I am working on HUVEC cells in order to make the Oxidative Stress Injury Model Induced by H2O2.I treated the cells with 0.3% H2O2(30%) for 16 hours, then IC50 was determined by MTT test, but in the validation stage of the model, the oxidative stress indicators were not as expected. what stock of hydrogen peroxide I should be used to induce oxidative stress?
the concentration of 0.3% (30%) H2O2 for 16 hours is relatively high and may potentially result in severe cellular damage or cytotoxicity, leading to unexpected oxidative stress indicators.
To optimize your oxidative stress model and achieve the desired level of stress without excessive cytotoxicity, it is advisable to perform a dose-response experiment with a range of H2O2 concentrations. The following steps can guide you in determining the appropriate concentration of H2O2:
By systematically evaluating the effects of different H2O2 concentrations, you can identify the appropriate concentration that induces oxidative stress while maintaining cellular viability and obtaining the expected oxidative stress indicators. It is also important to consider the specific characteristics and sensitivity of HUVEC cells when selecting the H2O2 concentration.
George Andrei Draghici Thank you for your beautiful and intelligent answer.
You said that the working solution of 0.3% is too much and causes cell toxicity. Is this the reason of the increased activity of superoxide dismutase enzyme? I expected the activity of this enzyme to decrease.
Oxidative stress can trigger various cellular responses, including the activation of antioxidant defense systems. One of the key enzymes involved in antioxidant defense is superoxide dismutase (SOD), which catalyzes the conversion of superoxide radicals into hydrogen peroxide and molecular oxygen. In the presence of high levels of H2O2, cells may upregulate the production of SOD as a protective mechanism to counteract the increased oxidative stress. The increased activity of SOD enzyme can help in the detoxification of superoxide radicals and reduce their potential damaging effects on cellular components.
George Andrei Draghici Yes, it is true, but at first the enzyme activity increases, but after some time and the creation of oxidative stress, the enzyme activity decreases.
You are correct. Initially, in response to oxidative stress, cells may upregulate the activity of antioxidant enzymes, including superoxide dismutase (SOD), as a protective mechanism to mitigate the harmful effects of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radicals. However, prolonged exposure to high levels of ROS can lead to oxidative damage to cellular components, including proteins and enzymes. This oxidative damage can potentially affect the activity and stability of enzymes, including SOD. As a result, the enzyme activity may decrease over time due to the cumulative effects of oxidative stress.
Additionally, excessive ROS production can overwhelm the antioxidant defense systems, leading to oxidative imbalance and further damage to cellular components, including enzymes. This can contribute to the observed decrease in SOD activity after prolonged exposure to high levels of H2O2.
George Andrei Draghici Yes, great. Thank you for giving me your valuable time. Have a wonderful day and night.
You're welcome! I'm glad I could assist you. If you have any more questions in the future, feel free to ask. Have a wonderful day and night as well! Take care!
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