The impact of comet assay data normalization in human biomonitoring studies outcomes
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Esteves, Filipa↗
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Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing
Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.
3D spheroids↗
Bystander effects in radiation-induced genomic instability
Exposure of GM10115 hamster-human hybrid cells to X-rays can result in the induction of chromosomal instability in the progeny of surviving cells. This instability manifests as the dynamic production of novel sub-populations of cells with unique cytogenetic rearrangements involving the "marker" human chromosome. We have used the comet assay to investigate whether there was an elevated level of endogenous DNA breaks in chromosomally unstable clones that could provide a source for the chromosomal rearrangements and thus account for the persistent instability observed. Our results indicate no significant difference in comet tail measurement between non-irradiated and radiation-induced chromosomally unstable clones. Using two-color fluorescence in situ hybridization we also investigated whether recombinational events involving the interstitial telomere repeat-like sequences in GM10115 cells were involved at frequencies higher than random processes would otherwise predict. Nine of 11 clones demonstrated a significantly higher than expected involvement of these interstitial telomere repeat-like sequences at the recombination junction between the human and hamster chromosomes. Since elevated levels of endogenous breaks were not detected in unstable clones we propose that epigenetic or bystander effects (BSEs) lead to the activation of recombinational pathways that perpetuate the unstable phenotype. Specifically, we expand upon the hypothesis that radiation induces conditions and/or factors that stimulate the production of reactive oxygen species (ROS). These reactive intermediates then contribute to a chronic pro-oxidant environment that cycles over multiple generations, promoting chromosomal recombination and other phenotypes associated with genomic instability.
NASA Discipline Radiation Health↗
Genotoxicity evaluation using primary hepatocytes isolated from rhesus macaque ( Macaca mulatta )
Non-human primates (NHPs) have played a vital role in fundamental, pre-clinical, and translational studies because of their high physiological and genetic similarity to humans. We report a method to isolate primary hepatocytes from the livers of rhesus macaques (Macaca mulatta) after in situ whole liver perfusion. Isolated primary macaque hepatocytes (PMHs) were treated with various compounds known to have different pathways of genotoxicity/carcinogenicity and the resulting DNA damage was evaluated using the high-throughput CometChip assay. The comet data were quantified using benchmark dose (BMD) modeling and the BMD50 values for treatments of PMHs were compared with those generated from primary human hepatocytes (PHHs) in our previous study. The results showed that despite varying CYP450 enzyme activities, PMHs had the same sensitivity and specificity as PHHs in detecting four indirect-acting (i.e., requiring metabolic activation) and seven direct-acting genotoxicants/carcinogens, as well as five non-carcinogens that are negative or equivocal for genotoxicity in vivo. The BMD50 estimates and their confidence intervals revealed species differences for DNA damage potency, especially for direct-acting compounds. The present study provides a practical method for maximizing the use of animal tissues by isolating primary hepatocytes from NHPs. Our data support the use of PMHs as a reliable surrogate of PHHs for evaluating the genotoxic hazards of chemical substances for humans.
59 BASIC BIOLOGICAL SCIENCES↗
Mechanistic Evaluation of Black Cohosh Extract-Induced Genotoxicity in Human Cells
Abstract Black cohosh extract (BCE) is marketed to women as an alternative to hormone replacement therapy for alleviating menopausal symptoms. Previous studies by the National Toxicology Program revealed that BCE induced micronuclei (MN) and a nonregenerative macrocytic anemia in rats and mice, likely caused by disruption of the folate metabolism pathway. Additional work using TK6 cells showed that BCE induced aneugenicity by destabilizing microtubules. In the present study, BCE-induced MN were confirmed in TK6 and HepG2 cells. We then evaluated BCE-induced DNA damage using the comet assay at multiple time points (0.5–24 h). Following a 0.5-h exposure, BCE induced significant, concentration-dependent increases in %tail DNA in TK6 cells only. Although DNA damage decreased in TK6 cells over time, likely due to repair, small but statistically significant levels of DNA damage were observed after 2 and 4 h exposures to 250 µg/ml BCE. A G1/S arrest in TK6 cells exposed to 125 µg/ml BCE (24 h) was accompanied by apoptosis and increased expression of γH2A.X, p-Chk1, p-Chk2, p53, and p21. Conditioning TK6 cells to physiological levels of folic acid (120 nM) did not increase the sensitivity of cells to BCE-induced DNA damage. BCE did not alter global DNA methylation in TK6 and HepG2 cells cultured in standard medium. Our results suggest that BCE induces acute DNA strand breaks which are quickly repaired in TK6 cells, whereas DNA damage seen at 4 and 24 h may reflect apoptosis. The present study supports that BCE is genotoxic mainly by inducing MN with an aneugenic mode of action.
Seo, Ji-Eun↗
A mission design for the Halley comet rendezvous using Ion Drive
The Ion Drive propulsion system, a derivative of the old Solar Electric Propulsion (SEP) technology is considered adequate to perform all mission objectives of a proposed Halley's comet rendezvous (scheduled for launch in 1982) except one: control of thermal energy from the concentrating solar arrays. This problem can be solved, however, by adding a separable tail probe to the baseline system. The system consists of an Ion Propulsion Module (IPM) and a Mission Module (MM). Scientific objectives include a determination of the structure of the comet nucleus, an evaluation of nucleus evolution, an assay of the comet's atmosphere and ionosphere, and a study of the interaction between the comet and the interplanetary medium. Attention is given to the navigation parameters necessary for heliocentric transfer and post-rendezvous circumnavigation of the comet.
Boain, R. J.↗
Clinostat rotation induces apoptosis in luteal cells of the pregnant rat
Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.
NASA Discipline Cell Biology↗
Space exploration and the history of solar-system volatiles
The thermochemical history of volatile substances in all solar-system planets, satellites, and planetoids is discussed extensively. The volatiles are viewed as an interface between the abiotic and biotic worlds and as a key to the history of bodies of the solar system. A flowsheet of processes and states is exhibited. Differences in bulk volatiles distribution between the planetary bodies and between the interior, surface, and atmosphere of each body are considered, as well as sinks for volatiles in degassing. The volatiles-rich Jovian and Saturnian satellites, the effect of large-planet magnetosphere sweeps on nearby satellites, volatiles of asteroids and comets, and the crucial importance of seismic, gravity, and libration data are treated. A research program encompassing analysis of the elemental and isotopic composition of rare gas in atmospheres, assay of volatiles-containing phases in regoliths, and examination of present or past atmospheric escape/accretion processes is recommended.
Fanale, F. P.↗
Design of a High-Assay Low-Enriched Uranium Tri-Structural Isotropic Critical Experiment for Advanced Reactor Validation
High-assay low-enriched uranium (HALEU) fuel is a key component of many small modular reactor designs. Critical experiments are an important way to understand the neutronic performance of systems by obtaining nuclear data validations through measurements. Data reduce uncertainty and risk by showing that systems respond as predicted to changes such as temperature, subsequently advancing the overall technology readiness level of the materials within. Numerous critical experiments have been performed at the National Criticality Experiments Research Center (NCERC) operated by Los Alamos National Laboratory at the Nevada National Security Site since it became operational in 2011. However, the first experiment with HALEU fuel did not occur until 2024. Through extensive engineering, the experiment described in this paper was successfully designed and executed for the Comet vertical lift assembly at NCERC to perform measurements with HALEU tri-structural isotropic fuel that will assist in validation of nuclear data and computational modeling of small modular reactors for years to come.
73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗