We previously have reported that the mitogen-activated protein kinase (MAPK) pathway is stimulated by adhesion of human chondrocytes to anti-␤ 1 -integrin antibodies or collagen type II in vitro. These mechanisms most likely prevent chondrocyte dedifferentiation to fibroblast-like cells and chondrocyte death. To investigate whether this pathway plays an essential role for the differentiation, phenotype, and survival of chondrocytes, we blocked mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) (MEK), a kinase upstream of the kinase Erk by using U0126. Exposure of chondrocytes to U0126 caused activation of caspase-3 in a dose-dependent manner. Western blot analysis with an antibody specific for dually phosphorylated Erk shows that collagen type II induced phosphorylation of Erk1/2 was specifically blocked by U0126 in a dose-dependent manner. Immunohistochemical analysis showed that treated chondrocytes were caspase-3 positive. In treated chondrocytes, the cleavage of 116-kDa poly(ADP-ribose)polymerase resulted in the 85-kDa apoptosis-related cleavage fragment and was associated with caspase-3 activity. Analysis by electron microscopy showed typical morphological signs of apoptosis, such as crescent-shaped clumps of heterochromatin, and a degraded pericellular matrix. Thus, these results indicate that the MEK/Erk signal transduction pathway is involved in the maintenance of chondrocytes differentiation and survival. These data stimulate further investigations on the role of mitogen-activated protein kinase pathways in human chondrocytes.Apoptosis, or programmed cell death, plays a key role in embryogenesis, immunological competence and tissue homeostasis for cell removal and can distinguished biochemically and morphologically from cell necrosis, which is a passive, energyindependent form of cell death. Chondrocyte degradation and death occurs in enchondral ossification as well as in age-associated athropathies such as osteoarthritis (1, 2). Chondrocyte apoptosis, can be induced in vitro by a variety of agents, such as nitric oxide, oxygen radical scavengers (3), tumor necrosis factor (4), and interleukin-1␤ (5).It is known that many cell types including chondrocytes require integrin mediated interactions with the extracellular matrix to survive, differentiate, and proliferate (6, 7). Cells undergo a specific cell death or apoptosis in the absence of specific matrix components (8). Interaction between chondrocytes and cartilage matrix components or anti-␤ 1 -integrin antibodies leads to a rearrangement of cytoskeletal and signaling proteins localized at focal adhesions and focal adhesion kinase (6 -9). These stimulate docking proteins such as Src-homology collagen. Src-homology collagen then associates with growth factor receptor-bound protein 2, and extracellular signal-regulated kinase (Erk) 1 (7). These mechanisms most likely prevent chondrocyte dedifferentiation to fibroblast-like cells and chondrocytes death (7)(8)10).It is well known that one of the early reactions occurring in the c...

The cellular changes which lead to the increase of drug-metabolizing enzymes following drug administration are explained by correlating new biochemical data with previously reported electron microscopic and pharmacologic observations. Repeated administration of phenobarbital and several other drugs results in a quantitative increase of the smooth endoplasmic reticulum of the liver cell. The marked increase in drug-metabolizing enzymes is found to occur in this enlarged smooth membrane fraction of the endoplasmic reticulum.

Quinolones accumulate in cartilage, and because they form chelate complexes with divalent cations, they possess the potential to induce a deficiency of functionally available magnesium. To test the hypothesis that quinolone-induced arthropathy is caused (or aggravated) by magnesium deficiency in cartilage, we induced magnesium deficiency by feeding juvenile rats a magnesium-deficient diet for 9 days and treated the rats with single oral doses of ofloxacin (0, 100, 300, 600, or 1,200 mg/kg of body weight) during this period. Additional groups of juvenile rats on a normal diet were treated with ofloxacin correspondingly. Typical cartilage lesions (e.g., swollen matrix, cleft formation) were found in knee joints of all magnesium-deficient rats, including those without ofloxacin treatment. Lesions in these groups were not distinguishable from lesions induced by a single dose of 600 mg of ofloxacin per kg of body weight or higher in rats on a normal diet. Ofloxacin levels in plasma after 600 mg/kg of body weight were approximately 10-fold higher than those in humans during therapy with this quinolone. Lesions in rats treated with ofloxacin plus magnesium deficiency were more pronounced than those in rats with normal magnesium concentrations. After intake of a magnesium-deficient diet for 9 days, the magnesium concentration in serum (mean ؎ standard deviation) was 0.18 ؎ 0.05 mmol/liter (control on normal diet, 0.82 ؎ 0.10 mmol/liter). Magnesium concentrations in bone (femur) and cartilage (processus xiphoideus) samples were 64.7 ؎ 10.5 and 14.3 ؎ 3.9 mmol/kg of dry weight, respectively, which corresponded to approximately 50% of the concentrations measured in controls on a normal diet. It was concluded that quinolone-induced arthropathy is probably caused by a deficit of available magnesium in joint cartilage due to the formation of quinolone-magnesium chelate complexes. If juvenile patients must be treated with quinolones for serious infections, it seems prudent to ensure that these patients do not have a disturbed magnesium balance.Quinolone-induced arthropathy is an unusual toxic effect observed with all known quinolones. Cartilage lesions are inducible in juvenile animals of multiple species, such as dogs (3,4,31), rats (14, 29), nonhuman primates (29), and others (9). The chondrotoxic potential of quinolones in humans under therapeutic conditions is low. Most juvenile patients have shown no signs of arthropathy after treatment with these drugs (1, 26). On the other hand, symptoms of arthropathy in juvenile and even adult patients have been described in a considerable number of case reports (e.g., references 5 and 24). Since most of these patients had cystic fibrosis-a disease which itself can be associated with arthropathy-the causal relationship in these observations and the indications for quinolone therapy in pediatrics remain matters for discussion (6,25,28).Recently, we hypothesized that quinolones might affect magnesium-dependent integrins of the ␤ 1 subfamily in articular cartilage as the primary event of...

Arthropathia in juvenile animals is the most important toxic effect induced by quinolones. We conducted pharmacokinetic and morphological studies with ofloxacin on non-human primates (Callithrix jacchus, Marmosets) and rats. In the marmoset, electron microscopy and the application of immuno-morphological methods proved to be suitable for the detection of specific alterations in cartilage (e.g. loss of proteoglycans and altered chondrocytes). Subsequently performed electron microscopic examinations in rats showed similar specific alterations of the femur cartilage surface after multiple oral applications of 600 mg ofloxacin/kg body wt. These results were correlated with pharmacokinetic data obtained for the same species. After single oral application of 100, 300 or 600 mg ofloxacin/kg body wt to 5 week-old rats peak plasma levels were achieved 15-45 min after administration indicating a rapid absorption of the drug. The following peak concentrations were measured for the three doses applied (mean +/- SD): 8.9 +/- 2.1, 22.6 +/- 7.5 mg/l and 33.5 +/- 9.8 mg/l, respectively. After 360 min the concentrations were 1.1 +/- 0.4, 5.9 +/- 2.5 and 15.9 +/- 5.1 mg/l, respectively. After subcutaneous injection of 100 mg ofloxacin/kg body wt the mean peak concentration was 27.7 +/- 2.6 mg/l after 45 min (0.5 +/- 0.2 mg/l after 360 min). In the marmoset higher plasma concentrations were measured with comparable doses. One, 3, and 6 h after the last of nine administrations of 200 mg ofloxacin/kg body wt, the mean (+/- SD) plasma concentrations were: 42.7 +/- 16.7, 40.6 +/- 9.5, and 26.5 +/- 3.6 mg ofloxacin/l plasma. Typical alterations of the joint cartilage of juvenile rats (e.g. opened chondrocyte cavities, swelling of rough endoplasmic reticulum and mitochondrial swelling in the chondrocytes) were induced by oral administration of ofloxacin at doses that were approximately 100 times higher than therapeutic ones, but led to peak plasma concentrations which were only approximately 10 times above the therapeutic level.

Various lipid soluble drugs act unspecifically on the endoplasmic reticulum of liver cells. They cause the development of new smooth membranes containing drug metabolizing enzymes.1.2 A report about this phenomenon should begin by illustrating the alteration in the image of the liver cells after treating animals with a number of unrelated drugs. In connection with the morphological description I shall attempt to show that the interpretation of the changes observed with the electron microscope correspond with results obtained with biochemical methods. Afterwards, evidence will be given for a typical enzyme induction, i.e., the enhanced synthesis of a particular microsonial cytochrome which proceeds the new formation of intracellular membranes. Finally we should think over the consequences of this peculiar side effect which should be taken into consideration in drug therapy and in evaluation of chronic toxicity studies. Morphological A Iterations in the Endoplasmic Reticulum of Liver Cells after Treatment with Various DrugsThe structures of normal liver cells which can be observed under the electron microscope are diagrammed in FIGURE 1. Besides the nucleus and mitochondria the endoplasmic reticulum (e.r.) is clearly visible. It consists mainly of two different types of membranes, the smooth and the rough.3 The latter are encrusted with small particles, the ribosomes. Moreover the cytoplasm is filled with dense granules which can be demonstrated after fixation with lead hydroxide. The nature and function of these particles are unknown (FIGURE 2). After treating rats and rahbits with several injections of 50-100 mg./kg. of three different and unrelated drugs such as phenobarbital, tolbutamide, or nikethamide, striking changes in the image of e x . are observed. The dense granules in the cytoplasma are replaced by numerous new smooth membranes which form a clearly visible tridimensional lattice work of tubules about 25-50 mp, in diameter bordered by membranes 6 m p thick (FIGURE 3 ) . However nucleus, mitochondria and the rough membranes of the ex. appear to remain unchanged, but the number of free ribosomes not associated with rough membranes is increased. The development of new smooth membranes is even detectable after the first injection of phenobarbital and achieved its maximum after the fifth dose given daily or on alternating days.Porter and Bruni4 observed a similar increase of smooth membranes after treating rats with the carcinogenic 3'-methyl-p-dimthylaminoazobenzol. However the appearance of new smooth membranes was followed by disorganization of rough membranes. Changes in the composition and in the Content of Enzymes in the Endoplastnic ReticulumIn order to elucidate the morphological alterations in the liver cells we isolated the ex., which, as it is well known, desintegrates to microsomes when liver tissue FIGURE 1. Diagram of the normal liver cell of rats. FIGURE 2. Electron micrograph of a normal rat liver cell. 0 rat, 60 g. of weight . ( 1 : 21,000).