Toxicity of Metal Ions Released from a Fixed Orthodontic Appliance to Gastrointestinal Tract Cell Lines.

The mechanism of toxicity and cellular response to metal ions present in the environment is still a very current area of research. In this work, which is a continuation of the study of the toxicity of metal ions released by fixed orthodontic appliances, eluates of archwires, brackets, ligatures, and bands are used to test the prooxidant effect, cytotoxicity, and genotoxicity on cell lines of the gastrointestinal tract. Eluates obtained after three immersion periods (3, 7, and 14 days) and with known amounts and types of metal ions were used. Four cell lines-CAL 27 (human tongue), Hep-G2 (liver), AGS (stomach) and CaCo-2 (colon)-were treated with each type of eluate at four concentrations (0.1×, 0.5×, 1.0×, and 2.0×) for 24 h. Most eluates had toxic effects on CAL 27 cells over the entire concentration range regardless of exposure time, while CaCo-2 proved to be the most resistant. In AGS and Hep-G2 cells, all samples tested induced free radical formation, with the highest concentration (2×) causing a decrease in free radicals formed compared to the lowest concentrations. Eluates containing Cr, Mn, and Al showed a slight pro-oxidant effect on DNA (on plasmid φX-174 RF I) and slight genotoxicity (comet assay), but these effects are not so great that the human body could not "resist" them. Statistical analysis of data on chemical composition, cytotoxicity, ROS, genotoxicity, and prooxidative DNA damage shows the influence of metal ions present in some eluates on the toxicity obtained. Fe and Ni are responsible for the production of ROS, while Mn and Cr have a great influence on hydroxyl radicals, which cause single-strand breaks in supercoiled plasmid DNA in addition to the production of ROS. On the other hand, Fe, Cr, Mn, and Al are responsible for the cytotoxic effect of the studied eluates. The obtained results confirm that this type of research is useful and brings us closer to more accurate in vivo conditions.

Studying the Functional Potential of Ground Ivy (Glechoma hederacea L.) Extract Using an In Vitro Methodology.

Glechoma hederacea L., known as ground ivy, has a long history of use in folk medicine. The main bioactive compounds in ground ivy are polyphenolic compounds known for their potent antioxidant and antimicrobial activities and thus have high potential as functional ingredients against bacterial infections and the occurrence of chronic diseases associated with oxidative stress in the human body. The aim of the present study was to determine the biological activity of ground ivy extract on selected human cell lines, including hepatic (HepG2), tongue (CAL 27), gastric (AGS) and colon (Caco-2) cancer cell lines by evaluating cytotoxicity, formation of reactive oxygen species and genotoxicity. The antioxidant capacity of the extract was additionally evaluated using cellular model macromolecules of protein and DNA, bovine serum album and plasmid phiX174 RF1 DNA. The effect of ground ivy extract on representatives of human microflora, including L. plantarum, E. coli and S. aureus, was also studied. The cytotoxicity of the extract depended on the type of cells treated, and the pro-oxidant effect generally decreased with increasing exposure time. The most pronounced genoprotective effect against hydroxyl radical damage was monitored in model plasmid DNA and occurred at the highest tested concentration (0.25 mg mL-1), with 95.89% preservation of the supercoiled form of the plasmid. This concentration also had the most significant antioxidant activity on the model protein-14.01% more than the positive control prepared using Trolox. The ground ivy extract showed high antimicrobial potential against the pathogenic bacteria E. coli and S. aureus.

Association of Vitamins D, B9 and B12 with Obesity-Related Diseases and Oral Microbiota Composition in Obese Women in Croatia.

Research background: Oral microbiota has become an important factor in obesity, but its association with obesity-related diseases and serum 25-hydroxy vitamin D [25(OH)D] and B complex amounts is still uncertain. The main aim of the paper is to determine the variation in oral microbiota composition as a response to the vitamin status and obesity-related diseases in obese females from Croatia. We hypothesized that the prevalence of probiotic or pathogenic bacteria in the oral cavity of obese women in Croatia depends on the amounts of vitamin B9 (folic acid), B12 and 25(OH)D in serum and/or hypertension, diabetes and prediabetes diagnosis. Experimental approach: To test the defined research hypothesis, female individuals with body mass index (BMI)≥30 kg/m2 (N=70) were recruited to participate in this study. Obese women were divided into groups according to BMI value, diagnosis of obesity-related diseases and amount of micronutrient in blood. For the quantitative determination of folic acid, vitamin B12 and 25(OH)D in serum, an electrochemiluminescence protein binding assay (ECLIA) was performed. Microorganisms isolated from the saliva of obese women were analyzed by MALDI-TOF mass spectrometer. Results and conclusions: The presented results do not support the hypothesis that the prevalence of probiotic or pathogenic bacteria in the oral cavity of obese women in Croatia depends on the amount of micronutrients. On the other hand, hypertension and diabetes/prediabetes favour the growth of oral pathogens, specifically increased levels of Candida sp. Novelty and scientific contribution: To the best of our knowledge, this is the first study showing the relationship between obesity, micronutrient amount, oral microbiota composition, and the incidence of obesity-related disease. We included only obese women from Croatia, so it is regionally specific. Also, we have shown that oral microbiota composition is not connected with micronutrient deficiencies but only with obesity-related diseases.

The functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit.

BACKGROUND: We evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality. RESULTS: A plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC® 19111™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl3-exposed rats before Lactobacillus treatment. CONCLUSION: The combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.

Synthesis, DNA/RNA-interaction and biological activity of benzo[k,l]xanthene lignans.

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants - presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring - influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.

Influence of Dehydrated Wheat/Rice Cereal Matrices on Probiotic Activity of Bifidobacterium animalis ssp. lactis BB-12®§.

Three novel dehydrated wheat/rice cereal functional products with an addition of well documented probiotic Bifidobacterium animalis ssp. lactis BB-12® (BB-12®) were developed in Podravka factory for the infants older than 4 months: instant rice cereal, instant rice cereal with fruits and instant wheat cereal with vanilla. Notably, the number of viable BB-12® cells in each of the novel products was higher than the required minimal number of probiotic cells per gram of product (106 CFU/g) during the storage period of 106 weeks. Therefore, BB-12® strain recovery and genome stability were evaluated by strain-specific polimerase chain reaction and amplified fragment length polymorphism fingerprinting analysis. Further aim was to evaluate the influence of these three different cereal food matrices on specific probiotic properties of BB-12® strain in vitro. Applied food matrices positively influenced the survival in the simulated conditions of the gastrointestinal tract and antagonistic activity against undesirable microorganisms, while no influence on auto- and coaggregation ability of B. animalis ssp. lactis BB-12® was observed. Adhesion to extracellular matrix proteins and intestinal epithelial Caco-2 cells together with antibacterial activity emphasized competitive pathogen exclusion from Caco-2 cells by probiotic strain BB-12®.

Toxicity assessment of untreated/treated electroplating sludge using human and plant bioassay.

The purpose of this work was to assess the risk to the environment arising from the electroplating sludge from both chemical and toxicological point of view. Both approaches were used for the assessment of the treatment efficiency which consisted of CaO based solidification followed by thermal treatment at 400°C. The elemental composition was determined in the bulk samples and the leachates of untreated sludge. The toxicity of the leachate was determined using two human colorectal adenocarcinoma cell lines (Caco-2 and SW 480) and Hordeum vulgare L. based plant bioassay. The same toxicity tests were employed to the leachate of the treated sludge. Untreated sludge showed extremely high cytotoxic effect to both human and plant bio-system in dose-dependent manner. The percentages higher than 0.5% and 0.05% of the leachate caused significant cytotoxic effect on Caco-2 and SW 480 cells, respectively. The percentages of the leachate higher than 0.05% also showed significant toxic effect to H. vulgare L. bio-system with complete arrest of seed germination following the treatment with 100% to 5% of the leachate. The leachate of the treated sludge showed no toxicity to any of the test systems confirming the efficiency and justification of the employed procedures for the detoxification of electroplating sludge.

Consumer acceptability of liquorice root (Glycyrrhiza glabra L.) as an alternative sweetener and correlation with its bioactive content and biological activity.

Consumer acceptability and sensory properties of liquorice root (Glycyrrhiza glabra L.) were evaluated. Quantitation of total polyphenolics and glycyrrhizic acid (GA), as well as the antioxidant capacity of liquorice extracts, was conducted and their biological effects (cytotoxic, antioxidative/pro-oxidative activity, lipid peroxidation on human laryngeal carcinoma cell line) compared to the ones of their predominant bioactive compound - GA. Conducted consumer survey revealed poor familiarity with liquorice (12.37% of correspondents), but willingness towards its use as an alternative sweetener (77.32% of consumers). Polyphenolic content of evaluated extracts ranged from 1018.18 to 1277.27 mg gallic acid equivalents (GAE)/l while GA content varied between 2179.53 and 2944.13 mg/l. The most pronounced cytotoxic effect (60%) and lipid peroxidation were exerted by treatment with the highest applied extract concentrations (10 mg/ml). Pure GA exhibited cytotoxic and pro-oxidative effects at concentrations of 0.12-0.6 mg/ml. Due to high GA content, coupled with its pronounced cytotoxic activity, the intake of liquorice root should be limited.

Nettle (Urtica dioica L.) extracts as functional ingredients for production of chocolates with improved bioactive composition and sensory properties.

Pursuant to the tendencies of producing functional foods, attractive to a wide range of consumers, in this study chocolates enriched with freeze dried (FD) and concentrated (CE) nettle extracts were formulated, and their polyphenolic and antioxidant capacity stability evaluated during 12 months of storage. A simple aqueous extraction procedure of nettle was developed, and the defined extract evaluated for its cytotoxic and antioxidant/prooxidant activity on human colon cancer cell line (SW 480). An increase in total polyphenolic content, chlorogenic acid and flavonoid derivatives (originating from nettle extract) contents was achieved in enriched chocolates. Implementation of FD extract enabled higher increase of polyphenolic content in comparison to CE extract. During storage, fluctuations of polyphenolic content were observed, but the final bioactive parameters did not differ (or increased) from the initial ones. Nettle enriched chocolates exhibited more intense bitterness and astringency, while dark chocolates were preferred over milk and semisweet ones.

In silico attempt to reveal the link between cancer development and combined exposure to the maize herbicides: Glyphosate, nicosulfuron, S-metolachlor and terbuthylazine.

Pesticides are crucial for crop protection and have seen a 50 % increase in use in the last decade. Besides preventing significant crop losses their use has raised health concerns due to consumer exposure through residues in food and water. The toxicity data from individual components is often used to assess overall mixture toxicity, but uncertainty persists in understanding the behaviors of individual chemicals within these mixtures. Assessing the risk of pesticide mixture exposure remains challenging, potentially leading to overestimation or underestimation of toxicity. This study aims to establish a possible link between exposure to a herbicide mixture and genotoxic effects, focusing on cancer development. Our analysis was focused on four herbicides glyphosate, nicosulfuron, S-metolachlor and terbuthylazine. To determine the link between genes associated with cancer development due to exposure to herbicide mixture, a CTD database tools were used. Through the ToppFun tool molecular function and biological process associated with genes common to the disease of interest and selected herbicides were evaluated. And finally, GeneMANIA was used in order to analyze the function and interaction between common genes of herbicide mixture. Among the 7 common genes for herbicide mixture and cancer development coexpression characteristics were dominant at 65.41 %, 22.14 % of annotated genes shared the same pathway and 7.88 % showed co-localization. Among six target genes involved in genetic disease development co-expression was dominant at 87.34 %, colocalization at 8.03 % and shared protein domains at 4.52 %. Comprehensive molecular analyses, encompassing genomics, proteomics, and pathway analysis, are essential to unravel the specific mechanisms involved in the context of the studied mixture and its potential carcinogenic effects.

Reduction of oral pathogens and oxidative damage in the CAL 27 cell line by Rosmarinus officinalis L. and Taraxacum officinale Web. extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Dandelion (Taraxacum officinale Web.) and rosemary (Rosmarinus officinalis L.) are treasured botanicals with a long usage history in traditional herbal practices worldwide. Dandelion was used to treat kidney, spleen, and liver disease, as well as cardiovascular disease, diabetes, and bacterial infections, whereas rosemary was used to treat pain, spasms, and to improve blood circulation. AIM OF THE STUDY: The aim of this study was to determine the influence of rosemary and dandelion leaves aqueous extracts on the human tongue epithelial carcinoma cell line (CAL 27) at the level of interaction between oral microbiota and tongue epithelial cells, genomic damage, and H2O2 - induced oxidative damage protection. MATERIALS AND METHODS: The polyphenolic composition of the extracts was determined by spectrophotometric and HPLC analyses. After extract treatment, cytotoxic impact and ROS generation in CAL 27 cells were measured using the MTT assay and the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay, respectively. Microdilutions were applied to investigate the antimicrobial and adhesive properties against representatives of the oral microbiota. The single-cell gel electrophoresis (comet assay) and cytokinesis-blocked micronucleus cytome assay (CBMN cyt) were used to detect induced genomic damages. RESULTS: Both extracts increased the adhesion of the lactic acid bacteria L. plantarum but decreased the adhesion of the bacterial pathogens S. enterica serovar Typhimurium LT21 and E. coli K-12 MG1655 adhesion onto CAL 27 cells. 1 h treatment with 5x concentrated dandelion extract and 1x, 2.5x, and 5x of rosemary extract caused an increase in comet tail intensity. CBMN cyt results demonstrated a significant increase in micronucleus formation even at concentrations several times lower than the usual bioactive compound concentrations found in a cup of beverage, with higher concentrations also inducing cell apoptosis and necrosis. Rosemary extract showed a protective effect against H2O2 - induced oxidative damage by decreasing the apoptotic cell number, probably preventing mutations leading to tumor aggressiveness, invasion, and metastasis. CONCLUSIONS: Both tested extracts demonstrated their usefulness in maintaining good oral bacteria balance and their protective capability as powerful antitumor agents by causing a protective apoptotic effect in tumor cell line already at the dosage of an average daily cup.

Cytotoxic activity of strawberry tree (Arbutus unedo L.) honey, its extract, and homogentisic acid on CAL 27, HepG2, and Caco-2 cell lines.

Strawberry tree (Arbutus unedo L.) honey (STH), also known as "bitter honey", is a traditional medicine widely used in the Mediterranean area. Regardless of geographical origin, it usually has a very high content of phenolic compounds and strong antioxidant capacity. Yet, little is still known about the effects of STH, its phenolic extract (STHE), and its main bioactive compound - homogentisic acid (HGA) - at the cell level. The aim of this study was to estimate total phenolic content, DPPH radical scavenging activity, and ferric reducing antioxidant power of STH made in Croatia and investigate cytotoxic and pro-oxidative effects of STH, STHE and HGA on three human cell lines: tongue squamous cell carcinoma (CAL 27), hepatocellular carcinoma (HepG2), and epithelial colorectal adenocarcinoma cells (Caco-2) cells. These substances were tested at four concentrations (0.5-5× average human daily intake of STH) and over 30 min and 1 and 2 h. Croatian STH had a total phenolic content of 1.67 g gallic acid equivalents (GAE) per kg of honey, DPPH radical scavenging activity of 2.96 mmol Trolox equivalents (TE) per kg of honey, and ferric reducing antioxidant power (FRAP) of 13.5 mmol Fe2+ per kg of honey. Our results show no clear and consistent time- or concentration-dependent cytotoxicity in any of the cell lines. ROS levels in all the three cell types at almost all exposure times were not significantly higher than control. The most important observation is that the tested substances have low cytotoxicity and high biocompatibility, regardless of concentration, which is a good starting point for further research of their biological effects in other models.

Red Beetroot and Banana Peels as Value-Added Ingredients: Assessment of Biological Activity and Preparation of Functional Edible Films.

In the present study, water extracts from banana and red beetroot peels were evaluated as a potential source of biologically active compounds for the formulation of edible films. Using spectrophotometric and HPLC-DAD methodologies, banana peel extract was found to be a valuable source of dopamine (156.08 mg L-1), while red beetroot peel extract was abundant in red-violet pigments betacyanins (90.1 mg betanin L-1). The biological activity of the extracts was studied by determining their effects on macromolecular models, including DNA (plasmid phiX RF1 DNA), protein (bovine serum albumin), and lipid (linoleic acid) models, as well as on continuous human cell lines of colon cancer Caco-2 and hepatocellular liver cancer Hep G2 at concentrations of 0.2 and 1 mg mL-1. Results showed that the extracts had no adverse effects and both were further used for the formulation of edible films using alginate in combination with three types of plant proteins-rice, peanut, and pumpkin. In general, edible films based on banana peel extract were characterized by better bioactive properties compared with the films based on red beetroot peel extract. The addition of peanut proteins into the formulations resulted in the most desirable bioactive profile of the formulated edible films, including total phenolic content and antioxidant capacity. Aside from the control sample prepared only with the alginate, the highest dopamine content was determined in the film with incorporated pumpkin proteins (10.72 mg g-1 dw), while the sample prepared with peanut proteins was richest in betacyanins (175.58 mg betanin g-1 dw).

Proteome changes in human bladder T24 cells induced by hydroquinone derived from Arctostaphylos uva-ursi herbal preparation.

ETHNOPHARMACOLOGICAL RELEVANCE: Arctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. AIM OF THE STUDY: This study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. MATERIALS AND METHODS: Quantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. RESULTS: Through in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 µg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. CONCLUSIONS: Despite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.

Non-dioxin-like PCBs in ten different fish species from the Danube river in Serbia.

This work has been developed to examine the level of non-dioxin-like (ndl) PCBs (28, 52, 101, 138, 153 and 180) in (a) ten different freshwater fish species from the Danube river, (b) two sampling points: up and downstream of the industrial zone of the city of Pancevo (ecological hot spot in Serbia) and (c) two time points i.e., in 2001 and 2006. Obtained results would serve to analyse spatial, temporal and congener profile characteristics of ndl PCBs cumulated in fish tissues due to environmental pollution. Sixty-four samples of the following species were collected: wels (Silirus glanus), pike (Esox lucius), bream (Abramis brama), crucian carp (Carassius carassius), pike pearch (Stizostedion lucioperca), barbel (Barbus barbus), tench (Tinca tinca), sterlet (Acipenser ruthenus L.), common carp (Cyprinus carpio) and bighead carp (Hypophthalmichthys nobilis). Gas chromatography coupled with electron capture detector was used for analysis of ndl PCBs. Total ndl PCBs content in upstream samples ranged from 2.7 to 98.1 ng/g and from 4.9 to 68.3 ng/g in 2001 and 2006, respectively. During the 5 years, ndl PCBs content increased significantly in downstream samples i.e., ndl PCBs varied from 13.7 to 46.1 ng/g and from 14.4 to 107.2 ng/g in 2001 and 2006, respectively. PCBs 138 and 180 were predominant congeners in 2001, while in 2006 the most abundant PCB congeners were 138 and 153. In 2006, the presence of PCB 28 and PCB 52 has indicated a recent contamination event. Data on continual monitoring of PCBs in all relevant environmental compartments together with appropriate biomonitoring data are expected to give comprehensive insight into the fate and behaviour profile of these contaminants.

PHYSICO chemical properties and toxicological effect of landfill groundwaters and leachates.

Waste landfills represent a global problem, which is more pronounced in developing countries because of the lack of resources to implement procedures that include separation and waste processing. The aim of this research was to analyze leachate and ground waters samples at the site, upstream and downstream from the landfill during different year seasons on a registered non-hazardous waste dump and to conduct physico-chemical and biological assays to determine potential risk for the ecosystem. Potential cytotoxic, prooxidative and mutagenic effects of leachates and water samples were evaluated on human laryngeal cell line (HEp2). Leachates collected at landfill site caused genotoxic effect and had a higher pH, chemical oxygen demand (COD), biochemical oxygen demand (BOD) and elevated concentrations of phosphorus, chloride, nitrogen compounds and sulphate. Genotoxicity of the leachate was increased in samples collected in dry and warm period of the year. These results are in accordance to the physico-chemical analysis which revealed that during summer period, because of intense degradation process at high temperatures increased concentrations of different chemicals can be found in leachate. Groundwater collected downstream and upstream from landfill did not show statistically significant (geno)toxic effect, irrespective of the sampling season. Chemical analysis revealed that all compounds in groundwater were below permitted values. Purification process at landfill is effective and compounds that reach groundwater do not represent a toxicological threat.

Reduced cytotoxicity in PCB-exposed Chinese Hamster Ovary (CHO) cells pretreated with vitamin E.

The aim of this study was to evaluate protective effects of vitamin E (50 -150 µM) in ovary cells upon cytotoxic effects induced by two structurally distinct PCB congeners - planar "dioxin-like" PCB 77 and non-planar di-ortho-substituted PCB 153 with an emphasis on identifying differences in the mechanism of vitamin E action depending on the structure of congeners. Application of three bioassays confirmed that PCBs decrease ovarian cell proliferation with slightly profound effects of PCB 77. PCB - induced ROS production and lipid peroxidation were significant for both congeners with also more noticeable effect for PCB 77. Vitamin E pre-incubation has improved viability of cells, reduced ROS formation and lipid peroxidation induced by PCBs' treatment. Preincubation with vitamin E was more effective when cells where treated with non-planar PCB 153. Altogether, vitamin E action was protective, congener specific and more effective when ovary cells were exposed to ortho-substituted PCB congener.

Mutagenicity and DNA-damaging potential of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid in vitro.

The aim of this study was to evaluate in vitro toxicity of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid. Cytotoxicity and pro-oxidative effect of both compounds were studied on human colon adenocarcinoma cell line SW 480. No significant cytotoxic effect of either compound was observed. Results of an Ames test on Salmonella typhimurium did not indicate mutagenic activity of clenbuterol on TA 98 and TA 100 strains, regardless of metabolic activation. Potential mutagenic effects of the highest clenbuterol concentration (2500 ng/ml) were observed on the TA 1535 strain. The obtained results of alkaline comet assay on isolated human lymphocytes suggested that both compounds induced an increase of primary DNA damage in a concentration-dependent manner. 4-ADBA was a slightly more potent inducer of primary DNA damage as compared to clenbuterol. Chromosomal aberration analysis showed that clenbuterol caused a statistically significant increase in the total number of aberrant cells only at the highest concentration tested (3% vs. 0.7% in the negative control). The results of this study might represent a solid frame for designing and planning future studies with both compounds, which should further clarify their mechanisms of action and genotoxic/cytogenetic effects relevant for human risk assessment.

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 Ucka, 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.

Molecular and cellular approach in the study of antioxidant/pro-oxidant properties of Micromeria croatica (Pers.) Schott.

Phytochemical composition and antioxidant activity of three wild populations of endemic Illyric-Balcanic species Micromeria croatica (Pers.) Schott have been evaluated with respect to plant organ and growing location. Multivariate analysis (principal component analysis) was performed to visualise (dis-)similarity among samples and identify the correlations between phytochemical variables that explain the most variability. The tested leaf extract from BaCic kuk locality exhibited protective effects against reactive oxygen species-induced damage of DNA and inhibition of lipid peroxidation, while it caused oxidative degradation of protein in the bovine serum albumin assay at higher concentrations. This extract also exhibited cytotoxic activity and facilitated the formation of reactive oxygen species in the HEp2 cell line, in a dose-dependent manner.