Characterization of Rapana thomasiana as an indicator of environmental quality of the Black Sea coast of Bulgaria.

The aim of this investigation was to determine the contents of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), minerals, trace elements and bioactivity in the gastropod Rapana thomasiana, which can be used as an environmental bioindicator organism. The chemical differences between Rapana thomasiana from polluted (RapaPol) and non-polluted (RapaNPol) sites of the Black Sea coast in Bulgarian were investigated. Chromatography and high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) were used for evaluation of PAHs, PCBs, minerals and trace elements. Methanol extracts from RapaPol and RapaNPol (to a lesser degree) contained relatively high amounts of free phenolics (2.50 +/- 0.3 and 1.57 +/- 0.18 mg GAE/g DW, respectively) and exhibited the following respective levels of antioxidant activities determined by two radical-scavenging assays (microMTE/g DW): 1.8 +/- 0.2 and 0.98 +/- 0.08 by 1-diphenyl-2-picrylhydrazyl method (DPPH); 1.74 +/- 0.17 and 1.04 +/- 0.12 by cupric reducing antioxidant capacity (CUPRAC). The total amounts of elements, PAHs and PCBs were higher in RapaPol than in RapaNPol. The obtained indices of Rapana thomasiana can serve as a bioindicator of the environmental ecological quality.

Interactions of oxidatively modified calf skin collagen with platelets and phagocytes.

OBJECTIVES: The effects of non-modified and oxidatively modified calf skin collagen type I on platelet aggregation and the oxidative burst of phagocytes were examined in the framework of a general hypothesis that collagen, platelets and phagocytes cooperate to modulate the oxidative burst of phagocytes and the extent of oxidative stress. MATERIALS AND METHODS: Calf skin collagen type I was subjected to oxidative modification by hydrogen peroxide or hydroxyl radical. Thermal denaturation of collagen was performed in a spectrophotometer equipped with a temperature gradient device. The aggregation of isolated human platelets obtained after differential centrifugation was measured using a dual-channel aggregometer. The production of reactive oxygen species by human whole blood phagocytes was evaluated by luminol-enhanced chemiluminescence. RESULTS: Oxidative modification of collagen samples was characterized by a decrease in denaturation transition temperature. Oxidatively modified samples showed a modified SDS-PAGE pattern, evidencing a significant destruction of the collagen. All oxidatively modified collagen samples, independent of the oxidation treatment applied, lost their platelet-aggregating and phagocyte oxidative burst-inducing activity. CONCLUSION: The results suggest that reactive oxygen species were able to modify collagen. On the other hand, oxidatively modified collagen lost its activating properties towards platelets and phagocytes.

Synthesis, Cytotoxicity and Antioxidant Activity of New Analogs of RC-121 Synthetic Derivatives of Somatostatin.

BACKGROUND: Based on the structure of RC-121 (D-Phe-c (Cys-Tyr-D-Trp-Lys-Val-Cys)-Thr-NH2, - synthetic derivatives of somatostatin), some analogs were synthesized and tested for in vitro cytotoxic and antioxidant activity. OBJECTIVES: The new analogs were modifyed at position 5 with Dap (diaminopropanoic acid), Dab (diaminobutanoic acid) and Orn and at position 6 with the unnatural amino acids Tle (t-leucine). METHODS: The in vitro cytotoxic effects of the substances were investigated against a panel of human tumor cell lines HT-29 (Human Colorectal Cancer Cell Line), MDA-MB-23 (Human Breast Cancer Cell Line), Hep G-2 (Human Hepatocellular Carcinoma Cell Line) and HeLa (cervical cancer cell line). The antioxidant capacities were tested by ORAC (Oxygen Radical Antioxidant Capacity) and HORAC (Hydroxyl Radical Averting Capacity) methods. RESULTS: All substances expressed significantly higher antioxidant capacity by comparison with galic acid and Trolox. All substances showed considerable antioxidant capacity as well. Compound 2T (D-Phe-c(Cys-Tyr-DTrp- Dap-Tle-Cys)-Thr-NH2)had the highest antioxidant effect. The compound 4T (D-Phe-c(Cys-Tyr-D-Trp- Orn-Tle-Cys)-Thr-NH2) displayed antiproliferative effect on HeLa cells with IC50 30 µM. The peptide analog 3T (D-Phe-c(Cys-Tyr-D-Trp-Lys-Tle-Cys)-Thr-NH2) exerted the most pronounced inhibition on the cell vitality up to 53%, 56% and 65% resp. against MDA-MB-23, Hep G-2, HeLa in the higher tested concentration. CONCLUSION: The somatostatin analogs showed moderate influence on the vitality of different tumor cells and could be used in changing their pathology.

Changes in mussel Mytilus galloprovincialis protein profile as a reaction of water pollution.

In the present report the changes in mussels Mytilus galloprovincialis protein profile, as a reaction of water pollution, is presented. The antioxidant capacity was determined by the efficacy to scavenge the derived radical in mussel samples using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. The highest percentage of inhibition was estimated in protein mussels from the dirty area (DM) in comparison with the clean samples (CM). The amounts of both zinc and copper determined by atomic spectroscopy were significantly higher in the protein mussels from the dirty than from the clean areas (P<0.05). Fourier Transform Infrared (FT-IR) spectroscopy and fluorescence revealed specific qualitative changes in secondary and tertiary structures of mussel proteins from the dirty area by the shift in the amides I and II positions and fluorescence intensity. Estimated temperature and enthalpy of denaturation in the protein mussels well correlated with fluorescence and spectroscopic measurements and showed the changes influenced by water pollution.

Relationship between seawater pollution and qualitative changes in the extracted proteins from mussels Mytilus galloprovincialis.

The aim of this study was to find a reliable biomarker of seawater pollution. For this purpose the contents of Zn and Cu, proteins and antioxidant activity in mussels Mytilus galloprovincialis collected from polluted and non-polluted sites of the Bulgarian Black Sea coast were compared. To determine the above-mentioned indices atomic spectroscopy, Fourier Transform Infrared (FT-IR) spectroscopy, fluorescence, differential scanning calorimetry (DSC), and two antioxidant tests were used. It was found that the amounts of Zn and Cu were significantly higher in the mussel proteins from the polluted than from the non-polluted sites (P<0.05). FT-IR spectroscopy and fluorescence revealed specific qualitative changes in secondary and tertiary structures in mussel proteins in the samples from polluted sites. The thermodynamic properties of proteins and the changes upon denaturation were correlated with the secondary structure of proteins and disappearance of alpha-helix. Purified protein scavenging activity against 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(*+)) was significantly higher in mussel samples from polluted than from non-polluted sites. Therefore, the changes in Zn and Cu concentration, in protein's secondary and tertiary structures and antioxidant activity in mussels M. galloprovincialis from polluted sites can be a reliable biomarker of the level of the seawater pollution.

Rapana venosa consumption improves the lipid profiles and antioxidant capacities in serum of rats fed an atherogenic diet.

In the recent years, the consumption of seafood has increased. There are no results on the studies of Rapana venosa (Rv) as a supplementation to the diets. We hypothesized that Rv would increase antioxidant capacity and reduce blood lipids, based on the composition of bioactive compounds and fatty acids. Therefore, the aim of this investigation was to evaluate in vitro and in vivo actions of Rv from contaminated (C) and non-C (NC) regions of collection on lipid profiles, antioxidant capacity, and enzyme activities in serum of rats fed an atherogenic diet. Twenty-four male Wistar rats were divided into 4 groups of 6 each and named control, cholesterol (Chol), Chol/RvC and Chol/RvNC. Rats of all 4 groups were fed the basal diet, which included wheat starch, casein, soybean oil, cellulose, vitamin (American Institute of Nutrition for laboratory animals vitamin mixtures), and mineral mixtures (American Institute of Nutrition for laboratory animals mineral mixtures). During 28 days of the experiment, the rats of the control group received the basal diet only, and the diets of the other 3 groups were supplemented with 1% of Chol, 1% of Chol, and 5% of Rv dry matter from C and NC areas. Dry matter from C and NC areas supplemented diets slightly hindered the rise in serum lipids vs. Chol group: total Chol, 13.18% and 11.63% and low-density lipoprotein Chol, 13.57% and 15.08%, respectively. Cholesterol significantly decreased the value of total antioxidant capacity. The supplementation of Rv to the Chol diet significantly affected the increase of antioxidant capacity in serum of rats, expressed by the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) method. The water extracts of Rv exhibited high binding properties with bovine serum albumin in comparison with quercetin. In conclusion, atherogenic diets supplemented with Rv from C and NC areas hindered both the rise in serum lipids levels and the decrease in the antioxidant capacity. Based on fluorescence and electrospray-ionization mass spectrometry profiles and in vivo studies, changes in the intensity of the found peaks were estimated in the serum samples after supplemented diets. These findings indicate that the supplementation of Rv to the atherogenic diets improve the lipid profiles and the antioxidant status in serum of rats.

Plasma circulating fibrinogen stability and moderate beer consumption.

MODERATE BEER CONSUMPTION (MBC) IS CARDIOPROTECTIVE: it positively influences plasma lipid levels and plasma antioxidant activity in beer-consuming individuals. The connection between MBC and blood coagulation is not clearly defined. Forty-two volunteers were equally divided into experimental (EG) and control (CG) groups following coronary bypass surgery. For 30 consecutive days, only patients of the EG consumed 330 mL of beer per day (about 20 g of alcohol). A comprehensive clinical investigation of 42 patients was done. Blood samples were collected before and after the investigation for a wide range of laboratory tests. The plasma fibrinogen was denatured with 8 M urea and intrinsic fluorescence (IF), hydrophobicity and differential scanning calorimetry (DSC) were used to reveal possible qualitative changes. After 30 days of moderate beer consumption, positive changes in the plasma lipid levels, plasma anticoagulant and plasma antioxidant activities were registered in patients of the EG group. In 17 out of 21 patients of the same group, differences in plasma circulating fibrinogen's (PCF), secondary and tertiary structures were found. The stability of fibrinogen, expressed in thermodynamic parameters, has shown that the loosening of the structure takes place under ethanol and urea denaturation. Also fluorescence stability of PCF was decreased. No changes in the lipid levels, anticoagulant and antioxidant activity or changes in PCF were detected in patients of CG. In conclusion, for the first time after a short term of moderate beer consumption some qualitative changes in the plasma circulating fibrinogen were detected: differences in the emission peak response, fluorescence intensity and all thermodynamic data. Together, with the decrease in the PCF concentration it may lead to an elevation of the blood anticoagulant activity.

Structural changes in plasma circulating fibrinogen after moderate beer consumption as determined by electrophoresis and spectroscopy.

The effects of short-term moderate beer consumption (MBC) on plasma circulating fibrinogen (PCF) in patients suffering from coronary atherosclerosis were investigated by use of 2-dimensional electrophoresis (2-DE), circular dichroism (CD), and Fourier transform infrared spectroscopy (FT-IR). Forty-eight volunteers after coronary bypass surgery were divided into experimental (EG) and control (CG) groups, each of 24. Patients of the EG group consumed 330 mL of beer/day (about 20 g of alcohol) for 30 consecutive days, and CG volunteers drank mineral water instead of beer. Blood samples were collected before and after the experiment. In 21 out of 24 patients after beer consumption the plasma circulating fibrinogen was compromised: changes in its secondary structure were found. These changes were expressed in relatively low electrophoretic mobility and charge heterogeneity, decrease in alpha-helix and increase in beta-sheet, and in slight shift of amide I and II bands. Our findings indicate that one of the positive benefits of moderate beer consumption is to diminish the production of fibrinogen and its stability, which reduces the potential risk exerted by this protein. Thus, in most of beer-consuming patients some qualitative structural changes in plasma circulating fibrinogen were detected.

Antioxidants in the black mussel (Mytilus galloprovincialis) as an indicator of Black Sea coastal pollution.

The biologically active compounds, antioxidant activities, and free radical scavenging effects of black mussel dry matter (Mytilus galloprovincialis)--(BMDM) were investigated. The extract from BMDM with absolute methanol (BMDMAMet) showed the strongest inhibition of lipid peroxidation as a function of its concentration, and was comparable to the antioxidant activity of butylated hydroxyanisole, at the same concentration of 0.2 mg/ml. The extract with 50%methanol/water (BMDM50%Met) had the weakest antioxidant activity, whereas other extracts such as 1.2 M HCl in 50%methanol/water (BMDM50%Met/HCl) exhibited an average inhibition of lipid peroxidation. BMDMAMet extract showed marked activities in free radical scavenging determined by beta-carotene bleaching (beta-carotene), nitric oxide (NO*) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS*+ methods. The antioxidant activities of BMDMAMet extract determined beta-carotene, NO* and ABTS*+ methods showed the highest correlation coefficients (R2) such as 0.998, 0.9976 and 0.6103, respectively, with the presence of total polyphenols. BMDM50%Met had lower antioxidant ability than BMDM50%Met/HCl in different scavenging methods, indicating that the major antioxidant components in these extracts must be derived from the polyphenols. Correlation between proteins and antioxidants was very low (0.0318 and 0.0433). The mussel extracts from polluted areas have shown by all using methods higher antioxidant activity than from the clean ones. The results presented in this report indicate that the antioxidant activities of mussel extracts could possibly be an additional index of pollution characterization.

Growth suppression of human breast carcinoma stem cells by lipid peroxidation product 4-hydroxy-2-nonenal and hydroxyl radical-modified collagen.

Breast cancer is a leading cause of mortality and morbidity in women, mostly due to high metastatic capacity of mammary carcinoma cells. It has been revealed recently that metastases of breast cancer comprise a fraction of specific stem-like cells, denoted as cancer stem cells (CSCs). Breast CSCs, expressing specific surface markers CD44(+)CD24(-/low)ESA(+) usually disseminate in the bone marrow, being able to spread further and cause late metastases. The fundamental factor influencing the growth of CSCs is the microenvironment, especially the interaction of CSCs with extracellular matrix (ECM). The structure and function of ECM proteins, such as the dominating ECM protein collagen, is influenced not only by cancer cells but also by various cancer treatments. Since surgery, radio and chemotherapy are associated with oxidative stress we analyzed the growth of breast cancer CD44(+)CD24(-/low)ESA(+) cell line SUM159 cultured on collagen matrix in vitro, using either native collagen or the one modified by hydroxyl radical. While native collagen supported the growth of CSCs, oxidatively modified one was not supportive. The SUM159 cell cultures were further exposed to a supraphysiological (35 microM) dose of the major bioactive lipid peroxidation product 4-hydroxynonenal (HNE), a well known as 'second messenger of free radicals', which has a strong affinity to bind to proteins and acts as a cytotoxic or as growth regulating signaling molecule. Native collagen, but not oxidised, abolished cytotoxicity of HNE, while oxidized collagen did not reduce cytotoxicity of HNE at all. These preliminary findings indicate that beside direct cytotoxic effects of anticancer therapies consequential oxidative stress and lipid peroxidation modify the microenvironment of CSCs influencing oxidative homeostasis that could additionally act against cancer.

Antioxidative properties of Jaffa sweeties and grapefruit and their influence on lipid metabolism and plasma antioxidative potential in rats.

The effective substances (polyphenols, phenolic and ascorbic acids, flavonoids and dietary fibers) and antioxidative activities, using different radical-scavenging tests, were determined for Jaffa sweeties and grapefruit. The antioxidative activities comprised the contributions from polyphenols, phenolic acids, flavonoids and ascorbate components, and were well-correlated with polyphenols and flavonoids. The correlation coefficient between the polyphenols and antioxidative activity varied from 0.73 to 0.99. All applied methods showed that sweeties had higher antioxidative activity than grapefruit. Experiments on laboratory animals show that diets supplemented with sweeties, and to a lesser extent with grapefruit, increased the plasma antioxidative potential and improved the lipid metabolism, especially in the rats fed with added cholesterol. These findings provide additional characterization of the nutritional value of citrus fruits and their influence on the lipid metabolism in rats.

Seasonal variability of phytoplankton at Varna Bay (Black Sea).

Intrinsic fluorescence and SDS-PAGE analysis were employed to study the seasonal qualitative and quantitative changes of phytoplankton composition at Varna Bay (Black Sea). Variation in the maximum emission wavelength (lambda(max)) of the phytoplankton proteins (398 nm in the summer and 340 nm in the spring) was observed. In addition, a decrease in fluorescence intensity, and a shift in lambda(max) as a result of changes in phytoplankton protein stability, according to the season, was noted. Similarly, SDS-PAGE analysis showed different protein patterns for each season, for example in summertime the major protein constituents were of 14, 37, 48 and 70 kDa, while in the springtime the sizes ranged between 38 and 48 kDa. In general, higher carbohydrate and protein contents correlated with larger phytoplankton biomass found during the summer. The dominant species, the Bacillariophyceae and Dinophyceae, were found to be present in the water body in an alternate pattern. All of these changes could be accounted for by the adaptation of the organism to seasonal variations that modify the sea environment at Varna Bay.