Launched in the autumn of 2014 by the Radionuclide Ecotoxicology Laboratory (LECO) and lasting three years, the ISATIS (Ionizing Radiation and Biological Mechanisms) project focused on analyzing, in living organisms, damage to proteins induced by chronic exposure to low doses of ionizing radiation. It especially concentrated on mechanisms of protein carbonylation and their impact on cell function, in relation to DNA damage and repair, epigenetic effects, and apoptosis (through other projects conducted at LECO), as well as major biological functions of organisms. The ultimate goal was a better understanding of the basic mechanisms involved in these biological responses, in order to improve radiation protection for animal species, including humans.
Context and aims
The conventional approach adopted in studies of the biological effects of ionizing radiation considers nuclear DNA to be the critical target for radiation-induced damage (UNSCEAR, 2012). Until recently, most of the work in this field has focused on DNA damage and repair. However, experimental studies (Daly et al., 2010, Krisko and Radman, 2010, Krisko et al., 2012) have shown that proteins, rather than DNA, are the critical targets of damage caused by cell irradiation in many biological models. These studies demonstrate that survival in many organisms is related to the extent of oxidative damage to proteins, which compromises their functions, including efficiency of enzymes performing DNA repair and replication. The carbonylation of proteins is irreversible. Damaged proteins may, however, be removed by the cell as long as the degradative mechanism targeting them (protein complexes) is sufficiently active (Figure 1). In certain cases, at high doses of irradiation (i.e., 200–1,000 Gy), a correlation exists between the production of carbonylated proteins and a decline in fertility—for example, in invertebrates, a lower egg hatching rate and thus less reproductive success—or even a greater mortality. At low doses (<0.1 Gy), however, protein carbonylation is poorly studied as a marker.
See the summary diagram of the role of protein carbonylation in the deleterious effects of ionizing radiation toxicity.
Figure 1: Diagram illustrating role of protein carbonylation in effects of ionizing radiation toxicity
The ISATIS project aimed to study and monitor such protein damage, in particular the phenomenon of carbonylation, which is poorly studied in these dose ranges. The objective was to better understand the mechanisms of radiotoxicity at low doses of ionizing radiation, by studying interactions between ionizing radiation and biomolecules. This involved establishing correlations between concentrations of carbonylated proteins, the nature of the proteins damaged, degradation of the damaged proteins, and ultimately, macroscopic effects such as reproductive consequences. In a comparative approach, two models with distinct radiosensitivity, the nematode
Caenorhabditis elegans and the zebra fish
Danio rerio, were exposed to low and high doses of acute and chronic external g radiation. In parallel, several parameters were monitored: damage to DNA (single-strand and double-strand breaks) and their repair mechanisms, epigenetic processes (transmissible changes in gene function not derived from altered DNA sequences) and cell death (apoptosis). The chosen approach aimed to be generic, irrespective of the biological model used, permitting reasonable extrapolation to diverse species.
Conduct of project
The ISATIS project relied on a "systems biology" approach, based on both a description of the interactions of ionizing radiation with molecules (e.g., proteins, DNA, and lipids) playing key roles in biological functions—especially reproduction—and comparisons between the selected animal models.
The biological models used in the laboratory are familiar (C. elegans (Figure 2) and
D. rerio) and have different degrees of radiosensitivity. Their genomes and proteomes are known, which makes it possible to study their molecular mechanisms and link them to reproduction.
Figure 2: Adult C. Elegans nematod
© IRSN/Kuzmic M. et al.
Exposure to ionizing radiation was achieved by external irradiation with a controlled dose. Irradiations (gamma,
Cs) were first carried out in the MIRRE irradiator (a mini-irradiator delivering an external gamma dose rate to small organisms, from a few hundred nGy.h-1
up to 100 mGy.h-1
) and then at both the MICADO Lab irradiation facility and the Curie Institute (for acute irradiation).
In addition to laboratory studies, a study was carried out on the tree frog
Hyla orientalis, taken from different sites within the Chernobyl exclusion zone contaminated with
90Sr. Amphibians, such as tree frogs, are ideal organisms to study for several reasons: (i) their skin is highly permeable to pollutants, (ii) they are exposed to pollutants from diverse aquatic and terrestrial habitats during their lifetimes, and (iii) they are among the animals listed by the International Commission for Radiological Protection (ICRP) for the study of radiological risk assessment.
Evaluation of protein carbonylation and proteolytic activity
Two methods for measuring carbonylation have been developed. The first method makes it possible to quantify and identify carbonylated proteins in a sample made up of several individuals having undergone the same treatment. This method relies on the extraction and chemical separation of proteins, whose carbonyls are specifically labelled. The second method measures the degree of carbonylation of biomolecules (i.e., proteins, DNA, RNA, and lipids) in the whole organism (Figure3). It relies on the use of specific carbonyl and biomolecule labelling with confocal imaging analysis.
In parallel, the level of activity of the proteasome (a proteic complex involved in the degradation of damaged proteins) was evaluated under different irradiation conditions to detect potential modifications in the capacity of cells to degrade oxidized proteins.
Figure 3: Confocal images (fluorescence) of C. elegans nematod adults (left) and eggs (right) stained with a multiplex carbonyls/lipids/DNA/proteins labelling
© IRSN/Kuzmic M. et al.
Evaluation of proteome modulations
Another approach to understanding radiotoxicity is to consider the impact of ionizing radiation on protein expression. Since proteins are involved in many biological processes, including cellular proteolytic activity (especially via the proteasome), antioxidant defense and DNA repair, their study is essential for an understanding of the processes involved in phenomena of radiotoxicity. Proteomics reveals changes in protein expression in exposed individuals—in comparison with controls—and thus makes it possible to profile such radiation-induced modifications.
Evaluation of DNA damage: oxidative damage and repair
Quantification of DNA damage (single-strand breaks, double-strand breaks, and modified bases) was considered, but could only be performed in
D. rerio using the comet assay, during other projects conducted in the laboratory.
Evaluation of epigenetic changes
Epigenetic changes were assessed in other laboratory projects using several markers, depending on both the level of observation and the biological model. These included DNA methylation, which contributes to chromosome structure and stability and to transcription.
Evaluation of the impact on major biological functions
Irradiation and the measurements made at the molecular and cellular levels were considered in parallel with macroscopic effects on major biological functions such as longevity and reproduction (e.g., number of eggs laid and hatching rate).
Presented below are the findings of evaluations of carbonylation, protein expression, and proteolytic activity in response to ionizing radiation (i.e.,
3H, and at polluted sites), obtained after chronic or acute exposure in various model organisms or environmentally-affected organisms at moderate doses. These data, which are non-exhaustive, are interpreted in light of macroscopic variables—including aspects of reproduction and longevity—as well as other molecular markers where possible.
(i) Relationship between protein carbonylation and reproduction after acute or chronic gamma irradiation of wild-type
In this study, after acute irradiation (0–200 Gy, 1–15 Gy·min-1), a significant decrease in total egg-laying per individual and a decrease in hatching success were observed starting at 30 Gy. After chronic irradiation (0–6.5 Gy, 7–100 mGy·h-1), only a significant decrease in total egg-laying per individual was observed, but starting at 3.3 Gy (50 mGy·h-1 for 65 h). In addition, it was shown that carbonylated protein levels increased after acute irradiation for certain doses (6.5 Gy in particular), but were rather stable after chronic irradiation. The level of carbonylated proteins is dependent on the concentration of reactive oxygen species (ROS) but also on the capacity of cells to eliminate modified proteins, which is related to proteasome activity. Depending on the nature of the substrate to be degraded, three proteasome forms may be involved (Figure 4A). Here the 20S form (thought to be dedicated to the degradation of oxidized proteins) seems to show the greatest drop in activity with acute irradiation. However, there appears to be little impact on its expression (Figure 4B). The activity and expression of the ATP/ubiquitin-dependent (30S and 26S) forms, the abundance of which reflects the overall activity of the proteasome, seem little impacted after acute irradiation at the lowest doses but are decreased at high doses. After chronic irradiation, however, it appears that the proteasome—especially the 20S form—is stimulated.
A/ Scheme of the proteasome and its constitutive subunits (Papaevgeniou and Chondrogianni., 2014 The ubiquitin proteasome system in C.elegans and its regulation). 26S and 30S units: ATP- and ubiquitin-dependent; 20S unit: ATP- and ubiquitin- independent.
B/ Native gel measurement of the 30S, 26S and 20S proteasome activity (left panel) after electrophoretic separation and their expression levels after Westernblot analysis (right panel). For each set of images, at the left: signal from control nematodes (defined as 100% activity) and at the right: signal from nematodes exposed to 1Gy of acute irradiation.
The proteomic analysis identified signaling pathways potentially involved in the response to acute and chronic ionizing radiation in
C. elegans. It was determined that the underlying mechanisms in the cases of acute and chronic exposure are distinct. This result implies that the chronic risks of ionizing radiation cannot be readily extrapolated from acute irradiation data. A number of protein targets related to reproduction (oogenesis, embryogenesis), the stress response, and proteasome activity constitute molecular parameters of potential interest that deserve further study in future experiments, at lower doses or over several generations. Indeed, these protein markers may even prove to be predictors for the effects of radiation.
One previous study noted the influence of external gamma irradiation on the number of male gametes (not directly demonstrated here through the proteomic study). In order to deepen our knowledge of the molecular mechanisms associated with reproductive toxicity, and to validate the markers identified in the global approach, it may prove interesting to carry out a specific comparative morphological and molecular study of the radiosensitivity of male and female gametes in
C. elegans to determine the causes of diminished reproduction.
(ii) Link between protein carbonylation and aging after chronic gamma irradiation of the
C. elegans Glp-1 mutant
In this study, our results indicate that chronic exposure to ionizing radiation shortens the lifetime of the sterile
C. elegans Glp-1 mutant, regardless of the duration of exposure (from egg stage to 65 hours stage or from egg stage to 19 days stage), the dose rate (7 or 50 mGy·h-1) or the overall dose (0.5, 3.5, or 24 Gy) tested. A related question is whether the results might be the same with even shorter exposures (egg stage at 24 h or 48 h), covering a specific stage of development, such as diakinesis (shown in the literature to be the most radiosensitive phase). Carbonylation has been shown to be a good biomarker of aging, older nematodes having higher carbonylation levels than young nematodes. Irradiation, on the other hand, reduces the extent of carbonylation whatever the dose or duration of exposure. This decrease in carbonylation could partly be explained by an increase in proteasome activity, which is already highly active in these mutants. In addition, the quantity of lipids has been studied and follows the same trends as carbonylation, i.e., increasing with age, but decreasing after irradiation (Figure 5). Lipids are very important signaling molecules involved in cell membrane building and energy storage. In addition, the link between reproductive function, lipid homeostasis, and longevity is described in the literature, although the mechanisms by which they impact each other are not clearly defined. Our findings therefore call for deeper investigation to acquire a better understanding of the possible connections. In any case, lipid levels and carbonylation may well be potential markers of irradiation and aging, respectively.
Positive colocalization between proteins and lipids at adult stage, irradiated 10d with a dose rate of 52 mGy.h-1 (cumulative dose 12.5 Gy), and at old stage (unirradiated), that excludes colocalization with carbonyls (Pearson’s coefficient carbonyl-protein ~ 0.2) – Thesis M. Kuzmic.
(iii) Determination of the concentration of carbonyl proteins (CP) in
g irradiation at 0.5 and 5 mGy·h-1 of juveniles (from eggs to larval stages)—from parents irradiated at 0.5 and 5 mGy·h-1 in another study conducted in the laboratory—no significant effect on the level of carbonylation could be observed after 4 days and 10 days of irradiation (though a tendency to increase at 10 days for 0.5 and 5 mGy·h-1 was observed). The analysis of cellular proteolytic activity under these conditions may provide keys for interpreting this response.
After exposure to 3.7 x 102 or 3.4 x 103 μGy.h-1 of tritiated water of juvenile stages (from eggs to larvae) for 7 and 10 days in another study conducted at LECO, a significant decrease in the level of carbonylated proteins was observed at 10 days, compared to controls. This might be explained by lower numbers of oxidative species in the medium (which is not apparent through in vivo analysis of the reactive oxygen species production index in the larvae) or by a significant stimulation of cellular defense systems such as proteolysis, which destroy damaged proteins and thereby promote their resynthesis. This last hypothesis could be the subject of a specific study.
(iv) Determination of carbonyl protein (CP) concentrations in the muscle of
Another study conducted in the laboratory considered the carbonylation marker and protein expression in the muscle of the tree frog
H. orientalis taken from three sites in the Chernobyl exclusion zone, each differentially contaminated (Cs and Sr) with ambient dose rates of 0.10, 3.7, and 32.4 μGy·h-1 respectively. Little difference was observed in protein carbonylation, although levels appear to be lower in individuals from the intermediate site. A comparative proteomic approach in the same frogs has revealed repression of several muscle proteins in individuals from the two most contaminated sites. Further analysis of these findings requires complementary investigations, including the identification of a reference genome—which currently only partially exists for
H. orientalis—in order to identify protein variants and characterize their biological functions. They must then be studied in light of the total dose (internal and external) received by each individual.
As a result of these very diverse analyses of protein carbonylation in developing organisms irradiated in the laboratory (D. rerio, C. elegans) and in muscle tissues of organisms taken from a contaminated environment (H. orientalis), it appears that this protein damage, a marker of aging in our studies, does not increase in direct response to chronic irradiation. On the contrary, it appears to decrease under certain conditions, which may be explained by a significant stimulation of cellular defense systems, such as proteolysis, by chronic exposure. This path of investigation must be considered alongside a proteomic analysis associated with the experimental conditions studied to make the findings more conclusive. This will ultimately improve the knowledge of protein markers potentially associated with chronic exposure to ionizing radiation, and in relation to radiation-induced effects on individuals, such as reproductive toxicity.
Dissemination of findings
Both methods developed for the measurement of carbonyls have been the topics of publications. Other manuscripts addressing these results are being prepared.