Induction, Effects, and Quantification of Sublethal Oxidative Stress by Hydrogen Peroxide on Cultured Human Fibroblasts

• Published in: Experimental cell research Vol. 216; no. 2; pp. 388 - 395
• Main Authors: Mocali, Alessandra, Caldini, Riccardo, Chevanne, Marta, Paoletti, Francesco
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-02-1995
• Subjects: Actin Cytoskeleton - drug effects; Adenosine Triphosphate - metabolism; Biomarkers; Calcium - metabolism; Cell Adhesion - drug effects; Cell Survival; Cells, Cultured; Cytoskeletal Proteins - metabolism; DNA - biosynthesis; Fibroblasts - drug effects; Fibroblasts - enzymology; Fibroblasts - metabolism; Fibroblasts - pathology; Humans; Hydrogen Peroxide - toxicity; NAD - metabolism; Oxidative Stress; Protein Biosynthesis
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.1995.1049

Conditions to induce and parameters to evaluate sublethal oxidative stress of cultured human fibroblasts have been investigated in the attempt to identify markers for a more accurate quantification of cell injury. Sublethal oxidative stress was obtained by treating fibroblasts with 0.5 m
M H
2O
2 in DMEM plus 5% FCS for times not exceeding 60 min. Under these conditions cells remained viable throughout long-term incubation, showing no appreciable release of cytosolic enzymes into the medium. On the contrary, exposures of fibroblasts to 0.5 m
M H
2O
2 for times >60 min induced a lethal cell injury which was fully expressed 2 days later by massive monolayer wasting and leakage of cytosolic components. Early metabolic effects of sublethal stress consisted of a rapid and significant fall of both ATP and NAD
+ pools. Concomitantly, there was a moderate increase (about threefold) in both ADP-ribosyl transferase activity and free [Ca
2+]
i, while the specific activity of glyceraldehyde-3-phosphate dehydrogenase was partially decreased upon treatment. Oxidative injury also caused delayed effects consisting of a large depression of both protein and DNA synthesis. However, while the former was partially restored within 10 days of incubation, the latter remained severely impaired, as encountered in a growth-arrested population. Microfilaments of H
2O
2-treated cells appeared to be morphologically altered due to partial fragmentation of cytoskeleton actin which, however, was still maintained in the polymerized form as F-actin. Moreover, sublethally injured fibroblasts exhibited a reduced adhesiveness to plastic once they were detached and reseeded into new dishes. Relative adhesion efficiencies (number of adherent cells at 16 h as a percentage of seeded cells) were found to correlate inversely with times of exposure to H
2O
2. This finding allowed the identification of a biological parameter which showed itself to be very sensitive to oxidative stress and was also useful for developing an assay to grade sublethal injury to fibroblasts.