Related Experiment Videos
Inactivation of transforming activity of plasmid DNA by lipid peroxidation
Abstract:
DNA damage due to NADPH-dependent lipid peroxidation of liposomes was examined using liposomes prepared from lipids, NADPH-cytochrome P-450 reductase and cytochrome P-450 isolated from rat liver microsomes. Plasmid pBR322 DNA was incubated in the reaction mixture for liposomal lipid peroxidation and introduced to Escherichia coli CSR603 (uvrArecA). More of the transforming activity of the DNA was lost as the lipid peroxidation progressed, and this inactivation was dependent on the extent of lipid peroxidation. Single strand breaks occurred in the plasmid DNA. Hydroxyl radical scavengers could not prevent most of the strand breaks or the lipid peroxidation reaction. Chloroform extracts from the reaction mixture of peroxidized microsomes also inactivated the transforming activity of pBR322 DNA but did not cause strand breaks. The 105 000 X g supernatant of the reaction mixture, which contained more than 85% of the thiobarbituric acid-reactive substances, did not inactivate the plasmid DNA. The degradative products of [U-14C]arachidonic acid in the liposomes did not bind to DNA. These results led to the conclusion that at least two types of DNA damaging agent are produced during NADPH-dependent microsomal lipid peroxidation. One induces single strand breaks of DNA and another inactivates the plasmid-transforming activity without inducing strand breaks.
Insights
NADPH-dependent lipid peroxidation generates DNA damaging agents. These agents cause single-strand breaks and inactivate DNA transforming activity, indicating distinct mechanisms of DNA damage.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Lipid peroxidation is a process involving oxidative degradation of lipids.
- NADPH-dependent enzymes in microsomes play a role in cellular oxidative stress.
- DNA integrity is crucial for cellular function and can be compromised by reactive oxygen species.
Purpose of the Study:
- To investigate DNA damage induced by NADPH-dependent lipid peroxidation.
- To identify the types of DNA damaging agents produced during this process.
- To elucidate the mechanisms by which these agents affect DNA.
Main Methods:
- Liposomes were prepared with lipids, NADPH-cytochrome P-450 reductase, and cytochrome P-450 from rat liver microsomes.
- Plasmid pBR322 DNA was incubated in the peroxidation reaction mixture.
- DNA transforming activity was assessed by introducing it to Escherichia coli CSR603 (uvrArecA).
- Single-strand breaks were analyzed, and the effects of hydroxyl radical scavengers were tested.
- Chloroform extracts and supernatant fractions were analyzed for DNA damaging potential.
Main Results:
- Lipid peroxidation progressively reduced the transforming activity of plasmid DNA.
- Single-strand breaks were observed in the plasmid DNA.
- Hydroxyl radical scavengers did not fully prevent lipid peroxidation or DNA strand breaks.
- Chloroform extracts inactivated DNA transforming activity without causing strand breaks.
- Supernatant fractions containing thiobarbituric acid-reactive substances did not inactivate DNA.
Conclusions:
- NADPH-dependent microsomal lipid peroxidation produces at least two distinct DNA damaging agents.
- One agent induces single-strand breaks in DNA.
- A second agent inactivates DNA transforming activity through a mechanism independent of strand breaks.