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Thiol oxidation and inhibition of Ca-ATPase by adriamycin in rabbit heart microsomes
1Department of Pathology, Christchurch School of Medicine, New Zealand.
Abstract:
Incubation of rabbit heart microsomes with Adriamycin and NADPH resulted in the oxidation of approximately 25% of protein thiols and 66% inhibition of Ca-ATPase activity. Thiol oxidation and Ca-ATPase inactivation were iron-dependent and could be catalysed by ferritin. Removal of contaminating catalase revealed that both processes required H2O2 which could be supplied by O2 under aerobic conditions. However, O2- was not involved. Butylated hydroxytoluene (BHT), alpha-tocopherol and beta-carotene inhibited lipid peroxidation of microsomes, but did not inhibit thiol oxidation or the inactivation of Ca-ATPase. Likewise, the hydroxyl radical scavengers benzoate, formate and mannitol were not inhibitory. Glutathione (GSH), however, prevented inactivation of Ca-ATPase. It is concluded that Adriamycin-enhanced redox reactions involving iron and H2O2 are responsible for oxidizing microsomal thiol groups and inhibition of Ca-ATPase. Disruption of Ca transport within the myocyte by this process could contribute to the cardiotoxicity of Adriamycin.
Insights
Adriamycin causes oxidative damage to heart microsomes by oxidizing protein thiols and inhibiting Ca-ATPase activity. This iron and hydrogen peroxide-dependent process may contribute to Adriamycin-induced cardiotoxicity.
Area of Science:
- Biochemistry
- Cardiology
- Toxicology
Background:
- Adriamycin (doxorubicin) is an effective chemotherapy agent but can cause cardiotoxicity.
- The precise mechanisms underlying Adriamycin-induced cardiotoxicity are not fully understood.
- Cardiac microsomes play a crucial role in regulating intracellular calcium levels.
Purpose of the Study:
- To investigate the biochemical mechanisms by which Adriamycin affects rabbit heart microsomes.
- To determine the role of oxidative stress in Adriamycin-induced damage to microsomal proteins and enzymes.
- To elucidate the specific reactive species involved in Adriamycin cardiotoxicity.
Main Methods:
- Incubation of rabbit heart microsomes with Adriamycin and NADPH.
- Measurement of protein thiol oxidation and Ca-ATPase activity.
- Iron-dependency studies and catalytic activity of ferritin.
- Investigation of hydrogen peroxide (H2O2) and superoxide anion (O2-) involvement.
- Assessment of antioxidant and radical scavenger effects (BHT, alpha-tocopherol, beta-carotene, benzoate, formate, mannitol, glutathione).
Main Results:
- Adriamycin/NADPH treatment oxidized protein thiols and inhibited Ca-ATPase activity in a dose-dependent manner.
- These effects were iron-dependent and catalyzed by ferritin.
- Hydrogen peroxide (H2O2), supplied by O2 under aerobic conditions, was required, but superoxide anion (O2-) was not involved.
- Lipid peroxidation inhibitors did not prevent thiol oxidation or Ca-ATPase inactivation.
- Hydroxyl radical scavengers were ineffective, but glutathione (GSH) protected Ca-ATPase activity.
Conclusions:
- Adriamycin-induced cardiotoxicity involves iron- and H2O2-dependent oxidation of microsomal thiol groups and Ca-ATPase inactivation.
- Disruption of calcium transport by Adriamycin contributes to its cardiotoxic effects.
- Understanding these mechanisms can inform strategies to mitigate Adriamycin-induced heart damage.