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Cellular DNA damage and lipid peroxidation after whole body gamma irradiation and treatment with Haberlea rhodopensis extract in rabbits

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Abstract

The aim of the study is to investigate radioprotective effects of total extract from Haberlea rhodopensis (a natural plant in the Balkan region) in vivo. For that, alkaline comet assay was performed on peripheral lymphocytes and plasma MDA (a marker of lipid peroxidation) concentrations were determined in New Zealand white rabbits (5 animals per group) 24 hours after whole body exposure to.-radiation (2.0 Gy) or sham irradiation eventually associated to intramuscular injection of 0.24 g/kg H. rhodopensis extract 2 hours before or 30 minutes after irradiation. The comet frequency, reflecting cellular DNA damage, was dramatically increased in lymphocytes from irradiated rabbits but this increase was significantly limited (by 40%) when H. rhodopensis extract was administered preliminary to irradiation. In addition, plasma MDA concentrations remained low in gamma-exposed rabbits treated before or after by H. rhodopensis extract whereas they were markedly elevated in animals only submitted to gamma-radiations. The results demonstrate the direct and indirect radioprotective effects in vivo of H. rhodopensis extract probably mediated by some antioxidant compounds.

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... Haberlea rhodopensis pretreatment in irradiated lymphocytes of New Zealand rabbits showed a decrease in chromosomal aberrations, micronucleus cells, and aberrant cells at a dose of 120 mg/kg of BW (Svetlana Yordanova Georgieva, 2004). The plant also decreases oxidative stress by scavenging free radicals and decreasing LPO (GEORGIEVA et al., 2012;Popov et al., 2013). These activities, along with the ability to regulate DNA repair genes with no significant toxicity, favor radioprotection. ...
... Human lymphocytes Free radical scavenging (Vrinda and Uma Devi, 2001) 3 Vitis vinifera 10 mL/day X-ray Rats Antioxidant activity (Andrade et al., 2011) 4 Allium cepa 5 mL/kg 1050 kV X-ray Rats Decreased oxidative stress (Kenneth et al., 2014) (GEORGIEVA et al., 2012;Popov et al., 2013;Georgieva, 2004) 12 Nigella sativa extract and oil 250 mg/kg 8 Gy γ-radiation ...
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Radiation can be lethal at high doses, whereas controlled doses are useful in medical applications. Other applications include power generation, agriculture sterilization, nuclear weapons, and archeology. Radiation damages genetic material, which is reflected in genotoxicity and can cause hereditary damage. In the medical field, it is essential to avoid the harmful effects of radiation. Radiation countermeasures and the need for radioprotective agents have been explored in recent years. Considering plants that evolve in radiative conditions, their ability to protect organisms against radiation has been studied and demonstrated. Crude extracts, fractioned extracts, isolated phytocompounds, and plant polysaccharides from various plants have been used in radioprotection studies, and their efficiency has been proven in various in vitro and in vivo experimental models. It is important to identify the mechanism of action to develop a potent plant-based radioprotective agent. To identify this protective mechanism, it is necessary to understand the damage caused by radiation in biological systems. This review intends to discuss the effects of ionizing radiation on biological systems and evaluate plant-based radioprotectants that have tested thus far as well as their mechanism of action in protecting against the toxic effects of radiation. From the review, the mechanism of radioprotection exhibited by the plant-based products could be understood. Meanwhile, we strongly suggest that the potential products identified so far should undergo clinical trials for critically evaluating their effects and for developing an ideal and compatible radioprotectant with no side-effects.
... These are important aspects to be considered whenever in vivo experiments are performed. Analogous results regarding radioprotection were obtained by Georgieva et al. (2012 , Table 1 Biological studies with leaf extracts of Haberlea rhodopensis. (continued on next page) Y.N. ...
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Ethnopharmacological relevance: Haberlea rhodopensis (HR) use dates back to the Thracian and Roman periods. Bulgarians call it Orpheus flower and exploit its leaves for making tea and extracts with detoxifying, tonic, restorative and rejuvenating effects. HR was traditionally applied in wound healing and treatment of cattle diseases. Aim of the study: The general aim of the review was to analyze the progress of phytochemical and pharmacological studies on HR, focusing on its radioprotective and immunomodulating effects. Materials and methods: The main source material for the review was collected using several global search engines with the phrase: Haberlea rhodopensis, as well as Bulgarian books and dissertations. Results: HR metabolite profile includes large amounts of free sugars, polyols, polysaccharides (PS), flavonoids, phenolic acids and carotenoids. The radioprotective effect of 70% ethanolic leaf extract (70HREE) is explained by preservation of lymphocytes, other blood cells and testicular tissue from aberration under γ-radiation via stimulation of antioxidant enzymes and neutralization of free radicals. The extract immunomodulating activity results from raised antibody response, stem and neutrophil cell count, complement system activation, anti-tumour and anti-inflammatory effects. The detoxifying, restorative, rejuvenating and wound healing plant properties known to ethnomedicine were supported by radioprotective and immunomodulating studies. Conclusions: Metabolites of phenolic origin involved in HR resurrection are supposed to contribute to its radioprotective, immunomodulatory, anti-mutagenic and anti-aging effects. However, there is no chemical characterization of 70HREE in the investigations with humans and animals. Structure-activity relationship studies on HR immunomodulating and radioprotective compounds, and on their mode of action are required. They should include not only phenols but PS and other unexplored molecules. The metabolic activity of phagocytes, platelets and lymphocytes triggered by HR extracts has to be examined to elucidate their immunostimulatory potential. HR formulations can be tested in cosmetic, food and medical products as adjuvants to treat infectious, chronic inflammatory and tumour diseases, and especially in patients undergoing radiotherapy.
... The results showed that after exposure to ionising radiation (2.0 Gy) a reliable increase in the percentage of comets was found, about sixfold higher than that in the control group, and 2.7 times higher than that in the group, treated with plant extract. The administration of H. rhodopensis extract before whole-body irradiation of rabbits induced a decrease in the comets with approximately 40% in comparison with the group, subject only to irradiation (Georgieva et al. 2012b). ...
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This review discusses the potential of Haberlea rhodopensis as a food additive. The following are described: plant distribution, reproduction, cultivation, propagation and resurrection properties; extraction, isolation and screening of biologically active compounds; metabolite changes during dehydration; phytotherapy-related properties such as antioxidant potential and free radical-scavenging activities, antioxidant skin effect, antibacterial activity, cytotoxic activity and cancer-modulating effect, radioprotective effect, chemoprotective effect, immunologic effect; present use in homoeopathy and cosmetics, pharmacological and economical importance; perspectives based on the ethnobotanical data for medicinal, cosmetic or ritual attributes. H. rhodopensis showed unique medical and pharmaceutical potential, related to antioxidant, antimicrobial, antimutagenic, anticancer, radioprotective, chemoprotective and immunological properties. H. rhodopensis extracts lack any cytotoxic activity and could be used in phytotherapy. The metabolic profiling of H. rhodopensis extracts revealed the presence of biologically active compounds, possessing antiradical and other physiological activities, useful for design of in vitro synthesised analogues and drugs.
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Georgieva, S., Gencheva, D., Popov, B., Grozeva, N. & Zhelyazkova, M. (2019). Radioprotective action of resurrection plant Haberlea rhodopensis Friv. (Gesneriaceae) and role of fl avonoids and phenolic acids. Bulg. J. Agric. Sci., 25 (Suppl. 3), 158-168 Haberlea rhodopensis Friv. (Gesneriaceae) is a Balkan endemic and resurrection glacial relict plant which is distributed in Bulgaria (Rhodope Mountains, Sredna Gora Mt. and Central Balkan). Currently, there is a huge interest in Haberlea rhodopen-sis, and several scientifi c studies aimed at the isolation and identifi cation of its active components as well as the investigation of the pharmacological eff ects and possibilities of the use of Haberlea rhodopensis as a medical plant have been conducted. The main eff ects of Haberlea rhodopensis include radioprotective, anti-mutagenic, antioxidant and anti-ageing properties. This review emphasises the radioprotective potential of Haberlea rhodopensis and focuses on the biological properties of its ingredients like fl avonoid aglycones and glycosides as well as phenolic acids in relation to their capacity to capture free radicals and reduce oxidative stress. More research оn animals and humans are needed for clarifi cation of the mechanisms of action and the eventual side eff ects of Haberlea thodopensis and its compounds as radioprotective agents.
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Disufiram (a drug used for the treatment of alcoholism) protected microsomal membranes and plasmid DNA against damages induced by gamma-radiation. The peroxidation of membrane lipids increased linearly with the radiation dose up to 600 Gy, and the presence of disulfiram inhibited membrane lipid peroxidation as assayed by the presence of thiobarbituric acid reacting substances. The reduction of the quantity of the supercoiled (ccc) form of plasmid pBR322 DNA is directly related to the radiation-induced damage, particularly to DNA strand breaks. There was a complete protection of plasmid DNA when exposed to gamma-radiation in the presence of disufiram (0.1 mM) at 300 Gy. This drug also protected deoxyribose against damages caused by hydroxyl radicals produced by the Fenton reaction. The administration of DSF to mice prior to whole-body radiation exposure (4 Gy) resulted in a reduction of peroxidation of membrane lipids in mice liver as well as a decrease in radiation-induced damage to cellular DNA, as assayed by single-cell gel electrophoresis (comet assay). The results thus suggest the possible use of DSF as a radioprotector.
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The radioprotective effects of citrus extract were investigated by using the micronucleus test for anticlastogenic and cell proliferation activity. A single intraperitoneal (i.p.) injection of citrus extract (Citrus aurantium var. amara) at 250, 500, 1000 mg/kg body weight 1 h prior to gamma-ray irradiation (1.5 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCE(S)) and normochromatic erythrocytes (MnNCE (S)). All three doses of citrus extract significantly reduced the frequencies of MnPCEs and MnNCEs in mice bone marrow compared to non-drug-treated irradiated control (p < 0.005-0.05). The optimum dose for protection in mouse was 250 mg/kg to protect mice bone marrow 2.2-fold against the side effects of gamma-irradiation with respect to the non-drug-treated irradiated control. The flavonoids were contained in citrus extract, probably to show protective activity, and reduced the clastogenic effect of radiation on mice bone marrow. Therefore fruits and vegetables contain flavonoids to be useful as protective effects under such stress conditions as irradiation.
Article
Chlorophyllin (CHL) has been examined as an antioxidant/radioprotector in splenic lymphocytes from BALB/c mice. CHL inhibited lipid peroxidation induced by 2,2'-azobis(2-propionimidinedihydrochloride) (AAPH) in lymphocytes in vitro. It also partially prevented radiation-induced suppression of mitogenic stimulation of lymphocytes in vitro. Generation of intracellular reactive oxygen species (ROS) by radiation or AAPH was measured as oxidation of dichlorodihydrofluorescein diacetate (H(2)DCF-DA) using flow cytometry. Addition of CHL to lymphocytes in vitro significantly inhibited the increase in intracellular ROS. Further, lymphocytes from mice treated with CHL (100-400 microg/gbw i. p.) showed varying levels of ROS depending on the dose and the time (24 to 72 h) after injection. The extent of radiation-induced apoptosis and suppression of concanavalin A (con A)-induced mitogenesis ex vivo corresponded with changes in ROS levels in CHL-administered mice. Antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) were also estimated in lymphocytes from CHL-treated mice. CHL offered protection against whole body irradiation (WBI)-induced lipid peroxidation and apoptosis in lymphocytes at all the time points studied. These results demonstrate antioxidant effect of CHL in vivo.
Article
To investigate the radioprotective activity of a leaf extract of bael leaf (Aegle marmelos) (AME) in mice exposed to different doses of gamma-radiation. The acute toxicity of AME was evaluated in Swiss albino male mice administered various intraperitoneal single doses of AME. For radioprotection studies, mice were administered different doses, 0, 5, 10, 15, 20 or 40 mg kg(-1), of AME or sterile physiological saline intraperitoneally once daily consecutively for 5 days before exposure to 10 Gy 60Co gamma-radiation or five doses of 15 mg kg(-1) AME before exposure to 6, 7, 8, 9, 10 or 11 Gy. The animals were monitored for symptoms of radiation sickness and mortality up to 30 days post-irradiation. Glutathione and lipid peroxidation were estimated in the surviving animals of both groups on day 31 post-irradiation. AME was non-toxic up to a single dose of 1750 mg kg(-1). The optimum radioprotective dose was five consecutive doses of 15 mg kg(-1) AME, where the highest survival to 10 Gy radiation was observed. The irradiation caused a dose-dependent decline in survival, while treatment of mice with AME enhanced survival. The dose reduction factor was 1.15. Irradiation caused a dose-dependent decline in the level of glutathione accompanied by an elevation in lipid peroxidation. AME pretreatment arrested glutathione decline and lipid peroxidation significantly. AME treatment reduced the symptoms of radiation-induced sickness and increased survival. The radioprotective action might be due to free-radical scavenging and arrest of lipid peroxidation accompanied by an elevation in glutathione.
Article
The flavanoid derivative troxerutin, used clinically for treating venous disorders, protected biomembranes and cellular DNA against the deleterious effects of gamma-radiation. The peroxidation of lipids (measured as thiobarbituric acid-reacting substances, or TBARS) in rat liver microsomal and mitochondrial membranes resulting from gamma-irradiation up to doses of 500 Gy in vitro was prevented by 0.2 mM troxerutin. The administration of troxerutin (175 mg/kg body weight) to tumor-bearing mice by ip one hour prior to 4 Gy whole-body gamma-irradiation significantly decreased the radiation-induced peroxidation of lipids in tissues such as liver and spleen, but there was no reduction of lipid peroxidation in tumor. The effect of troxerutin in gamma-radiation-induced DNA strand breaks in different tissues of tumor-bearing mice was studied by comet assay. The administration of troxerutin to tumor-bearing animals protected cellular DNA against radiation-induced strand breaks. This was evidenced from decreases in comet tail length, tail moment, and percent of DNA in the tails in cells of normal tissues such as blood leukocytes and bone marrow, and these parameters were not altered in cells of fibrosarcoma tumor. The results revealed that troxerutin could preferentially protect normal tissues against radiation-induced damages in tumor-bearing animals.
Article
Diethyldithiocarbamate (DDTC) is studied for its antioxidant and radioprotective abilities. DDTC at a concentration of 0.5 mM reduced DPPH radical. DDTC reduced the damage to deoxyribose resulting from hydroxyl radicals generated by Fenton reaction, indicating that the radioprotective abilities of this compound could be due to the free radical scavenging. DDTC protected rat liver microsomal membranes in vitro from peroxidative damage in lipids (measured as TBARS) resulting from 50 Gy gamma-radiation. It also protected plasmid pBR322 DNA from radiation-induced strand breaks. An oral administration of DDTC to mice before whole body gamma-radiation exposure (4 Gy) resulted in a reduction of radiation-induced lipid peroxides in the liver homogenates. An administration of DDTC to mice before gamma-radiation reduced the radiation-induced DNA damage as studied by single cell gel-electrophoresis (comet assay). The comet parameters such as tail length, tail moment, and percent of DNA in tail were found to increase in the blood leukocytes of mice exposed to 4 Gy gamma-radiation. When DDTC was administered to mice before the radiation exposure, the increase in the comet parameters as a result of radiation was prevented, indicating a protection of cellular DNA. The present study has implication for the potential use of DDTC as a radioprotector.
Article
The development of radioprotective agents has been the subject of intense research in view of their potential for use within a radiation environment, such as space exploration, radiotherapy and even nuclear war. However, no ideal, safe synthetic radioprotectors are available to date, so the search for alternative sources, including plants, has been on going for several decades. In Ayurveda, the traditional Indian system of medicine, several plants have been used to treat free radical-mediated ailments and, therefore, it is logical to expect that such plants may also render some protection against radiation damage. A systematic screening approach can provide leads to identifying potential new candidate drugs from plant sources, for mitigation of radiation injury. This article reviews some of the most promising plants, and their bioactive principles, that are widely used in traditional systems of medicine, and which have rendered significant radioprotection in both in vitro and in vivo model systems. Plants and their constituents with pharmacological activities that may be relevant to amelioration of radiation-mediated damage, including antiemetic, antiinflammatory, antioxidant, cell proliferative, wound healing and haemopoietic stimulatories are also discussed.
Article
The radioprotective effects of ethanolic extract of propolis (EEP) and quercetin on the white blood cells of the whole-body irradiated CBA mice were investigated. Irradiation was performed using a gamma-ray source ((60)Co), and absorbed dose was 9 Gy. The efficiency of test components was evaluated when given intraperitoneally (ip) at a dose of 100 mg kg(-1) for 3 consecutive days before and/or after irradiation. Moreover, possible genotoxic effects of test components were also assessed on non-irradiated animals. For each experimental group leukocyte count was determined and the primary DNA damage in leukocytes was assessed using the alkaline comet assay. The higher efficiency of EEP and quercetin was observed when given preventively. The results suggest that propolis and quercetin given to mice before irradiation protect their white blood cells from lethal effects of irradiation and diminish primary DNA damage as confirmed by the alkaline comet assay. Positive results obtained on gamma-irradiated mice given EEP and quercetin, complementary with our earlier observations on survival of irradiated mice, indicate that these compounds could be considered effective non-toxic radioprotectors. The exact mechanisms of radioprotection by these compounds and their effects on DNA repair processes are still to be elucidated.
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