Evaluation of the biological activity of naturally occurring 5,8-dihydroxycoumarin.

5,8-Dihydroxycoumarin (5,8-DHC) was isolated from aerial parts of sweet grass (Hierochloë odorata L.) and screened for antioxidant and genotoxic activities. A clear linear dependency of radical scavenging capacity in DPPH• and ABTS•+ assays was determined. 5,8-DHC was very efficient in retarding rapeseed oil oxidation (Oxipress test). TPC (total phenols content) and FRAP (the ability to reduce ferric ion to ferrous ion) assays revealed a somewhat lower antioxidant capacity of 5,8-DHC as compared with gallic acid. Genotoxic activity was tested using different genetic end-points: chromosome aberrations (CAs) and micronuclei (MN) in Wistar rat bone marrow in vivo, CAs and sister chromatid exchanges (SCEs) in human lymphocytes in vitro, and somatic mutations and recombination in Drosophila melanogaster wing cells in vivo. 5,8-DHC did not increase frequency of CAs in rat bone marrow cells, but induced a significant increase of MN. It was slightly mutagenic in the Drosophila melanogaster assay after 120 h of treatment, but not after 48 h of treatment. 5,8-DHC induced both CAs and SCEs in vitro in human lymphocytes in a clear dose-dependent manner. Thus, 5,8-DHC may be classified as weakly genotoxic both in vivo and in vitro.

Biomonitoring study of dry cleaning workers using cytogenetic tests and the comet assay.

Perchloroethylene (PCE) is the main solvent used in the dry cleaning industry worldwide. The aim of the present work was to evaluate the genotoxic potential of occupational exposure to PCE in dry cleaning workers. The study was carried out in 59 volunteers (30 workers, 29 controls). The genotoxic effect was evaluated by analyzing chromosome aberrations (CAs), and micronuclei (MN) and DNA damage (assessed by the comet assay) in peripheral blood lymphocytes. Environmental monitoring of exposure was carried out on personal breathing zone air samples collected during two consecutive working days by measuring the concentration of PCE air levels. The mean PCE concentration in workplace air of dry cleaning workers was 31.40 mg/m(3). There were no significant differences in CA frequency between dry cleaning workers and the controls, but analysis showed a significant association of CA frequency with employment duration and frequency of exposure to PCE. The MN frequency and DNA damage detected by alkaline comet assay were significantly increased in dry cleaning workers compared to the controls. The results suggest that (a) chronic occupational exposure to dry cleaning solvents below permissible occupational exposure limit of 70 mg/m(3) (i.e., ~10.3 ppm) may lead to an increased risk of genetic damage among dry cleaning workers, and (b) CA, MN tests, and comet assay are useful to monitor populations exposed to low doses of PCE.

Establishment and Characterization of a New Pancreatic Ductal Adenocarcinoma Cell Line Capan-26.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma is one of the deadliest forms of human cancer. Since only a vast panel of cell lines can fully recapitulate disease heterogeneity, our aim was to establish a new pancreatic cancer cell line. MATERIALS AND METHODS: Newly established pancreatic ductal adenocarcinoma cell line Capan-26 was characterized by assessing growth rate, tumor and stem cell marker expression, colony forming efficiency, mutations of KRAS and TP53 genes, karyotype and sensitivity to drug treatment. RESULTS: Cell doubling time was 74 h. We detected CA19-9, CEACAM6, CD44, OCT4 and ZEB1 expression in Capan-26 cell line. Cells formed colonies in soft agar, have a deletion of KRAS exon 3 and a point mutation V172F in TP53 exon 5. They are a mixed aneuploid/polyploid population with high sensitivity to gemcitabine. CONCLUSION: Capan-26 is a unique cell line that may be used to study the mechanism of pancreatic cancer.

Genotoxic properties of Betonica officinalis, Gratiola officinalis, Vincetoxicum luteum and Vincetoxicum hirundinaria extracts.

Genotoxicity of B. officinalis, G. officinalis, V. luteum and V. hirundinaria extracts, which demonstrated strong antioxidant capacity, was tested using chromosome aberration, sister chromatid exchange (SCE), cytokinesis-block micronucleus and alkaline single-cell gel electrophoresis (comet) assays in human lymphocytes in vitro and Ames Salmonella/microsome test. All tested extracts were not mutagenic in S. typhimurium strains TA98 and TA100 with and without metabolic activation and did not induce chromosome aberrations in human lymphocytes in vitro. Extract from G. officinalis was the only one, which induced significant increase in micronuclei, indicating possible aneugenic effect. All investigated plant extracts induced DNA damage evaluated by the comet assay, while B. officinalis and V. luteum extracts induced slight increase in SCE values. The determined variation in response might be due to the plant extract tested and donor susceptibility.

Genotoxicity and antioxidant activity of five Agrimonia and Filipendula species plant extracts evaluated by comet and micronucleus assays in human lymphocytes and Ames Salmonella/microsome test.

The species of Agrimonia and Filipendula have been traditionally used in folk medicine as anti-inflammatory herbs. This study extends the knowledge on bioactivities of F. palmata, A. eupatoria, A. procera, F. ulmaria and F. vulgaris by comprehensive characterization of their methanolic extracts. Antioxidant properties of extracts were evaluated by DPPH• (2,2-diphenyl-1-picrylhydrazyl), ABTS•+ 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) scavenging and oxygen radical absorbance capacities (ORAC). Genotoxicity of extracts was tested using alkaline single-cell gel electrophoresis (comet) and cytokinesis-block micronucleus assays in human lymphocytes in vitro and the Ames Salmonella/microsome test. All investigated Agrimonia and Filipendula extracts possessed strong antioxidant activity, which was comparable with that of a standard antioxidant trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thirty five compounds belonging to the classes of phenolic acids, flavonoids, phenylpropanoids and ellagitanins were detected by ultra-performance liquid chromatography - mass spectrometry (UPLC-Q-TOF-MS). Agrimonia and Filipendula extracts induced an increase in a DNA damage in the comet assay expressed as mean percentage of DNA in the comet tail. However, these extracts did not produce reverse mutation in bacterial cells in the Ames test and were not genotoxic in the micronucleus test. However, a slight though significant decrease of nuclear division index values was determined. In general, this study proved that Agrimonia and Filipendula species are a good source of bioactive compounds; their extracts may be classified as non-mutagenic and non-clastogenic in vitro under conditions of the current study. Consequently, the plants may be a promising material for nutraceuticals and natural medicines.

Antioxidant and Genotoxic Properties of Hispidin Isolated from the Velvet-Top Mushroom, Phaeolus schweinitzii (Agaricomycetes).

Antioxidant and genotoxic properties of hispidin isolated from the Phaeolus schweinitzii mushroom were evaluated with various assays. Hispidin demonstrated strong free radical scavenging, oxygen radical absorbance capacity, and ferric-reducing antioxidant power; in all applied assays, hispidin exhibited antioxidant capacity similar to or higher than that of the reference antioxidant Trolox. Genotoxic activity of hispidin was assessed using different end points: chromosome aberrations, micronuclei, sister chromatid exchanges, and primary DNA damage (detected by the comet assay) in human lymphocytes in vitro, and gene mutations in the Salmonella/microsome test. Hispidin did not increase the frequency of chromosome aberrations, micronuclei, or primary DNA damage in human lymphocytes in vitro and did not produce reverse mutation in bacterial cells. However, we identified in human lymphocytes a statistically significant dose-dependent increase in sister chromatid exchange frequency and a decrease in replication index and nuclear division index values.

Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil.

The induction of micronuclei and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analyzed in the erythrocytes of peripheral blood and cephalic kidney of turbot (Scophthalmus maximus) and Atlantic cod (Gadus morua), treated with crude oil (Statfjord B, Norway) and with nonylphenol. Significant increase in MN was observed in turbot kidney and blood after exposure to 30 ppb of nonylphenol, 0.5 ppm of oil, and after co-exposure to 0.5 ppm of oil spiked with additional mixture of alkylphenols and PAHs (P varied between 0.0054 and <0.0001). The induction of micronuclei was observed only in cod kidney after exposure to spiked oil (P=0.0317). Significant inter-specific differences after the exposure to 0.5 ppm of oil (P=0.0385) and after treatment with spiked oil (P=0.0067) were observed. In turbot cephalic kidney, the elevated levels of bi-nucleated cells were observed in all treatment groups (P values varied in a range from 0.05 to 0.0025) while the increase in cells with nuclear buds was noted after the exposure to 0.5 ppm of oil (P=0.05). The fragmented-apoptotic cells appeared after the exposure to nonylphenol (P=0.0039) and to spiked oil (P<0.0001). In turbot blood, only the significant induction in nuclear buds was detected. Statistically significant inter-tissue differences were found only in the induction of fragmented-apoptotic cells after the exposure to nonylphenol and to spiked oil.

Biomarker responses as indication of contaminant effects in blue mussel (Mytilus edulis) and female eelpout (Zoarces viviparus) from the southwestern Baltic Sea.

During a field study performed in spring and autumn 2001 and 2002, blue mussels (Mytilus edulis) and female eelpout (Zoarces viviparus) were collected at three locations in the Wismar Bay (Baltic Sea), and several biomarkers of contaminant effects were analysed. Besides seasonal and inter-annual variations, biomarker signals were most pronounced at the location closest to Wismar Harbour (Wendorf) in both species. Lysosomal membrane stability (LMS) was lowest and acetylcholinesterase activity (AChE) was significantly reduced. Frequency of micronuclei (MN) was significantly higher (in blue mussels), indicating mutagenic effects. In eelpout elevated levels of DNA adducts, EROD induction and PAH-metabolites were measured. Metallothionein (MT), biomarker for trace metal exposure, showed a gradient only in spring. Organochlorine contaminant analyses (PCBs, DDTs) corresponded to the observed biomarker levels. The results obtained clearly demonstrate pollution effects in the southwestern Baltic Sea. Moreover, they show that a multibiomarker approach is also applicable in a brackish water environment.

Biomarker responses in flounder (Platichthys flesus) and mussel (Mytilus edulis) in the Klaipeda-Butinge area (Baltic Sea).

During the EU project BEEP a battery of biomarkers was applied in flounder (Platichthys flesus) and the blue mussel (Mytilus edulis) collected at three locations off the Lithuanian coast (Baltic Sea) in June and September 2001 and 2002. The elevated biomarker responses in specimens sampled in September 2001 were apparently related to the extensive dredging activities in the Klaipeda port area and subsequent dumping of contaminated sediments. High concentrations of organic pollutants (organochlorines and PBDEs) were also measured in the tissues of both indicator species. In addition, response levels of genotoxicity, cytotoxicity, immunotoxicity as well as concentrations of PAH metabolites in the bile of flounder showed elevations in 2002 after an oil spill in the Butinge oil terminal in November 2001. In flounder, biomarker measurements 10 months after the spill indicated recovery processes but in mussels a high level of genotoxicity could still be observed 22 months later. The present study illustrates the usefulness of the multi-biomarker approach in the detection of biological effects of pollution in this region of the Baltic Sea.

A preliminary assessment of singlet oxygen scavenging, cytotoxic and genotoxic properties of Geranium macrorrhizum extracts.

Strong radical-scavenging activity of Geranium macrorrhizum extracts isolated by using various solvent systems has been reported previously. This study aimed at expanding the knowledge on the bioactivities of antioxidatively active G. macrorrhizum butanol fraction, which was isolated from ethanolic extract (EB), and water fraction, which was isolated from water extract (WW) by measuring their singlet oxygen scavenging properties, as well as preliminary assessment of cytotoxicity and genotoxicity toward mammalian cells. The cytotoxicity (necrosis induction) of the extracts in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by antioxidants and stimulated by the prooxidant BCNU (N,N'-bis(2-chloroethyl)-N-nitrosourea). This indicates that the cytotoxicity of G. macrorrhizum extracts is at least partly attributed to their prooxidant action, presumably due to the formation of quinoidal products of their (auto)oxidation. The latter was evidenced by the nature of the peroxidase-catalyzed oxidation products, which supported DT-diaphorase-catalyzed oxidation of NADPH and participated in conjugation reactions with reduced glutathione. The genotoxic properties were studied using chromosome aberration (CA) and sister chromatid exchange (SCE) tests in human lymphocytes in vitro and Drosophila melanogaster somatic mutation and recombination test (SMART) in vivo. In the CA test, only the highest doses of both fractions significantly increased chromosome aberration frequency. In the SCE test, both fractions induced SCEs in a clear dose-dependent manner. G. macrorrhizum extracts were not genotoxic in the SMART test in vivo. Our data indicate that in spite of the possible beneficial (antioxidant) effects of Geranium extracts, the possibilities of their use as ingredients of functional foods and/or food supplements should be further examined due to their cyto- and genotoxic effects resulting mainly from the action of quercetin-derived components abundant in the extracts.

Analysis of micronuclei in blue mussels and fish from the Baltic and North Seas.

Micronuclei (MN) were analyzed in erythrocytes of flounder (Platichthys flesus) and wrasse (Symphodus melops) and in gill cells of blue mussels (Mytilus edulis). The organisms were collected from three study stations in the Baltic Sea and from seven stations in the North Sea (Karmsund area, Norway) 4 times. The statistically significant differences obtained were related to the season, sex of the fish, and sampling locality. Higher MN frequencies were found in fish and mussels collected from the most polluted study stations in the North Sea. The same tendency could be described in the Baltic Sea; however, it was masked by the recent oil spill from the Butinge oil terminal. Our results showing higher MN frequencies in presumably what were the most polluted study locations suggest that MN tests in fish and mussels may be used for the detection of genotoxic effects in a marine environment. The endpoint is well characterized and can be easily recognized, and the technique is convenient to use in field samplings following standard procedures and protocols.