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Paraquat- and diquat-induced oxygen radical generation and lipid peroxidation in rat brain microsomes
Kunio Yumino1, Ikuo Kawakami, Mamoru Tamura
1Division of Biophysics, Institute for Electronic Science, Hokkaido University, Sapporo 060-8638, Japan.
Abstract:
NADPH-menadione reductase activity by rat brain microsomes (Ms) was decreased 40-50% by 10 microM dicumarol, a potent inhibitor of DT-diaphorase, whereas no change in NADPH-paraquat (PQ) and -diquat (DQ) reductase activity was observed. NADPH-DQ reductase activity in brain Ms was 2.5-fold higher than NADPH-PQ reductase activity. The formation of PQ and DQ radicals was verified optically and observed directly by ESR spectroscopy in the NADPH-PQ and -DQ reductase reactions by brain Ms under anaerobic conditions. PQ- and DQ-induced superoxide formation was confirmed by the detection of DMPO-OOH ESR signals and followed by chemiluminescence (CL) of a Cypridina luciferin analogue (CLA). The kinetics and intensity of the CL were consistent with the observations that the reduction in DQ is faster than that in PQ. Thiobarbituric acid reactive substances (TBARS) and phospholipid hydroperoxides in brain Ms increased in the presence of NADPH and Fe3+. The generation of both lipid peroxidation products derived from brain Ms decreased with increasing concentrations of PQ and DQ. The inhibitory effect of DQ is more pronounced than that of PQ. The formation of PQ- and DQ-induced reactive oxygen species was not associated with lipid peroxidation in rat brain Ms.
Insights
Dicumarol inhibits DT-diaphorase but not NADPH-paraquat (PQ) or -diquat (DQ) reductase activity in rat brain microsomes. PQ and DQ generate reactive oxygen species, but not through lipid peroxidation.
Area of Science:
- Biochemistry
- Neuroscience
- Toxicology
Background:
- NADPH-dependent reductases in brain microsomes play roles in xenobiotic metabolism and oxidative stress.
- DT-diaphorase is a key enzyme involved in the reduction of quinones, while paraquat (PQ) and diquat (DQ) are known pro-oxidants.
Purpose of the Study:
- To investigate the specific reductase activities involved in PQ and DQ metabolism in rat brain microsomes.
- To determine the role of DT-diaphorase in PQ and DQ reduction.
- To assess the generation of reactive oxygen species and lipid peroxidation products induced by PQ and DQ.
Main Methods:
- Enzyme activity assays for NADPH-menadione, NADPH-paraquat (PQ), and NADPH-diquat (DQ) reductases.
- Electron spin resonance (ESR) spectroscopy to detect PQ and DQ radicals and superoxide formation (DMPO-OOH).
- Chemiluminescence (CL) assays using a Cypridina luciferin analogue (CLA) to monitor superoxide kinetics.
- Quantification of thiobarbituric acid reactive substances (TBARS) and phospholipid hydroperoxides as markers of lipid peroxidation.
Main Results:
- Dicumarol, a DT-diaphorase inhibitor, significantly decreased NADPH-menadione reductase activity but did not affect NADPH-PQ or NADPH-DQ reductase activity.
- NADPH-DQ reductase activity was 2.5-fold higher than NADPH-PQ reductase activity in brain microsomes.
- PQ and DQ directly formed radicals and induced superoxide generation, confirmed by ESR and CL, with DQ reduction being faster than PQ.
- PQ and DQ inhibited NADPH and Fe3+-induced lipid peroxidation (TBARS and phospholipid hydroperoxides), with DQ showing a more pronounced effect.
- The generation of PQ- and DQ-induced reactive oxygen species was not linked to lipid peroxidation in rat brain microsomes.
Conclusions:
- DT-diaphorase is not involved in the reduction of PQ or DQ in rat brain microsomes.
- Brain microsomes possess distinct reductase activities for PQ and DQ, with higher capacity for DQ.
- PQ and DQ induce oxidative stress via reactive oxygen species generation, independent of direct lipid peroxidation.
- These findings contribute to understanding the neurotoxic mechanisms of PQ and DQ.