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1.
ACS Omega ; 4(9): 13902-13912, 2019 Aug 27.
Article in English | MEDLINE | ID: mdl-31497708

ABSTRACT

The clinical use of some drugs, such as carbamazepine, phenytoin, and allopurinol, is often associated with adverse cutaneous reactions. The bioactivation of drugs into immunologically reactive metabolites by the liver is postulated to be the first step in initiating a downstream cascade of pathological immune responses. Current mechanistic understanding and the ability to predict such adverse drug cutaneous responses have been partly limited by the lack of appropriate cutaneous drug bioactivation experimental models. Although in vitro human liver models have been extensively investigated for predicting hepatotoxicity and drug-drug interactions, their ability to model the generation of antigenic reactive drug metabolites that are capable of eliciting immunological reactions is not well understood. Here, we employed a human progenitor cell (HepaRG)-derived hepatocyte model and established highly sensitive liquid chromatography-mass spectrometry analytical assays to generate and quantify different reactive metabolite species of three paradigm skin sensitizers, namely, carbamazepine, phenytoin, and allopurinol. We found that the generation of reactive drug metabolites by the HepaRG-hepatocytes was sensitive to the medium composition. In addition, a functional assay based on the activation of U937 myeloid cells into the antigen-presenting cell (APC) phenotype was established to evaluate the immunogenicity potential of the reactive drug metabolites produced by HepaRG-derived hepatocytes. We showed that the reactive drug metabolites of known skin sensitizers could significantly upregulate IL8, IL1ß, and CD86 expressions in U937 cells compared to the metabolites from a nonskin sensitizer (i.e., acetaminophen). Thus, the extent of APC activation by HepaRG-hepatocytes conditioned medium containing reactive drug metabolites can potentially be used to predict their skin sensitization potential.

2.
Wound Repair Regen ; 27(6): 715-719, 2019 11.
Article in English | MEDLINE | ID: mdl-31276613

ABSTRACT

Evaluating interactions between dressing and wound is important for understanding wound management. This study quantitatively compared four polyurethane foam-based wound dressings for their absorption profile, cell penetration, and adherence using two novel in vitro assays. The dressing with uniform pore sizes varying from 25~75 µm showed the highest absorption of both culture media and serum. The same dressing showed a 1.2- to 3.6-fold lower cell adherence (3 hours) than the other dressings, and ~20-fold lower cell penetration (5 days) than dressings with pore sizes varying from 55 to 343 µm. Additionally, cell and dressing interactions using a 3-dimensional wound healing assay showed that the dressings with the smallest pore size of 25~75 µm maintained the highest cell viability (76.3%) and promoted cell migration into the wound site. This data suggest that polyurethane foam dressing with smaller and evenly distributed pores promotes wound healing with less cellular adhesion and penetration.


Subject(s)
Bandages , Polyurethanes/pharmacology , Skin Absorption/drug effects , Wound Healing/drug effects , Wounds and Injuries/therapy , Analysis of Variance , Bandages, Hydrocolloid , Cell Adhesion/physiology , Cell Survival/physiology , Cells, Cultured , Coculture Techniques , Culture Media , Evaluation Studies as Topic , Humans , In Vitro Techniques , Wounds and Injuries/physiopathology
3.
Biomaterials ; 216: 119221, 2019 09.
Article in English | MEDLINE | ID: mdl-31195301

ABSTRACT

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.


Subject(s)
Antimalarials/pharmacology , Hepatocytes/parasitology , Malaria/drug therapy , Plasmodium/drug effects , Animals , Cell Culture Techniques/methods , Cell Line , Cells, Cultured , Hepatocytes/cytology , Humans , Liver/cytology , Liver/parasitology , Macaca fascicularis , Macaca mulatta , Parasitic Sensitivity Tests/methods , Recurrence , Secondary Prevention , Spheroids, Cellular/cytology , Spheroids, Cellular/parasitology , Sporozoites/drug effects
4.
Toxicol In Vitro ; 50: 47-53, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29366910

ABSTRACT

Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver specific functions and maintaining hepatic polarity. However, the utility of hepatocyte co-culture models for studies, such as drug-drug interaction studies, has not been completely elucidated. In this study the induction of Cyp1a2, Cyp2b1/2, and Cyp3a2, the three major cytochrome P450 (CYP) isoforms in the rat liver, was evaluated in randomly mixed co-cultures and micropatterned co-cultures. We found that in both co-culture configurations, the drug-induced Cyp1a2, Cyp2b1/2, Cyp3a2 mRNA and activity were suppressed relative to those in monocultured hepatocytes. Further, we observed a significant increase in TGFß1 production in the co-cultures. Addition of 100 pg/ml TGFß1 to hepatocyte monocultures resulted in the suppression of Cyp1a2, Cyp2b1/2, and Cyp3a2 induction. These findings implicate TGFß1 as one of the important factors impairing drug induced CYP induction in co-cultures and suggests that caution needs to be exercised in the use of hepatocyte-fibroblast co-cultures for CYP induction studies.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Enzyme Induction , Fibroblasts/metabolism , Hepatocytes/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Cells, Cultured , Coculture Techniques , Cytochrome P-450 Enzyme Inducers/pharmacology , Cytochrome P-450 Enzyme System/genetics , Male , Mice , NIH 3T3 Cells , Rats, Wistar
5.
J Appl Toxicol ; 36(2): 320-9, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26201057

ABSTRACT

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.


Subject(s)
Cells, Cultured/drug effects , Chemical and Drug Induced Liver Injury/pathology , Cytochrome P-450 Enzyme System/metabolism , Drug Evaluation/methods , Drug Interactions/physiology , Hepatocytes/drug effects , Humans
6.
Toxicol Rep ; 3: 252-261, 2016.
Article in English | MEDLINE | ID: mdl-28959545

ABSTRACT

As legacy toxicogenomics databases have become available, improved data mining approaches are now key to extracting and visualizing subtle relationships between toxicants and gene expression. In the present study, a novel "aggregating bundles of clusters" (ABC) procedure was applied to separate cholestatic from non-cholestatic drugs and model toxicants in the Johnson & Johnson (Janssen) rat liver toxicogenomics database [3]. Drug-induced cholestasis is an important issue, particularly when a new compound enters the market with this liability, with standard preclinical models often mispredicting this toxicity. Three well-characterized cholestasis-responsive genes (Cyp7a1, Mrp3 and Bsep) were chosen from a previous in-house Janssen gene expression signature; these three genes show differing, non-redundant responses across the 90+ paradigm compounds in our database. Using the ABC procedure, extraneous contributions were minimized in comparisons of compound gene responses. All genes were assigned weights proportional to their correlations with Cyp7a1, Mrp3 and Bsep, and a resampling technique was used to derive a stable measure of compound similarity. The compounds that were known to be associated with rat cholestasis generally had small values of this measure relative to each other but also had large values of this measure relative to non-cholestatic compounds. Visualization of the data with the ABC-derived signature showed a very tight, essentially identically behaving cluster of robust human cholestatic drugs and experimental cholestatic toxicants (ethinyl estradiol, LPS, ANIT and methylene dianiline, disulfiram, naltrexone, methapyrilene, phenacetin, alpha-methyl dopa, flutamide, the NSAIDs--indomethacin, flurbiprofen, diclofenac, flufenamic acid, sulindac, and nimesulide, butylated hydroxytoluene, piperonyl butoxide, and bromobenzene), some slightly less active compounds (3'-acetamidofluorene, amsacrine, hydralazine, tannic acid), some drugs that behaved very differently, and were distinct from both non-cholestatic and cholestatic drugs (ketoconazole, dipyridamole, cyproheptadine and aniline), and many postulated human cholestatic drugs that in rat showed no evidence of cholestasis (chlorpromazine, erythromycin, niacin, captopril, dapsone, rifampicin, glibenclamide, simvastatin, furosemide, tamoxifen, and sulfamethoxazole). Most of these latter drugs were noted previously by other groups as showing cholestasis only in humans. The results of this work suggest that the ABC procedure and similar statistical approaches can be instrumental in combining data to compare toxicants across toxicogenomics databases, extract similarities among responses and reduce unexplained data varation.

7.
Pharmacogenomics ; 16(8): 779-801, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26067483

ABSTRACT

AIM: We release the Janssen Toxicogenomics database. This rat liver gene-expression database was generated using Codelink microarrays, and has been used over the past years within Janssen to derive signatures for multiple end points and to classify proprietary compounds. MATERIALS & METHODS: The release consists of gene-expression responses to 124 compounds, selected to give a broad coverage of liver-active compounds. A selection of the compounds were also analyzed on Affymetrix microarrays. RESULTS: The release includes results of an in-house reannotation pipeline to Entrez gene annotations, to classify probes into different confidence classes. High confidence unambiguously annotated probes were used to create gene-level data which served as starting point for cross-platform comparisons. Connectivity map-based similarity methods show excellent agreement between Codelink and Affymetrix runs of the same samples. We also compared our dataset with the Japanese Toxicogenomics Project and observed reasonable agreement, especially for compounds with stronger gene signatures. We describe an R-package containing the gene-level data and show how it can be used for expression-based similarity searches. CONCLUSION: Comparing the same biological samples run on the Affymetrix and the Codelink platform, good correspondence is observed using connectivity mapping approaches. As expected, this correspondence is smaller when the data are compared with an independent dataset such as TG-GATE. We hope that this collection of gene-expression profiles will be incorporated in toxicogenomics pipelines of users.


Subject(s)
Databases, Factual , Liver/metabolism , Toxicogenetics , Animals , Data Mining , Humans , Liver/drug effects , Oligonucleotide Array Sequence Analysis/methods , Rats , Transcriptome
8.
Toxicol Appl Pharmacol ; 275(3): 189-97, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24486436

ABSTRACT

Previously we reported a gene expression signature in rat liver for detecting a specific type of oxidative stress (OS) related to reactive metabolites (RM). High doses of the drugs disulfiram, ethinyl estradiol and nimesulide were used with another dozen paradigm OS/RM compounds, and three other drugs flutamide, phenacetin and sulindac were identified by this signature. In a second study, antiepileptic drugs were compared for covalent binding and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital produced robust OS/RM gene expression. In the present study, liver RNA samples from drug-treated rats from more recent experiments were examined for statistical fit to the OS/RM signature. Of all 97 drugs examined, in addition to the nine drugs noted above, 19 more were identified as OS/RM-producing compounds-chlorpromazine, clozapine, cyproterone acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole, methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, coumarin, ritonavir, amitriptyline, valproic acid, enalapril, and chloramphenicol. Importantly, all of the OS/RM drugs listed above have been linked to idiosyncratic hepatotoxicity, excepting chloramphenicol, which does not have a package label for hepatotoxicity, but does have a black box warning for idiosyncratic bone marrow suppression. Most of these drugs are not acutely toxic in the rat. The OS/RM signature should be useful to avoid idiosyncratic hepatotoxicity of drug candidates.


Subject(s)
Chemical and Drug Induced Liver Injury/genetics , Gene Expression Profiling , Liver/drug effects , Oxidative Stress/drug effects , Oxidative Stress/genetics , Toxicogenetics/methods , Animals , Databases, Genetic , Gene Expression Regulation/drug effects , Genetic Markers , Liver/metabolism , Male , Oligonucleotide Array Sequence Analysis , Rats , Rats, Sprague-Dawley
9.
Biomaterials ; 33(7): 2165-76, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22189144

ABSTRACT

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 µm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.


Subject(s)
Drug Evaluation, Preclinical/methods , Hepatocytes/cytology , Models, Biological , Spheroids, Cellular/physiology , Animals , Cells, Cultured , Collagen/metabolism , Hepatocytes/physiology , Humans , Male , Rats , Rats, Wistar , Spheroids, Cellular/cytology
10.
Biomaterials ; 32(29): 6982-94, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21741702

ABSTRACT

Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 µm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell-cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to ß-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.


Subject(s)
Cell Culture Techniques/instrumentation , Cellulose/chemistry , Galactose/chemistry , Hepatocytes/cytology , High-Throughput Screening Assays/methods , Hydrogels/chemistry , Spheroids, Cellular/cytology , Animals , Biocompatible Materials/chemistry , Cell Culture Techniques/methods , Cells, Cultured , Cytochrome P-450 Enzyme System/metabolism , Hepatocytes/metabolism , High-Throughput Screening Assays/instrumentation , Male , Materials Testing , Molecular Structure , Pharmaceutical Preparations/metabolism , Porosity , Rats , Rats, Wistar , Spheroids, Cellular/metabolism , Tissue Engineering/methods
11.
Biomaterials ; 32(4): 1229-41, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20971505

ABSTRACT

Hepatotoxicity evaluation of pharmaceutical lead compounds in early stages of drug development has drawn increasing attention. Sandwiched hepatocytes exhibiting stable functions in culture represent a standard model for hepatotoxicity testing of drugs. We have developed a robust and high-throughput hepatotoxicity testing platform based on the sandwiched hepatocytes for drug screening. The platform involves a galactosylated microfabricated membrane sandwich to support cellular function through uniform and efficient mass transfer while protecting cells from excessive shear. Perfusion bioreactor further enhances mass transfer and cellular functions over long period; and hepatocytes are readily transferred to 96-well plate for high-throughput robotic liquid handling. The bioreactor design and perfusion flow rate are optimized by computational fluid dynamics simulation and experimentally. The cultured hepatocytes preserved 3D cell morphology, urea production and cytochrome p450 activity of the mature hepatocytes for 14 days. When the perfusion-cultured sandwich is transferred to 96-well plate for drug testing, the hepatocytes exhibited improved drug sensitivity and low variability in hepatotoxicity responses amongst cells transferred from different dates of perfusion culture. The platform enables robust high-throughput screening of drug candidates.


Subject(s)
Drug Evaluation, Preclinical/methods , Hepatocytes/drug effects , Hepatocytes/metabolism , High-Throughput Screening Assays/methods , Pharmaceutical Preparations/chemistry , Animals , Cells, Cultured , Hepatocytes/cytology , High-Throughput Screening Assays/instrumentation , Male , Rats , Rats, Wistar
12.
Trends Biotechnol ; 29(3): 110-8, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21129798

ABSTRACT

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.


Subject(s)
Liver/cytology , Tissue Engineering , Animals , Biocompatible Materials , Embryonic Stem Cells/cytology , Hepatocytes/physiology , Humans , Liver/physiology , Liver Regeneration , Tissue Scaffolds
13.
Toxicol Sci ; 117(2): 493-504, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20616206

ABSTRACT

The unexpected observation of a hyperglycemic effect of some tricycle-based delta opioid receptor (DOR) agonists led to a series of studies to better understand the finding. Single administration of two novel tricyclic DOR agonists dose dependently elevated rat plasma glucose levels; 4-week toxicology studies confirmed the hyperglycemic finding and further revealed pancreatic ß-cell hypertrophy, including vacuole formation, as well as bone dysplasia and Harderian gland degeneration with regeneration. Similar diabetogenic effects were observed in dog. A review of the literature on the antiserotonergic and antihistaminergic drug cyproheptadine (CPH) and its metabolites revealed shared structural features as well as similar hyperglycemic effects to the present series of DOR agonists. To further evaluate these effects, we established an assay measuring insulin levels in the rat pancreatic ß-cell-derived RINm5F cell line, extensively used to study CPH and its metabolites. Like CPH, the initial DOR agonists studied reduced RINm5F cell insulin levels in a concentration-dependent manner. Importantly, compound DOR potency did not correlate with the insulin-reducing potency. Furthermore, the RINm5F cell insulin results correlated with the diabetogenic effect of the compounds in a 5-day mouse study. The RINm5F cell insulin assay enabled the identification of aryl-aryl-amine DOR agonists that lacked an insulin-reducing effect and did not elevate blood glucose in repeated dosing studies conducted over a suprapharmacologic dose range. Thus, not only did the RINm5F cell assay open a path for the further discovery of DOR agonists lacking diabetogenic potential but also it established a reliable, economical, and high-throughput screen for such potential, regardless of chemotype or target pharmacology. The present findings also suggest a mechanistic link between the toxicity observed here and that underlying Wolcott-Rallison Syndrome.


Subject(s)
Cyproheptadine/toxicity , Hyperglycemia/chemically induced , Insulin-Secreting Cells/drug effects , Narcotic Antagonists/toxicity , Pancreas/drug effects , Serotonin Antagonists/toxicity , Animals , Blood Glucose/analysis , Blood Glucose/drug effects , Cell Enlargement/drug effects , Cell Line, Tumor , Cyproheptadine/analogs & derivatives , Diabetes Mellitus, Type 1/metabolism , Dogs , Epiphyses/abnormalities , Epiphyses/metabolism , Female , High-Throughput Screening Assays , Hyperglycemia/metabolism , Insulin/blood , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Insulinoma/drug therapy , Insulinoma/metabolism , Male , Mice , Osteochondrodysplasias/metabolism , Pancreas/metabolism , Pancreas/pathology , Rats , Rats, Sprague-Dawley , Vacuoles/drug effects , Vacuoles/ultrastructure
14.
Toxicol Sci ; 103(1): 28-34, 2008 May.
Article in English | MEDLINE | ID: mdl-18281259

ABSTRACT

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.


Subject(s)
Carcinogenicity Tests/methods , Genomics , Animals , Gene Expression Profiling , Male , Rats , Rats, Sprague-Dawley
15.
Chem Res Toxicol ; 20(4): 600-8, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17381134

ABSTRACT

Felbamate is an antiepileptic drug that is associated with minimal toxicity in preclinical species such as rat and dog but has an unacceptable incidence of serious idiosyncratic reactions in man. Idiosyncratic reactions account for over half of toxicity-related drug failures in the marketplace, and improving the preclinical detection of idiosyncratic toxicities is thus of paramount importance to the pharmaceutical industry. The formation of reactive metabolites is common among most drugs associated with idiosyncratic drug reactions and may cause deleterious effects through covalent binding and/or oxidative stress. In the present study, felbamate was compared to several other antiepileptic drugs (valproic acid, carbamazepine, phenobarbital, and phenytoin), using covalent binding of radiolabeled drugs and hepatic gene expression responses to evaluate oxidative stress/reactive metabolite potential. Despite causing only very mild effects on covalent binding parameters, felbamate produced robust effects on a previously established oxidative stress/reactive metabolite gene expression signature. The other antiepileptic drugs and acetaminophen are known hepatotoxicants at high doses in the rat, and all increased covalent binding to liver proteins in vivo and/or to liver microsomes from human and rat. With the exception of acetaminophen, valproic acid exhibited the highest covalent binding in vivo, whereas carbamazepine exhibited the highest levels in vitro. Pronounced effects on oxidative stress/reactive metabolite-responsive gene expression were observed after carbamazepine, phenobarbital, and phenytoin administration. Valproic acid had only minor effects on the oxidative stress/reactive metabolite indicator genes. The relative ease of detection of felbamate based on gene expression results in rat liver as having potential oxidative stressor/reactive metabolites indicates that this approach may be useful in screening for potential idiosyncratic toxicity. Together, measurements of gene expression along with covalent binding should improve the safety assessment of candidate drugs.


Subject(s)
Anticonvulsants/toxicity , Epilepsy/drug therapy , Gene Expression Regulation/drug effects , Liver/drug effects , Liver/metabolism , Phenylcarbamates/toxicity , Propylene Glycols/toxicity , Animals , Cells, Cultured , Epilepsy/pathology , Felbamate , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Protein Binding , Rats
16.
Toxicol Appl Pharmacol ; 216(3): 416-25, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-16926038

ABSTRACT

Heme oxygenase-1 (HO-1) is one of several enzymes induced by hepatotoxicants, and is thought to have an important protective role against cellular stress during liver inflammation and injury. The objective of the present study was to evaluate the role of HO-1 in estradiol-induced liver injury. A single dose of ethinyl estradiol (500 mg/kg, po) resulted in mild liver injury. Repeated administration of ethinyl estradiol (500 mg/kg/day for 4 days, po) resulted in no detectable liver injury or dysfunction. Using RT-PCR analysis, we demonstrate that HO-1 gene expression in whole liver tissue is elevated (>20-fold) after the single dose of ethinyl estradiol. The number and intensity of HO-1 immunoreactive macrophages were increased after the single dose of ethinyl estradiol. HO-1 expression was undetectable in hepatic parenchymal cells from rats receiving Methocel control or a single dose of ethinyl estradiol, however cytosolic HO-1 immunoreactivity in these cells after repeated dosing of ethinyl estradiol was pronounced. The increases in HO-1 mRNA and HO-1 immunoreactivity following administration of a single dose of ethinyl estradiol suggested that this enzyme might be responsible for the observed protection of the liver during repeated dosing. To investigate the effect of HO-1 expression on ethinyl estradiol-induced hepatotoxicity, rats were pretreated with hemin (50 micromol/kg, ip, a substrate and inducer of HO-1), with tin protoporphyrin IX (60 micromol/kg, ip, an HO-1 inhibitor), or with gadolinium chloride (10 mg/kg, iv, an inhibitor/toxin of Kupffer cells) 24 h before ethinyl estradiol treatment. Pretreatment with modulators of HO-1 expression and activity had generally minimal effects on ethinyl estradiol-induced liver injury. These data suggest that HO-1 plays a limited role in antioxidant defense against ethinyl estradiol-induced oxidative stress and hepatotoxicity, and suggests that other coordinately induced enzymes are responsible for protection observed with repeated administration of high doses of this compound.


Subject(s)
Antioxidants/metabolism , Estrogens/pharmacology , Ethinyl Estradiol/pharmacology , Heme Oxygenase-1/biosynthesis , Liver/enzymology , Animals , Biomarkers , Enzyme Induction/drug effects , Female , Gadolinium/pharmacology , Gene Expression/drug effects , Heme Oxygenase-1/antagonists & inhibitors , Hemin/pharmacology , Immunohistochemistry , Liver/drug effects , Macrophages/drug effects , Metalloporphyrins/pharmacology , Protoporphyrins/pharmacology , RNA/biosynthesis , RNA/isolation & purification , Rats , Rats, Sprague-Dawley , Response Elements , Reverse Transcriptase Polymerase Chain Reaction
17.
Mol Carcinog ; 45(12): 914-33, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16921489

ABSTRACT

Toxicogenomics technology defines toxicity gene expression signatures for early predictions and hypotheses generation for mechanistic studies, which are important approaches for evaluating toxicity of drug candidate compounds. A large gene expression database built using cDNA microarrays and liver samples treated with over one hundred paradigm compounds was mined to determine gene expression signatures for nongenotoxic carcinogens (NGTCs). Data were obtained from male rats treated for 24 h. Training/testing sets of 24 NGTCs and 28 noncarcinogens were used to select genes. A semiexhaustive, nonredundant gene selection algorithm yielded six genes (nuclear transport factor 2, NUTF2; progesterone receptor membrane component 1, Pgrmc1; liver uridine diphosphate glucuronyltransferase, phenobarbital-inducible form, UDPGTr2; metallothionein 1A, MT1A; suppressor of lin-12 homolog, Sel1h; and methionine adenosyltransferase 1, alpha, Mat1a), which identified NGTCs with 88.5% prediction accuracy estimated by cross-validation. This six genes signature set also predicted NGTCs with 84% accuracy when samples were hybridized to commercially available CodeLink oligo-based microarrays. To unveil molecular mechanisms of nongenotoxic carcinogenesis, 125 differentially expressed genes (P<0.01) were selected by Student's t-test. These genes appear biologically relevant, of 71 well-annotated genes from these 125 genes, 62 were overrepresented in five biochemical pathway networks (most linked to cancer), and all of these networks were linked by one gene, c-myc. Gene expression profiling at early time points accurately predicts NGTC potential of compounds, and the same data can be mined effectively for other toxicity signatures. Predictive genes confirm prior work and suggest pathways critical for early stages of carcinogenesis.


Subject(s)
Carcinogens/toxicity , Cell Transformation, Neoplastic/chemically induced , Gene Expression Profiling , Genes, Neoplasm/drug effects , Liver Neoplasms, Experimental/chemically induced , Animals , Cell Transformation, Neoplastic/genetics , Gene Expression/drug effects , Liver/drug effects , Liver Neoplasms, Experimental/genetics , Male , Mutagenicity Tests , Oligonucleotide Array Sequence Analysis , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Toxicogenetics
18.
Basic Clin Pharmacol Toxicol ; 98(6): 537-46, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16700814

ABSTRACT

Understanding the response of biological systems to xenobiotics is fundamental to the evaluation of drug safety. Toxicologists have traditionally gathered pathological, morphological, chemical and biochemical information from in vivo studies of preclinical species in order to assess drug safety and to determine how new drugs can be safely administered to the human patient population. In recent years the emerging "-omics" technologies have been developed and integrated into preclinical studies in order to better assess drug safety by gaining information on the cellular and molecular events underlying adverse drug reactions. Genomics approaches in particular have become readily available and are being applied in several stages of drug development. The burgeoning literature on what has become known as "toxicogenomics" has for the most part highlighted successful applications of gene expression profiling in predictive toxicology, enabling decisions to be made on the developability of a compound early in the drug development process. It is also becoming apparent that toxicogenomic approaches are good starting points to develop experiments designed to gain a mechanistic insight into drug toxicities within and across species. Gene expression arrays permit the measurement of responses of essentially all the genes in the entire genome to be monitored, and knowledge of the function of the genes affected can identify the potential mechanisms to then be confirmed using conventional biochemical, toxicological and pathological approaches. As toxicologists put these technologies into practice they build up a knowledge base to better characterize toxicities at the molecular level and to make the search for much needed, novel biomarkers of toxicity more achievable.


Subject(s)
Drug Evaluation, Preclinical/methods , Drug-Related Side Effects and Adverse Reactions/genetics , Genomics , Gene Expression Profiling , Humans , Microarray Analysis , Risk Assessment
19.
Toxicol Mech Methods ; 16(2-3): 51-8, 2006.
Article in English | MEDLINE | ID: mdl-20020997

ABSTRACT

Since the identification in the 1950s of deoxyribonucleic acid as the building block of life, the impact of molecular biology has been far-reaching. Understanding the processes of how DNA is replicated, transcribed into RNA and then translated into protein products has not only provided a fundamental knowledge of life but has also spawned a plethora of applications. Molecular biology has been high profile and widespread in research into the biology of disease and in drug discovery. It has additionally found application in understanding the adverse effects, or toxicity, of candidate drugs and how they interfere with biochemical and biological processes. In recent times the biggest impact of molecular biology in toxicology has been through the study of differential gene expression, largely as a result of the advent of genomics. This review seeks to describe how toxicogenomics strategies have been implemented and integrated into nonclinical studies of drug safety.

20.
Toxicol Appl Pharmacol ; 207(2 Suppl): 171-8, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-15982685

ABSTRACT

Macrophage activators (MA), peroxisome proliferators (PP), and oxidative stressors/reactive metabolites (OS/RM) all produce oxidative stress and hepatotoxicity in rats. However, these three classes of hepatotoxicants give three distinct gene transcriptional profiles on cDNA microarrays, an indication that rat hepatocytes respond/adapt quite differently to these three classes of oxidative stressors. The differential gene responses largely reflect differential activation of transcription factors: MA activate Stat-3 and NFkB, PP activate PPARa, and OS/RM activate Nrf2. We have used gene signature profiles for each of these three classes of hepatotoxicants to categorize over 100 paradigm (and 50+ in-house proprietary) compounds as to their oxidative stress potential in rat liver. In addition to a role for microarrays in predictive toxicology, analyses of small subsets of these signature profiles, genes within a specific pathway, or even single genes often provide important insights into possible mechanisms involved in the toxicities of these compounds.


Subject(s)
Genomics , Liver/drug effects , Oxidative Stress , Toxicology , Animals , Gene Expression Profiling , Liver/metabolism , Oligonucleotide Array Sequence Analysis , Rats
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