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.     

Effects of oxygen-free radicals on proliferation kinetics of cultured rabbit articular chondrocytes

It can be postulated that among the factors implicated in cartilaginous lesions, oxygen-derived free radicals seem to have a prominent part. To investigate this hypothesis, rabbit articular chondrocyte cultures have been exposed to oxygen-derived reactive species generated by the hypoxanthine-xanthine oxydase system. We observed a dose-dependent decrease of cellular growth. In order to explain this result, cell cycle progression and binucleate cell fractions have been studied. A greater number of binucleate cells and an increase in cell volume were observed. Flow cytometry analysis revealed a perturbation in cell cycle progression leading to a significant increase in the proportion of cells in G2 phase and an important augmentation in cell protein content confirmed by biochemical assays. This model shows which type of alteration can be induced by oxygen-derived free radicals in vitro. In addition, we deem this model to be useful for studying degenerative processes and for screening drugs that can scavenge oxygen-free radicals.     

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.     

Modulation of fibroblast proliferation by oxygen free radicals.

The major unexplained phenomenon in fibrotic conditions is an increase in replicating fibroblasts. In this report we present evidence that oxygen free radicals can both stimulate and inhibit proliferation of cultured human fibroblasts, and that fibroblasts themselves release superoxide (O2.-) free radicals. Fibroblasts released O2.- in concentrations which stimulated proliferation, a finding confirmed by a dose-dependent inhibition of proliferation by free radical scavengers. Oxygen free radicals released by a host of agents may thus provide a very fast, specific and sensitive trigger for fibroblast proliferation. Prolonged stimulation may result in fibrosis, and agents which inhibit free radical release may have a role in the prevention of fibrosis.     

Proliferation kinetics of rabbit articular chondrocytes in primary culture and at the first passage

The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.     

Effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on rabbit articular chondrocytes in culture

Investigations were carried out to assess the effects of disodium 1-hydroxyethane-1,1-diphosphonate and disodium dichloromethanediphosphonate (compounds containing a P-C-P bond) on isolated rabbit articular chondrocytes in culture. Studies on growth behaviour showed that both diphosphonates displayed inhibitory actions, dichloromethanediphosphonate producing the larger effect. Both compounds inhibited the uptake of 2-deoxy-d-glucose, dichloromethanediphosphonate once more being the more potent of the two. The uptake of alpha-aminoisobutyrate was considerably increased by chondrocytes treated with dichloromethanediphosphonate, whereas 1-hydroxyethane-1,1-diphosphonate showed no effects. The biosynthesis of sulphated extracellular macromolecules secreted by the cells into the pericellular space as well as into the growth medium was greatly increased by dichloromethanediphosphonate but not by 1-hydroxyethane-1,1-diphosphonate. The stimulatory effect was dose-dependent. Short-term exposure of already confluent cells to dichloromethanediphosphonate as opposed to growing the cells to confluence in the presence of the diphosphonate revealed that the stimulatory effects were already evident after 24h, indicating that cell division is not necessarily required to produce the observed effects. The increment in proteoglycan synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture. Pulse-chase experiments together with studies on the enzyme arylsulphatase revealed that the appearance of increased amounts of proteoglycans was the result of a stimulation in synthesis and not due to an inhibition in turnover.     

Effects of sodium butyrate on growth and cell-cycle kinetics of cultured rabbit articular chondrocytes

The sodium salt of n-butyric acid was found to inhibit the growth of asynchronous cultures of rabbit articular chondrocytes. This inhibitory effect was dose-dependent between 1 mM and 5 mM, reversible, and accompanied by volume enhancement and modification of cellular morphology. Flow-cytometric analysis showed that drug exposure led to a slowing-down of the cell-cycle progression; after 1 day's exposure, cells accumulated in G1, and after 2 or 3 days' treatment, in G2, without a blockage in M; the increase of cells in G2 was in fact due to an enhancement of binculeated cells. The treated cells had an increased RNA content. Articular chondrocytes seem to be target cells for sodium butyrate and therefore it represents a valuable biological tool for studying the mechanisms of their growth regulation.     

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2) tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5g/L glucose (n=3). Lactate release was also independent from O(2) tension, but lower for cells at 4.5g/L glucose (n=3). Our observations indicated that O(2) consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O(2) and glucose.     

Synthesis of a "free" form of hyaluronic acid by articular chondrocytes in monolayer culture

Primary and first passage rabbit chondrocyte cultures synthesized a "free" form of hyaluronic acid (HA-f) previously characterized in rabbit cartilage. HA-f was isolated from the [3H] glcN/35SO4-labelled cell-associated-fraction (CAF) and from the culture medium by successive equilibrium centrifugations in Cs2SO4/CsCl/Cs2SO4 under low salt conditions. The culture medium HA-f appeared in the void volume of Sepharose CL-2B eluted with low salt, (0.5M sodium acetate), and was susceptible to digestion with Streptomyces hyaluronidase. HA-f aggregated purified rabbit cartilage proteoglycan monomer. These results indicated that HA-f probably subserves hyaluronic acid already complexed with proteoglycan monomer. Newly synthesized HA-f may be required for the continual formation of proteoglycan aggregates.     

模拟空气潜水对大鼠脾组织氧自由基生成的影响

目的:观察模拟空气潜水对大鼠脾组织氧自由基(OFR)生成的影响。方法:腹腔注射自旋捕捉剂,高气压处理后检测大鼠脾组织生成的OFR。结果:模拟空气潜水后大鼠脾组织OFR生成增多,并检测到了.OH信号。结论:空气模拟潜水时呼吸气中高分压氧可促进脾组织OFR生成,主要类型可能为羟自由基。     

Effects of sesamin on the biosynthesis of chondroitin sulfate proteoglycans in human articular chondrocytes in primary culture

Osteoarthritis (OA) is a degenerative joint disease that progressively causes a loss of joint functions and the impaired quality of life. The most significant event in OA is a high degree of degradation of articular cartilage accompanied by the loss of chondroitin sulfate-proteoglycans (CS-PGs). Recently, the chondroprotective effects of sesamin, the naturally occurring substance found in sesame seeds, have been proved in a rat model of papain-induced osteoarthritis. We hypothesized that sesamin may be associated with possible promotion of the biosynthesis of CS-PGs in human articular chondrocytes. The aim of the study was to investigate the effects of sesamin on the major CS-PG biosynthesis in primary human chondrocyte. The effects of sesamin on the gene expression of the PG core and the CS biosynthetic enzymes as well as on the secretion of glycosaminoglycans (GAGs) in monolayer and pellet culture systems of articular chondrocytes. Sesamin significantly increased the GAGs content both in culture medium and pellet matrix. Real-time-quantitative PCR showed that sesamin promoted the expression of the genes encoding the core protein (ACAN) of the major CS-PG aggrecan and the biosynthetic enzymes (XYLT1, XYLT2, CHSY1 and CHPF) required for the synthesis of CS-GAG side chains. Safranin-O staining of sesamin treated chondrocyte pellet section confirmed the high degree of GAG accumulation. These results were correlated with an increased level of secreted GAGs in the media of cultured articular chondrocytes in both culture systems. Thus, sesamin would provide a potential therapeutic strategy for treating OA patients.     

Effects by doxorubicin on the myocardium are mediated by oxygen free radicals

We hypothesized that doxorubicin (DOX) induces cardiotoxicity of myocardium via oxygen radicals. The present study is aimed at examining the membrane alterations by oxygen radicals generated by DOX in adult rats and cultured neonatal myocytes. Our results showed that DOX 1) decreased beta-adrenoceptor (BAR) density in the cell membrane, 2) increased the membrane permeability of cultured neonatal rat myocytes and 3) altered the ultrastructure of myofibrils and subplasmalemmal actin networks. These effects were reproducible by exogenous hydrogen peroxide. The antioxidant melatonin (MLT) inhibited enzyme leakage and peroxidation in a concentration-dependent manner. It is concluded that DOX induces cardiotoxicity through lipid peroxidation and melatonin is an effective antioxidant against the reactive oxygen intermediates generated by DOX.     

Dose effects of transfected c-Ha-ras oncogene in transformed cell clones

We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-ras^{Val 12} oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2–4, or 4–8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the tranformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-ras^{Val 12} grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.     

活性氧、自由基与植物的衰老

介绍近 1 0年来有关活性氧、自由基的产生 ,对植物的伤害及植物对活性氧、自由基清除的研究进展。     

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay, with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced free radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.     

Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation

By oxymetry and electron paramagnetic resonance (EPR), we investigated the effects of repeated anoxia/re-oxygenation (A/R) periods on the respiration and production of free radicals by synoviocytes (rabbit HIG-82 cell line and primary equine synoviocytes) and equine articular chondrocytes. Three periods of 20 min anoxia followed by re-oxygenation were applied to 10(7)cells; O(2) consumption was measured before anoxia and after each re-oxygenation. After the last A/R, cellular free radical formation was investigated by EPR spectroscopy with spin trapping technique (n=3 for each cell line). Both types of synoviocytes showed a high O(2) consumption, which was slowered after anoxia. By EPR with the spin trap POBN, we proved a free radical formation. Results were similar for equine and rabbit synoviocytes. For chondrocytes, we observed a low O(2) consumption, unchanged by anoxia, and no free radical production. These observations suggest an oxidant activity of synoviocytes, potentially important for the onset of osteoarthritis.     

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.     

家蝇幼虫提取物清除氧自由基的作用

探讨了家蝇Musca domesticaL.幼虫提取物对氧自由基的清除作用。采用脱氧核糖-铁体系和邻苯三酚自氧化体系分别测定了不同浓度的提取物对羟自由基和超氧阴离子自由基的清除率,同时采用H2O2氧化体系测定了提取物的抗氧化值。结果显示家蝇幼虫提取物对羟自由基和超氧阴离子自由基的IC50分别为1.93 mg/mL和3.26 mg/mL;浓度为0.5%的提取物的抗氧化值为9.01 mg/g,为同浓度维生素C抗氧化值的1.29倍。