Why this Project?

Radiation-induced change of mitochondrial import capacity is a uniquel physiological endpoint which can be used to study the role of endogenous oxidative metabolism in the cellular responses. This project will investigate the mitochondrial import response in cells exposed to low and high dose γ- radiation from a ¹³⁷Cs source.

Project Goals

To examine and compare the efficiency of import of protein into mitochondria isolated from control and irradiated human cells.
To compare the protein import capacity in quiescent density-inhibited cells in mitochondria from cells exposed to 4 Gy with that from the controls.
To determine how these changes in import efficiency are linked with different stages of the cell cycle.
To contrast the protein import in mitochondria isolated from cells exposed to low dose/low dose-rate γ-rays (10 cGy, 0.2 cGy/h) with high doses and controls.
To evaluate the mitochondrial membrane potential and correlate it with the expression of radiation induced adaptive responses.

Experimental Approach

Using normal human fibroblasts (AG1522), experimental conditions were optimized for the import of a protein, frataxin, into the mitochondrial matrix.
A toxic dose of 4 Gy (3.3 Gy/min) or lower doses of 10 cBy were given to the cells, and they were subsequently incubated at 37 °C for 1 h.
Cells will be harvested at different incubation periods following subculture to lower density and intercellular communication.
Mitochondrial membrane potential change as a function of radiation exposure will be quantified with a JC-1 fluorescent probe.

Expected Outcomes

Mitochondrial membrane potential and an ATP-regenerating system were found to be required for efficient import. Compared to controls, a significant decrease in import capacity was observed in mitochondria isolated from density-inhibited cell cultures exposed to a toxic dose Incubation for a longer period (12 h) attenuated inhibition of protein import, suggesting either repair or elimination of damaged mitochondria

These data can be used to determine if the dose response to γ-rays is non linear, and identify the distinct biochemical events that may underlie the cellular response to high and low doses of both low and high LET exposure.