Dihydroethidium-derived fluorescence in electrically stressed cells indicates intracellular microenvironment...

Area of Science:

• Biophysics
• Cell Biology
• Electrobiology

Background:

• Intracellular reactive oxygen species (ROS) are implicated in biological effects of electric fields.
• Dihydroethidium (DHE) is a common probe for detecting intracellular ROS.
• ROS contribute to membrane damage, genetic alterations, and cell death.

Purpose of the Study:

• To investigate the mechanism behind DHE-derived fluorescence changes in cells exposed to pulsed electric fields (PEFs).
• To differentiate between ROS generation and other cellular responses affecting DHE fluorescence post-PEF exposure.

Main Methods:

• Chinese hamster ovary (CHO) cells were exposed to 300-ns electric pulses.
• DHE fluorescence was monitored and compared to cells experiencing tert-butyl-hydroperoxide (t-BHP) induced oxidative stress.
• Fluorescence localization and kinetics were analyzed in both DHE-loaded and Ethidium-loaded cells.

Main Results:

• PEF exposure caused distinct changes in DHE-derived fluorescence localization, with decreased cytosolic and increased nucleolar intensity.
• tert-butyl-hydroperoxide (t-BHP) induced a uniform increase in DHE-derived fluorescence across the cell.
• Ethidium (Eth+) fluorescence patterns post-PEF exposure mirrored those of DHE, irrespective of DHE oxidation.

Conclusions:

• DHE-derived fluorescence changes after PEF exposure are not indicative of intracellular ROS generation.
• These fluorescence alterations reflect stress-induced modifications in the intracellular microenvironment affecting Eth+ fluorescence.
• Re-evaluation of DHE as a sole indicator for ROS in PEF studies is warranted.