Ksenija Durgo

Spectrophotometric analysis of flavonoid-DNA interactions and DNA damaging/protecting and cytotoxic potential of flavonoids in human peripheral blood lymphocytes.

The ability of luteolin, kaempferol and apigenin to bind to calf thymus (ct)-DNA, mode of action and stability of flavonoids in buffer were investigated. Spectrophotometric analysis revealed a rapid degradation of apigenin in an aqueous medium, while kaempferol and luteolin were stable for 24h upon dissolution in water. Spectrophotometric study of the interactions of kaempferol and luteolin with calf thymus DNA suggests classic intercalation as their dominant binding mode to DNA. Cytotoxicity/genotoxicity and cytoprotective/genoprotective effects of flavonoids in non-stressed and hydrogen peroxide stressed human peripheral lymphocytes were investigated using the fluorescent dye exclusion method and alkaline comet assay. Flavonoids revealed significant genoprotective effects in hydrogen peroxide stressed cells and in cells submitted to longer incubation in the cell culture medium. Luteolin, followed by apigenin and kaempferol, was shown to be the most effective in protecting DNA from oxidative damage induced by hydrogen peroxide. However, the investigated flavonoids also induced DNA damage, indicating their prooxidative capacity. The balance between the protection of DNA from oxidative damage and prooxidative effects was strongly dependent on flavonoid concentration and the incubation period.

The Bioactive Potential of Red Raspberry (Rubus idaeus L.) Leaves in Exhibiting Cytotoxic and Cytoprotective Activity on Human Laryngeal Carcinoma and Colon Adenocarcinoma

In this article, the bioactive potential of red raspberry leaves, a by-product of this widely spread plant, mostly valued for its antioxidant-rich fruits, was determined. The polyphenolic profile and antioxidative properties of red raspberry leaf extract were determined and examined for potential biological activity. Cytotoxic effect, antioxidative/prooxidative effect, and effect on total glutathione concentration were determined in human laryngeal carcinoma (HEp2) and colon adenocarcinoma (SW 480) cell lines. SW 480 cells are more susceptible to raspberry leaf extract in comparison with HEp2 cells. The antioxidative nature of raspberry leaf extract was detected in HEp2 cells treated with hydrogen peroxide, as opposed to SW 480 cells, where raspberry leaf extract induced reactive oxygen species formation. Raspberry leaf extract increased total glutathione level in HEp2 cells. This effect was reinforced after 24 hours of recovery, indicating that induction was caused by products formed during cellular metabolism of compounds present in the extract. Comparison of the results obtained on these two cell lines indicates that cellular response to raspberry extract will depend on the type of the cells that are exposed to it. The results obtained confirmed the biological activity of red raspberry leaf polyphenols and showed that this traditional plant can supplement the daily intake of valuable natural antioxidants, which exhibit beneficial health effects.

The assessment of genotoxic effects of wastewater from a fertilizer factory.

In this study we investigated cytotoxic, mutagenic and genotoxic effects of different concentrations of wastewater from the phosphoric gypsum depot near the factory for fertilizing agents ‘INA Petrokemija’ (Kutina, Croatia). The Ames test was performed on Salmonella typhimurium TA98 and TA100 strains, in the presence of S9 mix, glutathione and buffer, respectively. Cytotoxicity was studied on human laryngeal carcinoma cells (HEp2) and human cervical cells (HeLa). The level of lipid peroxidation in these two cell lines was evaluated in parallel. To establish the levels of primary DNA damage, the alkaline comet assay was performed on treated human peripheral blood leukocytes. No mutagenic effects of phosphoric gypsum on Salmonella typhimurium strains in the presence of S9 mix, GSH or PBS were observed. However, strong cytotoxic effect was observed on both human cell lines when they were treated with different concentrations of wastewater. Lipid peroxidation was induced and increased by prolonged time of incubation, highlighting that the damage was not repaired, but increased with the time of incubation. The results of the alkaline comet assay indicate significant DNA damaging potential of wastewater for human leukocytes. Since phosphoric gypsum transport water in its present composition and acidity is highly toxic and acts as prooxidant, causing free radicals formation and DNA damage, urgent neutralization/purification of the wastewater to a level acceptable for disposal into the environment is mandatory. Copyright

Phytochemical Attributes of Four Conventionally Extracted Medicinal Plants and Cytotoxic Evaluation of Their Extracts on Human Laryngeal Carcinoma (HEp2) Cells

The bioactive composition and cytotoxic and antioxidative/prooxidative effects of four medicinal plants: yarrow (Achillea millefolium L.), hawthorn (Crataegus oxyacantha L.), ground ivy (Glechoma hederacea L.), and olive (Olea europea L.) on human laryngeal carcinoma cell line (HEp2) were investigated. Water extracts of these plants obtained by infusion, maceration, and decoction were characterized for their polyphenol content and antioxidant capacity. Based on the extraction efficiency of polyphenols, the final extracts were obtained whose polyphenolic profile, polysaccharides, mineral content, and cytoprotective activities were determined. The overall highest content of polyphenols and antioxidant capacity was determined in hawthorn, followed by yarrow and ground ivy, and the lowest in olive leaves extract. Phytochemical screening revealed the presence of phenolic acids, as the most abundant bioactive compounds, followed by flavonoids, flavons, and flavonols. All examined medicinal plants reduced the cell viability and reactive oxygen species formation in a dose- and time-dependent manner. Ground ivy and yarrow containing a high content of phenolic acids and polysaccharides were more efficient to decrease the cell survival when compared to olive leaf and hawthorn. Experiments confirmed the importance of polyphenolic composition rather than content of investigated plants and revealed a relationship between the polyphenolic and polysaccharide contents and antioxidant/prooxidant characters of medicinal plants.

Combined effects of cadmium and decabrominated diphenyl ether on thyroid hormones in rats.

The aim of this study was to see how a mixture of cadmium (Cd) and decabrominated diphenyl ether (BDE209) affect thyroid function, namely thyroid-stimulating hormone (TSH), thyroxin (T4), free thyroxin (FT4), triiodothyronin (T3), and free triiodothyronin (FT3) in Wistar rats (eight per group) receiving either a single substance or their combination by gavage for 28 days. Three groups were receiving Cd alone in the doses of 2.5 mg kg-1, 7.5 mg kg-1, or 15 mg kg-1 b. w. a day, three groups were receiving BDE209 in the doses of 1000 mg kg-1, 2000 mg kg-1, or 4000 mg kg-1 b. w. a day, while nine groups were receiving different mixtures of Cd and BDE209 in these doses (3x3 design). The results have indicated that the Cd+BDE209 mixtures more potently disrupt thyroid hormone homeostasis than would be expected from these chemicals alone.

Cytotoxic and Genotoxic Effects of the Quercetin/Lanthanum Complex on Human Cervical Carcinoma Cells In Vitro

Cytotoxic and Genotoxic Effects of the Quercetin/Lanthanum Complex on Human Cervical Carcinoma Cells In Vitro Quercetin is the main flavonoid in diet with a potential in the treatment of cancer, cardiovascular, and neurodegenerative diseases. Due to its specific planar chemical structure, quercetin readily forms chelates with metal ions. Complexes of bioactive compounds and metal ions such as lanthanum often show strong cytotoxic and antitumour properties. The aim of this study was to compare the genotoxic effects of the quercetin/lanthanum complex on human cervical carcinoma cells with compare it to the effects of free ligands, quercetin, and lanthanum alone. The quercetin/lanthanum complex showed considerable cytotoxicity in the concentration range of (100 to 1000) mmol mL-1 and exposure time of three hours. The complex also induced a dose-dependent pro-oxidative effects and the formation of single-strand and double-strand DNA breaks. Although we obtained promising results on the cell level, future experiments should answer whether the quercetin/lanthanum complex is cancer-specific and stable enough in physiological conditions to make a potential new antitumour drug. Genotoksični učinak kompleksa kvercetina i lantana na ljudske stanice karcinoma grla maternice Kvercetin je jedan od najzastupljenijih flavonoida u prehrani. Zbog specifične kemijske strukture i bioloških svojstava jedan je od najproučavanijih flavonoida kao potencijalni lijek koji bi se rabio za liječenje kardiovaskularnih, neurodegenerativnih i tumorskih bolesti. Kvercetin zbog svoje planarne kemijske strukture vrlo lako stupa u interakciju s metalnim ionima te tvori kelate. Cilj ove studije bio je utvrditi potencijalni genotoksični učinak kompleksa kvercetina i lantana na ljudskoj staničnoj liniji karcinoma grla maternice i usporediti taj učinak s genotoksičnim učincima pojedinačnih spojeva, kvercetina i lantana. Istraživani kompleks izazvao je značajnu toksičnost u rasponu koncentracija od 100 mmol mL-1 do 1000 mmol mL-1 i u vremenu inkubacije od 3 sata. Kompleks je pokazao prooksidativnu aktivnost u ovisnosti o koncentraciji te je pri najvišim istraživanim koncentracijama izazvao lomove DNA. Ako bi u daljnjim istraživanjima kompleks kvercetina i lantana pokazao selektivno djelovanje prema stanicama raka i stabilnost u fiziološkim uvjetima, mogao bi se promatrati u svjetlu potencijalnog antitumorskog lijeka, što bi trebala rasvijetliti daljnja istraživanja.

Cadmium and decabrominated diphenyl ether mixture: In vitro evaluation of cytotoxic, prooxidative and genotoxic effects.

In order to look into the combined effects of Cd and BDE-209 in vitro, this study was aimed at examining cytotoxic and genotoxic effects using the human colon carcinoma cell line (SW 480) as a biological test system as well as to determine if ROS production was one of the possible mechanisms of their mixture action. This cell line was chosen since ingestion of contaminated food/water represents an important route of exposure to both Cd and BDE-209, which is why intestinal cells are a common target for the contaminants present in food and water. Cells were treated with single Cd in concentrations of 2.5, 7.5 or 15μg Cd/mL (corresponding to 22, 67 or 134μM), single BDE-209 in concentrations of 2.5, 5 or 10μg BDE209/mL (corresponding to 2.5, 5 or 10μM), and their mixtures (design 3×3). Mixture of Cd and BDE-209 has shown clear potential to reduce the viability of SW 480 cells, as evidenced by cytotoxicity associated with ROS generation. Factorial regression models used to identify type of interaction revealed synergism related to mixture citotoxicity and additive interaction for the effect on ROS production. The results from this introductory study could contribute to the issue of possible adverse effects associated with co-exposure and body burden with two persistent environmental pollutants, Cd and BDE-209.

Toxicological Characterization of the New Water Cleaning Product and its Waste By-Product

Abstract Water extracts genotoxicity of the coagulant produced from industrial waste (red mud and waste base) and its waste mud remained after waste water treatment by the coagulation/flocculation process were studied. Tests were conducted in order to confirm nontoxicity of this new product prior to commercial production and usage and also to recommend a safe way for a handling and disposal of the remaining waste material. The toxicity investigation included (i) determining frequency of the cell survival, (ii) the Ames assays, (iii) micronucleus assay, and (iv) cell proliferation kinetics (expressed as mitotic index). These techniques were also employed in toxicity testing of the different concentrations of metal salts, zinc chloride, and lead nitrate in this case since these two elements were present in high concentrations in the waste water intended for the purification with the new coagulant. Mixture of metal salts in the concentrations that represent maximum allowed values for water extracts of technological waste was also tested. Two strains of Salmonella typhimurium, TA98, and TA100 were used for determining cytotoxicity and for the Ames test, while the cytogenetic investigations were performed on human peripheral blood lymphocytes. Water extracts of the coagulant and its waste mud did not induce a significant increase of the micronuclei in human peripheral blood lymphocytes. They also did not disturb lymphocyte proliferation kinetics in vitro. As regards lead nitrate it proved not to be cytotoxic on bacterial strains in the tested concentration range (1–100 mg/L), whereas zinc chloride showed cytotoxic effect for the concentrations above 25 mg/L. The Ames test results for the noncytotoxic concentrations of these metals were negative. Comparing to the negative control a significantly higher number of the micronuclei was determined after the treatment of the whole blood with 100 mg/L of zinc chloride, as well as with 10 and 100 mg/L of lead nitrate. A linear, dose dependent increase was obtained for both salts. Similar results were obtained on the basis of the mitotic index.

Genetic and phytochemical variability of six Teucrium arduini L. populations and their antioxidant/prooxidant behaviour examined by biochemical, macromolecule- and cell-based approaches

Teucrium arduini L., an Ilyric-Balcanic endemic species, has been reported for decades as a valuable plant used in traditional medicine for treating digestive disorders. The present study evaluated genetic and phytochemical variability of six T. arduini populations in order to determine factors that influence an accumulation of polyphenolic compounds. Results strongly suggest that a phytochemical variation was caused by environmental rather than genetic factors. T. arduini leaf extract from the locality Učka, which accumulated significantly more polyphenolic phytochemicals in comparison to others, showed antioxidant activity in DNA and lipid bioassays. Furthermore, the same extract exhibited prooxidant behaviour at protein level and induce formation of reactive oxygen species in human laryngeal carcinoma cells causing cytotoxic activity, in a dose dependent manner. All the results of the present study suggested that T. arduini extract could be responsible for antioxidative/prooxidative mechanisms and would help in determination of optimal conditions for their ethnopharmacological use.

Citotoxicity status of electroplating wastewater prior/after neutralization/purification with alkaline solid residue of electric arc furnace dust

Toxicological safety of new procedure for the neutralisation/purification of wastewater originated from zinc plating facility was investigated. Wastewater was treated with alkaline solid residue-by-product of zinc recovery from electric arc furnace dust. For determination of citotoxic potential of untreated and purified wastewater MTT test on HEp2 (human laryngeal carcinoma) and HeLa (human cervical carcinoma) cells lines and alkaline comet assay on human leukocytes were used. Then 100% of the sample as well as different dilutions were tested. Compared to negative control 100, 75 and 50% of the sample of untreated wastewater significantly decreased survival of both HEp2 and HeLa cell lines. In the presence of undiluted sample survival percentage of HeLa and HEp2 cells were only 2.3 and 0.3% respectively. Only undiluted purified wastewater showed slight but insignificant decrease of the survival of both cell lines. Even 0.5% of the sample of original electroplating wastewater exhibited significantly higher value of all comet assay parameters compared to negative control. There was no significant difference between negative control and purified wastewater for any of comet assay parameters. Significantly lower level of primary DNA damage recorded after treatment with purified water, even comparable with negative control, confirmed effectiveness of the purification process.