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Aldehydic products of lipid peroxidation inactivate cytochrome P-450
V U Buko1, A A Artsukevich, K V Ignatenko
1Institute of Biochemistry, National Academy of Sciences, Grodno, Belarus. buko@biochem.belpak.grodno.by
Abstract:
The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.
Insights
Lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), damage rat liver microsomes. MDA and HNE decrease cytochrome P-450 activity and alter membrane properties, with protective effects observed from glutathione and cysteine.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Lipid peroxidation generates reactive aldehydes like MDA and HNE.
- These aldehydes can modify cellular macromolecules, including proteins and lipids.
- Cytochrome P-450 enzymes are crucial for xenobiotic metabolism and are susceptible to oxidative damage.
Purpose of the Study:
- To investigate the effects of MDA and HNE on rat liver microsomal membrane structure and cytochrome P-450 activity.
- To elucidate the mechanisms by which these aldehydes inactivate cytochrome P-450.
- To assess the protective potential of reduced glutathione and cysteine against aldehyde-induced damage.
Main Methods:
- Incubation of rat liver microsomes with varying concentrations of MDA and HNE.
- Measurement of cytochrome P-450 content and activity.
- Assessment of membrane microviscosity using spin labels (OTMB).
- Enzyme activity assays for NADPH oxidase and NADPH cytochrome c reductase.
- Spectrophotometric analysis of cytochrome P-450 inactivation.
Main Results:
- MDA and HNE significantly decreased cytochrome P-450 content and activity.
- MDA lowered lipid microviscosity, while HNE increased it, indicating differential effects on membrane structure.
- Both aldehydes reduced the activity of NADPH oxidase and NADPH cytochrome c reductase.
- Reduced glutathione and cysteine partially protected cytochrome P-450 from inactivation by MDA and HNE.
- Increased absorbance at 420 nm suggested cytochrome P-450 inactivation due to altered lipid hydrophobicity.
Conclusions:
- HNE and MDA exert detrimental effects on rat liver microsomes, leading to cytochrome P-450 inactivation.
- HNE appears to directly interact with cytochrome P-450 SH-groups, while MDA's effect may involve initial membrane surface modification.
- Antioxidants like glutathione and cysteine offer partial protection against these toxic aldehydes.
- These findings highlight the damaging potential of lipid peroxidation products on essential metabolic enzymes.