Domagoj Đikić

DNA-protective effects of quercetin or naringenin in alloxan-induced diabetic mice.

Diabetes mellitus is associated with a high production of reactive oxygen species, which may cause oxidative DNA damage. High levels of genomic damage have been associated with liver and renal failure as well as immune-system decline. Flavonoids are effective antioxidants and may protect against several chronic diseases including diabetes. This study used the comet assay to assess the levels of DNA damage in the blood, liver and kidney cells in untreated and quercetin (QU) or naringenin treated diabetic mice. In addition, the study was designed to establish whether QU or naringenin might have a biological effect in protecting diabetic mice against oxidative stress by using survival studies to observe total body injury at the level of the organism. QU or naringenin were injected to mice intraperitoneally (i.p.) at a dose of 50mg/kg for 7days starting 2days after a single dose (75mg/kg, i.v.) alloxan injection. These findings suggest that QU or naringenin treatment resulted in a significant increase in the body weight, the haematological and immunological parameters of blood, as well as leading to 100% survival of diabetic mice. The tested flavonoids have protective effects against alloxan-induced DNA-damage in peripheral lymphocytes but not in the liver and kidney cells of diabetic mice. It might be hypothesised that diabetic mice with a high intake of flavonoid-rich foods, and specifically foods rich in quercetin or naringenin, might be relatively protected against long-term complications of diabetes due to decreased oxidative stress. Various co-operative and synergistic action mechanisms of the tested flavonoids may lead to the protection of the whole organism against diabetes.

Radioprotective Effects of Quercetin and Ethanolic Extract of Propolis in Gamma-Irradiated Mice

Radioprotective Effects of Quercetin and Ethanolic Extract of Propolis in Gamma-Irradiated Mice The aim of this study was to assess radioprotective effects of quercetin and the ethanolic extract of propolis (EEP) in CBA mice exposed to a single radiation dose 4 Gy (60Co). The mice were treated with 100 mg kg-1 quercetin or EEP a day for three consecutive days either before (pre-treatment) or after gamma-irradiation (therapy). Leukocyte count was determined in blood drawn from the tail vein, and DNA damage in leukocytes was assessed using the alkaline comet assay. Genotoxic effects of the test compunds were also evaluated in non-irradiated mice. The levels of radioprotection provided by both test compounds were compared with those established in mice that were given chemical radioprotector S-(2-Aminoethy1)isothiouronium bromide hydrobromide (AET). Mice that received pre-treatment were less sensitive to irradiation. Mice given the post-irradiation therapy showed a slight but not significant increase in total leukocyte count over irradiated negative control. Quercetin showed better protective properties than EEP in both pre-treatment and therapy, and activated a higher number of leukocytes in non-irradiated mice. The alkaline comet assay suggests that both natural compounds, especially when given as pre-treatment, protect against primary leukocyte DNA damage in mice. At tested concentrations, EEP and quercetin were not genotoxic to non-irradiated mice. AET, however, caused a slight but not significant increase in DNA damage. Although the results of this study show the radioprotective potential of the test compounds, further investigation is needed to clarify the underlying protection mechanisms. Primjena alkalnog kometnog testa u istraživanju radioprotektivnih učinaka alkoholnog ekstrakta propolisa i kvercetina na miševima ozračenim gama-zračenjem

Synergistic Effects of Irinotecan and Flavonoids on Ehrlich Ascites Tumour-Bearing Mice

Swiss albino mice were given Ehrlich ascites tumour cells (1 × 10(6)) intraperitoneally. For survival analysis and tumour growth analysis, the mice were administered quercetin and naringin (100 mg/kg) daily for 3 consecutive days, beginning on the third day after intraperitoneal (i.p.) injection of Ehrlich ascites tumour cells (1 × 10(6)). Irinotecan was administered ip at a dose of 50 mg/kg on days 1, 13 and 19. For the analysis of cell types and differential count of cells present in the peritoneal cavity, peripheral whole-blood leucocyte count and the comet assay, the mice were treated therapeutically with quercetin and naringin (100 mg/kg) and irinotecan (50 mg/kg) daily for 3 consecutive days beginning on third day after i.p. injection of Ehrlich ascites tumour cells (1 × 10(6)). We observed the synergistic anti-tumour effect expressed as the median survival time of mice treated with naringin in combination with irinotecan. All test components inhibited tumour growth and increased lifespan of mice except quercetin. The total number of cells present in the peritoneal cavity of mice significantly decreased in all treatments except quercetin. Single irinotecan and irinotecan combined with naringin had the highest DNA-damaging potential on peripheral blood leucocytes and lowest primary DNA damage, both in the kidney and liver cells as measured by the alkaline comet assay. Our results showed enhanced anti-tumour activity of irinotecan in combined treatment with flavonoids to reduce the deteriorating reaction of cytostatic drugs.

Effect of competition on habitat utilization in two temperate climate gecko species

Competition over spatial niche utilisation is one of most common competitive interactions between species in sympatry. Moreover, competitive interactions may involve age classes, and can fluctuate temporally. Consequently, evasive strategies that enable co-existence are likely to be important in the evolution of species assemblages. Here we investigate a system of two co-existing species of temperate geckos with similar ecologies (the house gecko, Hemidactylus turcicus and the wall gecko, Tarentola mauritanica), providing an opportunity to study the effect of species interactions. Juveniles and adults of both species were investigated throughout their daily and annual cycle to explore the effect of inter- and intra-specific interactions on microhabitat use. The two species showed differences in habitat use for both age classes in sympatry. In sympatry, T. mauritanica uses more open habitats and is more active. In contrast, H. turcicus is found in more closed habitats, closer to the ground and to vegetation cover. In allopatry, H. turcicus was observed in more open habitats, closer to the ground, and to vegetation cover, when compared to the population in sympatry with T. mauritanica. These differences in habitat usage were significant for both age classes. Moreover, there were differences, both in sympatry and in allopatry, between age classes that were dependent on season. In conclusion, the presence of a competitor induces a spatial shift in individuals of both age classes of H. turcicus. Observed plasticity in habitat utilisation in both age classes of H. turcicus is used to argue for the invasive potential of this species.

Carbendazim combined with imazalil or cypermethrin potentiate DNA damage in hepatocytes of mice

Traces of pesticides imazalil, cypermethrin and carbendazim are detected in plants used for human consumption. To explore whether their application in oral combinations will induce DNA breaks in hepatocytes, a subchronic in vivo experiment was performed in Swiss mice. Doses of 10 mg kg–1 of imazalil (im) and cypermethrin (cy), and 20 mg kg–1 of carbendazim (car) and their combinations (im, 10 mg kg–1 + cy, 10 mg kg–1; im, 10 mg kg–1 + car, 20 mg kg–1; car, 20 mg kg–1 + cy, 10 mg kg–1) were applied daily for 28 days. Afterward, DNA damage in hepatocytes was evaluated by comet assay. Individually, imazalil and cypermethrin damaged DNA at alkali-labile sites, while the tail moment (TM) of carbendazim alone was similar to control but with higher tail length. In combination with carbendazim clastogen, properties of imazalils and cypermethrins were potentiated compared to all other treatments and control. There were pronounced sex differences in pattern of fragmentation between treated groups. Higher long tail nuclei (LTN) in females indicate that certain cells in females were especially prone to total nucleus disintegration. Due to synergistic effects, low environmentally present concentrations of imazalil and cypermethrin in food, and especially their mixtures with carbendazim have genotoxic potential that could be particularly dangerous over prolonged exposure in mammalian organism.

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.

Inhibitory effect of a propolis on Di-n-Propyl Disulfide or n-Hexyl salycilate-induced skin irritation, oxidative stress and inflammatory responses in mice

PURPOSE Thermal imaging has been utilised, both preclinically and clinically, as a tool for assessing inflammation. Psoriasis is a chronic inflammatory skin disease characterised by hyperkeratosis, dermal inflammatory infiltrate and increased angiogenesis. The aim of the present study was to assess the usefulness of thermography in psoriatic lesion regression after topical treatment with bee propolis, recognised as potent antioxidants and anti-inflammatory agents. METHODS We monitored the inflammation process induced by irritants such as n-Hexyl salycilate (HXS) or Di-n-Propyl Disulfide (PPD) by histopatological assessment of the skin, thermographic scanning, total number of inflammatory cells in the peritoneal cavity, differential analysis of cells in the peritoneal cavity, macrophage spreading index, haematological and biochemical parameters, frequencies of micronucleated reticulocytes, lipid peroxidation and glutathione assay in the skin. RESULTS Topically applied ethanolic extract of propolis (EEP) with HXS or PPD reduced the lipid peroxidation in the skin and total number of inflammatory cells in the skin and peritoneal cavity, functional activity of macrophages, the number of micronuclei in mouse peripheral blood reticulocytes and enzymatic activity of ALP and AST. CONCLUSION These results demonstrate that topical application of EEP may improve psoriatic-like skin lesions by suppressing functional activity of macrophages and ROS production. Taken together, it is suggested that EEP can safely be utilised in the prevention of psoriasis-related inflammatory changes without causing any toxic effect.

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.

Addition of propolis to irinotecan therapy prolongs survival in ehrlich ascites tumor-bearing mice.

We investigated possible synergistic action of anticancer drug Irinotecan (IRI) combined with ethanolic (EEP) and water-soluble (WSDP) derivate of propolis on Swiss albino mice injected with Ehrlich ascites tumor (EAT). For survival analysis mice were administered WSDP and EEP (100 mg/kg) daily for 3 consecutive days, beginning on 3rd day after EAT cell (1×10⁶) injection. IRI was administered at a dose of 50 mg/kg on days 1, 13, and 19. We simultaneously studied peripheral white blood cell count, cell types washed from the peritoneal cavity, functional activity of macrophages from peritoneal cavity, and the level of primary DNA damage in leukocytes, kidney, and liver cells using the alkaline comet assay. Three out of 9 mice per group survived the entire duration of the experiment (90 days) in groups treated with IRI combined with WSDP and EEP. All test components increased survival of mice by 7.53% to 231.54%. Combined treatment with IRI and/or WSDP and EEP significantly decreased percentage of tumor cells in the peritoneal cavity as compared to nontreated EAT-injected mice. All treated animals had significantly higher percentage of neutrophils in the peritoneal cavity in comparison to nontreated EAT-injected mice. We observed significantly higher value of DNA damage in leukocytes of mice treated with IRI and combination of IRI and/or WSDP and EEP as compared to nontreated EAT-injected mice, while the same treatment decreased DNA damage in kidney. Our results showed that addition of propolis to IRI treatment enhanced antitumor activity of IRI and prolongs survival in EAT-bearing mice, which definitely deserve further studies to clarify the possible mechanisms of antitumor actions of combined herb-drug treatments.

The age, growth and feeding habits of the European conger eel, Conger conger (L.) in the Adriatic Sea

Abstract This study determined basic biological data for the European conger eel Conger conger (L.) population in the coastal waters of the eastern Adriatic Sea. Juveniles and immature females dominated the coastal population, whereas males were relatively uncommon. The population structure determined by the study suggested spatial separation of sexes and spawning grounds in deeper waters. Both edge-type and marginal increment analyses confirmed the formation of a single growth annulus per year on the ground otoliths. The observed maximum age of the coastal conger eels was 8 years, although most of the sampled fish were 5 years old. The estimated parameters of the von Bertalanffy growth model suggested that the growth of the conger eels was relatively slow. C. conger is an opportunistic predator. Its diet was composed primarily of fishes, followed by crustaceans and cephalopods. Due to the evident site fidelity of the species, the wide prey spectrum of the conger eels (33 taxa) reflected the local benthic community structure.