Jana Navarová

Investigation of anti-oxidative, cytotoxic, DNA-damaging and DNA-protective effects of plant volatiles eugenol and borneol in human-derived HepG2, Caco-2 and VH10 cell lines

Plant volatiles, which can get into the human organism in food, medicines, or cosmetic preparations, frequently manifest antibacterial, antifungal, antiviral and other effects. We studied anti-oxidative, cytotoxic, genotoxic and possible DNA-protective effects of eugenol and borneol. Anti-oxidative activities of aqueous and ethanolic solutions of these two volatile compounds of plants were determined by a spectrophotometric method by the use of the stable DPPH radical. Borneol did not show any anti-oxidative activity even at the highest concentrations soluble in water or ethanol (<1000mM), while eugenol did manifest anti-oxidative activity, and at much lower concentrations (5-100 microM). The cytotoxicity of eugenol and borneol as well as their DNA-damaging effects and their influence on sensitivity of cells against the DNA-damaging effects of H(2)O(2) were investigated in three different cell lines, i.e. malignant HepG2 hepatoma cells, malignant Caco-2 colon cells, and nonmalignant human VH10 fibroblasts. The trypan-blue exclusion assay showed that in the three cell lines the cytotoxicity of eugenol was significantly higher than that of borneol. Single-cell gel electrophoresis revealed that borneol did not cause any DNA strand-breaks at the concentrations studied, but showed that all concentrations of eugenol (<600 microM) significantly increased the level of DNA breaks in human VH10 fibroblasts and to a lower degree in Caco-2 colon cells. The DNA-damaging effects of eugenol were not observed in metabolically active HepG2 hepatoma cells. Borneol and eugenol differed also with respect to their DNA-protective effects. While borneol protected HepG2 and, to a lesser extent, VH10 cells (but not Caco-2) against H(2)O(2)-induced DNA damage, eugenol either did not change the cellular sensitivity to H(2)O(2) (HepG2 cells) or it even increased the sensitivity (Caco-2 and VH10 cells). These results do not indicate any correlation between the DNA-protective and the anti-oxidative capacities of eugenol and borneol.

Protective effect of melatonin in acetic acid induced colitis in rats.

Abstract:  Possible protective effects of exogenous melatonin on colonic inflammation were studied in rats. Colitis was induced by intracolonic (i.c.) instillation of 4% acetic acid (AA) and the resulting injury was assessed after 1 and 48 hr. Diffuse hyperemia and bleeding with erosions and ulcerations were observed in the colons of vehicle‐treated rats. Melatonin administered in doses of 5 and 10 mg/kg reduced significantly the extent of gross mucosal damage after intraperitoneal as well as i.c. dosing. The inflammation induced increase in colonic wet weight was also reduced by melatonin treatment. In the early phase of colonic inflammation (60 min), melatonin partly prevented the decrease of reduced glutathione (GSH) content and limited lysosomal enzyme, N‐acetyl‐glucosaminidase and cathepsin D, activities induced by AA, with no changes in proteins or acid phosphatase activity. Increase of myeloperoxidase activity (MPO) caused by colonic inflammation was prevented by melatonin given i.c. As observed 48 hr after AA exposure, there was no difference between the effect of vehicle and melatonin on the content of GSH. Colitis did not influence the melatonin content of the colon. After administration of exogenous melatonin, plasma, pineal and gut melatonin tended to increase. The results indicate that melatonin participates in various defense mechanisms against colonic inflammatory processes by preserving the important endogenous antioxidant reserve of GSH, by preventing lysosomal enzyme disruption, by inhibiting enhanced MPO activity, thus reducing the extent of colonic damage, mainly in the early phase of colitis.

Assessment of Antioxidative, Chelating, and DNA-Protective Effects of Selected Essential Oil Components (Eugenol, Carvacrol, Thymol, Borneol, Eucalyptol) of Plants and Intact Rosmarinus officinalis Oil

Selected components of plant essential oils and intact Rosmarinus officinalis oil (RO) were investigated for their antioxidant, iron-chelating, and DNA-protective effects. Antioxidant activities were assessed using four different techniques. DNA-protective effects on human hepatoma HepG2 cells and plasmid DNA were evaluated with the help of the comet assay and the DNA topology test, respectively. It was observed that whereas eugenol, carvacrol, and thymol showed high antioxidative effectiveness in all assays used, RO manifested only antiradical effect and borneol and eucalyptol did not express antioxidant activity at all. DNA-protective ability against hydrogen peroxide (H2O2)-induced DNA lesions was manifested by two antioxidants (carvacrol and thymol) and two compounds that do not show antioxidant effects (RO and borneol). Borneol was able to preserve not only DNA of HepG2 cells but also plasmid DNA against Fe(2+)-induced damage. This paper evaluates the results in the light of experiences of other scientists.

Protective effect of sulfoethylglucan against hexavalent chromium

The effect of pretreatment with sulfoethylglucan (SEG) on the frequency of micronuclei and the liver alkaline phosphatase activity induced by potassium bichromate (Cr(VI)) in mice was evaluated. Simultaneous application of SEG and Cr(VI) decreased the frequency of micronuclei in bone marrow cells (P < 0.01) and the level of liver alkaline phosphatase activity in comparison to the Cr(VI) group. Pretreatment with SEG 24 h prior to the first Cr(VI) application resulted in a more pronounced decrease in the Cr(VI)-induced frequency of micronuclei. The mechanisms of the protective effects of sulfoethylglucan could be explained either by the formation of Cr ion complexes with sulfoethyl groups of glucan or by the scavenging ability of SEG to trap hydroxyl radicals.

Rosmarinic acid administration attenuates diabetes-induced vascular dysfunction of the rat aorta

Oxidative stress as well as inflammation processes are engaged in diabetic vascular complications. Rosmarinic acid, a natural phenol antioxidant carboxylic acid, was found to have multiple biological activity, including anti‐inflammatory and antitumour effects, which are a consequence of its inhibition of the inflammatory processes and of reactive oxygen species scavenging. The aim of this work was to study effects of rosmarinic acid administration on vascular impairment induced by experimental diabetes in rats.

Suppressing effects of glucan on micronuclei induced by cyclophosphamide in mice.

The effect of pretreatment with carboxymethylglucan (CMG) on the frequency of micronuclei induced by cyclophosphamide administration in mice was evaluated. Two doses of CMG (50 mg/kg body weight) injected either intraperitoneally 24 h or intravenously 1 h prior to two cyclophosphamide administrations (80 mg/kg) significantly decreased the frequency of micronucleated PCE in bone marrow. Of two evaluated derivatives of carboxymethylglucan, the K3 derivative was most efficient. The results show that it is possible to achieve a suppressive effect of soluble carboxymethylglucan prepared from Saccharomyces cerevisiae against cyclophosphamide mutagenicity. The notion may be useful for glucans effects against pharmacocarcinogenesis. Therapeutic application of glucan with cyclophosphamide therapy may provide a remarkable decrease of the secondary tumour risk. The utilization of these results for human patients needs to be considered.

Comparison of biological processes induced in HepG2 cells by tert-butyl hydroperoxide (t-BHP) and hydroperoxide (H2O2): The influence of carvacrol.

This paper presents comparisons of biological impacts of the oxidants H2O2 and t-BHP on human liver cells, and shows modulation of these effects by the phenolic compound carvacrol. To understand better how these oxidants exert their effect on DNA and on the activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), we measured intracellular antioxidant glutathione (iGSH) and intracellular reactive oxidative species (iROS). DNA lesions corresponded to single-strand DNA breaks, alkali-labile lesions and formamido-pyrimidine-DNA-glycosylase (FPG)-sensitive sites. Pre-treatment of cells with carvacrol substantially decreased the number of H2O2-induced DNA lesions, but the number of t-BHP-induced DNA lesions was not reduced. Activities of both SOD and GPx were stimulated significantly by carvacrol and were reduced by the combined effect of carvacrol and oxidants. H2O2 and t-BHP alone influenced the level of antioxidant enzymes differently. While H2O2 did not markedly change the activity of SOD or GPx, lower concentrations of t-BHP stimulated activity of SOD and mainly GPx. The level of iROS was increased by both oxidants and decreased by carvacrol applied either alone or with oxidants. The level of iGSH was not influenced in any of the treatments tested. Our results show that although both oxidants induced oxidative stress and damaged cellular DNA, their influences on other molecular processes were different. The protective effect of carvacrol against DNA-damaging effects of H2O2 was unambiguous, but reduction by carvacrol of the DNA-damaging effect of t-BHP was not observed. These results suggest that the phenolic compound carvacrol contributes to the defence mechanisms of the human organism, but these beneficial effects are dependent on the origin and source of the actual oxidative stress.

Protection of the vascular endothelium in experimental situations.

Protection of the vascular endothelium in experimental situations One of the factors proposed as mediators of vascular dysfunction observed in diabetes is the increased generation of reactive oxygen species (ROS). This provides support for the use of antioxidants as early and appropriate pharmacological intervention in the development of late diabetic complications. In streptozotocin (STZ)-induced diabetes in rats we observed endothelial dysfuction manifested by reduced endothelium-dependent response to acetylcholine of the superior mesenteric artery (SMA) and aorta, as well as by increased endothelaemia. Changes in endothelium-dependent relaxation of SMA were induced by injury of the nitric oxide radical (·NO)-signalling pathway since the endothelium-derived hyperpolarising factor (EDHF)-component of relaxation was not impaired by diabetes. The endothelial dysfunction was accompanied by decreased ·NO bioavailabity as a consequence of reduced activity of eNOS rather than its reduced expression. The results obtained using the chemiluminiscence method (CL) argue for increased oxidative stress and increased ROS production. The enzyme NAD(P)H-oxidase problably participates in ROS production in the later phases of diabetes. Oxidative stress was also connected with decreased levels of reduced glutathione (GSH) in the early phase of diabetes. After 10 weeks of diabetes, adaptational mechanisms probably took place because GSH levels were not changed compared to controls. Antioxidant properties of SMe1EC2 found in vitro were partly confirmed in vivo. Administration of SMe1EC2 protected endothelial function. It significantly decreased endothelaemia of diabetic rats and improved endothelium-dependent relaxation of arteries, slightly decreased ROS-production and increased bioavailability of ·NO in the aorta. Further studies with higher doses of SMe1EC2 may clarify the mechanism of its endothelium-protective effect in vivo.

Evaluation of developmental neurotoxicity: some important issues focused on neurobehavioral development

Evaluation of developmental neurotoxicity: some important issues focused on neurobehavioral development Exposure of the developing organism to industrial chemicals and physical factors represents a serious risk factor for the development of neurobehavioral disorders, such as attention-deficit hyperactivity disorder, autism and mental retardation. Appropriate animal models are needed to test potentially harmful effects and mechanisms of developmental neurotoxicity of various chemical substances. However, there are significant human vs. rat differences in the brain developmental profile which should be taken into account in neurotoxicity studies. Subtle behavioral alterations are hard to detect by traditional developmental toxicity and teratogenicity studies, and in many cases they remain hidden. They can however be revealed by using special behavioral, endocrine and/or pharmacological challenges, such as repeated behavioral testing, exposure to single stressful stimulus or drugs. Further, current neurobehavioral test protocols recommend to test animals up to their adulthood. However some behavioral alterations, such as anxiety-like behavior or mental deficiency, may become manifest in later periods of development. Our experimental and scientific experiences are highly suggestive for a complex approach in testing potential developmental neurotoxicity. Strong emphasis should be given on repeated behavioral testing of animals up to senescence and on using proper pharmacological and/or stressful challenges.

Melatonin attenuates hypertension-related proarrhythmic myocardial maladaptation of connexin-43 and propensity of the heart to lethal arrhythmias.

We hypothesized that the pineal hormone melatonin, which exhibits cardioprotective effects, might affect myocardial expression of cell-to-cell electrical coupling protein connexin-43 (Cx43) and protein kinase C (PKC) signaling, and hence, the propensity of the heart to lethal ventricular fibrillation (VF). Spontaneously hypertensive (SHR) and normotensive Wistar rats fed a standard rat chow received melatonin (40 μg/mL in drinking water during the night) for 5 weeks, and were compared with untreated rats. Melatonin significantly reduced blood pressure and normalized triglycerides in SHR, whereas it decreased body mass and adiposity in Wistar rats. Compared with healthy rats, the threshold to induce sustained VF was significantly lower in SHR (18.3 ± 2.6 compared with 29.2 ± 5 mA; p < 0.05) and increased in melatonin-treated SHR and Wistar rats to 33.0 ± 4 and 32.5 ± 4 mA. Melatonin attenuated abnormal myocardial Cx43 distribution in SHR, and upregulated Cx43 mRNA, total Cx43 protein, and its functional phosphorylated forms in SHR, and to a lesser extent, in Wistar rat hearts. Moreover, melatonin suppressed myocardial proapoptotic PKCδ expression and increased cardioprotective PKCε expression in both SHR and Wistar rats. Our findings indicate that melatonin protects against lethal arrhythmias at least in part via upregulation of myocardial Cx43 and modulation of PKC-related cardioprotective signaling.