[Show abstract][Hide abstract] ABSTRACT: Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of US adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα) and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/mL) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/mL. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187 and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/mL). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/mL) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2 and an antagonist for human PPARα.
[Show abstract][Hide abstract] ABSTRACT: In humans, microsomal epoxide hydrolase (mEH) contributes important biological functions that underlie both detoxification and bioactivation fates arising from exposures to foreign chemicals. Previously, we discovered that human mEH gene transcription is initiated from alternative promoters. The respective transcripts are programmed with tissue specificity and the upstream E1b promoter contributes predominantly to mEH expression. The results presented demonstrate that exposures to the Nrf2 activators, sulforaphane (SFN) and tert-butylhydroquinone (tBHQ), markedly activate E1b transcription in human lung and liver cells. Genomic analyses identified two major DNase I hypersensitive regions (HS-1 and HS-2) within the ~ 15 kb intervening sequence separating E1b from the downstream E1 promoter. In BEAS-2B cells, the Nrf2 effectors, SFN and tBHQ, selectively activated the more distal HS-2 through an antioxidant-response element (ARE). An activator protein 1/12-O-tetradecanoylphorbol-13-acetate interaction was further identified within the HS-2 enhancer that functioned to additionally contribute to ARE-mediated induction responsiveness of the E1b promoter. The results demonstrate that ARE modulation, integrated with additional transcriptional complexes, regulates the tissue-specific expression of mEH and that these processes likely coordinate both the protective and bioactivation functions contributed by mEH activities in human tissues.
[Show abstract][Hide abstract] ABSTRACT: Toxicogenomics (TGx) is employed frequently to investigate underlying molecular mechanisms of the compound of interest and, thus, has become an aid to mode of action determination. However, the results and interpretation of a TGx dataset are influenced by the experimental design and methods of analysis employed. This article describes an evaluation and reanalysis, by two independent laboratories, of previously published TGx mouse liver microarray data for a triazole fungicide, propiconazole (PPZ), and the anticonvulsant drug phenobarbital (PB). Propiconazole produced an increase incidence of liver tumors in male CD-1 mice only at a dose that exceeded the maximum tolerated dose (2500 ppm). Firstly, we illustrate how experimental design differences between two in vivo studies with PPZ and PB may impact the comparisons of TGx results. Secondly, we demonstrate that different researchers using different pathway analysis tools can come to different conclusions on specific mechanistic pathways, even when using the same datasets. Finally, despite these differences the results across three different analyses also show a striking degree of similarity observed for PPZ and PB treated livers when the expression data are viewed as major signalling pathways and cell processes affected. Additional studies described here show that the postulated key event of hepatocellular proliferation was observed in CD-1 mice for both PPZ and PB, and that PPZ is also a potent activator of the mouse CAR nuclear receptor. Thus, with regard to the events which are hallmarks of CAR-induced effects that are key events in the mode of action (MOA) of mouse liver carcinogenesis with PB, PPZ-induced tumours can be viewed as being promoted by a similar PB-like CAR-dependent MOA.
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The proximal E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide -300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.
[Show abstract][Hide abstract] ABSTRACT: The constitutive androstane receptor (CAR; NR1I3) is a critical xenobiotic sensor that regulates xenobiotic metabolism, drug clearance, energy and lipid homeostasis, cell proliferation and development. Although constitutively active, in hepatocytes CAR is normally held quiescent through a tethering mechanism in the cytosol, anchored to a protein complex that includes several components, including heat shock protein 90. Release and subsequent nuclear translocation of CAR is triggered through either direct binding to ligand activators such as 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), or through indirect chemical activation, such as with phenobarbital (PB). In this study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR's interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR and its interaction with the SUG1 subunit of the 26S proteasome. Together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity.
[Show abstract][Hide abstract] ABSTRACT: Abstract The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.
Critical Reviews in Toxicology 11/2013; · 6.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of the constitutive androstane receptor (CAR, NR1I3) is enriched in the mature mammalian liver and increasingly recognized for its prominent role in regulating a myriad of processes including biotransformation, chemical transport, energy metabolism and lipid homeostasis. Previously, we demonstrated that CAR levels were markedly enhanced during the differentiation of hepatic-like cells derived from hESCs, prompting the hypothesis that CAR contributes a key functional role in directing human hepatogenesis. Here we demonstrate that over-expression of CAR in human embryonic stem cells (ESCs), transduced by a lentiviral vector, accelerates the maturation of hepatic-like cells, with CAR over-expressing cells exhibiting a 2.5-fold increase in albumin secretion by day 20 in culture differentiation, and significantly enhanced levels of mRNA expression of several liver-selective markers, including hepatic transcription factors, plasma proteins, biotransformation enzymes, and metabolic enzymes. CAR over-expressing cells also exhibited enhanced CITCO-inducible CYP3A7 enzymatic activity. Knockdown of CAR via siRNA attenuated the differentiation-dependent expression programs. In contrast, expression levels of the pregnane X receptor (PXR), a nuclear receptor most similar to CAR in primary sequence, were negligible in human fetal liver tissues or in the differentiating hESCs, and stable over-expression of PXR in hepatic-induced hESCs failed to enhance expression of hepatic phenotype markers. Together, these results define a novel role for human CAR in hepatic lineage commitment.
[Show abstract][Hide abstract] ABSTRACT: Δ(9)-Tetrahydrocannabinol (Δ(9)-THC) has been reported as possessing anti-estrogenic activity, although the mechanisms underlying these effects are poorly delineated. In this study, we used the estrogen receptor α (ERα)-positive human breast cancer cell line, MCF-7, as an experimental model and show that Δ(9)-THC exposures markedly suppress 17β-estradiol (E2)- induced MCF-7 cell proliferation. We demonstrate that these effects result from Δ(9)-THC's ability to inhibit E2-liganded ERα activation. Mechanistically, the data obtained from biochemical analyses revealed that: i) Δ(9)-THC up-regulates ERβ, a repressor of ERα, inhibiting the expression of E2/ERα-regulated genes that promote cell growth, and that ii) Δ(9)-THC induction of ERβ modulates E2/ERα signaling in the absence of direct interaction with the E2 ligand binding site. Therefore, the data presented support the concept that Δ(9)-THC's anti-estrogenic activities are mediated by the ERβ disruption of E2/ERα signaling. αααααααααα
Chemical Research in Toxicology 05/2013; · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH, EPHX1) is a critical xenobiotic-metabolizing enzyme, catalyzing both detoxification and bioactivation reactions that direct the disposition of chemical epoxides, including the carcinogenic metabolites of several polycyclic aromatic hydrocarbons. Recently, we discovered that a previously unrecognized and primate-specific EPHX1 transcript, termed E1-b, was actually the predominant driver of EPHX1 expression in all human tissues. In this study, we identify another human EPHX1 transcript, designated as E1-b'. Unusually, both the E1-b and E1-b' mRNA transcripts are generated from the use of a far upstream gene promoter, localized ∼18.5 kb 5'-upstream of the EPHX1 protein-coding region. Although expressed at comparatively lower levels than E1-b, the novel E1-b' transcript is readily detected in all tissues examined, with highest levels maintained in human ovary. The E1-b' mRNA possesses unusual functional features in its 5'-untranslated region, including a GC-rich leader sequence and two upstream AUGs that encode for short peptides of 26 and 17 amino acids in length, respectively. Results from in vitro transcription/translation assays and direct transfection in mammalian cells of either the E1-b' transcript or the encoded peptides demonstrated that the E1-b' upstream open reading frames (uORFs) are functional, with their presence markedly inhibiting the translation of EPHX1 protein, both in cis and in trans configurations. These unique uORF peptides exhibit no homology to any other known uORF sequences but likely function to mediate post-transcription regulation of EPHX1 and perhaps more broadly as translational regulators in human cells.
[Show abstract][Hide abstract] ABSTRACT: Previously, we reported that (-)-xanthatin, a naturally occurring xanthanolide present in the Cocklebur plant, exhibits potent anti-proliferative effects on human breast cancer cells, accompanied by an induction of the growth arrest and DNA damage-inducible gene 45γ (GADD45γ), recognized recently as a novel tumor suppressor gene. However, the mechanisms mediating this activation were unknown. Topoisomerase IIα (Topo IIα) inhibition has been reported to produce a cell death response accompanied by an atypical DNA laddering fragmentation profile, similar to that noted previously for (-)-xanthatin. Therefore we hypothesized that (-)-xanthatin's GADD45γ activation was mediated through the Topo IIα pathway. Here, we identify that (-)-xanthatin does function as a catalytic inhibitor of Topo IIα, promoting DNA damage. In addition, reactive oxygen species (ROS) were elevated in cells treated with this agent. Mechanistically, it was determined that the induced levels of GADD45γ mRNA resulting from (-)-xanthatin exposures were stabilized by coordinately produced ROS, and that the consequent induction of GADD45γ mRNA, GADD45γ protein and ROS generation were abrogated by co-treatment with N-acetyl-L-cysteine. Taken together, the data support the concept that Topo IIα inhibition by (-)-xanthatin is a trigger that stimulates expression of DNA damage-inducible GADD45γ mRNA and that concomitantly produced ROS act downstream to further enhance the GADD45γ mRNA/GADD45γ protein induction process, resulting in breast cancer cell death.
[Show abstract][Hide abstract] ABSTRACT: To investigate gene(s) being regulated by ∆(9)-tetrahydrocannabinol (∆(9)-THC), we performed DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are poorly differentiated breast cancer cells, treated with ∆(9)-THC for 48 hr at an IC50 concentration of approximately 25 µM. Among the highly up-regulated genes (> 10-fold) observed, fatty acid 2-hydroxylase (FA2H) was significantly induced (17.8-fold). Although the physiological role of FA2H has not yet been fully understood, FA2H has been shown to modulate cell differentiation. The results of Oil Red O staining after ∆(9)-THC exposure showed the distribution of lipid droplets (a sign of the differentiated phenotype) in cells. Taken together, the results obtained here indicate that FA2H is a novel ∆(9)-THC-regulated gene, and that ∆(9)-THC induces differentiation signal(s) in poorly differentiated MDA-MB-231 cells.
The Journal of Toxicological Sciences 01/2013; 38(2):305-308. · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cannabidiol (CBD), a major non-psychotropic constituent of fiber-type cannabis plant, has been reported to possess diverse biological activities, including anti-proliferative effect on cancer cells. Although CBD is obtained from non-enzymatic decarboxylation of its parent molecule, cannabidiolic acid (CBDA), few studies have investigated whether CBDA itself is biologically active. Results of the current investigation revealed that CBDA inhibits migration of the highly invasive MDA-MB-231 human breast cancer cells, apparently through a mechanism involving inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase, RhoA. It is established that activation of the RhoA signaling pathway leads to inhibition of the mobility of various cancer cells, including MDA-MB-231 cells. The data presented in this report suggest for the first time that as an active component in the cannabis plant, CBDA offers potential therapeutic modality in the abrogation of cancer cell migration, including aggressive breast cancers.
[Show abstract][Hide abstract] ABSTRACT: Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately with coregulator proteins. The human constitutive androstane receptor (CAR; NR1I3) is expressed primarily in liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy, and lipid homeostasis. In this report, DAX-1, a nuclear receptor family member with corepressor properties, was identified as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1's downstream LXXLL and its PCFQVLP motifs were critical contributors to DAX-1's corepression activities, although two other LXXM/LL motifs located nearer the N terminus had no impact on the CAR functional interaction. Deletion of DAX-1's C-terminal transcription silencing domain restored CAR1 transactivation activity in reporter assays to approximately 90% of control, demonstrating its critical function in mediating the CAR repression activities. Furthermore, results obtained from mammalian two-hybrid experiments assessing various domain configurations of the respective receptors showed that full-length DAX-1 inhibited the CAR-SRC1 interaction by approximately 50%, whereas the same interaction was restored to 90% of control when the DAX-1 transcription silencing domain was deleted. Direct interaction between CAR and DAX-1 was demonstrated with both alpha-screen and coimmunoprecipitation experiments, and this interaction was enhanced in the presence of the CAR activator 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Results obtained in primary human hepatocytes further demonstrated DAX-1 inhibition of CAR-mediated CITCO induction of the CYP2B6 target gene. The results of this investigation identify DAX-1 as a novel and potent CAR corepressor and suggest that DAX-1 functions as a coordinate hepatic regulator of CAR's biological function.
[Show abstract][Hide abstract] ABSTRACT: The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays, obscuring the detection of ligand activators. However, a human splice variant of CAR, termed CAR3, exhibits negligible constitutive activity. In addition, CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study, we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses, thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse, rat, and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays, the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: exo-Methylene lactone group-containing compounds, such as (--)-xanthatin, are present in a large variety of biologically active natural products, including extracts of Xanthium strumarium (Cocklebur). These substances are reported to possess diverse functional activities, exhibiting anti-inflammatory, antimalarial, and anticancer potential. In this study, we synthesized six structurally related xanthanolides containing exo-methylene lactone moieties, including (--)-xanthatin and (+)-8-epi-xanthatin, and examined the effects of these chemically defined substances on the highly aggressive and farnesyltransferase inhibitor (FTI)-resistant MDA-MB-231 cancer cell line. The results obtained demonstrate that (--)-xanthatin was a highly effective inhibitor of MDA-MB-231 cell growth, inducing caspase-independent cell death, and that these effects were independent of FTase inhibition. Further, our results show that among the GADD45 isoforms, GADD45γ was selectively induced by (--)-xanthatin and that GADD45γ-primed JNK and p38 signaling pathways are, at least in part, involved in mediating the growth inhibition and potential anticancer activities of this agent. Given that GADD45γ is becoming increasingly recognized for its tumor suppressor function, the results presented here suggest the novel possibility that (--)-xanthatin may have therapeutic value as a selective inducer of GADD45γ in human cancer cells, in particular in FTI-resistant aggressive breast cancers.
Chemical Research in Toxicology 06/2011; 24(6):855-65. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant.
[Show abstract][Hide abstract] ABSTRACT: 15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein (ox-LDL), a major causal factor for atherosclerosis. Both enzymatic (15-LOX) and non-enzymatic (Cu(2+)) mechanisms have been proposed for the production of ox-LDL. We have recently reported that cannabidiol-2',6'-dimethyl ether (CBDD) is a selective and potent inhibitor of 15-LOX-catalyzed linoleic acid oxygenation (Takeda et al., Drug Metab. Dispos., 37, 1733-1737 (2009)). In the LDL, linoleic acid is present as cholesteryl linoleate, the major fatty acid esterified to cholesterol, and is susceptible to oxidative modification by 15-LOX or Cu(2+). In this investigation, we examined the efficacy of CBDD on i) 15-LOX-catalyzed oxygenation of cholesteryl linoleate, and ii) ox-LDL formation catalyzed by 15-LOX versus Cu(2+)-mediated non-enzymatic generation of this important mediator. The results obtained demonstrate that CBDD is a potent and selective inhibitor of ox-LDL formation generated by the 15-LOX pathway. These studies establish CBDD as both an important experimental tool for characterizing 15-LOX-mediated ox-LDL formation, and as a potentially useful therapeutic agent for treatment of atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: The limited availability of hepatic tissue suitable for the treatment of liver disease and drug discovery research advances the generation of hepatic-like cells from alternative sources as a valuable approach. In this investigation we exploited a unique hepatic differentiation approach to generate hepatocyte-like cells from human embryonic stem cells (hESCs). hESCs were cultured for 10-20 days on collagen substrate in highly defined and serum free hepatocyte media. The resulting cell populations exhibited hepatic cell-like morphology and were characterized with a variety of biological endpoint analyses. Real-time PCR analysis demonstrated that mRNA expression of the 'stemness' marker genes NANOG and alkaline phosphatase in the differentiated cells was significantly reduced, findings that were functionally validated using alkaline phosphatase activity detection measures. Immunofluorescence studies revealed attenuated levels of the 'stemness' markers OCT4, SOX2, SSEA-3, TRA-1-60, and TRA-1-81 in the hepatic-like cell population. The hepatic character of the cells was evaluated additionally by real-time PCR analyses that demonstrated increased mRNA expression of the hepatic transcription factors FOXA1, C/EBPα, and HNF1α, the nuclear receptors CAR, RXRα, PPARα, and HNF4α, the liver-generated plasma proteins α-fetoprotein, transthyretin, transferrin, and albumin, the protease inhibitor α-1-antitrypsin, metabolic enzymes HMGCS2, PEPCK, and biotransformation enzymes CYP3A7, CYP3A4, CYP3A5, and CYP2E1. Indocyanine green uptake albumin secretion and glycogen storage capacity further confirmed acquisition of hepatic function. These studies define an expeditious methodology that facilitates the differentiation of hESCs along a hepatic lineage and provide a framework for their subsequent use in pharmacological and toxicological research applications requiring a renewable supply of human hepatocytes.
[Show abstract][Hide abstract] ABSTRACT: We report a synergistic method using bioassay-directed liquid chromatography fractionation and time-of-flight mass spectrometry to guide and accelerate bioactive compound discovery. To steer purification and assays toward anticipated neutral lipid activators of a constitutive androstane receptor splice variant, a relative mass defect filter was calculated, based on the ratio of the mass defect to the measured ion mass, and used to reduce the number of candidate ion masses. Mass measurements often lack sufficient accuracy to provide unambiguous assignments of elemental compositions, and since the relative mass defect reflects fractional hydrogen content of ions, this value is largely determined by the hydrogen content of a compound's biosynthetic precursors. A relative mass defect window ranging from 600-1000 ppm, consistent with an assortment of lipids, was chosen to assess the number of candidate ions in fractions of fetal bovine serum. This filter reduced the number of candidate ion m/z values from 1345 to 892, which was further reduced to 21 by intensity and isotope filtering. Accurate mass measurements from time-of-flight mass spectrometry and fragment ion masses generated using nonselective collision-induced dissociation suggested dioctyl phthalate as one of few neutral lipid constituents in the active fraction. The identity of this compound was determined to be di(2-ethylhexyl) phthalate using GC/MS, and it was ranked as a promising candidate for reporter assay screening.
Rapid Communications in Mass Spectrometry 12/2010; 24(24):3578-84. · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To commemorate the 50th anniversary of the Society of Toxicology, this special edition article reviews the history and current scope of xenobiotic metabolism and transport, with special emphasis on the discoveries and impact of selected "xenobiotic receptors." This overall research realm has witnessed dynamic development in the past 50 years, and several of the key milestone events that mark the impressive progress in these areas of toxicological sciences are highlighted. From the initial observations regarding aspects of drug metabolism dating from the mid- to late 1800's, the area of biotransformation research witnessed seminal discoveries in the mid-1900's and onward that are remarkable in retrospect, including the discovery and characterization of the phase I monooxygenases, the cytochrome P450s. Further research uncovered many aspects of the biochemistry of xenobiotic metabolism, expanding to phase II conjugation and phase III xenobiotic transport. This led to hallmark developments involving integration of genomic technologies to elucidate the basis for interindividual differences in response to xenobiotic exposures and discovery of nuclear and soluble receptor families that selectively "sense" the chemical milieu of the mammalian cell and orchestrate compensatory changes in gene expression programming to accommodate complex xenobiotic exposures. This review will briefly summarize these developments and investigate the expanding roles of xenobiotic receptor biology in the underlying basis of toxicological response to chemical agents.