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.
Acetaminophen overdose is the most frequent cause of acute liver injury. The main mechanism of acetaminophen toxicity has been attributed to oxidation of acetaminophen. The oxidation product is very reactive and reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). Although this conjugate has been recognized to be generally nontoxic, we have found recently that APAP-SG could produce a toxic effect. Therefore, the aim of our study was to estimate the toxicity of purified APAP-SG by characterizing the inhibitory effect in human glutathione reductase (GR) and comparing that to the inhibitory effect of the natural inhibitor reduced glutathione. We used two types of human GR: recombinant and freshly purified from red blood cells. Our results show that GR was significantly inhibited in the presence of both APAP-SG and reduced glutathione. For example, the enzyme activity of recombinant and purified GR was reduced in the presence of 4 mm APAP-SG (with 0.5 mm glutathione disulfide) by 28% and 22%, respectively. The type of enzyme inhibition was observed to be competitive in the cases of both APAP-SG and glutathione. As glutathione inhibits GR activity in cells under physiological conditions, the rate of enzyme inhibition ought to be weaker in the case of glutathione depletion that is typical of acetaminophen overdose. Notably, however, enzyme activity likely remains inhibited due to the presence of APAP-SG, which might enhance the pro-oxidative status in the cell. We conclude that our finding could reflect some other pathological mechanism that may contribute to the toxicity of acetaminophen.
& A method is described here for the determination of total glutathione and glutathione disulfide in human whole blood and plasma with a suitable sample preparation procedure to prevent glutathione oxidation and glutathione disulfide hydrolysis. Whole blood samples were obtained from a group of blood donors. After adequate sample preparation, the samples were derivatized with ortho-phthaldialdehyde to form a stable, highly fluorescent tricyclic derivative. Reverse-phase column chromatography was used for the separation and fluorimetric detection to monitor the effluent. The analytical performance of this method was satisfactory for the determination of total glutathione and glutathione disulfide. The intra-assay and inter-assay coefficients of variation were below 10%. The recoveries were as follows: 96.1% (CV 1.4%) and 104.3% (CV 2.4%) for whole blood, 92.3% (CV 2.4%) and 107.4% (CV 2.6%) for plasma. We found no significant differences in both total glutathione and glutathione disulfide concentration between the women and men. We developed a selective high-performance liquid chromatography method for the determination of total glutathione and glutathione disulfide in human whole blood and plasma. Our sophisticated sample preparation procedure prevents significant glutathione oxidation and glutathione disulfide hydrolysis.
The kidneys are one of the organ that can be commonly damaged by a number of toxic compounds (heavy metals, xenobiotics, drugs, etc.). To characterize the mechanism of toxicity, a variety of methods have been developed. The in vitro methods belong among the mostly used. Especially, the use of cell lines seems to be the leading approach to test and to characterize the toxicity mechanisms. At present, several cell lines of animal (from rat, dog, pig) or human origin are available. A detailed evaluation must go before any selection of a suitable cell line for experiments. Therefore, the aim of this review was to describe and to evaluate the mostly used renal cell lines.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.