New methods for determination of 2-butoxyethanol, butoxyacetaldehyde and butoxyacetic acid in aqueous systems, with special reference to cell culture conditions.

Ethylene glycol ethers, especially 2-ethoxyethanol and 2-butoxyethanol (BE) are frequently used in industry and household as solvents and detergents because of their excellent hydrophilic and lipophilic properties. BE and its oxidation products, butoxyacetaldehyde (BAL) and butoxyacetic acid (BAA), are mainly associated with haemolytic toxicity. No method to determine BAL in aqueous systems (e.g. urine or blood) has been published up to now. BAL was synthesized by dehydration of BE and identified by gas chromatography-mass spectrometry. For determination of BAL and BE with head space-capillary gas chromatography, water and HCl or sodium dihydrogen phosphate were added to the sample. No further extraction or derivatization were necessary. For BAA determination after adding HCl and sodium dihydrogen phosphate the samples were extracted with ethyl acetate and derivatized with 2,2,2-trichloroethanol/HCl. The analytical methods presented here are reliable, sensitive and rapid. The new methods were developed for mammalian cell culture systems, because such in vitro systems are especially useful for metabolic studies and have the advantage of choosing species and organ specificity. In the cell culture experiments presented here it was demonstrated that Opossum kidney cells are able to metabolize BAL to BAA within 24 h. After this interval, in the cells neither BAL nor BAA were accumulated, whereas BAA was found in the cell culture media.

Sodium monochloroacetate causes cytotoxic effects, an increased lactate and pyruvate level and induces ultra structural and cytoskeletal alterations in cultured kidney and liver epithelial cells.

1. Monochloroacetic acid (MCAA) and its sodium salt, sodium monochloroacetate (SMCA) are widely used in chemical industries as intermediates in the synthesis of carboxymethylcellulose, phenoxyacetic acid, thioglycolic acid, glycine, indigoid dyes and others. Moreover, MCAA has been found as a by-product of the chlorination disinfection of drinking water and as an environmental contaminant of the atmosphere from the photodechlorination reactions of chlorinated hydrocarbons. Little is known about the mode of action of both compounds on the cellular level. From cases of accidental poisoning of man it is known that MCAA accumulates in liver and kidney. 2. In this study, the cytotoxicity of SMCA on cultured liver (Chang liver cells) and kidney epithelial cells of the proximal tubule (Opossum kidney cells) was investigated and its effect on metabolism, ultrastructure and organization of cytoskeleton was examined. 3. Independent from the growth state of the cells (proliferating or quiescent), the results clearly show that SMCA causes a dose-dependent decrease in cell viability after an exposure period of 24 h. In all experiments, proliferating cells were more sensitive than quiescent and confluent cells. Liver cells were less sensitive against SMCA treatment than kidney epithelial cells. In contrast to liver cells, kidney cells exhibited a dose-dependent decrease in cell volume. The decrease in cell viability was accompanied by an increase of lactate and pyruvate concentrations released into the culture medium. In the case of Opossum kidney cells, lactate and pyruvate levels increased 5 - 6-fold, whereas in the case of Chang liver cells the increase was approximately twofold. While the ultrastructure of liver cells remained unaltered after drug treatment, kidney cells exhibited cytoplasmic vacuolization, membraneous disruption and especially mitochondrial alterations. In accordance with the changes in the ultrastructure of Opossum cells, was the reorganization of cytoskeletal elements with an increased stress fiber network at the basolateral surface as well as a partial depolymerization of microtubules and vimentin filaments. A cytoskeletal reorganization was not observed for Chang liver cells after SMCA treatment. 4. The results demonstrate that SMCA causes a dose-dependent cytotoxicity which is accompanied by metabolic, mitochondrial and cytoskeletal alterations in the cells.

[Insufficient ventilation as the etiology of illness perception in an elementary school]. / Mangelhafte Lüftung als Auslöser von Befindlichkeitsstörungen in einer Grundschule.

PURPOSE: Two years after renovation of the classrooms in a primary school (installation of insulated windows and closing of the ventilation shaft) pupils and teachers complained about offensive odours, irritation of the eyes and of the nose, complaints of the respiratory tract, headaches and disturbed mental concentration. The presented study determines the causes, suggests measures for help and checks their effectiveness by means of measurements. METHODS: Before starting our measurements, the air quality had already been assessed by an expert. There was no evidence of elevated concentrations of air contaminants. Because of the content of phthalate plasticizers and flame retardants in the linoleum sealants there was an offensive odour. To determine the cause, the air in the subjective mostly affected classroom was analysed for phthalate plasticizers, their metabolites and alkyl phosphates. We also made aerosol measurements with a cascade impactor, determined bacterial counts in the air, and measured the indoor climate and the internal air flow. RESULTS: The concentrations of phthalate plasticizers and their metabolites in the air were not elevated significantly. The screening for alkyl phosphates was negative. The amount of inhalable particles was 0.046 mg/m3. The bacterial count in the air was negligible. On the other hand the indoor climate during the heating period in winter was remarkably changed. The average room temperature was 26 degrees C (reaching a maximum of 36 degrees C with direct sunlight in the classroom), the average humidity was 21% (minimum 7%) and the change of air was approximately 0.5 per hour. Reopening the ventilation shaft and tilting of only one window resulted in a much greater rate of air change. After installation of temperature regulators and regular use of the venetian blinds in the classroom, the room temperature and the relative humidity during the morning lessons were, as a rule, normalised. Among both pupils and teachers the reports of offensive odours and health disorders were subsequently clearly reduced. CONCLUSIONS: To determine the cause of health disorders indoors, it is apparently to be of great importance to carry out measurements of the climate as well as to assess the level of air contaminants. By use of modern energy-saving construction possible effects on the indoor climate should be be taken into account during the planning stage of changes to avoid health disorders resulting from changed interior climate conditions.

Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume.

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel (IK channel; Pflügers Arch 432:R87-R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca2+ concentration ([Ca2+]i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca2+]i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca2+]i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca2+]i. We therefore suggest that cell volume - possibly via [Ca2+]i - is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells.

Investigations on the nephrotoxicity and hepatotoxicity of trivalent and hexavalent chromium compounds.

In contrast to trivalent chromium (Cr(III)) compounds, hexavalent chromium ((Cr(VI)) compounds are oxidizing agents capable of directly inducing tissue damage and possessing carcinogenic, mutagenic and teratogenic potency. After oral or dermal absorption of Cr(VI), the kidney is the main target organ for chromium accumulation, which might result in acute tubular necrosis in humans. In contrast, an acute toxic effect of Cr(VI) on the liver has not yet been described. Therefore, we used two established epithelial cell lines from the kidney (Opossum kidney cells) and the liver (Hep G2 cells) to design an in vitro-assay which is able to examine acute toxic effects of chromium compounds. Cells of both cell lines were treated with various concentrations of Cr(III) and Cr(VI) ranging from 0.01 micromol/l to 1 mmol/l for 24 h. Thereafter, cell morphology, organization of the intracellular cytoskeleton, number of viable cells and mean cell volume were examined. The results show that Cr(VI), but not Cr(III), has an acute cytotoxic effect and causes a dose-dependent loss in cell viability. The effective dose that caused 50% of cell death was 5 micromol/l for kidney epithelial cells and 50 micromol/l for liver epithelial cells. This means that kidney epithelial cells are 10 times more sensitive towards Cr(VI) treatment than liver epithelial cells and this might explain the known nephrotoxicity in vivo. The loss in cell viability was accompanied by a rounding and detachment of the cells and a marked reduction of intracellular F-actin-containing stress fibers. Microtubules and intermediate-sized filaments were observed to be unaffected. Only in the case of kidney epithelial cells, a dose-dependent cell volume increase was observed after Cr(VI) treatment at concentrations up to 50 micromol/l. At higher concentrations, the cell volume decreased due to the high number of cells undergoing lysis and the appearance of cellular fragments. Various chloride channel blockers with different specificities, molecular structures and inhibitory potentials were tested for their ability to prevent Cr(VI)-induced cell damage. None of the channel blockers was able to inhibit cell damage, suggesting that the uptake of Cr(VI) through the general anion transport system of the cell membrane might be only one facet of cellular uptake and toxification. The data presented here not only confirm the different organ-specific effects of Cr(III) and Cr(VI), but also provide a basis for future experiments on the understanding of acute toxicity of Cr(VI) compounds. Moreover, the results demonstrate that the designed in vitro-assay might be a useful tool to prove whether non-toxic Cr(III) can be oxidized to Cr(VI) under specific industrial conditions (for example, in the leather or chrome industry).

Nephrotoxic effects of di-(2-ethylhexyl)-phthalate (DEHP) hydrolysis products on cultured kidney epithelial cells.

1. Di-(2-ethylhexyl)-phthalate (DEHP) possesses a great industrial value as a plasticizing agent and has become an ubiquitous environmental contaminant. In most species it is rapidly metabolized to mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (2-EHA). Evaluation of toxicity of DEHP and its primary metabolites has been focussed on reproductive toxicity and hepatocarcinogenic properties. The aim of this study was to determine the nephrotoxic potential of both DEHP metabolites by use of cultured kidney epithelial cells (Opossum kidney cells; OK cells). 2. For this purpose, OK cells were exposed for 3 days to MEHP and 2-EHA at concentrations ranging from 0.1 -500 micromol/L and the toxicity as well as the effects on migratory activity and intracellular cytoskeleton were studied by cell biological, morphological and morphometric methods. 3. When compared with corresponding controls, treatment of OK cells with MEHP and 2-EHA, respectively, showed marked differences in cell viability between both DEHP metabolites. MEHP caused a dose-dependent decrease in cell viability (ED50 = 25 micromol/L) accompanied by a moderate swelling of the cells at concentrations up to 25 micromol/L. MEHP concentrations higher than 25 micromol/L caused a dose-dependent shrinkage of the cells and the occurrence of a high amount of cell debris as a result of cell lysis. 2-EHA did not cause a reduced viability or an altered cell volume. The migratory activity of OK cells was not significantly influenced by both metabolites. Moreover, MEHP toxicity resulted in a largely reduced and altered organization of F-actin (stress fibers), but not of myosin, microtubules and vimentin. 4. The study indicates that cultured epithelial cells can be used as a prescreening system to assess the nephrotoxicity of hazardous substances such as DEHP. As demonstrated in this study, only MEHP, but not 2-EHA, has a marked nephrotoxic effect in vitro.

Activation of Na+/H(+)-exchanger by transforming Ha-ras requires stimulated cellular calcium influx and is associated with rearrangement of the actin cytoskeleton.

Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts (+ras cells) results in growth factor-independent proliferation and marked alteration of cytoskeletal architecture including breakdown of actin stress fiber network. Compared to identical cells not expressing the oncogene (-ras cells), +ras cells exhibit a more alkaline intracellular pH (pHi) and a larger cell volume (CV), both of which are important mitogenic elements. They are due to a set point shift for activation of the Na+/H(+)-exchanger. Moreover +ras cells respond to stimuli like 0.5% fetal calf serum or bradykinin with sustained oscillation of the cell membrane potential (PD) due to stimulated Ca2+ entry which triggers pulsatile release of calcium from internal stores and subsequent activation of calcium-sensitive K+ channels. 10 mumol/l bepridil inhibit oscillations of PD and protect +ras cells against actin stress fiber depolymerization. It is shown that bepridil blocks both cellular calcium entry as measured by Mn2+ quenching of fura-2 fluorescence and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene. Inhibition of the Na+/H(+)-exchange with 10 mumol/l HOE 694, on the other hand, does not significantly alter Ha-ras stimulated calcium entry or cytoskeletal rearrangement. In -ras cells ionomycin (0.1 mumol/l) leads to a transient increase in Cai. This effect is paralleled by a transient depolymerization of actin stress fiber network which cannot be inhibited by HOE 694. Disruption of the actin cytoskeleton in -ras cells by cytochalasin D does not alter steady state cell volume or Na+/ H(+)-exchange activity. However, stimulation of cytochalasin-treated -ras cells with bradykinin leads to cell swelling which can be blunted by HOE 694. The results show that both cytoskeletal rearrangement and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene require stimulated calcium influx and Cai oscillations. The depolymerization of the actin cytoskeleton is permissive for the Na+/ H(+)-exchanger to cause cell swelling upon stimulation with bradykinin.

A prescreening system for potential antiproliferative agents: implications for local treatment strategies of postangioplasty restenosis.

BACKGROUND: Recent advances in the understanding of the biology of restenosis indicate that it is predominantly caused by a multifactorial stimulation of smooth muscle cell proliferation. The aim of this study was to investigate the in vitro effect of five potential antiproliferative agents on smooth muscle cells from human atherosclerotic femoral arteries. METHODS AND RESULTS: Primary stenosing plaque material of 24 patients (aged 63 +/- 14 years) and restenosing plaque material of 7 patients (aged 65 +/- 9 years) was selectively extracted from femoral arteries by the Simpson atherectomy device. Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells by positive reaction with smooth muscle alpha-actin. Dalteparin sodium (0.001-100 anti-Xa units/ml), cyclosporine A (0.005-500 micrograms/ml), colchicine (0.00004-4 pg/ml), etoposide (0.002-200 micrograms/ml), and doxorubicin (0.0005-50 micrograms/ml) were added to the cultures. Six days after seeding, cells were trypsinized and cell number was measured by a cell counter. All five agents tested exhibited a significant inhibition of smooth muscle cell proliferation (P < 0.001). After an incubation time of 48 h, the cytoskeletal components, alpha-actin, vimentin, and microtubules were investigated. At peak concentrations, all five tested agents except dalteparin sodium caused severe damage to the cytoskeleton. CONCLUSIONS: All five potential antiproliferative agents exhibited a significant inhibition of smooth muscle cell proliferation. The development of new intravascular delivery systems may open the way for local antiproliferative treatment strategies in interventional cardiology.

Effects of calcium channel blockers on NIH 3T3 fibroblasts expressing the Ha-ras oncogene.

NIH 3T3 fibroblasts expressing the ras oncogene (+ras cells) respond to bradykinin, bombesin or serum with sustained oscillations of cell membrane potential reflecting oscillations of intracellular calcium activity and subsequent activation of calcium-sensitive K+ channels. In contrast, identical cells not expressing the oncogene (-ras cells) respond to bradykinin with a single, transient hyperpolarization of the cell membrane. Furthermore, +ras cells are characterized by a serum-independent proliferation, an increase in cell volume and a marked reorganization of the cytoskeleton. It has been shown previously that the calcium channel blocker nifedipine, but not verapamil and diltiazem, inhibits oscillations of cell membrane potential as well as proliferation. In this study, we have examined the effect of several calcium channel blockers (bepridil, nifedipine, verapamil, diltiazem) on the proliferation, volume and cytoskeletal reorganization of +ras cells. Bepridil (10 mumol/l), which is also shown here to inhibit oscillations of cell membrane potential, and nifedipine (10 mumol/l) caused a decrease in cell number, whereas verapamil and diltiazem (10 mumol/l each) resulted in growth rates which did not differ from untreated +ras cells. The increase in cell volume as observed in untreated +ras cells was also observed for cells treated with verapamil and diltiazem, whereas cell volumes of +ras cells treated with bepridil and nifedipine were markedly reduced and similar to the values obtained for -ras cells. In addition, bepridil and nifedipine markedly inhibited cytoskeletal rearrangement, i.e depolymerization of actin-containing stress fibers. This inhibitory effect was not observed for verapamil and diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminium phthalocyanines-induced photolysis of human vascular wall cells in culture and the effect of fluoride on photodynamic action.

BACKGROUND: Phthalocyanines, second-generation photosensitizers with several attractive properties for use in photodynamic therapy, have been shown to accumulate in malignant lesions and atherosclerotic plaques. After exposure of phthalocyanines-loaded tissues to visible light, the targeted cells become injured and eventually die. In vitro, when fluoride is added before exposure to the light, it can protect some cell types against photodynamic action sensitized by chloroaluminium phthalocyanine (AIPc) and its derivatives. METHODS: The effect of 50 mumol/l chloroaluminium phthalocyanine tetrasulfonate (AIPcS4) and 5 mumol/l AIPc, with and without the addition of 5 mumol/l NaF, on the viability of cultured human endothelial cells (HuEC), human smooth muscle cells (HuSMC), and skin fibroblasts (HuSF) was examined. A 150 W quartz halogen light bulb equipped with a cut-off filter (lambda > 605 nm) was used to activate the phthalocyanines. Cell viability was examined 24 h after irradiation by staining of the cells with fluorescein diacetate/ethidiumbromide and by counting the number of attached cells with a cell counter. RESULTS: In the case of AIPc, the viability of all cell types tested was reduced, in a dose-dependent manner, to about 50% of that of the corresponding controls for a maximum irradiation time of 600 s. When we used AIPcS4, both HuEC and HuSF were considerably less sensitive than HuSMC. The addition of fluoride to AIPc-loaded cells before exposure to light protected HuEC and HuSF, but not HuSMC. In the case of AIPcS4, with and without fluoride, only HuSMC were sensitive. CONCLUSIONS: The addition of fluoride to AIPc or the use of AIPcS4 without fluoride could be a valuable approach, selectively destroying HuSMC without affecting HuEC and HuSF, for the reduction of restenosis rates after recanalization of stenosed or occluded arteries. Moreover, because neither type of phthalocyanines causes cutaneous phototoxicity, these second-generation photosensitizers seem to be best suited to the photodynamic treatment of atherosclerosis.

Human fibroblasts in culture exhibit a low sensitivity against cyclosporin A treatment.

The nephrotoxicity of cyclosporin A (CSA) after chronic treatment is well known and includes in later stages tubular atrophy associated with interstitial fibrosis. In order to examine whether interstitial fibrosis due to CSA treatment in vivo is related to a hyperproliferative activity of fibroblasts, the effects of CSA on the growth characteristics of cultured human skin fibroblasts (HUSF) were investigated at CSA concentrations ranging from 10 ng/ml to 50 micrograms/ml. We found that CSA at concentrations higher than 7.5 micrograms/ml inhibited cell proliferation (p < 0.05 at concentrations above 5 micrograms/ml; n = 3) and cloning efficiency (p < 0.05 at concentrations above 5 micrograms/ml; n = 3) in a dose-dependent manner and caused a promotion of cell attachment at concentrations above 10 micrograms/ml (p < 0.05; n = 4), but did not influence cell spreading. At lower concentrations CSA-treated HUSF did not differ in their growth characteristics from the corresponding controls. A 50% inhibition of proliferation was calculated by extrapolation for a CSA concentration of 70 micrograms/ml for HUSF. The inhibition of HUSF proliferation was reversible even at the highest CSA concentration of 50 micrograms/ml. Under the same experimental conditions, a 50% inhibition of proliferation was observed for Madin-Darby canine kidney (MDCK) cells to be 5.5 micrograms/ml, e.g. at a 15-fold lower CSA concentration. Moreover, CSA caused a dose-dependent and reversible cell elongation of HUSF and a significant increase in the average cell diameter from 19.2 +/- 0.3 microns (control; mean +/- SEM, n = 4) to 22.2 +/- 0.2 microns for 50 micrograms/ml CSA (mean +/- SEM, n = 4) and in median cell volume from 4,210 +/- 160 fl (control; mean +/- SEM, n = 4) to 7,020 +/- 190 fl for 50 micrograms/ml CSA (mean +/- SEM; n = 4). These alterations described above were not correlated with a cytotoxic effect as checked by a fluorescent staining for cell vitality. Alterations in the organization of cytoskeletal components such as stress fibers, intermediate-sized filaments and microtubules directly due to CSA treatment were not observed. In contrast, the amount of fibronectin present on the cell surface was considerably increased by CSA. Although HUSF in culture do not respond to CSA treatment by an increased proliferative activity, they are much less affected by CSA than other cell types (i.e MDCK cells).(ABSTRACT TRUNCATED AT 400 WORDS)

Involvement of microtubules in the link between cell volume and pH of acidic cellular compartments in rat and human hepatocytes.

Cell swelling is shown to induce an increase in acridine orange fluorescence intensity, an effect pointing to the alkalinization of acidic vesicles. Since autophagic hepatic proteolysis is accomplished by pH-sensitive proteinases within acidic lysosomes, this effect may contribute to the well-known inhibitory effect of cell swelling on proteolysis. In the present study, the role of microtubules in volume-dependent alterations of pH in acidic vesicles of rat and human hepatocytes was studied. Colcemid and colchicine were used to depolymerize microtubules and vesicular pH was monitored using two different fluorescent dyes, fluorescein isothiocyanate conjugated-dextran and acridine orange. Colcemid and colchicine, but not the inactive stereoisomer gamma-lumicolchicine, blunted the increase of pH during osmotic cell swelling. The alkalinization of acidic vesicles by NH4Cl was not significantly modified by colcemid or colchicine, indicating that the vesicles were still sensitive to alkalinizing procedures other than cell swelling. Further, colchicine, but not gamma-lumicolchicine, inhibited the antiproteolytic action of osmotic cell swelling. The present observations point to an involvement of the microtubule network in the link of cell volume, lysosomal pH, and proteolysis.

Morphological alterations and cytoskeletal reorganization in opossum kidney (OK) cells during osmotic swelling and volume regulation.

Cells from a variety of tissues regulate their volume when exposed to anisotonic conditions. After exposure of cells to hypotonic conditions, the rapid phase of cell swelling is followed by a slower phase of cell shrinkage towards the initial volume. The present study investigates morphological alterations of adherent and fully spread cells after exposure to hypotonic conditions and the reorganization of cytoskeletal components such as F-actin, actin-binding proteins, microtubules and intermediate-sized filaments. We used cells of a continuous epithelial cell line from the opossum kidney (OK cells), which were exposed to hypotonic conditions for a period of 60 min at 25 degrees C. The osmolarity was reduced by 40% from 320 mosmol/l (isotonic conditions) to 192 mosmol/l (hypotonic conditions). The initial swelling after exposure of OK cells to hypotonic conditions caused enhanced ruffling membrane activity, formation of lamellipodia and an extended space between adjacent cells which was caused by a more rounded cell shape. Moreover, the height of cells located in the centre of cell clusters increased by 32 +/- 8% (mean value +/- SEM) as checked by morphometric analysis of the vertical distance between the apical and basolateral F-actin domain. Although the fluorescence intensity and organization of F-actin in a horizontal direction remained unaltered during cell swelling, we observed a loss of periodicity and irregular distribution of myosin aggregates and a partial rearrangement of vimentin filaments in the form of short fragments. In all experiments the organization of microtubles was observed to be unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum-free cultivation of bovine lens epithelial cells.

Mammalian cells in culture have individual nutritional requirements which are mainly fulfilled by the addition of fetal calf serum to the basic culture medium. Since many of the serum components are as yet poorly understood or even completely unknown, a number of difficulties arise in the evaluation of the effect of exogenously added factors or drugs on the growth of the cells. This paper presents data demonstrating the successful adaptation, routine cultivation and cryopreservation of bovine lens epithelial cells (BLEC) under serum-free culture conditions by use of the commercially available serum substitutes BMS, BM-86 Wissler and Ultroser G. Moreover, a culture medium especially designed for cell quiescence, named LR-1, is presented. Cells cultivated in culture medium containing 10% fetal calf serum served as controls. While BM-86 Wissler caused significantly reduced growth rates within 2 days and, finally, cell death after 12 days of incubation, the use of BMS resulted in growth rates which did not differ from the corresponding controls. Ultroser G resulted in a significant increase of proliferative activity of BLEC. LR-1 medium caused cell quiescence and kept the cells alive for a number of days. Thus, LR-1 allowed evaluation of the response of the cells to a mitogenic mixture from bovine brain mainly containing endothelial cell growth factor. The results demonstrate that cultivation of BLEC is possible under serum-free culture conditions. Moreover, the medium LR-1, which causes cell quiescence, is useful for the evaluation of growth factor-induced effects in vitro.

[Growth characteristics of bovine retinal pericytes in culture]. / Wachstumscharakteristika von bovinen retinalen Perizyten in Kultur.

The loss of retinal pericytes is one of the earliest changes in diabetic retinopathy. In order to study this phenomenon in vitro, an optimal isolation and cultivation system has to be established. Therefore, pericytes from bovine retinae were isolated enzymatically with 0.4% collagenase in phosphate-buffered saline and identical immunologically by positive staining with antibodies against smooth muscle alpha-actin. Routine cultivation of pericytes was performed by using DMEM supplemented with 10% fetal calf serum. Dependent on the in vitro age of cells, the effect of the following reagents on proliferative activity was determined: fetal calf serum, heparin, ECGF, ECGF+heparin, and glucose. Increasing serum concentrations stimulated the proliferation of pericytes, although the degree of stimulation was reduced with increasing in vitro age. Heparin inhibited the growth in a dose-dependent manner; the achieving 50% inhibition was extrapolated to be 25 micrograms/ml. ECGF increased pericyte proliferation significantly, with a maximum at 10 microliters/ml. In addition, ECGF reversed the inhibitory effect of heparin. Furthermore, all tested glucose concentrations (5.5-27.75 mmol/l) did not show any influence on growth rates of pericytes. The results demonstrate that routine cultivation of retinal pericytes is possible. Moreover, they indicate that enhanced blood glucose concentrations, as observed in diabetic patients, are not the only important factor in the loss of retinal pericytes.

Cytoskeletal reorganization in NIH 3T3 fibroblasts expressing the ras oncogene.

Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts leads to a set point shift of cell volume regulation and causes an increase in cell volume by activation of Na+/H+ exchange and Na+, K+, 2Cl- cotransport. Since both ion transport systems are thought to be governed by the cytoskeleton, the aim of this study was to examine the alterations in growth characteristics and cytoskeletal organization due to the expression of the oncogene. The experiments were performed on NIH 3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct and expressing the oncogene after treatment with low serum medium and 1 mumol/l dexamethasone (+ras cells). Transfected cells not expressing the oncogene (-ras cells) and treated with low serum medium, but without the addition of dexamethasone, served as controls. The growth characteristics were examined and the cytoskeletal architecture was visualized by indirect immunofluorescence microscopy using specific antibodies and fluorescent dyes. Expression of the ras oncogene was accompanied by a significant and serum-independent increase in proliferative activity irrespective from the coating of the dishes with attachment factors (poly-L-lysine, collagen type I). Both, -ras and +ras cells, proliferated slower on substrates coated with poly-L-lysine than on tissue culture plastic or collagen type I. Expression of the ras oncogene also resulted in a significant increase in cell volume which was independent from the substrate. +ras Cells became more elongated, exhibited long cytoplasmic protrusions and tended to detach when compared with -ras cells. Examination of the cytoskeletal architecture in +ras and -ras cells revealed marked differences such as a depolymerization of the stress fiber network to strongly fluorescent "focals" as well as the absence of vinculin-containing attachment plaques (focal contacts), a disorganization of non-muscle myosin and of cell surface fibronectin in +ras cells. In addition, a retraction of microtubules and vimentin filaments to the perinuclear region was also observed in +ras cells. For comparison, NIH 3T3 fibroblasts which were not transfected with the ras oncogene (0ras cells) and which were also subjected to the experimental conditions described above (low serum medium +/- dexamethasone), did not exhibit the cytoskeletal alterations as observed for +ras cells. The results demonstrate that the expression of ras oncogene causes not only profound alterations in the proliferative activity, cell volume and cell morphology, but also a marked reorganization of cytoskeletal architecture, which may participate in the altered regulation of volume-regulatory ion transporters in the cell membrane.

Corticosteroid agents inhibit proliferation of smooth muscle cells from human atherosclerotic arteries in vitro.

We studied the in vitro effect of steroid agents on smooth muscle cells from human atherosclerotic arteries. Recent advances in the understanding of the biology of restenosis indicate that restenosis is predominantly caused by a multifactorial stimulation of smooth muscle cell proliferation. Primary stenosing plaque material of 24 patients (aged 63 +/- 14 years) and restenosing plaque material of 7 patients (aged 65 +/- 9 years) was selectively extracted from femoral arteries by the Simpson atherectomy device. Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells by positive reaction with smooth muscle alpha-actin. The steroid agents prednisolone (0.0075-750 micrograms/ml), hydrocortisone (0.0125-1250 micrograms/ml), and dexamethasone (0.0004-40 micrograms/ml) were added to the cultures. Six days after seeding the cells were trypsinized and the cell number was measured by a cell counter. All three steroid agents exhibited a significant antiproliferative effect on smooth muscle cell proliferation. At high concentrations of hydrocortisone, cytoskeletal elements of smooth muscle cells such as actin, microtubules, and vimentin, were largely altered. Our data indicate that the proliferation of smooth muscle cells from human atherosclerotic arteries in vitro can be inhibited by steroid agents and thus may open the way for local post-angioplasty treatment strategies.

The in-vitro effect of antineoplastic agents on proliferative activity and cytoskeletal components of plaque-derived smooth-muscle cells from human coronary arteries.

BACKGROUND: Restenosis after successful percutaneous transluminal coronary angioplasty remains the major clinical problem limiting the long-term efficacy of the treatment. Recent advances in the understanding of the biology of restenosis indicate that its cause is predominantly a multifactorial stimulation of smooth-muscle cell proliferation. The aim of this study was to investigate the in-vitro effect of antineoplastic agents on smooth-muscle cells isolated from human coronary plaque material. METHODS: Atherosclerotic tissue from coronary arteries was extracted from 15 patients of both sexes by thrombendarterectomy. Cells were isolated using enzymatic disaggregation and identified to be smooth-muscle cells with fluorescent antibodies for smooth-muscle-specific alpha-actin. The antineoplastic agents cytarabine (500-0.005 micrograms/ml), doxorubicin (50-0.0005 micrograms/ml), and vincristine (10-0.0001 micrograms/ml) were added to the cultures. Six days after seeding, the cells were trypsinized and then counted. RESULTS: All three antineoplastic agents had a strong dose-dependent antiproliferative effect on cultured smooth-muscle cells. After the application of cytostatic agents, cells either became rounded or underwent complete lysis. Cytoskeletal elements, such as actin, microtubules, and vimentin, were largely altered. CONCLUSION: This investigation examined the potential role of antineoplastic therapy in the prevention of restenosis after coronary angioplasty. The development of new intravascular delivery systems, such as coated stents, may open the way for local antiproliferative strategies in interventional cardiology.

Natural and synthetic estrogens and prostacyclin production in human endothelial cells from umbilical cord and leg veins.

We have evaluated the effects of 17 beta-estradiol and 17 alpha-ethinylestradiol on prostacyclin production (measured as prostaglandin-6-keto-F1 alpha) in human endothelial cell cultures that had been obtained from umbilical cord and from leg veins. Endothelial cultures were treated with the estrogens at 3 different concentrations (10(-6), 10(-7), 10(-8) M) for up to 72h. 17 beta-estradiol did not affect basal 6-keto-PGF1 alpha production in these cell cultures. With 17 alpha-ethinylestradiol the synthesis of 6-keto-PGF1 alpha was not statistically significant changed. At least in our in vitro system we could not find an effect of natural and synthetic estrogens on 6-keto-PGF1 alpha production. This does, of course not rule out an effect in the more complex in vivo-situation.

Photodynamic therapy of vascular stenosis: results from cell culture studies on human endothelial cells.

BACKGROUND: Experimental results from human cell culture studies have shown that smooth muscle cells derived from human atherosclerotic plaques react more sensitively to photoactivated dihematoporphyrin-ester and -ether (DHE) than smooth muscle cells from human nonatherosclerotic arteries. A therapeutic concept of photodynamic therapy of vascular stenosis appears, therefore, to be promising. The prerequisite for an intravascular application is a relatively harmless application of the photosensitizing agent to the luminal lining of endothelial cells. METHODS: The effect of DHE with and without photoactivation was examined on endothelial cell cultures from human saphenous veins. The cellular uptake of DHE in relation to the serum content of the culture medium was evaluated, as well as its effect on proliferative activity, cell size, cell volume, and cellular viability. RESULTS: Intracellular uptake of DHE decreased significantly when higher serum concentrations were present in the culture medium. Incubation of cells with the photosensitizer for 9 days in the dark without light activation resulted in a significant decrease of endothelial cell proliferation only at concentrations higher than 2.5 micrograms/mL (= 2.5 mg/kg body weight for systemic application in vivo). Additional photoactivation caused no reduction of cell viability at DHE concentrations of 1 microgram/mL, but at 2.5 micrograms/mL and 5 micrograms/mL, a reduction of viable cells within 24 hours was observed in relation to the energy densities used for irradiation. CONCLUSIONS: Because smooth muscle cells from atherosclerotic plaques are, however, much more sensitive to photodynamic treatment, the concept of a photodynamic therapy of vascular stenosis may provide a good tool in the reduction of restenosis rates after recanalization of severely stenosed or even occluded arteries.