Rudolf Schwabe

Ultrastructure of Achilles Tendons of Rats Treated with Ofloxacin and Fed a Normal or Magnesium-Deficient Diet

ABSTRACT Fluoroquinolones can cause tendinitis and tendon rupture. However, toxicological as well as clinical information on quinolone-induced tendopathy is scarce. We performed extensive electron microscopic studies with Achilles tendon specimens from ofloxacin-treated rats. The drug was given at a dose of 1,200 mg/kg (body weight) orally. Juvenile Wistar rats received one or three oral doses each of 1,200 mg of ofloxacin/kg (body weight)/day. Three days after treatment, the tenocytes of their Achilles tendons showed degenerative alterations, such as multiple vacuoles and vesicles in the cytoplasm that had developed due to swellings and dilatations of cell organelles. Other indications of cell degradation were the occurrence of cell debris and cell detachment from the extracellular matrix accompanied by a loss of cell-matrix interaction. The tenocytes of juvenile Wistar rats that had been treated at day 36 with a single oral dose of 1,200 mg of ofloxacin/kg (body weight) and sacrificed either 3 or 6 months later exhibited similar degenerative alterations. The number of degenerative alterations of tenocytes after ofloxacin treatment was considerably higher in rats that had received a magnesium-deficient diet than in rats with normal magnesium status. Of the adult rats that had been treated once, 5 times, and 10 times with ofloxacin and killed 1 day later, only those with the 10-times treatment showed a significantly increased number of degeneratively altered tenocytes. In summary, effects observed in tendons show similar pathological features as described earlier in cartilage, indicating that quinolone-induced arthropathy and quinolone-induced tendopathy probably are different clinical manifestations of the same toxic effect on cellular components of connective tissue structures.

Chondrotoxicity of ciprofloxacin in immature beagle dogs: immunohistochemistry, electron microscopy and drug plasma concentrations.

Abstract The systemic effects of ciprofloxacin in immature Beagles were studied. Dogs of 10–11 weeks were dosed orally for 5 days with 0 (n=3), 30 (n=5) and 200 (n=5) mg ciprofloxacin/kg body wt. Plasma concentrations were measured by high-performance liquid chromatography (HPLC) 1 h after dosing (assuming to be peak concentrations). In view of the high doses used, the plasma concentrations were rather low and declined during the study period. For example, plasma concentrations in the high dose group were 6.6 ± 0.9 mg/l (day 1), 3.9 ± 1.4 mg/l (day 3), and 2.6 ± 1.6 mg/l (day 5). In control dogs and in dogs treated with the low dose of ciprofloxacin no pathological changes were seen by light microscopy. However, cleft formation and erosions were observed in joint cartilage from two of five dogs treated with 200 mg/kg. It is noteworthy that despite the high dose used cartilage lesions were not detectable in all five dogs of this group by light microscopy. Using antibodies against cell membrane receptors (e.g. the α5β1-integrin) or matrix components (fibronectin, collagen II) the articular cartilage effects were studied in detail by immunohistochemistry. The most sensitive alteration was an increase in fibronectin which was detectable in the vicinity of the lesions in cartilage samples from the group of dogs administered the high dose. No clear-cut changes were seen with the use of antibodies against other matrix components. Electron microscopy revealed typical alterations in chondrocytes from dogs treated with ciprofloxacin: e.g., swollen mitochondria and enlarged rough endoplasmic reticulum. These changes were much more pronounced in dogs from the high dose group than in dogs from the low dose group. Our main conclusion is that after oral administration ciprofloxacin exhibits rather low chondrotoxicity, even in the most sensitive species known to date. This correlates with the findings in humans that ciprofloxacin seems to be less chondrotoxic than pefloxacin or other quinolones.

Supplementation with Magnesium and Tocopherol Diminishes Quinolone-induced Chondrotoxicity in Immature Rats

Quinolones induce joint cartilage lesions in immature animals, but very little is known about the mechanism of this toxic effect.We have previously reported that identical joint cartilage lesions can be induced either by quinolone treatment or by feeding a magnesium-deficient diet to juvenile rats. We also demonstrated that themost sensitive developmental phase for the induction of such lesions seems to be identical for both treatments. Based on these results, we concluded that the lack of functionally available magnesium in joint cartilage is the primary event in the pathogenesis of quinolone-induced chondrotoxicity.[1-3] The objective of the present study was to assess whether cartilage lesions can be diminished if quinolone-treated immature rats are supplemented with magnesium in addition to their regular dietary magnesium supply. As some evidence exists that free radical formationmight play a role in the pathogenesis of quinolone-induced cartilage defects,[4] we also added tocopherol to the diet.

Synergistic Effect of Ofloxacin and Magnesium Deficiency on Joint Cartilage in Immature Rats

ABSTRACT Single high oral doses of fluoroquinolones (e.g., 1,200 mg of ofloxacin/kg of body weight) are chondrotoxic in juvenile rats. Characteristic cartilage lesions are detectable as early as 12 h after treatment. Since this dosing regimen does not reflect the therapeutic situation, we studied the effects of a 5- or 7-day treatment with ofloxacin at lower oral doses (10, 30, and 100 mg/kg twice a day [b.i.d.]) on joint cartilage in 4-week-old rats. We additionally investigated whether the effects of ofloxacin under these conditions are enhanced in animals kept on a magnesium-deficient diet during treatment. Knee joints were examined histologically. The concentrations of ofloxacin and magnesium were determined in plasma and cartilage. The lowest ofloxacin dose at which cartilage lesions occurred in animals on a standard diet was 100 mg/kg b.i.d. for 5 days. Peak plasma ofloxacin levels were approximately 10 mg/liter in these rats and thus were in the same range as the levels in the plasma of humans during therapy with high doses of ofloxacin. Treatment with 30 mg of ofloxacin/kg b.i.d. for 7 days caused no cartilage lesions in rats on a standard diet, but lesions did occur in 10 of 12 rats that were simultaneously fed a magnesium-deficient diet. Magnesium concentrations in bone, plasma, and cartilage from animals on an Mg2+-deficient diet were significantly lower than those in the controls. The concentration in plasma from animals on an Mg2+-deficient diet was 0.27 ± 0.03 mmol/liter, whereas it was 0.88 ± 0.08 mmol/liter in plasma from rats on a standard diet (means ± standard deviations). Ofloxacin treatment did not change the total magnesium concentrations in tissues, as determined with ashed samples. The incidence of ofloxacin-induced lesions was higher in the magnesium-deficient animals, suggesting a synergistic effect. These results must be taken into account for a benefit-risk evaluation if ofloxacin is considered for use in the pediatric population.

Quinolone-induced arthropathy : exposure of magnesium-deficient aged rats or immature rats, mineral concentrations in target tissues and pharmacokinetics

Abstract Quinolone treatment or magnesium deficiency induce identical cartilage lesions in juvenile rats and show additive arthropathogenic effects. It has been shown previously that neither condition is arthropathogenic in 8-week-old rats. Joint cartilage from aged individuals is rather prone to pathological alterations but information on prolonged quinolone treatment and/or dietarily induced magnesium deficiency in aged animals is not available. We treated magnesium-deficient (n = 9) aged Wistar rats (age 15 months) and age-matched controls with daily doses of 600 mg ofloxacin/kg body wt. by gastric intubation for 28 days. Further groups of magnesium-deficient and control rats (n = 9 and n = 10, respectively) received the vehicle only. Peak plasma concentrations of ofloxacin in adult rats were 20.5 ± 5.6 mg/l (mean ± SD) following treatment with a single dose of 600 mg/kg body wt. At the end of the experiment the degree of magnesium deficiency was most pronounced in plasma (Mg2+-def., 0.33 ± 0.12 mmol/l; control, 0.97 ± 0.08 mmol/l) and less pronounced in sternal cartilage (Mg2+-def., 10.8 ± 3.6 mmol/kg dry wt; control, 13.3 ± 2.8 mmol/kg dry wt), whereas the magnesium concentration in femoral bone remained unchanged (Mg2+-def., 201 ± 13 mmol/kg dry wt; control, 204 ± 11 mmol/kg dry wt). Histological investigation of the knee joints revealed no cartilage lesions following ofloxacin treatment, magnesium deficiency or a combination of both conditions. By contrast, cartilage lesions such as scars and erosions of the joint surface, chondrocyte clusters within acellular areas of the cartilage matrix and persisting clefts were detectable in knee joints from 7 of 10 adult rats (age 9 months) which had been treated with 4 × 600 mg fleroxacin/kg body wt. at 5 weeks of age. Mean plasma concentration of fleroxacin in juvenile rats was approx. 50 mg/l between 1.5 and 6 h after dosing and the drug was still detectable in plasma 48 h after dosing (0.4 ± 0.1 mg/l). Our data indicate that joint cartilage in aged rats is not altered by a 4-week quinolone treatment, even during magnesium deficiency. Cartilage lesions in adult rats were only detectable if the animals had been treated during the sensitive phase at 5 weeks postnatally.

Chondrotoxicity and Target Tissue Kinetics of Ofloxacin in Immature Rats after Multiple Doses

Fluoroquinolones can cause lesions in joint cartilage of immature rats and other animals (reviewed by Stahlmann & Lode[1]). It is well known from studies in various species that these drugs reach rather high concentrations in joint cartilage which exceed those found in plasma,[2-4]but toxicological studies correlating target tissue kineticswith the pathological findings under clinically relevant conditions are lacking. Lesions in the joint cartilage of juvenile rats can be induced by a single high dose of ofloxacin (e.g. 600 mg/kg bodyweight), but anti-infective treatment with ofloxacin is usually performed with much lower doses (e.g. 5 to 10 mg/kg bodyweight) given twice daily for 5 to 10 days.We therefore studied a multiple-dose regimen of twice-daily administration of relatively low doses of ofloxacin for 5 days to immature rats to provide data which more closely resemble the therapeutic situation. We used doses of 30 and 100 mg/kg bodyweight, taking into account that kinetics in humans and small laboratory animals differ considerably because of the more rapid elimination in small animals.

Toxicity of β-blockers in a rat whole embryo culture: concentration-response relationships and tissue concentrations

Beta-adrenoceptor blockers are widely used drugs for the treatment of cardiovascular diseases. Since β-blockers cross the placenta, it is essential to consider possible adverse effects on the embryo. Six β-adrenoceptor blockers were tested at various concentrations (10–5000 μM) in a rat whole embryo culture. Although inducing a very similar pattern of dysmorphogenetic effects (incomplete flexure, disturbed development of the neural tube, the head, the heart and the tail bud), the compounds exhibited a wide range of embryotoxic potency. Estimation of the EC50 (median-concentration producing dysmorphogenesis in 50% of the embryos) for the six compounds revealed differences of more than two orders of magnitude: propranolol 25 μM, alprenolol 30 μM, atenolol 4000 μM. Measurements of the concentrations of the various drugs in the cultured embryos at corresponding EC50 levels showed differing values: metoprolol 4.5 μM, propranolol 5.2 μM, alprenolol 8.4 μM, pindolol 9.0 μM, acebutolol 12.5 μM and atenolol 77.0 μM. With regard to the EC50 and the degree of substance transfer to the embryo it can be stated that propranolol and metoprolol show a much higher intrinsic potency to interfere with normal in vitro embryonic development than, e.g. atenolol.

DNA modification induced by 6-mercaptopurine riboside in murine embryos.

The cytostatic drug 6-mercaptopurine riboside (6-MPr) was investigated in mice in order to test the hypothesis that the teratogenicity of this antimetabolite is paralleled by an incorporation into the DNA of the embryos during organogenesis. DNA modification in the embryos was analysed 4 h following s.c. administration of [35S]-labelled 6-MPr to the dams on day 11 of pregnancy. The DNA of the embryos was isolated and hydrolysed to the bases by formic acid. Following separation by cation-exchange HPLC 6-thioguanine was found in the hydrolysate. Quantitation was performed by liquid scintillation counting. Evaluations of 6 doses in the range of 8-25 mg/kg were performed. An incorporation rate of 6-thioguanine from 32-56 pmol per mumol guanine was found in the DNA of the embryos. These findings suggest that, similar to the previously studied alkylating agents, the teratogenicity of 6-MPr may be, at least in part, induced via DNA modification of the embryos.

Combined prenatal toxicity of 6‐mercaptopurine riboside and hydroxyurea in mice

Hydroxyurea (HU) and 6-mercaptopurine riboside (6-MPr) are used as cytostatic chemotherapeutics. Their teratogenic potential in experimental animals has been well known for several decades. Generally, it is assumed that the toxicity of both agents is due to an interference with enzymes of DNA synthesis. In the case of 6-MPr, it was speculated that the teratogenicity in rodents might be paralleled by or even correlated to an incorporation of 6-thioguanine into the DNA of the embryos. In this study, the interaction between these two compounds with regard to teratogenicity in NMRI mice was investigated. Dose-response data of 6-MPr (s.c.-treatment) were published earlier. First, a dose-response study with HU alone (i.p.-treatment) was performed. From these data, the doses for the combination study were derived: HU 250 mg/kg (NOAEL dose) and 6-MPr 16 mg/kg (strongly teratogenic dose). In all experimental groups the substances were administered to the dams once on day 11 of gestation. Combination effects were investigated applying various dosing regimens. In group I treatment was simultaneous, in group II HU was administered 2 h before 6-MPr, in group III 2 h after 6-MPr. The differences in the overall frequency of gross structural abnormalities were moderate. However, when analysing the effects in more detail (single abnormalities), group III exhibited great differences: 1) 6-MPr co-treatment increased the frequency of HU effects (skull defects) and 2) HU co-treatment decreased the frequency of 6-MPr effects (limb defects) when compared to the findings of the dose-response studies. In addition, the influence of HU on the 6-MPr-induced DNA modification was determined by measuring the incorporation of 6-thioguanine into the DNA of day-11 embryos. As expected, the HU co-treatment corresponding to the group III dosing regimen of the teratogenicity experiment decreased the incorporation rate by ca. 40%. This was in parallel to the decrease in the frequency of 6-MPr effects in the teratogenicity III group. This finding may be considered as a further indication that in the case of 6-MPr, DNA modification is accompanying teratogenicity.

Effects of Ofloxacin on the Ultrastructure of Achilles Tendon in Rats

Cases of tendinitis and tendon ruptures have been described after treatment with quinolones.[1,2] However, clinical and morphological information on quinolone-induced tendinopathy is scarce. In contrast to the well known effects of quinolones on joint cartilage, cases of tendinopathy occurred in adult rather than in juvenile patients. Therefore, we performed extensive electron microscopic studies with Achilles tendon specimens from ofloxacin-treated juvenile and adult rats.