Cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol (TFM) in tadpole cell lines from North American frogs.

The common field lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), is used to treat streams and creeks infested with highly invasive and destructive sea lamprey (Petromyzon marinus) in the tributaries of the Great Lakes. Unfortunately, amphibian deaths have been reported following stream treatments with TFM. Larval amphibians (tadpoles) are more susceptible to TFM toxicity than adult amphibians. The aim of this study was to test the toxicity of TFM in eight new tadpole cell lines from the green frog (Lithobates clamitans), wood frog (Lithobates sylvaticus), and American toad (Anaxyrus americanus). A cell viability bioassay using two fluorescent dyes, Alamar Blue and CFDA-AM, was performed following 24-h and 72-h exposures to a range of TFM concentrations. In general, TFM exposure reduced Alamar Blue fluorescence more rapidly than CFDA-AM fluorescence in tadpole cells, suggesting that Alamar Blue is perhaps a better diagnostic indicator of cell health for acute TFM cytotoxicity. At present, the in vivo 96-h LC50s of TFM are only available for L. clamitans and they correlated well with the in vitro EC50 values for the green frog tadpole cell lines in this study. The eight tadpole cell lines with different relative sensitivities to TFM cytotoxicity could prove to be useful tools in assessing next-generation lampricides in high-throughput bioassays to ensure safety in frogs before their sea lamprey-targeted application in the field.

Hypersensitive response to interferon-stimulated gene (ISG)-inducing double-stranded RNA in American bullfrog tadpole fibroblasts.

American bullfrogs are thought to be carriers of ranaviruses and contribute to their global spread via trade. Bullfrog tadpoles succumb to ranaviral infection's more severe and deadly effects than bullfrog adults. Presently, little is known about bullfrog tadpoles' innate antiviral immunity, possible due to the lack of available bullfrog tadpole cell lines. In this study, we describe a novel bullfrog tadpole fibroblast cell line named BullTad-leg. Its general cellular attributes, gene expression and function of class-A scavenger receptors (SR-As), and responses to poly IC (a synthetic dsRNA mimicking viral dsRNAs and a potent inducer of the interferon (IFN)-mediated antiviral responses) are investigated. Its abundant expression of vimentin corroborated with the cells' fibroblast morphology. BullTad-leg cells expressed transcripts of four SR-A members: SR-AI, SCARA3, SCARA4, and SCARA5, but transcripts of MARCO, the fifth SR-A member, were not detected. BullTad-leg cells expressed functional SR-As and could bind AcLDL. BullTad-leg cells exhibited cytotoxicity in response to poly IC treatment via SR-As. Additionally, very low doses of poly IC were able to induce dose-dependent expressions of ISGs including Mx, PKR, ISG20, and IFI35. This research sheds new light on the innate immune response, particularly SR-A biology and dsRNA responsiveness, in bullfrog tadpoles.

X-ray-induced bio-acoustic emissions from cultured cells.

PURPOSE: We characterize for the first time the emission of acoustic waves from cultured cells irradiated with X-ray photon radiation. METHODS AND MATERIALS: Human cancer cell lines (MCF-7, HL-60) and control cell-free media were exposed to 1 Gy X-ray photons while recording the sound generated before, during and after irradiation using custom large-bandwidth ultrasound transducer. The effects of dose rate and cell viability were investigated. RESULTS: We report the first recorded acoustic signals captured from a collective pressure wave response to ionizing irradiation in cell culture. The acoustic signal was co-terminous with the radiation pulse, its magnitude was dependent on radiation dose rate, and live and dead cells showed qualitatively and quantitatively different acoustic signal characteristics. The signature of the collective acoustic peaks was temporally wider and with higher acoustic power for irradiated HL-60 than for irradiated MCF-7. CONCLUSIONS: We show that X-ray irradiation induces two cultured cancer cell types to emit a characteristic acoustic signal for the duration of the radiation pulse. The rapid decay of the signal excludes acoustic emissions themselves from contributing to the inter-organism bystander signal previously reported in intact animals, but they remain a potential component of the bystander process in tissues and cell cultures. This preliminary study suggests that further work on the potential role of radiation-induced acoustic emission (RIAE) in the inter-cellular bystander effect is merited.

Environmental radiobiology of amphibians - knowledge gaps to be filled using cell lines.

Amphibians are facing an unprecedented level of population declines worldwide. The causes run the gamut from habitat loss and succumbing to opportunistic pathogen infections to vulnerability to toxic pollutants and ultraviolet (UV)-B radiation exposure. Anthropogenic activities including Chernobyl and Fukushima nuclear disasters and radioactive waste leakage into the environment raise the background radiation levels. Their immediate and chronic effects on amphibian populations are still being studied. However, the literature on environmental radiation effects on amphibian health still requires a lot more work. Laboratory and field works need to be conducted hand in hand in order to make informative and conclusive analyses to distinguish bad from good and harm from risk or to argue for or against the linear no-threshold model in radioprotection programs. Amphibian cell lines can help seek answers to important questions pertaining environmental radiobiology and amphibian health wherever they can suitably and effectively. The purpose of this work is to show that amphibian cell lines can 'rescue' important knowledge gaps in the literature, especially in the low-dose radiation mechanisms. Presently, there are 142 amphibian cell lines developed from six urodelans and 17 anurans. Amphibian cell lines can help expand and enrich the limited literature on environmental radiation effects on amphibians. They can be used to study mechanisms of radiation actions and discover reliable biomarkers for low-dose exposure. They can be used in environmental radiation monitoring and radioprotection programs. They can be used to determine the effects of co-exposure of IR and other stressors in the environment on amphibian health. They represent an ethical choice for amphibian conservation efforts in the current global amphibian declines. Lessons learned from cellular data can be useful guides to gain a better picture of effects occurring at the amphibian population and ecosystem levels.

The sine qua non of the fish invitrome today and tomorrow in environmental radiobiology.

Fish cell lines, collectively referred to as the fish invitrome, are useful diagnostic tools to study radiation impacts on aquatic health and elucidate radiation mechanisms in fish. This paper will highlight the advantages, discuss the challenges, and propose possible future directions for uses of the fish invitrome in the field of environmental radiobiology. The fish invitrome contains at least 714 fish cell lines. However, only a few of these cell lines have been used to study radiation biology in fish and they represent only 10 fish species. The fish invitrome is clearly not yet explored for its full potential in radiation biology. Evidence suggests that they are useful and, in some cases, irreplaceable in making underlying theories and fundamental concepts in radiation responses in fish. The debate of whether environmental radiation is harmful, presents risks, has no effect on health, or is beneficial is on-going and is one that fish cell lines can help address in a time-effective fashion. Any information obtained with fish cell lines is useful in the framework of environment radiation risk assessments. Radiation threats to aquatic health will continue due to the very likely rise of nuclear energy and medicine in the future. The fish invitrome, in theory, lives forever and can meet new challenges at any given time to provide diagnostic risk analyses pertaining to aquatic health and environmental radiation protection.

From tangled banks to toxic bunnies; a reflection on the issues involved in developing an ecosystem approach for environmental radiation protection.

The objective of this paper is to present the results of discussions at a workshop held as part of the International Congress of Radiation Research (Environmental Health stream) in Manchester UK, 2019. The main objective of the workshop was to provide a platform for radioecologists to engage with radiobiologists to address major questions around developing an Ecosystem approach in radioecology and radiation protection of the environment. The aim was to establish a critical framework to guide research that would permit integration of a pan-ecosystem approach into radiation protection guidelines and regulation for the environment. The conclusions were that the interaction between radioecologists and radiobiologists is useful in particular in addressing field versus laboratory issues where there are issues and challenges in designing good field experiments and a need to cross validate field data against laboratory data and vice versa. Other main conclusions were that there is a need to appreciate wider issues in ecology to design good approaches for an ecosystems approach in radioecology and that with the capture of 'Big Data', novel tools such as machine learning can now be applied to help with the complex issues involved in developing an ecosystem approach.

A pilot study of radiation-induced bystander effect in radio-adapting frogs at a radiologically contaminated site located on the chalk river laboratories property.

PURPOSE: To measure medium borne bystander effects, to study the influence of radioadaptive response (RAR) on bystander response, and to discover reliable radioresponsive biomarkers in radio-adapting frogs from Duke Swamp contaminated with an above-background radiation level and in naïve frogs from Twin Lake as the background control site. MATERIALS AND METHODS: Frogs were captured at Duke Swamp and Twin Lake and brought to the lab at the Canadian Nuclear Laboratories facility. Half of the frogs from each site were irradiated with 4 Gy while the other half of the frogs were left with no further radiation treatment. Frog bladders were removed and placed in sterile culture media. Upon arrival at McMaster University, the bladders were processed for tissue cultures. After 48 h, the culture media conditioned by the bladder explants were harvested for clonogenic reporter survival assay and calcium flux measurements for assessing bystander effects. HPV-G cells were used as bystander reporter cells in all radiation-induced bystander effect (RIBE) assays. The frog bladder cultures were incubated for another 10-12 days followed by immunochemical staining for bcl-2 and c-myc expressions to analyze cellular anti-apoptotic (pro-survival) and pro-apoptotic (pro-death) responses, respectively. RESULTS: Only culture media conditioned by bladders from 4-Gy-irradiated naïve frogs from Twin Lake induced bystander effects (reduction of HPV-G reporter cells' clonogenic survival and presence of strong calcium flux activities). The 4 Gy irradiation dose increased pro-apoptotic c-myc expression in naïve frogs' bladder explants. Culture media conditioned by bladders from radio-adapting frogs from Duke Swamp enhanced HPV-G's clonogenic survival and a 4 Gy irradiation challenge did not change the enhanced clonogenic survival nature nor induce calcium flux. In bladder explants from both control and 4-Gy-irradiated radio-adapting frogs, anti-apoptotic bcl-2 expression for pro-survival responses was ubiquitous while c-myc expression for pro-death responses was limited to a small fraction of cells. CONCLUSION: The clonogenic RIBE reporter assay using HPV-G and calcium flux measurements are useful diagnostic tools for RIBE assessment of field biological samples, specifically those from frogs. RAR induced by environmentally relevant low-dose radiation induces protective bystander response. Bcl-2 and c-myc are reliable biomarkers for evaluating low dose radiation responses in wild populations of amphibians. Overall, this pilot study emphasizes the importance of looking at non-targeted effects (NTEs) in natural populations of non-human biota that could be vulnerable to chronic low-dose radiation exposures.

Influence of common dietary supplements (curcumin, andrographolide, and d-limonene) on the radiobiological responses of p53-competent colonic cancer epithelial cells.

PURPOSE: The main goal of the research was to determine whether commercially available common dietary phytochemical supplements (curcumin, andrographolide, and d-limonene) have radiomodulatory effects on p53-competent human colonic epithelial cells. METHODS: Clonogenic survival assays were used to characterize effects of the phytochemicals on cultured colonic epithelial cells (HCT116 p53+/+) in direct irradiation or upon receipt of irradiated-cell conditioned media (for bystander effects). In direct irradiation, feeding regimen experiments included compound administration pre- and post-irradiation, which was used as a basis to define effects as radioprotective and radiomitigative, respectively. In the bystander effect experiments, either donor or recipient cell cultures were fed with the phytochemicals and bystander-induced clonogenic cell death was quantitatively evaluated. Dose challenge was in the range of 0.5 - 5 Gy using the gamma source (Cs-137). RESULTS: Curcumin, andrographolide, and d-limonene appeared to not exhibit radioprotective and radiomitigative properties in HCT116 p53+/+ cells. D-limonene was found to induce radiosensitization in post-irradiation administration. All three compounds appeared not to modulate the radiation-induced bystander signal production and response in HCT116 p53+/+ cells. CONCLUSIONS: Curcumin, andrographolide, and d-limonene are known to have many chemoprotective benefits. This work shows that they, however, did not protect colonic epithelial HCT116 p53+/+ cells from radiation killing. As HCT116 p53+/+ cells are tumourigenic in nature, this finding implies that these three dietary compounds would not reduce the killing efficacy of radiation in gastrointestinal tumorigenesis. The post-irradiation radiosensitizing effect of d-limonene was an intriguing observation worth further investigation.

Xela DS2 and Xela VS2: Two novel skin epithelial-like cell lines from adult African clawed frog (Xenopus laevis) and their response to an extracellular viral dsRNA analogue.

The skin epithelial layer acts as an important immunological barrier against pathogens and is capable of recognizing and responding to pathogen-associated molecular patterns (PAMPs) in human and mouse models. Although presumed, it is unknown whether amphibian skin epithelial cells exhibit the ability to respond to PAMPs such as viral double-stranded RNA (dsRNA). To address this, two cell lines from the dorsal skin (Xela DS2) and ventral skin (Xela VS2) of the African clawed frog (Xenopus laevis) were established. Xela DS2 and Xela VS2 cells have an epithelial-like morphology, express genes associated with epithelial cells, and lack senescence-associated beta-galactosidase activity. Cells grow optimally in 70% Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 26 °C. Upon treatment with poly(I:C), a synthetic analogue of viral dsRNA and known type I interferon inducer, Xela DS2 and Xela VS2 exhibit marked upregulation of key antiviral and pro-inflammatory transcripts suggesting frog epithelial cells participate in the recognition of extracellular viral dsRNA and production of local inflammatory signals; similar to human and mouse models. Currently, these are the only known Xenopus laevis skin epithelial-like cell lines and will be important for future research in amphibian epithelial cell biology, initial host-pathogen interactions, and rapid screening of the effects of environmental stressors, including contaminants, on frog skin epithelial cells.

Quantifying Biophoton Emissions From Human Cells Directly Exposed to Low-Dose Gamma Radiation.

Biophoton emission leading to bystander effects (BEs) was shown in beta-irradiated cells; however, technical challenges precluded the analysis of the biophoton role in gamma-induced BEs. The present work was to design an experimental approach to determine if, what type, and how many biophotons could be produced in gamma-irradiated cells. Photon emission was measured in HCT116 p53+/+ cells irradiated with a total dose of 22 mGy from a cesium-137 source at a dose rate of 45 mGy/min. A single-photon detection unit was used and shielded with lead to reduce counts from stray gammas reaching the detector. Higher quantities of photon emissions were observed when the cells in a tissue culture vessel were present and being irradiated compared to a cell-free vessel. Photon emissions were captured at either 340 nm (in the ultraviolet A [UVA] range) or 610 nm. At the same cell density, radiation exposure time, and radiation dose, HCT116 p53+/+ cells emitted 2.5 times more UVA biophotons than 610-nm biophotons. For the first time, gamma radiation was shown to induce biophoton emissions from biological cells. As cellular emissions of UVA biophotons following beta radiation lead to BEs, the involvement of cellular emissions of the same type of UVA biophotons in gamma radiation-induced BEs is highly likely.

5-HT2A and 5-HT3 receptors contribute to the exacerbation of targeted and non-targeted effects of ionizing radiation-induced cell death in human colon carcinoma cells.

Purpose: Serotonin (5-HT) is implicated in the underlying mechanisms which mediate cell death following ionizing radiation exposure, however, effects appear to be cell type-dependent. We sought to further characterize the role of 5-HT and 5-HT receptors (5-HTRs) in the exacerbation of cell death following ionizing radiation exposure in human colon carcinoma cells.Materials and methods: We examined the clonogenic survival of colon carcinoma HCT116 cells treated with 5-HT and the selective 5-HTR antagonists ketanserin (5-HT2A) and ondansetron (5-HT3), following exposure to direct ionizing radiation and irradiated cell-conditioned medium (ICCM). The relative expression of these target receptors was measured using western blotting.Results: Western blotting results revealed that relative protein levels of the 5-HT2A and 5-HT3 receptors were similar. 5-HT concentration-dependent increases in cell death that occurred following direct ionizing radiation exposure were abolished by both 5-HTR antagonists. Death of nonirradiated cells recipient of ICCM was increased in a concentration-dependent manner by 5-HT when present during donor cell irradiation. Both 5-HTR antagonists completely abolished the increases in bystander-induced cell death generated by 5-HT. Finally, we show that exposure of cells to 5-HT prior to receipt of ICCM can also dictate the degree of bystander-induced cell death.Conclusions: Our findings demonstrate a definitive role for 5-HT in the exacerbation of cell death following ionizing radiation exposure in colon carcinoma cells and highlight 5-HTRs as potential markers for predicting cellular radiosensitivity.

One-Decade-Spanning transgenerational effects of historic radiation dose in wild populations of bank voles exposed to radioactive contamination following the chernobyl nuclear disaster.

The concept of historic radiation doses associated with accidental radioactive releases and their role in leading to radiation-induced non-targeted effects on affected wild animals are currently being evaluated. Previous research studying Fukushima butterfly, Chernobyl bird and fruit fly populations shows that the effects are transgenerational, underlined by the principles of genomic instability, and varied from one species to another. To further expand on the responses of and their sensitivity in different taxonomically distinct groups, the present study sought to reconstruct historic radiation doses and delineate their effects on bank voles (Clethrionomys glareolus) found within a 400-km radius of the Chernobyl Nuclear Power Plant meltdown site. Historic dose reconstruction from the whole-body dose rates for the bank vole samples for their parental generation at the time of radioactive release was performed. Relationships between the historic doses and cytogenetic aberrations and embryonic lethality were examined via graphical presentations. Results suggest that genomic instability develops at the historic dose range of 20-51 mGy while a radioadaptive response develops at the historic dose range of 51-356 mGy. The Linear No-Threshold (LNT) relationship was absent at historic doses of lower than 356 mGy at all generations. However, LNT was apparent when the very high historic dose of 10.28 Gy in one sampling year was factored into the dose response curve for the bank vole generation 21-22. It is worth being reminded that natural mutation accumulation and other environmental stressors outside the realm of dose effects could contribute to the observed effects in a multiple-stressor environment. Nevertheless, the consistent development of genomic instability and radio-adaptive response across generations and sampling sites unearths the utmost fundamental radiobiological principle of transgenerational non-targeted effects. As a result, it calls for better attention and regulation from global governing bodies of environmental health protection.

Serotonin and 5-HT3 receptors sensitize human skin cells to direct irradiation cell death but not to soluble radiation-induced bystander signals.

Ionizing radiation (IR) is an environmental carcinogen and the biological damages it elicits are mechanistically distinct between high and low doses. Non-targeted effects occurring in nonirradiated cells such as the radiation-induced bystander effect predominate at low doses of IR. However, the role of non-targeted effects in environmental radiation protection is often overlooked because the governing mechanisms are complex and multifactorial. An improved understanding of the signaling molecules and their capacity to sensitize specific cell types are essential in establishing environmental IR risks. In particular, serotonin (5-HT) has been identified to exacerbate both direct irradiation and bystander-induced cell death (CD) in certain cell types, although not all cell types are responsive to 5-HT in this respect. In this study, we further characterize the role of 5-HT and 5-HT receptors (5-HTR) in the amplification of CD following IR exposure in human keratinocytes. We examined the survival of HaCaT cells treated with 5-HT and the 5-HTR antagonists ketanserin (5-HT2A) and ondansetron (5-HT3) following exposure to direct IR and irradiated cell condition medium (ICCM). Nonirradiated cell survival was consistent with the vehicle control among 5-HT concentrations ranging from 0.001 to 100 µM. Significant 5-HT concentration-dependent increases in CD occurred following direct IR exposure. Nonirradiated ICCM-recipient CD was not altered by 5-HT (0.001-100 µM) when present during donor cell irradiation among all IR doses. Increases in direct irradiation CD evoked by 5-HT were significantly attenuated by ondansetron, blocking the effect of 5-HT, whereas ketanserin did not alter CD. Western blotting of these target 5-HTRs revealed protein expression of the 5-HT3 receptor, while the 5-HT2A receptor was not detected. We have demonstrated a definitive role for 5-HT in the exacerbation of CD following direct IR exposure and identified the 5-HT3 receptor as a potential target for ameliorating radiation damage in keratinocytes.

Regulation of endogenous antigen presentation in response to suboptimal temperatures in a walleye skin fibroblast cell line.

A skin fibroblast cell line WE-skin11f from walleye (Sander vitreus) was used to study the impact of temperature (26 °C, 20 °C, 14 °C, or 4 °C) on the transcript levels of genes involved in the endogenous antigen processing and presentation pathway (EAPP), which is an important antiviral pathway of vertebrates. Partial coding sequences were found for 4 previously unidentified walleye EAPP members, calreticulin, calnexin, erp57, and tapasin, and the constitutive transcript levels of these genes in WE-skin11f was unchanged by culture incubation temperature. The viral mimic poly (I:C) and viral haemorrhagic septicaemia virus (VHSV) IVb were used to study possible induction of EAPP transcripts (b2m, mhIa, and tapasin). The walleye cells were exquisitely sensitive to poly (I:C), losing adherence and viability at concentrations greater than 100 ng/mL, particularly at suboptimal temperatures. VHSV IVb viral particles were produced from infected WE-skin11f cells at 20 °C, 14 °C, and 4 °C but with much lower production at 4 °C. Under conditions where their impact on the viability of WE-skin11f cultures was slight, poly (I:C) and VHSV IVb were shown to induce b2m, mhIa, and tapasin transcript°s at 26 °C and 20 °C respectively. However, at 4 °C, the up-regulation of EAPP transcript levels was either delayed or completely impaired when compared to the 26 °C and 20 °C control temperatures of the respective experiments. These in vitro results suggest that suboptimal temperatures may be capable of modulating the regulation of the EAPP in walleye cells during viral infection.

Characterization of the continuous skin fibroblastoid cell line, WE-skin11f, from walleye (Sander vitreus).

A walleye dermal fibroblastoid cell line, WE-skin11f, was established and characterized. WE-skin11f was immunocytochemically positive for two known dermal fibroblast protein markers: vimentin and collagen I. At passage 26, WE-skin11f cultures contained both diploid and aneuploid populations. Ascorbic acid was required to produce extracellular collagen I fibres. Both of the skin fibroblastoid cell lines, WE-skin11f and rainbow trout-derived RTHDF, were not as good as the walleye caudal fin fibroblastoid cell line, WE-cfin11f, at forming abundant dense extracellular collagen matrices. The thermobiology of WE-skin11f was similar to that of other walleye cell lines with 26°C showing best temperature for growth and 4°C showing no growth but 100% viability. The transcript levels of b2m and mhIa genes of the major histocompatibility class I receptor in WE-skin11f were largely similar at all temperatures examined (4, 14, 20 and 26°C). Cortisol had a variety of effects on WE-skin11f cells: growth inhibition, morphological change from fibroblastoid to epithelioid, and enhancement of barrier function. Treatment of WE-skin11f cells with the physiologically relevant concentration of 100 ng/ml cortisol inhibited collagen I synthesis and matrix formation. Thus, WE-skin11f cell line could be useful in fish dermatology, endocrinology, and immunology research.

Improving a fish intestinal barrier model by combining two rainbow trout cell lines: epithelial RTgutGC and fibroblastic RTgutF.

An in vitro model of the fish intestine is of interest for research and application in diverse fields such as fish physiology, aquaculture and chemical risk assessment. The recently developed epithelial barrier model of the fish intestine relies on the RTgutGC cell line from rainbow trout (Oncorhynchus mykiss), cultured in inserts on permeable membranes. Our aim was to extend the current system by introducing intestinal fibroblasts as supportive layer in order to reconstruct the epithelial-mesenchymal interface as found in vivo. We therefore initiated and characterized the first fibroblast cell line from the intestine of rainbow trout, which has been termed RTgutF. Co-culture studies of RTgutGC and RTgutF were performed on commercially available electric cell substrate for impedance sensing (ECIS) and on newly developed ultrathin, highly porous alumina membranes to imitate the cellular interaction with the basement membrane. Cellular events were examined with non-invasive impedance spectroscopy to distinguish between barrier tightness and cell density in the ECIS system and to determine transepithelial electrical resistance for cells cultured on the alumina membranes. We highlight the relevance of the piscine intestinal fibroblasts for an advanced intestinal barrier model, particularly on ultrathin alumina membranes. These membranes enable rapid crosstalk of cells cultured on opposite sides, which led to increased barrier tightening in the fish cell line-based epithelial-mesenchymal model.

Class A scavenger receptor expression and function in eight novel tadpole cell lines from the green frog (Lithobates clamitans) and the wood frog (Lithobates sylvatica).

A total of eight tadpole cell lines were established from green frogs (Lithobates clamitans) and wood frogs (Lithobates sylvatica). The five green frog cell lines were named GreenTad-HF1, GreenTad-HF2, GreenTad-HF3, GreenTad-HE4, and GreenTad-gill. The three wood frog cell lines were named WoodTad-HE1, WoodTad-Bone, and WoodTad-rpe. DNA barcoding confirmed the cell lines to be from the correct species and the growth characteristics (optimal temperature and FBS requirement) were elucidated. In order to begin studying the innate immune capacity for each cell line, class A scavenger receptor expression and function were next explored. All cell lines expressed genes for at least 3 of the 5 class A scavenger receptor (SR-A) family members, but the gene expression patterns varied between cell lines. MARCO was only expressed in GreenTad-HE4 and WoodTad-Bone, while only GreenTad-HF3 did not express SCARA5 and only WoodTad-rpe did not express SR-AI. Acetylated low density lipoprotein (AcLDL) is a well-defined ligand for SR-As and WoodTad-rpe was the only cell line to which it was unable to bind. In the other seven tadpole cell lines, the SR-A competitive ligands (dextran sulfate, fucoidan, polyinosinic acid) blocked AcLDL binding whereas the SR-A non-competitive ligand counterparts (chondroitin sulfate, fetuin, polycytidylic acid, respectively) did not. Overall, these new eight cell lines can become important tools in the study of innate immunity in general and SR-A functions in particular in green frogs and wood frogs.

Effects of Dose Rate on the Reproductive Cell Death and Early Mitochondrial Membrane Potential in Different Human Epithelium-Derived Cells Exposed to Gamma Rays.

Dose rate is one of the most varied experimental parameters in radiation biology research. In this study, effects of dose rates on the radiation responses of 2 different types of human epithelium-derived cells, immortalized keratinocytes (HaCaT), and colorectal cancer cells (HCT116 p53+/+ and HCT116 p53-/-) were systematically studied. Cells were γ-irradiated at one of the 4 dose rates (24.6, 109, 564, and 1168 mGy/min) to a total dose of 0.5 to 2 Gy. Clonogenic survival and mitochondrial membrane potential (MMP) were measured to assess the levels of reproductive cell death and damage to mitochondrial physiology, respectively. It was found that clonogenic survival was similar at all 4 tested dose rates in the 3 cell lines. The loss of MMP occurred at all tested dose rates in all 3 cell lines except for one case where the MMP increased in HCT116 p53+/+cells after exposure to 0.5 Gy at 24.6 mGy/min. In HCT116 cells, the loss of MMP was the most severe at high dose/dose rate combination exposure and when p53 was expressed. In contrast, no effect in dose rate was observed with HaCaT cells as the reduction level of MMP was similar at the tested dose rates.

Characterization of Radioprotective, Radiomitigative and Bystander Signaling Modulating Effects of Endogenous Metabolites - Phenylacetate, Ursodeoxycholate and Tauroursodeoxycholate - on HCT116 Human Colon Carcinoma Cell Line.

Exposures to ionizing radiation can cause depletion in stem cell reservoirs and lead to chronic injury processes that exacerbate carcinogenic and inflammatory responses. Therefore, radioprotective measures, against both acute and chronic biological effects of radiation, require frequent intake of nontoxic natural products, which have practical oral administration. The goal of this study was to characterize the radioprotective, radiomitigative and radiation-induced bystander effect-inhibiting properties of endogenous metabolites: phenylacetate, ursodeoxycholate and tauroursodeoxycholate. Compounds were administered pre- and postirradiation as well as in donor and recipient bystander flasks to analyze whether these might adequately protect against radiation injury as well as facilitate recovery from the exposures. The clonogenic HCT116 p53 wild-type cancer cell line in this study shares characteristics of stem cells, such as high reproductive viability, which is an effective marker to demonstrate compound effectiveness. Clonogenic assays were therefore used to characterize radioprotective, radiomitigative and bystander inhibiting properties of treatment compounds whereby cellular responses to radiation were quantified with macroscopic colony counts to measure cell survival in flasks. The results were statistically significant for phenylacetate and tauroursodeoxycholate when administered preirradiation, conferring radioprotection up to 2 Gy, whereas administration postirradiation and in bystander experiments did not confer radioprotection in vitro. These findings suggest that phenylacetate and tauroursodeoxycholate might be effective radioprotectors, although they possess no radiomitigative properties.