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.

• Keywords
• Aminophenol, Cell toxicity, Cysteine conjugates, Glutathione conjugation, Kidney injury,
• MeSH
• Aminophenols * toxicity   MeSH
• Cysteine MeSH
• Glutathione MeSH
• Kidney MeSH
• Humans MeSH
• Acetaminophen * toxicity   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 4-aminophenol MeSH Browser
• Aminophenols * MeSH
• Cysteine MeSH
• Glutathione MeSH
• Acetaminophen * 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
• Apoptosis drug effects   MeSH
• Bisbenzimidazole chemistry   MeSH
• Cell Death drug effects   MeSH
• Cell Nucleus drug effects   metabolism   MeSH
• Cell Line MeSH
• Hep G2 Cells MeSH
• Cisplatin pharmacology   MeSH
• Microscopy, Fluorescence methods   MeSH
• Spectrometry, Fluorescence methods   MeSH
• DNA Fragmentation drug effects   MeSH
• Camptothecin pharmacology   MeSH
• Humans MeSH
• Antineoplastic Agents pharmacology   MeSH
• Flow Cytometry methods   MeSH
• Reproducibility of Results MeSH
• Staurosporine pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bisbenzimidazole MeSH
• Cisplatin MeSH
• Camptothecin MeSH
• Antineoplastic Agents MeSH
• Staurosporine 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
• Cell Culture Techniques methods   MeSH
• Cell Line MeSH
• Cisplatin toxicity   MeSH
• Cadmium toxicity   MeSH
• Humans MeSH
• Analgesics, Non-Narcotic toxicity   MeSH
• Acetaminophen toxicity   MeSH
• Antineoplastic Agents toxicity   MeSH
• Kidney Tubules, Proximal drug effects   pathology   MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cisplatin MeSH
• Cadmium MeSH
• Analgesics, Non-Narcotic MeSH
• Acetaminophen MeSH
• Antineoplastic Agents MeSH