New method detects biological oxidant derived from CO2 in cells

The presence of high levels of carbon dioxide (CO₂) in our atmosphere doesn’t just impact the climate; it also affects the very functioning of our cells. This gas interacts with hydrogen peroxide (H₂O₂), a vital substance in the human body, giving rise to a powerful oxidant known as peroxymonocarbonate.

“More and more evidence is emerging that peroxymonocarbonate is important in both cells’ adaptive responses via redox signaling and in cellular dysfunction. There is also epidemiological evidence that the levels of CO₂ our cities are close to reaching cause a number of physiological problems. And the mechanisms underlying the toxicity of CO2 are poorly understood,” said Ohara Augusto, full professor at the University of São Paulo‘s Chemistry Institute (IQ-USP) in Brazil.

A study led by Augusto, introduces an innovative method for detecting peroxymonocarbonate in cells using fluorescent molecular probes. For the first time ever, this elusive substance has been successfully identified within cells. The study, conducted under the auspices of the Center for Research on Redox Processes in Biomedicine (Redoxome), a Research, Innovation and Dissemination Center (RIDC) funded by FAPESP, marks a significant leap forward in our understanding of cellular processes.

“This research is important both because it has produced a method to show that peroxymonocarbonate is produced under certain conditions, including cellular conditions, and because it has raised this as a topic for discussion, a boon considering the scant attention paid to CO₂ in the redox field,” Augusto said.

To unveil the presence of peroxymonocarbonate, the researchers embarked on a captivating journey of measuring fluorescence from boronate probes. They ingeniously orchestrated an enzyme reaction to unleash steady-state physiological concentrations of hydrogen peroxide, then meticulously scrutinized boronate probe fluorescence in the presence and absence of CO₂.

These remarkable boronate probes have the remarkable ability to detect a range of substances, including hydrogen peroxide, peroxynitrite, hypochlorous acid, and peroxymonocarbonate, each eliciting distinct responses that unveil their true nature.

Delving deeper into the study, the researchers activated macrophages to unleash hydrogen peroxide. These macrophages, the guardians of our immune system, wield the power to produce an array of oxidants, each playing a unique role dependent on their activation state.

Amidst a series of meticulous control experiments, the researchers made a resounding discovery: the cells did not yield peroxynitrite or hypochlorous acid but unequivocally generated peroxymonocarbonate in the presence of CO₂. This groundbreaking revelation opens new frontiers in our understanding of cellular oxidants and their intricate interplay with the environment.

“This is a relatively simple method for detecting peroxymonocarbonate in physiological concentrations of hydrogen peroxide and CO₂. It used to be impossible, but researchers can now conclude that some effects observed in cells, such as higher oxidation of certain proteins or cell responses, may be due to peroxymonocarbonate, and they will be able to test this finding,” Augusto said.

Peroxymonocarbonate, a compound known to chemists since the 1960s, has surprising implications for biological systems. Despite initial skepticism due to low concentrations of its precursors and slow formation rate, research in the first decade of the millennium has revealed its presence in cells and its potential role in oxidative damage. Furthermore, redox signaling is now recognized as an adaptive response with far-reaching implications.

“When stress increases slightly, the cell adapts. Formation of oxidants, for example, can lead to the expression of genes for antioxidant enzymes, responding to oxidative stress in this case. Moreover, many pathways that lead to cellular responses involve thiol proteins, which peroxymonocarbonate oxidizes faster than hydrogen peroxide,” Augusto said, adding that irreversible cellular damage occurs only when oxidant formation is very substantial.

Carbon dioxide serves as a crucial precursor to peroxymonocarbonate, alongside hydrogen peroxide. It naturally occurs in the atmosphere and within the human body, with the average person exhaling approximately 1.0 kg of CO₂ per day as a metabolic byproduct.

From a redox standpoint, CO₂ influences the reactivity of hydrogen peroxide and peroxynitrite, essential metabolites of molecular oxygen. It also exerts an impact on gene expression, including genes associated with inflammation. Additionally, it plays a role in protein nitration via peroxynitrite and in protein carbamylation, a post-translational modification that can significantly affect the biological function of proteins.

While further evidence of its function as a biological oxidant is necessary, peroxymonocarbonate seems to be a potential intermediary for the detrimental effects of elevated CO₂ levels in our bodies. Furthermore, CO₂ operates through non-redox mechanisms, as highlighted by Augusto.

Journal reference:

1. Edlaine Linares, Divinomar Severino, Daniela R. Truzzi, Natalia Rios, Rafael Radi, Ohara Augusto. Production of Peroxymonocarbonate by Steady-State Micromolar H2O2 and Activated Macrophages in the Presence of CO2/HCO3– Evidenced by Boronate Probes. Chemical Research in Toxicology, 2024; DOI: 10.1021/acs.chemrestox.4c00059