Diploid Fibroblasts Research Articles

Medullins A-H, Sesquiterpenes from Perenniporia medulla-panis, and Their Cellular Signaling Mechanism in HDF Cells.

A chemical investigation of an ethyl acetate-soluble layer in the culture broth of Perenniporia medulla-panis resulted in the isolation of eight novel sesquiterpenes conjugated Gly (1), l-Val (2), l-Ala (3), l-Tyr (4), l-Thr (5), l-Ile (6), l-Leu (7), and l-Phe (8). Elucidation of their structures was performed through comprehensive spectroscopic analysis. The absolute configuration of the sesquiterpene skeleton was ascertained using modified Mosher's methods. The configurations of the amino acid units in compounds 2-8 were identified through acid hydrolysis followed by LC-MS analysis employing Marfey's method. Compounds 1-3 and 5-8 showed significant regulating effect on MAP kinase activity (p-ERK and p-JNK) in human diploid fibroblast (HDF) cells.

Histone H3.3 chaperone HIRA renders stress-responsive genes poised for prospective lethal stresses in acquired tolerance.

Appropriate responses to environmental challenges are imperative for the survival of all living organisms. Exposure to low-dose stresses is recognized to yield increased cellular fitness, a phenomenon termed hormesis. However, our molecular understanding of how cells respond to low-dose stress remains profoundly limited. Here we report that histone variant H3.3-specific chaperone, HIRA, is required for acquired tolerance, where low-dose heat stress exposure confers resistance to subsequent lethal heat stress. We found that human HIRA activates stress-responsive genes, including HSP70, by depositing histone H3.3 following low-dose stresses. These genes are also marked with histone H3 Lys-4 trimethylation and H3 Lys-9 acetylation, both active chromatin markers. Moreover, depletion of HIRA greatly diminished acquired tolerance, both in normal diploid fibroblasts and in HeLa cells. Collectively, our study revealed that HIRA is required for eliciting adaptive stress responses under environmental fluctuations and is a master regulator of stress tolerance.

A Lombard Variety of Sweet Pepper Regulating Senescence and Proliferation: The Voghera Pepper.

Aging and its related disorders are important issues nowadays and the first cause of this physio-pathological condition is the overproduction of ROS. Ascorbic acid is an antioxidant mediator and its anti-aging proprieties are well known. Our previous data demonstrated that Voghera sweet pepper (VP), a distinctive type of pepper cultivated in Italy, is particularly rich in ascorbic acid. Based on these data, the anti-aging effect mediated by extracts of the edible part of VP was evaluated on an in vitro model of both young and old Normal Human Diploid Fibroblasts (NHDF). Using phase contrast microscopy, we observed that VP may help cells in the maintenance of physiological morphology during aging. Cytofluorimetric analyses revealed that VP extracts led to an increase in DNA synthesis and percentage of living cells, linked to a consequent increase in mitotic events. This hypothesis is supported by the enhancement of PCNA expression levels observed in old, treated fibroblasts, corroborating the idea that this extract could recover a young phenotype in adult fibroblasts, confirmed by the study of p16 and p53 expression levels and TEM analyses. Based on these results, we may suppose that VP can lead to the partial recovery of "young-like" phenotypes in old fibroblasts.

MLKL, a new actor of UVB-induced apoptosis in human diploid dermal fibroblasts

Ultraviolet radiation (UVR) is a major environmental mutagen. In skin, UVR can initiate cancer through the induction of mutagenic DNA damage and promote its progression. An important cancer prevention mechanism is the regulated cell death (RCD), which can safely dispose of damaged cells. Apoptosis, a well-known RCD, is known to be activated by UVR, but part of the mechanism and proteins involved in UVR-induced apoptosis are still to be discovered. Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) are two proteins involved in necroptosis, a form of RCD. Here, we have evaluated the implication of RIPK3 and MLKL in UVB-induced cell death in human diploid dermal fibroblasts. Our results show that RIPK3 and MLKL play opposite roles in UVB-induced cell death, in a necroptosis independent pathway. We showed that RIPK3 protects cells from UVB cell death, while MLKL sensitizes cells to UVB-induced apoptosis. Taken together these results are the first to show the implication of RIPK3 and MLKL in survival and apoptosis, respectively, bringing two new actors in UVB-induced cell death pathway.

SRSF7 downregulation induces cellular senescence through generation of MDM2 variants.

Alternative splicing (AS) enables a pre-mRNA to generate different functional protein variants. The change in AS has been reported as an emerging contributor to cellular senescence and aging. However, it remains to be elucidated which senescent AS variants are generated in and regulate senescence. Here, we observed commonly down-regulated SRSF7 in senescent cells, using publicly available RNA-seq datasets of several in vitro senescence models. We further confirmed SRSF7 deregulation from our previous microarray datasets of time-series replicative senescence (RS) and oxidative stress-induced senescence (OSIS) of human diploid fibroblast (HDF). We validated the time-course changes of SRSF mRNA and protein levels, developing both RS and OSIS. SRSF knockdown in HDF was enough to induce senescence, accompanied by p53 protein stabilization and MDM2 variants formation. Interestingly, expression of MDM2 variants showed similar patterns of p53 expression in both RS and OSIS. Next, we identified MDM2-C as a key functional AS variant generated specifically by SRSF7 depletion. Finally, we validated that MDM2-C overexpression induced senescence of HDF. These results indicate that SRSF7 down-regulation plays a key role in p53-mediated senescence by regulating AS of MDM2, a key negative regulator of p53, implying its critical involvement in the entry into cell senescence.

Features of SARS-CoV-2 Replication in Various Types of Reptilian and Fish Cell Cultures.

SARS-CoV-2 can enter the environment from the feces of COVID-19 patients and virus carriers through untreated sewage. The virus has shown the ability to adapt to a wide range of hosts, so the question of the possible involvement of aquafauna and animals of coastal ecosystems in maintaining its circulation remains open. the aim of this work was to study the tropism of SARS-CoV-2 for cells of freshwater fish and reptiles, including those associated with aquatic and coastal ecosystems, and the effect of ambient temperature on this process. In a continuous cell culture FHM (fathead minnow) and diploid fibroblasts CGIB (silver carp), SARS-CoV-2 replication was not maintained at either 25 °C or 29 °C. At 29 °C, the continuous cell culture TH-1 (eastern box turtle) showed high susceptibility to SARS-CoV-2, comparable to Vero E6 (development of virus-induced cytopathic effect (CPE) and an infectious titer of 7.5 ± 0.17 log10 TCID50/mL on day 3 after infection), and primary fibroblasts CNI (Nile crocodile embryo) showed moderate susceptibility (no CPE, infectious titer 4.52 ± 0.14 log10 TCID50/mL on day 5 after infection). At 25 °C, SARS-CoV-2 infection did not develop in TH-1 and CNI. our results show the ability of SARS-CoV-2 to effectively replicate without adaptation in the cells of certain reptile species when the ambient temperature rises.

Epigenetic activation of secretory phenotypes in senescence by the FOXQ1-SIRT4-GDH signaling

Although metabolic reprogramming is characterized as a hallmark of aging, implications of the crucial glutamate dehydrogenase (GDH) in human senescence remain poorly understood. Here, we report that GDH activity is significantly increased in aged mice and senescent human diploid fibroblasts. This enzymatic potentiation is associated with de-repression of GDH from its functionally suppressive ADP-ribosylation modification catalyzed by NAD-dependent ADP-ribosyltransferase/deacetylase SIRT4. A series of transcription analyses led to the identification of FOXQ1, a forkhead family transcription factor (TF), responsible for the maintenance of SIRT4 expression levels in juvenile cells. However, this metabolically balanced FOXQ1-SIRT4-GDH axis, is shifted in senescence with gradually decreasing expressions of FOXQ1 and SIRT4 and elevated GDH activity. Importantly, pharmaceutical inhibition of GDH suppresses the aberrantly activated transcription of IL-6 and IL-8, two major players in senescence-associated secretory phenotype (SASP), and this action is mechanistically associated with erasure of the repressive H3K9me3 (trimethylation of lysine 9 on histone H3) marks at IL-6 and IL-8 promoters, owing to the requirement of α-ketoglutaric acid (α-KG) from GDH-mediated glutamate dehydrogenase reaction as a cofactor for histone demethylation. In supplement with the phenotypic evidence from FOXQ1/SIRT4/GDH manipulations, these data support the integration of metabolism alterations and epigenetic regulation in driving senescence progression and highlight the FOXQ1-SIRT4-GDH axis as a novel druggable target for improving human longevity.

P06-038-23 Effect of Allomyrina dichotoma Larvae Ethyl Acetate Fraction on Ultraviolet B-Induced Aging in Human Diploid Fibroblasts

P06-038-23 Effect of Allomyrina dichotoma Larvae Ethyl Acetate Fraction on Ultraviolet B-Induced Aging in Human Diploid Fibroblasts

ANT2 Accelerates Cutaneous Wound Healing in Aged Skin by Regulating Energy Homeostasis and Inflammation

An effective healing response is critical to healthy aging. In particular, energy homeostasis has become increasingly recognized as a factor in effective skin regeneration. Adenine nucleotide translocase 2 (ANT2) is a mediator of ATP import into mitochondria for energy homeostasis. Although energy homeostasis and mitochondrial integrity are critical for wound healing, the role played by ANT2 in the repair process had not been elucidated to date. In our study, we found that ANT2 expression decreased in aged skin and cellular senescence. Interestingly, overexpression of ANT2 in aged mouse skin accelerated the healing of full-thickness cutaneous wounds. In addition, upregulation of ANT2 in replicative senescent human diploid dermal fibroblasts (HDFs) induced their proliferation and migration, which are critical processes in wound healing. Regarding energy homeostasis, ANT2 overexpression increased the ATP production rate by activating glycolysis and induced mitophagy. Notably, ANT2-mediated upregulation of HSPA6 in aged HDFs downregulated proinflammatory genes that mediate cellular senescence and mitochondrial damage. This study demonstrates a previously uncharacterized physiological role of ANT2 in skin wound healing via regulating cell proliferation, energy homeostasis and inflammation. Thus, our study links energy metabolism to skin homeostasis and reports to our knowledge previously unreported genetic factor that enhances wound healing in an aging model.

Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest.

Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-β-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy.

Single-cell transcriptomic analysis uncovers diverse and dynamic senescent cell populations

Senescence is a state of enduring growth arrest triggered by sublethal cell damage. Given that senescent cells actively secrete proinflammatory and matrix-remodeling proteins, their accumulation in tissues of older persons has been linked to many diseases of aging. Despite intense interest in identifying robust markers of senescence, the highly heterogeneous and dynamic nature of the senescent phenotype has made this task difficult. Here, we set out to comprehensively analyze the senescent transcriptome of human diploid fibroblasts at the individual-cell scale by performing single-cell RNA-sequencing analysis through two approaches. First, we characterized the different cell states in cultures undergoing senescence triggered by different stresses, and found distinct cell subpopulations that expressed mRNAs encoding proteins with roles in growth arrest, survival, and the secretory phenotype. Second, we characterized the dynamic changes in the transcriptomes of cells as they developed etoposide-induced senescence; by tracking cell transitions across this process, we found two different senescence programs that developed divergently, one in which cells expressed traditional senescence markers such as p16 (CDKN2A) mRNA, and another in which cells expressed long noncoding RNAs and splicing was dysregulated. Finally, we obtained evidence that the proliferation status at the time of senescence initiation affected the path of senescence, as determined based on the expressed RNAs. We propose that a deeper understanding of the transcriptomes during the progression of different senescent cell phenotypes will help develop more effective interventions directed at this detrimental cell population.

Sesamin Metabolites Suppress the Induction of Cellular Senescence

Cellular senescence induces inflammation and is now considered one of the causes of organismal aging. Accumulating evidence indicates that age-related deterioration of mitochondrial function leads to an increase in reactive oxygen species (ROS) and DNA damage, which in turn causes cellular senescence. Thus, it is important to maintain mitochondrial function and suppress oxidative stress in order to inhibit the accumulation of senescent cells. Sesamin and its isomer episesamin are types of lignans found in sesame oil, and after being metabolized in the liver, their metabolites have been reported to exhibit antioxidant properties. However, their effects on cellular senescence remain unknown. In this study, the effects of sesamin, episesamin, and their metabolites SC1 and EC1-2 on replicative senescence were evaluated using human diploid lung fibroblasts, and TIG-3 cells. The results showed that sesamin and episesamin treatment had no effect on proliferative capacity compared to the untreated late passage group, whereas SC1 and EC1-2 treatment improved proliferative capacity and mitigated DNA damage of TIG-3 cells. Furthermore, other cellular senescence markers, such as senescence-associated secretory phenotype (SASP), mitochondria-derived ROS, and mitochondrial function (ROS/ATP ratio) were also reduced by SC1 and EC1-2 treatment. These results suggest that SC1 and EC1-2 can maintain proper mitochondrial function and suppress the induction of cellular senescence.

Heterogeneity of Cellular Senescence: Cell Type-Specific and Senescence Stimulus-Dependent Epigenetic Alterations

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

Voghera Sweet Pepper: A Potential Ally against Oxidative Stress and Aging.

In the present study, the potential functional properties of the extracts from the edible part of Capsicum annuum L. var. Peperone di Voghera (VP) were studied. The phytochemical analysis revealed a high amount of ascorbic acid, paralleled by a low carotenoid content. Normal human diploid fibroblasts (NHDF) were chosen as the in vitro model models to investigate the effects of the VP extract on oxidative stress and aging pathways. The extract of Carmagnola pepper (CP), another important Italian variety, was used as the reference vegetable. The cytotoxicity evaluation was performed firstly, using a 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, while the VP potential antioxidant and antiaging activity was investigated by immunofluorescence staining focusing on specifically selected proteins. The MTT data revealed the highest cell viability at a concentration of up to 1 mg/mL. The immunocytochemical analyses highlighted an increased expression of transcription factors and enzymes involved in redox homeostasis (Nrf2, SOD2, catalase), improved mitochondrial functionality, and the up-regulation of the longevity gene SIRT1. The present results supported the functional role of the VP pepper ecotype, suggesting a feasible use of its derived products as valuable food supplements.

Evaluation of the Cytotoxic, Antioxidative and Antimicrobial Effects of Dracocephalum moldavica L. Cultivars.

The aim of the present study was to correlate the antioxidant, antimicrobial, and cytotoxic activities of hydroalcoholic extracts obtained from the aerial parts of three Dracocephalum moldavica L. cultivars with their polyphenolic compositions. The polyphenols were identified and quantified using spectrophotometrical methods and LC-MS analysis. Their antioxidant capacities were assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. Their in vitro antimicrobial efficacies were assessed using the agar well diffusion and broth microdilution methods. Their cytotoxicity was investigated on normal diploid foreskin fibroblasts (BJ) and on colorectal adenocarcinoma (DLD-1) cell lines. The results pointed out significant amounts of polyphenolic compounds in the compositions of the tested cultivars, with rosmarinic acid as the main compound (amounts ranging between 5.337 ± 0.0411 and 6.320 ± 0.0535 mg/mL). All three cultivars displayed significant antioxidant (IC50 ranging between 35.542 ± 0.043 and 40.901 ± 0.161 µg/mL for the DPPH assay, and for the FRAP assay 293.194 ± 0.213 and 330.165 ± 0.754 µmol Trolox equivalent/mg dry vegetal material) and antimicrobial potential (especially towards the Gram-positive bacteria), as well as a selective toxicity towards the tumoral line. A significant positive correlation was found between antioxidant activity and the total phenolic acids (r2 = 0.987) and polyphenols (r2 = 0.951). These findings bring further arguments for strongly considering D. moldavica cultivars as promising vegetal products, which warrants further investigation.

Photosensitization of human skin fibroblasts by vemurafenib promotes pleiotropic effects on membrane-enclosed organelles and apoptosis.

Vemurafenib (VB), a BRAF inhibitor and a first-line treatment for unresectable or metastatic melanoma, is strongly phototoxic towards normal skin cells. Herein, we show that in cultured HS 68 human diploid dermal fibroblasts, low concentrations of VB suffice to promote photosensitization to low doses of UVA (∼ 5J/cm2), as evidenced by a significant decrease in cell viability. In contrast to data obtained in chemico our results support a role for ROS (reactive oxygen species). Indeed, peroxidation of cellular lipids was observed which could be alleviated by the lipophilic antioxidant BHT (2,6-di-tert-butyl-4-methylphenol). Using in vivo confocal laser scanning microscopy and vital fluorescent probes it was shown at the single cell level that the plasma membrane and lipid-rich organelles, namely mitochondria, endoplasmic reticulum, and lysosomes, as well as actin filaments, were severely damaged by the UVA-induced VB-photosensitization. Finally, we showed that mitochondrial impairment was concurrent with caspase 3/7 activation and cell death by apoptosis.

Anti-skinaging effects of Gryllus bimaculatus on ERM-CZ100-exposed human diploid fibroblasts

Anti-skinaging effects of <i>Gryllus bimaculatus</i> on ERM-CZ100-exposed human diploid fibroblasts

A dual-vector phospholipid nanosystem of doxorubicin: accumulation and cytotoxic effect in breast cancer cells in vitro.

Various chemotherapeutic agents are used to treat breast cancer (BC); one of them is the anthracycline antibiotic doxorubicin (Dox), which, in addition to its cytostatic effect, has serious side effects. In order to reduce its negative impact on healthy organs and tissues and to increase its accumulation in tumors, Dox was incorporated into phospholipid nanoparticles. The additional use of vector molecules for targeted delivery to specific targets can increase the effectiveness of Dox due to higher accumulation of the active substance in the tumor tissue. The integrin αvβ3, which plays an important role in cancer angiogenesis, and the folic acid receptor, which is responsible for cell differentiation and proliferation, have been considered in this study as targets for such vector molecules. Thus, a phospholipid composition of Dox containing two vector ligands, cRGD peptide and folic acid (NPh-Dox-cRGD-Fol(3,4)), was prepared. Study of the physical properties of the developed composition NPh-Dox-cRGD-Fol(3,4) showed that the average particle size was 39.62±4.61 nm, the ζ-potential value was 4.17±0.83 mV. Almost all Dox molecules were incorporated into phospholipid nanoparticles (99.85±0.21%). The simultaneous use of two vectors in the composition led to an increase in the Dox accumulation in MDA-MB-231 BC cells by almost 20% as compared to compositions containing each vector separately (folic acid or the cRGD peptide). Moreover, the degree of Dox internalization was 22% and 24% higher than in the case of separate use of folic acid and cRGD peptide, respectively. The cytotoxic effect on MDA-MB-231 cells was higher during incubations with the compositions containing folic acid as a single vector (NPh-Dox-Fol(3,4)) and together with the RGD peptide (NPh-Dox-cRGD-Fol(3,4)). Experiments on the Wi-38 diploid fibroblast cell line have shown a significantly lower degree of cytotoxic effect of the phospholipid composition, regardless of the presence of the vector molecules in it, as compared to free Dox. The results obtained indicate the potential of using two vectors in one phospholipid composition for targeted delivery of Dox.

Suppressive effects of pterostilbene on human cytomegalovirus (HCMV) infection and HCMV-induced cellular senescence

BackgroundHuman cytomegalovirus (HCMV), a member of the β-herpesvirus family, causes the establishment of a latent infection that persists throughout the life of the host and can be reactivated when immunity is weakened. To date, there is no vaccine to prevent HCMV infection, and clinically approved drugs target the stage of viral replication and have obvious adverse reactions. Thus, development of novel therapeutics is urgently needed.MethodsIn the current study, we identified a naturally occurring pterostilbene that inhibits HCMV Towne strain replication in human diploid fibroblast WI-38 cells through Western blotting, qPCR, indirect immunofluorescence assay, tissue culture infective dose assays. The time-of-addition experiment was carried out to identify the stage at which pterostilbene acted. Finally, the changes of cellular senescence biomarkers and reactive oxygen species production brought by pterostilbene supplementation were used to partly elucidate the mechanism of anti-HCMV activity.ResultsOur findings revealed that pterostilbene prevented lytic cytopathic changes, inhibited the expression of viral proteins, suppressed the replication of HCMV DNA, and significantly reduced the viral titre in WI-38 cells. Furthermore, our data showed that pterostilbene predominantly acted after virus cell entry and membrane fusion. The half-maximal inhibitory concentration was determined to be 1.315 μM and the selectivity index of pterostilbene was calculated as 26.73. Moreover, cell senescence induced by HCMV infection was suppressed by pterostilbene supplementation, as shown by a decline in senescence-associated β-galactosidase activity, decreased production of reactive oxygen species and reduced expression of p16, p21 and p53, which are considered biomarkers of cellular senescence.ConclusionTogether, our findings identify pterostilbene as a novel anti-HCMV agent that may prove useful in the treatment of HCMV replication.

Effects of oxygen on the response of mitochondria to X-irradiation and reactive oxygen species-mediated fibroblast activation

Purpose In living organisms, sensitivity to radiation increases in the presence of oxygen (O2) compared with that under anoxic or hypoxic conditions. Here, we investigated whether O2 concentration affected the response of mitochondria to X-rays radiation, which is associated with tumor microenvironment formation via fibroblast activation in radiation-related tumors. Materials and methods O2 concentrations were controlled at <5% (internal environmental oxygen condition) or anoxic levels during culture of normal human diploid lung fibroblasts TIG-3 and MRC-5. Protein expression associated with the response of mitochondria to radiation was assessed using immunostaining or western blotting. Results Induction of DNA damage (marker: γ-H2A histone family member X) and mitochondrial signaling (AMP-activated protein kinase), suppression of mitochondrial metabolic activity, and generation of reactive oxygen species occurred with radiation in cells cultured under 5% and 20% O2 conditions. However, reducing O2 concentration mitigated the effects of radiation on cell growth, mitochondrial damage (parkin), induction of antioxidant responses (nuclear factor E2-related factor 2), and fibroblast activation (α-smooth muscle actin). Radiation did not affect the markers used in this study in the absence of O2. Conclusion O2 concentration affected the response of mitochondria to radiation and reactive oxygen species-mediated fibroblast activation. Higher O2 concentrations enhanced the effects of radiation on mitochondria in human fibroblasts. In vitro studies may overestimate in vivo radiation effects due to high O2 concentrations.