Oxidative Stress leads to Increased, LPO

The biological plausibility for this key event relationship is strong: The relationship and mechanism between oxidative stress leading to lipid peroxidation is very well established and studied.

The empirical evidence for this key event relationship is Strong. There are many studies linking oxidative stress to lipid peroxidation within both in vivo and in vitro research articles and most articles describe the mechanisms behind this KER.

Dose concordance:

An additional study that demonstrates great dose concordance in vivo is the administration of acrolein at concentration of 0-100 uM to human vascular endothelial cells. It was shown that exogenous acrolein can initiate oxidative stress and as a down stream result, lipid peroxidation through the creation of reactive oxygen species. A recent study in 2022 measured reactive oxygen species through two ROS specific dyes (MitoSox and DCH-DA) which revealed a significant increase in ROS at 50 uM of acrolein when compared to the control. In addition, the concentration of acrolein needed to induce a significant change in MDA measured cells was 100 uM thus proving that a lower dose was required to induce oxidative stress in human vascular endothelial cells than with lipid peroxidation via MDA measurement (Zhou et al, 2022).

One new dose concordance example follows the administration the ROS generating pharmaceutical- cyclophosphamide at concentrations of 0, 10, and 20 ug/ml to testicular Leydig cells. In this study it was seen that as the concentration of cyclophosphamide increased, the concentration of reactive oxygen species measured (via a ROS assay kit) very significantly increased at 10ug/ml. In addition, the concentration of MDA (measured via an MDA assay kit) followed a similar trend however only slightly significantly increased after 10 ug/ml of cyclophosphamide. This study showed that oxidative stress via reactive oxygen species required the same dose of stressor to illicit change in lipid peroxidation (Liao et al, 2024). 

Another example of dose concordance with this KER is paraquat and hydrogen peroxide application to Vibrio cholerae (Abrashev et al., 2011).  This study also demonstrated that the dose required for oxidative stress was less than/ equal to that needed to induce lipid peroxidation but through indirect markers. Cells were exposed to paraquat and hydrogen peroxide separately for one hour at concentrations of  0, 0.1, 0.3, 0.5, 1.0, 2.0, 3.0 mM. This resulted in increasing amounts of reactive oxygen species (specifically superoxide radicals and hydrogen peroxide) at 0.3 mM and higher. Similarly, lipid peroxidation and overall oxidative damage through protein carbonylation was measured at similar doses of 0, 0.1, 0.5, and 1 mM and showed the most change at 0.5 mM of paraquat (Abrashev et al., 2011, Rodríguez-García et al., 2020).

 One final example of dose concordance is an in vitro study which exposed purified rat liver microsomal lipids to paraquat (a well studied oxidative stress inducer) in the presences of a NADPH-cytochrome c reductase. The cytochrome enzyme was included to interact with paraquat and include radicals which could then be measured against Malondialdehyde (MDA) concentrations as a result of lipid peroxidation. It was seen that as the concentration of paraquat increased from 0-0.0001 M the concentration of MDA also increased from 0.37 nmole/min/ml to 1.21 nmole/min/ml (Bus et al, 1976). To make this study a perfect dose concordance experiment, including the concentration of reduced paraquat radicals would further the explanation of oxidative stress leading to lipid peroxidation.  

Temporal concordance:

One example of temporal concordance regarding the relationship between oxidative stress and lipid peroxidation is the application of paraquat to mouse fibroblasts (Peter et al., 1991). As the concentration of paraquat increased from 0-2.5 mM, and as a result radicals increased, the concentration of MDA also increased. MDA is a known metabolite of lipid peroxidation which was measured from 0-4 hours (Peter et al., 1991). To make this a perfect temporal concordance experiment depiction, one would also need to include the measurement of an oxidative stress marker like reactive oxygen species production. This could be done by measuring superoxide radicals similar to the study done by Abrashev in 2011. This study with these modifications would be very fundamental in depicting oxidative stress preceding lipid peroxidation. 

In addition, reactive oxygen species as a result of hyperglycemia in a study conducted in humans has been recommended to depict in vivo temporal concordance for future studies. Where an increase in glucose plasma levels overtime occurs before the occurrence of lipid peroxidation markers like MDA and 8-isoPGF2α (Ito et al., 2020)

Incidence concordance:

Much of the data found regarding incidence concordance was imperfect (just lacking population effects or 1/2 key event measurments) however one could expose a population of cells to an oxidative stress inducers like paraquat and measure the amount of lipid peroxidation and oxidative stress through ROS and oxidized lipid specific dyes with microscopy. Following this, one could measure the frequency of cells that show signs of oxidative stress (ex through ROS fluorescent dyes, which would show high fluorescence) and compare that to cells showing signs of lipid peroxidation (for instance a higher amount of membrane damage). A singular probe that can achieve this is Lipid Peroxidation Probe -BDP and has been used by multiple studies for similar experiments (Ma et al., 2023, Yang et al., 2023). Hypothetically one should see a higher amount of cells conveying oxidative stress than cells conveying lipid peroxidation for incidence concordance to be true.  

The mechanism for this KER is very well understood and there is a high degree of concordance between many species, so far no large uncertainties or inconsistencies have been found. 

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