Animal testing has been prohibited for the safety assessment of cosmetic ingredients or finished products. Thus, alternative non-animal methods, followed by confirmatory clinical studies on human volunteers, should be used as the sole legally acceptable approach within the EU. The safety assessment of cosmetic products requires the involvement of multiple scientific disciplines, including analytical chemistry and biomedicine, as well as in chemico, in vitro and in silico toxicology. Recent data suggest that fragrance components may exert multiple adverse biological effects, e.g. cytotoxicity, skin sensitisation, (photo)genotoxicity, mutagenicity, reprotoxicity and endocrine disruption. Therefore, a pilot study was conducted with selected samples of fragrance-based products, such as deodorant, eau de toilette and eau de parfum, with the aim of integrating results from a number of alternative non-animal methods suitable for the detection of the following toxicological endpoints: cytotoxicity (with 3T3 Balb/c fibroblasts); skin sensitisation potential (in chemico method, DPRA); skin sensitisation potential (LuSens in vitro method, based on human keratinocytes); genotoxicity potential (in vitro Comet assay with 3T3 Balb/c cells); and endocrine disruption (in vitro YES/YAS assay). The presence of twenty-four specific known allergens in the products was determined by using GC-MS/MS. The strategies for estimation of the NOAEL of a mixture of allergens, which were proposed by the Scientific Committee on Consumer Products in their 'Opinion on Tea tree oil' document and by the Norwegian Food Safety Authority in their 'Risk Profile of Tea tree oil' report, were used as models for the NOAEL estimation of the mixtures of allergens that were identified in the individual samples tested in this study.
- MeSH
- alergeny toxicita analýza MeSH
- kosmetické přípravky * toxicita MeSH
- lidé MeSH
- parfém * analýza MeSH
- pilotní projekty MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- tandemová hmotnostní spektrometrie MeSH
- tea tree oil * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Health care facilities and hospitals generate significant amounts of wastewater which are released into the sewage system, either after a preliminary treatment or without any further treatment. Hospital wastewater may contain large amounts of hazardous chemicals and pharmaceuticals, some of which cannot be eliminated entirely by wastewater treatment plants. Moreover, hospital effluents may be loaded with a plethora of pathogenic microorganisms or other microbiota and microbiome residues. The need to monitor hospital effluents for their genotoxic hazard is of high importance, as detailed information is scarce. DNA-based information can be acquired directly from samples through the application of various molecular methods, while cell-based biomonitoring assays can provide important information about impaired cellular pathways or mechanisms of toxicity without prior knowledge of the identity of each toxicant. In our study, we evaluated samples of chlorinated hospital wastewater discharged into the sewage system after this disinfection process. The assessment of cytotoxicity, genotoxicity and mutagenicity of the hospital effluents was performed in vitro by using a broad battery of biomonitoring assays that are relevant for human health effects. All the tested hospital wastewater samples could be classified as potentially genotoxic, and it is concluded that the microbiota present in hospital wastewater might contribute to this genotoxic potential.
- MeSH
- chemické látky znečišťující vodu * analýza toxicita MeSH
- lidé MeSH
- nemocnice MeSH
- odpadní voda * toxicita MeSH
- poškození DNA MeSH
- testy genotoxicity MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Cells grown in monocultures are widely used to model lung tissue. As a result of these culture conditions, these cells exhibit poor morphological similarity to those present in in vivo lung tissue. MucilAir™, a 3-D in vitro model comprising human basal, goblet and ciliated cells, represents a fully differentiated respiratory epithelium that can be used as an alternative and a more realistic system. The aim of our study was to compare the effects of short-term and long-term exposure to two polycyclic aromatic hydrocarbons (PAHs) - benzo[a]pyrene (B[a]P) and 3-nitrobenzanthrone (3-NBA) - using MucilAir as a model of human lung tissue. Two concentrations (0.1 μM and 1 μM) were tested at three time points (24 hours, 7 days and 28 days). Several aspects were assessed: cytotoxicity (lactate dehydrogenase (LDH) release), integrity of the cell layer (transepithelial electrical resistance (TEER)), induction of oxidative stress (reactive oxygen species production) and changes in the expression of selected genes involved in PAH metabolism (CYP1A1 and AKR1C2) and the antioxidant response (ALDH3A1, SOD1, SOD2, GPX1, CAT, HMOX1 and TXNRD1). The results showed that exposure to B[a]P caused a spike in LDH release at day 5. Exposure to 3-NBA caused a number of spikes in LDH release, starting at day 5, and a decrease in TEER after 11 days. CYP1A1 gene expression was upregulated after the 7-day and 28-day B[a]P exposures, as well as after the 24-hour and 7-day 3-NBA exposures. HMOX1 and SOD1 were downregulated after both 24-hour PAH treatments. HMOX1 was upregulated after a 1-week exposure to 3-NBA. There were no significant changes in the messenger RNA (mRNA) levels of AKR1C2, ALDH3A1, TXNRD1, SOD2, GPX1 or CAT. These results illustrate the potential use of this 3-D in vitro lung tissue model in studying the effects of chronic exposure to PAHs.
Morphology, motility, proliferation rate and markers of oxidative stress in primary human gingival fibroblasts (GF) and periodontal ligamental fibroblasts (PDL-F) grown in zinc-deficient cultivation medium (ZDM), were studied over a 5-week culture period. A low-zinc environment effectively reduced the total, as well as the free, intracellular zinc content in both cell types, over the course of the experiment. Decreased intracellular zinc content resulted in altered cellular morphology, reduced motility, and rearrangement of actin and tubulin in the cytoskeleton. In addition, fibroblasts with low zinc content exhibited decreased proliferation, accompanied by changes in cell cycle distribution, expression of specific biochemical markers, increased oxidative stress and the activation of caspase-3. Supplementation of ZDM with exogenous zinc prevented the loss of intracellular zinc, while also restoring the morphology, cell proliferation and mitogenic signalling of the cultured cells. Moreover, such supplemented cells were protected against oxidative stress and cell death. Of the two primary cell cultures examined, GF were more sensitive to decreased intracellular zinc content, when compared to PDL-F. The results obtained suggest that the human primary cell cultures can be useful for the longer-term evaluation of the effects of nutritional factors originating from the environment.
- MeSH
- buněčné dělení účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- fibroblasty cytologie účinky léků fyziologie MeSH
- gingiva cytologie účinky léků fyziologie MeSH
- glutathion metabolismus MeSH
- kaspasy metabolismus MeSH
- lidé MeSH
- periodontální vaz cytologie účinky léků fyziologie MeSH
- pohyb buněk účinky léků MeSH
- replikace DNA účinky léků MeSH
- signální transdukce účinky léků fyziologie MeSH
- zinek nedostatek farmakologie toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An express (3-minute) test for acute toxicity determination by using the oligochaete annelid, Tubifex tubifex, is described. The EC50(Tubifex tubifex) [EC50(Tt)] for movement inhibition was calculated by using a concentration-response dependence. The reproducibility of the test was checked over several years and by several workers. Its applicability is limited to compounds which are soluble in water. The calculated EC50(Tt) indices correlate with LC50 values determined by using the fish, Pimephales promelas (96-hour assay), and with ICG50 values determined by using the ciliate, Tetrahymena pyriformis (48-hour assay) with high statistical significance (r = 0.822, n = 35, and r = 0.927, n = 80, respectively). The correlation between the EC50(Tt) indices and rat oral LD50 values (48-hour assay) was r = 0.519 (n = 67). The correlation within organic compounds was closer (r = 0.635, n = 60) than with the heterogeneous series of chemicals. A similar trend was noticed for the correlation with mouse oral LD50 values (r = 0.479, n = 56) with the heterogeneous series of chemicals, as compared that with the series without inorganic salts (r = 0.605, n = 42), and similarly with mouse intraperitoneal LD50 values, where r = 0.543 (n = 50) with the heterogeneous series of chemicals and r = 0.893 (n = 33) with the series of organic chemicals.
- MeSH
- anorganické látky * toxicita MeSH
- LD50 MeSH
- modely u zvířat * MeSH
- Oligochaeta * účinky léků MeSH
- organické látky * toxicita MeSH
- testy akutní toxicity * metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hallmarks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P-glycoprotein.
- MeSH
- apoptóza účinky záření MeSH
- buňky K562 enzymologie patologie účinky léků MeSH
- cyklosporin toxicita MeSH
- DNA nádorová účinky léků MeSH
- financování organizované MeSH
- glutathion metabolismus MeSH
- glutathiondisulfid metabolismus MeSH
- HL-60 buňky enzymologie patologie účinky léků MeSH
- imunosupresiva toxicita MeSH
- kaspasa 3 biosyntéza MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- methylmethansulfonát toxicita MeSH
- nekróza chemicky indukované MeSH
- oxidační stres účinky záření MeSH
- P-glykoprotein genetika metabolismus MeSH
- poškození DNA MeSH
- RNA nádorová analýza MeSH
- synergismus léků MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
S-adenosylmethionine (SAMe) has been shown to protect hepatocytes from toxic injury, both experimentally-induced in animals and in isolated hepatocytes. The mechanisms by which SAMe protects hepatocytes from injury can result from the pathways of SAMe metabolism. Unfortunately, data documenting the protective effect of SAMe against mitochondrial damage from toxic injury are not widely available. Thioacetamide is frequently used as a model hepatotoxin, which causes in vivo centrilobular necrosis. Even though thioacetamide-induced liver necrosis in rats was alleviated by SAMe, the mechanisms of this protective effect remain to be verified. The aim of our study was to determine the protective mechanisms of SAMe on thioacetamide-induced hepatocyte injury by using primary hepatocyte cultures. The release of lactate dehydrogenase (LDH) from cells incubated with thioacetamide for 24 hours, was lowered by simultaneous treatment with SAMe, in a dose-dependent manner. The inhibitory effect of SAMe on thioacetamide-induced lipid peroxidation paralleled the effect on cytotoxicity. A decrease in the mitochondrial membrane potential, as determined by Rhodamine 123 accumulation, was also prevented. The attenuation by SAMe of thioacetamide-induced glutathione depletion was determined after subsequent incubation periods of 48 and 72 hours. SAMe protects both cytoplasmic and mitochondrial membranes. This effect was more pronounced during the development of thioacetamide-induced hepatocyte injury that was mediated by lipid peroxidation. Continuation of the SAMe treatment then led to a reduction in glutathione depletion, as a potential consequence of an increase in glutathione production, for which SAMe is a precursor.
- MeSH
- alternativy testů na zvířatech MeSH
- antagonismus léků MeSH
- financování organizované MeSH
- glutathion metabolismus MeSH
- hepatocyty enzymologie účinky léků MeSH
- karcinogeny toxicita MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- nekróza chemicky indukované prevence a kontrola MeSH
- ochranné látky farmakologie MeSH
- peroxidace lipidů účinky léků MeSH
- potkani Wistar MeSH
- S-adenosylmethionin farmakologie MeSH
- thioacetamid toxicita MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.
- MeSH
- alternativy testů na zvířatech MeSH
- financování organizované MeSH
- hepatocyty metabolismus patologie účinky léků MeSH
- jaterní mitochondrie metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- kyslík analýza metabolismus MeSH
- manometrie MeSH
- membránový potenciál mitochondrií fyziologie účinky léků MeSH
- oxidační stres MeSH
- oxidancia toxicita MeSH
- potkani Wistar MeSH
- spotřeba kyslíku MeSH
- terc-butylhydroperoxid toxicita MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zduření mitochondrií fyziologie účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
The role of biomembranes in the chronic toxicity of environmentally occurring chromium acetate hydroxide was investigated by using primary human fibroblasts. Transport of chromium acetate hydroxide across the plasma membrane of the cell, and the effects of chromium (III) ions on the plasma membrane as well as other intracellular membranes, were determined during six weeks of continuous exposure by using atomic absorption spectrometry, observation of cell morphology, membrane integrity assays (for lactate dehydrogenase leakage and lysosomal membrane disruption), and mitochondrial assays (for mitochondrial dehydrogenase activity and mitochondrial transmembrane potential analysis). The type of cell death induced by long-term exposure was determined in terms of phosphatidylserine externalisation, caspase-3 activation, and chromatin fragmentation. Chromium acetate hydroxide, at a concentration of 100 micromol/l, accumulated in exposed cells, inflicting plasma membrane damage and suppressing mitochondrial function. Antioxidant co-enzyme Q, at a concentration of 10 micromol/l, partially prevented plasma membrane damage and mitochondrial dysfunction. Exposure to chromium acetate hydroxide produced apoptosis, necrosis and an intermediate type of cell death in primary human fibroblasts. These results show that the plasma membrane and mitochondrial membrane are important targets for chronic chromium acetate hydroxide toxicity, and that this in vitro system holds promise for studying the toxicity resulting from long-term exposure to metal ions.
- MeSH
- apoptóza účinky záření MeSH
- fibroblasty metabolismus patologie účinky léků MeSH
- financování vládou MeSH
- intracelulární membrány enzymologie účinky léků MeSH
- kultivované buňky MeSH
- kůže metabolismus patologie účinky léků MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- látky znečišťující životní prostředí toxicita MeSH
- lidé MeSH
- mitochondrie enzymologie účinky léků MeSH
- nekróza MeSH
- organokovové sloučeniny toxicita MeSH
- sloučeniny chromu toxicita MeSH
- ubichinon farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
Exploring alternatives to laboratory animals in education is less important in the context of the number of animals used than from the point of view of society's attitude toward this issue. The situation at the Faculty of Medicine in Hradec Králové has changed, mainly as a result of a Joint European TEMPUS Project. The most important result of this project was general incorporation of alternatives into the curricula at our faculty. There are several standard features of the curricula at the Faculty of Medicine in Hradec Králové: no use of dead animals for dissections; no use of animals for surgical training; no harmful use of living animals; clear and thorough consideration of the advantages and disadvantages of alternatives; and practical experience with alternative in vitro methods for each student.
- MeSH
- alternativy testů na zvířatech * MeSH
- laboratorní zvířata MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
