Characterization of acetaminophen toxicity in human kidney HK-2 cells.

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.

The response of human ectomesenchymal dental pulp stem cells to cisplatin treatment.

AIM: To determine the response of dental pulp stem cells (DPSCs) to DNA-damaging cytostatic cisplatin and compare it with the response of normal human dermal fibroblasts (HDFs). METHODOLOGY: Dental pulp stem cells were exposed to 5, 10, 20 or 40 µmol L(-1) of cisplatin. The proliferation of affected cells was assessed by a Z2 Counter and viability was assessed by means of a Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis and induction of apoptosis were performed by flow cytometry. Induction of apoptosis was determined by monitoring the activities of caspases. The expression of proteins was detected by electrophoresis and Western blotting. The descriptive statistics of the results was analyzed by Student's t-test. RESULTS: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. All three main Mitogen-activated protein kinases (MAPK) families - extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinase (JNK) and p38 were activated after treatment of DPSCs with cisplatin. The activation of MAPK pathways was not observed in HDFs exposed to cisplatin. The exposure of DPSCs and HDFs to cisplatin provoked an increase in p53 and p21 expression and p53 phosphorylation of serine 15. Higher concentrations of cisplatin reduced the viability of DPSCs and HDFs and induced the activation of caspases 3/7 and 9. CONCLUSION: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. Cisplatin in higher concentrations triggered activation of MAPK and apoptosis in DPSCs but not in HDFs.

Role of transplanted bone marrow cells in response to skeletal muscle injury.

The recently discovered capacity of bone marrow cells (BMCs) to contribute to injury-induced skeletal muscle regeneration has brought new possibilities in the treatment of skeletal muscle diseases. However, a suitable method of BMC transplantation usable for such therapy has to be established. In this work, recipient mice were intramuscularly injected with cardiotoxin, then whole-body lethally irradiated to eradicate satellite cells in their injured tibialis anterior (TA) muscles and to suppress haematopoiesis, and subsequently intravenously transplanted with lacZ+ BMCs with the aim to investigate the role of exogenous BMCs in response to skeletal muscle injury. Seven to 33 days after grafting, recipient TA muscles were examined to detect donor-derived X-gal+ cells and analysed by quantitative PCR. In injured recipients' muscles, X-gal positivity was identified 14 and 33 days after grafting in some infiltrating neutrophils and macrophages, infrequently in fibroblasts of endomysium, and in many large multinucleated cells (devoid of myogenic markers desmin and nestin) resembling foreign body giant cells situated in the vicinity of necrotic muscle fibres. qPCR confirmed the presence of transplanted lacZ+ BMCs in injured recipients' muscles. Our results proved the ability of intravenously transplanted adult BMCs to settle in injured muscles and generate blood cells that infiltrated endomysium and took part in the cleaning reaction. After inhibition of endogenous myogenesis, BMCs were not able to participate in formation of new muscle fibres due to persisting necrosis of degenerated muscle fibres. Instead, BMCs attempted to resorb necrotic structures, which confirmed the indispensable role of bone marrow-derived macrophages in skeletal muscle regeneration.