Characterization of multidrug transporters in a normal renal tubular cell line resistant to doxorubicin. Multidrug transporters in the LLC-PK(1) cell line and its resistant counterpart.

LLC-PK(1) is a proximal tubular cell line derived from normal pig kidney which has a structure and function similar to those of renal proximal tubular cells and which expresses baseline levels of P-glycoprotein. We isolated by drug selection a doxorubicin-resistant cell line (LLC-PK(1)/ADR) that exhibited a multidrug-resistant phenotype; this cell line was characterized by reduced intracellular drug concentrations, an increased drug extrusion, and increased expression of a 170-kDa P-glycoprotein detected by Western blot analysis with monoclonal antibody C219. In addition, an increased expression of MDR1 mRNA was seen by reverse transcriptase-polymerase chain reaction. These results suggest that it is possible to induce the overexpression of P-glycoprotein by chronic treatment with doxorubicin in a normal cell line that physiologically expresses low levels of this protein. This multi-resistant cell line could provide an interesting model for studying the role of P-glycoprotein and the consequence of its induction in a normal tissue.

Effect of low temperature on mast cell exocytosis. Early cytoplasmic vesicle formation and the role of cytoskeleton.

Mast cells stimulated with adriamycin at 4 degrees C underwent a unique exocytotic reaction. Rat peritoneal cells including mast cells were stimulated in vitro with adriamycin (100 micrograms/ml) for 0, 10, 30 or 60 sec and observed by transmission electron microscopy. Early changes could be observed after 10 sec stimulation and consisted in an approximately 5-fold increase (p < 0.001) of 0.05-0.2 micron diameter cytoplasmic vesicles. The Golgi apparatus showed signs of activation and vacuolization. From 10 to 30 sec, cytoplasmic vesicles fused with the perigranular membranes and with the membranes of developing secretory channels. At 60 sec, the number of vesicles and vacuoles diminished to nearly two-fold starting levels. The exocytotic reaction characteristically resulted in the formation of enormously dilated granular cavities. The secretory process appeared incomplete; after 60 sec, in fact, maximal histamine release was 20% and exocytosis could be found in approximately 30% of mast cells. Pre-incubation with vinblastine followed by adriamycin stimulation at 37 degrees C determined a dose-dependent inhibition of histamine release which was accompanied by the ultrastructural appearance of numerous 0.05-0.5 micron cytoplasmic vesicles and by signs of inhibited exocytosis. Our results support the concept that hyperstability of the cortical cytoskeleton coupled with microtubule perturbation would be responsible for the depressed pattern of mast cell exocytosis observed at 4 degrees C. Although stimulation at 4 degrees C induces a paradoxal secretory process, we believe that this approach may represent a useful model for understanding some basic mechanisms of exocytosis in mast cells.

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation.

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studies showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators.

Effect of paclitaxel and Cremophor EL on mast cell histamine secretion and their interaction with adriamycin.

The effect of paclitaxel and of its solvent Cremophor EL, a polyoxyethylated castor oil on histamine release in rat peritoneal mast cells has been tested. Paclitaxel dissolved in Cremophor EL/ethanol and Cremophor EL alone induced a moderate histamine release; this secretory activity is provoked by the solvent Cremophor EL but is so scanty that it seems most unlikely to be responsible of the severe hypersensitivity reactions frequently caused by this drug. Since in a number of protocols paclitaxel is used in combination with anthracyclines, and since anthracyclines are well known histamine releasing agents, the effect of the combination of these substances was also studied. Cremophor EL did not increase the exocytotic activity of adriamycin; on the contrary, the release of histamine induced by this anthracycline was significantly limited. This is an interesting observation since it has been suggested that anthracycline-induced histamine release is involved in the pathogenesis of the cardiotoxicity caused by these drugs. Low concentrations of paclitaxel in Cremophor EL and Cremophor EL alone inhibited adriamycin uptake into the granules of mast cells, a process mediated by an active transport system, recently identified with the P-170 glycoprotein pump.

Effect of the carboxylic ionophore monensin on histamine release from rat peritoneal mast cells.

The effect of the monovalent carboxylic ionophore monensin, which mediates a one-for-one exchange of intracellular H+ for extracellular Na+, was investigated in purified rat peritoneal mast cells. Monensin inhibited histamine secretion induced by compound 48/80, adriamycin and the calcium ionophore A23187; the inhibitory effect was maximal when the compound was added at least 10 min before the secretagogues. Washing of cells before addition of the secretagogues did not abolish the inhibitory effect of monensin. On the contrary the carboxylic ionophore was completely ineffective in preventing concanavalin A-induced histamine release. When rat peritoneal mast cells were incubated in the presence of monensin for longer period (up to 5 hours), the substance induced a slow, progressive and dose dependent histamine release, which, at least for lower doses was noncytotoxic. The secretory effect of monensin was still present if the ionophore was washed away after 10 min of incubation, and the incubation continued in drug-free medium. Monensin stimulated histamine secretion was strictly dependent on extracellular Na+ concentrations, and independent on extracellular Ca++.

Evidence for the lack of involvement of free radicals in adriamycin-induced histamine release from rat peritoneal cells.

The antineoplastic drug adriamycin, at a concentration of 100 micrograms/ml, caused a significant histamine release from rat peritoneal mast cells. This exocytotic process was unaffected by pretreatment with various concentrations of catalase, superoxide-dismutase, D-mannitol, alpha-tocopherol and reduced glutathione. Only very high concentrations of n-acetylcysteine significantly limited this release; this substance was also active in limiting histamine secretion induced by a classic mast cell secretagogue, compound 48/80.

Characterization of histamine secretion induced by anthracyclines in rat peritoneal mast cells.

The histamine-releasing activity of three anthracyclines, adriamycin, daunomycin and epirubicin, has been tested on rat peritoneal mast cells. The three drugs induced a marked and dose-dependent histamine secretion, in a noncytotoxic manner. The release was not sustained by extracellular calcium but was largely dependent on intracellular stores of this cation. This function was blocked by extremes of temperature (0 and 45 degrees), was very rapid and virtually complete within 10 sec. Treatment of mast cells with theophylline or disodium cromoglycate significantly reduced the secretory response to anthracyclines. On the basis of these results it is clear that the stimulant effect of anthracyclines is a true exocytotic response and thus is very similar to that of the classic mast cell secretagogue, compound 48/80.

Anticonvulsant activity of two polyethylene glycols in mice.

The anticonvulsant activity of two polyethylene glycols of different molecular weight (400 and 4000) was tested in mice. The two substances were inactive in the maximal electroshock test, but, when intraperitoneally administered, were efficacious in the pentetrazole and strychnine seizure tests. Polyethylene glycol 4000 showed the most potent anticonvulsant action. On the contrary, when polyethylene glycols were orally administered, they did not alter the time for seizure onset and for death.

Impairment of motor coordination induced by doxorubicin in mice.

The neurotoxic effects of a single intraperitoneal or intravenous injection of doxorubicin were evaluated in CD1 mice by means of the rotarod test. The test was performed daily for nine weeks after treatment. For both routes of administration, animals were treated with various doxorubicin dosages ranging from 18 to 5.9 mg/kg. The three higher i.v. doses (18, 14.4 and 12.5 mg/kg) of doxorubicin induced a severe motor coordination impairment. The histopathological analysis of these animals showed severe damage of sensory nerves. On the contrary, all the i.p. treated animals did not show any sign of motor impairment and of appreciable neurohistological lesion.

Effects of ICRF 159 on adriamycin-induced cardiomyopathy in rats.

The effect of ICRF 159 on adriamycin (ADR) cardiotoxicity and on total myocardial calcium content was examined in rats. ICRF 159 did not increase the survival time of ADR-treated animals; however the histological findings showed a significant prevention of ADR-induced cardiomyopathy (CMP) by ICRF. The total myocardial calcium content of animals treated with ADR was significantly higher, while no significant difference was seen in animals pretreated with ICRF 159 as compared with controls. As these findings suggested a role of calcium in ADR CMP and in the pharmacological action of ICRF in this disease, we also tested a closely related chelating agent, EDTA. This molecule decreased myocardial calcium levels in ADR-treated animals almost to normal values; however the histological cardiac alterations were not prevented.

[Myocardial disease caused by adriamycin. Experimental animal models and possible pharmacologic prevention]. / Miocardiopatia da adriamicina. Modelli sperimentali animali e possibilità di prevenzione farmacologica.

Adriamycin (ADR) is one of the most useful drugs in oncologic practice; it is effective not only against leukemias and lymphomas, but also against many solid tumors. A severe, frequent and dose dependent cardiomyopathy (CMP) limits its clinical use, therefore the cumulative dose usually administered is less than 550 mg/m2; however this procedure limits the therapeutic effectiveness of the drug. As this substance causes regression of a variety of neoplasms, during the past years a number of authors have studied the pathogenesis of the CMP. Data available on this subject are numerous but often in contrast. This review considers the experimental animal models used to study drug induced CMP; data on the biochemical mechanism underlying this complication and the efficacy of different antidotal procedures are also reviewed.

In vitro mutagenic activity of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) in eukaryotic and prokaryotic cells.

The in vitro mutagenic activity of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC), has been studied in bacteria and Chinese hamster cells with and without metabolic activation by rat liver microsomes. DTIC was found to be highly mutagenic in the two systems. It is noteworthy that DTIC in the prokaryotic systems did not require metabolic activation to be effective. By comparing the mutagenic activity on bacteria of DTIC and of its monomethyl-and hydroxy-methyl-derivatives (MIC and HMIC), it is evident that MIC and HMIC display a pattern of mutagenicity different from DTIC. It suggests that neither MIC nor HMIC are the direct responsible metabolites for the mutagenic activity of DTIC in bacteria.

Mutagenic activity of 3 diazoacetylglycine derivatives on V79 Chinese hamster cells.

3 diazoacetylglycine derivatives, diazoacetylglycine amide (DGA), diazoacetylglycine ethyl ester (DGE) and diazoacetylglycine hydrazide (DGH), known as antitumour agents, and shown to be mutagenic in bacteria, were studied for their mutagenic activity in the HGPRT system of V79 Chinese hamster cells in culture (V79/HGPRT system). All 3 drugs were highly effective in inducing 6-thioguanine-resistant mutants at concentrations that were not significantly cytotoxic.