A systems biology analysis of reproductive toxicity effects induced by multigenerational exposure to ionizing radiation in C. elegans.

Understanding the effects of chronic exposure to pollutants over generations is of primary importance for the protection of humans and the environment; however, to date, knowledge on the molecular mechanisms underlying multigenerational adverse effects is scarce. We employed a systems biology approach to analyze effects of chronic exposure to gamma radiation at molecular, tissue and individual levels in the nematode Caenorhabditis elegans. Our data show a decrease of 23% in the number of offspring on the first generation F0 and more than 40% in subsequent generations F1, F2 and F3. To unveil the impact on the germline, an in-depth analysis of reproductive processes involved in gametes formation was performed for all four generations. We measured a decrease in the number of mitotic germ cells accompanied by increased cell-cycle arrest in the distal part of the gonad. Further impact on the germline was manifested by decreased sperm quantity and quality. In order to obtain insight in the molecular mechanisms leading to decreased fecundity, gene expression was investigated via whole genome RNA sequencing. The transcriptomic analysis revealed modulation of transcription factors, as well as genes involved in stress response, unfolded protein response, lipid metabolism and reproduction. Furthermore, a drastic increase in the number of differentially expressed genes involved in defense response was measured in the last two generations, suggesting a cumulative stress effect of ionizing radiation exposure. Transcription factor binding site enrichment analysis and the use of transgenic strain identified daf-16/FOXO as a master regulator of genes differentially expressed in response to radiation. The presented data provide new knowledge with respect to the molecular mechanisms involved in reproductive toxic effects and accumulated stress resulting from multigenerational exposure to ionizing radiation.

Gamma radiation induces life stage-dependent reprotoxicity in Caenorhabditis elegans via impairment of spermatogenesis.

The current study investigated life stage, tissue and cell dependent sensitivity to ionizing radiation of the nematode Caenorhabditis elegans. Results showed that irradiation of post mitotic L4 stage larvae induced no significant effects with respect to mortality, morbidity or reproduction at either acute dose ≤6 Gy (1500 mGy·h-1) or chronic exposure ≤15 Gy (≤100 mGy·h-1). In contrast, chronic exposure from the embryo to the L4-young adult stage caused a dose and dose-rate dependent reprotoxicity with 43% reduction in total brood size at 6.7 Gy (108 mGy·h-1). Systematic irradiation of the different developmental stages showed that the most sensitive life stage was L1 to young L4. Exposure during these stages was associated with dose-rate dependent genotoxic effects, resulting in a 1.8 to 2 fold increase in germ cell apoptosis in larvae subjected to 40 or 100 mGy·h-1, respectively. This was accompanied by a dose-rate dependent reduction in the number of spermatids, which was positively correlated to the reprotoxic effect (0.99, PCC). RNAseq analysis of nematodes irradiated from L1 to L4 stage revealed a significant enrichment of differentially expressed genes related to both male and hermaphrodite reproductive processes. Gene network analysis revealed effects related to down-regulation of genes required for spindle formation and sperm meiosis/maturation, including smz-1, smz-2 and htas-1. Furthermore, the expression of a subset of 28 set-17 regulated Major Sperm Proteins (MSP) required for spermatid production was correlated (R2 0.80) to the reduction in reproduction and the number of spermatids. Collectively these observations corroborate the impairment of spermatogenesis as the major cause of gamma radiation induced life-stage dependent reprotoxic effect. Furthermore, the progeny of irradiated nematodes showed significant embryonal DNA damage that was associated with persistent effect on somatic growth. Unexpectedly, these nematodes maintained much of their reproductive capacity in spite of the reduced growth.

Current evidence for a role of epigenetic mechanisms in response to ionizing radiation in an ecotoxicological context.

The issue of potential long-term or hereditary effects for both humans and wildlife exposed to low doses (or dose rates) of ionising radiation is a major concern. Chronic exposure to ionising radiation, defined as an exposure over a large fraction of the organism's lifespan or even over several generations, can possibly have consequences in the progeny. Recent work has begun to show that epigenetics plays an important role in adaptation of organisms challenged to environmental stimulae. Changes to so-called epigenetic marks such as histone modifications, DNA methylation and non-coding RNAs result in altered transcriptomes and proteomes, without directly changing the DNA sequence. Moreover, some of these environmentally-induced epigenetic changes tend to persist over generations, and thus, epigenetic modifications are regarded as the conduits for environmental influence on the genome. Here, we review the current knowledge of possible involvement of epigenetics in the cascade of responses resulting from environmental exposure to ionising radiation. In addition, from a comparison of lab and field obtained data, we investigate evidence on radiation-induced changes in the epigenome and in particular the total or locus specific levels of DNA methylation. The challenges for future research and possible use of changes as an early warning (biomarker) of radiosensitivity and individual exposure is discussed. Such a biomarker could be used to detect and better understand the mechanisms of toxic action and inter/intra-species susceptibility to radiation within an environmental risk assessment and management context.

Interplay between ionizing radiation effects and aging in C. elegans.

Living species are chronically exposed to environmental ionizing radiations from sources that can be overexpressed by nuclear accidents. In invertebrates, reproduction is the most radiosensitive studied endpoint, likely to be connected with aging. Surprisingly, aging is a sparsely investigated endpoint after chronic ionizing radiation, whereas understanding it is of fundamental interest in biology and medicine. Indeed, aging and aging-related diseases (e.g., cancer and degenerative diseases) cause about 90% of deaths in developed countries. Therefore, glp-1 sterile Caenorhabditis elegans nematode was used to assess the impact of chronic gamma irradiation on the lifespan. Analyses were performed, at the individual level, on aging and, in order to delve deeper into the mechanisms, at the molecular level, on oxidative damage (carbonylation), biomolecules (lipids, proteins and nucleic acids) and their colocalization. We observed that ionizing radiation accelerates aging (whatever the duration (3-19 days)/dose (0.5-24 Gy)/dose rate (7 and 52 mGy h-1) tested) leading to a longevity value equivalent to that of wt nematode (∼25-30 days). Moreover, the level of protein oxidative damage (carbonylation) turned out to be good cellular biomarker of aging, since it increases with age. Conversely, chronic radiation treatments reduced carbonylation levels and induced neutral lipid catabolism whatever the dose rate and the final delivered dose. Finally, under some conditions a lipid-protein colocalization without any carbonyl was observed; this could be linked to yolk accumulation in glp-1 nematodes. To conclude, we noticed through this study a link between chronic gamma exposure, lifespan shortening and lipid level decrease associated with a decrease in the overall carbonylation.

A dynamic energy-based model to analyze sublethal effects of chronic gamma irradiation in the nematode Caenorhabditis elegans.

Understanding how toxic contaminants affect wildlife species at various levels of biological organization (subcellular, histological, physiological, organism, and population levels) is a major research goal in both ecotoxicology and radioecology. A mechanistic understanding of the links between different observed perturbations is necessary to predict the consequences for survival, growth, and reproduction, which are critical for population dynamics. In this context, experimental and modeling studies were conducted using the nematode Caenorhabditis elegans. A chronic exposure to external gamma radiation was conducted under controlled conditions. Results showed that somatic growth and reproduction were reduced with increasing dose rate. Modeling was used to investigate whether radiation effects might be assessed using a mechanistic model based upon the dynamic energy budget (DEB) theory. A DEB theory in toxicology (DEB-tox), specially adapted to the case of gamma radiation, was developed. Modelling results demonstrated the suitability of DEB-tox for the analysis of radiotoxicity and suggested that external gamma radiation predominantly induced a direct reduction in reproductive capacity in C. elegans and produced an increase in costs for growth and maturation, resulting in a delay in growth and spawning observed at the highest tested dose rate.

In situ visualization of carbonylation and its co-localization with proteins, lipids, DNA and RNA in Caenorhabditis elegans.

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 precludes quantification. Since carbonylated proteins correlate with and become a suspected cause of morbidity and mortality in some organisms, there is a need for their accurate quantification and localization. Using appropriate fluorescent probes, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism. In C. elegans, we found that after UV irradiation carbonylation co-localizes mainly with proteins and, to a lesser degree, with DNA, RNA and lipids. The method efficiency was illustrated by carbonylation induction assessment over 5 different UV doses. The procedure enables the monitoring of carbonylation in the nematode C. elegans during stress, aging and disease along its life cycle including the egg stage.

Effects of radionuclide contamination on leaf litter decomposition in the Chernobyl exclusion zone.

The effects of radioactive contamination on ecosystem processes such as litter decomposition remain largely unknown. Because radionuclides accumulated in soil and plant biomass can be harmful for organisms, the functioning of ecosystems may be altered by radioactive contamination. Here, we tested the hypothesis that decomposition is impaired by increasing levels of radioactivity in the environment by exposing uncontaminated leaf litter from silver birch and black alder at (i) eleven distant forest sites differing in ambient radiation levels (0.22-15µGyh(-1)) and (ii) along a short distance gradient of radioactive contamination (1.2-29µGyh(-1)) within a single forest in the Chernobyl exclusion zone. In addition to measuring ambient external dose rates, we estimated the average total dose rates (ATDRs) absorbed by decomposers for an accurate estimate of dose-induced ecological consequences of radioactive pollution. Taking into account potential confounding factors (soil pH, moisture, texture, and organic carbon content), the results from the eleven distant forest sites, and from the single forest, showed increased litter mass loss with increasing ATDRs from 0.3 to 150µGyh(-1). This unexpected result may be due to (i) overcompensation of decomposer organisms exposed to radionuclides leading to a higher decomposer abundance (hormetic effect), and/or (ii) from preferred feeding by decomposers on the uncontaminated leaf litter used for our experiment compared to locally produced, contaminated leaf litter. Our data indicate that radio-contamination of forest ecosystems over more than two decades does not necessarily have detrimental effects on organic matter decay. However, further studies are needed to unravel the underlying mechanisms of the results reported here, in order to draw firmer conclusions on how radio-contamination affects decomposition and associated ecosystem processes.

Nested interactions in the combined toxicity of uranium and cadmium to the nematode Caenorhabditis elegans.

Uranium is a natural, ubiquitous radioactive element for which elevated concentrations can be found in the vicinity of some nuclear fuel cycle facilities or intensive farming areas, and most often in mixtures with other contaminants such as cadmium, due to co-occurrence in geological ores (e.g. U- or P-ore). The study of their combined effects on ecosystems is of interest to better characterize such multi-metallic polluted sites. In the present study, the toxicity of binary mixture of U and Cd on physiological parameters of the soil nematode Caenorhabditis elegans was assessed over time. Descriptive modeling using concentration and response addition reference models was applied to compare observed and expected combined effects and identify possible synergistic or antagonistic interactions. A strong antagonism between U and Cd was identified for length increase and brood size endpoints. The study revealed that the combined effects might be explained by two nested antagonistic interactions. We demonstrate that the first interaction occurred in the exposure medium. We also identified a significant second antagonistic interaction which occurred either during the toxicokinetic or toxicodynamic steps. These findings underline the complexity of interactions that may take place between chemicals and thus, highlight the importance of studying mixtures at various levels to fully understand underlying mechanisms.

Assessment of growth, genotoxic responses and expression of stress related genes in the Pacific oyster Crassostrea gigas following chronic exposure to ionizing radiation.

Marine organisms are exposed to low doses of anthropogenic contaminants during their entire life. Authorized amounts of radionuclides are discharged in the Channel by nuclear facilities. The Pacific oyster was used to investigate the potential impact of chronic exposure to ionizing radiation. Though we exposed larvae and spat for two weeks to much higher concentrations than those encountered near nuclear facilities, oyster growth and expression of 9 selected stress genes were not significantly changed. To determine potential DNA damage, 2year old oysters were exposed for two weeks to tritiated water. The comet assay was used to evaluate the level of DNA strand breaks in haemocytes, whilst the 'clearance rate' was used as a measure of physiological effects. Whilst other parameters did not alter, DNA damage significantly increased. Our results highlight the significance of the observed DNA damage and their potential consequences at higher levels of biological organization.

Effects of chronic gamma irradiation: a multigenerational study using Caenorhabditis elegans.

The effects of chronic exposure to (137)Cs gamma radiation (dose rate ranging from 6.6 to 42.7 mGy h(-1)) on growth and reproductive ability were carried out over three generations of Caenorhabditis elegans (F0, F1, and F2). Exposure began at the egg stage for the first generation and was stopped at the end of laying of third-generation eggs (F2). At the same time, the two subsequent generations from parental exposure were returned to the control conditions (F1' and F2'). There was no radiation-induced significant effect on growth, hatchability, and cumulative number of larvae within generations. Moreover, no significant differences were found in growth parameters (hatching length, maximal length, and a constant related to growth rate) among the generations. However, a decrease in the cumulative number of larvae across exposed generations was observed between F0 and F2 at the highest dose rate (238.8 ± 15.4 and 171.2 ± 13.1 number of larvae per individual, respectively). Besides, the F1' generation was found to lay significantly fewer eggs than the F1 generation for tested dose rates 6.6, 8.1, 19.4, and 28.1 mGy h(-1). Our results confirmed that reproduction (here, cumulative number of larvae) is the most sensitive endpoint affected by chronic exposure to ionizing radiation. The results obtained revealed transgenerational effects from parental exposure in the second generation, and the second non-exposed generation was indeed more affected than the second exposed generation.

Heavy metal accumulation by recombinant mammalian metallothionein within Escherichia coli protects against elevated metal exposure.

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins known to provide protection against toxic heavy metals such as cadmium. In an attempt to increase the ability of bacterial cells to accumulate heavy metals, sheep MTII was produced in fusion with the maltose binding protein (MBP) and localized to the cytoplasmic or periplasmic compartments of Escherichia coli. For all metals tested, higher levels of bioaccumulation were measured with strains over-expressing MBP-MT in comparison with control strains. A marked bioaccumulation of Cd, As, Hg and Zn was observed in the strain over-expressing MBP-MT in the cytoplasm, whereas Cu was accumulated to higher levels when MBP-MT was over-expressed in the periplasm. Metal export systems may also play a role in this bioaccumulation. To illustrate this, we over-expressed MBP-MT in the cytoplasm of two mutant strains of E. coli affected in metal export. The first, deficient in the transporter ZntA described to export numerous divalent metal ions, showed increasing quantities of Zn, Cd, Hg and Pb being bioaccumulated. The second, strain LF20012, deficient in As export, showed that As was bioaccumulated in the form of arsenite. Furthermore, high quantities of accumulated metals, chelated by MBP-MT in the cytoplasm, conferred greater metal resistance levels to the cells in the presence of added toxic metals, such as Cd or Hg, while other metals showed toxic effects when the export systems were deficient. The strain over-expressing MBP-MT in the cytoplasm, in combination, with disruption of metal export systems, could be used to develop strategies for bioremediation.

Transfer modelling and toxicity evaluation of the effluent from an installation of cleansing and uranium recovery using a battery of bioassays.

On July 7, 2008, a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) led to the spillage of uranium in a stream. The acute toxicity of the effluent was evaluated, and compared to the toxicity of uranium nitrate in bioassays using several organisms: Chlamydomonas reinhardtii, Daphnia magna, Chironomus riparius and Danio rerio. A sediment bioassay was also performed on C. riparius using water and sediment sampled along the river. Results showed that effluent EC(50) 72 h was 0.65 mg U/l for algae and LC(50) 48 h was 1.67 mg U/l for daphnia, while values obtained for uranium nitrate were higher. The LC(50) 96 h of effluent to C. riparius was 22.7 mg U/l, similar to value for uranium nitrate; the sediment collected was not toxic to C. riparius larvae. The LOEC of effluent and uranium nitrate on HT(50) of D. rerio were similar (0.03 mg U/l), but larvae were more sensitive to uranium nitrate than to effluent. Our results suggest that other substances contained in the effluent could potentially be toxic to wildlife in association with uranium. In parallel, the modelling of the transfers based on uranium measurements in the surface water was used to fill data gaps and assess the impact along the river. These results provided an estimate of exposure conditions that occurred along the river. This approach allowed us to see that the risk to ecosystem during this incident was certainly low and concerned a short period of time, but it could have existed at least for some species.

Effects of waterborne uranium on survival, growth, reproduction and physiological processes of the freshwater cladoceran Daphnia magna.

Acute uranium toxicity (48 h immobilisation test) for Daphnia magna was determined in two different exposure media, differing in pH and alkalinity. LC(50) varied strongly between media, from 390+/-40 microgL(-1)U at pH 7 to 7.8+/-3.2 mgL(-1)U at pH 8. According to the free ion activity model uranium toxicity varies as a function of free uranyl concentration. This assumption was examined by calculating uranium speciation in our water conditions and in those reported in the literature. Predicted changes in free uranyl concentration could not solely explain observed differences in toxicity, which might be due to a competition or a non-competitive inhibition of H(+) for uranium transport and/or the involvement of other bioavailable chemical species of uranium. Chronic effects of uranium at pH 7 on mortality, ingestion and respiration, fecundity and dry mass of females, eggs and neonates were investigated during 21-day exposure experiments. A mortality of 10% was observed at 100 microgL(-1)U and EC(10) for reproduction was 14+/-7 microgL(-1)U. Scope for growth was affected through a reduction in feeding activity and an increase in oxygen consumption at 25 microgL(-1)U after 7 days of exposure. This had strong consequences for somatic growth and reproduction, which decreased, respectively, by 50% and 65% at 50 microgL(-1)U after 7 days and at 25 microgL(-1)U after 21 days. Uranium bioaccumulation was quantified and associated internal alpha dose rates from 2.1 to 13 microGyh(-1) were estimated. Compared to the toxicity of other alpha-emitting radionuclides and stable trace metals, our results confirmed the general assumption that uranium chemical toxicity predominates over its radiotoxicity.

Enhanced toxic metal accumulation in engineered bacterial cells expressing Arabidopsis thaliana phytochelatin synthase.

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.