1. Introduction
Ionizing radiation produces different types of DNA damage, with double-strand breaks (DSB) being especially important. In this sense, the repair capacity contributes not only to the maintenance of the genome integrity but also to the resistance to radiation [
1,
2,
3]. In addition, the response to radiation to ensure cell homeostasis activates other mechanisms involved in cell cycle blockage, free radicals’ formation, and apoptosis inhibition [
1].
DSB can also be produced by products generated during cellular metabolism, occurring mainly during replication. Hydrolysis, oxidation, and non-enzymatic methylation phenomena can produce DNA modifications that contribute to the appearance of DSB [
4]. The cellular state, young or old, the metabolic state, the phase of the cell cycle, etc., contribute to the ability to repair the induced damage.
Spontaneous or metabolic-induced lesions that appear during cell replication are repaired predominantly by homologous recombination (HR) due to the proximity of the two sister strands. However, the breaks induced by external agents that appear in distant places between homologous sequences are repaired by non-homologous end-joining (NHEJ) [
5,
6]. HR is free of errors, but NHEJ frequently produces errors. It is preferred to induce an error rather than not repair the damage, in order to guarantee the stability of the genome and cell viability [
7]. In
S. cerevisiae, the role of HR in DSB repair predominates [
8,
9].
Mutations that appear in the DSB repair genes can trigger processes of radiosensitivity, premature aging, immunodeficiency, and carcinogenesis [
7]. The appearance of reactive oxygen species (ROS) after exposure to DNA-damaging agents (such as ionizing radiation) activates the signaling pathways that repair the induced damage. Chronic exposure to ROS is associated with the induction of cancer and with the development and progression of neurodegenerative and cardiovascular diseases. A connection between ROS exposure and the phenomenon of aging has also been described [
10].
The aging phenomenon is a multifactorial process in which a high energy metabolism is essential to maintain cellular homeostasis, repair damage, and slow down the process of cellular deterioration. Thus, the decline in energy metabolism caused by DNA-damaging agents accelerates the aging process and, conversely, high glycolytic and respiratory activity increase the cellular resistance to environmental agents that cause stress [
11].
Recent studies show that not only are the damages caused to the DNA molecule important, but so are those produced on the proteins. In this sense, the response of proteins to stress caused by radiation also activates molecular response mechanisms, especially chaperones. Heat shock proteins (HSP) play an important role in the maintenance of proteostasis through the correct folding of damaged proteins [
12]. However, when misfolding proteins are oxidized, the damage is fixed and therefore chaperones cannot restore folded proteins correctly. Cellular aging also produces the appearance of proteins damaged by oxidation, which accumulate with age, causing the loss of cellular homeostasis. Therefore, there is a connection between aging, damage caused by ROS, and radiation. This connection can be at the genomic or proteomic level or at both levels at the same time [
13].
The mechanisms of response to ionizing radiation and to agents causing oxidative damage, as well as to damage and alterations in the proteome during cellular aging, are highly dynamic processes. During the peak period of cellular response, the number and type of active molecules is more important than during the initiation and attenuation stages [
14]. This overexpression of biomolecules highlights the multiple roles that many of them play together in the processes of radiation response, aging, and response to oxidative stress.
The yeast
Saccharomyces cerevisiae constitutes a model organism whose gene characteristics and functions are well described in multiple curated databases. Systems biology and the use of bioinformatics tools allows us to address the need to know the common molecular mechanisms between different phenomena, to study their genetic basis, and their regulation, with the ultimate need being to search for molecular markers common to different phenomena [
15].
The aim of this work is to study the relationship between resistance/response to ionizing radiation, cellular aging, and the response mechanisms to oxidative stress, free radicals, reactive oxygen species (ROS), and antioxidant activity in the yeast S. cerevisiae, in order to understand the mechanisms of interaction and regulation between them and to search for molecular markers related to these phenomena.
4. Discussion
For many years, there has been speculation that there is an important relationship between resistance to radiation and cellular aging. Such a relationship could change the course of research and the treatment of tumors with radiotherapy [
7].
The molecular mechanisms of response to physical and chemical agents that cause cell damage are conserved, presenting on numerous occasions the molecular pathways involved in the repair of damage of multiple natures to restore cell homeostasis (genomics and proteomics), maintain cell integrity, and ensure survival with maximum energy savings [
6].
Model organisms, such as S. cerevisiae, and the use of bioinformatics tools allow us to compare well-known and established molecular mechanisms from curated databases. This methodology provides very valuable information, without the need for experimental laboratory work, in the search for common intra- and inter-species response mechanisms and molecular markers. In this way, the proposed work represents an advance in the subsequent definition, through experimental testing, of the utility of said markers.
Currently, research at the molecular level is guided based on the information obtained in the literature and especially in multiple molecular databases. The methodology used in this work allows obtaining new data from crossing information in curated databases. The works carried out in silico allow use to obtain very valuable information in a very short time and with very few resources. This information, obtained from crossing data in different curated databases, allows us to channel the research work and in some cases, as in this one, discover molecular markers common to various phenomena, which would go undetected in in vitro studies. The data that appear in the databases have been duly validated and accepted for inclusion. This fact reinforces a priori the validity of the markers found. Even so, it is important to carry out new validations of the markers under different exposure conditions. However, the objective of this work is not this but to provide new evidence for further studies. The validation of the proposed markers is left for later studies, where different conditions of exposure to radiation, aging, and response to oxidizing agents are tested. The present work saves time and effort in the search for common markers from data that have already been duly validated for their inclusion in the used curated databases.
Starting from the idea that each phenomenon (resistance/response to radiation, aging, oxidative stress, free radicals, antioxidant activity, ROS) triggers multiple responses with the participation of multiple biological processes, molecular functions, and metabolic pathways, this work demonstrates that among all of them there are common mechanisms to ensure cell homeostasis. This fact facilitates the definition of common molecular markers of response.
Table 6 shows a description of the genes identified with MCODE, strongly related to resistance/response to radiation and cellular aging. Their functions, as well as their main role and the effect of their inhibition on the lifespan of
S. cerevisiae, are also described. It is worth noting a minimum lifespan reduction range of 15–30% for the
MRE11 gene and a maximum value of 70% reduction for
RAD50 and
RAD52. The average lifespan reduction was 50%. Among the main functions involved in both phenomena, depending on the greater participation of genes and the greater effect on lifespan were as follows: DNA repair, genomic stability, replication stress, DNA damage, DNA damage response, strand exchange, meiotic recombination, and the maintenance of genome integrity. It is important to highlight that the
HSP104 gene, common to the three phenomena studied, did present a relationship with cellular aging, observing a 40% reduction in life expectancy.
The genes described in S. cerevisiae showed homologous genes in mammalian cells of different species. They constitute groups of genes that are very well conserved during evolution, since they are essential for the maintenance of life. They are mainly responsible for repairing the lesions produced in the DNA by physical and chemical agents, and guaranteeing the stability of the genome and telomeres. Specifically, most of the homologous human genes found are involved in DNA DSB repair mechanisms, followed by genes that provide stability to the genome. It is important to highlight in yeast cells the HSP104 disaggregase protein and its human homologue, CLPB, which are both involved in protein degradation and the reactivation of misfolded proteins in response to stress.
The results obtained from the comparison of common genes show that cell aging has a strong relationship with the response/resistance to ionizing radiation. Although the response to oxidative stress is important in the cell when addressing resistance to radiation, there are more common genes with the aging phenomenon. The existence of one single gene (HSP104) common to the three phenomena could indicate the possibility of defining a good common biomarker.
The aging phenotype can be progressed or prevented depending on the effectiveness of the damage repair at the protein level. Since radiation damage occurs mainly at the genomic level, the mechanism common to aging must also be at the genomic level, as it has been observed in the gene set enrichment analysis. However, when the three phenomena are considered together (response to radiation, oxidative stress, and aging), the joint response mechanisms must ensure resistance to oxidative damage caused to proteins. In this sense, the HSP104 protein plays a predominant role [
13].
Cellular aging is a multifactorial phenomenon at the organism, tissue, and cellular levels. In this sense, it is logical to think about its interrelation with other processes, such as those described for the effects of radiation obtained from the GO enrichment analysis and protein–protein interactions data. This point of view shows that the response/resistance to radiation is also a multifactorial phenomenon that interacts with phenomena that participate in aging and also in the response to ROS, free radicals, oxidative stress, etc.
The increase in damage to DNA causes an increase in ROS levels, causing damage to numerous molecules, which causes an alteration of their functions [
6]. These alterations reveal the activation of response mechanisms that are also activated during cell aging, to prevent protein oxidation. In this way, ROS levels activate processes to maintain both genome and proteome integrity [
10].
Exposure to ionizing radiation activates all available cellular machinery to prevent/repair DNA and protein damage [
12]. The repair of DNA damage is mainly carried out by HR and NHEJ. The mechanisms that prevent the activity of free radicals and ROS also come into play, hence the found interaction with the common response genes
NTG1,
SIT4,
OGG1, and
HSP104. The last damage caused is on the proteins, mainly by oxidation. Since both the response/resistance to radiation and cell aging are multifactorial phenomena, it is logical to think that there are common molecular mechanisms at the level of DNA and protein damage repair. This fact could explain the large number of common processes and proteins that interact with each other in both phenomena (
Table 1). However, this work shows that all the proteins involved in the response/resistance to radiation are mainly involved in processes that seek cellular homeostasis at the genomic level (DNA repair and genomic stability). The homeostasis of the genome is therefore the common phenomenon between the effect of radiation and aging, against the protein homeostasis that would mainly dominate as the majority mechanism to avoid the phenomenon of aging.
However, if we look for a common mechanism of response to the three phenomena, it seems that, according to the data obtained, guaranteeing the correct activity of the proteome is key to maintaining cell survival and controlling aging by minimizing oxidative damage to proteins. In this sense, it is logical that the HSP104 protein is the only common one obtained involved in various metabolic pathways responsible for maintaining longevity and avoiding misfolded proteins. HSP play an important role in cellular proteostasis, also maintaining the correct folding and stabilization of the protein complexes involved in DNA repair and thus contributing to the effectiveness of response mechanisms against DNA-damaging agents [
12]. Krisko and Radman [
13] used HSP104-GFP aggregates as a marker of aging in
S. cerevisiae due to the accumulation of misfolded protein aggregates during the yeast lifespan.
The observed regulatory mechanisms highlight the importance of regulation by transcription factors in response to heat. This type of response mediated by HSP constitutes a common response mechanism against different agents that cause physical stress (misfolded and oxidized proteins). The HSP104 protein presents 31 regulators. This characteristic shows the importance that it has in multiple processes to maintain the homeostasis of the proteome, especially the one in charge of repairing DNA damage, as evidenced by the relationship found in this work. Molon and Zadrag-Tecza [
29] treated the protein HSP104 as a marker of yeast aging due to its accumulation with age and with the exposure to thermal and oxidative stress, indicating that it is a necessary protein to maintain normal cell life.
This work focused on the phenomenon of aging. Only genes involved in aging and not in senescence were considered. Discrimination was performed in the gene search process using the GenAge database [
30]. This database only provides information on aging genes (both replicative and chronological aging) and not on senescence. In this way, although senescence genes could be obtained in the search process for genes related to radiation response/resistance, they were eliminated as they did not coincide with the respective aging genes obtained in the GenAge database after crossing the data from both phenomena.
The results obtained in this work indicate that the HSP104 protein participates in regulatory pathways related to aging, response/resistance to ionizing radiation, oxidative stress, free radical activity, and the response to ROS, as well as antioxidant activity; therefore, they suggest that it could be a good biomarker of these processes.
This study shows that the proposed markers are similar in human, mouse, and rat due to the homology found in almost all genes found for S. cerevisiae.
After comparing with specific databases for ionizing radiation (RadBioBase), a different response to irradiation was observed in human and mouse cells. It is striking that human genes (EXO1, RAD51, and FEN1) after exposure to X-rays were down-regulated while mouse genes (Exo1, Rad51 and Clpb) were up-regulated. Although the data found in RadBioBase regarding the genes of interest in this study are scarce, this could indicate a specific response of the species and, to a lesser extent, are specific to the gene evaluated, as is the case of EXO1 and RAD51 genes whose expression state is opposite in human compared with mouse.
After comparing the mouse homologous genes with the MGI database, only the Rad52, Wrn, and Ogg1 genes were obtained as genes involved in the response to oxidative stress. Since these genes are also involved in the response to radiation, this fact suggests that these genes could be the main mouse genes involved in the response to the oxidative damage produced by ionizing radiation. However, searching for these genes in RadBioBase did not produce any results.
5. Conclusions
The results found in this work indicate that the response/resistance to ionizing radiation has a strong relationship with the cellular aging phenomenon. In this sense, the HR and NHEJ DNA repair pathways are the most important common processes necessary to maintain cellular homeostasis. On the other hand, the base excision-repair pathway is the main common response mechanism to oxidative stress, free radicals, ROS, and antioxidant activity caused by ionizing radiation. The metabolic routes of longevity regulation are those that jointly contribute to the three phenomena studied.
The existence of common genes between the studied phenomena indicate the possibility of defining common potential biomarkers in S. cerevisiae. The study carried out proposes 11 common biomarkers for the response/resistance to ionizing radiation and aging (EXO1, MEC1, MRE11, RAD27, RAD50, RAD51, RAD52, RAD55, RAD9, SGS1, YKU70) and two biomarkers for response/resistance to radiation and oxidative stress, free radicals, ROS, and antioxidant activity (NTG1, OGG1). In addition, it is important to highlight that the HSP104 protein could be a good biomarker common to the three phenomena studied.
The experimental validation of the proposed biomarkers has not been considered in this work because it is outside the context of the proposed objectives. Said validation is therefore left for later studies, where different conditions of exposure to radiation, aging, and the response to oxidizing agents are tested. Further studies could define its potential use.