Brain DNA damaging effects of volatile anesthetics and 1 and 2 Gy gamma irradiation in vivo: Preliminary results.

Although both can cause DNA damage, the combined impact of volatile anesthetics halothane/sevoflurane/isoflurane and radiotherapeutic exposure on sensitive brain cells in vivo has not been previously analyzed. Healthy Swiss albino male mice (240 in total, 48 groups) were exposed to either halothane/sevoflurane/isoflurane therapeutic doses alone (2 h); 1 or 2 gray of gamma radiation alone; or combined exposure. Frontal lobe brain samples from five animals were taken immediately and 2, 6, and 24 h after exposure. DNA damage and cellular repair index were analyzed using the alkaline comet assay and the tail intensity parameter. Elevated tail intensity levels for sevoflurane/halothane were the highest at 6 h and returned to baseline within 24 h for sevoflurane, but not for halothane, while isoflurane treatment caused lower tail intensity than control values. Combined exposure demonstrated a slightly halothane/sevoflurane protective and isoflurane protective effect, which was stronger for 2 than for 1 gray. Cellular repair indices and tail intensity histograms indicated different modes of action in DNA damage creation. Isoflurane/sevoflurane/halothane preconditioning demonstrated protective effects in sensitive brain cells in vivo. Owing to the constant increases in the combined use of radiotherapy and volatile anesthetics, further studies should explore the mechanisms behind these effects, including longer and multiple exposure treatments and in vivo brain tumor models.

Potential radioprotective properties of arbutin against ionising radiation on human leukocytes in vitro.

Arbutin is a simple phenolic glucoside biosynthesised in many plant families. Some of the everyday foods that contain arbutin are species of the genus Origanum, peaches, cereal products, coffee and tea and Arctostaphyllos uva ursi L. leaves. Arbutin possesses various beneficial effects in the organism, and was confirmed effective in the treatment of urinary tract infections as well as in preventing skin hyperpigmentation. It shows antioxidant and anti-inflammatory properties, and antitumor activity. The aim of this study was to explore potential radioprotective properties of arbutin in concentrations of 11.4 µg/mL, 57 µg/mL, 200 µg/mL and 400 µg/mL administered as a pre-treatment for one hour before exposing human leukocytes to ionising radiation at a therapeutic dose of 2 Gy. The alkaline comet assay was used to establish the levels of primary DNA damage, and cytokinesis-block micronucleus (CBMN) cytome assay to determine the level of cytogenetic damage. None of the tested concentrations of single arbutin showed genotoxic and cytotoxic effects. Even at the lowest tested concentration, 11.4 µg/mL, arbutin demonstrated remarkable potential for radioprotection in vitro, observed both at the level of primary DNA damage, and using CBMN cytome assay. The best dose reduction compared with amifostine was observed after pre-treatment with the highest concentration of arbutin, corresponding to 400 µg/mL. Promising results obtained on the leukocyte model speak in favour of extending similar experiments on other cell and animal models.

DNA damage assessment in peripheral blood of Swiss albino mice after combined exposure to volatile anesthetics and 1 or 2 Gy radiotherapy in vivo.

PURPOSE: Patient immobilization by general volatile anesthesia (VA) may be necessary during medical radiology treatment, and its use has increased in recent years. Although ionizing radiation (IR) is a well-known genotoxic and cytotoxic agent, and VA exposure has caused a range of side effects among patients and occupationally exposed personnel, there are no studies to date comparing DNA damage effects from combined VA and single fractional IR dose exposure. MATERIAL AND METHODS: We investigate whether there is a difference in white blood cells DNA damage response (by the alkaline comet assay) in vivo in 185 healthy Swiss albino mice divided into 37 groups, anesthetized with isoflurane/sevoflurane/halothane and exposed to 1 or 2 Gy of IR. Blood samples were taken after 0, 2, 6 and 24 h after exposure, and comet parameters were measured: tail length, tail intensity and tail moment. The cellular DNA repair index was calculated to quantify the efficiency of cells in repairing and re-joining DNA strand breaks following different treatments. RESULTS: In combined exposures, halothane caused higher DNA damage levels that were dose-dependent; sevoflurane damage increase did not differ significantly from the initial 1 Gy dose, and isoflurane even demonstrated a protective effect, particularly in the 2 Gy dose combined exposure. Nevertheless, none of the exposures reached control levels even after 24 h. CONCLUSION: Halothane appears to increase the level of radiation-induced DNA damage, while sevoflurane and isoflurane exhibited a protective effect. DNA damage may have been even greater in target organs such as liver, kidney or even the brain, and this is proposed for future study.

Carbendazim impends hepatic necrosis when combined with imazalil or cypermethrin.

Imazalil, cypermethrin and carbendazim are detected in plants for human nutrition. To explore whether their combinations, applied orally in low doses, would induce changes in metabolic patterns and hepatotoxicity, a subchronic in vivo experiment was conducted. Doses of 10mg/kg of imazalil (im) and cypermethrin (cy) and 20 mg/kg of carbendazim (car) and their combinations (im, 10 mg/kg+cy, 10mg/kg; im, 10mg/kg+car, 20mg/kg; car, 20 mg/kg + im, 10 mg/kg) were given to Swiss mice daily over 28 days. After 24 hr from the last dose, the relationships of cytotoxicity biomarkers were analysed: serum lactate dehydrogenase, aspartate transaminase, alanine transferase, amylase, alkaline phosphatase, creatine kinase, creatinine and total proteins. Individual pesticides showed different toxic potential (cy>im car) generally characterized by increase in enzyme activities. Histological analysis showed that cypermethrin, but not imazalil or carbendazim, alone can cause mild necrosis. Combinations generally caused decrease in the activity of enzymes, indicating liver damage. Low doses of carbendazim in combination with low doses of imazalil or cypermethrin caused very pronounced hepatic necrosis, more than any of the three individually applied pesticides or combination of imazalil and cypermethrin. In fruits and vegetables for human consumption, residues of these three pesticides and prolonged combined intake of low doses, which by themselves acutely would not cause any effect, may have similar hepatotoxic effects.

The in vivo genotoxicity of cisplatin, isoflurane and halothane evaluated by alkaline comet assay in Swiss albino mice.

The aim of this study was to evaluate the genotoxicity of repeated exposure to isoflurane or halothane and compare it with the genotoxicity of repeated exposure to cisplatin. We also determined the genotoxicity of combined treatment with inhalation anaesthetics and cisplatin on peripheral blood leucocytes (PBL), brain, liver and kidney cells of mice. The mice were divided into six groups as follows: control, cisplatin, isoflurane, cisplatin-isoflurane, halothane and cisplatin-halothane, and were exposed respectively for three consecutive days. The mice were treated with cisplatin or exposed to inhalation anaesthetic; the combined groups were exposed to inhalation anaesthetic after treatment with cisplatin. The alkaline comet assay was performed. All drugs had a strong genotoxicity (P<0.05 vs. control group) in all of the observed cells. Isoflurane caused stronger DNA damage on the PBL and kidney cells, in contrast to halothane, which had stronger genotoxicity on brain and liver cells. The combination of cisplatin and isoflurane induced lower genotoxicity on PBL than isoflurane alone (P<0.05). Halothane had the strongest effect on brain cells, but in the combined treatment with cisplatin, the effect decreased to the level of cisplatin alone. Halothane also induced the strongest DNA damage of the liver cells, while the combination with cisplatin increased its genotoxicity even more. The genotoxicity of cisplatin and isoflurane on kidney cells were nearly at the same level, but halothane caused a significantly lower effect. The combinations of inhalation anaesthetics with cisplatin had stronger effects on kidney cells than inhalation anaesthetics alone. The observed drugs and their combinations induced strong genotoxicity on all of the mentioned cells.

Genotoxicity and cytotoxicity of cisplatin treatment combined with anaesthetics on EAT cells in vivo.

In this study, DNA damage in tumour cells, as well as irreversible cell damage leading to apoptosis induced in vivo by the combined application of cisplatin and inhalation anaesthetics, was investigated. The genotoxicity of anaesthetics on Ehrlich ascites tumour (EAT) cells of mice, alone or in combined application with cisplatin, was estimated by using the alkaline comet assay. The percentage of EAT cell apoptosis was quantified by flow cytometry. Groups of EAT-bearing mice were (i) treated intraperitoneally with cisplatin, (ii) exposed to repeated anaesthesia with inhalation anaesthetic, and (iii) subjected to combined treatment of exposure to anaesthetics after cisplatin for 3 days. Sevoflurane, halothane and isoflurane caused strong genotoxic effects on tumour cells in vivo. The tested anaesthetics alone showed no direct effect on programmed cell death although sevoflurane and especially halothane decreased the number of living EAT cells in peritoneal cavity lavage. Repeated anaesthesia with isoflurane had stimulatory effects on EAT cell proliferation and inhibited tumour cell apoptosis (6.11%), compared to the control group (10.26%). Cisplatin caused massive apoptosis of EAT cells (41.14%) and decreased the number of living EAT cells in the peritoneal cavity. Combined cisplatin and isoflurane treatment additionally increased EAT cell apoptosis to 51.32%. Combined treatment of mice with cisplatin and all anaesthetics increased the number of living tumour cells in the peritoneal cavity compared to cisplatin treatment of mice alone. These results suggest that the inhalation of anaesthetics may protect tumour cells from the cisplatin-induced genotoxic and cytotoxic effects.

Prevention of peritoneal carcinomatosis in mice with combination hyperthermal intraperitoneal chemotherapy and IL-2.

PURPOSE: The purpose of this study was to investigate the effect of local chemoimmunotherapy and hyperthermal intraperitoneal chemotherapy (HIPEC) in a mouse model of induced peritoneal carcinomatosis. MATERIAL AND METHODS: Peritoneal carcinomatosis in mice was produced by intraperitoneal implantation of MCa cells (5 x 10(3)). Interleukin-2 (4.1 x 10(4) IU/mouse) was injected into the abdominal cavity of mice at day 7 and 3 before implantation of tumour cells. Immediately after implantation of MCa cells mice were treated twice with 2 ml of saline that was heated either at 37 degrees C or 43 degrees C and cytostatics (doxorubicin 20 mg kg(-1), cisplatin 10 mg kg(-1), mitomycin 5 mg kg(-1), or 5-FU 150 mg kg(-1)). We followed the survival of animals and side effects appearing with different forms of treatment. RESULTS: Combined treatment with Interleukin-2 (IL-2) and cytostatics (5-FU, CIS or MIT) significantly affected the development of peritoneal carcinomatosis and increased the survival of mice (ILS% - 37 degrees C = 29.88, 199.32, and 108.52, ILS% - 43 degrees C = 62.69, 260.50, and 178.05, respectively). However, intraperitoneal chemotherapy on survival time of mice with DOX + IL-2 was ineffective as compared with DOX alone. CONCLUSION: We would like to stress that treatment with IL-2 prior to tumour diagnosis is not clinically practical, rather, the manuscript attempts to describe an experimental proof of principle. Results suggest the synergistic effect of hyperthermia, chemotherapy and immunotherapy; IL-2 significantly increases antitumor activity of hyperthermic chemotherapy and survival rate of mice with peritoneal carcinomatosis.

The effects of prometryne on subchronically treated mice evaluated by SCGE assay.

Prometryne is a methylthio-s-triazine herbicide used to control annual broadleaf and grass weeds in many cultivated plants. Significant traces are documented in environment, mainly water, soil and plants used for human and domestic animal nutrition. Data on the toxic effects of prometryne and other methylthio-s-triazine have scorcely been published. The goal of this study was to investigate if prometryne, applied orally, could induce DNA damage in mouse leukocytes, in subchronical in vivo experimental design. Three different doses of prometryne were applied per os repeatedly every 48 hours. After the 7th dose (day 14) and the 14th dose (day 28) blood leucocytes were analyzed by alkaline Single Cell Gel Electrophoresis (Comet) assay. The results of three different comet parameters showed general increase in Olive tail moment, tail length and tail intensity values in treated groups of animals. The increase in measured values was almost proportional to the dose received and the time of exposure. We conclude that prometryne or its metabolic residues have the potential to induce processes that cause genotoxic effects on leukocytes on mice in in vivo repeated exposure.

Effects of prometryne on apoptosis and necrosis in thymus, lymph node and spleen in mice.

Prometryne is a methylthio-s-triazine herbicide. Significant traces are documented in environment, mainly waters, soil and plants used for nutrition. The aim of this study was to estimate prometryne immunotoxic properties through induction of apoptotic and/or necrotic changes in thymocytes, splenocytes and lymph node cells after repeated subchronical exposure. Three different doses of prometryne (185, 375, 555mgkg(-1)) were applied per os every 48h, over 28 days. Flow cytometry assay (annexinV-FITC and PI) was conducted to record apoptotic and necrotic damage. In the spleen significant changes in the percentage of apoptotic cells were not detected between treated and control groups respectively. In thymus and lymph node, within the lowest dose group (185mgkg(-)1), an increase in percentage of early apoptosis without any significant increase in necrosis was detected. Medium (375mgkg(-1)) as well as high dose triggered increase in late apoptosis in lymph node while in thymus; late apoptosis was increased only in animals exposed to the highest dose (555mgkg(-1)). The highest applied dose, in thymus and lymph node respectively, caused a general decrease in percentage of vital cells in favour of marked increase of percentages of all types of dying cells (apoptotic, late apoptotic/early necrotic and necrotic). Prometryne caused disbalance in major organs of immune system, markedly lymph nodes and thymus, by induction of early apoptotic changes in dose/time specific manner.

Evaluation of radioprotective effects of propolis and quercetin on human white blood cells in vitro.

This in vitro study aimed at investigating the possible radioprotective effects of natural substances propolis and quercetin on gamma-irradiated human white blood cells. The levels of primary DNA damage were studied by the alkaline comet assay, while the cytogenetic damage was evaluated using the analysis of structural chromosome aberration and cytokinesis-block micronucleus assay. The results obtained by all endpoints indicate acceptable toxicity profiles of propolis and quercetin in vitro, and also confirmed their radioprotective abilities. Propolis was found to be more effective in diminishing the levels of primary and more complex cytogenetic DNA damage in gamma-irradiated white blood cells. Data gathered in present study support the use of propolis and quercetin as non-toxic protective substances. However, to clarify the underlying mechanisms of their cyto/radioprotective activities, additional studies are necessary at both in vitro and in vivo levels.

Assessment by survival analysis of the radioprotective properties of propolis and its polyphenolic compounds.

The radioprotective effects of propolis and polyphenolic compounds from propolis on the radiation-induced mortality of mice exposed to 9 Gy of gamma-irradiation were studied. Intraperitoneal (i.p.) treatment of mice at doses of 100 mg kg(-1) body weight of propolis (water or ethanolic extract; WSDP or EEP) or its polyphenolic compounds (quercetin, naringin caffeic acid, chrysin) consecutively for 3 d before irradiation, delayed the onset of mortality and reduced the symptoms of radiation sickness. All test compounds provided protection against hematopoietic death (death within 30 d after irradiation). The greatest protection was achieved with quercetin; the number of survivors at the termination of the experiment was 63%. According to statistical analyses by the Kaplan-Meier method and the log-rank test, a significant difference between test components and control was found (p<0.001). Treatment with test components after lethal irradiation was ineffective. These results suggest that propolis and its polyphenolic compounds given to mice before irradiation protect mice from the lethal effects of whole-body irradiation.

Histological types of polypoid cutaneous melanoma II.

The aim of this study was to ascertain which histological types of melanoma can clinically and morphologically appear as polypoid melanomas. In 645 cases of primary cutaneous melanoma we have analyzed criteria for diagnosis of polypoid cutaneous melanoma and afterwards we have analyzed growth phase in each polypoid melanoma, histological type of atypical melanocytes, the number of epidermal ridges which are occupied by atypical melanocytes, and distribution according to age, sex and location, as well as the disease free survival. According to the criteria for polypoid melanomas we have found 147 (22.8%) polypoid cutaneous melanomas. Analyzing the growth phases, histological types of atypical melanocytes and the number of affected epidermal ridges in the group of polypoid melanomas we have ascertained 2 (1.4%) ALMs, 4 (2.8%) LMMs, 42 (28.6%) SSMs and 99 (67.2%) NMs. Our conclusion is that polypoid cutaneous melanomas are morphological forms of various histological melanoma types (ALM, LMM, SSM and NM) and they can all display polypoid morphological form. Polypoid cutaneous melanomas are most often of nodular histological type.