Related Experiment Videos
NADPH- and adriamycin-dependent microsomal release of iron and lipid peroxidation
1Institute of General Pathology, Catholic University School of Medicine, Largo F. Vito 1, Rome, Italy.
Abstract:
In a previous study (Minotti, G., 1989, Arch. Biochem. Biophys. 268, 398-403) NADPH-supplemented microsomes were found to reduce adriamycin (ADR) to semiquinone free radical (ADR-.), which in turn autoxidized at the expense of oxygen to regenerate ADR and form O2-. Redox cycling of ADR was paralleled by reductive release of membrane-bound nonheme iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. In the present study, iron release was found to increase with concentration of ADR in a superoxide dismutase- and catalase-insensitive manner. This suggested that membrane-bound iron was reduced by ADR-. with negligible contribution by O2-. or interference by its dismutation product H2O2. Following release from microsomes, Fe2+ was reconverted to Fe3+ via two distinct mechanisms: (i) catalase-inhibitable oxidation by H2O2 and (ii) catalase-insensitive autoxidation at the expense of oxygen, which occurred upon chelation by ADR and increased with the ADR:Fe2+ molar ratio. Malondialdehyde formation, indicative of membrane lipid peroxidation, was observed when approximately 50% of Fe2+ was converted to Fe3+. This occurred in presence of catalase and low concentrations of ADR, which prevented Fe2+ oxidation and favored only partial Fe2+ autoxidation, respectively. Lipid peroxidation was inhibited by superoxide dismutase via increased formation of H2O2 from O2-. and excessive Fe2+ oxidation. Lipid peroxidation was also inhibited by high concentrations of ADR, which favored maximum Fe2+ release but also caused excessive Fe2+ autoxidation via formation of very high ADR:Fe2+ molar ratios. These results highlighted multiple and diverging effects of ADR, O2-., and H2O2 on iron release, iron (auto-)oxidation and lipid peroxidation. Stimulation of malondialdehyde formation by catalase suggested that lipid peroxidation was not promoted by reaction of Fe2+ with H2O2 and formation of hydroxyl radical. The requirement for both Fe2+ and Fe3+ was indicative of initiation by some type of Fe2+/Fe3+ complex.
Insights
Adriamycin (ADR) redox cycling releases iron from cell membranes. This iron then undergoes oxidation, contributing to lipid peroxidation, with complex interactions between ADR, oxygen, and hydrogen peroxide influencing these processes.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Adriamycin (ADR) undergoes redox cycling, generating semiquinone free radicals (ADR-.) and superoxide radicals (O2-.).
- Previous studies indicated ADR redox cycling is linked to the release of membrane-bound iron (Fe2+).
- The interplay between ADR, iron metabolism, and oxidative stress in cellular damage is not fully understood.
Purpose of the Study:
- To investigate the mechanisms of iron release and subsequent oxidation following ADR treatment.
- To elucidate the roles of ADR, O2-., and hydrogen peroxide (H2O2) in lipid peroxidation.
- To determine the relationship between ADR concentration, iron redox state, and membrane damage.
Main Methods:
- Utilized NADPH-supplemented microsomes and varying concentrations of adriamycin (ADR).
- Measured iron release using bathophenanthroline assay for Fe2+ mobilization.
- Assessed iron oxidation (Fe2+ to Fe3+) and malondialdehyde formation as an indicator of lipid peroxidation.
- Investigated the effects of superoxide dismutase (SOD) and catalase (CAT) on these processes.
Main Results:
- Iron release increased with ADR concentration, independent of SOD and CAT, suggesting direct reduction by ADR-.
- Released Fe2+ was oxidized to Fe3+ via H2O2-dependent (CAT-inhibitable) and oxygen-dependent (CAT-insensitive) pathways.
- Lipid peroxidation (malondialdehyde formation) occurred when Fe2+ was partially oxidized to Fe3+ and was stimulated by CAT, indicating it was not driven by hydroxyl radical from Fe2+/H2O2.
Conclusions:
- ADR redox cycling directly causes iron release from microsomes.
- Both H2O2 and oxygen contribute to iron re-oxidation, with ADR chelation influencing the latter.
- Lipid peroxidation is initiated by a complex involving both Fe2+ and Fe3+, and its modulation by ADR concentration and antioxidant enzymes highlights complex cellular defense and damage mechanisms.