Developmental delay with hypotrophy associated with homozygous functionally relevant REV3L variant.

REV3L encodes a catalytic subunit of DNA polymerase zeta (Pol zeta) which is essential for the tolerance of DNA damage by inducing translesion synthesis (TLS). So far, the only Mendelian disease associated with REV3L was Moebius syndrome (3 patients with dominant REV3L mutations causing monoallelic loss-of-function were reported). We describe a homozygous ultra-rare REV3L variant (T2753R) identified with whole exome sequencing in a child without Moebius syndrome but with developmental delay, hypotrophy, and dysmorphic features who was born to healthy parents (heterozygous carriers of the variant). The variant affects the amino acid adjacent to functionally important KKRY motif. By introducing an equivalent mutation (S1192R) into the REV3 gene in yeasts, we showed that, whereas it retained residual function, it caused clear dysfunction of TLS in the nucleus and instability of mitochondrial genetic information. In particular, the mutation increased UV sensitivity measured by cell survival, decreased both the spontaneous (P < 0.005) and UV-induced (P < 0.0001) mutagenesis rates of nuclear DNA and increased the UV-induced mutagenesis rates of mitochondrial DNA (P < 0.0005). We propose that our proband is the first reported case of a REV3L associated disease different from Moebius syndrome both in terms of clinical manifestations and inheritance (autosomal recessive rather than dominant). KEY MESSAGES: First description of a human recessive disorder associated with a REV3L variant. A study in yeast showed that the variant affected the enzymatic function of the protein. In particular, it caused increased UV sensitivity and abnormal mutagenesis rates.

Increase of mtDNA number and its mutant copies in rat brain after exposure to 150 MeV protons.

Proton beam therapy is widely used for treating brain tumor. Despite the efficacy of treatment, the use of this therapy has met some limitations associated with possible damage to normal brain tissues located beyond the tumor site. In this context, the exploration of the harmful effects of protons on the normal brain tissues is of particular interest. We have investigated changes in the total mitochondrial DNA (mtDNA) copy number and identified mtDNA mutant copies in three brain regions (the hippocampus, cortex and cerebellum) of rats after irradiation their whole-head with 150 MeV protons at doses of 3 and 5 Gy. The study was performed in 2-months old male Spraque Dawley rats (n = 5 each group). The mtDNA copy numbers were determined by real-time PCR. The level of mtDNA heteroplasmy was estimated using Surveyor nuclease technology. Our results show that after head exposure to protons, levels of mtDNA copy number in three rat brain regions increase significantly as the levels of mtDNA mutant copies increase. The most significant elevation is observed in the hippocampus. In conclusion, an increase in mtDNA mutant copies may contribute to mitochondrial dysfunction accompanied by increased oxidative stress in different brain regions and promote the development of neurodegenerative diseases and the induction of carcinogenesis.

Deciphering and simulating models of radiation genotoxicity with CRISPR/Cas9 systems.

This short review explores the utility and applications of CRISPR/Cas9 systems in radiobiology. Specifically, in the context of experimentally simulating genotoxic effects of Ionizing Radiation (IR) to determine the contributions from DNA targets and 'Complex Double-Stranded Breaks' (complex DSBs) to the IR response. To elucidate this objective, this review considers applications of CRISPR/Cas9 on nuclear DNA targets to recognize the respective 'nucleocentric' response. The article also highlights contributions from mitochondrial DNA (mtDNA) - an often under-recognized target in radiobiology. This objective requires accurate experimental simulation of IR-like effects and parameters with the CRISPR/Cas9 systems. Therefore, the role of anti-CRISPR proteins in modulating enzyme activity to simulate dose rate - an important factor in radiobiology experiments is an important topic of this review. The applications of auxiliary domains on the Cas9 nuclease to simulate oxidative base damage and multiple stressor experiments are also topics of discussion. Ultimately, incorporation of CRISPR/Cas9 experiments into computational parameters in radiobiology models of IR damage and shortcomings to the technology are discussed as well. Altogether, the simulation of IR parameters and lack of damage to non-DNA targets in the CRISPR/Cas9 system lends this rapidly emerging tool as an effective model of IR induced DNA damage. Therefore, this literature review ultimately considers the relevance of complex DSBs to radiobiology with respect to using the CRISPR/Cas9 system as an effective experimental tool in models of IR induced effects.

Evaluation of the role of mitochondria in the non-targeted effects of ionizing radiation using cybrid cellular models.

Radiobiology is moving towards a better understanding of the intercellular signaling that occurs upon radiation and how its effects relate to the dose applied. The mitochondrial role in orchestrating this biological response needs to be further explored. Cybrids (cytoplasmic hybrids) are useful cell models for studying the involvement of mitochondria in cellular processes. In the present study we used cybrid cell lines to investigate the role of mitochondria in the response to radiation exposure. Cybrid cell lines, derived from the osteosarcoma human cell line 143B, harboring, either wild-type mitochondrial DNA (Cy143Bwt), cells with mitochondria with mutated DNA that causes mitochondrial dysfunction (Cy143Bmut), as well as cells without mitochondrial DNA (mtDNA) (143B-Rho0), were irradiated with 0.2 Gy and 2.0 Gy. Evaluation of the non-targeted (or bystander) effects in non-irradiated cells were assessed by using conditioned media from the irradiated cells. DNA double stranded breaks were assessed with the γH2AX assay. Both directly irradiated cells and cells treated with the conditioned media, showed increased DNA damage. The effect of the irradiated cells media was different according to the cell line it derived from: from Cy143Bwt cells irradiated with 0.2 Gy (low dose) and from Cy143Bmut irradiated with 2.0 Gy (high dose) induced highest DNA damage. Notably, media obtained from cells without mtDNA, the143B-Rho0 cell line, produced no effect in DNA damage. These results point to a possible role of mitochondria in the radiation-induced non-targeted effects. Furthermore, it indicates that cybrid models are valuable tools for radiobiological studies.

Individual and combined effects of the infrared, visible, and ultraviolet light components of solar radiation on damage biomarkers in human skin cells.

The ability of solar ultraviolet (UV) to induce skin cancer and photoaging is well recognized. The effect of the infrared (IR) and visible light (Vis) components of solar radiation on skin and their interaction with UV is less well known. This study compared the effects of physiologically relevant doses of complete (UV + Vis + IR) solar-simulated light and its individual components on matched primary dermal fibroblasts and epidermal keratinocytes from human donors on three biomarkers of cellular damage (reactive oxygen species (ROS) generation, mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) damage). There was a greater induction of ROS, mtDNA, and nDNA damage with the inclusion of the visible and IR components of solar-simulated light in primary fibroblast cells compared to primary keratinocytes (P < .001). Experiments using exposure to specific components of solar light alone or in combination showed that the UV, Vis, and IR components of solar light synergistically increased ROS generation in primary fibroblasts but not primary keratinocytes (P < .001). Skin cell lines were used to confirm these findings. These observations have important implications for different skin cell type responses to the individual and interacting components of solar light and therefore photodamage mechanisms and photoprotection interventions.

Targeting reduced mitochondrial DNA quantity as a therapeutic approach in pediatric high-grade gliomas.

BACKGROUND: Despite increased understanding of the genetic events underlying pediatric high-grade gliomas (pHGGs), therapeutic progress is static, with poor understanding of nongenomic drivers. We therefore investigated the role of alterations in mitochondrial function and developed an effective combination therapy against pHGGs. METHODS: Mitochondrial DNA (mtDNA) copy number was measured in a cohort of 60 pHGGs. The implication of mtDNA alteration in pHGG tumorigenesis was studied and followed by an efficacy investigation using patient-derived cultures and orthotopic xenografts. RESULTS: Average mtDNA content was significantly lower in tumors versus normal brains. Decreasing mtDNA copy number in normal human astrocytes led to a markedly increased tumorigenicity in vivo. Depletion of mtDNA in pHGG cells promoted cell migration and invasion and therapeutic resistance. Shifting glucose metabolism from glycolysis to mitochondrial oxidation with the adenosine monophosphate-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) or the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA) significantly inhibited pHGG viability. Using DCA to shift glucose metabolism to mitochondrial oxidation and then metformin to simultaneously target mitochondrial function disrupted energy homeostasis of tumor cells, increasing DNA damage and apoptosis. The triple combination with radiation therapy, DCA and metformin led to a more potent therapeutic effect in vitro and in vivo. CONCLUSIONS: Our results suggest metabolic alterations as an onco-requisite factor of pHGG tumorigenesis. Targeting reduced mtDNA quantity represents a promising therapeutic strategy for pHGG.

Mitochondrial DNA damage in the hair bulb: can it be used as a noninvasive biomarker of local exposure to low LET ionizing radiation?

Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.

Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.

Increased mitochondrial DNA4977-bp deletion in catheterization laboratory workers with long-term low-dose exposure to ionizing radiation.

AIMS: Ionizing radiation may lead to mitochondrial DNA (mtDNA) mutations and changes in mtDNA content in cells, major driving mechanisms for carcinogenesis, vascular aging and neurodegeneration. The aim of this study was to investigate the possible induction of common mitochondrial deletion (mtDNA4977) and mtDNA copy number (mtDNA-CN) changes in peripheral blood of personnel working in high-volume cardiac catheterization laboratories (Cath Labs). METHODS: A group of 147 Cath Lab workers (median individual effective dose = 16.8 mSv, for the 41 with lifetime dosimetric record) and 74 unexposed individuals were evaluated. The occupational radiological risk score was computed for each subject on the basis of the length of employment, individual caseload and proximity to the radiation source. mtDNA4977 deletion and mtDNA-CN were assessed by using quantitative real-time polymerase chain reaction. RESULTS: Increased levels of mtDNA4977 deletion were observed in high-exposure Cath Lab workers compared with unexposed individuals ( p < 0.0001). Conversely, mtDNA-CN was significantly greater in the low-exposure workers ( p = 0.003). Occupational radiological risk score was positively correlated with mtDNA4977 deletion (Spearman's r = 0.172, p = 0.03) and inversely correlated with mtDNA-CN (Spearman's r = -0.202, p = 0.01). In multiple regression model, occupational radiological risk score emerged as significant predictor of high levels of mtDNA4977 deletion (ß coefficient = 0.236, p = 0.04). CONCLUSION: mtDNA4977 deletion is significantly high in Cath Lab personnel. Beyond the well-recognized nuclear DNA, mtDNA damage might deserve attention as a pathogenetic molecular pathway and a potential therapeutic target of ionizing radiation damage.

The first in vivo multiparametric comparison of different radiation exposure biomarkers in human blood.

The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0-2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.

Improving mitochondrial function significantly reduces metabolic, visual, motor and cognitive decline in aged Drosophila melanogaster.

Mitochondria play a major role in aging. Over time, mutations accumulate in mitochondrial DNA leading to reduced adenosine triphosphate (ATP) production and increased production of damaging reactive oxygen species. If cells fail to cope, they die. Reduced ATP will result in declining cellular membrane potentials leading to reduced central nervous system function. However, aged mitochondrial function is improved by long wavelength light (670 nm) absorbed by cytochrome c oxidase in mitochondrial respiration. In Drosophila, lifelong 670-nm exposure extends lifespan and improves aged mobility. Here, we ask if improved mitochondrial metabolism can reduce functional senescence in metabolism, sensory, locomotor, and cognitive abilities in old flies exposed to 670 nm daily for 1 week. Exposure significantly increased cytochrome c oxidase activity, whole body energy storage, ATP and mitochondrial DNA content, and reduced reactive oxygen species. Retinal function and memory were also significantly improved to levels found in 2-week-old flies. Mobility improved by 60%. The mode of action is likely related to improved energy homeostasis increasing ATP availability for ionic ATPases critical for maintenance of neuronal membrane potentials. 670-nm light exposure may be a simple route for resolving problems of aging.

Effect of densely ionizing radiation on cardiomyocyte differentiation from human-induced pluripotent stem cells.

The process of human cardiac development can be faithfully recapitulated in a culture dish with human pluripotent stem cells, where the impact of environmental stressors can be evaluated. The consequences of ionizing radiation exposure on human cardiac differentiation are largely unknown. In this study, human-induced pluripotent stem cell cultures (hiPSCs) were subjected to an external beam of 3.7 MeV α-particles at low mean absorbed doses of 0.5, 3, and 10 cGy. Subsequently, the hiPSCs were differentiated into beating cardiac myocytes (hiPSC-CMs). Pluripotent and cardiac markers and morphology did not reveal differences between the irradiated and nonirradiated groups. While cell number was not affected during CM differentiation, cell number of differentiated CMs was severely reduced by ionizing radiation in a dose-responsive manner. ß-adrenergic stimulation causes calcium (Ca2+) overload and oxidative stress. Although no significant increase in Ca2+ transient amplitude was observed in any group after treatment with 1 µmol/L isoproterenol, the incidence of spontaneous Ca2+ waves/releases was more frequent in hiPSC-CMs of the irradiated groups, indicating arrhythmogenic activities at the single cell level. Increased transcript expression of mitochondrial biomarkers (LONP1, TFAM) and mtDNA-encoded genes (MT-CYB, MT-RNR1) was detected upon differentiation of hiPSC-CMs suggesting increased organelle biogenesis. Exposure of hiPSC-CM cultures to 10 cGy significantly upregulated MT-CYB and MT-RNR1 expression, which may reflect an adaptive response to ionizing radiation. Our results indicate that important aspects of differentiation of hiPSCs into cardiac myocytes may be affected by low fluences of densely ionizing radiations such as α-particles.

Effects of proton beam irradiation on mitochondrial biogenesis in a human colorectal adenocarcinoma cell line.

Proton beam therapy has recently been used to improve local control of tumor growth and reduce side-effects by decreasing the global dose to normal tissue. However, the regulatory mechanisms underlying the physiological role of proton beam radiation are not well understood, and many studies are still being conducted regarding these mechanisms. To determine the effects of proton beams on mitochondrial biogenesis, we investigated: mitochondrial DNA (mtDNA) mass; the gene expression of mitochondrial transcription factors, functional regulators, and dynamic-related regulators; and the phosphorylation of the signaling molecules that participate in mitochondrial biogenesis. Both the mtDNA/nuclear DNA (nDNA) ratio and the mitochondria staining assays showed that proton beam irradiation increases mitochondrial biogenesis in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced aggressive HT-29 cells. Simultaneously, proton beam irradiation increases the gene expression of the mitochondrial transcription factors PGC-1α, NRF1, ERRα, and mtTFA, the dynamic regulators DRP1, OPA1, TIMM44, and TOM40, and the functional regulators CytC, ATP5B and CPT1-α. Furthermore, proton beam irradiation increases the phosphorylation of AMPK, an important molecule involved in mitochondrial biogenesis that is an energy sensor and is regulated by the AMP/ATP ratio. Based on these findings, we suggest that proton beam irradiation inhibits metastatic potential by increasing mitochondrial biogenesis and function in TPA-induced aggressive HT-29 cells.

Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900MHz radiofrequency fields.

HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900MHz radiofrequency fields (RF) at 120µW/cm2 power intensity for 4h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2'-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells.

[Deletions in Mitochondrial DNA from the Peripheral Blood of Mayak PA Workers Exposed to Long-Term Ionizing Radiation].

The number of large deletions of mitochondrial DNA in whole peripheral blood of the former Mayak PA workers occupationally exposed to prolonged γ-radiation has been determined in the long term period after irradiation (mean cumulative dose 135.40 ± 22.03 cGy, age range at the time of blood sampling 67-76 years) and compared with the number of deletions in groups of "young" (19-33 years) and "adult" (66-73 years) individuals who had no contact with radiation sources. Samples of the total DNA from the peripheral blood were obtained from the Radiobiological Human Tissue Repository of the Southern Urals Biophysics Institute (Ozyorsk, Chelyabinsk region) and used for carrying out a long-distance PCR. The analysis of the data showed a statistically significant increase in the number of large deletions in the peripheral blood of "adult" donors of the control group as compared with the control group of "young" donors (51.6 and 14.3%, respec- tively). No statistically significant difference in the number of large deletions in the group of former Mayak PA workers occupationally subjected to prolonged exposure to γ-radiation as compared with the control do- nors of similar age was found (53.6 and 43.8% respectively).

Cell-free DNA in the urine of rats exposed to ionizing radiation.

Investigation of cell-free DNA (cf-DNA) in body fluids, as a potential biomarker for assessing the effect of ionizing radiation on the organism, is of considerable interest. We investigated changes in the contents of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) in the urine of X-ray-exposed rats. Assays of cf-mtDNA and cf-nDNA were performed by a real-time PCR in rat urine collected before and after irradiation of animals with doses of 3 and 5 Gy. We also determined the presence of mutations in urine cf-mtDNA, as recognized by Surveyor nuclease. A sharp increase in cf-mtDNA and cf-nDNA in the urine of irradiated rats was observed within 24 h after exposure, followed by a decrease to normal levels. In all cases, the contents of cf-mtDNA fragment copies (estimated by gene tRNA) were significantly higher than those of cf-nDNA estimated by gene GAPDH. A certain portion of mutant cf-mtDNA fragments was detected in the urine of exposed rats, whereas they were absent in the urine of the same animals before irradiation. These preliminary data also suggest that the increased levels of urine cf-mtDNA and cf-nDNA may be a potential biomarker for noninvasive assessment of how the organism responds to ionizing radiation influence.

High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage.

Long-term biological effects induced by ionizing radiation--implications for dose mediated risk.

Ionizing radiations are considered to be risk agents that are responsible for the effects on interaction with living matter. The occurring biological effects are due to various factors such as: dose, type of radiation, exposure time, type of biological tissue, health condition and the age of the person exposed. The mechanisms involved in the direct modifications of nuclear DNA and mitochondrial DNA are reviewed. Classical target theory of energy deposition in the nucleus that causes DNA damages, in particular DNA double-strand breaks and that explanation of the biological consequences of ionizing radiation exposure is a paradigm in radiobiology. Recent experimental evidences have demonstrated the existence of a molecular mechanism that explains the non-targeted effects of ionizing radiation exposure. Among these novel data, genomic instability and a variety of bystander effects are discussed here. Those bystander effects of ionizing radiation are fulfilled by cellular communication systems that give rise to non-targeted effects in the neighboring non irradiated cells. This paper provides also a commentary on the synergistic effects induced by the co-exposures to ionizing radiation and various physical agents such as electromagnetic fields and the co-exposures to ionizing radiation and chemical environmental contaminants such as metals. The biological effects of multiple stressors on genomic instability and bystander effects are also discussed. Moreover, a brief presentation of the methods used to characterize cyto- and genotoxic damages is offered.

Mitochondrial genetic abnormalities after radiation exposure.

Because mitochondria are prone to oxidative stress, damage to their DNA might provide a record of radiation exposure. We measured the effect of gamma radiation on mitochondrial DNA (mtDNA) copy number and common deletion (mito-CD) mutations using Beas-2B and HFL-1 cells lines and C3H/HeJ mice exposed to total-body irradiation (TBI) and sub-TBI. DNA was extracted 5 days after cell irradiation or 12 months after animal exposure. We found that: (1) natural ratios of mtDNA/nDNA and mito-CD/mtDNA varied between cell lines; (2) mtDNA copy number decreased in Beas-2B and increased in HFL-1 following 2 Gy; (3) mito-CD in both cell lines increased after 2 Gy; (4) in aged mice, the natural ratios of mtDNA/nDNA varied from 0.723 to 8.146 in different tissues; (5) in kidney tissue, TBI and sub-TBI mildly increased mtDNA copy number but substantially increased mtDNA-CD; and (6) in liver tissue, TBI and sub-TBI induced a slight increase in mtDNA copy number and a larger increase in mtDNA-CD. These findings indicate that mtDNA copy number varies in time by cell type, but there is a substantial and sustained increase in mtDNA mutations that occurs to different degrees in different tissues and cells following irradiation.

Mitochondrial D310 D-Loop instability and histological subtypes in radiation-induced cutaneous basal cell carcinomas.

BACKGROUND: Basal cell carcinoma (BCC) is the most frequent skin cancer. An elevated prevalence of BCC has been associated with radiation, namely after the Tinea capitis epilation treatment, being these tumors described as more aggressive. Mitochondrial DNA (mtDNA) mutations have been reported in many human tumors, but their occurrence in BCC is poorly documented. OBJECTIVE: The purpose of this work was to evaluate BCC histological subtypes in individuals subjected to X-ray epilation for Tinea capitis treatment when compared to non-irradiated patients. Moreover we also wanted to evaluate mitochondrial D-Loop instability in both groups of BCCs in order to compare the frequency of D-Loop mutations in post-irradiation BCC versus sporadic BCC. METHODS: 228 histological specimens corresponding to BCCs from 75 irradiated patients and 60 non-irradiated patients were re-evaluated for histological subtype. Subsequently, we sequenced the D-Loop 310 repeat in blood, oral mucosa, tumor lesions and, whenever available, non-tumoral adjacent tissue from these patients. RESULTS: The infiltrative subtype of BCC, considered to be more aggressive, was significantly more frequent in irradiated patients. BCC D-Loop D310 mutation rate was significantly higher in irradiated BCCs than in the non-irradiated ones. Moreover, it was associated with a higher irradiation dose. The presence of mtDNA heteroplasmy in patients' blood was associated with a higher mutation rate in the BCCs suggesting that a more unstable genotype could predispose to mtDNA somatic mutation. CONCLUSIONS: Our results suggest that radiation-induced BCCs may be considered to be more aggressive tumors. Further studies are needed to clarify the role of mtDNA D-Loop mutations in tumors from irradiated patients.