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Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria
1Department of Pathology and Internal Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112.
Abstract:
Agents that affect mitochondrial respiration have been shown to enhance the generation of reactive oxygen metabolites. On the basis of the well-demonstrated ability of gentamicin to alter mitochondrial respiration (stimulation of state 4 and inhibition of state 3), it was postulated that gentamicin may enhance the generation of reactive oxygen metabolites by renal cortical mitochondria. The aim of this study was to examine the effect of gentamicin on the production of hydrogen peroxide (measured as the decrease in scopoletin fluorescence) in rat renal cortical mitochondria. The hydrogen peroxide generation by mitochondria was enhanced from 0.17 +/- 0.02 nmol . mg-1 . min-1 (n = 14) in the absence of gentamicin to 6.21 +/- 0.67 nmol . mg-1 . min-1 (n = 14) in the presence of 4 mM gentamicin. This response was dose dependent with a significant increase observed at even the lowest concentration of gentamicin tested, 0.01 mM. Production of hydrogen peroxide was not increased when gentamicin was added to incubation media in which mitochondria or substrate was omitted or heat-inactivated mitochondria were used. The gentamicin-induced change in fluorescence was completely inhibited by catalase (but not by heat-inactivated catalase), indicating that the decrease in fluorescence was due to hydrogen peroxide. Thus this study demonstrates that gentamicin enhances the production of hydrogen peroxide by mitochondria. Because of their well-documented cytotoxicity, reactive oxygen metabolites may play a critical role in gentamicin nephrotoxicity.
Insights
Gentamicin significantly increases hydrogen peroxide production in kidney mitochondria. This finding suggests reactive oxygen metabolites may contribute to gentamicin-induced kidney damage.
Area of Science:
- Biochemistry
- Nephrology
- Mitochondrial Biology
Background:
- Mitochondrial respiration agents can increase reactive oxygen species (ROS).
- Gentamicin alters mitochondrial respiration, stimulating state 4 and inhibiting state 3.
- This suggests gentamicin might enhance ROS generation in renal mitochondria.
Purpose of the Study:
- To investigate the effect of gentamicin on hydrogen peroxide production in rat renal cortical mitochondria.
Main Methods:
- Measurement of hydrogen peroxide generation via scopoletin fluorescence.
- Incubation of isolated rat renal cortical mitochondria with varying gentamicin concentrations.
- Control experiments included omitted mitochondria, substrate, or heat-inactivated mitochondria.
- Inhibition assays using catalase and heat-inactivated catalase.
Main Results:
- Gentamicin significantly enhanced hydrogen peroxide generation in a dose-dependent manner.
- Increased production was observed from 0.17 nmol/mg/min to 6.21 nmol/mg/min at 4 mM gentamicin.
- Significant increases were noted even at the lowest tested concentration (0.01 mM).
- No increase in hydrogen peroxide was observed in control conditions without mitochondria, substrate, or with heat-inactivated mitochondria.
- Catalase completely inhibited the gentamicin-induced fluorescence decrease, confirming hydrogen peroxide involvement.
Conclusions:
- Gentamicin administration enhances hydrogen peroxide production by renal cortical mitochondria.
- Increased reactive oxygen metabolites due to gentamicin may play a crucial role in its nephrotoxicity.