Search NASA⌕ Search

SEARCH · Search NASA

Results for “genotoxicity”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Control of transcription elongation and DNA repair by alarmone ppGpp

Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Gene Expression Thresholds Derived From Short-term Exposures Identify Rat Liver Tumorigens

Abstract Traditional methods for cancer risk assessment are resource-intensive, retrospective, and not feasible for the vast majority of environmental chemicals. In this study, we investigated whether quantitative genomic data from short-term studies may be used to set protective thresholds for potential tumorigenic effects. We hypothesized that gene expression biomarkers measuring activation of the key early events in established pathways for rodent liver cancer exhibit cross-chemical thresholds for tumorigenesis predictive for liver cancer risk. We defined biomarker thresholds for 6 major liver cancer pathways using training sets of chemicals with short-term genomic data (3–29 days of exposure) from the TG-GATES (n = 77 chemicals) and DrugMatrix (n = 86 chemicals) databases and then tested these thresholds within and between datasets. The 6 pathway biomarkers represented genotoxicity, cytotoxicity, and activation of xenobiotic, steroid, and lipid receptors (aryl hydrocarbon receptor, constitutive activated receptor, estrogen receptor, and peroxisome proliferator-activated receptor α). Thresholds were calculated as the maximum values derived from exposures without detectable liver tumor outcomes. We identified clear response values that were consistent across training and test sets. Thresholds derived from the TG-GATES training set were highly predictive (97%) in a test set of independent chemicals, whereas thresholds derived from the DrugMatrix study were 96%–97% predictive for the TG-GATES study. Threshold values derived from an abridged gene list (2/biomarker) also exhibited high predictive accuracy (91%–94%). These findings support the idea that early genomic changes can be used to establish threshold estimates or “molecular tipping points” that are predictive of later-life health outcomes.

Hill, III,, Thomas↗
DOE OSTI2025

DNA adducts form in mouse lung and liver after oral naphthalene exposure

Naphthalene is a ubiquitous combustion product and environmental contaminant with known human exposure. Chronic exposure to naphthalene vapor leads to respiratory tumor formation in rodents. Naphthalene forms DNA adducts (precursors to genotoxicity) in tissue explants but it is unclear if this occurs in vivo. Wild-type C57BL/6 mice were orally exposed to 50 mg/kg 14 C-naphthalene. Naphthalene-DNA adducts were detected by accelerator mass spectrometry at 2- to 72-h post-exposure in both lung and liver, with decreasing abundance over time. Adducts persisted even at 72-h after exposure, which indicates possible evasion of DNA repair and potential to contribute to mutagenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin

Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS–PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS–PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein–protein interactions supports a predicted molecular basis for enzyme–carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii , an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3′−5′ exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5′−3′ exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identifiedidentifiedidentifiedin the archaeon Haloferax volcanii , which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3′−5′ exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5′−3′ exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana

Background: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. Results: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. Conclusions: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Roundup causes embryonic development failure and alters metabolic pathways and gut microbiota functionality in non-target species

Background: Research around the weedkiller Roundup is among the most contentious of the twenty-first century. Scientists have provided inconclusive evidence that the weedkiller causes cancer and other life-threatening diseases, while industry-paid research reports that the weedkiller has no adverse effect on humans or animals. Much of the controversial evidence on Roundup is rooted in the approach used to determine safe use of chemicals, defined by outdated toxicity tests. We apply a system biology approach to the biomedical and ecological model species Daphnia to quantify the impact of glyphosate and of its commercial formula, Roundup, on fitness, genome-wide transcription and gut microbiota, taking full advantage of clonal reproduction in Daphnia. We then apply machine learning-based statistical analysis to identify and prioritize correlations between genome-wide transcriptional and microbiota changes. Results: We demonstrate that chronic exposure to ecologically relevant concentrations of glyphosate and Roundup at the approved regulatory threshold for drinking water in the US induce embryonic developmental failure, induce significant DNA damage (genotoxicity), and interfere with signaling. Furthermore, chronic exposure to the weedkiller alters the gut microbiota functionality and composition interfering with carbon and fat metabolism, as well as homeostasis. Using the “Reactome,” we identify conserved pathways across the Tree of Life, which are potential targets for Roundup in other species, including liver metabolism, inflammation pathways, and collagen degradation, responsible for the repair of wounds and tissue remodeling. Conclusions: Our results show that chronic exposure to concentrations of Roundup and glyphosate at the approved regulatory threshold for drinking water causes embryonic development failure and alteration of key metabolic functions via direct effect on the host molecular processes and indirect effect on the gut microbiota. The ecological model species Daphnia occupies a central position in the food web of aquatic ecosystems, being the preferred food of small vertebrates and invertebrates as well as a grazer of algae and bacteria. The impact of the weedkiller on this keystone species has cascading effects on aquatic food webs, affecting their ability to deliver critical ecosystem services.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1993

Toxicity study of dimethylethoxysilane (DMSES), the waterproofing agent for the Orbiter heat protective system

DMES, a volatile liquid, is used by NASA to waterproof the Orbiter thermal protective system. During waterproofing operations at the Oribter Processing Facility at KSC, workers could be exposed to DMES vapor. To assess the toxicity of DMES, acute and subchronic (2-week and 13-week) inhalation studies were conducted with rats. Studies were also conducted to assess the potential of DMES. Inhalation exposure concentrations ranged from 40 ppm to 4000 ppm. No mortality was observed during the studies. Exposures to 2100 ppm produced narcosis and ataxia. Post-exposure recovery from these CNS effects was rapid (less than 1 hr). These effects were concentration-dependent and relatively independent of exposure length. Exposure to 3000 ppm for 2 weeks (5 h/d, 5 d/wk) produced testicular toxicity. The 13-week study yielded similar results. Results from the genotoxicity assays (in vivo/in vitro unscheduled DNA synthesis in rat primary heptaocytes, chromosomal aberrations in rat bone marrow cells; reverse gene mutation in Salmonella typhimurium; and forward mutation in Chinese hamster culture cells) were negative. These studies indicated that DMES is mildly to moderately toxic but not a multagen.

Lam, Chiu-Wing↗
NASA NTRS1999

A Novel Technique for Performing Space Based Radiation Dosimetry Using DNA: Results from GRaDEx-I and the Design of GRaDEx-II

Because of the large amounts of cosmic radiation in the space environment relative to that on earth, the effects of radiation on the physiology of astronauts is of major concern. Doses of radiation which can cause acute or chronic biological effects are to be avoided, therefore determination of the amount of radiation exposure encountered during space flight and assessment of its impact on biological systems is critical. Quantifying the radiation dosage and damage to biological systems, especially to humans during repetitive high altitude flight and during long duration space flight is important for several reasons. Radiation can cause altered biosynthesis and long term genotoxicity resulting in cancer and birth defects, etc. Radiation damage to biological systems depends in a complex way on incident radiation species and their energy spectra. Typically non-biological, i.e. film or electronic monitoring systems with narrow energy band sensitivity are used to perform dosimetry and then results are extrapolated to biological models. For this reason it may be desirable to perform radiation dosimetry by using biological molecules e.g. DNA or RNA strands as passive sensors. A lightweight genotoxicology experiment was constructed to determine the degree to which in-vitro naked DNA extracted from tissues of a variety of vertebrate organisms is damaged by exposure to radiation in a space environment. The DNA is assayed by means of agarose gel electrophoresis to determine damage such as strand breakage caused by high momentum particles and photons, and base oxidation caused by free radicals. The length distribution of DNA fragments is directly correlated with the radiation dose. It is hoped that a low mass, low cost, passive biological system to determine dose-response relationship (increase in strand breaks with increase in exposure) can be developed to perform radiation dosimetry in support of long duration space flight, and to predict negative effects on biological systems (e.g. astronauts and greenhouses) in space. The payload was flown in a 2.5 cubic foot Get Away Special (GAS) container through NASA's GAS program. It was subjected to the environment of the space shuttle cargo bay for the duration of the STS-91 mission (9 days). Results of the genotoxicology and radiation dosimetry experiment (GRaDEx-I) as well as the design of an improved follow on payload are presented.

Ritter, Joe↗
NASA NTRS1999

A Novel Technique for Performing Space Based Radiation Dosimetry Using DNA-Results from GRaDEx-I and the Design of GRaDEx-II

Because of the large amounts of cosmic radiation in the space environment relative to that on earth, the effects of radiation on the physiology of astronauts is of major concern. Doses of radiation which can cause acute or chronic biological effects are to be avoided, therefore determination of the amount of radiation exposure encountered during space flight and assessment of its impact on biological systems is critical. Quantifying the radiation dosage and damage to biological systems, especially to humans during repetitive high altitude flight and during long duration space flight is important for several reasons. Radiation can cause altered biosynthesis and long term genotoxicity resulting in cancer and birth defects etc. Radiation damage to biological systems depends in a complex way on incident radiation species and their energy spectra. Typically non-biological, i.e. film or electronic monitoring systems with narrow energy band sensitivity are used to perform dosimetry and then results are extrapolated to biological models. For this reason it may be desirable to perform radiation dosimetry by using biological molecules e.g. DNA or RNA strands as passive sensors. A lightweight genotoxicology experiment was constructed to determine the degree to which in vitro naked DNA extracted from tissues of a variety of vertebrate organisms is damaged by exposure to radiation in a space environment. The DNA is assayed by means of agarose gel electrophoresis to determine damage such as strand breakage caused by high momentum particles and photons, and base oxidation caused by free radicals. The length distribution of DNA fragments is directly correlated with the radiation dose. It is hoped that a low mass, low cost, passive biological system to determine dose response relationship (increase in strand breaks with increase in exposure) can be developed to perform radiation dosimetry in support of long duration space flight, and to predict negative effects on biological systems (e.g. astronauts and greenhouses) in space. The payload was flown in a 2.5 cubic foot Get Away Special (GAS) container through NASA's GAS program. It was subjected to the environment of the space shuttle cargo bay for the duration of the STS-91 mission (9 days). Results of the genotoxicology and radiation dosimetry experiment (GRaDEx-1) as well as the design of an improved follow on payload are presented.

Ritter, Joe↗
NASA NTRS2004

Ionization Cross Sections and Dissociation Channels of the DNA Sugar-Phosphate Backbone by Electron Collisions

It has been suggested that the genotoxic effects of ionizing radiation in living cells are not caused by the highly energetic incident radiation, but rather are induced by less energetic secondary species generated, the most abundant of which are free electrons.' The secondary electrons will further react to cause DNA damage via indirect and direct mechanisms. Detailed knowledge of these mechanisms is ultimately important for the development of global models of cellular radiation damage. We are studying one possible mechanism for the formation cf DNA strand breaks involving dissociative ionization of the DNA sugar-phosphate backbone induced by secondary electron co!lisions. We will present ionization cross sections at electron collision energies between threshold and 10 KeV using the improved binary encounter dipole (iBED) formulation' Preliminary results of the possible dissociative ionization pathways will be presented. It is speculated that radical fragments produced from the dissociative ionization can further react, providing a possible mechanism for double strand breaks and base damage.

Dateo, Christopher↗
NASA NTRS2002

Radiation-induced gene expression in the nematode Caenorhabditis elegans

We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by RT-PCR differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density.

Non-NASA Center↗
NASA NTRS2003

Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in mice

The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51. Copyright 2003 Wiley-Liss, Inc.

Non-NASA Center↗
NASA NTRS2002

An ATM-independent S-phase checkpoint response involves CHK1 pathway

After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

Non-NASA Center↗
NASA NTRS1988

Micronucleation in the lens epithelium following in vivo exposure to physical and chemical mutagens

Rats were exposed to cataractogenic doses of known physical and chemical genotoxic agents in order to study the efficacy of using micronuclei to monitor mutagenicity in the lens epithelium. The total numbers of micronuclei were counted in lens epithelia from rats exposed to graded doses of either 250 kVp X-rays or the anti-leukemic drug, 1,4 dimethanesulfonoxybutane (Myleran (R)). The results indicate a dose-dependent incidence of micronucleation in the lens epithelium following exposure. The findings are consistent with the hypothesis that the cataractogenicity of certain agents may be related to their effect on the genome of lens epithelial cells.

NASA Discipline Number 22-70↗
NASA NTRS1998

Heavy-ion radiobiology: new approaches to delineate mechanisms underlying enhanced biological effectiveness

Shortly after the discovery of polonium and radium by Marie Curie and her husband and colleague, Pierre Curie, it was learned that exposure to these alpha-particle emitters produced deleterious biological effects. The mechanisms underlying the increased biological effectiveness of densely ionizing radiations, including alpha particles, neutrons and highly energetic heavy charged particles, remain an active area of investigation. In this paper, we review recent advances in several areas of the radiobiology of these densely ionizing radiations, also known as heavy ions. Advances are described in the areas of DNA damage and repair, chromosome aberrations, mutagenesis, neoplastic transformation in vitro, genomic instability, normal tissue radiobiology and carcinogenesis in vivo. We focus on technical innovations, including novel applications of pulsed-field gel electrophoresis, fluorescence in situ hybridization (FISH), linkage analysis, and studies of gene expression and protein expression. We also highlight the use of new cellular and animal systems, including those with defined DNA repair deficiencies, as well as epithelial cell model systems to assess neoplastic transformation both in vitro and in vivo. The studies reviewed herein have had a substantial impact on our understanding of the genotoxic effects of heavy ions as well as their distinct effects on tissue homeostasis. The use of these radiations in cancer therapy is also discussed. The use of both heavy-ion and proton therapy is on the upswing in several centers around the world, due to their unique energy deposition characteristics that enhance the therapeutic effect and help reduce damage to normal tissue.

Review, Academic↗
NASA NTRS2005

Microfabricated Genomic Analysis System

Genetic sequencing and many genetic tests and assays require electrophoretic separation of DNA. In this technique, DNA fragments are separated by size as they migrate through a sieving gel under the influence of an applied electric field. In order to conduct these analyses on-orbit, it is essential to acquire the capability to efficiently perform electrophoresis in a microgravity environment. Conventional bench top electrophoresis equipment is large and cumbersome and does not lead itself to on-orbit utilization. Much of the previous research regarding on-orbit electrophoresis involved altering conventional electrophoresis equipment for bioprocessing, purification, and/or separation technology applications. A new and more efficient approach to on-orbit electrophoresis is the use of a microfabricated electrophoresis platform. These platforms are much smaller, less expensive to produce and operate, use less power, require smaller sample sizes (nanoliters), and achieve separation in a much shorter distance (a few centimeters instead of 10 s or 100 s of centimeters.) In contrast to previous applications, this platform would be utilized as an analytical tool for life science/medical research, environmental monitoring, and medical diagnoses. Identification of infectious agents as well as radiation related damage are significant to NASA s efforts to maintain, study, and monitor crew health during and in support of near-Earth and interplanetary missions. The capability to perform genetic assays on-orbit is imperative to conduct relevant and insightful biological and medical research, as well as continuing NASA s search for life elsewhere. This technology would provide an essential analytical tool for research conducted in a microgravity environment (Shuttle, ISS, long duration/interplanetary missions.) In addition, this technology could serve as a critical and invaluable component of a biosentinel system to monitor space environment genotoxic insults to include radiation.

Gonda, Steve↗
NASA NTRS2002

Development of Three-Dimensional Multicellular Tissue-Like Constructs for Mutational Analysis Using Macroporous Microcarriers

A three-dimensional (3-D), tissue-like model was developed for the genotoxic assessment of space environment. In previous experiments, we found that culturing mammalian cells in a NASA-designed bioreactor, using Cytodex-3 beads as a scaffold, generated 3-D multicellular spheroids. In an effort to generate scaffold-free spheroids, we developed a new 3-D tissue-like model by coculturing fibroblast and epithelial cell in a NASA bioreactor using macroporous Cultispher-S(TradeMark) microcarriers. Big Blue(Registered Trademark) Rat 2(Lambda) fibroblasts, genetically engineered to contain multiple copies (>60 copies/cell) of the Lac I target gene, were cocultured with radio-sensitive human epithelial cells, H184F5. Over an 8-day period, samples were periodically examined by microscopy and histology to confirm cell attachment, growth, and viability. Immunohistochemistry and western analysis were used to evaluate the expression of specific cytoskeletal and adhesion proteins. Key cell culture parameters (glucose, pH, and lactate concentrations) were monitored daily. Controls were two-dimensional mono layers of fibroblast or epithelial cells cultured in T-flasks. Analysis of 3-D spheroids from the bioreactor suggests fibroblast cells attached to and completely covered the bead surface and inner channels by day 3 in the bioreactor. Treatment of the 3-day spheroids with dispase II dissolved the Cultisphers(TradeMark) and produced multicellular, bead-less constructs. Immunohistochemistry confirmed the presence of vi.mentin, cytokeratin and E-cadherin in treated spheroids. Examination of the dispase II treated spheroids with transmission electron microscopy (TEM) also showed the presence of desmosomes. These results suggest that the controlled enzymatic degradation of an artificial matrix in the low shear environment of the NASA-designed bioreactor can produce 3-D tissue-like spheroids. 2

Jordan, Jacqueline A.↗