Lada Rumora

Cytotoxicity and apoptosis induced by fumonisin B1, beauvericin and ochratoxin A in porcine kidney PK15 cells: effects of individual and combined treatment

The objective of this study was to determine individual and combined effects of fumonisin B1 (FB1), beauvericin (BEA) and ochratoxin A (OTA) on porcine kidney epithelial PK15 cell survival by measuring lactate dehydrogenase (LDH) activity, apoptotic index and caspase-3 activity. Cells were treated with 0.05, 0.5 and 5xa0μg/ml of each mycotoxin or with the combinations of two or all three mycotoxins for 24 and 48xa0h. Changes in LDH and caspase-3 activity, and in apoptotic index showed that the cytotoxic and apoptotic effects of these mycotoxins were concentration- and time- dependent. Significant increase of LDH activity was observed after 48xa0h of exposure to the highest concentration of FB1 (45%), BEA (84%) and OTA (77%), as compared to control. OTA increased caspase-3 activity after 24xa0h of treatment with 0.5xa0μg/mL (84%), while BEA (319%) and FB1 (419%) significantly affected this enzyme activity after 48xa0h (Pxa0<xa00.05). Increase of caspase-3 activity preceded significant morphological apoptotic changes, which were detected after 48xa0h of exposure to a single toxin. Combined treatment with FB1, BEA and OTA resulted mostly in additive effects on LDH activity, and additive and synergistic effects on caspase-3 activity and apoptotic index.

A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin.

The aim of this study was to establish the involvement of calcium signalling in genotoxicity, apoptosis and necrosis evoked by ochratoxin A (OTA) and citrinin (CTN) alone or in combination in porcine kidney PK15 cells. Cell proliferation test (MTT) and trypan blue assays (24xa0h) demonstrated that CTN (IC50xa0=xa073.5xa0±xa01.0, 75.4xa0±xa01.4xa0μM, respectively) was less toxic than OTA (IC50xa0=xa014.0xa0±xa02.4, 20.5xa0±xa01.0xa0μM, respectively). To test their cytotoxic interactions, two doses of single OTA (6 and 10xa0μM) and CTN (30 and 50xa0μM) and their combinations were applied. Combined treatment showed additive cytotoxic effects. OTA and CTN induced dose-dependent increase in cytosolic calcium level (assessed with Fura-2 AM). However, combined treatment did not provoke additional increase in calcium signal. The rate of apoptosis and necrosis (DAPI-antifade staining) was significantly higher after 12xa0h than 24xa0h, while the frequencies of micronuclei (MNs) and nuclear buds (NBs) were higher after 24xa0h than 12xa0h treatment. Combined exposure resulted in apoptotic and necrotic synergism, while genotoxic effects of OTAxa0+xa0CTN were noted as antagonistic or additive. Co-exposure of cells to calcium chelator BAPTA-AM significantly reduced CTN and OTAxa0+xa0CTN-evoked apoptosis. Twenty-four hour after co-exposure to BAPTA-AM and a single OTA and CTN, MNs significantly decreased while NBs dropped significantly after co-treatment with BAPTA-AM and OTAxa0+xa0CTN. In conclusion, disturbance of Ca2+ homeostasis caused by OTA and CTN plays a significant role in cell genotoxicity and death.

Cytotoxic and genotoxic effects of fumonisin B1 on rabbit kidney RK13 cell line.

Abstract Fumonisins, mycotoxins produced by certain strains of Fusarium moniliforme, could induce various diseases in animals and are suspected human carcinogens. Fumonisin B1 (FB1), the most commonly found fumonisin, has been characterised as a tumour initiator and a tumour promoter, a mitogen and an anti-proliferative agent. In this study we examined the cytotoxicity and genotoxicity of FB1 in rabbit kidney RK13 cells. To evaluate the effects of FB1 on survival of this cell line we analysed cell viability, membrane integrity, DNA fragmentation and overall morphology of the cells. The genotoxic potential of FB1 was estimated by monitoring the ability of this mycotoxin to induce micronuclei in RK13 cells. Exposure to FB1 caused a significant increase in micronucleus frequency in a concentration- and in a time-dependent manner. Nanomolar concentrations of FB1 decreased cell viability after 24xa0h and even more so after 48xa0h of exposure. The morphological changes observed suggested that an increased number of RK13 cells were dying by the process of apoptosis. However, FB1 also induced impairments of cell and mitochondrial membrane integrity, as assessed by lactate dehydrogenase and glutamate dehydrogenase leakage. These results could imply that nanomolar concentrations of FB1 induced apoptosis, which subsequently may proceed to secondary necrosis. In summary, our observations suggest that FB1 is genotoxic and cytotoxic to RK13 cells.

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.

Impairments of heat shock protein expression and MAPK translocation in the central nervous system of follitropin receptor knockout mice

The central nervous system is exposed to the chronic oxidative stress during aging when the endogenous defence weakens and the load of reactive oxygen species enhances. Sex hormones and heat shock proteins (Hsps) participate in these responses to stress. Their regulation is disturbed in aging. We assessed the expression of Hsps in hippocampus and cortex of follitropin receptor knockout (FORKO) mice, known to exhibit gender and age-dependent imbalance in sex steroids and gonadotropins. These imbalances could contribute to an impaired regulation of Hsps thereby increasing the risk of developing neurodegenerative disorders. Our study shows that, in the hippocampus the expression of Hsp70 and Hsp25 was reduced in 20-month-old FORKO mice. However, in the cortex both Hsps were significantly down regulated only in elderly females. There is a well-established co-regulation between Hsps and mitogen-activated protein kinases (MAPKs). Significant, gender-specific impairments in the translocation of phosphorylated ERK and JNK were found in the CNS structures in aged FORKO mice. Our results suggest that hormonal imbalances lead to a disturbed subcellular distribution of activated MAPKs which contribute to the impairments of signal transduction networks maintaining normal physiological functions in the cortex and hippocampus that are associated with neurodegenerative changes in aging.

Effect of non-genetic factors on paraoxonase 1 activity in patients undergoing hemodialysis

OBJECTIVESnHemodialyzed patients have lower paraoxonase 1 (PON1) activity. Higher mortality risk from cardiovascular disease observed in these patients could be due to the low antiathetrogenic activity of PON1. Understanding the mechanism that causes lower PON1 activity could provide the possibility for modulation of enzyme activity in purpose of preventing and/or decreasing development of atherosclerosis.nnnDESIGN AND METHODSn87 healthy individuals and 71 hemodialyzed patients were enrolled in this study.nnnRESULTSnHemodialyzed patients had reduced PON1 paraoxonase and arylesterase activity, concentrations of HDL, HDL(3) and HDL(2) and concentrations of free thiol groups. Distribution of HDL subfractions and distribution of PON1 phenotypes as well as concentrations of MDA were not different between two study groups. In the in vitro experiment high concentrations of urea, creatinine, uric acid and addition of patients sera ultrafiltrate did not significantly affect PON1 paraoxonase activity.nnnCONCLUSIONnDecreased HDL concentration as well as lower PON1 concentration (shown indirectly by the enzyme arylesterase activity) might contribute, at least partly, to the reduced PON1 activity observed in hemodialyzed patients. Decreased concentration of free thiol groups in sera suggest that free thiol group (Cys284) in PON1 might also be oxidized, which can affect PON1 activity.

Glutathione cycle in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low‐molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (nu2009=u2009109) and healthy subjects (nu2009=u200951). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never‐smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase‐dependent regulation of the glutathione redox state is vital for protection against oxidative stress. Copyright

Quantification of malondialdehyde by HPLC-FL - application to various biological samples.

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2-thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15-3.0u2009µmol/L) the method was linear (R(2) u2009=u20090.9963), the between-day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within-day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non-smokers, 46.3u2009±u20094.7u2009years; Nu2009=u200938) was 2.2u2009±u20091.4u2009µmol/L; that in liver tissue of common carp (Cyprinus carpio; Nu2009=u200912) was 0.02u2009±u20090.004u2009µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; Nu2009=u200910) it was 0.18u2009±u20090.02u2009nmol/mg proteins. The HPLC-FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory.

Differential activation of MAPKs by individual and combined ochratoxin A and citrinin treatments in porcine kidney PK15 cells

The aim of this study was to investigate the underlying mechanisms of OTA and CTN individual and combined toxicity in porcine kidney PK15 cells of proximal tubule origin. Activation and expression of mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 were determined by Western blot analysis. MAPKs were differentially activated by single or dual OTA and CTN treatments. Single OTA and CTN stimulated transient ERK and prolonged JNK activation, while phospho-p38 signal was more persistent after OTA treatment. Mycotoxin mixture provoked significant down-regulation of ERK activation, more prolonged phospho-p38 signal, and two-stage JNK phosphorylation pattern. In order to define the role of particular MAPKs in mycotoxin(s) cytotoxicity, we performed MTT assay with specific MAPKs inhibitors. In both individual and combined treatments JNK and p38 inhibition significantly induced cell survival. When cells were exposed to toxin mixture, inhibition of ERK also promoted cell survival, although to a lesser extent that JNK and p38 inhibition. Next we investigated the association between calcium (Ca(2+)) and MAPKs after OTA and/or CTN treatments, and we employed Ca(2+) chelator BAPTA-AM. We demonstrated that p38 activation was significantly down-regulated in cells treated with CTN alone or OTA + CTN suggesting the role of Ca(2+) in mycotoxin-induced cell death.

Association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-alpha (+489G/A) polymorphisms with COPD in Croatian population.

OBJECTIVEnTo test for possible association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-α (+489G/A) polymorphisms with chronic obstructive pulmonary disease (COPD) in Croatian population.nnnMETHODSnGenotyping of DNA isolated from whole blood of 130 COPD patients (as defined by spirometry) and 95 healthy controls was performed. Fragment size analysis upon restriction enzyme digestion and/or sequencing was used for genotype/allele definition. Significance of findings was tested using χ(2) test.nnnRESULTSnhsp70-2 (+1267A/G) polymorphism was significantly associated with COPD. Results of genotyping analysis indicated that a genotype carrying G allele was preferentially associated with COPD; odds ratio (OR)=1.50, 95% confidence interval (CI)=1.00-2.24 and P=0.061. OR for the GG genotype was 3.47 with CI=1.26-9.56 and P=0.04. No association for hsp70-hom (+2437T/C), TNF-α (+489G/A) and HMOX-1 (number of GT repeats) polymorphisms were found. In addition, comparison of genotype frequencies among different stages of disease severity (GOLD II-IV) revealed no discrimination for any of the tested polymorphisms.nnnCONCLUSIONnThis study is supporting the association of hsp70-2 (+1267A/G) polymorphism and COPD. Higher frequency of G allele and GG genotype in Croatian COPD patients was observed. There was no evidence for the association of hsp70-hom (+2437T/C), TNF-α (+489G/A) SNPs and HMOX-1 (number of GT repeats) polymorphism with COPD. Allele and genotype frequencies for all of the tested polymorphisms show no association with disease severity (GOLD II-IV).