Assessment of epigenetic mechanisms and DNA double-strand break repair using laser micro-irradiation technique developed for hematological cells.

BACKGROUND: Certain tumors rely heavily on their DNA repair capability to survive the DNA damage induced by chemotherapeutic agents. Therefore, it is important to monitor the dynamics of DNA repair in patient samples during the course of their treatment, in order to determine whether a particular drug regimen perturbs the DNA repair networks in cancer cells and provides therapeutic benefits. Quantitative measurement of proteins and/or their posttranslational modification(s) at DNA double strand breaks (DSBs) induced by laser microirradiation provides an applicable diagnostic approach to examine DNA repair and its dynamics. However, its use is restricted to adherent cell lines and not employed in suspension tumor cells that include the many hematological malignancies. METHODS: Here, we report the development of an assay to laser micro-irradiate and quantitatively measure DNA repair transactions at DSB sites in normal mononuclear cells and a variety of suspension leukemia and lymphoma cells including primary patient samples. FINDINGS: We show that global changes in the H3K27me3-ac switch modulated by inhibitors of Class I HDACs, EZH2 methyltransferase and (or) H3K27me3 demethylases do not reflect the dynamic changes in H3K27me3 that occur at double-strand break sites during DNA repair. INTERPRETATION: Results from our mechanistic studies and proof-of-principle data with patient samples together show the effectiveness of using the modified micro-laser-based assay to examine DNA repair directly in suspension cancer cells, and has important clinical implications by serving as a valuable tool to assess drug efficacies in hematological cancer cells that grow in suspension.

The Phase-Shifting Effect of Bright Light Exposure on Circadian Rhythmicity in the Human Transcriptome.

Light is a potent synchronizer of the central circadian clock; however, the effect of light exposure on peripheral gene expression is largely unknown. The objective of this study was to explore the effect of bright light exposure on genome-wide peripheral gene expression levels during a 4-day simulated night shift protocol in which the habitual sleep period is delayed by 10 h. Eleven healthy participants (mean age, 24 years; range, 18-30; 10 men/1 woman) were studied under controlled laboratory conditions. Three participants were exposed to bright light (~6,500 lux) for 8 h during the nightly waking period, while the other 8 were maintained in dim-light conditions (~10 lux). At baseline and on the fourth day after the shift in the sleep period, blood samples were collected during two 24-h measurement periods. RNA was extracted from peripheral blood mononuclear cells (PBMCs) and used to obtain transcriptomic data. Using 2 independent approaches to determine phase shifts among rhythmically expressed genes after the shifted sleep schedule compared with baseline, we found that the average phase delay in the bright light group was approximately 8 to 9 h, whereas the average phase delay in the control group was approximately 1 to 2 h, both at the group level and at the individual level. In line with these findings, further analysis using partial least squares regression indicated that the peripheral circadian transcriptome of PBMCs was predictive of the phase of the central circadian pacemaker after only 3 days of bright light exposure. These results indicate that bright light exposure exerts a phase-shifting effect on the circadian transcriptome in PBMCs with a magnitude similar to its effect on the central circadian clock.

Effects of exposure to electromagnetic field from 915 MHz radiofrequency identification system on circulating blood cells in the healthy adult rat.

We investigated whether exposure to the 915 MHz radiofrequency identification (RFID) signal affected circulating blood cells in rats. Sprague-Dawley rats were exposed to RFID at a whole-body specific absorption rate of 2 W/kg for 8 h per day, 5 days per week, for 2 weeks. Complete blood counts were performed after RFID exposure, and the CD4+ /CD8+ ratio was determined by flow cytometry. The number of red blood cells (RBCs) and the values of hemoglobin, hematocrit, and RBC indices were increased in the RFID-exposed group compared with those in the cage-control and sham-exposed groups (P < 0.05). However, the RBCs and platelet numbers were within normal physiologic response ranges. The number of white blood cells, including lymphocytes, was decreased in RFID-exposed rats. However, there was no statistically significant difference between the sham-exposed and RFID-exposed groups in terms of T-cell counts or CD4+ /CD8+ ratio (P > 0.05). Although the number of circulating blood cells was significantly altered by RFID exposure at a whole-body specific absorption rate of 2 W/kg for 2 weeks, these changes do not necessarily indicate that RFID exposure is harmful, as they were within the normal physiological response range. Bioelectromagnetics. 39:68-76, 2018. © 2017 Wiley Periodicals, Inc.

Gamma radiation at a human relevant low dose rate is genotoxic in mice.

Even today, 70 years after Hiroshima and accidents like in Chernobyl and Fukushima, we still have limited knowledge about the health effects of low dose rate (LDR) radiation. Despite their human relevance after occupational and accidental exposure, only few animal studies on the genotoxic effects of chronic LDR radiation have been performed. Selenium (Se) is involved in oxidative stress defence, protecting DNA and other biomolecules from reactive oxygen species (ROS). It is hypothesised that Se deficiency, as it occurs in several parts of the world, may aggravate harmful effects of ROS-inducing stressors such as ionising radiation. We performed a study in the newly established LDR-facility Figaro on the combined effects of Se deprivation and LDR γ exposure in DNA repair knockout mice (Ogg1(-/-)) and control animals (Ogg1(+/-)). Genotoxic effects were seen after continuous radiation (1.4 mGy/h) for 45 days. Chromosomal damage (micronucleus), phenotypic mutations (Pig-a gene mutation of RBC(CD24-)) and DNA lesions (single strand breaks/alkali labile sites) were significantly increased in blood cells of irradiated animals, covering three types of genotoxic activity. This study demonstrates that chronic LDR γ radiation is genotoxic in an exposure scenario realistic for humans, supporting the hypothesis that even LDR γ radiation may induce cancer.

Effects of 10-GHz microwaves on hematological parameters in Swiss albino mice and their modulation by Prunus avium.

The objective of this study was to investigate the modulatory role of Prunus avium fruit extract (PAE) on several blood parameters after exposure to 10-GHz microwaves. Swiss albino mice from an inbred colony were selected and divided into 3 groups. Mice in group I served as the control; they were placed in a Plexiglas cage (without energizing the system) for 2 hours/day for 30 consecutive days. Group II mice were exposed to 10-GHz microwaves for 2 hours/day for 30 consecutive days. Mice in group III received PAE (500 mg/kg/body weight) orally once daily 1 hour before exposure to 10-GHz microwaves (2 hours/day) for 30 consecutive days. After 30 days of treatment, blood samples were collected from mice in all groups and analyzed. Hemoglobin, monocytes, packed cell volume, red blood cells, mean corpuscular hemoglobin concentration declined significantly (P ≤ 0.01), whereas white blood cells, lymphocytes, erythrocyte sedimentation rate, and mean corpuscular volume increased significantly (P ≤ 0.01) compared to the control group (group I). Cholesterol, alkaline phosphatase, and lipid peroxidation also increased significantly (P ≤ 0.01). Depletion in blood sugar, total protein, acid phosphatase, and glutathione levels was noted after microwave exposure compared with levels in the sham-exposed (control) mice. Histopathological alterations in blood cells also were seen. Signs of improvements in the hematological, biochemical, and histopathological parameters were recorded in group III, where PAE was supplemented before exposure. Exposure to microwaves influences hematological parameters, which could be ameliorated by the supplementation of PAE.

Production of interleukin-5, -10 and interferon-γ in cord blood is strongly associated with the season of birth.

BACKGROUND: The effect of labour and different labour-related factors on the cord blood (CB) cell cytokine production is still relatively unknown. OBJECTIVE: To study the relationships between the production of IL-5, IL-10 and IFN-γ in CB samples and maternal, early neonatal and birth-related factors. METHODS: Whole-blood samples were collected after birth (n=423) and they were stimulated for 24 and 48 h with a combination of phorbol ester and ionomycin. Production of IL-5, IL-10 and IFN-γ was determined using ELISA. Maternal, early neonatal and birth-related variables were recorded prospectively during pregnancy, and during and after delivery. RESULTS: After multivariable adjustment for confounders, the strongest predictor of IL-5, IL-10 and IFN-γ production in CB cell samples was the season of birth. Children born in the spring had significantly lower cytokine responses compared with those born in the fall. IL-5 production was inversely associated with female gender of the child and maternal smoking. If corrections for white blood cell (WBC) counts were not performed, IL-5 production was also significantly associated with the mode of delivery. Respectively, the production of IL-10 and IFN-γ was inversely associated with prostaglandin induction before birth. CONCLUSION: Environmental exposure to pollen and ultraviolet irradiation during gestation may have an effect on the cytokine profile of the offspring in CB because children born in the spring or winter showed the lowest IL-5, IL-10 and IFN-γ responses. The production of IL-10 and IFN-γ was also inversely associated with prostaglandin labour induction before birth. Other labour-related factors were not significantly associated with production of IL-5, IL-10 and IFN-γ after WBC count correction.

Antioxidant and radiation antagonistic effect of Saraca indica.

The present study was undertaken to investigate the radiation mitigating effect of hydro-alcoholic extract of Saraca indica (SIE) against mice exposed to whole body gamma radiation. Free radical scavenging ability by SIE was demonstrated using hydroxyl, superoxide anion, ABTS*+ and DPPH radicals generated in vitro. A significant increase in the animal survival (dose reduction factor = 1.39), inhibition in the decline of hematological parameters as well as increased number of spleen colony-forming units was observed when SIE was administered prior to radiation. SIE pretreatment increased the levels of glutathione, glutathione S- trans-ferase, catalase and lowered lipid peroxidation. Our findings for the first time demonstrate the protective potential of SIE against radiation induced syndromes with an optimal dose of 400 mg/kg b.wt. The radiation mitigating effect may be attributed to the mechanisms such as free radical scavenging, elevation in antioxidant status, and reduction in lipid peroxidation.

Radiomodifying effect of organic grape juice supplementation on hematological parameters and organ weight in whole-body X-irradiation in rats.

The aim of this study is testing black grape juice as a radiomodifier against whole body X-irradiation using an animal model. Sixteen male Wistar rats were divided into four groups where two were irradiated by X-rays from a 200 kV machine specially designed to biological samples. Animals were fed ad libitum and drank voluntarily 2-10 ml a day of grape juice or placebo (isocaloric glucose and fructose solution) for one week before and two weeks after 6 Gy X-irradiation when they were sacrificed. Results have shown a significant liver weight loss in irradiated placebo group only while grape juice one has presented no losses. Hematological analysis showed typical abnormalities for ionizing radiation exposure, including early leucopenia and anemia. The intake of grape juice induced an increase in granulocyte percent count.

Biological effects of Echinacea purpurea on human blood cells.

The aim of this study was to investigate radioprotective properties of Echinacea purpurea tablets in vivo. We analysed lymphocyte chromosome aberrations (CA), micronuclei (MN), apoptosis of leukocytes and haematological parameters in a group of radiation workers who were identified as carrying dicentric chromosomes in their lymphocytes. All radiation workers were taking two 275 mg Echinacea tablets b.i.d., according to a pharmacist's recommendation. All parameters were analysed before and after the two-week treatment. At the end of the treatment lymphocyte CA frequency dropped significantly, and the number of apoptotic cells increased. The inverse lymphocyte-to-granulocyte ratio at the beginning of the study changed to normal at its end. In conclusion, biological effects observed after administration of Echinacea purpurea preparation suggest that it may be beneficial for the prevention of adverse health effects in workers exposed to ionising radiation.

Radioprotective effect of Lyophyllum decastes and the effect on immunological functions in irradiated mice.

In this study, to explore the radiation protection effects of Lyophyllum Decastes Sing (LDS), a hot distilled-water extract of LDS was orally administered at a dosage of 250mg/kg every other day for a period of 2 weeks in irradiated mice. An automatic blood cell counter was used to measure white blood cells (lymphocytes, monocyte, and granulocytes) one day before X-ray irradiation, and 3 hours, 12 hours, 24 hours, 3 days, 7 days, 15 days and 30 days after irradiation. The Dunnett test was used to examine statistical significance of differences. The peripheral blood cell counts in the Lyophyllum-administered non-irradiation group revealed an increase in the numbers of leukocytes, lymphocytes and monocytes. For 2 Gy whole body radiation, a significant statistical difference was found between the X-ray group and the Lyophyllum plus X-ray group in the numbers of leukocytes, lymphocytes and monocytes. The results suggest that Lyophyllum restrains blood cell-count falling after irradiation, which is probably mediated at least in part by hemopoietic function, and NK and LAK activities seems to play a role in preventing secondary infections associated with irradiation.

Oral oxymetholone reduces mortality induced by gamma irradiation in mice through stimulation of hematopoietic cells.

Oxymetholone is a 17alpha -alkylated anabolic-androgenic steroid. This drug can stimulate bone marrow cells and increase the blood cells in the peripheral blood vessels. It has been used for the treatment of anemia caused by low red cell production. Since oxymetholone has hematopoietic effect, we studied radioprotective effects of this drug in mice. In this study, we determined percentage of survival, dose-reduction factor (DRF) and hematological parameters in irradiated mice which treated with or without oxymetholone. Oxymetholone administrated at different doses 80, 160, 320, 640 mg/kg by gavages at 24 h before 8 Gy gamma irradiation. At 30 days after treatment, the following percentage of animals survival in each group was as: 80 mg/kg, 50%; 160 mg/kg, 50%; 320 mg/kg, 55%; 640 mg/kg, 75% and vehicle, 15%. Percentage of survival increased in all of treated groups statistically compared with irradiated-vehicle group. In the groups treated by oxymetholone, maximum protection was realized at 640 mg/kg. In order to calculate the DRF for oxymetholone, mice were exposed to whole-body gamma irradiation with dose ranges between 5.83 and 11.23 Gy. The probit line for oxymetholone-treated mice was shifted to the right with a DRF of 1.14. In mice exposed to whole-body gamma-irradiation (4 Gy), an oral administration of 640 mg/kg oxymetholone ameliorated radiation-induced decreases in circulating platelets and erythrocytes, but had a less effect on total number of WBC. These results demonstrate that oxymetholone stimulates myelopoiesis and thrombocytopenia and enhances survival in mice after ionizing radiation.

Effects of Aegle marmelos (L.) Correa on the peripheral blood and small intestine of mice exposed to gamma radiation.

The radioprotective effect of bael (Aegle marmelos, AME) extract was studied in Swiss albino mice against radiation-induced changes in the peripheral blood, spleen colony forming units, and intestinal mucosa. The mice were treated with 250 mg/kg body weight of AME orally once daily for five consecutive days before exposure to an acute dose of 7 Gy of gamma radiation after the last administration. The peripheral blood was collected and evaluated for red blood cell (RBC), hemoglobin, total leukocyte count (TLC), and lymphocyte count on days one and seven postirradiation. The nucleated bone marrow cells were isolated and tested for colony-forming units (CFUs) in spleen at days one and seven. AME protected mice against the radiation-induced decline in hemoglobin, total leukocyte, and lymphocytes counts and the clonogenicity of hemopoietic progenitor cells assessed by the exogenous spleen colony-forming assay. Irradiation of mice caused a significant decline in the villus height and crypt number with an increase in goblet and dead cells in the small intestine, where the maximum changes were observed on day one postirradiation, indicating a severe damage, and signs of recovery at day seven postirradiation. Treatment of mice with AME before irradiation elevated the peripheral cell count as well as villus height and the crypt number accompanied by a decline in goblet and dead cells when compared with the irradiation control. The recovery and regeneration were faster in AME pretreated animals than the irradiation alone. AME pretreatment significantly decreased lipid peroxidation accompanied by a significant elevation in the GSH concentration in the mouse intestine. The data clearly indicate that the AME significantly reduced the deleterious effect of radiation in the intestine and bone marrow of mouse and could be a useful agent in reducing the side effects of therapeutic radiation.

[Chronic radiation damage by uranium and plutonium nuclear fission products].

Chronical radiation sickness is a special form of the radiation damage. It occurs when doses of chronical irradiation exceed their values established for professionals. The sickness is well studied in clinical observations. It may take place also if uranium and plutonium nuclear fission products (NFP) enter the organism. In the last case the chronical radiation sickness practically is not investigated. In the article we present the results of the experimental studies on dogs of the damage caused by NFP.

Molecular evidence of tumour cell removal from salvaged blood after irradiation and leucocyte depletion.

Intra-operative autologous blood recovery offers many advantages. However, blood salvage during cancer surgery is of limited use due to the potential presence of circulating tumour cells. It was the aim of this study to show that intra-operative salvage blood can be freed of cells and cellular DNA after leucoreduction by filtration and irradiation of washed blood. Known amounts of tissue culture derived from carcinoma, melanoma and osteosarcoma were added to whole blood bags. This mixture was then submitted to washing, leucoreduction and irradiation. Samples were studied stepwise in relation to the integrity and size of DNA by the polymerase chain reaction (PCR). After filtration and irradiation, PCR targeting the beta-globin gene (268 bp amplicon) was negative. Our results were corroborated by studying plasma samples added with tumoural cells. Using PCR methodology, we showed the absence of DNA from cells in experimentally contaminated blood and plasma bags after filtration and irradiation. This experimental study is an effort to ensure the safety of intra-operative autologous transfusion.

Inhibition of angiotensin I-converting enzyme induces radioprotection by preserving murine hematopoietic short-term reconstituting cells.

Angiotensin I-converting enzyme (ACE) inhibitors can affect hematopoiesis by several mechanisms including inhibition of angiotensin II formation and increasing plasma concentrations of AcSDKP (acetyl-N-Ser-Asp-Lys-Pro), an ACE substrate and a negative regulator of hematopoiesis. We tested whether ACE inhibition could decrease the hematopoietic toxicity of lethal or sublethal irradiation protocols. In all cases, short treatment with the ACE inhibitor perindopril protected against irradiation-induced death. ACE inhibition accelerated hematopoietic recovery and led to a significant increase in platelet and red cell counts. Pretreatment with perindopril increased bone marrow cellularity and the number of hematopoietic progenitors (granulocyte macrophage colony-forming unit [CFU-GM], erythroid burst-forming unit [BFU-E], and megakaryocyte colony-forming unit [CFU-MK]) from day 7 to 28 after irradiation. Perindopril also increased the number of hematopoietic stem cells with at least a short-term reconstitutive activity in animals that recovered from irradiation. To determine the mechanism of action involved, we evaluated the effects of increasing AcSDKP plasma concentrations and of an angiotensin II type 1 (AT1) receptor antagonist (telmisartan) on radioprotection. We found that the AT1-receptor antagonism mediated similar radioprotection as the ACE inhibitor. These results suggest that ACE inhibitors and AT1-receptor antagonists could be used to decrease the hematopoietic toxicity of irradiation.

[The role of erythrocyte rheological determinants in the regulation of bloodflow structure]. / Rol' reologicheskikh determinant éritrotsitov v reguliatsii struktury krovotoka.

Correlations of rheological determinants of the erythrocyte viscous characteristics were studied in normal subjects and coronary patients treated traditionally and with photohemotherapy. A rigid relationship between erythrocyte deformability and aggregation was detected in the patients. Blood exposure to He-Ne laser and UV is a potent method for correcting the blood rheology. Physiological significance of the rigid relationship of erythrocyte deformability and aggregation consists in the maintenance of the structure of flowing blood, characteristic of its native status, and represents an additional mechanism of realization of the adaptive potential of blood viscosity regulation in mammals.

[Transfusion-associated graft-versus-host disease guideline on gamma irradiation of blood components]. / Doença do enxerto contra hospedeiro pós-transfusional-guia para irradiação gama de hemocomponentes.

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare and usually fatal syndrome. Clinical manifestations are fever, maculopapular skin rash, nausea, vomiting, diarrhea, hepatitis and pancytopenia owing to bone marrow hypoplasia. It can occur in recipients with severe immunosuppression and in immunocompetent recipients after transfusion of cellular components from HLA homozygous donor to recipients heterozygous for that HLA haplotype. The diagnosis is made by clinical manifestation and skin biopsy. Antithymocyte globulin and high dose systemic corticosteroids are both the most used therapy. The back of knowledge about this syndrome, the rapid evolution and the absence of treatment response are related to patients bad evolution. Gamma irradiation of blood products has been the mainstay of TA-GVHD prevention. Dose of 2500 cGy is required to completely inactivate T cells. Irradiation damage red cells membrane and the red celis units can not be storage for long time after irradiation. High potassium levels is the mainly change in red cells units. White cell-reduction filters do not prevent TA-GVHD and gamma irradiation does not prevent alloimmunization or blood reactions. Only cellular components like whole blood, red cells, platelets and granulocytes need be irradiated. Ali blood components should be irradiated to: first or second-degree relatives, patients need HLA-matched platelets, recipients of allogeneic or autologous bone marrow transplantation, patients with Hodgkin's disease, patients treated with purine analogue drugs, intrauterine transfusion, pre-term infants and when congenítal immunodeficiency states is suspected. It is recommended irrradiated blood to patients with neoplastic disease when they receive intensive chemotherapy.

[Indometofen causes biochemical changes in the blood cells characteristic for a radioresistant state of the body]. / Indometofen vyzyvaet v kletkakh krovi biokhimicheskie izmeneniia, kharakternye dlia radiorezistentnogo sostoianiia organizma.

The DNA and RNA contents, RNA/DNA ratio, and spontaneous and latex-induced oxidant activity indices of the whole blood were studied in the nitroblue tetrazolium test of mono- and polymorphonuclear blood leucocytes of intact dogs after injection of lipopolysaccharide pyrogenal. Significant changes in the above parameters were revealed for radioresistant (survived) and radiosensitive (lost) animals exposed to a subsequent prolonged gamma irradiation with a lethal dose of 7.64 Gy (LD75/45). Peroral introduction of 30 mg/kg indometofen (an indole analog of tamoxifen), which is a potential radioprotector, to dogs increased the survival rates of the irradiated dogs up to 93% and aided in the adaptive biochemical changes in the nuclear cell compartment of blood to induce a radioresistant status of the organism.