Cold light fluorimetry: a microtitration technology for cell culture to evaluate anethole dithiolethione and other biothiols.
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Acetaminophen↗
Biomedical subjects
M Adolphe
Publications and source records attributed to M Adolphe.
Rabbit articular chondrocyte functional toxicity test.
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Alcian Blue↗
[Alternative methods to animal experimentation. Scientific and ethical problems].
The alternative methods include all the technologies able to replace animal experimentation. This denomination has been much debated and several researchers prefer the term of complementary methods. Alternative methods consist mainly of methods based on organ and cell culture but also includes cell organelles. These methods have been introduced gradually over the years particularly in toxicology but also in biology, physiology, pathology and pharmacology. The reasons for this development are from technological and ethical sources. This last point was due to the consciousness of industrial countries on the animal suffering which is at the origin of groups for animal welfare, able to influence european governments. The results of the development of the alternative methods are an increase in fundamental and applied research under the influence of various organisations such as in England: FRAME (Fund for the Replacement of Animals in Medical Experimentals), in USA: John Hopkins Center and in ECC: ECVAM (European Center for the Validation of Alternative Methods). This last Center is particularly devoted to validation which are defined as "the process whereby the reliability and relevance of a procedure are established for a particular purpose". This involves several stages. Some validations procedures are now in progress mainly in the aim of evaluating potential alternative methods to the Draize eye irritation test. Alternative methods are able to decrease the use of animal experiments and consequently improve animal ethics although they could not replace totally animal experiments. However they are complementary and very useful for the screening of drugs and mechanistic areas.
Animal Testing Alternatives↗
Transient mitochondrial transcript level decay in oxidative stressed chondrocytes.
Steady-state levels of 12S rRNA and NADH dehydrogenase subunit 4 mRNA (ND4) mitochondrial transcripts were measured on rabbit articular chondrocyte in culture. In pseudosynchronized chondrocytes, changes of mitochondrial RNA levels were observed during the progression of the cells in the cell cycle. Oxidative stress generated by the hypoxanthine-xanthine oxidase system (HX-XO) induced a transient decrease in the levels of both ND4 and 12S rRNA. Mitochondrial RNA levels recovered 24 h after the oxidative stress. These RNA level changes were not associated with modifications in the structure or the copy number of the mitochondrial genome. Furthermore, the decrease in the amount of the mitochondrial transcripts observed may be related to a transient inhibition of mitochondrial transcription since the treatment of cells with ethidium bromide (a mitochondrial transcription inhibitor) resulted in the same decrease in 12S rRNA level as HX-XO treatment alone.
Animals↗
New in vitro fluorimetric microtitration assays for toxicological screening of drugs.
Flow cytometry has been widely used to quantify fluorescent probes in cell culture. However, FCM is not adapted to toxicological screenings due to the cost, the length and the poor reproducibility of this technique. Moreover, several multicenter studies have preferred microtitration methodologies for drug screening. A new fluorimetric technology has been designed that is sensitive and adapted to direct screening in 96-well microplates. This fluorimeter uses cold light technology (CLF) with chemical and physical modifications of the lighting system (Rat et al., 1995). CLF allows reading of UV, visible and near infrared fluorescence by increasing light energy (from 1000 to 2300 lumens) and reducing the calorific part of light (IR > 900 nm, Joule effect). It induces a decrease in background and a 500- to 1000-fold improvement of detection limit of probes in comparison with classical fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy evaluation of cell injury induced by physical agents (UVA) or chemical toxins (CCl4). Four biological endpoints for cytotoxicity evaluation have been tested with several probes: proliferation (H33258); viability (fluorescent Neutral Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects (Rhodamine 123). Rh123 assay appeared more sensitive than fluorimetric or photometric detection of Neutral Red assay. Cold light fluorimetry (CLF) permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF is shown to improve classical cytotoxicity assays and, owing to its adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri dishes), it is thus a promising alternative to flow cytometry for drug cytotoxicity screening.
Carbon Tetrachloride↗
Cytotoxicity of sulphur mustard on a human keratinocyte cell line: direct effects compared to conditioned-medium effects.
Direct toxic effects of sulphur mustard (SM) were compared to SM-conditioned medium effects in a human keratinocyte cell line. Cytotoxicity was evaluated by measurement of cell viability with the Neutral Red uptake assay. Culture directly exposed to SM exhibited cytotoxic dose-response curves after 24, 48 or 72 h. Inhibitory concentration 50 (IC50) appeared to be more than 10 times lower after 72 h than the corresponding value after 24 h. In contrast, no cytotoxic activity was observed in media which were collected 4 h or 24 h after SM exposure. Cell viability was unchanged even if observation was extended to 48 h. Results suggested that SM cytotoxicity was not due to potential mediators secreted by human keratinocytes.
Cell Line↗
[Human articular chondrocyte culture. Value in pharmacotoxicology].
Inflammatory and degenerative joint diseases are a major health problem of today. Research is aimed at understanding the processes that bring about degeneration and at the pharmacotoxicological studies of new compounds. Articular cartilage is a connective tissue consisting of an organized extracellular matrix, composed of collagen and proteoglycan aggregates, in which chondrocytes are embedded. Chondrocyte culture has been developed since 1971. However, the use of human chondrocytes for pharmacotoxicology studies is much more recent. The effect of agents can be studied on a number of aspects of chondrocyte metabolism. The most studied are: cell proliferation, proteoglycan degradation and release of matrix proteolytic enzymes or cytokines in the culture medium. Several types of chondrocyte culture can be used for in vitro studies. Organ culture of cartilage slices permits investigation on intact tissue. Chondrocytes can also be released from the articular tissue by serial enzymatic digestions and cultured in a monolayer or tridimensional system. Monolayer culture is easy to set up and allows the culture of large amounts of cells. However, this type of culture is hindered by the phenotypic instability of the chondrocytes. Culture in suspension or within a gel (agarose, collagen) or alginate beads is more suitable for the maintenance of the differentiated phenotype. Chondrocyte immortalization by transfection with oncogenes has been described, but the immortalizing agent still needs to be improved to permit the maintenance of some differentiative properties. The most studied agents are non-steroidal anti-inflammatory drugs. Chondroprotective drugs should also be mentioned. The type of chondrocyte culture for pharmacotoxicological study should be chosen keeping in mind the expected goals of the research undertaken.
Cartilage, Articular↗
Development of immortalized human articular cartilage cell lines.
Human articular cartilage cells were transfected with the t.-sensitive polyomavirus large T antigen of SV40. Several immortalized chondrocyte cell lines were obtained. The types of acidic polysaccharides and of collagen synthesized suggest dedifferentiation in the in vitro culture system used afterwards to obtain large numbers of cells.
Antigens, Polyomavirus Transforming↗
Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions.
Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.
Alkaline Phosphatase↗
SV40-immortalization of rabbit articular chondrocytes: alteration of differentiated functions.
Cell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue-stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that pro alpha 1 (II) mRNA was undetectable, whereas pro alpha 1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re-express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.
Animals↗
Differential temperature sensitivity of cultured cells from cartilaginous or bone origin.
The effects of long-term exposure to hyperthermia were studied on several cell cultures of cartilaginous or bone origin after a 4-day treatment at 40 degrees C. Chondrocytes proliferation, as well as mitochondrial activity were not modified by these culture conditions (40 degrees C) but protein content and cell volume were increased. In contrast, the proliferative capacity of osteoblasts, MC3T3.E1 a and ROS 17/2.8 was decreased and their protein content, cell volume and mitochondrial activity were increased. Chondrocytes appeared to be thermoresistant, and osteoblastic cells thermosensitive. Furthermore, temperature sensitivity was greater for the continuous established osteoblastic cell line MC3T3.E1 and for the cancerous established osteoblastic cell line ROS 17/2.8 than for chondrocytes.
Animals↗
Chromosomal, mitochondrial and metabolic alterations in SV40-transformed rabbit chondrocytes.
Karyotype, mitochondrial ultrastructure and several enzymatic activities were studied in two clones, D22 and D27, from SV40-transformed rabbit chondrocytes. Similar chromosome alterations, with recurrent losses and gains were observed at the various passages. Mitochondria were rare, with increase in size and crest alterations. By comparison to non-transformed rabbit chondrocytes, activities of superoxide dismutase 1 and 2 (SOD) and glutathione peroxidase were increased, those of glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase fluctuated according to passages, thymidylate synthase decreased, thymidine kinase and hypoxanthine-phosphoribosyl-transferase increased and the ratio lactate dehydrogenase B/A increased. In most cases, these variations were correlated with the number of chromosomes carrying the genes encoding for corresponding enzymes. These results, compared to those obtained in SV40-transformed human fibroblasts, demonstrate that the two cell types behave differently for detoxication systems against oxygen radicals, in particular for SOD2 activity, and have opposite imbalances of chromosomes carrying the corresponding genes.
Animals↗
Toxic effects and detection of oxygen free radicals on cultured articular chondrocytes generated by menadione.
The aim of this work was to study the proliferation pathological perturbations of cultured chondrocytes in response to menadione, an oxygen free radicals producing drug. Rabbit articular chondrocytes in monolayer culture were treated with 10(-5) M, 1.5.10(-5) M and 2.10(-5) M of menadione during three days. A dose dependent decrease of the proliferative capacity was observed. Flow cytometry analysis revealed a perturbation of the cell cycle progression consisting in an accumulation of cells in the S and G2 + M phases. This growth perturbation was due to oxygen radicals production since a treatment with catalase suppressed these toxic effects. Furthermore, to identify oxygen derived radicals in the cellular suspension of cultures treated with menadione, we used a technique of spin-trapping coupled with electron spin resonance (ESR). The ESR signal corresponding to the DMPO hydroxyl radical adduct (DMPO-OH) has been detected. The spectra observation indicated the actual production of hydroxyl radical. However, superoxide anions have not been identified; this fact can be explained by the low reactivity of these anions with DMPO and by the decomposition of signal DMPO-OOH to DMPO-OH.
Animals↗
Cross contamination associated with the use of multiwell culture plates for cytotoxicity assessment of volatile chemicals.
In vitro toxicity testing can involve technical problems due to the evaporation of volatile test chemicals. The cytotoxicity of two volatile chemicals (butanol and ethanol) has been assessed with neutral red assay in conventional microtiter plates. The non volatile DMSO chemical is used as a negative control. Under these conditions, an important cross contamination between test concentration groups has been observed. This affects cytotoxicity estimation which is overestimated. This cross contamination is prevented when special plates containing removable bars are used.
Butanols↗
Effects of oxygen free radicals on articular chondrocytes in culture: c-myc and c-Ha-ras messenger RNAs and proliferation kinetics.
Since oxygen free radicals are believed to play an important role in cartilage degradation, we studied the effects of these radicals generated by the hypoxanthine xanthine oxidase system on rabbit articular chondrocytes in culture. Among the damages induced by these radicals, cell proliferation inhibition and G2 arrest were observed. To elucidate the mechanisms involved in this phenomenon, the expression of c-myc and c-Ha-ras genes whose products are associated with cell growth control was studied. Results showed that in chondrocytes, c-myc and c-Ha-ras expression was particularly important during the G1 phase of the cell cycle and that oxygen reactive species, especially H2O2, induced an important decrease of c-myc and c-Ha-ras mRNA levels. Chondrocytes cell cycle analysis revealed an accumulation of cells in G2 phase. It led us to suggest that the chondrocyte cell cycle perturbations observed after oxygen free radicals treatment could be associated with the decrease of c-myc and c-Ha-ras expression.
Animals↗
Mitochondrial biogenesis in rabbit articular chondrocytes transferred to culture.
The effect of the switch to aerobic growth conditions was examined in rabbit articular chondrocytes transferred to culture. Spectroscopic analysis of the cytochromes of the respiratory chain shows that only cytochrome b is present in chondrocytes from cartilage, cytochromes c, c1, and a.a3 being undetectable as compared with the typical spectrum found in a primary cell culture on day 4. Steady state levels of RNA transcripts of nuclear (cytochrome c) and mitochondrial genes (cytochrome b and cytochrome oxidase subunits II and III) involved in the oxidative metabolism were determined relative to the RNA transcripts of the nuclear gene for glyceraldehyde phosphate dehydrogenase involved in the glycolytic pathway and to mitochondrial ribosomal RNAs. Chondrocytes transferred to culture showed a general increase in the levels of all transcripts, but the effect on mitochondrial transcripts was much greater (x 20) than the effect on nuclear transcripts (x 3-4). These results show the absence of a coordinate regulation of the expression of mitochondrial and nuclear genes coding for components of the respiratory chain. The increase in mitochondrial DNA triggered by culture conditions does not appear to be sufficient to account for the enhanced transcription. Concomitant with these mitochondrial changes, the level of transcripts for the collagen II gene involved in the differentiation function decreases dramatically (3% of the control on day 3).
Animals↗
[Hexosaminidase and alkaline phosphatase in cartilage and chondrocyte cultures obtained from normal and arthritic rabbit joints].
Hexosaminidase and alkaline phosphatase activities were studied in a rabbit model of osteoarthritis. Enzyme activities were determined in cartilage slices and cultures of chondrocytes from normal and arthritic joints. Alkaline phosphatase activity was increased in cartilage slices from rabbits with osteoarthritis, as compared with normal cartilage, whereas no difference was seen for hexosaminidase activity. Alkaline phosphatase activity was not found in chondrocyte cultures. Hexosaminidase activity was significantly higher in chondrocytes from joints with arthritis, as compared with chondrocytes from normal joints, regardless of the mode of expression of results (enzyme activity normalized for cell protein content of for number of cells). Chondrocyte hexosaminidase activity can be proposed as an enzyme marker for osteoarthritis in chondrocyte culture models.
Alkaline Phosphatase↗