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Multiple mechanisms for doxorubicin cytotoxicity on glomerular epithelial cells 'in vitro'
G M Ghiggeri1, R Bertelli, F Ginevri
1Department of Nephrology, G. Gaslini Institute, Genoa, Italy.
Abstract:
This study was planned to define the metabolic pathways for free radical production by isolated glomeruli and glomerular epithelial cells in vitro after exposure to cytotoxic doses of doxorubicin. A net increment in glomerular superoxide anion (O2.) synthesis was observed at doxorubicin doses between 10 and 30 micrograms/ml, a drug level which also induced a parallel increment in uric acid synthesis. Since the synthesis of O2. with production of uric acid implies an activity of xanthine oxidase, a few experiments were performed with glomeruli which had been deprived of xanthine oxidase activity. In this case doxorubicin-inducible O2. and uric acid synthesis by glomeruli was practically nil. A similar stimulatory effect of O2. synthesis was induced by doxorubicin on glomerular epithelial cells and also in this case O2. synthesis was suppressed by pre-treating cells with deoxyconformicin, a selective inhibitor of adenosine deaminase. Finally, equimolar amounts of the drug were equally cytotoxic even when kept constantly outside the cell by a stable linkage with an agarose macroporous bed. In summary, these data demonstrate that O2. is generated by isolated glomeruli and glomerular epithelial cells 'in vitro' when exposed to cytotoxic amounts of doxorubicin and that purine degradation to uric acid furnish the metabolic pathways for glomerular O2. generation. However, doxorubicin is comparably cytotoxic on glomerular epithelial cells from outside cells thus suggesting that also a membrane perturbation may activate the series of events leading to cell injury.
Insights
Doxorubicin induces free radical production in kidney cells via purine degradation. This study identifies xanthine oxidase as a key enzyme in this superoxide anion (O2.) generation process.
Area of Science:
- Nephrology
- Biochemistry
- Cell Biology
Background:
- Doxorubicin is a potent chemotherapy agent with known nephrotoxic effects.
- The mechanisms underlying doxorubicin-induced kidney damage, particularly free radical production, require further elucidation.
Purpose of the Study:
- To define the metabolic pathways responsible for free radical generation in isolated glomeruli and glomerular epithelial cells exposed to doxorubicin.
- To investigate the role of xanthine oxidase and adenosine deaminase in doxorubicin-induced oxidative stress in kidney cells.
Main Methods:
- Isolated glomeruli and glomerular epithelial cells were exposed to cytotoxic doses of doxorubicin in vitro.
- Superoxide anion (O2.) and uric acid synthesis were measured.
- Experiments involved glomeruli depleted of xanthine oxidase activity and cells treated with deoxycoformicin, an adenosine deaminase inhibitor.
- Cytotoxicity was assessed using doxorubicin linked to an agarose bed to confine it extracellularly.
Main Results:
- Doxorubicin treatment led to increased glomerular superoxide anion (O2.) and uric acid synthesis at specific concentrations (10-30 µg/ml).
- Inhibition of xanthine oxidase activity in glomeruli abolished doxorubicin-induced O2. and uric acid production.
- Doxorubicin also stimulated O2. synthesis in glomerular epithelial cells, which was suppressed by deoxycoformicin.
- Doxorubicin exhibited comparable cytotoxicity when confined outside the cells, suggesting membrane perturbation plays a role.
Conclusions:
- Purine degradation through xanthine oxidase is a primary metabolic pathway for doxorubicin-induced superoxide anion (O2.) generation in isolated glomeruli and glomerular epithelial cells.
- Adenosine deaminase may also be involved in the oxidative stress response of glomerular epithelial cells to doxorubicin.
- Doxorubicin-induced cytotoxicity in glomerular epithelial cells can occur via mechanisms initiated by membrane perturbation, independent of intracellular drug entry.