All key biological macromolecules are susceptible to carbonylation – an irreparable oxidative damage with deleterious biological consequences. Carbonyls in proteins, lipids and DNA from cell extracts have been used as a biomarker of oxidative stress and aging, but formation of insoluble aggregates by carbonylated proteins sometimes precludes their quantification. Since carbonylated proteins can correlate with morbidity and mortality, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism of the nematode C. elegans. The method enabled us to show that after UV irradiation, carbonylation colocalizes mainly with proteins, and was applied to improve the knowledge of mechanisms of E. coli cell death and C. elegans aging.
There is a growing body of evidence that carbonylation could be the root cause of cell death and aging. To explore this possibility, we have monitored in E. coli DNA damage and protein carbonylation after UVC irradiation. A method that detects in real time all emerging mutations allows the exclusion of mutations as the leading cause of UVC-induced cell death. The recovery process of DNA is nearly complete within two hours after irradiation, while UVC-induced protein carbonylation remains doubled. Our new method reveals that UVC induces also an increase in lipid levels and changes in lipid pattern which could indicate membrane permeabilization. Since cell death by membrane disintegration follows the increase in protein carbonylation, we have concluded that cell function (proteome activity) is the primary target in cell death. This is not surprising given the fact that all cellular structures and functions (e.g., DNA and membrane integrity) depend on proteome activity.
Moreover, we applied this method to explore the effects of chronic ionizing radiation of C. elegans on oxidative damage to its proteins. To follow the lifespan of rapidly reproducing animals after irradiation we used a conditional sterile glp-1 mutant. Our results confirmed that: (i) increase in protein carbonylation with C. elegans’ age establishes carbonylation as a good biomarker of aging and (ii) low-dose chronic ionizing radiation reduces the lifespan of sterile C. elegans whatever the dose/dose rate tested. However, there is also a rather unexpected result of reduced carbonylation after irradiation which could be explained by increased activity of proteasome in the germline ablated glp-1 worms. In addition, radiations induced a decrease in lipid levels, whatever the dose or the dose rate. Fat composition and fat content together could induce a beneficial or detrimental effect on longevity. All these findings are of particular importance. These parameters could become predictive biomarkers of biological consequences of radiation and aging.
In conclusion, our new procedure enables us to monitor carbonylation in E. coli during death and in the nematode C. elegans during its life cycle, including the egg stage, through all the stress and aging. That opens up a possibility that the method might be applied for ex vivo diagnostic detection of oxidative carbonylation in the samples of human biopsies.