Lysosomal oxidative stress cytotoxicity induced by nitrofurantoin redox cycling in hepatocytes

Area of Science:

• Biochemistry
• Cell Biology
• Toxicology

Background:

• The enzymes mediating NFT reductive activation remain unidentified.
• Subcellular mechanisms of NFT-induced oxidative stress and cytotoxicity are not fully understood.

Purpose of the Study:

• To elucidate the enzymatic pathways involved in NFT reductive activation.
• To determine the subcellular targets and mechanisms of NFT-induced oxidative stress and cytotoxicity.

Main Methods:

• Investigated NFT cytotoxicity and reactive oxygen species (ROS) formation using enzyme inhibitors (cytochrome P450, P450 reductase, DT diaphorase).
• Assessed subcellular organelle integrity (mitochondrial membrane potential, lysosomal damage) and protease activation in hepatocytes.
• Utilized endocytosis inhibitors, lysosomotropic agents, lysosomal protease inhibitors, a ferric chelator (desferoxamine), antioxidants, and ROS scavengers.

Main Results:

• Inhibitors of cytochrome P450 or P450 reductase, but not DT diaphorase, prevented NFT-induced cytotoxicity and ROS formation, suggesting reductive activation by these enzymes.
• NFT induced lysosomal damage and hepatocyte protease activation, while mitochondrial membrane potential remained largely unaffected prior to cell death.
• Lipid peroxidation preceded cytotoxicity, and was prevented by desferoxamine, antioxidants, and ROS scavengers.

Conclusions:

• NFT is reductively activated by cytochrome P450 and/or P450 reductase under aerobic conditions.
• Lysosomal damage, protease activation, and subsequent lipid peroxidation mediated by ROS are key events in NFT-induced cytotoxicity.
• Hydrogen peroxide (H2O2) reacts with lysosomal Fe(+2) to generate ROS, initiating a cascade of events leading to cell death.