Edward LeCluyse - Academia.edu (original) (raw)

Papers by Edward LeCluyse

Research paper thumbnail of Induction of cytochrome P450 3A4 activity by cyclophosphamide, alone and in combination with dexamethasone

Clinical Pharmacology & Therapeutics, 1999

Research paper thumbnail of Troglitazone induces CYP3A4 in cultured human hepatocytes

Clinical Pharmacology & Therapeutics, 1999

Research paper thumbnail of Dexamethasone induction of cytochrome P450 3A4 activity in human hepatocytes and healthy volunteers

Clinical Pharmacology & Therapeutics, 1999

Research paper thumbnail of Incorporating High-Throughput Exposure Predictions with Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing

Toxicological Sciences, 2015

We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance t... more We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast(™) HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast(™) efforts expand (i.e., Phase II) beyond food-use pesticides towards a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. EPA ExpoCast(™) program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study, three or 13 chemicals possessed AERs <1 or <100, respectively. Diverse bioactivities across a range of assays and concentrations were also noted across the wider chemical space surveyed. The availability of HT exposure estimation and bioactivity screening tools provides an opportunity to incorporate a risk-based strategy for use in testing prioritization.

Research paper thumbnail of Evaluation of the in vitro Induction Potential of Xenobiotics known to be Transactivators of Human Pregnane X Receptor (hPXR)

The human transcription factor pregnane X receptor (PXR) is a key regulator of enzyme expression ... more The human transcription factor pregnane X receptor (PXR) is a key regulator of enzyme expression of cytochrome P450 3A4 and 2B6 (CYP3A4 and CYP2B6). Several clinical compounds that have the ability to activate hPXR also induce CYP3A4 enzyme activity. We recently showed that the drugs terbinafine, diclofenac, sildenafil, glimepiride, montelukast and ticlopidine transactivate PXR, even though there are no reports of CYP3A4 clinical induction. Thus far, none of these drugs have been evaluated in vitro for CYP induction. To assess the potential of these therapeutics to contribute to drug interactions mediated by CYP3A4 and CYP2B6 induction, we used primary cultures of human hepatocytes from 3 donors and treated these for 3 days with each test compound and rifampicin (positive control) at 8 concentrations (0.78, 1.25, 3.12, 6.25, 12.5, 25, 50 and 100 mM). Enzyme activity was assessed using media-based assays and mRNA expression quantitated using qRT-PCR (Taqman) analysis. Our data reveal...

Research paper thumbnail of Dexamethasone differentially regulates expression of carboxylesterase genes in humans and rats

Drug metabolism and disposition: the biological fate of chemicals, 2000

Carboxylesterases play important roles in the metabolism of endogenous and foreign compounds, the... more Carboxylesterases play important roles in the metabolism of endogenous and foreign compounds, therefore, xenobiotic regulation of carboxylesterase gene expression has both physiological and pharmacological significance. We previously reported that liver microsomal esterase activity was significantly decreased in rats treated with dexamethasone accompanied by a decrease in immunoreactive proteins of rat hydrolase A, B, and C. The aim of this study was to determine whether the suppressed expression of these enzymes was linked to the change of the mRNA levels, and whether cultured hepatocytes responded similar to whole animals to this chemical. Northern blotting analyses demonstrated that the levels of the corresponding mRNA were markedly decreased in rats treated with dexamethasone, suggesting that the suppressed expression is achieved through trans-suppression and/or increased degradation of the transcripts. Exposure of cultured rat hepatocytes to nanomolar levels of dexamethasone ma...

Research paper thumbnail of A map of the PPAR alpha transcription regulatory network for primary human hepatocytes

Chemico-biological interactions

Nuclear receptor activation in liver leads to coordinated alteration of the expression of multipl... more Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARa) in primary human hepatocytes using the selective PPARa ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIPseq studies at 2 and 24 h to assess genomic binding of PPARa. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARa binding-either directly at PPARa response elements or via alternative mechanisms. Almost half of regulated genes had no PPARa binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARa and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERa, and HNF4a. The linkage from PPARa binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10 lM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways.

Research paper thumbnail of Toxicity of selected bioactivated compounds in primary rat hepatocytes cultured in micropatterned co-cultures

Research paper thumbnail of Investigation of the Induction Properties of HMG-CoA Reductase Inhibitors Cerivastatin, Simvastatin, Atorvastatin, Pravastatin, and Fluvastatin in Cultures of Primary Human Hepatocytes

Research paper thumbnail of Response to Comments on "Anti-Influenza Prodrug Oseltamivir Is Activated by Carboxylesterase Human Carboxylesterase 1, and the Activation Is Inhibited by Antiplatelet Agent Clopidogrel

Journal of Pharmacology and Experimental Therapeutics, 2007

Research paper thumbnail of Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration

Pharmaceutical Research, 1998

Research paper thumbnail of In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone

Clinical Pharmacology and Therapeutics, 2000

The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethas... more The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethasone on cytochrome P4503A4 (CYP3A4) activity in healthy volunteers; and the concentration-effect relationship between dexamethasone and CYP3A4 activity in primary human hepatocyte cultures. The effect of dexamethasone (8 mg administered by mouth two times a day for 5 days) on CYP3A4 activity in 12 healthy volunteers was assessed with the erythromycin breath test and urinary ratio of dextromethorphan to 3-methoxymorphinan. Concentration-effect of dexamethasone on CYP3A4-dependent testosterone 6-beta-hydroxylation was determined in human hepatocytes treated with 2 to 250 micromol/L dexamethasone. The percent of erythromycin metabolized per hour increased from 2.20% +/- 0.60% (mean +/- SD) at baseline to 2.67% +/- 0.55% on day 5 of dexamethasone (mean increase in hepatic CYP3A4 activity 25.7% +/- 24.6%; P = .004). The mean urinary ratio of dextromethorphan to 3-methoxymorphinan was 28 (4.8 to 109) and 7 (1 to 23) at baseline and on day 5 of dexamethasone (mean decrease = 49%; P = .06). Substantial intersubject variability was observed in the extent of CYP3A4 induction. The extent of CYP3A4 induction was inversely correlated with baseline erythromycin breath test (r2 = 0.58). In hepatocytes, dexamethasone 2 to 250 micromol/L resulted in an average 1.7-fold to 6.9-fold increase in CYP3A4 activity, respectively. The extent of CYP3A4 induction with dexamethasone in hepatocyte preparations was inversely correlated with baseline activity (r2 = 0.59). These data demonstrate that dexamethasone at doses used clinically increased CYP3A4 activity with extensive intersubject variability and that the extent of CYP3A4 induction was, in part, predicted by the baseline activity of CYP3A4 in both healthy volunteers and human hepatocyte cultures.

Research paper thumbnail of Cytochrome P450 Induction

Enzyme Inhibition in Drug Discovery and Development: The Good and the Bad, 2010

... In this chapter, we have discussed the key model systems and study design considerations for ... more ... In this chapter, we have discussed the key model systems and study design considerations for ... There have been successes combining computational and experimental data for different classes of molecules. ... CYP1A enzymes in a similar fashion; however, it is not a member of ...

Research paper thumbnail of Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

Toxicology and Applied Pharmacology, 2013

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role... more The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.

Research paper thumbnail of Hepatic Stem Cells and Hepatoblasts: Identification, Isolation, and Ex Vivo Maintenance

Stem Cell Culture, 2008

This review is an update of a methods review published previously (Macdonald et al., 2002) combin... more This review is an update of a methods review published previously (Macdonald et al., 2002) combined with some information taken from a lengthy review on hepatic stem cells (HpSCs) (Schmelzer et al., 2006b) and then providing current protocols on HpSCs, especially ...

Research paper thumbnail of Evaluating and Predicting Human Cytochrome P450 Enzyme Induction

Drug Metabolism Handbook, 2009

Cytochrome P450 (CYP) isoforms are the predominate enzymes involved in the biotransformation and ... more Cytochrome P450 (CYP) isoforms are the predominate enzymes involved in the biotransformation and elimination of xenobiotics. Since their identification and characterization in 1962, they have become one of the most published enzyme systems in ...

Research paper thumbnail of Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes

Xenobiotica, 2000

1. Troglitazone was the first thiazolidinedione approved for clinical use in the treatment of non... more 1. Troglitazone was the first thiazolidinedione approved for clinical use in the treatment of non-insulin-dependent diabetes mellitus. During clinical investigations of drug-drug interactions with therapeutics (terfenadine and cyclosporine) known to be metabolized by CYP3A4, pharmacokinetic interactions were noted upon troglitazone multiple-dose treatments. The nature of the interactions suggested induction of CYP3A enzymes. 2. Primary cultures of human hepatocytes were used to investigate the induction potential of troglitazone with respect to CYP3A4, CYP2B6 and CYP1A1/2. In human hepatocytes, troglitazone induced both immunoreactive CYP3A4 protein and testosterone 6beta-hydroxylase activity in a dose-dependent fashion (EC50 = 5-10 microM), accompanied by an increase in CYP3A4 mRNA. The capacity of troglitazone to induce CYP3A4 was between that of rifampin (EC50 = 0.8 microM) and dexamethasone (40-50 microM). Troglitazone increased CYP2B6 immunoreactive protein but did not significantly effect CYP1A1/2 activity, immunoreactive protein or mRNA. 3. Troglitazone produced significant increases in CYP3A message, protein and activity in primary rat hepatocytes, a slight increase in CYP2B1/2 activity and no change in CYP1A1/2 message or activity. 4. These results provide evidence that troglitazone can induce CYP3A and CYP2B enzymes while apparently not altering CYP1A. This provides a rationale for the clinically observed interactions of troglitazone with selected CYP3A4 substrates.

Research paper thumbnail of Liver biomarker and in vitro assessment confirm the hepatic origin of aminotransferase elevations lacking histopathological correlate in beagle dogs treated with GABAA receptor antagonist NP260

Toxicology and Applied Pharmacology, 2014

NP260 was designed as a first-in-class selective antagonist of α4-subtype GABAA receptors that ha... more NP260 was designed as a first-in-class selective antagonist of α4-subtype GABAA receptors that had promising efficacy in animal models of pain, epilepsy, psychosis, and anxiety. However, development of NP260 was complicated following a 28-day safety study in dogs in which pronounced elevations of serum aminotransferase levels were observed, although there was no accompanying histopathological indication of hepatocellular injury. To further investigate the liver effects of NP260, we assayed stored serum samples from the 28-day dog study for liver specific miRNA (miR-122) as well as enzymatic biomarkers glutamate dehydrogenase and sorbitol dehydrogenase, which indicate liver necrosis. Cytotoxicity assessments were conducted in hepatocytes derived from dog, rat, and human liver samples to address the species specificity of the liver response to NP260. All biomarkers, except ALT, returned toward baseline by Day 29 despite continued drug treatment, suggesting adaptation to the initial injury. In vitro analysis of the toxicity potential of NP260 to primary hepatocytes indicated a relative sensitivity of dog>human>rat, which may explain, in part, why the liver effects were not evident in the rodent safety studies. Taken together, the data indicate that a diagnostic biomarker approach, coupled with sensitive in vitro screening strategies, may facilitate interpretation of toxicity potential when an adaptive event masks the underlying toxicity.

Research paper thumbnail of Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes

Toxicology and Applied Pharmacology, 2004

Liver is a prime site for conversion of inorganic arsenic (iAs) to methylated metabolites, includ... more Liver is a prime site for conversion of inorganic arsenic (iAs) to methylated metabolites, including methylarsenicals (MAs) and dimethylarsenicals (DMAs). To assess interindividual variation in the capacity of liver to metabolize iAs, we examined the metabolic fate of arsenite (iAs(III)) in normal primary human hepatocytes obtained from eight donors and cultured under standard conditions. Methylation rates, yields, and distribution of arsenicals were determined for hepatocytes exposed to 0.3-30 nmol of iAs(III)/mg of protein for 24 h. Although the accumulation of arsenic (As) by cells was a linear function of the initial concentration of iAs(III) in culture, the concentration of As retained in cells varied several fold among donors. DMAs was the major methylated metabolite found in cultures exposed to low concentrations of iAs(III); at higher concentrations, MAs was always predominant. Maximal rates for methylation of iAs(III) were usually attained at 3 or 9 nmol of iAs(III)/mg of protein and varied about 7-fold among donors. For most donors, the methylation rate decreased at the highest iAs(III) concentrations. MAs was the major methylated metabolite retained in cells regardless of exposure level. DMAs was the major methylated metabolite found in medium. The interindividual differences in rates for iAs(III) methylation were not strictly associated with variations in basal mRNA levels for cyt19, an As-methyltransferase. Analysis of the coding sequence of cyt19 identified one heterozygote with Met287Thr mutation in a single allele. Thus, genetic polymorphism of cyt19 along with other cellular factors is likely responsible for interindividual differences in the capacity of primary human hepatocytes to retain and metabolize iAs(III).

Research paper thumbnail of Evaluation of an in vitro toxicogenetic mouse model for hepatotoxicity

Toxicology and Applied Pharmacology, 2010

Numerous studies support the fact that a genetically diverse mouse population may be useful as an... more Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to determine whether viable cells may be isolated from different mouse inbred strains, evaluate the reproducibility of cell yield, viability and functionality over subsequent isolations, and assess the utility of the model for toxicity screening. Hepatocytes were isolated from 15 strains of mice (A/J, B6C3F1, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, BALB/cByJ, AKR/J, MRL/MpJ, NOD/LtJ, NZW/LacJ, PWD/PhJ and WSB/EiJ males) and cultured for up to 7 days in traditional 2-dimensional culture. Cells from B6C3F1, C57BL/6J, and NOD/LtJ strains were treated with acetaminophen, WY-14,643 or rifampin and concentration-response effects on viability and function were established. Our data suggest that high yield and viability can be achieved across a panel of strains. Cell function and expression of key liver-specific genes of hepatocytes isolated from different strains and cultured under standardized conditions are comparable. Strain-specific responses to toxicant exposure have been observed in cultured hepatocytes and these experiments open new opportunities for further developments of in vitro models of hepatotoxicity in a genetically diverse population.

Research paper thumbnail of Induction of cytochrome P450 3A4 activity by cyclophosphamide, alone and in combination with dexamethasone

Clinical Pharmacology & Therapeutics, 1999

Research paper thumbnail of Troglitazone induces CYP3A4 in cultured human hepatocytes

Clinical Pharmacology & Therapeutics, 1999

Research paper thumbnail of Dexamethasone induction of cytochrome P450 3A4 activity in human hepatocytes and healthy volunteers

Clinical Pharmacology & Therapeutics, 1999

Research paper thumbnail of Incorporating High-Throughput Exposure Predictions with Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing

Toxicological Sciences, 2015

We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance t... more We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast(™) HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast(™) efforts expand (i.e., Phase II) beyond food-use pesticides towards a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. EPA ExpoCast(™) program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study, three or 13 chemicals possessed AERs <1 or <100, respectively. Diverse bioactivities across a range of assays and concentrations were also noted across the wider chemical space surveyed. The availability of HT exposure estimation and bioactivity screening tools provides an opportunity to incorporate a risk-based strategy for use in testing prioritization.

Research paper thumbnail of Evaluation of the in vitro Induction Potential of Xenobiotics known to be Transactivators of Human Pregnane X Receptor (hPXR)

The human transcription factor pregnane X receptor (PXR) is a key regulator of enzyme expression ... more The human transcription factor pregnane X receptor (PXR) is a key regulator of enzyme expression of cytochrome P450 3A4 and 2B6 (CYP3A4 and CYP2B6). Several clinical compounds that have the ability to activate hPXR also induce CYP3A4 enzyme activity. We recently showed that the drugs terbinafine, diclofenac, sildenafil, glimepiride, montelukast and ticlopidine transactivate PXR, even though there are no reports of CYP3A4 clinical induction. Thus far, none of these drugs have been evaluated in vitro for CYP induction. To assess the potential of these therapeutics to contribute to drug interactions mediated by CYP3A4 and CYP2B6 induction, we used primary cultures of human hepatocytes from 3 donors and treated these for 3 days with each test compound and rifampicin (positive control) at 8 concentrations (0.78, 1.25, 3.12, 6.25, 12.5, 25, 50 and 100 mM). Enzyme activity was assessed using media-based assays and mRNA expression quantitated using qRT-PCR (Taqman) analysis. Our data reveal...

Research paper thumbnail of Dexamethasone differentially regulates expression of carboxylesterase genes in humans and rats

Drug metabolism and disposition: the biological fate of chemicals, 2000

Carboxylesterases play important roles in the metabolism of endogenous and foreign compounds, the... more Carboxylesterases play important roles in the metabolism of endogenous and foreign compounds, therefore, xenobiotic regulation of carboxylesterase gene expression has both physiological and pharmacological significance. We previously reported that liver microsomal esterase activity was significantly decreased in rats treated with dexamethasone accompanied by a decrease in immunoreactive proteins of rat hydrolase A, B, and C. The aim of this study was to determine whether the suppressed expression of these enzymes was linked to the change of the mRNA levels, and whether cultured hepatocytes responded similar to whole animals to this chemical. Northern blotting analyses demonstrated that the levels of the corresponding mRNA were markedly decreased in rats treated with dexamethasone, suggesting that the suppressed expression is achieved through trans-suppression and/or increased degradation of the transcripts. Exposure of cultured rat hepatocytes to nanomolar levels of dexamethasone ma...

Research paper thumbnail of A map of the PPAR alpha transcription regulatory network for primary human hepatocytes

Chemico-biological interactions

Nuclear receptor activation in liver leads to coordinated alteration of the expression of multipl... more Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARa) in primary human hepatocytes using the selective PPARa ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIPseq studies at 2 and 24 h to assess genomic binding of PPARa. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARa binding-either directly at PPARa response elements or via alternative mechanisms. Almost half of regulated genes had no PPARa binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARa and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERa, and HNF4a. The linkage from PPARa binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10 lM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways.

Research paper thumbnail of Toxicity of selected bioactivated compounds in primary rat hepatocytes cultured in micropatterned co-cultures

Research paper thumbnail of Investigation of the Induction Properties of HMG-CoA Reductase Inhibitors Cerivastatin, Simvastatin, Atorvastatin, Pravastatin, and Fluvastatin in Cultures of Primary Human Hepatocytes

Research paper thumbnail of Response to Comments on "Anti-Influenza Prodrug Oseltamivir Is Activated by Carboxylesterase Human Carboxylesterase 1, and the Activation Is Inhibited by Antiplatelet Agent Clopidogrel

Journal of Pharmacology and Experimental Therapeutics, 2007

Research paper thumbnail of Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration

Pharmaceutical Research, 1998

Research paper thumbnail of In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone

Clinical Pharmacology and Therapeutics, 2000

The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethas... more The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethasone on cytochrome P4503A4 (CYP3A4) activity in healthy volunteers; and the concentration-effect relationship between dexamethasone and CYP3A4 activity in primary human hepatocyte cultures. The effect of dexamethasone (8 mg administered by mouth two times a day for 5 days) on CYP3A4 activity in 12 healthy volunteers was assessed with the erythromycin breath test and urinary ratio of dextromethorphan to 3-methoxymorphinan. Concentration-effect of dexamethasone on CYP3A4-dependent testosterone 6-beta-hydroxylation was determined in human hepatocytes treated with 2 to 250 micromol/L dexamethasone. The percent of erythromycin metabolized per hour increased from 2.20% +/- 0.60% (mean +/- SD) at baseline to 2.67% +/- 0.55% on day 5 of dexamethasone (mean increase in hepatic CYP3A4 activity 25.7% +/- 24.6%; P = .004). The mean urinary ratio of dextromethorphan to 3-methoxymorphinan was 28 (4.8 to 109) and 7 (1 to 23) at baseline and on day 5 of dexamethasone (mean decrease = 49%; P = .06). Substantial intersubject variability was observed in the extent of CYP3A4 induction. The extent of CYP3A4 induction was inversely correlated with baseline erythromycin breath test (r2 = 0.58). In hepatocytes, dexamethasone 2 to 250 micromol/L resulted in an average 1.7-fold to 6.9-fold increase in CYP3A4 activity, respectively. The extent of CYP3A4 induction with dexamethasone in hepatocyte preparations was inversely correlated with baseline activity (r2 = 0.59). These data demonstrate that dexamethasone at doses used clinically increased CYP3A4 activity with extensive intersubject variability and that the extent of CYP3A4 induction was, in part, predicted by the baseline activity of CYP3A4 in both healthy volunteers and human hepatocyte cultures.

Research paper thumbnail of Cytochrome P450 Induction

Enzyme Inhibition in Drug Discovery and Development: The Good and the Bad, 2010

... In this chapter, we have discussed the key model systems and study design considerations for ... more ... In this chapter, we have discussed the key model systems and study design considerations for ... There have been successes combining computational and experimental data for different classes of molecules. ... CYP1A enzymes in a similar fashion; however, it is not a member of ...

Research paper thumbnail of Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

Toxicology and Applied Pharmacology, 2013

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role... more The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.

Research paper thumbnail of Hepatic Stem Cells and Hepatoblasts: Identification, Isolation, and Ex Vivo Maintenance

Stem Cell Culture, 2008

This review is an update of a methods review published previously (Macdonald et al., 2002) combin... more This review is an update of a methods review published previously (Macdonald et al., 2002) combined with some information taken from a lengthy review on hepatic stem cells (HpSCs) (Schmelzer et al., 2006b) and then providing current protocols on HpSCs, especially ...

Research paper thumbnail of Evaluating and Predicting Human Cytochrome P450 Enzyme Induction

Drug Metabolism Handbook, 2009

Cytochrome P450 (CYP) isoforms are the predominate enzymes involved in the biotransformation and ... more Cytochrome P450 (CYP) isoforms are the predominate enzymes involved in the biotransformation and elimination of xenobiotics. Since their identification and characterization in 1962, they have become one of the most published enzyme systems in ...

Research paper thumbnail of Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes

Xenobiotica, 2000

1. Troglitazone was the first thiazolidinedione approved for clinical use in the treatment of non... more 1. Troglitazone was the first thiazolidinedione approved for clinical use in the treatment of non-insulin-dependent diabetes mellitus. During clinical investigations of drug-drug interactions with therapeutics (terfenadine and cyclosporine) known to be metabolized by CYP3A4, pharmacokinetic interactions were noted upon troglitazone multiple-dose treatments. The nature of the interactions suggested induction of CYP3A enzymes. 2. Primary cultures of human hepatocytes were used to investigate the induction potential of troglitazone with respect to CYP3A4, CYP2B6 and CYP1A1/2. In human hepatocytes, troglitazone induced both immunoreactive CYP3A4 protein and testosterone 6beta-hydroxylase activity in a dose-dependent fashion (EC50 = 5-10 microM), accompanied by an increase in CYP3A4 mRNA. The capacity of troglitazone to induce CYP3A4 was between that of rifampin (EC50 = 0.8 microM) and dexamethasone (40-50 microM). Troglitazone increased CYP2B6 immunoreactive protein but did not significantly effect CYP1A1/2 activity, immunoreactive protein or mRNA. 3. Troglitazone produced significant increases in CYP3A message, protein and activity in primary rat hepatocytes, a slight increase in CYP2B1/2 activity and no change in CYP1A1/2 message or activity. 4. These results provide evidence that troglitazone can induce CYP3A and CYP2B enzymes while apparently not altering CYP1A. This provides a rationale for the clinically observed interactions of troglitazone with selected CYP3A4 substrates.

Research paper thumbnail of Liver biomarker and in vitro assessment confirm the hepatic origin of aminotransferase elevations lacking histopathological correlate in beagle dogs treated with GABAA receptor antagonist NP260

Toxicology and Applied Pharmacology, 2014

NP260 was designed as a first-in-class selective antagonist of α4-subtype GABAA receptors that ha... more NP260 was designed as a first-in-class selective antagonist of α4-subtype GABAA receptors that had promising efficacy in animal models of pain, epilepsy, psychosis, and anxiety. However, development of NP260 was complicated following a 28-day safety study in dogs in which pronounced elevations of serum aminotransferase levels were observed, although there was no accompanying histopathological indication of hepatocellular injury. To further investigate the liver effects of NP260, we assayed stored serum samples from the 28-day dog study for liver specific miRNA (miR-122) as well as enzymatic biomarkers glutamate dehydrogenase and sorbitol dehydrogenase, which indicate liver necrosis. Cytotoxicity assessments were conducted in hepatocytes derived from dog, rat, and human liver samples to address the species specificity of the liver response to NP260. All biomarkers, except ALT, returned toward baseline by Day 29 despite continued drug treatment, suggesting adaptation to the initial injury. In vitro analysis of the toxicity potential of NP260 to primary hepatocytes indicated a relative sensitivity of dog>human>rat, which may explain, in part, why the liver effects were not evident in the rodent safety studies. Taken together, the data indicate that a diagnostic biomarker approach, coupled with sensitive in vitro screening strategies, may facilitate interpretation of toxicity potential when an adaptive event masks the underlying toxicity.

Research paper thumbnail of Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes

Toxicology and Applied Pharmacology, 2004

Liver is a prime site for conversion of inorganic arsenic (iAs) to methylated metabolites, includ... more Liver is a prime site for conversion of inorganic arsenic (iAs) to methylated metabolites, including methylarsenicals (MAs) and dimethylarsenicals (DMAs). To assess interindividual variation in the capacity of liver to metabolize iAs, we examined the metabolic fate of arsenite (iAs(III)) in normal primary human hepatocytes obtained from eight donors and cultured under standard conditions. Methylation rates, yields, and distribution of arsenicals were determined for hepatocytes exposed to 0.3-30 nmol of iAs(III)/mg of protein for 24 h. Although the accumulation of arsenic (As) by cells was a linear function of the initial concentration of iAs(III) in culture, the concentration of As retained in cells varied several fold among donors. DMAs was the major methylated metabolite found in cultures exposed to low concentrations of iAs(III); at higher concentrations, MAs was always predominant. Maximal rates for methylation of iAs(III) were usually attained at 3 or 9 nmol of iAs(III)/mg of protein and varied about 7-fold among donors. For most donors, the methylation rate decreased at the highest iAs(III) concentrations. MAs was the major methylated metabolite retained in cells regardless of exposure level. DMAs was the major methylated metabolite found in medium. The interindividual differences in rates for iAs(III) methylation were not strictly associated with variations in basal mRNA levels for cyt19, an As-methyltransferase. Analysis of the coding sequence of cyt19 identified one heterozygote with Met287Thr mutation in a single allele. Thus, genetic polymorphism of cyt19 along with other cellular factors is likely responsible for interindividual differences in the capacity of primary human hepatocytes to retain and metabolize iAs(III).

Research paper thumbnail of Evaluation of an in vitro toxicogenetic mouse model for hepatotoxicity

Toxicology and Applied Pharmacology, 2010

Numerous studies support the fact that a genetically diverse mouse population may be useful as an... more Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to determine whether viable cells may be isolated from different mouse inbred strains, evaluate the reproducibility of cell yield, viability and functionality over subsequent isolations, and assess the utility of the model for toxicity screening. Hepatocytes were isolated from 15 strains of mice (A/J, B6C3F1, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, BALB/cByJ, AKR/J, MRL/MpJ, NOD/LtJ, NZW/LacJ, PWD/PhJ and WSB/EiJ males) and cultured for up to 7 days in traditional 2-dimensional culture. Cells from B6C3F1, C57BL/6J, and NOD/LtJ strains were treated with acetaminophen, WY-14,643 or rifampin and concentration-response effects on viability and function were established. Our data suggest that high yield and viability can be achieved across a panel of strains. Cell function and expression of key liver-specific genes of hepatocytes isolated from different strains and cultured under standardized conditions are comparable. Strain-specific responses to toxicant exposure have been observed in cultured hepatocytes and these experiments open new opportunities for further developments of in vitro models of hepatotoxicity in a genetically diverse population.