Hans-Joachim Merker

Ofloxacin in juvenile non-human primates and rats. Arthropathia and drug plasma concentrations

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 proteoglycanes 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. Since we found corresponding cartilage alterations in marmosets and rats, this species provides a convenient model for additional studies on chondrotoxic effects of quinolones.

INTEGRIN EXPRESSION AND COLLAGEN TYPE II IMPLICATED IN MAINTENANCE OF CHONDROCYTE SHAPE IN MONOLAYER CULTURE: AN IMMUNOMORPHOLOGICAL STUDY

Chondrocytes grown in monolayer culture at low density, with serum added, either dedifferentiate after several days whereby their cell shape changes or they are overgrown by fibroblast‐like cells. The aim of this study was to optimize the cultivation of chondrocytes in monolayer culture and to slow down their transformation or their overgrowth by fibroblast‐like cells. For this purpose freshly isolated chondrocytes of cartilage anlagen from 17‐day‐old mouse embryos were grown on plastic or collagen type II‐coated substrates. With this model: (a) chondrocytes grown on plastic substrates had almost completely changed to fibroblast‐like cells after 5 days in culture. (b) When grown on collagen type II, the chondrocytes maintained their round phenotype for more than 2 weeks in culture. (c) Immunomorphological investigations showed that chondrocytes produce collagen type II and fibronectin and express specific surface receptors (integrins of the β1‐group) on the membrane from day 1 until the end of the culture period when grown on collagen type II. (d) Treatment with β1‐integrin antibodies clearly reduces chondrocyte adhesion on collagen type II by about 70%. Hence, these data indicate that the most probable influence of collagen type II on cellular behaviour depends on the integrins participating in a chondrocyte—collagen type II interaction, and this model represents a pure chondrocyte culture which allows cell growth for an extended period.

Magnesium deficiency induces joint cartilage lesions in juvenile rats which are identical to quinolone-induced arthropathy.

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.

Reduction in left ventricular messenger RNA for transforming growth factor beta(1) attenuates left ventricular fibrosis and improves survival without lowering blood pressure in the hypertensive TGR(mRen2)27 rat

Angiotensin II recruits transforming growth factor &bgr;1 (TGF&bgr;1) and is related to left ventricular fibrosis. However, it is unclear whether chronic in vivo reduction in left ventricular TGF&bgr;1 expression blunts fibrosis and improves outcome in angiotensin II–dependent hypertension. Four-week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg · kg−1 · d−1), or tranilast (a nonspecific TGF&bgr; inhibitor; 400 mg · kg−1 · d−1) (n=10 for each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1±0.16 versus 2.1± 0.06 mg/g body wt;P <0.05) was significantly (P <0.05) blunted by both tranilast (2.7±0.05) and losartan (2.7±0.07). Both drugs prevented the increase in left ventricular TGF&bgr;1 mRNA and fibronectin mRNA and blunted the increase in hydroxyproline content and the increase in perivascular fibrosis. The perivascular fibrosis score correlated significantly with the level of expression of TGF&bgr;1 (r =0.62;P =0.019). In situ hybridization demonstrated increases in TGF&bgr;1 mRNA, predominantly in perivascular and nonmyocyte areas. Both drugs did not prevent the decrease in systolic or diastolic dP/dt, but tranilast significantly improved the survival of untreated Ren2 rats (P =0.029). In conclusion, TGF&bgr;1 mRNA expression is increased predominantly in nonmyocyte regions in the hypertrophied left ventricle in this angiotensin II–dependent model of hypertension. This increase is probably due to high angiotensin II levels rather than to hypertension. This is the first study to suggest that chronic inhibition of TGF&bgr;1 expression attenuates left ventricular hypertrophy and fibrosis, even without lowering blood pressure.

Evaluation of the genetic and embryotoxic effects of bis(tri-n-butyltin―oxide (TBTO), a broad-spectrum pesticide, in multiple in vivo and in vitro short-term tests

The genetic and embryotoxic effects of bis(tri-n-butyltin)oxide (TBTO) were evaluated in multiple in vivo and in vitro short-term tests preparatory to its potential wide use as a molluscicide in control of schistosomiasis. When tested in the rec assay in Bacillus subtilis, TBTO was not mutagenic and it did not induce reverse mutations in Klebsiella pneumoniae. Neither in the presence nor in the absecne of rat liver activation system did TBTO produce point mutations in Salmonella typhimurium strains TA1530, TA1535, TA1538, TA97, TA98 or TA100. TBTO was matagenic in strain TA100 in a fluctuation test, but only in the presence of rat liver S9 (Aroclor-induced). TBTO did not induce gene mutations in the yeast Schizosaccharomyces pombe, mitotic gene conversions in the yeast Saccharomyces cerevisiae, nor sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of rat or mouse liver S9. In the latter cells, structural chromosomal aberrations, endoreduplicated and polyploid cells were induced. TBTO did not induce gene mutations in V79 Chinese hamster cells (to 8-azaguanine-, ouabain- or 6-thioguanine-resistance) in the presence of a rat liver postmitochondrial fraction or in cell (hamster embryo cells and human and mouse epidermal keratinocyte)-mediated assays. In mouse lymphoma cells, TBTO did not induce 6-thioguanine- or BUdR-resistant mutations. As many tumour promoters inhibit metabolic cooperation between V79 Chinese hamster 6-thioguanine-resistant/-sensitive cells, TBTO was tested but showed no such activity. TBTO was examined for the induction of recessive lethal mutations in adult Berlin K male Drosophila melanogaster, either by feeding or by injection. Doses of 0.37 or 0.74 mM did not increase the number of X-linked recessive lethal mutations. An increased number of micronuclei was observed in the polychromatic erythrocytes of male BALB/c mice 48 h after a single oral dose of TBTO (60 mg/kg bw), while a lower dose (30 mg/kg bw) was ineffective. Neither of the two doses had induced micronuclei 30 h after treatment. The reproductive toxicity of TBTO was studied in NMRI mice. In a 10-day toxicity study, the LD50 and LD10 were 74 and 34 mg/kg bw, respectively. An increased frequency of cleft palates was seen in the fetuses of mice (compared with controls, 0.7%) treated orally during pregnancy with 11.7 mg/kg TBTO (7%), 23.4 mg/kg (24%) or 35 mg/kg (48%).(ABSTRACT TRUNCATED AT 400 WORDS)

Reproductive toxicity and toxicokinetics of lindane in the male offspring of rats exposed during lactation.

1 Reproductive toxicity and toxicokinetics of lindane during lactation were studied. For the reproductive toxicity study the dams were treated with a single dose of 6 mg/kg on day 9 or 14 of lactation, or with 1 mg/kg on days 9 to 14 of lactation. The male offspring were investigated at puberty and adulthood. For the toxicokinetic study, two groups of dams were treated with 6 mg/kg on day 9 or 14 of lactation. The concentration of lindane was measured in maternal plasma and milk, as well as in male offspring organs. 2 At adulthood, testicular weight and the number of sperm and spermatids were significantly reduced in all treated groups. 3 The testosterone level of the groups treated with 6 mg/ kg was significantly reduced to approximately 50% at puberty as well as in adulthood. In the group treated with 1 mg/kg, the testosterone level was in both age periods reduced, however, only at puberty was the reduction statistically significant. 4 The concentration oflindane in the testis was similar to that found in brain and was half the concentration found in the liver. 5 Histologically some areas of the testis exhibited distinct alterations ranging from small changes to a pronounced effect. 6 Exposure to lindane during lactation induces repro ductive hazards to male offspring rats which are detectable at adulthood.

Reproductive toxicity and tissue concentrations of lindane in adult male rats

1 The effects of lindane on the reproductive system and its concentration in different tissues were investigated. Groups were dosed orally with 6 mg lindane/kg for 5 days or with a single dose of 30 mg/kg body weight. 2 The results indicate that the number (mio) of sperma tids counted in the testes of both treated groups 2 weeks after treatment were significantly reduced compared to the control rats. Effects on the number of sperms were observed in both treated groups, but significant differences were only revealed on those with 30 mg/kg. 3 The concentration (μg/g tissue) of lindane in adipose tissue, liver, brain and testis was determined 24 h and 2 weeks after the last treatment. Lindane was detected in the testis of both groups 24 h and 2 weeks after the last treatment. 4 Histological investigation by electron microscopy revealed a pronounced ballooning of Sertoli cells accompanied by fragmentation or complete loss of organelles. 5 According to the result obtained, it is evident that lindane passes through the testis and exerts its toxic effect.

Nude Mice Are Not Hairless

In the present study, the morphological aspect of the skin and the hairs of athymic, macroscopically nude mice (NMRI, nu/nu) was investigated by descriptive light- and electron-microscopical methods a

Ganciclovir induces reproductive hazards in male rats after short-term exposure

1 The effect of short-term treatment of ganciclovir on male reproduction in adult rats was studied. The animals were treated subcutaneously with either a single dose of 60 mg/kg daily for 5 days (Gan5day) or with 100 mg/kg administered three times at 4 h- intervals (Gan1day). The effects were investigated every 2 weeks up to 8 weeks, followed by investigations 16 and 24 weeks after treatment to detect the potential of recovery. 2 Time to mating was significantly increased in Gan1day group. The pregnancy index and outcome were only decreased 8 weeks (Gan5day and Gan1day) or 16 weeks (Gan1day) after treatment. 3 The lowest values of sperm variables studied were registered 8 weeks after treatment: The number of spermatid was reduced up to 4% (Gan5day) or 2% (Gan1day) of control; the sperm number was 5% and 8% of control in Gan5day and Gan1day, respectively. Over 80% of sperm were abnormal in Gan5day group, and only few normal sperm was detected in Gan1day group. 4 Morphological investigation of testes revealed a clear- cut time-dependency effect. Four weeks after treat ment distinct alterations were located exclusively in the peripheral part of the tubuli which included fat inclusions, cell and pyknotic nuclear debris and swellings of Sertoli cells. The effect was reversible 24 weeks after treatment. 5 Ganciclovir induces testicular damage and affects sperm variables after short-term exposure. The in tensity and degree of the hazards varied in between the time of investigation after treatment.

Integrins on joint cartilage chondrocytes and alterations by ofloxacin or magnesium deficiency in immature rats

Abstract Recently, we showed that magnesium deficiency induces lesions in knee joint cartilage from 5-week-old rats that are very similar to ofloxacin-induced cartilage defects. We concluded that quinolone-induced arthropathy is probably due to chelation of magnesium and thus a deficit in functionally available magnesium in joint cartilage (Stahlmann et al. 1995). As magnesium deficiency in joint cartilage could impair chondrocyte-matrix interaction which is mediated by cation-dependent integrin receptors of the β1-subfamily, we investigated integrin expression in joint cartilage from untreated, ofloxacin-treated and magnesium-deficient Wistar rats. With immunohistochemical methods using monoclonal and polyclonal antibodies, we showed that the integrin pattern in joint cartilage from rats corresponded largely to integrin expression described for human cartilage tissue: β1, α1, α3 and αν subunits and the α5β1 and ανβ3 heterodimers were consistently expressed. Joint cartilage lesions were detected in ofloxacin-treated and magnesium-deficient rats. Lesions were more pronounced in the quinolone-treated group. Expression of several integrins was reduced in the vicinity of lesions after oral treatment with 2 × 600 mg ofloxacin/kg for 1 day. Gross-structural lesions (e. g., cleft formation, unmasked collagen fibres) in magnesium-deficient rats were very similar but changes in integrin expression were less pronounced. On the other hand, changes in cartilage matrix composition showed similar alterations in ofloxacin-treated and magnesium-deficient rats: fibronectin deposition in the cartilage matrix increased in both groups while glycosaminoglycan content decreased. In summary, similar defects occur in ofloxacin-treated and magnesium-deficient rats and with immunohistochemical methods subtle differences are demonstrable.