Most cited article - PubMed ID 28642085
Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells
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
Alzheimer's disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds' effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.
- Keywords
- Alzheimer’s disease, N-methyl-d-aspartate receptor, acetylcholinesterase, enzyme inhibition, monoamine oxidase A/B, multi-target directed ligands,
- MeSH
- Acetylcholinesterase metabolism MeSH
- Alzheimer Disease * drug therapy MeSH
- Cholinesterase Inhibitors therapeutic use MeSH
- Monoamine Oxidase Inhibitors therapeutic use MeSH
- Humans MeSH
- Monoamine Oxidase metabolism MeSH
- Neuroblastoma * drug therapy MeSH
- Drug Design MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Acetylcholinesterase MeSH
- Cholinesterase Inhibitors MeSH
- Monoamine Oxidase Inhibitors MeSH
- Monoamine Oxidase MeSH
PURPOSE: Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO2 P25 for biological testing in A549 cells and to evaluate their biological effects. METHODS: We determined the size and crystallinity of TiO2 P25. We used four techniques for TiO2 P25 dispersion. We estimated the colloid stability of TiO2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO2 P25 (0-100 µg.mL-1) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. RESULTS: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO2 P25. Under these conditions, TiO2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO2 P25 in A549 cells were met, similar results on the biological effects of TiO2 P25 were obtained in two independent cell laboratories. CONCLUSION: We optimized the experimental conditions of TiO2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO2 P25 as a reference material.
- Keywords
- A549 cells, P25, dispersion, nanoparticles, nanotoxicity, titanium dioxide,
- MeSH
- A549 Cells MeSH
- Sodium Chloride MeSH
- Glutathione MeSH
- Metal Nanoparticles MeSH
- Humans MeSH
- Nanoparticles * chemistry MeSH
- Oxidoreductases MeSH
- Lung MeSH
- Serum Albumin, Bovine * MeSH
- Titanium MeSH
- Water MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Sodium Chloride MeSH
- Glutathione MeSH
- Oxidoreductases MeSH
- Serum Albumin, Bovine * MeSH
- Titanium MeSH
- titanium dioxide MeSH Browser
- Water 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