepatic drug metabolizing enzymes (DMEs) by OFI and its flavonoid constituents (kaempferol, quercetin, isorhamnetin, and their glycosidic forms) were investigated using the liver microsomal fractions prepared from ovariectomized (OVX) rats, human liver microsomes, and human hepatocarcinoma cell line (HepG2). As a result, the oral administration of extracts of OFI (OFIE) in OVX rats induced hepatic CYP2B1, CYP3A1, and UGT2B1. OFIE, hydrolyzed (hdl) OFIE, and several flavonols induced the transcriptional activities of both CYP2B6 and CYP3A4 genes in HepG2 cells. Finally, OFIE did not inhibit activities of cytochrome P450 (CYPs) or uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), whereas hdl OFIE or flavonol treatment inhibited CYP1A2 and CYP3A1/3A4 in rat and human liver microsomes. Our data demonstrate that OFIE may induce or inhibit certain types of DMEs and indicate that drug-OFI interaction may occur when the substrate or inhibitor drugs of specific CYPs or UGTs are taken concomitantly with OFI-containing products.

Ten Years' Experience with the CYP2D6 Activity Score: A Perspective on Future Investigations to Improve Clinical Predictions for Precision Therapeutics.

The seminal paper on the CYP2D6 Activity Score (AS) was first published ten years ago and, since its introduction in 2008, it has been widely accepted in the field of pharmacogenetics. This scoring system facilitates the translation of highly complex CYP2D6 diplotype data into a patient’s phenotype to guide drug therapy and is at the core of all CYP2D6 gene/drug pair guidelines issued by the Clinical Pharmacogenetics Implementation Consortium (CPIC). The AS, however, only explains a portion of the variability observed among individuals and ethnicities. In this review, we provide an overview of sources in addition to CYP2D6 genotype that contribute to the variability in CYP2D6-mediated drug metabolism and discuss other factors, genetic and non-genetic, that likely contribute to the observed variability in CYP2D6 enzymatic activity.

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

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.

The FDA-approved anti-cancer drugs, streptozotocin and floxuridine, reduce the virulence of Staphylococcus aureus.

In Staphylococcus aureus, an important Gram-positive human pathogen, the SaeRS two-component system is essential for the virulence and a good target for the development of anti-virulence drugs. In this study, we screened 12,200 small molecules for Sae inhibitors and identified two anti-cancer drugs, streptozotocin (STZ) and floxuridine (FU), as lead candidates for anti-virulence drug development against staphylococcal infections. As compared with STZ, FU was more efficient in repressing Sae-regulated promoters and protecting human neutrophils from S. aureus-mediated killing. FU inhibited S. aureus growth effectively whereas STZ did not. Intriguingly, RNA-seq analysis suggests that both compounds inhibit other virulence-regulatory systems such as Agr, ArlRS, and SarA more efficiently than they inhibit the Sae system. Both compounds induced prophages from S. aureus, indicating that they cause DNA damages. Surprisingly, a single administration of the drugs was sufficient to protect mice from staphylococcal intraperitoneal infection. Both compounds showed in vivo efficacy in a murine model of blood infection too. Finally, at the experimental dosage, neither compound showed any noticeable side effects on blood glucose level or blood cell counts. Based on these results, we concluded that STZ and FU are promising candidates for anti-virulence drug development against S. aureus infection.

CYP2D6 Protein Level Is the Major Contributor to Interindividual Variability in CYP2D6-Mediated Drug Metabolism in Healthy Human Liver Tissue.

CYP2D6 genetic polymorphisms are considered a major contributor to the large interindividual variability in CYP2D6-mediated drug metabolism, but fail to explain a significant portion of the variability. The aim of this study was to assess the ability of the CYP2D6 activity score (AS) estimated from CYP2D6 genotype to predict CYP2D6 expression and enzyme activity. The CYP2D6 gene region was sequenced in 115 healthy human liver tissue samples to determine their CYP2D6 AS. Additionally, CYP2D6 enzyme activity, protein, and mRNA levels were estimated. CYP2D6 AS explained 23% of the interindividual variability in CYP2D6 activity, but only 7.5% in tissues assigned AS 1-2. The CYP2D6 protein level was found to be the major determinant of CYP2D6 activity, explaining 59% of variability. These findings suggest that while CYP2D6 AS is a good predictor of poor metabolizer phenotype, additional nongenetic factors may govern the rate of CYP2D6-mediated metabolism in those without the poor metabolizer phenotype.

Daikenchuto (TU-100) alters murine hepatic and intestinal drug metabolizing enzymes in an in vivo dietary model: effects of gender and withdrawal.

Herbal medicines and natural products used for maintenance of health or treatment of diseases have many biological effects, including altering the pharmacokinetics and metabolism of other medications. Daikenchuto (TU-100), an aqueous extract of ginger, ginseng, and Japanese green pepper fruit, is a commonly prescribed Kampo (Japanese herbal medicine) for postoperative ileus or bloating. The effects of TU-100 on drug metabolism have not been investigated. In this study, we analyzed the effect of TU-100 on expression of key drug-metabolizing enzymes (DMEs) and drug transporters (DTs) in murine liver and gastrointestinal tract using a dietary model. Liver, jejunum, and proximal colon were analyzed for phase I and II DMEs and DT mRNA expression by reverse transcription (RT) first by nonquantitative and followed by quantitative polymerase chain reaction (PCR) and protein expression. Liver, jejunum, and proximal colon expressed some identical but also unique DMEs and DTs. TU-100 increased the greatest changes in cytochrome (Cyp) 2b10 and Cyp3a11 and Mdr1a. Basal and TU-100 stimulated levels of DME and DT expression were gender-dependent, dose-dependent and reversible after cessation of TU-100 supplementation, except for some changes in the intestine. Quantitative Western blot analysis of protein extracts confirmed the quantitative PCR results.