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

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

Záměrem projektu je popis zcela nového mechanismu, který se dle našich jak pilotních, tak i již publikovaných výsledků účastní toxického poškození hepatocytů při předávkování acetaminofenem (APAP). Během tohoto předávkování vzniká v buňce specifický APAP metabolit, tzv. APAP-SG konjugát, který byl až dosud považován za zcela netoxický. My jsme ale prokázali, že je schopný inhibice glutathionreduktasy, tj. nezastupitelného intracelulárního enzymu. V rámci projektu chceme nejprve tento konjugát APAP-SG syntetizovat a purifikovat v dostatečném množství (k tomuto účelu je plánován nákup preparativní sestavy) a následně ho využít k podrobnému studiu jeho účinků v podmínkách in vitro na kultivovaných hepatocytech a in vivo na myších a potkanech. Kromě toho se plánujeme zaměřit také na možnost hepatoprotekce před účinky tohoto konjugátu. Třetí část projektu je zaměřena na studium toxicity štěpného produktu APAP-SG konjugátu, kterým je APAP-cystein, na buněčných liniích renálních tubulárních buněk.; The aim of the project is the description of a newly found toxic mechanism of acetaminophen toxicity that contributes to hepatotoxic effect of acetaminophen (APAP); our aim has been stated regarding our published and also unpublished data. After APAP overdose, the specific APAP metabolite, APAP-SG conjugate, is produced in the cell. This compound has been considered as non-toxic generally. However, we proved that APAP-SG is a potent inhibitor of glutathione reductase, a crucial intracellular enzyme. In this project, we intend to synthesize and purify APAP-SG in sufficient quantity. Therefore, we require purchase of preparative system. Consequently, we will use APAP-SG to detailed study of its effects in cultured hepatocytes in vitro and in vivo in mice and rats. As well, we will estimate the hepatoprotection against the conjugate effects. The last part of the project has been focused on the study of toxicity of APAP-SG cleavage product, APAP-Cys. This effect will be tested in renal tubular cell lines.

• MeSH
• experimenty na zvířatech MeSH
• glutathionreduktasa antagonisté a inhibitory   MeSH
• hepatocyty účinky léků   MeSH
• krysa rodu rattus MeSH
• kultivační techniky MeSH
• lékové postižení jater MeSH
• myši MeSH
• paracetamol škodlivé účinky   MeSH
• předávkování léky MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zneužívání léků na předpis MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Glutathione is an important tripeptide involved in a variety of cellular processes. Thus, precise knowledge of its levels is essential. Glutathione exists in two free forms-reduced and oxidized-and a number of methods exist to measure its levels. The aim of our work was to optimize a spectrofluorometric assay for reduced glutathione based on the reaction between glutathione and o-phthalaldehyde. We found that a change of excitation wavelength to 340 nm and modification of pH to 6.0 enhance sensitivity and specificity of the method (intraassay coefficient of variation CV < 3%, interassay CV = 5.1%, recovery = 98-102%, linearity = 0-1000 μM GSH, calibration R2 = 1.00). We also anticipated possible effect of various amino acids on the fluorescence signal, but no interference was found. We compared the optimized fluorometric method with a popular enzymatic recycling glutathione assay and found very strong correlation of results (r = 0.99, n = 45). We introduce here an optimized fluorometric method possessing sufficient sensitivity and specificity that is comparable to the enzymatic glutathione assay. Because the fluorometric assay procedure is faster and lower in cost, it could be ideal for routine analysis of reduced glutathione levels in a large number of samples.

• MeSH
• enzymy metabolismus   MeSH
• fluorometrie MeSH
• glutathion analýza   MeSH
• koncentrace vodíkových iontů MeSH
• o-ftalaldehyd chemie   MeSH
• oxidace-redukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Acetaminophen overdose is the most often cause of acute liver injury. The toxic mechanism is linked to formation of an active metabolite that reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). This compound has been recognized to be non-toxic generally. Our preliminary results showed, however, that APAP-SG could possess a toxic effect too. Therefore, the aim of our study was to prepare, purify and to test possible toxicity of APAP-SG. We prepared APAP-SG using organic synthesis. The conjugate was purified by preparative HPLC and its structure was confirmed using mass spectrometry. Final purity of APAP-SG was >98 %. We estimated a toxic effect of APAP-SG in isolated rat liver mitochondria using a fluorescent ROS probe. We assessed ROS production in presence of complex I or complex II substrates. The increase of ROS-dependent fluorescence in presence of glutamate/malate was 104±13 % and 130±10 % in 1 mM and 5 mM APAP-SG, respectively, in comparison with controls. ROS production related to presence of complex II substrate was enhanced 4-times in APAP-SG (5 mM) treated mitochondria (compared to controls). We conclude, we proved our hypothesis that APAP-SG conjugate is able to induce a mitochondrial impairment leading to enhanced ROS production.

• MeSH
• jaterní mitochondrie * metabolismus   účinky léků   MeSH
• játra metabolismus   MeSH
• krysa rodu rattus MeSH
• kyselina glutamová metabolismus   MeSH
• lékové postižení jater * metabolismus   patologie   MeSH
• maláty metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• oxidační stres * MeSH
• paracetamol * analogy a deriváty   chemická syntéza   izolace a purifikace   toxicita   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

• Publikační typ
• abstrakty MeSH

• MeSH
• financování organizované MeSH
• játra účinky léků   MeSH
• myši inbrední ICR MeSH
• paracetamol toxicita   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• abstrakty MeSH

The aim of the present work was to investigate a new mechanism likely contributing to the toxic action of acetaminophen, especially to explore the possible inhibition of glutathione reductase through an acetaminophen-glutathione conjugate (APAP-SG). APAP-SG conjugate was synthesized by organic synthesis and purified by column chromatography. The inhibitory effect of the conjugate on two types of glutathione reductase (from yeasts and rat hepatocytes) was tested spectrophotometrically. We found that the enzyme activity was reduced similarly after the treatment with 2.96 mM acetaminophenglutathione conjugate in both yeast and hepatocyte glutathione reductases (GR); the enzyme activity was inhibited to 52.7±1.5 % (2.4±0.3 mU/ml) in yeast GR (control activity was 5.6±0.3 mU/ml) and to 48.1±8.8 % (2.2±0.2 mU/ml) in rat hepatocytes lysate GR (control activity was 5.2±0.2 mU/ml). In addition, the enzyme activity (from hepatocytes lysate) was decreased to 79±7 %, 67±2 % and 39±7 %, in 0.37, 1.48 and 3.7 mM concentration of the conjugate, respectively. We found that glutathione reductase, the essential enzyme of the antioxidant system, was dose-dependently inhibited by the product of acetaminophen metabolism – the conjugate of acetaminophen and glutathione.

• MeSH
• aktivace enzymů účinky léků   MeSH
• financování organizované MeSH
• fixní kombinace léků MeSH
• glutathion chemická syntéza   toxicita   MeSH
• glutathionreduktasa antagonisté a inhibitory   metabolismus   MeSH
• hepatocyty enzymologie   patologie   účinky léků   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• neopioidní analgetika chemická syntéza   toxicita   MeSH
• paracetamol chemická syntéza   toxicita   MeSH
• potkani Wistar MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

• Publikační typ
• abstrakty MeSH