Tritiated thymidine induces developmental delay, oxidative stress and gene overexpression in developing zebrafish (Danio rerio).

Tritium is a betta emitter radionuclide. Being an isotope of hydrogen, it is easily transferred to different environmental compartments, and to human and non-human biota. Considering that tritium levels are expected to rise in the upcoming decades with the development of nuclear facilities producing tritium using fission processes, investigating the potential toxicity of tritium to human and non-human biota is necessary. Tritiated thymidine, an organic form of tritium, has been used in this study to assess its toxicity on fish embryo development. Zebrafish embryos (3.5 hpf; hours post fertilization) have been exposed to tritiated thymidine at three different activity concentrations (7.5; 40; 110 kBq/mL) for four days. These experiments highlighted that zebrafish development was affected by the exposure to organic tritium, with smaller larvae at 3 dpf after exposure to the two lowest dose rates (22 and 170 µGy/h), a delayed hatching after exposure to the two highest dose rates (170 and 470 µGy/h), an increase in the spontaneous tail movement (1 dpf) and a decrease in the heartbeat (3 dpf) after exposure to the highest dose rate. The results also highlighted an increase in ROS production in larvae exposed to the intermediate dose rate. A dysregulation of many genes, involved in apoptosis, DNA repair or oxidative stress, was also found after 1 day of exposure to the lowest tritium dose rate. Our results thus suggest that exposure to tritiated thymidine from a dose rate as low as 22 µGy/h can lead to sublethal effects, with an effect on the development, dysregulation of many genes and increase of the ROS production. This paper provides valuable information on toxic effects arising from the exposure of fish to an organic form of tritium, which was the main objective of this study.

Two distinct mechanisms lead to either oocyte or spermatocyte decrease in C. elegans after whole developmental exposure to γ-rays.

Wildlife is subject to various sources of pollution, including ionizing radiation. Adverse effects can impact the survival, growth, or reproduction of organisms, later affecting population dynamics. In invertebrates, reproduction, which directly impacts population dynamics, has been found to be the most radiosensitive endpoint. Understanding the underlying molecular pathways inducing this reproduction decrease can help to comprehend species-specific differences in radiosensitivity. From our previous studies, we found that decrease in reproduction is life stage dependent in the roundworm Caenorhabditis elegans, possibly resulting from an accumulation of damages during germ cell development and gamete differentiation. To go further, we used the same experimental design to assess more precisely the molecular determinants of reproductive toxicity, primarily decreases in gamete number. As before, worms were chronically exposed to 50 mGy·h-1 external gamma ionizing radiation throughout different developmental periods (namely embryogenesis, gametogenesis, and full development). To enable cross species extrapolation, conserved molecular pathways across invertebrates and vertebrates were analysed: apoptosis and MAP kinase Ras/ERK (MPK-1), both involved in reproduction and stress responses. Our results showed that these pathways are life-stage dependent, resulting from an accumulation of damages upon chronic exposure to IR throughout the life development. The Ras/ERK pathway was activated in our conditions in the pachytene region of the gonad where it regulates cell fate including apoptosis, but not in the ovulation zone, where it controls oocyte maturation and ovulation. Additionally, assessment of germ cell proliferation via Ras/ERK pathway showed no effect. Finally, a functional analysis of apoptosis revealed that while the decrease of the ovulation rate is caused by DNA-damaged induced apoptosis, this process does not occur in spermatocytes. Thus, sperm decrease seems to be mediated via another mechanism, probably a decrease in germ cell proliferation speed that needs further investigation to better characterize sex-specific responses to IR exposure. These results are of main importance to describe radio-induced reprotoxic effects and contribute as weight of evidence for the AOP #396 "Deposition of ionizing energy leads to population decline via impaired meiosis".

Tritiated Thymidine Internalization in Zebrafish Early Life Stages: Joint Use of Experimental Procedures and Microdosimetry.

Tritium is found in the environment under three forms: free in the water, gaseous, and bound to organic matter. Once internalized in living organisms, it can be found in two forms: tissue free water tritium (TFWT) and organically bound tritium (OBT). This study aims to better understand OBT internalization in living organisms and to show the complementarity between experimental procedures and microdosimetry simulations that have often been used to obtain more information on imparted energy to cell nuclei. To do so, tritiated thymidine, an organic form of tritium, was chosen and zebrafish embryos [3.5 h post fertilization (hpf)] were exposed to a range of activity concentrations (2.21 × 103 to 5.95 × 105 Bq/mL). First, individual zebrafish embryos were sampled after different exposure times (1 to 96 h) to qualify the internalization kinetics. Then, the barrier role of the chorion was assessed after 2 days of exposure. Lastly, individual zebrafish embryos were sampled after 1 and 4 days of exposure to measure the internalization in the whole fish and its DNA, but also to highlight a possible link between the internal dose rate and the external activity concentration. Microdosimetry simulations were also made to quantify the imparted energy that could occur in the zebrafish cells after exposure to tritium. Results showed that when bound to thymidine, tritium rapidly incorporates in zebrafish early life stages, with the internalization being almost complete after 24 h. Results also showed that while the chorion acted as a barrier to prevent thymidine from entering the embryos, significant levels could still be measured in the whole organisms as well as in DNA. This study also highlighted that when the external activity concentration increased, the internal dose rate increased as well, following a sigmoidal trend. Microdosimetry simulations highlighted that the size and shape of the cell matters, and that the smallest cells seem to be at the greater risk, with only low-energy electrons inducing energy depositions. A linear fit was also found between the mean energy deposited and the logarithm of the radius of the cell, thus showing that the quantity of deposited energy is proportional to the radius of the cell. While this study highlighted important internalization pattern, it will also be used as the starting point of a study focusing on the toxic effects of tritiated thymidine on zebrafish in its early life stages.

Insights into the modes of action of tritium on the early-life stages of zebrafish, Danio rerio, using transcriptomic and proteomic analyses.

In the environment, populations are exposed to different kinds of ionizing radiation. Little is known about their modes of action on non-human species, and whether or not they are similar for alpha, beta and gamma radiations, considered as the reference. In this context, tritium effects (beta emitter) under the form of tritiated water (HTO) were investigated in zebrafish, a common model in toxicology and ecotoxicology with a fully sequenced genome. Experiments were conducted on early life stages, considered to be highly sensitive to pollutants, by exposing eggs to 0.4 mGy/h of HTO until 10 days post fertilization. Tritium internalization was quantified, and effects were investigated using a combined approach of transcriptomic and proteomic analyses. Results highlighted similarities in the biological pathways affected by HTO by both techniques, such as defence response, muscle integrity and contraction, and potential visual alterations. These results correlated well with previous data obtained on earlier developmental stages (1 and 4 dpf). Interestingly, HTO effects were partly overlapping those obtained after gamma irradiation, underlying potential common modes of action. This study, therefore, brought a body of evidence on the effects of HTO observed at the molecular level on zebrafish larvae. Further studies could investigate if the effects persist in adult organisms.

Revealing the Increased Stress Response Behavior through Transcriptomic Analysis of Adult Zebrafish Brain after Chronic Low to Moderate Dose Rates of Ionizing Radiation.

High levels of ionizing radiation (IR) are known to induce neurogenesis defects with harmful consequences on brain morphogenesis and cognitive functions, but the effects of chronic low to moderate dose rates of IR remain largely unknown. In this study, we aim at defining the main molecular pathways impacted by IR and how these effects can translate to higher organizational levels such as behavior. Adult zebrafish were exposed to gamma radiation for 36 days at 0.05 mGy/h, 0.5 mGy/h and 5 mGy/h. RNA sequencing was performed on the telencephalon and completed by RNA in situ hybridization that confirmed the upregulation of oxytocin and cone rod homeobox in the parvocellular preoptic nucleus. A dose rate-dependent increase in differentially expressed genes (DEG) was observed with 27 DEG at 0.05 mGy/h, 200 DEG at 0.5 mGy/h and 530 DEG at 5 mGy/h. Genes involved in neurotransmission, neurohormones and hypothalamic-pituitary-interrenal axis functions were specifically affected, strongly suggesting their involvement in the stress response behavior observed after exposure to dose rates superior or equal to 0.5 mGy/h. At the individual scale, hypolocomotion, increased freezing and social stress were detected. Together, these data highlight the intricate interaction between neurohormones (and particularly oxytocin), neurotransmission and neurogenesis in response to chronic exposure to IR and the establishment of anxiety-like behavior.

Unusual evolution of tree frog populations in the Chernobyl exclusion zone.

Despite the ubiquity of pollutants in the environment, their long-term ecological consequences are not always clear and still poorly studied. This is the case concerning the radioactive contamination of the environment following the major nuclear accident at the Chernobyl nuclear power plant. Notwithstanding the implications of evolutionary processes on the population status, few studies concern the evolution of organisms chronically exposed to ionizing radiation in the Chernobyl exclusion zone. Here, we examined genetic markers for 19 populations of Eastern tree frog (Hyla orientalis) sampled in the Chernobyl region about thirty years after the nuclear power plant accident to investigate microevolutionary processes ongoing in local populations. Genetic diversity estimated from nuclear and mitochondrial markers showed an absence of genetic erosion and higher mitochondrial diversity in tree frogs from the Chernobyl exclusion zone compared to other European populations. Moreover, the study of haplotype network permitted us to decipher the presence of an independent recent evolutionary history of Chernobyl exclusion zone's Eastern tree frogs caused by an elevated mutation rate compared to other European populations. By fitting to our data a model of haplotype network evolution, we suspected that Eastern tree frog populations in the Chernobyl exclusion zone have a high mitochondrial mutation rate and small effective population sizes. These data suggest that Eastern tree frog populations might offset the impact of deleterious mutations because of their large clutch size, but also question the long-term impact of ionizing radiation on the status of other species living in the Chernobyl exclusion zone.

Assessing tritium internalisation in zebrafish early life stages: Importance of rapid isotopic exchange.

"It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% for both stages (24 hpf and 96 hpf)." Should be read as "It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% and 14% at 24 hpf and 96 hphf respectively".

Chronic toxicity of uranium to three benthic organisms in laboratory spiked sediment.

Due to mining activities, concentration of uranium (U) in the environment nearby former and operating sites can be higher than in other areas. The derivation of quality criteria for U in freshwater ecosystems, rivers and lakes includes the consideration of contaminated sediments and the associated risk to the benthic life. Therefore, the derivation of a quality criteria for sediment has been viewed as a logical and necessary extension of the work already done to establish water quality criteria. In order to contribute to the determination of a Quality Standard for sediment (QSsediment) according to the European recommendations, this study focuses on the acquisition of a new toxicity dataset, to enrich the few rare existing data, most often unsuitable. A basic set of organisms, including three complementary benthic organisms (Chironomus riparius, Hyalella azteca, Myriophyllum aquaticum), was chronically exposed to U spiked to a standard laboratory-formulated sediment, according to the related bioassay guidelines (ISO/FDIS16303, OECD 218/9, ISO/DIS 16191). We looked to determine when possible both NOEC and EC10 values for each organism. For C. riparius, a NOEC (emergence rate) value was estimated at 62 mgU, kg-1, dm and the EC10 value reached 188 mgU, kg-1, dm (CI95% 40-885 mgU kg-1, dm). For H. azteca, a NOEC (survival rate) value of 40 mgU kg-1, dm was observed while the EC10 value at 296 mgU kg-1, dm (CI95% = 155-436 mgU kg-1, dm) was slightly higher than for growth at 199 mgU kg-1, dm (CI95% = 107-291 mgU kg-1 dm). Finally, the less sensitive organism seemed to be the plant, M. aquaticum, for which we determined a NOEC value of 100 mgU kg-1, dm. Results obtained regarding the toxicity of U made it possible to suggest a preliminary QSsediment value of 4 mgU kg-1, dry mass. This value was shown conservative compared to U sediment quality criteria derived by other jurisdictions.

Disruption of AtHAK/KT/KUP9 enhances plant cesium accumulation under low potassium supply.

Understanding the molecular mechanisms that underlie cesium (Cs+ ) transport in plants is important to limit the entry of its radioisotopes from contaminated areas into the food chain. The potentially toxic element Cs+ , which is not involved in any biological process, is chemically closed to the macronutrient potassium (K+ ). Among the multiple K+ carriers, the high-affinity K+ transporters family HAK/KT/KUP is thought to be relevant in mediating opportunistic Cs+ transport. Of the 13 KUP identified in A. thaliana, only HAK5, the major contributor to root K+ acquisition under low K+ supply, has been functionally demonstrated to be involved in Cs+ uptake in planta. In the present study, we showed that accumulation of Cs+ increased by up to 30% in two A. thaliana mutant lines lacking KUP9 and grown under low K+ supply. Since further experiments revealed that Cs+ release from contaminated plants to the external medium is proportionally lower in the two kup9 mutant alleles, we proposed that KUP9 disruption could impair Cs+ efflux. By contrast, K+ status in kup9 mutants is not affected, suggesting that KUP9 disruption does not alter substantially K+ transport in experimental conditions used. The putative primary role of KUP9 in plants is further discussed.

Effects of gamma ionizing radiation exposure on Danio rerio embryo-larval stages - comparison with tritium exposure.

The objective was to investigate the effects of ionizing radiation induced in zebrafish early life stages by coupling responses obtained at the molecular (genotoxicity, ROS production, gene expression) and phenotypic (tissue alteration, embryo-larval development) levels. Here we present results obtained after exposure of 3 hpf larvae to 10 days of gamma irradiation at 3.3 × 101, 1.3 × 102 and 1.2 × 103 µGy/h, close to and higher than the benchmark for protection of ecosystems towards ionizing radiations of 101 µGy/h. Dose rates used in these studies were chosen to be in the 'derived consideration reference level' (DCRL) for gamma irradiation where deleterious effects can appear in freshwater fish. Also, these dose rates were similar to the ones already tested on tritium (beta ionizing radiation) in our previous work, in order to compare both types of ionizing radiation. Results showed that gamma irradiation did not induce any effect on survival and hatching. No effect was observed on DNA damages, but ROS production was increased. Muscle damages were observed for all tested dose rates, similarly to previous results obtained with tritium (beta ionizing radiation) at similar dose rates. Some molecular responses therefore appeared to be relevant for the study of gamma ionizing radiation effects in zebrafish.

Dose-dependent genomic DNA hypermethylation and mitochondrial DNA damage in Japanese tree frogs sampled in the Fukushima Daiichi area.

The long-term consequences of the nuclear disaster at the Fukushima Daiichi Nuclear Power Plant (FDNPP) that occurred on March 2011, have been scarcely studied on wildlife. We sampled Japanese tree frogs (Dryophytes japonicus), in a 50 -km area around the FDNPP to test for an increase of DNA damages and variation of DNA methylation level. The ambient dose rate ranged between 0.4 and 2.8 µGy h-1 and the total estimated dose rate absorbed by frogs ranged between 0.3 and 7.7 µGy h-1. Frogs from contaminated sites exhibited a dose-dependent increase of global genomic DNA methylation level (5-mdC and 5-hmdC) and of mitochondrial DNA damages. Such DNA damages may indicate a genomic instability, which may induce physiological adaptations governed by DNA methylation changes. This study stresses the need for biological data combining targeted molecular methods and classic ecotoxicology, in order to better understand the impacts on wildlife of long term exposure to low ionizing radiation levels.

Effects of tritiated water on locomotion of zebrafish larvae: a new insight in tritium toxic effects on a vertebrate model species.

Tritium (3H), a radioactive isotope of hydrogen, is ubiquitously present in the environment. In a previous study, we highlighted a mis-regulation of genes involved in muscle contraction, eye transparency and response to DNA damages after exposure of zebrafish embryo-larvae from 3 hpf to 96 hpf at 0.4 and 4 mGy/h of tritiated water (HTO). The present study aimed to link this gene mis-regulation to responses observed at higher biological levels. Analyses on spontaneous tail movement, locomotor activity and heart rate were performed. Histological sections of eyes were made to evaluate the impact of HTO on eye transparency and whole embryo immunostainings were realized to assess DNA double strand breaks repair using gamma-H2AX foci. We found a decrease of basal velocity as well as a decrease of response in 96 hpf larvae exposed at 0.4 mGy/h after a tactile stimulus as compared to controls. Histological sections of larvae eyes performed after the exposure to 4 mGy/h did not show obvious differences in lens transparency or retinal development between contaminated and control organisms. Gamma-H2AX foci detection revealed no differences in the number of foci between contaminated organisms and controls, for both dose rates. Overall, results highlighted more detrimental effects of HTO exposure on locomotor behavior in 96 hpf larvae exposed at the lowest dose rate. Those results could be linked to mis-regulation of genes involved in muscle contraction found in a previous study at the same dose rate. It appears that not all effects found at the molecular scale were confirmed using higher biological scales. These results could be due to a delay between gene expression modulation and the onset of physiological disruption or homeostatic mechanisms to deal with tritium effects. However, crossing data from different scales highlighted new pathways to explore, i.e. neurotoxic pathways, for better understanding HTO effects on organisms.

Tritiated Water Exposure in Zebrafish (Danio rerio): Effects on the Early-Life Stages.

Tritium, a radioactive isotope of hydrogen of natural and anthropogenic origin, is ubiquitously present in the environment. Effluents of nuclear centers of production are significant anthropogenic sources. With the upcoming project of thermonuclear fusion, tritium releases in the environment may increase. It is therefore important to characterize the ecological risk linked to tritium. The effects of tritiated water (HTO) were therefore studied in zebrafish larvae exposed for 10 d to different dose rates, 1.1 × 102 , 4.1 × 102 , and 3.8 × 103 µGy/h for larvae corresponding, respectively, to a water contamination of 104 , 105 , and 106 Bq/mL of HTO. Those dose rates were higher than 10 µGy/h, which is the threshold recommended to start monitoring ecosystems where radiological contaminants are present. Mortality, embryo-larval development, immune toxicity, genotoxicity, neurotoxicity, and alterations of tissues were investigated. The results showed that HTO exposure induced DNA damage and reactive oxygen species production and modulated the expression of genes involved in detoxification processes. Moreover, modifications of the muscular tissues (degradation of myofibrils at 4 d post fertilization and disorganization of mitochondria at later stages) were observed. The results differed with HTO dose rates and with developmental stages. These results will drive future research for the development of new HTO-sensitive biomarkers and will allow us to progress in the characterization of the modes of action of tritium in fish. Environ Toxicol Chem 2020;39:648-658. © 2019 SETAC.

Adverse effects induced by chronic gamma irradiation in progeny of adult fish not affecting parental reproductive performance.

Multigenerational studies have become of great interest in ecotoxicology since the consequence of parental exposure to contaminants on offspring generations was established in situ or in laboratory conditions. The present study mainly examined the chronic effects of external Cs-137 gamma irradiation exposure at 4 dose rates (control, 0.5, 5, and 50 mGy h-1 ) on adult zebrafish (F0) exposed for 10 d and their progeny (F1) exposed or unexposed for 4 to 5 d. The main endpoints investigated included parental reproductive performance, embryo-larval survival, DNA alterations, and reactive oxygen species (ROS) production in F0 and F1. No effects on reproductive success, fecundity, or egg fertilization rate were observed. However, drastic effects were observed on F1 exposed to 50 mGy h-1 , resulting in a mortality rate of 100%. The drastic effects were also observed when the progeny was not irradiated. It was demonstrated that the sensitivity of the embryos was mainly attributable to parental irradiation. Moreover, these drastic effects induced by adult irradiation disappeared over time when 10 d-irradiated adults were placed in a nonirradiated condition. Alterations in larval DNA were observed for the 3 dose rates, and an increase of ROS production was also shown for the 2 lowest dose rates. The present study improves our understanding of the consequences of parental exposure conditions to the progeny. Furthermore, it provides an incentive to take transmitted generational effects into account in ecological risk assessments. Environ Toxicol Chem 2019;38:2556-2567. © 2019 SETAC.

Assessing tritium internalisation in zebrafish early life stages: Importance of rapid isotopic exchange.

Tritium (3H) is mainly released into the environment in the form of tritiated water (HTO) by nuclear power plants and nuclear fuel reprocessing plants. To better understand how organisms may be affected by contamination to 3H it is essential to link observed effects to a correct estimation of absorbed dose rates. Due to quick isotopic exchanges between 3H and hydrogen, 3H measurement is difficult in small organisms such as zebrafish embryo, a model in ecotoxicological assay. This work aimed to optimise tritium measurement protocol to better characterise internalisation by early life stages of zebrafish. Zebrafish eggs were exposed at one HTO activity concentration of 1.22 × 105 Bq/mL. This activity was calculated to correspond to theoretical dose rates of 0.4 mGy/h, where some deleterious effects are expected on young fish. A protocol for the preparation of biological samples was adapted from the method classically used to segregate the different forms of tritium in organisms. To deal with very quick isotopic exchanges of 3H with hydrogen, the impacts of washing by non-tritiated water as well as the bias induced by absorbed tritium around organisms on the measured activity concentration were studied. We managed to develop protocols to perform total tritium and total organically bound tritium (OBT) activity concentrations measurements in zebrafish eggs and larvae. The measurement of these both forms allowed the calculation of tissue-free-water-tritium (TFWT). To better understand total tritium internalisation, a study of total tritium kinetics from 4 hpf (hour post-fertilization) to 168 hpf was performed. OBT and TFWT were also assessed to complete the total tritium internalisation kinetics. The internalisation is a rapid phenomenon reaching a steady-state within 24 h after the beginning of contamination for total tritium and TFWT, with concentration factors and TFWT/HTO close to unity. OBT formation seemed to be slower. It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% for both stages (24 hpf and 96 hpf). To verify absorbed dose rates at key developmental stages (24 hpf eggs and 96 hpf larvae), they were calculated from total tritium activity concentrations after exposure at 1.22 × 105 and 1.22 × 106 Bq/mL, as these two activity concentrations were used to assess effects of tritium in another part of the study. Dose rates calculated from total tritium activity concentrations measured in 24 hpf eggs and 96 hpf larvae were consistent with the nominal ones, which validates the robustness of the protocol developed in the present study.

Uranium transfer and accumulation in organs of Danio rerio after waterborne exposure alone or combined with diet-borne exposure.

Uranium (U) toxicity patterns for fish have been mainly determined under laboratory-controlled waterborne exposure conditions. Because fish can take up metals from water and diet under in situ exposure conditions, a waterborne U exposure experiment (20 µg L-1 , 20 d) was conducted in the laboratory to investigate transfer efficiency and target organ distribution in zebrafish Danio rerio compared with combined waterborne exposure (20 µg L-1 ) and diet-borne exposure (10.7 µg g-1 ). 233 Uranium was used as a specific U isotope tracer for diet-borne exposure. Bioaccumulation was examined in the gills, liver, kidneys, intestine, and gonads of D. rerio. Concentrations in the organs after waterborne exposure were approximately 500 ng g-1 fresh weight, except in the intestine (> 10 µg g-1 fresh wt) and the kidneys (200 ng g-1 fresh wt). No significant difference was observed between waterborne and diet-borne conditions. Trophic U transfer in organs was found but at a low level (< 10 ng g-1 fresh wt). Surprisingly, the intestine appeared to be the main target organ after both tested exposure modalities. The gonads (57% at 20 d) and the liver (41% at 20 d) showed the highest accumulated relative U burdens. Environ Toxicol Chem 2019;38:90-98. © 2018 SETAC.

Carotenoid distribution in wild Japanese tree frogs (Hyla japonica) exposed to ionizing radiation in Fukushima.

The nuclear accident in the Fukushima prefecture released a large amount of artificial radionuclides that might have short- and long-term biological effects on wildlife. Ionizing radiation can be a harmful source of reactive oxygen species, and previous studies have already shown reduced fitness effects in exposed animals in Chernobyl. Due to their potential health benefits, carotenoid pigments might be used by animals to limit detrimental effects of ionizing radiation exposure. Here, we examined concentrations of carotenoids in blood (i.e. a snapshot of levels in circulation), liver (endogenous carotenoid reserves), and the vocal sac skin (sexual signal) in relation to the total radiation dose rates absorbed by individual (TDR from 0.2 to 34 µGy/h) Japanese tree frogs (Hyla japonica). We found high within-site variability of TDRs, but no significant effects of the TDR on tissue carotenoid levels, suggesting that carotenoid distribution in amphibians might be less sensitive to ionizing radiation exposure than in other organisms or that the potential deleterious effects of radiation exposure might be less significant or more difficult to detect in Fukushima than in Chernobyl due to, among other things, differences in the abundance and mixture of each radionuclide.

Tritiated water exposure disrupts myofibril structure and induces mis-regulation of eye opacity and DNA repair genes in zebrafish early life stages.

Tritium (3H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H2O2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae.

Influence of root exudation of white lupine (Lupinus albus L.) on uranium phytoavailability in a naturally uranium-rich soil.

Mechanisms of uranium (U) transfer from soil to plants remain poorly understood. The kinetics of supply of U to the soil solution from solid phases could be a key point to understand its phytoavailability and implications for environmental risk assessment. Root activity, particularly the continuous release of organic acids in the rhizosphere, could have an effect on this supply. We tested the impact of citrate exudation by roots of Lupinus albus, either P-sufficient (P+) or P-deficient (P-), on the phytoavailability of U from a naturally contaminated soil (total content of 413 mg U kg-1) using a rhizotest design. Combined effects of P (P-/P+ used to modulate plant physiology) and citrate (model exudate) on the solubilization of U contained in the soils were tested in closed reactors (batch). The batch experiment showed the existence of a low U available pool (0.4% total U) and high accessibility (kd' around 20 L kg-1) which was not significantly affected by P treatment or citrate concentrations. Analysis of U, Fe, Ca, P and citrate concentrations in the batches suggested a complex combination of mechanisms and factors including desorption, resorption, precipitation, co-sorption. On rhizotest, L. albus plants extracted 0.5-0.75% of the total U and between 25 and 40% of the estimated available U present in the rhizotest in 5 days. Uranium accumulation at the whole plant level (20 mg U kg-1d.w., shoot to root ratio around 10-3) seemed to be dependent neither on the plant P nutrition status nor citrate exudation level, possibly in relation with the equivalent accessibility of U whatever the growth conditions. Yet differential translocation to shoots seemed to be positively correlated to citrate exudation.

Toxicokinetic and toxicodynamic of depleted uranium in the zebrafish, Danio rerio.

This study investigated the accumulation pattern and biological effects (genotoxicity and histopathology) to adult zebrafish (male and female) exposed to a nominal waterborne concentration of 20 µg L-1 of depleted uranium (DU) for 28 days followed by 27 days of depuration. Accumulation pattern showed that (i) DU accumulated in brain, (ii) levels in digestive tract were higher than those measured in gills and (iii) levels remained high in kidney, brain and ovary despite the 27 days of depuration period. Genotoxicity, assessed by comet assay, was significant not only during DU exposure, but also during depuration phase. Gonads, in particular the testes, were more sensitive than gills. The histology of gonads indicated severe biological damages in males. This study improved knowledge of ecotoxic profile of uranium, for which a large range of biological effects has already been demonstrated.