Jasna Franekić

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.

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.

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.

Identification of ABC Transporter Genes in Gonad Tissue of Two Mediterranean Sea Urchin Species: Black, Arbacia lixula L., and Rocky, Paracentrotus lividus L.

Multixenobiotic resistance (MXR) represents an important cellular detoxification mechanism in aquatic organisms as it provides them robustness toward natural and man-made contaminants. Several ABC transporters have major roles in the MXR phenotype – P-gp/ABCB1, MRP1–3/ABCC1–3 and BCRP/ABCG2. In this study, we identified the presence of ABC transporters involved in the MXR mechanism of Arbacia lixula and Paracentrotus lividus. AlABCB1/P-gp, AlABCC3/MRP3, AlABCC9/SUR-like and AlABCG-like transcripts were identified in A. lixula; and PlABCC1/P-gp, PlABCC3/MRP3, PlABCC5/MRP5, and PlABCC9/SUR-like transcripts in P. lividus. For each of the new partial sequences, we performed detailed phylogenetic and identity analysis as a first step toward full characterization and understanding of the ecotoxicological role of these ABC transporters.

Effect of 905 MHz microwave radiation on colony growth of the yeast Saccharomyces cerevisiae strains FF18733, FF1481 and D7

Effect of 905 MHz microwave radiation on colony growth of the yeast Saccharomyces cerevisiae strains FF18733, FF1481 and D7 Background. The aim of this study was to evaluate the effect of weak radiofrequency microwave (RF/MW) radiation emitted by mobile phones on colony growth of the yeast Saccharomyces cerevisiae. Materials and methods.S. cerevisiae strains FF18733 (wild-type), FF1481 (rad1 mutant) and D7 (commonly used to detect reciprocal and nonreciprocal mitotic recombinations) were exposed to a 905 MHz electromagnetic field that closely matched the Global System for Mobile Communication (GSM) pulse modulation signals for mobile phones at a specific absorption rate (SAR) of 0.12 W/kg. Results. Following 15-, 30- and 60-minutes exposure to RF/MW radiation, strain FF18733 did not show statistically significant changes in colony growth compared to the control sample. The irradiated strains FF1481 and D7 demonstrated statistically significant reduction of colony growth compared to non-irradiated strains after all exposure times. Furthermore, strain FF1481 was more sensitive to RF/MW radiation than strain D7. Conclusions. The findings indicate that pulsed RF/MW radiation at a low SAR level can affect the rate of colony growth of different S. cerevisiae strains.

Genotoxic Effects of Green Tea Extract on Human Laryngeal Carcinoma Cells In Vitro

Genotoxic Effects of Green Tea Extract on Human Laryngeal Carcinoma Cells In Vitro Green tea (Camellia sinensis) contains several bioactive compounds which protect the cell and prevent tumour development. Phytochemicals in green tea extract (mostly flavonoids) scavenge free radicals, but also induce pro-oxidative reactions in the cell. In this study, we evaluated the potential cytotoxic and prooxidative effects of green tea extract and its two main flavonoid constituents epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) on human laryngeal carcinoma cell line (HEp2) and its cross-resistant cell line CK2. The aim was to see if the extract and its two flavonoids could increase the sensitivity of the cisplatin-resistant cell line CK2 in comparison to the parental cell line. The results show that EGCG and green tea extract increased the DNA damage in the CK2 cell line during short exposure. The cytotoxicity of EGCG and ECG increased with the time of incubation. Green tea extract induced lipid peroxidation in the CK2 cell line. The pro-oxidant effect of green tea was determined at concentrations higher than those found in traditionally prepared green tea infusions. Genotoksični učinak ekstrakta zelenog čaja na ljudske stanice raka grkljana u kulturi Zeleni čaj, koji je vrlo popularno piće, proizvodi se iz biljke Camellia sinensis i bogat je flavonoidima za koje se smatra da imaju važnu ulogu u sprečavanju nastanka raka. Kao najvažniji mehanizmi djelovanja flavonoida najčešće se spominju vezanje slobodnih radikala te sprečavanje nastanka reaktivnih kisikovih skupina. Cilj ovog rada bio je istražiti potencijalni genotoksični učinak ekstrakta zelenog čaja te epigalokatehin galata (EGCG) i epikatehin galata (ECG), flavonoida koji se u zelenom čaju nalaze u najvišoj koncentraciji. Dobiveni su rezultati pokazali da EGCG i ekstrakt zelenog čaja izazivaju povećanu citotoksičnost otporne stanične linije raka grkljana CK2 nakon kraće inkubacije. Produljenjem vremena inkubacije povećava se citotoksičnost istraživanih spojeva. Također, ekstrakt zelenog čaja izaziva lipidnu peroksidaciju u CK2-stanicama. Prooksidativni učinak ekstrakta zelenog čaja u koncentracijama višim od onih prisutnih u infuzijskoj otopini dobivenoj tradicionalnim načinom, imaju prooksidativno djelovanje.

Comparative study of cytotoxic and cytoprotective activities of cocoa products affected by their cocoa solids content and bioactive composition

Polyphenolic profile and antioxidant properties of water extracts of milk, semisweet and dark chocolates, as well as cocoa liquor, were determined and examined for potential biological activity. Non-fat cocoa solids (NFCS), phenolic compounds content and the antioxidant capacity of cocoa product extracts were determined using UV/Vis spectrophotometric methods and HPLC analysis. The increase in NFCS was consistent with the increase in polyphenol content and antioxidant properties. Methylxanthines, theobromine and caffeine constituted the most abundant bioactive compounds, followed by flavan-3-ols epicatechin and procyanidin B2. Cytotoxic and antioxidative/prooxidative effects of cocoa product extracts were determined on human laryngeal carcinoma cell line (HEp2). Cocoa liquor containing the highest NFCS exhibited the lowest HEp2 cell viability, while milk chocolate characterized by the lowest NFCS exhibited no cytotoxic effect. Experiments revealed a strong relationship between the type of product/concentration/time of exposure and antioxidant/prooxidant character of cocoa products. Lower concentrations of semisweet, dark chocolate and cocoa liquor induced an increase in ROS formation, while the higher concentrations resulted in a decrease in ROS formation when compared with control (growth medium). Principal component analysis of the obtained results revealed specific grouping of samples (milk chocolate and cocoa liquor), while the observed dispersions indicated that the outcome of cytotoxic and cytoprotective activities of cocoa products are greatly affected by their concentration.

Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors

Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors Background. Testicular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs. Materials and methods. Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method. Results. LOH of the CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component. Conclusions. The higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior.

Effect of overexpression of E. coli 3-methyladenine-DNA glycosylase I (Tag) on survival and mutation induction in Salmonella typhimurium

Salmonella typhimurium, compared to Escherichia coli, is deficient in an inducible glycosylase activity harbouring only constitutive glycosylase functions. 3-Methyladenine-DNA glycosylase I encoded by the E. coli tag gene is a constitutively expressed repair enzyme that primarily removes N3-methyladenine but also N3-methylguanine from DNA by glycosylic cleavage in the first step of the base excision repair. In order to investigate in vivo effect of the overexpressed glycosylase I activity on survival capacity and mutation induction in S. typhimurium, and thereby elucidate the significance of both 3-methylpurines in cellular sensitivity to methylating agents (e.g., DMS), we transformed four his- S. typhimurium strains with the plasmid pCY5 carrying the E. coli tag gene under the control of the lac promoter. Although the 3-methyladenine-DNA glycosylase activity in cells carrying pCY5 was only 10-fold higher on exposure to IPTG compared to the TA1535 control strain carrying pUC8, the overexpression of the Tag protein completely suppressed deficiency in an inducible glycosylase activity, rendering cells resistance to toxic effects of DMS. The suppression was not influenced by the nucleotide excision repair pathway since there was no difference in recovered survival among NER-proficient and NER-deficient strains. The yield of mutation induction in the reversion assay was decreased to the level of spontaneous (his-->his+) revertant colonies showing that in the overall population in overexpressed conditions in vivo 3-methyl-guanine, in addition to 3-methyladenine, must have been removed from DNA by the E. coli Tag protein and therefore accounts for the second most important cytotoxic lesion.

P IV.10 - P IV.10 Stable yeast but not human apurinic/apyrimidinic en-donuclease transfectants of Chinese hamster cells are more resistant to genotoxic agents

Apurinic/apyrimidinic (AP) endonucleases play a main role in excisiion repair of either spontaneously arisen or induced abasic sites. In order to examine the biological significance of Ap endonucleases in protection of mammalian cells against genotocix agents, we have generated stable transfectants of Chinese hamster ovary (CHO) cells harbouring human (APE) and yeast (APN1) Ap endonuclease, and compared their effects as to protection against mutagen-induced cell killing and formation of chromosomal aberrations. Although APE was markedly expressed on RNA and protein level, nuclear extracts of human APE transfectants showed an activity equal to that of the parental cell line and did not become more resistant to the cell killing and clastogenic effect of methyl methanesulfonate and hydrogen peroxide. On the other hand, CHO cells stably transfected with the yeast APN1 gene expressed higher Ap endonuclease activity and became more resistant tot the cytotoxic and clastogenic effect of the agents. From the resulsts the conclusions have been drawn that i) excision repair capacity as well as mutagen resistance in mammalian cells can be enhanced by introducing the yeast APN1 gene and ii) Ap sites are both cytotoxic and clastogenic lesions.