Thursday 30 August 2018

The Interactions Of Antioxidants With Living Cells?

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Since the concept of oxidative stress” was coined by Helmut Sies in 1985, it has become so far-reaching that it is now considered a causative factor of many disease processes, from cardiovascular to neurodegenerative diseases, including the inexorable ageing process. This concept has permeated society so deeply that now all people are familiar with the ad-hoc term “antioxidant,” an attribute that supposedly counteracts the noxious effect of oxidative damage.
Nowadays, a myriad of products is marketed to consumers, including foods and diet supplements that carry labels with the term “antioxidant.” This labelling generates the impression that the intake of these products provides health-promoting benefits for the consumer. However, is this association true?
All living organisms obtain their energetic needs by breaking down carbohydrates, fatty acids, and amino acids, all processes integrated into metabolism. For aerobic organisms like us, the metabolic activity must be understood as the sequential oxidation of nutrients to obtain COand H2O from molecular oxygen, O2, a phenomenon known as cellular respiration that, for eukaryotes, takes place in a specialized structure, the mitochondria. This marvellous organic machine allows a highly efficient electron flow from carbon atoms present in the structure of energy substrates, such as glucose to O2. In principle, the energy power for living organisms rests in the electron flow as many machines, motors, and batteries do.
The mitochondrial machine is not perfect, and its functioning brings a marginal but measurable leak of electrons that ends in the formation of unstable molecules called reactive oxygen species (ROS). Those molecules were first identified in 1930 during vigorous research in the field of chemistry, but the notion that they were an intrinsic aspect of life functions occurred fifty years later.
Today, we understand that metabolism and enzymatic machinery of living cells generate ROS, such as superoxide anion, which is then transformed to hydrogen peroxide. Intracellular production of hydrogen peroxide is functionally confined to its immediate surroundings where it can act locally, modifying the activity of ionic channels and cytoskeleton components to promote cell movement or initiating adjustments in the global machinery by activating/deactivating the expression of genes. The reversibility of these cellular phenomena exists thanks to the hydrogen peroxide that oxidizes proteins and, more specifically, reacts with exposed sulfhydryl groups of cysteine residues. Our current vision of what happens inside of a living cell can be resumed as a constant generation of oxidant molecules derived from metabolic processes counteracted continually by a ready-to-act antioxidant machinery able to neutralize ROS and restore undesirable oxidation events, which establish the cellular redox homeostasis, an equilibrium between oxidant and reducing reactions.
In our work, recently published in the journal Redox Biology, we explored the redox processes occurring in living cells by taking advantage of a redox biosensor called HyPer, a fluorescent protein that holds a pair of cysteine which forms a disulfide bond only by surrounding hydrogen peroxide whereas its reduction back to sulfhydryl groups is achieved by the intrinsic antioxidant machinery that every cell possesses. This molecular tool was developed in 2006 by Belousov et al. and it has been extensively used to describe the oxidant nature of some stimuli or cellular conditions. We followed an alternative pathway, showing that by tracking HyPer signal in real-time, we could measure the antioxidant activity in living cells by giving short and moderate pulses of hydrogen peroxide to cells, which for the case of mammalian cells correspond to exposures of micromolar for a few minutes.
After observing the biosensor behaviour in more than ten cell lines, including endothelial cells obtained from umbilical cordon or fibroblast from gingival samples, for instance, we noted that after a transient oxidative challenge, every living cell was able to recover the fluorescence of the biosensor to a previous state. This observation was key to formulate a simple question: is this biosensor a good tool to monitor the antioxidant capacity of living cells?
Our published work covers this question by targeting HyPer at three subcellular regions very well-characterized by its redox properties: endoplasmic reticulum, mitochondria, and cytoplasm; and measuring the recovery of the biosensor. The results clearly showed that faster reducing in the subcellular environment related to the recovery of oxidized biosensor HyPer.
In this paper, we also considered another basic question about the feasibility to modify the intrinsic antioxidant machinery by adding an exogenous compound; more importantly, perhaps, asking whether that modification is translated to the performance of any cellular task. To tackle this question, we compared the performance of ten cell lines in terms of HyPer signal parameters that included baseline, response upon hydrogen peroxide pulses, and recovery from an oxidized state against their capacity to migrate. Curiously, the only parameter of HyPer that showed a proper correlation with migration was the recovery of an oxidized biosensor. We pushed this point further by modifying the antioxidant capacity with N-acetylcysteine, a well-known compound used to boost the intracellular bioproduction of glutathione in the research field and popular in diet supplement stores as well. The results indicated that by accelerating the recovery of the biosensor, we also improved the migratory capacity of the cells.
To conclude, we presented a new facet of a known biosensor HyPer for measuring the antioxidant capacity of living cells. In addition, we connect the migratory capacity with the capacity to reduce disulfide bonds. The type of cells we used belongs to the carcinoma category, which has deep implications in the way that we conceive the intake of antioxidant supplements.
We have learned that the intrinsic antioxidant capacity of any living cell is tightly connected to its metabolism and that this capacity effectively responds to an external input; but we still do not know to what extent normal processes inside of the cell are perturbed, or, more worryingly, which type of cell is preferably activated by antioxidant supplements. We are happy to contribute to a better understanding of how potential antioxidant compounds present in food interact with the complex antioxidant machinery of cells.
These findings are described in the article entitled Insights into the HyPer biosensor as a molecular tool for monitoring cellular antioxidant capacity, recently published in the journal Redox Biology.

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