title: Application of the TGx-28.65 transcriptomic biomarker to classify genotoxic and non-genotoxic chemicals in human TK6 cells in the presence of rat liver S9.
abstract: In vitro transcriptional signatures that predict toxicities can facilitate chemical screening. We previously developed a transcriptomic biomarker (known as TGx-28.65) for classifying agents as genotoxic (DNA damaging) and non-genotoxic in human lymphoblastoid TK6 cells. Because TK6 cells do not express cytochrome P450s, we confirmed accurate classification by the biomarker in cells co-exposed to 1% 5,6 benzoflavone/phenobarbital-induced rat liver S9 for metabolic activation. However, chemicals may require different types of S9 for activation. Here we investigated the response of TK6 cells to higher percentages of Aroclor-, benzoflavone/phenobarbital-, or ethanol-induced rat liver S9 to expand TGx-28.65 biomarker applicability. Transcriptional profiles were derived 3 to 4 hr following a 4 hr co-exposure of TK6 cells to test chemicals and S9. Preliminary studies established that 10% Aroclor- and 5% ethanol-induced S9 alone did not induce the TGx-28.65 biomarker genes. Seven genotoxic and two non-genotoxic chemicals (and concurrent solvent and positive controls) were then tested with one of the S9s (selected based on cell survival and micronucleus induction). Relative survival and micronucleus frequency was assessed by flow cytometry in cells 20 hr post-exposure. Genotoxic/non-genotoxic chemicals were accurately classified using the different S9s. One technical replicate of cells co-treated with dexamethasone and 10% Aroclor-induced S9 was falsely classified as genotoxic, suggesting caution in using high S9 concentrations. Even low concentrations of genotoxic chemicals (those not causing cytotoxicity) were correctly classified, demonstrating that TGx-28.65 is a sensitive biomarker of genotoxicity. A meta-analysis of datasets from 13 chemicals supports that different S9s can be used in TK6 cells, without impairing classification using the TGx-28.65 biomarker.
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Carole L
authors.initials: CL
authors.lastname: Yauk
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Buick
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Andrew
authors.initials: A
authors.lastname: Williams
authors.affiliation: Integrated Laboratory Systems Inc, Research Triangle Park, North Carolina.
authors.forename: Carol D
authors.initials: CD
authors.lastname: Swartz
authors.affiliation: Integrated Laboratory Systems Inc, Research Triangle Park, North Carolina.
authors.forename: Leslie
authors.initials: L
authors.lastname: Recio
authors.affiliation: Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, District of Columbia.|Department of Oncology, Georgetown University Medical Center, Washington, District of Columbia.
authors.forename: Heng-Hong
authors.initials: HH
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, District of Columbia.|Department of Oncology, Georgetown University Medical Center, Washington, District of Columbia.
authors.forename: Albert J
authors.initials: AJ
authors.lastname: Fornace
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Errol M
authors.initials: EM
authors.lastname: Thomson
authors.affiliation: Drug Safety Research and Development, Pfizer Inc, Groton, Connecticut.
authors.forename: Jiri
authors.initials: J
authors.lastname: Aubrecht
mesh_terms: D065767:Activation, Metabolic / Q000187:drug effects; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001140:Aroclors / Q000633:toxicity; D001571:Benzoflavones / Q000633:toxicity; D002460:Cell Line; D000431:Ethanol / Q000633:toxicity; D020869:Gene Expression Profiling / Q000379:methods*; D005819:Genetic Markers; D006801:Humans; D008099:Liver / Q000187:drug effects; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D010634:Phenobarbital / Q000633:toxicity; D051381:Rats; D059467:Transcriptome / Q000187:drug effects
chemical_list: D001140:Aroclors; D001571:Benzoflavones; D005819:Genetic Markers; D009153:Mutagens; D000431:Ethanol; D010634:Phenobarbital
keywords: S9; TGx-28.65 biomarker; biomarker; furan; genetic toxicology; metabolic activation; micronucleus; toxicogenomics