Jozsef Petrik

Antioxidant activities and polyphenolic contents of three selected Micromeria species from Croatia.

Antioxidant activities of three selected Micromeria species growing in Croatia (M. croatica, M. juliana and M. thymifolia) were evaluated using five different antioxidant assays, in comparison with plant polyphenolic constituents and reference antioxidants. All studied ethanolic extracts exhibited considerable activity to scavenge DPPH and hydroxyl free radicals, reducing power, iron chelating ability and total antioxidant capacity in the order: M. croatica > M. juliana > M. thymifolia. Total polyphenol (9.69–13.66%), phenolic acid (5.26–6.84%), flavonoid (0.01–0.09%) and tannin (3.07–6.48%) contents in dried plant samples were determined spectrophotometrically. A strong positive correlation between antioxidant activities and contents of phenolic acids and tannins was found, indicating their responsibility for effectiveness of tested plants. Our findings established Micromeria species as a rich source of antioxidant polyphenols, especially the endemic M. croatica.

Flavonoid aglycones can compete with Ochratoxin A for human serum albumin: A new possible mode of action

The mycotoxin Ochratoxin A (OTA) appears worldwide in cereals, plant products, different foods and drinks. Ochratoxin A binds to plasma albumin with a very high affinity. However, it is well known that natural flavonoids can also bind to human serum albumin (HSA) at the same binding site as OTA does (site I, subdomain IIA). A few experimental literature data suggest that reducing the bound fraction of OTA speeds up its elimination rate with a potential decrease in its toxicity. In our experimental model competitive binding properties of flavonoid aglycones were examined with a fluorescence polarization based approach. Our data show that some of the flavonoids are able to remove the toxin from HSA. We conclude that among the 13 studied flavonoid aglycones galangin and quercetin were the most effective competitors for OTA.

Fluorescence spectroscopic investigation of competitive interactions between ochratoxin A and 13 drug molecules for binding to human serum albumin

Ochratoxin A (OTA) is a highly toxic mycotoxin found worldwide in cereals, foods, animal feeds and different drinks. Based on previous studies, OTA is one of the major causes of the chronic tubulointerstitial nephropathy known as Balkan endemic nephropathy (BEN) and exerts several other adverse effects shown by cell and/or animal models. It is a well-known fact that OTA binds to various albumins with very high affinity. Recently, a few studies suggested that reducing the bound fraction of OTA might reduce its toxicity. Hypothetically, certain drugs can be effective competitors displacing OTA from its albumin complex. Therefore, we examined 13 different drug molecules to determine their competing abilities to displace OTA from human serum albumin (HSA). Competitors and ineffective chemicals were identified with a steady-state fluorescence polarization-based method. After characterization the competitive abilities of individual drugs, drug pairs were formed and their displacing activity were tested in OTA-HSA system. Indometacin, phenylbutazone, warfarin and furosemide showed the highest competing capacity but ibuprofen, glipizide and simvastatin represented detectable interaction too. Investigations of drug pairs raised the possibility of the presence of diverse binding sites of competing drugs. Apart from the chemical information obtained in our model, this explorative research might initiate future designs for epidemiologic studies to gain further in vivo evidence of long-term (potentially protective) effects of competing drugs administered to human patients.

Expression of Hsp70 in kidney cells exposed to ochratoxin A

Abstract. Ochratoxin A (OTA) is a possible etiological agent of endemic nephropathy, a chronic renal disease with high prevalence in limited geographic areas. Ochratoxicosis has many characteristics of different pathological states in which heat shock proteins (Hsps) are usually induced. The most inducible heat shock proteins belong to the Hsp70 family. We determined the level of expression of Hsp70 by the Western blot analysis in kidneys of rats treated with low doses of OTA and in LLC-PK1 and MDCK cells exposed to OTA. Estimation of cell viability and release of lactate dehydrogenase (LDH) confirmed the toxic effects of OTA on cultured cells. OTA affects the relative distribution of two Hsp70 isoforms (68-kDa and 74-kDa isoforms), but does not change total amount of Hsp70 in rat kidney. No changes in the Hsp70 level were detected in LLC-PK1 and MDCK cells treated with OTA, although the cells were seriously injured, as was seen from the reduced cell viability and increased release of LDH. Both cell lines were capable of having Hsp70 induced following a heat shock. However, exposure of the cells to OTA before the heat shock challenge prevented Hsp70 induction. Results of the study show that OTA does not induce Hsp70 in rat kidney or in cultured kidney cells. The absence of Hsp70 protective effects in the cells and tissues might be a possible explanation for the cumulative destructive effects of OTA and a silent onset of endemic nephropathy in humans and of OTA-induced experimental nephrotoxicity in animals.

Flavonoid diosmetin increases ATP levels in kidney cells and relieves ATP depleting effect of ochratoxin A

Diosmetin (DIOS) is a flavone aglycone commonly occurring in citrus species and olive leaves, in addition it is one of the active ingredients of some medications. Based on both in vitro and in vivo studies several beneficial effects are attributed to DIOS but the biochemical background of its action seems to be complex and it has not been completely explored yet. Previous investigations suggest that most of the flavonoid aglycones have negative effect on ATP synthesis in a dose dependent manner. In our study 17 flavonoids were tested and interestingly DIOS caused a significant elevation of intracellular ATP levels after 6- and 12-h incubation in MDCK kidney cells. In order to understand the mechanism of action, intracellular ATP and protein levels, ATP/ADP ratio, cell viability and ROS levels were determined after DIOS treatment. In addition, impacts of different enzyme inhibitors and effect of DIOS on isolated rat liver mitochondria were also tested. Finally, the influence of DIOS on the ATP depleting effect of the mycotoxin, ochratoxin A was also investigated. Our major conclusions are the followings: DIOS increases intracellular ATP levels both in kidney and in liver cells. Inhibition of glycolysis or citric acid cycle does not decrease the observed effect. DIOS-induced elevation of ATP levels is completely abolished by the inhibition of ATP synthase. DIOS is able to completely reverse the ATP-depleting effect of the mycotoxin, ochratoxin A. Most probably the DIOS-induced impact on ATP system does not originate from the antioxidant property of DIOS. Based on our findings DIOS may be promising agent to positively influence ATP depletion caused by some metabolic poisons.

Synergism Between Propolis and Hyperthermal Intraperitoneal Chemotherapy with Cisplatin on Ehrlich Ascites Tumor in Mice

We investigated antitumor, genotoxic, chemopreventive, and immunostimulative effects of local chemoimmunotherapy and hyperthermal intraperitoneal chemotherapy (HIPEC) in a mouse-bearing Ehrlich ascites tumor (EAT). Mice were treated with water-soluble derivative of propolis (WSDP) at a dose of 50 mg kg(-1) , 7 and 3 days before implantation of EAT cells, whereas cisplatin (5 or 10 mg kg(-1) ) was injected 3 days after implantation of EAT cells at 37°C and 43°C. The following variables were analyzed: the total number of cells, differential count of the cells present in the peritoneal cavity, functional activity of macrophages, comet assay, and micronucleus assay. The combination of WSDP + CIS 5 mg kg(-1) at 37°C resulted in tumor growth inhibition and increased the survival of mice by additional 115.25%. WSDP with HIPEC increased the survival of mice by additional 160.3% as compared with HIPEC. WSDP reduced cisplatin toxic and genotoxic effect to normal cells without affecting cisplatin cytotoxicity on EAT cells. In addition, WSDP with HIPEC increased the cytotoxic actions of macrophages to tumor cells. Water-soluble derivative of propolis increases macrophage activity and sensitivity of tumor cells to HIPEC and reduces cisplatin toxicity to normal cells.

Endothelin-1, big endothelin-1, and nitric oxide in patients with chronic renal disease and hypertension

The complex pathogenesis of chronic renal disease (CRD) depends on endothelin (ET) axis (ETs and ET receptors) and nitric oxide (NO) because of their vasoactive effects and their role in general modulation of vascular homeostasis. Various renal cells synthesize ETs and NO that play a significant role in renal hemodynamics as well as in water and salt excretion via urine. ET‐1 is a strong vasoconstrictor. Besides its vasoactive effects, ET‐1 modulates mitosis and apoptosis in a cell type‐dependent manner, and may play an important role in CRD pathogenesis. The aims of this study were to emphasize the role and interactions of ET‐1, Big ET‐1, and NO in CRD. Concentrations of these vasoactive molecules were measured in plasma/serum and/or urine of 57 patients with diabetic nephropathy (subgroup 1), arterial hypertension (subgroup 2) or CRD with chronic renal insufficiency (subgroup 3), and in healthy control subjects (n=18). In comparison with control group, urine concentration of Big ET‐1 was significantly increased (13.13 pmol/L vs. 11.34 pmol/L; P<0.001) in CRD patients, whereas plasma and urine concentrations of ET‐1 did not differ significantly. NO concentrations were also significantly increased in CRD patients (serum, 72.55 µmol/L; P<0.001, and urine 141.74 µmol/L; P<0.05) as compared to control group. Study results indicated that Big ET‐1 and NO could be useful diagnostic parameters in CRD for their diagnostic sensitivity and diagnostic specificity (Big ET‐1 in urine: 56.1 and 88.9%, and NO in serum: 66.7 and 83.3%, respectively). In addition, Big ET‐1 may prove useful in the differential diagnosis of diabetic nephropathy (78.6% diagnostic sensitivity and 88.9% diagnostic specificity). J. Clin. Lab. Anal. 23:347–356, 2009.