Interleukin-6 and tumor necrosis factor alpha levels after bisphosphonates treatment in vitro and in patients with malignancy.

Bisphosphonates are potent inhibitors of bone resorption and are widely used in the treatment of bone diseases. One of the side effects of administered aminobisphosphonates is transient fever and some biological changes that are suggestive of an acute phase response. Pamidronate [(3-amino-1-hydroxypropylidene).1, 1-bisphosphonate] and ibandronate [1-hydroxy-3-(methylpentylamino) propylidenebisphosphonate] incubated in heparinized whole blood at doses of 10(-4) and 10(-5) mol/L, induced the production of tumor necrosis factor alpha (TNFalpha). Moreover, pamidronate was found to slightly stimulate interleukin-6 IL-6 production. In contrast, clodronate (dichloromethylenebisphosphonate) did not increase IL-6 or TNFalpha. To investigate these phenomena in vivo, acute phase reaction was assessed in patients with malignant disease treated with 60 mg of pamidronate (n = 29), 1500 mg of clodronate (n = 8), or 0.5-2 mg of ibandronate (n = 6), all given intravenously. A significant decrease in lymphocyte and leukocyte count was observed in the pamidronate group. In the same group, seven patients (24%) showed a transient increase of body temperature above 37 degrees C with an increase > or = 0.5 degrees C at 24 h. These changes were not found in the patients treated with clodronate or ibandronate. Plasma IL-6 and TNFalpha levels increased significantly after pamidronate treatment, whereas no change was seen after clodronate infusion. The peak of IL-6 level (53.7 +/- 14.1 [SEM] pg/mL) was observed at 24 h, and that of TNFalpha level (26.9 +/- 3.4 pg/mL) at 48 h after the beginning of pamidronate administration (values before treatment, respectively: 28.6 +/- 7.1 pg/mL, p < 0.006; and 13.1 +/- 1.5 pg/mL, p = 0.0001). The peak of C-reactive protein (CRP) level was found at 48 h (41.0 +/- 7.8 vs. 25.5 +/- 5.6 mg/L before treatment, p < 0.01) and CRP levels were strongly correlated with IL-6 levels (p = 0.65,p < 0.001). Only one patient treated with ibandronate showed an increase in IL-6 and CRP levels. Patients treated with pamidronate, whose body temperatures were increased at 24 h, had a greater increases of circulating IL-6, TNFalpha, and CRP at 24 h and 48 h than patients without temperature increase. These results suggest that pamidronate treatment, but not clodronate and possibly not ibandronate at the doses used, induced an increase in the plasma levels of IL-6 and TNFalpha, which may be responsible for the acute phase reaction observed clinically.

Tributyltin biomonitoring using prosobranchs as sentinel organisms.

Tributyltin (TBT) compounds, some of the most toxic xenobiotics, produce a variety of pathological reactions in animals. A reliable biomonitoring method to assess the degree of environmental TBT pollution has been described based on investigations of virilization phenomena in prosobranch snails (Mollusca: Gastropoda). Examples are the imposex phenomenon in marine and freshwater species, the intersex reaction in littorinids and the reduction of female sexual glands and offspring numbers in further species resulting mainly in a sterilization of females. The degree of imposex or intersex in populations is determined by different biomonitoring indices which allow to assess the TBT pollution of the environment at low costs with high precision. The effectiveness of TBT legislations is analysed by extensive surveys in France and Ireland indicating that there is still a continuing threat to sensitive marine organisms. TBT disturbs the biosynthesis of steroid hormones on the level of estrogen biosynthesis. The observed virilization phenomena seem due to an inhibition of the cytochrome P-450 dependent aromatase by this organotin compound.

The use of Littorina littorea for tributyltin (TBT) effect monitoring - Results from the German TBT survey 1994/1995 and laboratory experiments.

Field investigations and laboratory experiments were performed to test the applicability of the intersex phenomenon in Littorina littorea for biological TBT-effect monitoring and to give further insights into toxicological aspects of intersex development. The intensity of the phenomenon is correlated with the degree of TBT contamination in the environment. Statistically highly significant correlations of biological indices and TBT concentrations in tissue and sediment exist. Intersex can be induced only in juvenile and sexually immature females and its intensity depends on the ontogenetic stage of development during TBT exposure. For the first time, malformations in male periwinkles closely related to the TBT contamination can be described; the reduction of male mamilliform penial glands shows highly significant correlations to TBT concentrations in tissues. The intersex phenomenon in Littorina littorea is proposed for a biological TBT-effect monitoring in those areas where the dogwhelk Nucella lapillus, as the more sensitive species in European surveys, is absent. The intersex index (ISI) as the mean value of the intersex stages in a sample should be used as the most sensitive biological parameter for the assessment of the TBT contamination in Littorina littorea.

Cell differentiation during early development of Nassarius reticulatus L. (Gastropoda Prosobranchia). I. Zygote to 16-cell stage.

The ultrastructure of the zygote and of early segmentation stages, up to the 16-cell stage, was investigated in normal Nassarius reticulatus from Roscoff (France). This study deals predominantly with structures that remain morphologically unchanged throughout this period, such as yolk granules, lipid droplets, and multimembranous vesicles. These organelles do not change in position, fine structure, or quantity from the egg to the 16-cell stage. The cortex and the vitelline layer also remain almost unchanged until the 16-cell stage. Cortical granules could not be observed and thus the thin vitelline layer is not transformed to a thick fertilization membrane. This phenomenon seems to be related to the facts that the eggs are protected by a tough capsule and that the cells of the embryo have to incorporate extraembryonic nutrient substances. From the 2-cell stage until the 16-cell stage micropinocytotic vesicles 500-2000 A in diameter are associated with the plasmalemma. Perhaps they help to incorporate the extraembryonic nutrient reserves. Desmosomes first appear at the 4-cell stage and are common at the 8-cell stage, when a small transitory blastocoel appears. Normally the cell borders are in close proximity during interphase. Syncytial connections between the blastomeres were not observed. Beginning at the trefoil stage the mitochondria increase in number and many apparent division stages are observed. The increase in mitochondria occurs in the perinuclear region throughout the embryo and does not result in a polar lobe especially rich or poor in mitochondria. From the 4-cell stage onwards, the number of ribosomes increases differentially in different blastomers.

Cell differentiation during early development of Nassarius reticulatus L. (Gastropoda, Prosobranchia). II. Morphological changes of nuclei and nucleoi.

In Nassarius reticulatus the nuclei and nucleoli undergo important morphological changes from the zygote to the 16-cell stage. In the zygote and in the trefoil (2-cell) and 4-cell stage, several agranular, fibrillar nucleolus-like bodies 1 mum diameter are present in the interphase nucleus. Granular nucleoli first appear at the 8-cell stage. These nucleoli have fibrillar and granular regions. The granular regions are made up predominantly of ribosome-liek osmiophilic granules. From the 16- and 32-cell stage onwards, the one or two spherical nucleoli of each nucleus measure 2.5 mum in diameter and show a concentric organization with a very dense central region surrounded by a broad peripheral zone containing numerous granules, possibly of ribonucleoprotein. At the same time the number of ribosome-like particles increase in the karyoplasm and that of ribosomes in the cytoplasm. These findings are surprising because in eggs with radial cleavage, which have been subjected to more detailed analysis, the first granular nucleoli appear after the end of the cleavage, at the blastula or gastrula stage. The early appearance of granular nucleoli which seem to be characteristic of several eggs with spiral cleavage is discussed in connection with biochemical data on RNA-synthesis.

Marisa cornuarietis (Gastropoda, prosobranchia): a potential TBT bioindicator for freshwater environments.

: The ramshorn snail Marisa cornuarietis (L., 1767) exhibits imposex (occurrence of male parts in addition to the female genital duct) under the influence of TBT (tributyltin) in laboratory experiments and accumulates this biocide in a time-and concentration-dependent manner. A comparison of BCF (bioconcentration factors) demonstrates that this limnic species accumulates more TBT than marine species. Evidence is given that TBT causes hormonal disorders which are responsible for imposex development. The testosterone/oestradiol quotient reflects the imposex development which can be described by a classification scheme differentiating four stages in Marisa cornuarietis. In the background of the constantly increasing organotin concentration in the limnic environment Marisa cornuarietis could be a well-suited bioindicator for TBT pollution. No other limnic species with a lower threshold concentration for measurable TBT effects is reported within the literature.

Characterization of the enzyme involved in the processing of big endothelin-1 in human lung epithelial cells.

Biosynthesis of active endothelin-1 (ET-1) implies an enzymatic processing of the inactive precursor Big ET-1 (1-39) into the mature, 21 amino acid peptide. The aim of this study was to characterize in airway and alveolar epithelial cells the enzymes responsible for this activation. BEAS-2B and A549 cells, which both produce ET-1, were studied in vitro as models for bronchiolar and alveolar cells, respectively. Both cell lines were able to convert exogenously added Big ET-1 (0.1 microM) into ET-1, suggesting a cell surface or an extracellular processing. The conversion was inhibited by phosphoramidon in both cell lines with an IC50 approximately 1 microM, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP). The endogenous production of serum-stimulated BEAS-2B and A549 cells was not inhibited by thiorphan, and phosphoramidon showed inhibition only at high concentration (>100 microM). Western blotting following electrophoresis in reducing conditions demonstrated a protein of MR 110 corresponding to the ECE-1 monomer in both BEAS-2B and A549 cells, as well as in whole lung extracts. By RT-PCR we revealed the mRNA encoding for the ECE-1b and/or -1c subtype, but not ECE-1a, in both cell lines. We conclude that BEAS-2B and A549 cells are able to process either endogenous or exogenous Big ET-1 by ECE-1 and that isoforms 1b and 1c could be involved in this processing with no significant role of NEP.

Function of human alveolar macrophages after a 3-day course of azithromycin in healthy volunteers.

Azithromycin (AZM) is a new macrolide antibiotic with a high intracellular/extracellular concentration ratio. Immunomodulatory and antiinflammatory properties have been reported with other macrolides, especially erythromycin. The aim of the present study was to evaluate the effect of AZM on the production of proinflammatory mediators by alveolar macrophages (AM) up to 4 weeks after a 3-day course of AZM (500 mg, once a day). Nineteen non-smoking healthy male subjects were investigated with bronchoscopy and bronchoalveolar lavage. Group 1 received no treatment. Groups 2, 3, and 4 were bronchoscoped 1, 7 and 30 days, respectively, after AZM administration. AZM concentrations were simultaneously measured in plasma and in AM extracts. In serum, AZM levels were higher in group 2 (32.8 +/- 14.2 micrograms/l), at the lower limit of detection in group 3 (2.8 +/- 1.7 micrograms/l), and no longer detectable in group 4. In AM extracts, the highest concentrations were measured in group 2 (51.6 +/- 28.3 ng/microliter) and in group 3 (31.8 +/- 17.2 ng/microliter), and were detected up to 30 days after treatment in group 4 (2.9 +/- 2.3 ng/microliter). There was no significant differences between groups for blood or BAL proinflammatory cytokines levels (TNF-alpha, IL-1 beta, IL-6), and for superoxide generation by AM. We conclude that a 3-day course of AZM 500 mg/day in healthy subjects does not alter the proinflammatory cytokine profile in blood and in AM despite the prolonged tissue impregnation by this drug.