DEVELOPMENTAL TOXICOLOGY Christian FoÈrster á Marcus RuÈcker á Mehdi Shakibaei Irmela Baumann-Wilschke á JuÈrgen Vormann Ralf Stahlmann Effects of ¯uoroquinolones and magnesium de®ciency in murine limb bud cultures Received: 7 October 1997 / Accepted: 25 February 1998 Abstract Quinolone-induced arthropathy is probably caused by a lack of functionally available magnesium in immature joint cartilage. We used an in vitro assay to study the eects of ¯uoroquinolones on cartilage for- mation in mouse limb buds from 12-day-old mouse embryos in regular and in magnesium-de®cient medium. Omission of magnesium from the medium had no ad- verse eect on the outcome of the culture: limb buds grew and dierentiated well in regular and in magne- sium-de®cient Bigger's medium. Lack of calcium, how- ever, severely impaired the development of the explants; this result was even more enhanced when both minerals (magnesium and calcium) were omitted. Electron mi- croscopy revealed cell necrosis and deposition of elec- tron-dense material in the vicinity of chondrocytes from limb buds after 6 days in a magnesium-free medium. A series of seven ¯uoroquinolones was tested at 30, 60, and 100 mg/l medium. At a concentration of 30 mg/l spar- ¯oxacin only had a slight eect on limb development. At concentrations of 60 and 100 mg/l spar¯oxacin, tema- ¯oxacin and cipro¯oxacin impaired limb development in vitro concentration-dependently. The eects were enhanced in a magnesium-de®cient medium (concen- tration of magnesium <10 lmol/l). Fleroxacin, lo- me¯oxacin and o¯oxacin impaired limb development only slightly; no signi®cant dierences were recognizable between the outcome in regular and in magnesium-de- ®cient medium. Pe¯oxacin did not show any eect on limb development in both media. Using electron mi- croscopy, very similar alterations as described above for the limbs cultured in magnesium-de®cient medium were observed with o¯oxacin at a concentration of 30 mg/l, which had no eect on the growth of the explants when evaluated macroscopically. The anity of six ¯uoro- quinolones to magnesium was determined by the use of a ¯uorescence assay. The anity to magnesium correlated with the activity of the drugs in the limb bud assay. We conclude that ¯uoroquinolones have no eect on murine limb development in vitro at concentrations that are achieved under therapeutic conditions (peak concentra- tions approx. 1±5 mg/l in plasma). Eects at higher concentrations (60 and 100 mg/l) are slightly enhanced (factor 2) if the magnesium concentration in the medium is low. Macroscopically, limbs develop regularly in a magnesium-free medium, but ultrastructurally typical alterations are exhibited (e.g. cell necrosis and pericel- lular deposition of electron-dense material). Key words Fluoroquinolones á Chondrotoxicity á Magnesium á Calcium á Limb bud culture Introduction Fluoroquinolones are contraindicated in paediatric pa- tients because all compounds tested to date induce joint cartilage lesions in juvenile animals such as dogs (Ing- ham et al. 1977; Tatsumi et al. 1978; Burkhardt et al. 1990), rats (Kato and Onodera 1988 a, b), non-human primates (Stahlmann et al. 1990) and others (for review see Gough et al. 1992; Stahlmann et al. 1993a). The chondrotoxic potential of the drugs is probably related to their ability to form stable chelate complexes with magnesium ions. We showed that by feeding a magne- sium-de®cient diet, joint cartilage lesions could be in- duced in juvenile rats which were indistinguishable by histology, electron microscopy and immunohistochem- istry from cartilage lesions observed after quinolone treatment. From these results it was concluded that the Arch Toxicol (1998) 72: 411±419 Ó Springer-Verlag 1998 C. FoÈrster á M. RuÈcker á I. Baumann-Wilschke á R. Stahlmann (&) Institut fuÈr Klinische Pharmakologie und Toxikologie, Freie UniversitaÈt Berlin, Garystrasse 5, D-14195 Berlin, Germany M. Shakibaei Institut fuÈr Anatomie, Freie UniversitaÈt Berlin, KoÈnigin-Luise-Strasse 15, D-14195 Berlin, Germany J. Vormann Institut fuÈr Molekularbiologie und Biochemie, Freie UniversitaÈt Berlin, Arnimallee 22, D-14195 Berlin, Germany