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The stress response resolution assay. I. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium.

Environmental and molecular mutagenesis (2013-04-05)
Dale M Walker, J Patrick O'Neill, Frederick L Tyson, Vernon E Walker
ABSTRACT

The events or factors that lead from normal cell function to conditions and diseases such as aging or cancer reflect complex interactions between cells and their environment. Cellular stress responses, a group of processes involved in homeostasis and adaptation to environmental change, contribute to cell survival under stress and can be resolved with damage avoidance or damage tolerance outcomes. To investigate the impact of environmental agents/conditions upon cellular stress response outcomes in epithelium, a novel quantitative assay, the "stress response resolution" (SRR) assay, was developed. The SRR assay consists of pretreatment with a test agent or vehicle followed later by a calibrated stress conditions exposure step (here, using 6-thioguanine). Pilot studies conducted with a spontaneously-immortalized murine mammary epithelial cell line pretreated with vehicle or 20 µg N-ethyl-N-nitrososurea/ml medium for 1 hr, or two hTERT-immortalized human bronchial epithelial cell lines pretreated with vehicle or 100 µM zidovudine/lamivudine for 12 days, found minimal alterations in cell morphology, survival, or cell function through 2 weeks post-exposure. However, when these pretreatments were followed 2 weeks later by exposure to calibrated stress conditions of limited duration (for 4 days), significant alterations in stress resolution were observed in pretreated cells compared with vehicle-treated control cells, with decreased damage avoidance survival outcomes in all cell lines and increased damage tolerance outcomes in two of three cell lines. These pilot study results suggest that sub-cytotoxic pretreatments with chemical mutagens have long-term adverse impact upon the ability of cells to resolve subsequent exposure to environmental stressors.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Lamivudine, ≥98% (HPLC), powder
Sigma-Aldrich
N-Nitroso-N-ethylurea, Bulk package
Sigma-Aldrich
N-Nitroso-N-ethylurea, ISOPAC®
Sigma-Aldrich
Amphotericin B solubilized, powder, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
Amphotericin B from Streptomyces sp., ~80% (HPLC), powder
Sigma-Aldrich
Amphotericin B from Streptomyces sp., BioReagent, suitable for cell culture, ~80% (HPLC)
Supelco
Amphotericin B trihydrate, VETRANAL®, analytical standard
Sigma-Aldrich
Amphotericin B solution, 250 μg/mL in deionized water, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
3′-Azido-3′-deoxythymidine, ≥98% (HPLC)
Amphotericin B for peak identification, European Pharmacopoeia (EP) Reference Standard
Supelco
Zidovudine, Pharmaceutical Secondary Standard; Certified Reference Material