Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept.

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.

Clean feed as countermeasure to reduce the 90Sr and 137Cs levels in fish from contaminated lakes.

Glubokoye Lake situated within the Chernobyl Exclusion Zone is highly contaminated with respect to radioactive caesium and strontium isotopes, which also is reflected in the contaminated fish. To utilize the fish resources in contaminated lakes, the present work presents for the first time the effectiveness of using clean feed to counteract contamination of radionuclides in fish. The study is based on a series of repeated experiments with Prussian carp (Carassius gibelio (Bloch, 1782)) kept in cages in the contaminated Glubokoye Lake during summer 2018-2021. By the addition of clean feed, the activity concentration of 137Cs in fish muscle tissues was lowered with a factor of 2-5 due to biodilution. Surprisingly, additional clean feed did not lead to further decrease in the uptake of 137Cs in fish. In contrast to 137Cs, the addition of clean feed increased the 90Sr activity concentration in fish by a factor of 2-4 compared to fish fed with naturally occurring feed items. Radioactive strontium accumulated mainly in the fish bones and the muscle tissue level was 2 orders of magnitude lower, similar to the distribution observed for stable Sr. By utilizing a new kinetic model describing the dynamics of strontium isotopes in bone tissues of fish, predictions fitted well with site-specific data, taking growth rates and aging into account. Results showed that clean feeding can be used to counteract high activity concentration of 137Cs in fish due to biodilution, but cannot counteract bioaccumulation of 90Sr. Findings highlighted that it is essential to understand underlying factors influencing the uptake pathways for contaminants, as access to clean feed could increase the growth and thereby reduce the body activity concentration of dietary associated radionuclides such as 137Cs (biodilution), as well as increase the transfer of dissolved compounds such as 90Sr directly from water to fish.

In vitro cellular and proteome assays identify Wnt pathway and CDKN2A-regulated senescence affected in mesenchymal stem cells from mice after a chronic LD gamma irradiation in utero.

Reliable data on the effects of chronic prenatal exposure to low dose (LD) of ionizing radiation in humans are missing. There are concerns about adverse long-term effects that may persist throughout postnatal life of the offspring. Due to their slow cell cycle kinetics and life-long residence time in the organism, mesenchymal stem cells (MSCs) are more susceptible to low level genotoxic stress caused by extrinsic multiple LD events. The aim of this study was to investigate the effect of chronic, prenatal LD gamma irradiation to the biology of MSCs later in life. C3H mice were exposed in utero to chronic prenatal irradiation of 10 mGy/day over a period of 3 weeks. Two years later, MSCs were isolated from the bone marrow and analyzed in vitro for their radiosensitivity, for cellular senescence and for DNA double-strand break recognition after a second acute gamma-irradiation. In addition to these cellular assays, changes in protein expression were measured using HPLC-MS/MS and dysregulated molecular signaling pathways identified using bioinformatics. We observed radiation-induced proteomic changes in MSCs from the offspring of in utero irradiated mice (leading to ~ 9.4% of all detected proteins being either up- or downregulated) as compared to non-irradiated controls. The proteomic changes map to regulation pathways involved in the extracellular matrix, the response to oxidative stress, and the Wnt signaling pathway. In addition, chronic prenatal LD irradiation lead to an increased rate of in vitro radiation-induced senescence later in life and to an increased number of residual DNA double-strand breaks after 4 Gy irradiation, indicating a remarkable interaction of in vivo radiation in combination with a second acute dose of in vitro radiation. This study provides the first insight into a molecular mechanism of persistent MSC damage response by ionizing radiation exposure during prenatal time and will help to predict therapeutic safety and efficacy with respect to a clinical application of stem cells.

Seasonal changes in uptake and depuration of 137Cs and 90Sr in silver Prussian carp (Carassius gibelio) and common rudd (Scardinius erythrophthalmus).

Dynamic transfer of radionuclides to fish was studied in a series of experiments under field condition in two lakes within the Chernobyl exclusion zone during 2016-2020. "Clean" common rudd (Scardinius erythrophthalmus) and silver Prussian carp (Carassius gibelio) were transported to the contaminated Glubokoye Lake and kept in cages during several months of exposure, while contaminated Glubokoye fish were kept in cages in the "clean" Starukha Lake. Radiocaesium (137Cs) and radiostrontium (90Sr) were determined in intestine contents, muscle and bone tissues based on repeated samples during several months of exposure. During summer, the activity concentrations of 137Cs and 90Sr increased with time of exposure in clean fish caged in the contaminated lake. During autumn and winter, however, minor changes in fish uptake occurred during several weeks of exposure to the contaminated water. Furthermore, depuration in the contaminated fish was significant during summer, while insignificant during winter when exposed in the «clean¼ water. The rate constant of 137Cs uptake in muscle was between 8.0 and 22 day-1 during summer, while 0.2 to 1.0 day-1 during autumn-winter. Similarly, the rate constant of 90Sr uptake in bone was between 1.4 and 1.6 day-1, while 0.08-0.52 day-1 during autumn-winter. Biological half-lives of 137Cs in fish muscle tissue in summer were 77 ± 10 days, while exceeded 230 days during seasons at low water temperature. The results demonstrated that the transfer of 137Cs and 90Sr to fish was highly dependent upon seasons, in particular the water temperature. The transfer data obtained during low water temperature seasons deviated significantly from transfer data in literature and handbooks. Thus, seasonal changes in radionuclide transfer to fish should be taken into account when radiological impact to fish is assessed.

In vivo assessment of reactive oxygen species production and oxidative stress effects induced by chronic exposure to gamma radiation in Caenorhabditis elegans.

In the current study, effects of chronic exposure to ionizing gamma radiation were assessed in the radioresistant nematode Caenorhabditis elegans in order to understand whether antioxidant defences (AODs) could ameliorate radical formation, or if increased ROS levels would cause oxidative damage. This analysis was accompanied by phenotypical as well as molecular investigations, via assessment of reproductive capacity, somatic growth and RNA-seq analysis. The use of a fluorescent reporter strain (sod1::gfp) and two ratiometric biosensors (HyPer and Grx1-roGFP2) demonstrated increased ROS production (H2O2) and activation of AODs (SOD1 and Grx) in vivo. The data showed that at dose-rates ≤10 mGy h-1 defence mechanisms were able to prevent the manifestation of oxidative stress. In contrast, at dose-rates ≥40 mGy h-1 the continuous formation of radicals caused a redox shift, which lead to oxidative stress transcriptomic responses, including changes in mitochondrial functions, protein degradation, lipid metabolism and collagen synthesis. Moreover, genotoxic effects were among the most over-represented functions affected by chronic gamma irradiation, as indicated by differential regulation of genes involved in DNA damage, DNA repair, cell-cycle checkpoints, chromosome segregation and chromatin remodelling. Ultimately, the exposure to gamma radiation caused reprotoxic effects, with >20% reduction in the number of offspring per adult hermaphrodite at dose-rates ≥40 mGy h-1, accompanied by the down-regulation of more than 300 genes related to reproductive system, apoptosis, meiotic functions and gamete development and fertilization.

A call for action: Improve reporting of research studies to increase the scientific basis for regulatory decision-making.

This is a call for action to scientific journals to introduce reporting requirements for toxicity and ecotoxicity studies. Such reporting requirements will support the use of peer-reviewed research studies in regulatory decision-making. Moreover, this could improve the reliability and reproducibility of published studies in general and make better use of the resources spent in research.