Derivations of Tissue Slice Technology as Toxicological Screening Systems

Persistent Link:
http://hdl.handle.net/10150/195419
Title:
Derivations of Tissue Slice Technology as Toxicological Screening Systems
Author:
Catania, Jeffrey Mark
Issue Date:
2006
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
In vitro toxicology studies are hindered by the use of specific cellular systems which solely examine one cell type. Precision-cut tissue slices mimic specific organ toxicity as normal cellular heterogeneity and organ architecture are retained. Experiments were performed using tissue slices from transgenic mice with enzyme reporter proteins for rapid analysis. CYP 1A1/β-galactosidase transgenic mouse liver and kidney slices challenged with 20 μM BNF for 24 hr remain viable and display organ-specific induction of β-galactosidase (~30-fold in liver and 3-fold in kidney). AP-1/luciferase transgenic mouse tissue slices incubated with 9 μM TPA also remained viable while exhibiting a tissue- and time-dependent induction of luciferase. In kidney slices, luciferase induction was approximately 1.5-fold at 2 hr, which increased to 2.5-fold at 4 hr. Liver slices displayed a rapid increase in luciferase at 2 hr (approximately 3-fold) which was abolished at 4 hr. To quicken experimental design via decreased sample preparation, a custom transgenic mouse was created based upon a fluorescent reporter protein. Subsequent studies with slices from this strain and another fluorescent-based transgenic strain did not display reporter induction. For optimization of the smaller tissues of mice and to create an easily deployable method of rapid detection, a tissue chip based system was created for generating large numbers of samples from a single organ and coupled with fluorescent indicators to maximize detection sensitivity for specific cellular processes. Fluorescence of 5-carboxyfluorescein increased at high concentrations of iodoacetamide (IAM), a quick-acting toxicant, indicating disruption of cellular membranes. The mitochondrial probe, TMRE, exhibited an increase in fluorescence with increasing IAM concentrations. Monobromobimane, a sulfhydryl probe, displayed a decrease in fluorescent intensity at higher IAM challenge; a finding confirmed with Ellman’s reagent. A probe used for calcium measurement, FURA-2, demonstrated an increase in fluorescence with increasing IAM concentrations. Importantly, the number of samples per organ/mouse was increased at least 3-fold and a significant time reduction per analysis was realized. These results suggest that both transgenic-based tissue slice studies and studies with fluorescent probes in naïve tissue chips are two methods of higher-throughput analyses to evaluate toxicant perturbations with in vitro studies.
Type:
text; Electronic Dissertation
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Pharmacology & Toxicology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Gandolfi, A. Jay
Committee Chair:
Gandolfi, A. Jay

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleDerivations of Tissue Slice Technology as Toxicological Screening Systemsen_US
dc.creatorCatania, Jeffrey Marken_US
dc.contributor.authorCatania, Jeffrey Marken_US
dc.date.issued2006en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractIn vitro toxicology studies are hindered by the use of specific cellular systems which solely examine one cell type. Precision-cut tissue slices mimic specific organ toxicity as normal cellular heterogeneity and organ architecture are retained. Experiments were performed using tissue slices from transgenic mice with enzyme reporter proteins for rapid analysis. CYP 1A1/β-galactosidase transgenic mouse liver and kidney slices challenged with 20 μM BNF for 24 hr remain viable and display organ-specific induction of β-galactosidase (~30-fold in liver and 3-fold in kidney). AP-1/luciferase transgenic mouse tissue slices incubated with 9 μM TPA also remained viable while exhibiting a tissue- and time-dependent induction of luciferase. In kidney slices, luciferase induction was approximately 1.5-fold at 2 hr, which increased to 2.5-fold at 4 hr. Liver slices displayed a rapid increase in luciferase at 2 hr (approximately 3-fold) which was abolished at 4 hr. To quicken experimental design via decreased sample preparation, a custom transgenic mouse was created based upon a fluorescent reporter protein. Subsequent studies with slices from this strain and another fluorescent-based transgenic strain did not display reporter induction. For optimization of the smaller tissues of mice and to create an easily deployable method of rapid detection, a tissue chip based system was created for generating large numbers of samples from a single organ and coupled with fluorescent indicators to maximize detection sensitivity for specific cellular processes. Fluorescence of 5-carboxyfluorescein increased at high concentrations of iodoacetamide (IAM), a quick-acting toxicant, indicating disruption of cellular membranes. The mitochondrial probe, TMRE, exhibited an increase in fluorescence with increasing IAM concentrations. Monobromobimane, a sulfhydryl probe, displayed a decrease in fluorescent intensity at higher IAM challenge; a finding confirmed with Ellman’s reagent. A probe used for calcium measurement, FURA-2, demonstrated an increase in fluorescence with increasing IAM concentrations. Importantly, the number of samples per organ/mouse was increased at least 3-fold and a significant time reduction per analysis was realized. These results suggest that both transgenic-based tissue slice studies and studies with fluorescent probes in naïve tissue chips are two methods of higher-throughput analyses to evaluate toxicant perturbations with in vitro studies.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmacology & Toxicologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorGandolfi, A. Jayen_US
dc.contributor.chairGandolfi, A. Jayen_US
dc.contributor.committeememberChen, Qinen_US
dc.contributor.committeememberErickson, Robert P.en_US
dc.contributor.committeememberFutscher, Bernard W.en_US
dc.contributor.committeememberMcQueen, Charleneen_US
dc.identifier.proquest1436en_US
dc.identifier.oclc752259897en_US
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