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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.

SUBMITTER: Clewell RA

PROVIDER: S-EPMC4226763 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

Clewell Rebecca A RA Sun Bin B Adeleye Yeyejide Y Carmichael Paul P Efremenko Alina A McMullen Patrick D PD Pendse Salil S Trask O J OJ White Andy A Andersen Melvin E ME

Toxicological sciences : an official journal of the Society of Toxicology 20140730 1

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 ...[more]

PMID: 25078064

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Project description:Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T4) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gavage with 18 different polyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T4 was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose-response functions (based on seven to nine doses per chemical) were determined for individual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmental concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED30), as well as predicted mixture outcomes were calculated using a flexible single-chemical-required method applicable to chemicals with differing dose thresholds and maximum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimentally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the first results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs.

Dataset Information

Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

Publications

Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

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