title: Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.
abstract: As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116). At chemical concentrations causing similar increases in p53 protein expression, p53-mediated protein expression and cellular processes showed substantial chemical-specific differences. These chemical-specific differences in the p53 transcriptional response appear to be determined by augmentation of the p53 response by co-regulators. More importantly, dose-response data for each of the chemicals indicate that the p53 transcriptional response does not prevent micronuclei induction at low concentrations. In fact, the no observed effect levels and benchmark doses for micronuclei induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53's post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. This effort demonstrates the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype.
authors: The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709 rclewell@thehamner.org.
authors: Rebecca A
authors: RA
authors: Clewell
authors: The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709.
authors: Bin
authors: B
authors: Sun
authors: Unilever, Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK.
authors: Yeyejide
authors: Y
authors: Adeleye
authors: Unilever, Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK.
authors: Paul
authors: P
authors: Carmichael
authors: The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709.
authors: Alina
authors: A
authors: Efremenko
authors: The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709.
authors: Patrick D
authors: PD
authors: McMullen
authors: The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709.
authors: Salil
authors: S
authors: Pendse
authors: The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709.
authors: O J
authors: OJ
authors: Trask
authors: Unilever, Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK.
authors: Andy
authors: A
authors: White
authors: The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709.
authors: Melvin E
authors: ME
authors: Andersen
mesh_terms: D023401:Animal Use Alternatives; D017209:Apoptosis / Q000187:drug effects; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D045325:HCT116 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced*; D009153:Mutagens / Q000737:chemistry / Q000633:toxicity*; D019055:No-Observed-Adverse-Effect Level; D018570:Risk Assessment; D015398:Signal Transduction / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D009153:Mutagens; D016159:Tumor Suppressor Protein p53
keywords: DNA damage response; animal alternatives; dose-response; p53; three Rs; toxicity pathways