In-vitro cell confluency affects primary human dermal fibroblast sensitivity to low concentrations of t-butylhydroperoxide

Introduction: Mitochondria play a central role in ATP production, radical oxygen species (ROS) generation, calcium signaling and apoptosis. Oxidative stress derived from mitochondria is associated with intrauterine growth restriction (IUGR), cardiovascular disease, cancer and aging. The molecular link between oxidative stress and these conditions is, however, poorly understood. Interestingly, elevated levels of intracellular S-adenosylhomocysteine (SAH), a potent inhibitor of most cellular methyltransferases, is also associated with IUGR, cardiovascular disease, cancer and aging. The molecular mechanism underlying these associations is thought to be reduced methylation capacity, leading to alterations in gene expression, cell differentiation, chromatin conformation and cell phenotype. At present, accumulation of intracellular SAH is believed to be due to nutritional deficiencies or genetic polymorphisms in the folate pathway. We now suggest that oxidative stress to mitochondria may increase intracellular SAH. Accumulation of intracellular SAH following oxidative stress to mitochondria may be an important link between oxidative stress and IUGR, cardiovascular disease, cancer and aging.

In order to test our hypothesis, we have begun defining sub-lethal concentrations of t-butylhydroperoxide (t-buOOH), a compound that selectively induces ROS formation in mitochondria. The second step will be to measure levels of SAH and homocysteine in treated and non-treated living cells using high perfomance liquid chromatography.

Methods and Materials: Stock cultures of primary human dermal fibroblasts were grown at 37°C in 100-mm tissue culture dishes in DMEM medium supplemented with 10% bovine calf serum and 1% streptomycin. Experimental cultures (40,000 cells per well) were plated in 35-mm tissue culture dishes and allowed to grow for 24 hours. The medium was then replaced with medium containing concentrations of 1ul, 10ul or 50uM t-buOOH. Light microscopy was used to evaluate cell apoptosis, characterized by cell shrinkage and detachment from the tissue culture dish, every 12 hours for 3 days.

Results: After 24 hours, experimental cell cultures exposed to 50 uM t-buOOH were uniformly killed. Experimental cultures exposed to both 10 and 1 uM t-buOOH, however, displayed apoptosis only in the periphery of the tissue culture dishes. Careful observation of control cell culture dishes revealed that after 24 hours, cell confluency was 100% centrally, while only 50% in the periphery. These findings suggest that in-vitro cell confluency affects human dermal fibroblast sensitivity to low concentrations of t-butylhydroperoxide.

Discussion: In order to test our hypothesis that oxidative stress to mitochondria increases intracellular SAH, we first need to define sub-lethal concentrations of t-buOOH, a compound that selectively induces ROS formation in mitochondria. We assumed that high concentrations of t-buOOH (50 uM) would induce apoptosis of all the cells in an experimental tissue culture dish. Exposure of primary human dermal fibroblasts to 50 uM did kill all of the observed cells. We also assumed that low concentrations of t-buOOH (1 and 10 uM) might not kill any of the cells in the tissue culture dishes. We observed, however, that low concentrations of t-buOOH induced apoptosis of partially confluent peripheral cells, but not of completely confluent central cells. Therefore, in-vitro cell confluency appears to affect human dermal fibroblast sensitivity to low concentrations of t-butylhydroperoxide. Future experiments will use cell cultures that have been allowed to grow to uniform, 100% confluency before being exposed to t-buOOH test concentrations.