Teresa Gabryelak

Antioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus.

Bivalve molluscs, particularly mussels, are sensitive biomarkers of aquatic ecosystem pollution. The tannins, water-soluble plant polyphenols, may play an important role in this environment and, mainly as a consequence of interaction with pollutants, their toxicity may change. We studied three naturally occurring compounds, tannic acid, ellagic acid and gallic acid, for their ability to modulate DNA damage produced by these tannins alone and in the presence of the oxidative stress inducer H(2)O(2), in cells of the digestive gland of mussels (Unio tumidus). After the treatment of the cells with polyphenols at different concentrations (1, 5, 15, 30, 60, 80, 100, 120, 180, 240 microM) and with hydrogen peroxide in the range of 0.04 and 0.1mM, single-strand breaks (ssb) in DNA were investigated, using the comet assay. The ability of phenolic acids to decrease DNA damage through their antioxidant properties was also assessed. The results show that the phenols, which are known as antioxidative agents, could also act as pro-oxidants. They induced ssb in DNA of the digestive gland at concentrations higher that 10 microM, but lower doses (1 and 5 microM) did not contribute to the DNA damage. This study was also designed to evaluate the protective effect of these tannins against H(2)O(2)-mediated DNA damage in the cells. In this treatment, the two concentrations (1 and 5 microM) significantly decreased the amount of lesions induced by H(2)O(2) (0.04 and 0.1mM). In conclusion, our results demonstrate that antioxidative properties of tannins may change to pro-oxidative activities at the higher concentrations. This suggests that the biologic actions of these compounds may be rather complicated.

Effects of tannins on Chinese hamster cell line B14.

Tannins, naturally occurring plant phenols, have been recognized as antioxidants, but toxic effects have also been observed. In the current investigation, the interaction of this type of compounds with Chinese hamster cells (cell line B14) has been examined. This study reports on the results of experiments in which B14 cells were exposed to tannins: tannic, ellagic and gallic acids in the concentration range 15-240 microM. The cytotoxic and genotoxic effects of these compounds were studied. The colorimetric MTT assay to assess cytotoxicity and the Comet assay for detection of DNA damage were used. In this paper, we also demonstrated the influence of tannins on the fluidity of the plasma membrane. This experiment was carried out by a spectrofluorometric method using two fluorescent probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 12-(9-anthroyloxy)stearic acid (12-AS). The tannins increased the fluidity in the internal region of the lipid bilayer, but no changes at the surface of the plasma membrane were observed. The results of the MTT assay showed that tannins could decrease the viability of cells and that their cytotoxicity was highest at the concentration of 60 microM. The degree of toxicity of these compounds was not correlated with the concentration used. The data obtained from the Comet assay showed that the tannins could also contribute to formation of DNA single-strand breaks.

Interaction between PAMAM-NH2 G4 dendrimer and 5-fluorouracil in aqueous solution

The formation equilibrium of poly(amidoamine) dendrimer (PAMAM-NH₂ G4) complex with an oncologic drug such as 5-fluorouracil (5-FU) in water at room temperature was examined. Using the results of the drug solubility in dendrimer solutions and the method of equilibrium dialysis, the maximal number of drug molecules in the dendrimer-drug complex and its equilibrium constant were evaluated. Solubility results show that PAMAM-NH₂ G4 dendrimer can transfer tens 5-fluorouracil molecules in aqueous solution. The number of active sites in a dendrimer macromolecule being capable of combining the drug, determined by the separation method, amounts to n=30 ± 4. The calculated equilibrium constant of the 5-FU-active site bonding is equal to K=(400 ± 120).

Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo.

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 μM) for 24 and 48 h and their antioxidant and pro‐oxidant effects were determined. The number of SH‐groups was quantified spectrophotometrically using Ellmans reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose‐ and time‐dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double‐strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1 h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24 h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH‐DA (dichlorofluorescein—diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 μM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 μM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water‐soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).

PAMAM G4 dendrimers affect the aggregation of α-synuclein.

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinsons disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.

Viologen-Phosphorus Dendrimers Inhibit α-Synuclein Fibrillation

Inhibition of α-synuclein (ASN) fibril formation is a potential therapeutic strategy in Parkinsons disease and other synucleinopathies. The aim of this study was to examine the role of viologen-phosphorus dendrimers in the α-synuclein fibrillation process and to assess the structural changes in α-synuclein under the influence of dendrimers. ASN interactions with phosphonate and pegylated surface-reactive viologen-phosphorus dendrimers were examined by measuring the zeta potential, which allowed determining the number of dendrimer molecules that bind to the ASN molecule. The fibrillation kinetics and the structural changes were examined using ThT fluorescence and CD spectroscopy. Depending on the concentration of the used dendrimer and the nature of the reactive groups located on the surface, ASN fibrillation kinetics can be significantly reduced, and even, in the specific case of phosphonate dendrimers, the fibrillation can be totally inhibited at low concentrations. The presented results indicate that viologen-phosphorus dendrimers are able to inhibit ASN fibril formation and may be used as fibrillar regulating agents in neurodegenerative disorders.

Phosphorus-containing dendrimers against α-synuclein fibril formation

The aim of this work was to study the effect of phosphorus-containing dendrimers (generations G3 and G4) on the fibrillation of α-synuclein (ASN). The inhibition of fibril formation (filamentous and aggregates) is a potential therapeutic strategy for neurodegenerative disorders such as Parkinsons and other motor disorder neurodegenerative diseases. The interaction between phosphorus-containing dendrimers and ASN was studied by fluorescence spectroscopy. The decrease in the fluorescence intensity of intrisinic tyrosine was the most marked change in the fluorescence intensity observed upon addition of dendrimers. Furthermore, the effect of dendrimers on ASN fibril formation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by fluorescence assays using the dye thioflavin T (ThT). The results showed that phosphorus-containing dendrimers G3 and G4 inhibited fibril formation, when they were used in the ASN/dendrimer ratios 1:0.1 and 1:0.5. However, the higher concentrations of dendrimers did not show this effect.

Study of interactions between phenolic compounds and H2O2 or Cu(II) ions in B14 Chinese hamster cells

The antioxidant and prooxidant effects of tannic, ellagic and gallic acids (1–60 μM) in the presence of hydrogen peroxide (40 and 100 μM) or copper ions (50 μM) on B14 Chinese hamster cells were examined.

Dendrimers—revolutionary drugs for infectious diseases

Over recent years innovative nanomolecules in a form of dendrimers have been gaining increasing interest. These compounds can be designed and modified in many ways giving a molecule which meets required expectations. For this reason dendrimers are the object of intensive studies in many fields of nanoscience including one of the most thriving--biomedicine. Numerous studies provide evidence that some dendrimers exhibit activities against many species/strains of viruses, bacteria, fungi, and prions. These types of dendritic nanostructures which are distinguished by antipathogenic properties and low cytotoxicity to eukaryotic cells may be potentially applied in medicine as novel drugs for various infectious diseases, especially those which are persistent, marked by high mortality rate, or untreatable. Dendrimers can exert their effect via different mechanisms of action, which are, in most cases, related to multivalency of the nanomolecule. The application of dendrimers is likely to be a breakthrough in prevention and treatment of infectious diseases which still beset humanity and may significantly improve the quality of peoples life.

PAMAM G4 dendrimers lower high glucose but do not improve reduced survival in diabetic rats.

For nearly a decade poly(amidoamine) (PAMAM) dendrimers G4 were claimed unnegligible cytotoxic agents. Here we monitored whether in vivo cytotoxic effect of PAMAM G4 (0.5 micromol kg(-1) day(-1)) may be compromised by its ameliorating effect on severe hyperglycaemia in chronic streptozotocin-diabetic Wistar rats. PAMAM G4 significantly reduced the 60-day overall survival in long-term experimental diabetes: treated animals were 6.7 times more likely to die than control animals (p<0.025). PAMAM G4 significantly reduced numerous biochemical parameters in blood, including glucose, glycated haemoglobin or protein oxidation, cholesterol and triglycerides, but apparently unchanged plasma insulin peptide C. Terminal blood glucose in PAMAM-treated animals was significantly higher in survivors, pointing to the possible preventive role of glycation in reducing of PAMAM G4 cytotoxicity. Our results provide the first in vivo evidence that PAMAM G4 is able to lower plasma glucose and suppress long-term markers of diabetic hyperglycaemia. Nevertheless, this beneficial influence cannot override PAMAM G4 cytotoxic effects in the increased mortality of streptozotocin-diabetic rats.