Acetaminophen (APAP) overdose causes liver injury, but in some cases it is associated also with renal impairment. While several studies exist in relation to acetaminophen nephrotoxicity, no reports have been published describing intracellular changes related to APAP nephrotoxicity in vitro. Because proximal tubular cells are considered to constitute a secondary site of drug-induced injury after hepatocytes, our study's aim was to estimate the toxicity in the human HK-2 cell line. We used a range of APAP concentrations (1-10 mM) to examine toxicity in the cells (1-48 h). We evaluated cell viability using the WST-1 and LDH tests. Cells impairment was also determined by monitoring ROS production, glutathione levels. We proved that HK-2 cells are able to metabolize acetaminophen. We observed moderate impairment of cells already after 1 h of treatment based on a finding of increased ROS production and decreased cell viability. After 24 h, the results showed significant cellular impairment at all tested concentrations except for 1 mM APAP, but no glutathione depletion was found. We conclude that HK-2 cells are susceptible to acetaminophen toxicity but, unlike hepatocytes, it might be not linked to glutathione depletion.

• MeSH
• buněčné linie MeSH
• glutathion metabolismus   MeSH
• ledviny účinky léků   metabolismus   MeSH
• lidé MeSH
• neopioidní analgetika škodlivé účinky   MeSH
• oxidační stres účinky léků   MeSH
• paracetamol škodlivé účinky   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Neutrophil gelatinase-associated lipocalin is an extracellular protein produced mostly in kidney. Recently, it has become a promising biomarker of renal damage in vivo. On the other hand, the validation of NGAL as a biomarker for nephrotoxicity estimation in vitro has not been characterized in detail yet. Since the HK-2 cells are frequently used human kidney cell line, we aimed to characterize the production of NGAL in these cells and to evaluate NGAL as a possible marker of cell impairment. We used heavy metals (mercury, cadmium), peroxide, drugs (acetaminophen, gentamicin) and cisplatin to mimic nephrotoxicity. HK-2 cells were incubated with selected compounds for 1-24h and cell viability was measured together with extracellular NGAL production. We proved that HK-2 cells possess a capacity to produce NGAL in amount of 2pg/ml/h. We found a change in cell viability after 24h incubation with all tested toxic compounds. The largest decrease of the viability was detected in mercury, acetaminophen, cisplatin and gentamicin. Unexpectedly, we found also a significant decrease in NGAL production in HK-2 cells treated with these toxins for 24h: to 11±5%, 54±5%, 57±6% and 76±9% respectively, compared with controls (=100%). Our results were followed with qPCR analysis when we found no significant increase in LCN2 gene expression after 24h incubation. We conclude that extracellular NGAL production negatively correlates with HK-2 cell impairment.

• MeSH
• akutní poškození ledvin chemicky indukované   metabolismus   MeSH
• biologické markery metabolismus   MeSH
• buněčné linie MeSH
• cisplatina toxicita   MeSH
• gentamiciny toxicita   MeSH
• kadmium toxicita   MeSH
• lidé MeSH
• lipokalin-2 genetika   metabolismus   MeSH
• paracetamol toxicita   MeSH
• rtuť toxicita   MeSH
• terc-butylhydroperoxid toxicita   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The human proximal tubular HK-2 cell line is an immortalized cell line commonly used for studying proximal tubular toxicity. Even as their use is presently increasing, there unfortunately are no studies focused on functional changes in HK-2 cells associated with passaging. The aim of the present study, therefore, was to evaluate the functional stability of HK-2 cells during 13 weeks of continuous passaging after 6 and 24 h of treatment with model nephrotoxic compounds (i.e., acetaminophen, cisplatin, CdCl(2)). Short tandem repeat profile, the doubling time, cell diameter, glutathione concentration, and intracellular dehydrogenase activity were measured in HK-2 cells at each tested passage. The results showed that HK-2 cells exhibit stable morphology, cell size, and cell renewal during passaging. Mean doubling time was determined to be 54 h. On the other hand, we observed a significant effect of passaging on the susceptibility of HK-2 cells to toxic compounds. The largest difference in results was found in both cadmium and cisplatin treated cells across passages. We conclude that the outcomes of scientific studies on HK-2 cells can be affected by the number of passages even after medium-term cultivation and passaging for 13 weeks.

• MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• cisplatina toxicita   MeSH
• kadmium toxicita   MeSH
• lidé MeSH
• neopioidní analgetika toxicita   MeSH
• paracetamol toxicita   MeSH
• protinádorové látky toxicita   MeSH
• proximální tubuly ledvin účinky léků   patologie   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Iron oxide nanoparticles (IONPs) have a great potential with regard to cell labelling, cell tracking, cell separation, magnetic resonance imaging, magnetic hyperthermia, targeted drug and gene delivery. However, a growing body of research has raised concerns about the possible unwanted adverse cytotoxic effects of IONPs. In the present study, the in vitro cellular uptake, antiproliferative activity, cytotoxicity, genotoxicity, prooxidant, microtubule-disrupting and apoptosis-inducing effect of Fe3O4@SiO2 and passivated Fe3O4@SiO2-NH2 nanoparticles on human renal proximal tubule epithelial cells (HK-2) have been studied. Both investigated silica coated IONPs were found to have cell growth-inhibitory activity in a time- and dose-dependent manner. Determination of cell cycle phase distribution by flow cytometry demonstrated a G1 and G2/M phase accumulation of HK-2 cells. A tetrazolium salt cytotoxicity assay at 24 h following treatment demonstrated that cell viability was reduced in a dose-dependent manner. Microscopy observations showed that both Fe3O4@SiO2 and Fe3O4@SiO2-NH2 nanoparticles accumulated in cells and appeared to have microtubule-disrupting activity. Our study also revealed that short term 1 h exposure to 25 and 100 μg/mL of silica coated IONPs causes genotoxicity. Compared with vehicle control cells, a significantly higher amount of γH2AX foci correlating with an increase in DNA double-strand breaks was observed in Fe3O4@SiO2 and Fe3O4@SiO2-NH2-treated and immunestained HK-2 cells. The investigated nanoparticles did not trigger significant ROS generation and apoptosis-mediated cell death. In conclusion, these findings provide new insights into the cytotoxicity of silica coated IONPs that may support their further safer use.

• MeSH
• apoptóza účinky léků   MeSH
• buněčné linie MeSH
• buněčný cyklus účinky léků   MeSH
• dvouřetězcové zlomy DNA MeSH
• epitelové buňky účinky léků   MeSH
• geny p53 MeSH
• histony genetika   MeSH
• lidé MeSH
• magnetické nanočástice toxicita   MeSH
• mikrotubuly účinky léků   MeSH
• oxid křemičitý toxicita   MeSH
• oxid železnato-železitý toxicita   MeSH
• poškození DNA * MeSH
• povrchové vlastnosti MeSH
• proximální tubuly ledvin cytologie   MeSH
• reaktivní formy kyslíku MeSH
• testy genotoxicity MeSH
• virová transformace buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Acetaminophen (APAP) belong among the most used analgesics and antipyretics. It is structurally derived from p-aminophenol (PAP), a potent inducer of kidney toxicity. Both compounds can be metabolized to oxidation products and conjugated with glutathione. The glutathione-conjugates can be cleaved to provide cysteine conjugates considered as generally nontoxic. The aim of the present report was to synthesize and to purify both APAP- and PAP-cysteine conjugates and, as the first study at all, to evaluate their biological effects in human kidney HK-2 cells in comparison to parent compounds. HK-2 cells were treated with tested compounds (0-1000 μM) for up to 24 h. Cell viability, glutathione levels, ROS production and mitochondrial function were determined. After 24 h, we found that both APAP- and PAP-cysteine conjugates (1 mM) were capable to induce harmful cellular damage observed as a decrease of glutathione levels to 10% and 0%, respectively, compared to control cells. In addition, we detected the disappearance of mitochondrial membrane potential in these cells. In the case of PAP-cysteine, the extent of cellular impairment was comparable to that induced by PAP at similar doses. On the other hand, 1 mM APAP-cysteine induced even larger damage of HK-2 cells compared to 1 mM APAP after 6 or 24 h. We conclude that cysteine conjugates with aminophenol are potent inducers of oxidative stress causing significant injury in kidney cells. Thus, the harmful effects cysteine-aminophenolic conjugates ought to be considered in the description of APAP or PAP toxicity.

• MeSH
• aminofenoly * toxicita   MeSH
• cystein MeSH
• glutathion MeSH
• ledviny MeSH
• lidé MeSH
• paracetamol * toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Oxime cholinesterase reactivators (oximes) are used to counteract organophosphate intoxication. Charged oximes are administered via intramuscular or intravenous injection when the majority of dose is unmetabolized and is excreted as urine. In this study, the effects of selected double charged oximes were determined in the HK-2 cell line as a model for renal toxicity screening. Some effects on dehydrogenase activity were found for obidoxime, asoxime (syn. HI-6), K027, and K203. The effects of K868 and K869 were found to be unreliable due to rapid degradation of both chlorinated oximes in the assay medium, resulting for K868 in an isoxazole-pyridinium product.

• MeSH
• buněčné linie MeSH
• ledviny účinky léků   metabolismus   MeSH
• lidé MeSH
• molekulární struktura MeSH
• oximy aplikace a dávkování   škodlivé účinky   chemie   MeSH
• reaktivátory cholinesterasy aplikace a dávkování   škodlivé účinky   chemie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We have identified through sequencing of amplified DNA a GCC-->GAC mutation in codon 115 of the beta-globin gene in a mother and daughter of a small Czech family. This base change was confirmed by hybridization with a 32P-labeled specific oligonucleotide probe and by gene mapping because it creates a new Ava II site. The mutation results in an Ala-->Asp replacement at beta 115(G17); this beta chain is severely unstable and could not be identified either as chain or as hemoglobin variant by isoelectrofocusing and various high performance liquid chromatography methods. Stability tests were mildly positive in freshly prepared lysates, but an unstable hemoglobin could not be detected in older lysates with these methods. Its presence results in a dominant type of beta-thalassemia in the two heterozygotes, with moderate anemia, reticulocytosis, nucleated red cells, target cells, and other red cell changes, Heinz body formation, and splenomegaly; the oldest of the two patients was splenectomized. Both subjects had a marked increase in fetal hemoglobin synthesis.

• MeSH
• beta-talasemie genetika   MeSH
• bodová mutace MeSH
• dominantní geny MeSH
• dospělí MeSH
• fetální hemoglobin biosyntéza   MeSH
• globiny genetika   MeSH
• hemoglobiny abnormální genetika   izolace a purifikace   MeSH
• heterozygot MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• předškolní dítě MeSH
• sekvence nukleotidů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Geografické názvy
• Česká republika MeSH

We have identified through sequencing of amplified DNA a GCC-->GAC mutation in codon 115 of the beta-globin gene in a mother and daughter of a small Czech family. This base change was confirmed by hybridization with a 32P-labeled specific oligonucleotide probe and by gene mapping because it creates a new Ava II site. The mutation results in an Ala-->Asp replacement at beta 115(G17); this beta chain is severely unstable and could not be identified either as chain or as hemoglobin variant by isoelectrofocusing and various high performance liquid chromatography methods. Stability tests were mildly positive in freshly prepared lysates, but an unstable hemoglobin could not be detected in older lysates with these methods. Its presence results in a dominant type of beta-thalassemia in the two heterozygotes, with moderate anemia, reticulocytosis, nucleated red cells, target cells, and other red cell changes, Heinz body formation, and splenomegaly; the oldest of the two patients was splenectomized. Both subjects had a marked increase in fetal hemoglobin synthesis.

• MeSH
• beta-talasemie * genetika   MeSH
• bodová mutace MeSH
• dominantní geny MeSH
• dospělí MeSH
• fetální hemoglobin biosyntéza   MeSH
• globiny * genetika   MeSH
• hemoglobiny abnormální * genetika   izolace a purifikace   MeSH
• heterozygot MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• předškolní dítě MeSH
• sekvence nukleotidů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Geografické názvy
• Česká republika MeSH

At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

• MeSH
• apoptóza účinky léků   MeSH
• bisbenzimidazol chemie   MeSH
• buněčná smrt účinky léků   MeSH
• buněčné jádro účinky léků   metabolismus   MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• cisplatina farmakologie   MeSH
• fluorescenční mikroskopie metody   MeSH
• fluorescenční spektrometrie metody   MeSH
• fragmentace DNA účinky léků   MeSH
• kamptothecin farmakologie   MeSH
• lidé MeSH
• protinádorové látky farmakologie   MeSH
• průtoková cytometrie metody   MeSH
• reprodukovatelnost výsledků MeSH
• staurosporin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cadmium is a heavy metal causing toxicity especially in kidney cells. The toxicity is linked also with enhanced oxidative stress leading to cell death. On the other hand, our recent experiments have shown that an increase of total intracellular dehydrogenases activity can also occur in kidney cells before declining until cell death. The aim of the present study, therefore, was to evaluate this transient enhancement in cell viability after cadmium treatment. The human kidney HK-2 cell line was treated with CdCl(2) at concentrations 0-200 microM for 2-24 h and intracellular dehydrogenase activity was tested. In addition, we measured reactive oxygen species (ROS) production, glutathione levels, mitochondrial membrane potential, and C-Jun-N-terminal kinase (JNK) activation. We found that significantly increased dehydrogenase activity could occur in cells treated with 25, 100, and 200 microM CdCl(2). Moreover, the results showed an increase in ROS production linked with JNK activation following the enhancement of dehydrogenase activity. Other tests detected no relationship with the increased in intracellular dehydrogenase activity. Hence, the transient increase in dehydrogenase activity in HK-2 cells preceded the enhancement of ROS production and our finding provides new evidence in cadmium kidney toxicity.

• MeSH
• aktivace enzymů účinky léků   fyziologie   MeSH
• buněčné linie MeSH
• kadmium toxicita   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   fyziologie   MeSH
• oxidační stres účinky léků   fyziologie   MeSH
• oxidoreduktasy metabolismus   MeSH
• proximální tubuly ledvin účinky léků   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH