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.
- Klíčová slova
- Aminophenol, Cell toxicity, Cysteine conjugates, Glutathione conjugation, Kidney injury,
- MeSH
- aminofenoly * toxicita MeSH
- cystein MeSH
- glutathion MeSH
- ledviny MeSH
- lidé MeSH
- paracetamol * toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 4-aminophenol MeSH Prohlížeč
- aminofenoly * MeSH
- cystein MeSH
- glutathion MeSH
- paracetamol * MeSH
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.
Glutathione, the very important intracellular antioxidant, is present in intracelullar environment in milimolar concentrations. Glutathione is a tripeptide molecule, which plays an essential role in the antioxidant system, as well as in maintenance of the intracellular redox state. This thiol compound exists in two forms, the reduced (GSH) and the oxidized (GSSG), and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. Number of analytical methods have been developed for the measurement of the glutathione. Especially, High Performance Liquid Chromatography methods (HPLC) are mostly used linked to different types of detection, including electrochemical, UV/VIS or fluorimetric detection. Another approach for glutathione assay is using the spectral methods, either fluorimetric or spectrophotometric assays. In enzymatic assay, glutathione reductase reduces GSSG with simultaneous oxidation of specific substrate, which is sequentially photometrically detected. The fluorimetric method is based on the detection of derivatized GSH molecule.
- MeSH
- fluorescenční spektrometrie MeSH
- glutathion analýza biosyntéza fyziologie MeSH
- lidé MeSH
- spektrofotometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- Názvy látek
- glutathion MeSH
