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Effect of selected nonsteroidal anti-inflammatory drugs on the viability of canine osteosarcoma cells of the D-17 line: in vitro studies
2019
Poradowski, Dominik | Obmińska-Mrukowicz, Bożena
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in veterinary medicine. They are used in pain control and in anti-inflammatory and antipyretic therapies. Some NSAIDs, e.g piroxicam, also have a documented anticancer effect. The objective of this study was to evaluate which of the commonly used NSAIDs (etodolac, flunixin, tolfenamic acid, carprofen, and ketoprofen) are cytotoxic to the D-17 cell line of canine osteosarcoma. The viability of the cells was evaluated using the MTT assay. Four independent repetitions were performed and the results are given as the average of these values; EC₅₀ values (half maximal effective concentration) were also calculated. The analysis of results showed that carprofen and tolfenamic acid displayed the highest cytotoxicity. Other drugs either did not provide such effects or they were very poor. For carprofen, it was possible to determine an EC₅₀ which fell within the limits of concentrations obtainable in canine serum after the administration of routinely used doses. The results are promising but further studies should be conducted to confirm them, since this study is only preliminary. The possibility of introducing carprofen and tolfenamic acid into the routine treatment of osteosarcoma in dogs should be considered.
Afficher plus [+] Moins [-]Cell viability in normal fibroblasts and liver cancer cells after treatment with iron (III), nickel (II), and their mixture
2018
Terpiłowska, Sylwia | Siwicka-Gieroba, Dorota | Siwicki, Andrzej Krzysztof
Introduction: Nickel and iron are very commonly occurring metals. Nickel is used in industry, but nowadays it is also used in medical biomaterials. Iron is an element necessary for cell metabolism and is used in diet supplements and biomaterials, whence it may be released along with nickel. Material and Methods: BALB/3T3 and HepG2 cells were incubated with iron chloride or nickel chloride at concentrations ranging from 100 to 1,400 µM. The following mixtures were used: iron chloride 200 µM plus nickel chloride 1,000 µM, or iron chloride 1,000 µM plus nickel chloride 200 µM. The cell viability was determined with MTT, LHD, and NRU tests. Results: A decrease in cell viability was observed after incubating the BALB/3T3 and HepG2 cells with iron chloride or nickel chloride. A synergistic effect was observed after iron chloride 1,000 μM plus nickel chloride 200 μM treatment in all assays. Moreover, the same effect was observed in the pair iron chloride 200 μM plus nickel chloride 1,000 μM in the LDH and NRU assays. Conclusions: Iron (III) and nickel (II) decrease cell viability. Iron chloride at a concentration of 200 µM protects mitochondria from nickel chloride toxicity.
Afficher plus [+] Moins [-]Ex vivo effects of corticosteroids on equine deep digital flexor and navicular fibrocartilage explant cell viability
2021
Sullivan, Stasia N. | Cole, Sara L. | Stewart, Matthew C. | Brokken, Matthew T. | Durgam, Sushmitha
OBJECTIVE To investigate the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on the viability of resident cells within the fibrocartilage on the dorsal surface of the deep digital flexor tendon (FC-DDFT) and fibrocartilage on the flexor surface of the navicular bone (FC-NB) of horses. SAMPLE 12 to 14 explants of FC-DDFT and of FC-NB from grossly normal forelimbs of 5 cadavers of horses aged 9 to 15 years without evidence of musculoskeletal disease. PROCEDURES Explants were incubated with culture medium (control) or TA-supplemented (0.6 or 6 mg/mL) or MPA-supplemented (0.5 or 5 mg/mL) medium for 6 or 24 hours. Explant metabolic activity and percentage of dead cells were assessed with a resazurin-based assay and live-dead cell staining, respectively, at each time point. Drug effects were assessed relative to findings for the respective control group. RESULTS Application of TA (at both concentrations) did not significantly change the cell viability of FC-DDFT explants. For FC-NB explants, TA at 6 mg/mL significantly reduced the metabolic activity and increased the percentage of dead cells at both time points. With either MPA concentration, FC-DDFT and FC-NB explants had reduced metabolic activity and an increased percentage of dead cells at 24 hours, whereas only MPA at 5 mg/mL was cytotoxic at the 6-hour time point. CONCLUSIONS AND CLINICAL RELEVANCE In ex vivo explants, TA was less cytotoxic to equine FC-DDFT and FC-NB cells, compared with MPA. Further work is warranted to characterize the drugs' transcriptional and translational effects as well as investigate their cytotoxicity at lower concentrations.
Afficher plus [+] Moins [-]Effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine primary hepatocytes
2015
LaDuke, Kathleen E. | Ehling, Sarah | Cullen, J. M. (John M.) | Bäumer, Wolfgang
OBJECTIVE To investigate the cytotoxic effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine hepatocytes. SAMPLE Commercially available cryopreserved canine primary hepatocytes. PROCEDURES The study consisted of 2 trials. In trial 1, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, or 15.000 μmol/L) for 24, 48, or 72 hours. At each time, cell viability and lactate dehydrogenase (LDH) activity were determined for each thiopurine-concentration combination, and alanine aminotransferase (ALT) activity was determined for cells incubated with each thiopurine at a concentration of 15 μmol/L. In trial 2, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 3 concentrations (18.75, 37.50, or 75.00 μmol/L) for 24 hours, after which the free glutathione concentration was determined for each thiopurine-concentration combination and compared with that for hepatocytes incubated without a thiopurine (control). RESULTS Incubation of hepatocytes with each of the 3 thiopurines adversely affected cell viability in a time- and concentration-dependent manner; however, this decrease in cell viability was not accompanied by a concurrent increase in LDH or ALT activity. Likewise, free glutathione concentration for hepatocytes incubated for 24 hours with supratherapeutic thiopurine concentrations (> 18.75 μmol/L) did not differ significantly from that of control cells.CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that thiopurines adversely affected the viability of canine hepatocytes in a time- and concentration-dependent manner but had a nonsignificant effect on the LDH and ALT activities and free glutathione depletion of those hepatocytes.
Afficher plus [+] Moins [-]Antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells
2014
Schwartz, Anita M. | McCrackin, Mary Ann | Schinazi, Raymond F. | Hill, Peter B. | Vahlenkamp, Thomas W. | Tompkins, Mary B. | Hartmann, Katrin
Objective-To compare cytotoxic effects and antiviral efficacy of 9 nucleoside reverse transcriptase inhibitors (NRTIs) against FIV in feline peripheral blood mononuclear cells. Sample-Peripheral blood mononuclear cells obtained from 3 specific pathogen-free cats. Procedures-3 of the 9 NRTIs had not been previously assessed in feline cell lines. Cytotoxic effects were determined by colorimetric quantification of a formazan product resulting from bioreduction of a tetrazolium reagent by viable peripheral blood mononuclear cells; uninfected cells from 1 cat were used in these assays. Cells from all 3 cats were infected with a pathogenic clone of FIV, and in vitro antiviral efficacy of each NRTI was assessed with an FIV p24 antigen capture ELISA. Results-Cytotoxic effects in feline peripheral blood mononuclear cells were observed only at concentrations > 10 μM for all 9 NRTIs. Comparison of the cytotoxic effect at the highest concentration investigated (500μM) revealed that didanosine and amdoxovir were significantly less toxic than abacavir. All drugs induced a dose-dependent reduction of FIV replication. At the highest concentration investigated (10μM), there was no significant difference in antiviral efficacy among the test compounds. Conclusions and Clinical Relevance-The evaluated NRTIs had low cytotoxicity against feline peripheral blood mononuclear cells and appeared to be safe options for further in vivo evaluation for the treatment of FIV-infected cats. There was no evidence suggesting that the newly evaluated compounds would be superior to the existing NRTIs for reducing FIV burden of infected cats.
Afficher plus [+] Moins [-]Analysis of the transport of and cytotoxic effects for nalbuphine solution in corneal cells
2012
Spatola, Ronald A. | Thangavelu, Mirunalni | Upadhyayula, Vijayasaradhi | Lee, Seungsoo | Phelps, Mitch A. | Chandler, Heather L.
Objective: To assess the in vitro effects of various nalbuphine concentrations on viability and wound healing ability of corneal cells and potential drug transport through the corneal epithelium. Sample: Cultured canine and human corneal epithelial cells (CECs) and cultured canine corneal stromal fibroblasts. Procedures: CECs and stromal fibroblasts were exposed to nalbuphine (concentration of solutions ranged from 0% to 1.2%) for up to 30 minutes, and viability was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A standard scratch test technique was used. Wound healing of CECs and stromal fibroblasts was evaluated following treatment with nalbuphine solutions < 0.1%. Liquid chromatography–mass spectrometry–mass spectrometry analysis was used to evaluate drug transport across a monolayer and a multilayer of human CECs. Results: A progressive decrease in viability was detected in canine CECs for all nalbuphine treatment groups, whereas treatment with only 0.5% or 1.2% nalbuphine significantly reduced corneal stromal fibroblast viability, compared with results for control cells. Within 24 hours, treatment with 0.1% nalbuphine solution significantly altered the healing rate of both canine CECs and stromal fibroblasts. Continuous increases in transport rates of nalbuphine were detected with time for both the monolayer and multilayer of human CECs. Conclusions and Clinical Relevance: In vitro, nalbuphine potentially could penetrate through corneal tissue, but it may cause damage to the corneal epithelium and stromal fibroblasts. Therefore, nalbuphine potentially may impair corneal wound healing.
Afficher plus [+] Moins [-]In vitro cytoprotective effects of acetylsalicylic acid, carprofen, meloxicam, or robenacoxib against apoptosis induced by sodium nitroprusside in canine cruciate ligament cells
2012
Waldherr, Katrin | Zurbriggen, Andreas | Spreng, David E. | Forterre, Simone
Objective: To determine whether incubation of cruciate ligament cells with acetylsalicylic acid, carprofen, meloxicam, or robenacoxib provides protection against apoptosis induced by sodium nitroprusside (SNP). Sample: Explants of cranial (CCL) and caudal (CaCL) cruciate ligaments from eight 1-day-old Beagles. Procedures: Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of carprofen/mL; 0.1, 1, or 10 μg of meloxicam/mL; or 0.1, 1, or 10 μg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E2 concentrations were measured via an ELISA. Results: Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 μg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE2 production from SNP-damaged cells. Conclusions and Clinical Relevance: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments.
Afficher plus [+] Moins [-]Enhancement of Pasteurella haemolytica leukotoxic activity by bovine serum albumin
1994
Waurzyniak, B.J. | Clinkenbeard, K.D. | Confer, A.W. | Srikumaran, S.
Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (BSA) for 2.5 hours enhanced culture supernatant leukotoxic activity [30,700 +/- 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 +/- 40 toxic units/ml)]. Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + BSA) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + BSA to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that P haemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of BSA, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.
Afficher plus [+] Moins [-]Toxin production by Pasteurella multocida isolated from rabbits with atrophic rhinitis
1993
DiGiacomo, R.F. | Deeb, B.J. | Brodie, S.J. | Zimmerman, T.E. | Veltkamp, E.R. | Chrisp, C.E.
Naturally acquired turbinate atrophy in rabbits was associated with Pasteurella multocida infection. Several in vitro and in vivo studies were conducted to document toxin production from P. multocida isolates and to determine the relation of toxin to atrophic rhinitis in rabbits. Ten isolates of P. multocida serotype A:12 were obtained from adult New Zealand White rabbits with noninduced atrophic rhinitis. Specific-pathogen-free rabbits inoculated intranasally with isolates of P. multocida developed rhinitis and turbinate atrophy. However, inoculation with filtrates of the same bacteria failed to induce turbinate atrophy. Cytotoxicity was observed in assays, using bovine embryonic turbinate cell cultures with extracts of P. multocida, but not in agar overlay cytotoxicity assays, using bovine embryonic turbinate, bovine embryonic lung, or Vero cell cultures, or in a sandwich ELISA, using monoclonal antibodies to purified P. multocida toxin. Thus, turbinate atrophy was experimentally reproduced in rabbits with isolates of P. multocida, but toxin was only detected in vitro by cell culture assay of P. multocida extracts.
Afficher plus [+] Moins [-]Effects of Pasteurella haemolytica leukotoxin on cultured bovine lymphoma cells
1989
Clinkenbeard, K.D. | Mosier, D.A. | Timko, A.L. | Confer, A.W.
Leukotoxin activity from culture supernatants of Pasteurella haemolytica serotype 1 in logarithmic growth phase caused rapid (less than 5 min) release of intracellular K+, uptake of extracellular Ca2+, and swelling of cultured bovine lymphoma cells (BL3 cells). Release of 51CrO(4)(2-) and lactate dehydrogenase (LDH) from BL3 cells began after 15 minutes of incubation with leukotoxin at 37 C and was completed between 60 and 120 minutes of incubation. In addition, leukotoxin exposure of BL3 cells resulted in cell aggregation and adherence to glass surfaces. Scanning electron microscopy indicated that after 10 minutes of leukotoxin exposure, BL3 cells increased in size, and large membrane defects developed between 20 and 60 minutes of exposure. The rate of release of LDH from leukotoxin-exposed BL3 cells was proportional to the amount of leukotoxin added. At high cell concentrations, the activity of LDH released at completion was directly proportional to the amount of leukotoxin added. Leukotoxin-induced release of LDH required a divalent cation, whereas K+ release and cell swelling did not. The addition of Ca2+, Mn2+, and Ba2+ resulted in increased leukotoxin-induced release of LDH. Divalent cation concentrations of 0.5 to 2.5 mM resulted in 50% of maximal stimulation. Ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid blocked increased release of LDH caused by Ca2+ addition, but had no effect on K+ release or cell swelling. Leukotoxin action on BL 3 cells (K+ release, cell swelling, Ca2+ uptake, and release of LDH) was prevented by incubation at 4 C.
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