Effect of sorafenib combined with cytostatic agents on hepatoblastoma cell lines and xenografts.

BACKGROUND: Sorafenib has recently been shown to reduce tumour growth in hepatoblastoma (HB) xenografts. The effect of a combined administration with cytostatic agents was now investigated. METHODS: Cell viability after treatment with sorafenib and different cytostatic agents was evaluated in two HB cell lines (HUH6 and HepT1) using MTT assay. ERK signalling was investigated by western blot, NOXA expression by rt-PCR, and formation of DNA adducts using immunocytology. NMRI mice bearing subcutaneous HUH6-derived tumours were treated with sorafenib alone or in combination with cisplatin. Tumour progression, viability, apoptosis, and vascularisation were monitored by tumour volume, AFP levels, TUNEL assay, and CD31 immunostaining, respectively. RESULTS: The combination of sorafenib and cisplatin led to a remarkable decrease in cell viability. The cisplatin-induced enhanced ERK1/2 activation, but not NOXA expression and the formation of DNA adducts was partly abrogated by sorafenib. In HB xenografts, both, sorafenib and alternated application of sorafenib and cisplatin significantly reduced tumour growth (P<0.05). Levels of AFP were lower in both treated groups (P=0.08). Relative apoptotic areas were increased (P=0.003). Mean vascular density was the lowest in the sorafenib/CDDP group (P=0.02). CONCLUSION: The combination of sorafenib with cisplatin might be a promising treatment option for high risk or recurrent HB.

DEHP and its active metabolites: leaching from different tubing types, impact on proinflammatory cytokines and adhesion molecule expression. Is there a subsumable context?

INTRODUCTION: Di(2-ethylhexyl)phthalate (DEHP) is suspected to be toxic for several reasons. During contact with a lipophilic medium, DEHP leaks from polyvinylchloride (PVC), but its influence on inflammatory reactions remains unknown. We examined specific DEHP leaching out of different tubing types, the possibly modulated liberation of proinflammatory cytokines and the induction of adhesion molecule expression in primary endothelial cells. MATERIALS AND METHODS: Blood samples were circulated in traditional PVC, nodioctyl phthalate (DOP) PVC and heparin-coated PVC tubing within a Chandler loop model. The blood was tested for the concentration of DEHP and its active metabolites as well as the liberation of the proinflammatory cytokines TNFα and IL1ß. Furthermore, we exposed human endothelial cells to circulated blood and analysed them for the expression of the adhesion molecules ICAM-1, VCAM-1 and E-selectin. RESULTS: In contrast to the other tubing, PVC tubing showed significantly elevated DEHP levels, but no alteration was observed concerning a potential up-regulation of the cytokines or activation of the endothelial adhesion molecule receptors. CONCLUSIONS: Our data conclude that there is no correlation between DEHP leaching and the inflammatory response after ECC support, but this study showed that even DEHP-free material is leaching DEHP and its toxic metabolites.

[(Inter)national and regional health goals in academic social-medical education conception for teaching medical students at the Eberhard Karls University Tuebingen]. / (Inter)nationale und regionale Gesundheitsziele in der sozialmedizinischen LehreLehrkonzeption für Medizinstudierende an der Eberhard Karls Universität Tübingen.

BACKGROUND: Social medicine deals with the specific interactions between medicine and society within a constantly changing social environment. The Institute of Occupational and Social Medicine, University Hospital Tuebingen, focuses on this relationship within the academic teaching of the Medical Faculty. Many of the issues thus directly affect the national health objectives and especially the health targets of the state of Baden-Württemberg, summarised in the Health Strategy Baden-Wuerttemberg. MATERIAL AND METHODS: In addition to the recommendations of the German Society for Social Medicine and Prevention (DGSMP) for the social medicine curriculum and the specific definition of the content by the Tuebingen medical faculty, national and regional health-care goals are also taken into account in the teaching conception. RESULTS: Classes are increasingly offered as training courses in small groups (seminars, group work with practical training), instead of classic lectures. These teaching methods allow the students to take part more actively in social medicine issues and to think and act within a comprehensive understanding of health management based on societal goals and the needs of a good health system. The concept is supported by the curriculum design element "log-book skills" of the Medical Faculty of Tuebingen. Feedback elements for teachers and students shape the further development of the concept. In dealing with real system data, practical experience on site and case vignettes, the students experience the links between societal influences, political objectives and medical action as well as the importance of accessibility of medical services for equity in health chances. CONCLUSIONS: The fact that advice and expertise play a crucial role in accessibility is a component to which too little attention is paid and calls for emphasis in the teaching concept. This teaching approach will deepen the understanding of the influence of psychosocial context factors and the conditions of the structural framework on the medical outcome. Furthermore there is a need for providing knowledge and special skills, which enable medical doctors to guide their patients optimally within the healthcare system and to make their contribution to a good system.

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.

[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.

Cytotoxic effects of 2-butoxyethanol in vitro are related to butoxyacetaldehyde, an intermediate oxidation product.

Ethylene glycol ethers belong to a group of solvents with a wide spectrum of applications, particularly because of their compatibility to both hydrophilic and lipophilic systems. Especially ethylene glycol monobutyl ether (2-butoxyethanol, BE) is widely used as a key ingredient in many industrial and consumer cleaning products. Therefore, the risk of human exposure and toxicity by BE as well as its potential for environmental contamination have to be carefully evaluated. By using an established kidney epithelial cell line from the proximal tubule (opossum kidney cells), we investigated the effects of BE on viability, proliferative activity, volume and the organization of the intracellular cytoskeleton of the cells. The experiments were performed with freshly used BE and BE that had been stored at room temperature in the original packing for 3 months after use. After this period of storage the latter BE contained-besides butyraldehyde and n-butanol-0.5 vol% butoxyacetaldehyde (BAL) as measured by capillary gas chromatography and mass spectrometry. Freshly used BE did not cause a toxic effect in the in vitro assays at all concentrations tested (up to 1 mg/ml). In contrast, stored BE which contained BAL reduced cell viability and mitotic activity in a dose-dependent manner. The effective concentration of stored BE causing a 50% loss in cell viability (EC(50/24h)) was calculated to be 1 mg/ml. The toxic effect of stored BE also resulted in alterations of cell morphology and a depolymerization of actin-containing stress fibers. Moreover, administration of stored BE also caused a dose-dependent cell volume increase by the uptake of water, pointing to a necrotic process. In addition, synthesized BAL with a purity of 73.5% (gas chromatography) was also tested and caused an EC(50/24h) of 15 µg/ml, which is a 70-fold lower concentration when compared with stored BE. The present study provides evidence that BE possesses only a low cytotoxic potential in vitro, whereas the corresponding BAL, an intermediate in the oxidation process of BE to butoxyacetic acid, has marked toxic effects. The occurrence of the aldehyde might explain the predominant hematological effects of BE observed in vivo.

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.