Progressive DNA damage in live cells by oxidants is the key factor contributing to cell aging and preconditioning to neoplastic transformation. The strategies to slow aging or prevent cancer rely on protection of DNA from the damage. Since cells reside within intercellular matrix it is of interest to know whether matrix constituents possess properties of modulating oxidative DNA damage. We explored, therefore, the effect of hyaluronate (HA), the ubiquitous component of the matrix, on extent of DNA damage induced by exogenous and endogenously generated oxidants. WI-38 and A549 cells were exposed to 200 µM H2O2 in the absence or presence of HA and induction of histone H2AX phosphorylation and activation of ATM, the reporters of DNA damage, was assessed by multiparameter cytometry. Also explored was effect of HA on constitutive H2AX phosphorylation that reflects DNA damage caused by endogenous oxidants generated during aerobic metabolism. HA of average MW 5.4 million (high MW) and 2 million (medium MW) at 0.1% (w/v) in culture medium totally prevented the H2O2-induced H2AX phosphorylation in both cell types whereas effect of 60,000 average MW (low MW) HA was somewhat less pronounced. Constitutive H2AX phosphorylation in WI-38 cells growing in the presence of 0.1% HA of low MW and medium MW was reduced by about 35 and 30%, respectively; no reduction was observed in A549 cells. The data indicate that HA protected DNA from damage caused by the exogenous oxidant H2O2. In WI-38 fibroblasts, the cells that express the HA-receptor CD44, HA also protected DNA from damage caused by endogenous oxidants. We postulate that expression of CD44 in some cell types such as stem cells may provide the means to internalize HA by endocytosis and one of the functions of the internalized HA may be protection of DNA from oxidants. The mechanism of protective effect of HA may either: i) involve entrapment of iron ions thereby inhibiting the Fenton's reaction that produces secondary oxidative species, and/or: ii) directly scavenging of primary and secondary ROIs, as an antioxidant, resulting in HA degradation. Since no significant degradation of HA upon its exposure in tissue culture medium to H2O2 was detected the scavenging may occur intracellularly.

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