Intranuclear concentration measurements of doxorubicin in living leucocytes from patients treated for a lympho-proliferative disorder.

The accumulation of doxorubicin (DOX) in white blood cells of treated patients has been studied by quantitative microspectrofluorometry. From blood samples of treated patients, leucocyte subpopulations were separated by the gradient method. Emission fluorescence spectra from a microvolume of a single living cell nucleus were analysed in terms of spectral shape and fluorescence yield between free and DNA-bound doxorubicin. With this non-destructive analysis technique, intranuclear doxorubicin concentrations were determined within +/- 10%. Doxorubicin concentrations were measured in patients treated with bolus injection. After an accumulation of DOX in leucocytes during the first 30 min, intranuclear doxorubicin concentration did not vary significantly for 24 h, whereas its concentration in plasma decreased. Despite large differences between patients, monocytes accumulated significantly more doxorubicin than granulocytes or lymphocytes did.

Concomitant decrease of resistance and modifications of the cytoskeleton after all-trans retinoic acid and phorbol ester treatments in a navelbine-resistant bladder carcinoma cell line.

The bladder carcinoma cell line J82-NVB was selected for resistance to the new vinca alkaloid Navelbine. These cells possessed a non-MDR phenotype and were cross-resistant to vinca alkaloids and taxoids. Some morphological differences between sensitive (J82) and resistant (J82-NVB) cells were observed J82 cells had a heterogeneous population morphology with both epithelial and spindle shaped cells, while J82-NVB cells were almost all of the epithelial type. Vimentin intermediate filaments were less organized in J82-NVB than in J82 cells. Moreover, desmosomes were present in the membranes of J82NVB cells but not in J82 cells. These findings suggest that J82 cells are poorly differentiated epithelial cells while J82-NVB cells possess some characteristics of a more differentiated epithelial cell line. After a two-week treatment with all-trans retinoic acid, all the cells became spindle shaped, vimentin filaments reappeared in the cytoplasm of J82-NVB cells and desmosomes disappeared from the membranes of these cells. These changes were accompanied by a decrease from 17 to 4.6 of the resistance factor of J82-NVB cells to Navelbine. This decrease in resistance was concomitant with modifications of microtubules assembly regulation mechanisms. After Navelbine treatment, microtubule reassembly occurred in resistant but not in sensitive nor in retinoic acid treated cells. Okadaic acid, a protein phosphatase inhibitor, inhibited microtubule reassembly in resistant cells, and 2-aminopurine, a protein kinase inhibitor, induced microtubule reassembly in sensitive cells after Navelbine treatment. These findings show that microtubule reassembly after depolymerization is regulated by the kinase/phosphatase systems. A treatment with phorbol myristate acetate (PMA), a protein kinase C (PKC) agonist, induced the same morphological modifications and resistance decrease as retinoic acid treatment. A specific PKC inhibitor (Bisindolymaleimide) prevented these PMA-induced morphological modifications and resistance decrease in J82-NVB cells, showing that these effects were mediated by PKC. This study suggests that, in part by acting on some properties of the cytoskeleton, the differentiation modulator, retinoic acid, and the signal transduction modulator, phorbol myristate acetate, can decrease the resistance of J82-NVB cells to microtubule poisons.

[Characterization of the mechanism of cross-resistance to vinca alkaloids and taxoids in the human J82 bladder tumor cell line]. / Caractérisation du mécanisme de résistance croisée entre vincaalcaloïdes et taxoïdes dans la lignée de carcinome de vessie humain J82.

A phenotype of resistance to the new vinca alkaloid Navelbine was induced in the J82 human bladder carcinoma cells. The resistance factor of the resistant cell line (J82-NVB) to Navelbine was 17. The resistance phenotype of these cells is not a multidrug-resistance (MDR) phenotype. J82-NVB cells lack overexpression of P-glycoprotein and cross-resistance to MDR drugs like doxorubicin, epipodophyllotoxins or colchicine. Navelbine efflux was similar in sensitive and resistant cells, and resistance could not be explained by a difference of drug accumulation in these two cell lines. The cells were cross-resistant to vinca alkaloids and taxoids whose targets are microtubules. Immunofluorescence study of microtubules showed that depolymerization occured for the same Navelbine concentration in sensitive and resistant cells. This concentration induced growth inhibition in sensitive but not in resistant cells. Moreover, depolymerization induced by Navelbine treatment was reversible, after drug removal, in resistant cells only. This study suggests that J82-NVB cell resistance mechanism involves alterations of microtubule dynamics, allowing recovery of microtubules functions after treatment.

Clinoptilolite as an ammonia binder in pig slurry.

Six gilts and six barrows are used in a 2 x 2 experiment with two phase feeding versus multiphase feeding and celite addition versus clinoptilolite addition. Nitrogen and ammonia concentration in the slurry on dry matter basis was reduced by 30% and 35% respectively by multiphase feeding, together with a decreased ratio of ammonia over nitrogen. Ammonia fixation in pig slurry by clinoptilolite increased from 21% after 14 days of supplementation upto 35% at 8 weeks after the supplementation period.

Determination of vinorelbine (Navelbine) in tumour cells by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the determination within tumour cells of a new vinca alkaloid, vinorelbine. Extractions of vinorelbine from cells were carried out using absolute ethanol. The extracts were injected into a reversed-phase system consisting of two Novapak C18 columns connected in series. The mobile phase was acetonitrile-phosphate buffer, pH 2.7 (60:40, v/v). Using a fluorescence detection, the limit of determination was 8 pmol injected. This method would be suitable for studying the cellular pharmacokinetics of vinorelbine in patients.

L-carnitine supplementation in breeding pigeons: impact on zootechnical performance and carnitine metabolism.

In the first experiment (Exp1), three consecutive breeding rounds were performed by two groups of six pigeon couples in order to study the impact of L-carnitine supplementation (80 mg x d(-1)) of parent pigeons on zootechnical performance. Both in the second and third experiments (Exp2, Exp3), one breeding round was performed by two groups of six pigeon couples to reveal the biochemical background of the increase in squab growth, the limitation of body weight decrease in male parent birds and the tendency for an improved cumulative feed efficiency due to L-carnitine supplementation in Exp1. Growth improvement of the squabs with L-carnitine was only seen when the parent pigeons were supplemented, together with a marked rise in the body weight of the parent birds around hatching. Based on the results of the crop milk analysis, growth improvement was probably due to a quantitative impact on crop milk production. The crop milk from the supplemented groups in both Exp2 and Exp3 had increased levels of carnitine. Carnitine, gamma-butyrobetaine and acetylcarnitine were increased in plasma samples of the supplemented parent pigeons. No differences were present in the squabs' plasma for these parameters. In the squabs of Exp3, no changes were seen in the proportional growth or the protein content of the heart, breast muscle and liver, but the breast muscle of the squabs from the supplemented group in Exp3 showed a considerable rise in carnitine and a marked decrease in gamma-butyrobetaine.

Characterisation of a navelbine-resistant bladder carcinoma cell line cross-resistant to taxoids.

A bladder carcinoma cell line (J82) was selected for resistance to the new vinca alkaloid navelbine. The resistance factor of the resistant subline (J82-NVB) to navelbine was 17. P-glycoprotein was not detected in the membrane of J82-NVB cells. The lack of cross-resistance to multidrug-resistant (MDR) drugs such as doxorubicin, epipodophyllotoxins and colchicine, the absence of increase in navelbine efflux and the fact that a reduced accumulation of the drug cannot account for the resistance level confirmed that the phenotype of resistance of J82-NVB cells is not a classical MDR phenotype. Moreover, verapamil did not reverse the resistance of J82-NVB cells. The cells were cross-resistant to vinca alkaloids and taxoids which share the same target protein: tubulin. Analysis of microtubules using immunofluorescence showed that disassembly of the microtubular network occurred for the same concentration of navelbine in sensitive and resistant cells. However, after treatment with a concentration of navelbine inducing depolymerisation in both sensitive and resistant cells, reassembly of the microtubular network was observed only in resistant cells. This study suggests that the mechanism of resistance of J82-NVB cells involves recovery from the inhibition of microtubule dynamics induced by drug treatment.