Eva Horváthová

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.

Rosemary-stimulated reduction of DNA strand breaks and FPG-sensitive sites in mammalian cells treated with H2O2 or visible light-excited Methylene Blue.

In this study possible protective effects of rosemary against oxidative DNA damage induced by H2O2- and visible light-excited Methylene Blue in colon cancer cells CaCo-2 and hamster lung cells V79 were investigated. The level of DNA damage (DNA strand breaks) was measured using the classical and modified single cell gel electrophoresis, so-called comet assay. Our findings showed that an ethanol extract from rosemary reduced the genotoxic activity of both agents after a long-term (24 h; 0.3 microg/ml) or short-term (2 h; 30 microg/ml) pre-incubation of cells. We suggest that the extract of rosemary exhibits a protective effect against oxidative damage to DNA as a consequence of scavenging of both *OH radicals and singlet oxygen ((1)O2).

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.

Effects of borneol on the level of DNA damage induced in primary rat hepatocytes and testicular cells by hydrogen peroxide.

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H2O2 in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H2O2-induced DNA damage and interfered only partly with rejoining of H2O2-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H2O2, i.e. enhanced DNA-damaging effects of H2O2. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H2O2 in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H2O2 in both hepatocytes and testicular cells.

Borneol administration protects primary rat hepatocytes against exogenous oxidative DNA damage

Experimental evidences suggest that most essential oils possess a wide range of biological and pharmacological activities that may protect tissues against oxidative damage. In this study, we investigated DNA-protective effect of borneol, a component of many essential oils, against oxidative DNA damage induced in primary cultures of rat hepatocytes. Borneol was added to drinking water of Sprague-Dawley rats and DNA resistance against oxidative agents was compared in hepatocytes originated from control and borneol-treated rats. Oxidative stress induced by visible light-excited methylene blue (MB/VL) or 2,3-dimethoxy-1,4-naphthoquionone (DMNQ) resulted in increased levels of DNA lesions measured by the modified single cell gel electrophoresis. Borneol (17 or 34 mg/kg body weight) added to drinking water of rats for 7 days reduced the level of oxidative DNA lesions induced in their hepatocytes by MB/VL or DMNQ. To explain the increased resistance of DNA towards oxidative stress, we measured the base-excision repair (BER) capacity in liver cell extracts of control and borneol-supplemented rats on DNA substrate of HepG2 cells containing oxidative damage. Our results showed that administration of borneol in drinking water had no effect on incision activity of hepatocytes isolated from supplemented rats. The spectrophotometric assessment of enzymatic antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the flow cytometric assessment of total intracellular glutathione (iGSH) in primary hepatocytes of borneol-supplemented rats showed no changes in SOD and GPx activities but higher iGSH content particularly in hepatocytes of higher borneol dose (34 mg/kg) supplemented rats in comparison to control animals. Despite the fact that borneol had no effect either on BER of oxidative DNA damage or on the levels of antioxidant enzymes and manifested no reducing power and radicals scavenging activity, it increased significantly the level of non-enzymatic antioxidant iGSH which could reduce the oxidative DNA lesions induced by MB/VL or DMNQ.

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.

Reduction of carcinogenesis by bio-based lignin derivatives

Abstract The potential medicinal application of lignin component of biomass derived from chemical treatment of wood for paper production was examined with regard to its ability to bind N -nitrosoamines and bile acids. Correlation between adsorption affinity of lignins towards N -nitrosodiethylamine and their cross-linking density was revealed. In contrast to lignin, all the tested carbohydrate preparations were poor adsorbents. It was revealed that the most effective lignin adsorbents—modified kraft and prehydrolysis lignins inhibit mutagenicity and SOS response induced by 4-nitroquinoline- N -oxide. Moreover, these lignin preparations exhibited a protective effect on deoxyribonucleic acid (DNA) in hamster V79 cells and in human VH10 and Caco-2 colon carcinoma cells exposed to hydrogen peroxide (H 2 O 2 ) treatment due to their antioxidant nature. In the case of N -methyl- N ′-nitro- N -nitrosoguanidine—treatment lignins reduced alkylation of DNA due to their high affinity for adsorption of mutagenic N -nitroso compounds. This dual ability of lignin tested to decrease genotoxic activity of chemicals seems to be very promising for their application as natural antimutagenic and anticarcinogenic agents.

Modulation of cisplatin sensitivity in human ovarian carcinoma A2780 and SKOV3 cell lines by sulforaphane

Cisplatin resistance is one of the major obstacles in the treatment of ovarian cancer. In an effort to look for new possibilities of how to overcome this difficulty, we studied the mechanisms of the interactions between sulforaphane (SFN) and cisplatin (cisPt) in combined treatment of human ovarian carcinoma A2780 and SKOV3 cell lines. Synergy (A2780) and antagonism (SKOV3) found in MTT assay was confirmed by apoptosis. While SFN significantly potentiated cisPt-induced DNA damage in A2780 cells, it protected SKOV3 cells against cisPt-crosslinking. We revealed a less efficient Nrf-2 pathway inducibility by SFN in A2780 compared to SKOV3 cells, when activation of the Nrf-2 pathway incites its protectivity against cisPt. Thus, different activation of the Nrf-2 pathway may explain the dual effects of SFN.

Assessment of toxicity, clastogenicity, mutagenicity and transforming activity of pentoxifylline in mammalian cells cultured in vitro

We tested the possible cytotoxic, clastogenic and genotoxic effects of pentoxifylline on different lines of mammalian cells cultured in vitro. This study was part of the developmental research of agapurin, since pentoxifylline represents an effective compound of this drug. Cells treated for a short time manifested a relatively high resistance to the toxic effects of pentoxifylline. Generally, only cells treated for a long time (18 h) or a short time (2 h) with high concentrations of drug manifested sensitivity to the toxic effects of pentoxifylline. Although the tested drug induced DNA synthesis inhibition in V79 and EUE cells and clastogenic effects in V79 cells, it was not able to induce either 6-TGr mutations in the HGPRT locus of V79 cells or morphological transformation of Syrian hamster embryo cells. Adding of microsomal fraction S9 to the treated cells did not markedly change the effects of pentoxifylline on different studied endpoints. We suggest that pentoxifylline has no genotoxic effects, and that the cytotoxicity and induction of chromosomal aberrations were induced by inhibition of cellular DNA replication.

Biological effects of four frequently used medicinal plants of Lamiaceae.

Cancer is one of the leading causes of death characterized by uncontrolled growth and spread of cancer cells. There are several hundred thousands of new cases of cancer worldwide. Clinical oncology is still challenged by toxicity and side effects of multimodal therapy strategies in which it is associated with poor prognosis for patients. There is an urgent necessity to develop novel therapy strategies and to utilize preventive potential of natural compounds. As the majority of anticancer drugs are of natural origin, natural products represent a valuable source for the identification and development of novel treatment options and chemopreventive mechanisms for cancer. This review is focused on the summary of published knowledges on the antioxidant and potential chemopreventive effects of biologically active substances present in the extracts of four plants of the family Lamiaceae (sage, thyme, rosemary and lavander) in different animal and in vitro systems. It is assumed that the chemopreventive and chemotherapeutic potential of natural compounds is the result of a combined action of several mechanisms.