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Background: Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000). Methods: MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs. Results: At day 3 all MWCNTs increased the number of neutrophils in BALF. C hemical purification had a greater effect on pro-inflammatory cytokines (IL-1β, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-β1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy. Conclusions: These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

Question Certain long and straight multiwalled carbon nanotubes (MWCNTs) have shown carcinogenic potential in animal models after intraperitoneal application. Nonetheless, the underlying mechanisms are still unclear. Owing to their fiber-like structure, the long and straight MWCNTs seem to cause more adverse effects than e.g. tangled types. These effects include the potential to cause malignant mesothelioma, a cancer originating from mesothelial cells, and often associated with asbestos exposure. Methods To gain insights into pertinent disease mechanisms, we analyzed the biological effects of two tailor-made MWCNTs (CNT2: slightly curved, diameter 0.050 mm, length 4.18 mm; CNT 6: straight, diameter 0.049 mm, length 10.9 mm), and a reference MWCNT (JRC NM-401: straight, diameter 0.067 mm, length 4.04 mm). These MWCNTs were tested for sterility and endotoxin content, and were free of relevant contamination. As a disease-relevant cell model, we used primary human peritoneal mesothelial cells (LP9) treated as submerged cultures for 24 h with 5 mg/cm2 of the different fibers. Results Immunofluorescence of alpha-tubulin, a protein that forms part of microtubules (a major cytoskeleton component), showed extensive abnormalities. Similarly, we observed depletion of lamin B1 (LMNB1), which is one of the structural proteins of the nuclear lamina and important in the maintenance of nuclear structure and function. After microarray analysis, we found many differentially-expressed genes relative to untreated control, i.e. 7609, 7478, 5742 for NM-401, CNT2, and CNT6, respectively. Bioinformatic analyses revealed many common significantly activated pathways and molecular functions, associated with inflammation and carcinogenesis, notably lung cancer. The top activated canonical pathways include IL6, IL8, and p38 MAPK signaling. Conclusion In conclusion, our results show that exposure to certain long and straight MWCNTs can lead to marked disturbance of cellular structure and the transcriptome of mesothelial cells, eventually contributing to carcinogenesis.

The present study examines the incorporation into ceramic compositions of certain additives to coat the surface of the quartz contained in the compositions, thus reducing quartz toxicity. The selected substances were nanoalumina, aluminium lactate, and organosilanes. Organosilanes are a very extensive family of compounds that simultaneously enable quartz toxicity to be reduced and the quartz surface to be functionalised, providing it with various properties. In this study, the incorporation of these compounds was conceived such that no substantial alteration of the ceramic process would be required. In addition, compounds were selected that would not adversely affect product behaviour during the process.

Das MCTDH-Verfahren hat sich als mächtiges Werkzeug zur Simulation mehrdimensionaler Systeme bewährt, insbesondere im Bereich der Molekülphysik. Es wurde auch bereits für gezielte Kontrolle molekularer Systeme eingesetzt, hauptsächlich unter Verwendung der bekannten Optimierungsschemata nach Krotov und Rabitz. In dieser Arbeit wird MCTDH mit dem vergleichsweise neuen Optimierungsverfahren CRAB kombiniert. Eigenzustände in verschiedenen eindimensionalen Systeme werden selektiv mit optimierten Laserpulsen angeregt. Hauptaugenmerk liegt auf einem Morseoszillator, dessen Vibrationszustände gezielt angeregt werden. Das dafür genutzte Feld wird in einer endlichen Fourierbasis dargestellt und mit Hilfe von CRAB optimiert. CRAB erweist sich für diese Anwendung als mindestens gleichwertig zu dem etablierten Krotov-Schema, indem die Zielzustände trotz geringem Rechenaufwand zu fast 100% besetzt werden. The multi-configurational time-dependent Hartree method (MCTDH) has been established as a powerful tool for the simulation of multi-dimensional systems, especially in the realm of molecular physics. It has also been used for optimal control of molecular systems, mostly employing the well-known Rabitz and the Krotov optimization schemes. In this thesis, the MCTDH software is combined with the relatively new optimization method CRAB (chopped random basis). State selective excitation of different one-dimensional systems is performed with optimized laser pulses. The main objective was vibrational excitation of a Morse oscillator with a light field expanded in a finite Fourier basis for CRAB optimization. In this case CRAB performed at least as good as the stablished Krotov scheme, with target populations near 100% despite smaller computational effort.

Einleitung: Die Thermoablation eines malignen Lebertumors birgt im Falle einer oberflächlichen Tumorlokalisation das Risiko der thermischen Schädigung unmittelbar angrenzender Organe. Interventionell-radiologische Techniken wie Luftinsufflation oder künstlicher Aszites sind als Lösungsmöglichkeit[for full text, please go to the a.m. URL], 131. Kongress der Deutschen Gesellschaft für Chirurgie

Introduction: Systemic chemotherapy (SYS) remains the gold standard in the treatment of irresectable colorectal liver metastases. The addition of the monoclonal antibodies (mAb) cetuximab or bevacizumab has led to further improvement of outcome. However, a hepatic-arterial infusion (HAI) as locoregional[for full text, please go to the a.m. URL], 128. Kongress der Deutschen Gesellschaft für Chirurgie

Objectives: Objective of the present study was to evaluate whether TEGDMA exhibits a genotoxic potential in human oral epithelial cells (OKF6/TERT2). Methods: TEGDMA-dependent cytotoxicity (0.025 to 5.0 mM), intracellular glutathione (GSH) content, and reactive oxygen species (ROS)-formation were assessed by means of fluorospectrophotometric analyses using propidium iodide, monobromobimane, and 2x,7x-dichlorodihydrofluorescein diacetate, respectively. All readings were performed with an FLX 800 microplate fluorescence reader (BioTec, Neufahrn, Germany) and were repeated at least 6 times with 6 replicates each. The alkaline comet assay ± human 8 hydroxyguanine DNA-glycosylase 1 (hOGG1), which is able to detect oxidative DNA-lesions, was used to determine the genotoxic potential of TEGDMA. One hundred cells were scored per sample. Results are expressed as median values of the olive tail moments (OTM) of 3 independent experiments. The Tukey ANOVA and Friedman test (p < 0.05) were applied for statistical analysis. Results: Incubation with TEGDMA (0.25 - 2.5 mM) resulted in significant GSH-depletion in OKF6/TERT2 cells in a time- and concentration-dependent manner (p

Einleitung: Die Addition monoklonaler Antikörper zur systemischen Chemotherapie kolorektaler Lebermetastasen regt erneut die Diskussion über lokoregionäre Therapieverfahren wie die hepato-arterielle Infusion (HAI) an. Mittels HAI können höhere Dosen eines Medikaments appliziert[for full text, please go to the a.m. URL], 126. Kongress der Deutschen Gesellschaft für Chirurgie

Purpose: The aim of the present investigation was to determine the incidence of micronuclei in peripheral blood erythrocytes of B6C3F1 mice that had been chronically exposed to radiofrequencies (RF) used for mobile communication. Materials and methods: 'Ferris wheels' were used to expose tube-restrained male and female mice to simulated environmental RF signals of the Global System for Mobile Communications (GSM, 902 MHz) or Digital Cellular System (DCS, 1747 MHz). RF signals were applied to the mice for 2 hours/day on 5 days/week for two years, at maximal whole-body-averaged specific absorption rates of 0.4, 1.3, and 4.0 W/kg body weight. Concurrent sham-exposed mice, cage controls, and positive controls injected with mitomycin C were included in this investigation. At necropsy, peripheral blood smears were prepared, and coded slides were stained using May-Grunwald-Giemsa or acridine orange. The incidence of micronuclei was recorded for each mouse in 2000 polychromatic and 2000 normochromatic erythrocytes. Results: There were no significant differences in the frequency of micronuclei between RF-exposed, sham-exposed, and cage control mice, irrespective of the staining/counting method used. Micronuclei were, however, significantly increased in polychromatic erythrocytes of the positive control mice. Conclusions: In conclusion, the data did not indicate RF-induced genotoxicity in mice after two years of exposure.

This paper describes the work carried out during the development of the SILICERAM project, executed during the years 2004 to 2007, explaining both its goals and the actions carried out. It presents the equipment designed and constructed for sampling airborne particulate matter in the workplaces, and also the physicochemical determintions and toxicological tests carried out on the dust collected. Finally, it outlines the most important findings and conclusions that have been obtained in this project, as well as some measures and good practices that could be implemented in the ceramics industries to avoid or minimise the presence of dust in the atmosphere.

In the course of standardisation of the in vitro micronucleus test for analysis of effluents according to ISO, a national round-robin study was organised by the German Federal Institute of Hydrology (BfG), involving 10 laboratories of private companies, universities and public authorities. The micronucleus assay was performed with the permanently growing Chinese hamster lung fibroblast cell line V79. All participants tested four encoded samples from one municipal and one industrial wastewater treatment plant with and without metabolic activation by S9-mix. Two of these samples were spiked in advance with defined concentrations of the clastogenic substances cyclophosphamide and mitomycin C, respectively. Cyclophosphamide and ethyl methanesulfonate were used as positive controls. The defined assessment criterion for genotoxicity was the lowest dilution of a sample that does not show any significant induction of micronuclei. Cytotoxicity was judged by determining the cell-survival index, i.e. the percentage growth rate of the cells compared with the corresponding negative controls. As supplementary qualitative criteria, the mitotic index and the proliferation index were assessed. All participants successfully established the method within a few weeks and generated viable test results in time. The two non-genotoxic samples were detected as negative by 90% (with S9-mix) and 95% (without S9-mix) of the participants. The mitomycin C-spiked wastewater sample (expected to be positive without S9-mix supplementation) was correctly judged as positive by all laboratories. The cyclophosphamide-spiked sample (expected to be positive with S9-mix addition) was evaluated correctly as genotoxic by 80% of the laboratories. A post-test analysis found evidence that the false negative results were due to technical failure, but not of a methodological nature. In 94% of all tests the sample LID values (lowest ineffective dilution = dilution stage of the sample in the test at which a statistically significant increase in the micronucleus rate was not detectable any more) varied by no more than one dilution step around the median LID value. The survival index was proven to be a robust measure for estimation of toxicity. This round-robin study is the first inter-laboratory comparison of the in vitro micronucleus test using wastewater samples. The test system is intended to complement the already DIN- and ISO-standardised bacterial tests, i.e. the umu-test and the Ames plate-incorporation assay. The data provide evidence that the robust and practicable in vitro micronucleus test is suitable as a routine method for wastewater testing.

Background, Aim and Scope: ZIEL UND HINTERGRUND. Im Zuge der internationalen Standardisierung des in vitro-Mikrokerntests mit V79-Zellen für die Untersuchung und Bewertung von Wasserproben auf Gentoxizität (ISO/DIS 21427-2) wurde unter Federführung der Bundesanstalt für Gewässerkunde (BfG) ein nationaler Ringversuch durchgeführt. Es beteiligten sich 10 Laboratorien aus der freien Wirtschaft, von Hochschulen und Behörden. Beim Mikrokerntest handelt es sich um die Testung auf eine spezielle Form von Chromosomenaberration. Um die Standardisierung des Verfahrens abschließen zu können, mussten codierte Abwasserproben in Form eines Ringtests untersucht werden. Es sollten damit die Durchführbarkeit des Verfahrens dokumentiert und Validitätsdaten erhoben werden. Materials and Methods: MATERIAL UND METHODEN. Der Mikrokerntest wurde mit V79-Zellen, einer permanent wachsenden Zelllinie von Lungenfibroblasten des Chinesischen Hamsters durchgeführt. Es wurden vier kodierte Proben von einer kommunalen und einer industriellen Kläranlage mit und ohne metabolische Aktivierung durch S9-Mix untersucht. Zwei der Proben waren im Vorfeld mit den Klastogenen Cyclophosphamid und Mitomycin C in definierten Konzentrationen aufdotiert worden. Bewertungskriterium für Gentoxizität war die Verdünnungsstufe der Probe im Test bei der keine statistisch signifikante Erhöhung der Mikrokernrate mehr auftrat (LID; 'Lowest Ineffective Dilution'). Kriterium für Zytotoxizität war der Survival-Index, also die prozentuale Wachstumsrate der Zellen im Vergleich zur Kontrolle. Als zusätzliche qualitative Merkmale wurden der Mitoseindex und der Proliferationsindex der Zellen bestimmt. Results: ERGEBNISSE. Obwohl mehrere der teilnehmenden Labore zuvor wenig bis keinerlei praktische Erfahrung mit dem in vitro- Mikrokerntest nach dem ISO-Entwurf hatten, können die Resultate als sehr gut bezeichnet werden. Es kam zu maximal 20% ungültigen Ergebnissen. Die beiden nicht-gentoxischen Proben wurden zu 100% bzw. zu 90% als negativ erkannt. Die mit Mitomycin C dotierte Probe (Soll: positiv ohne S9-Mix) wurde von allen Laboren richtig bewertet. Bei der Cyclophosphamid-Probe (Soll: positiv mit S9-Mix) erkannten 80% der Labore die Probe korrekt als positiv. In der Nachauswertung wurde festgestellt, dass die beiden falsch-negativen Ergebnisse auf technischen, nicht aber auf methodischen Mängeln beruhten. Die Resultate schwankten bei allen Proben maximal um eine Verdünnungsstufe bezogen auf den medianen LID-Wert. Der Survival-Index zeigte sich als robustes Maß für die Zytotoxizitätsabschätzung bei der Abwassertestung. Discussion: DISKUSSION. Die Detektion von Mikrokernen ist ein häufig verwendeter Parameter für das Erkennen zytogenetischer Schäden. Der Mikrokerntest ist anders als beispielsweise die Einzelzell-Gelelektrophorese (Comet-Assay) kein Indikatortestverfahren sondern zeigt bereits Mutagenität an, da irreparable DNA-Schäden analysiert werden. Treten Mikrokerne mit erhöhter Frequenz auf, ist in nachfolgenden Zellgenerationen mit schweren genetischen Schäden zu rechnen. Im Hinblick auf einen vorbeugenden Umwelt- und Gesundheitsschutz bedeutet dies, dass das Untersuchungsgut (Ab)Wasserprobe keine signifikante Erhöhung der Mikrokernrate hervorrufen sollte. Conclusions: SCHLUSSFOLGERUNGEN. Beim vorliegenden Ringversuch handelt es sich um den ersten Interlaboratoriums-Vergleich des Mikrokerntests mit Abwasserproben. Das Verfahren soll als eukaryontisches Pendant die bereits nach DIN und ISO standardisierten bakteriellen Verfahren umu-Test und Ames-Test komplementieren. Recommendations and Perspectives: AUSBLICK. Die erfolgreiche Durchführung dieses Ringversuchs rechtfertigt die Überführung des ISO-Entwurf ('Draft International Standard') in den 'Final Draft International Standard' (FDIS), die letzte Vorstufe zur internationalen Norm. Es steht damit ein valides eukaryontisches Testsystem für die Wassertestung zur Verfügung

Multidrug resistance (mdr) proteins of the mdr1 type function as multispecific xenobiotic transporters in hepatocytes. In the liver, mdr1 overexpression occurs during regeneration, cirrhosis, and hepatocarcinogenesis and may contribute to primary chemotherapy resistance. Cultured rat hepatocytes exhibit a time-dependent "intrinsic" increase in functional mdr1b expression, which depends on cyclooxygenase-catalyzed prostaglandin E(2) release. In the present study, the prostaglandin E (EP) receptor agonist misoprostol (1-10 mug/ml) further enhanced intrinsic mdr1b mRNA expression in primary rat hepatocytes. On the other hand, [1alpha(z),2beta,5alpha]-(+)-7-[5-[1,1'-(biphenyl)-4-yl]methoxy]-2-(4-morpholin yl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848B) (30 muM), an antagonist of the cAMP-coupled EP4 receptor, and the protein kinase A (PKA) inhibitor, N-(2-[bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H89) (10 nM), repressed intrinsic mdr1b mRNA up-regulation, whereas the stable cAMP analog 8-bromo-cAMP (10 muM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (100 muM) further enhanced intrinsic mdr1b expression. Primary rat hepatocytes, transiently transfected with reporter gene constructs controlled by mdr1b 5'-gene-flanking regions [-1074 to +154 base pairs (bp) or -250 to +154 bp], demonstrated pronounced mdr1b promoter activity, already without the addition of exogenous modulators. Nevertheless, activity was further stimulated by misoprostol, 8-bromo-cAMP, or IBMX. Cotransfection with expression vectors for PKI, an inhibitor protein of cAMP-dependent PKA, or KCREB, a dominant-negative mutant of the cAMP-responsive element-binding protein (CREB), decreased high-intrinsic mdr1b promoter activity. KCREB also counteracted misoprostol-induced mdr1b promoter activation. In conclusion, these data provide evidence for a pivotal role of EP receptor-stimulated, cAMP-dependent activation of PKA and CREB or CREB-related proteins in mdr1b gene activation in primary rat hepatocytes. Thus, these data might offer potential new target structures for the reversal of primary drug resistance, for example, of liver tumors.

Inhalation of respirable crystalline silica (RCS) may lead to lung diseases, e.g. chronic inflammation, silicosis and cancer. In 1997 RCS has been classified as human carcinogen by the IARC, but the multifactorial aspects of RCS toxicity were acknowledged and industry and science were encouraged to characterise the properties of RCS-containing dusts from various industrial sectors by physicochemical methods and to discriminate their toxic potential by appropriate in vitro/in vivo tests. The SILICERAM project has thus been initiated as a European Collective Research Project to provide those, responsible for setting legislation, with data that will ensure definition of appropriate RCS air limits for the ceramic industry. Within the SILICERAM project, the suitability of the in vitro alkaline comet assay (CA) for screening of the genotoxic potential of ceramic dusts was investigated. Primary rat alveolar macrophages, pre-cultured for 24h, were used as a model system. Mid-size DQ12 quartz was chosen as a positive control, demonstrating an almost linear, concentration-dependent (25-200µg/cm2) induction of DNA strand breaks (SB) after 2h of incubation. SB formation was inhibited by co-treatment with 10µM aluminium lactate, indicating a quartz-specific, surface-dependent clastogenic effect. In addition, DQ12 induced PGE2 liberation and LDH release. TiO2 Bayertitan T (rutile) was investigated as a potential negative control, but was shown to be inappropriate. Unexpectedly, TiO2 induced a significant increase in SB formation, not counteracted by aluminium lactate. Unlike TiO2, Al2O3 could subsequently be identified as an appropriate negative control for the CA. Contrived samples (representative of mineral mixtures employed in ceramic production) composed of different amounts of DQ12, China clay, and feldspar were investigated and could be ranked by their RCS content. Using the optimised CA, different ceramic dust samples collected in the tile, table- and sanitary ware, refractory and brick sector, will be screened and ranked concerning their genotoxic potential. Aluminium lactate will be used to differentiate quartz-dependent and -independent effects.

Natural protein phosphatase inhibitors have been characterised in various reports as cytotoxic agents, which due to apoptosis induction display anti-tumour activity. We have recently shown that synthetic nitrostyrene derivatives are also inhibitors of ser/thr phosphatases with a pronounced pro-apoptotic effect in different cell types. Thus nitrostyrenes might represent a new class of cytostatic compounds. Their pro-apoptotic potential resembled that of natural phosphatase inhibitors, e.g. cantharidic acid, but in comparison with cantharidic acid, apoptosis induction by nitrostyrene progressed much faster. Therefore, we were interested in additional mechanisms, participating in apoptosis by nitrostyrene derivatives. In the present study, we could demonstrate that nitrostyrene and its pro-apoptotic triaminopyrimidine adduct (each 10-25µM), but not the non-pro-apoptotic diaminopyridine adduct (10-25µM), displayed concentration-dependent rapid DNA damage in HL60-leukaemia cells. As assessed by the alkaline comet assay, this effect became already evident after 30 min of incubation. In contrast, the natural phosphatase inhibitor cantharidic acid did not induce DNA damage within the first 3 hours of incubation. We further investigated induction of apoptosis and DNA damage by nitrostyrene derivatives in comparison with that of doxorubicin (DOX) in two HL60 sublines, one, equivalent to the parental line (HL60-neo) and the other exhibiting over-expression of the anti-apoptotic protein Bcl-xL (HL60-Bcl-xL). HL60-neo cells displayed DNA damage and apoptosis induction (as assessed by DNA-fragmentation and caspase 3/7 activation) by pro-apoptotic nitrostyrene derivatives as well as by DOX (lµM), whereas HL60-Bcl-xL cells exhibited nearly no responsiveness towards DOX. Unresponsive-ness of HL60-Bcl-xL cells was not due to over-expression of the multidrug transporter MDR1, as there was no evidence for enhanced MDR1 mRNA expression. Interestingly, the pro-apoptotic effect of nitrostyrene derivatives as well as the early DNA damage was retained in the HL60-Bcl-xL subline. In conclusion, we could demonstrate a correlation between early DNA damage and the fast and prominent pro-apoptotic effect of nitrostyrene derivatives, which even persists in apoptosis compromised and thus chemotherapy resistant leukaemia cells.

Multidrug Resistance (MDR) is known as a serious problem in the treatment of acute myeloid leukaemias (AML). MDR is frequently associated with decreased drug accumulation in cancer cells caused by the ATP-dependent multidrug transporter MDR1. Unfortunately the anthracycline Doxorubicin (Dox), an important drug hi the therapy of pediatric AML, is a known substrate and strong inducer of mdrl-type P-glycoprotein (P-gp) expression. Since previous studies with rat heapto-cytes indicated that cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is involved in mdrl gene up-regulation, we were interested in whether Dox-induced MDR1 overexpression in human leukaemia cells is also dependent on COX-2-activity and thus might be overcome with selective COX-2-inhibitors. In the present study, the human AML cell line HL-60 exhibited basal MDR1 and COX-2 mRNA and functional P-gp expression. As expected, P-gp expression and MDR1-dependent transport activity were markedly increased (~15-fold) in the presence of 0.15µg/ml Dox. Interestingly, Dox-mediated up-regulation of functional P-gp expression was almost reduced to basal levels by pre-incubation with the COX-2-preferential inhibitor meloxicam (10, 100 and 500 nM), indicating that Dox-induced MDR1 up-regulation might be COX-2-dependent. Subsequent PGE2-liberation studies indeed demonstrated significant induction of PGE2-liberation by Dox (from undetectable PGE2-levels to -800 pg/ml after 3d of incubation). In addition, MDR1 mRNA expression in HL-60 cells was induced in the presence of PGEa (3 µg/ml). Furthermore, MTT-assays demonstrated dose-dependent enhancement of the cyto-static effect of Dox by pre-incubation with meloxicam in HL-60 cells (maximum 76% in the presence of l µM meloxicam at day 3) and in primary blasts of a child with AML. Meloxicam alone had no effect on cell viability. In contrast, in mono-nuclear cells from a healthy donor and in isolated CD34+ stem cells no modulating effect of meloxicam on cytostatic potential of Dox was evident. In conclusion, these data suggest that COX-dependent liberation of PGE2 is involved in Dox-mediated MDR1 up-regulation. COX-inhibitors thus could help to overcome MDR in tumor cells, specifically in the therapy of pediatric AML. Additional experiments with AML-blasts are currently underway to verify the results in primary tumor cells.

Although formaldehyde (HCHO) may be a key molecule in cell proliferation of biological systems, high concentrations of HCHO in aqueous solution display geno-toxic, carcinogenic, proliferation inhibiting and pro-apoptotic potential. In the respiratory tract, irritant and sensitizing effects of HCHO as well as induction of chronic inflammatory alterations have also been documented. Unfortunately, little is know about the effects of low HCHO concentrations on human respiratory epithelial cells of the nasal cavity. In the present study, primary human nasal epithelial cells (HUNEC) and human nasal carcinoma cells (RPMI-2650) were exposed, as model systems, for Ih to 0.0001-1000µM HCHO to investigate concentration-dependent effects on structural and functional cell parameter. With high HCHO concentrations (>10µM) the expected inhibition of cell proliferation (HUNEC: ~18%, RPMI-2650: ~57%) and formation of DNA-protein cross-links could be demonstrated. In addition, high HCHO concentrations reduced PGE2-synthesis of exposed cells (HUNEC: ~6.7%, RPMI-2650: ~14.7%) and inhibited expression of the intermediate filament cytokeratin 14 (HUNEC: up to ~5%, RPMI-2650 up to ~90%). Interestingly, low HCHO concentrations (

Mdrl-type P-glycoproteins mediate ATP-dependent elimination of various amphi-philic cations. Since intrinsic mdrl overexpression can contribute to chemotherapy resistance in tumour cells, it is important to elucidate endogenous mechanisms of mdr1 gene regulation. In previous studies, primary rat hepatocyte cultures have been characterized as a model for intrinsic mdrl overexpression, as they reveal intrinsic, time-dependent mdrlb up-regulation, which is due to transcriptional activation of the mdr1b gene. Using rat hepatocytes cultures, we have formerly demonstrated that cyclooxygenase-2-dependent liberation of prostaglandin E2 (PGE2) contributes to intrinsic mdrlb overexpression and that overexpression is further enhanced by the stable cAMP analogue 8-Br-cAMP or the phosphodiesterase inhibitor IBMX. Since stimulation of the PGE2-receptors EP2 and EP4 is associated with an increase in intracellular cAMP levels, we addressed the question whether these PGE2-receptors participate in intrinsic mdr1b gene activation. In the present study, rat hepatocytes cultured with the specific EP4-receptor antagonist AH23848B indeed demonstrated a concentration-dependent decrease in intrinsic mdrlb mRNA overexpression (in Northern blot analyses). This decrease was maximal (~62% inhibition) at 15-30µM after 24h of incubation. The potent EPl/EP2-receptor antagonist AH6809 was less effective (~25% inhibition at 5-10µM). To further elucidate the signal transduction cascade leading to intrinsic, PGE2-receptor-mediated mdr1b gene activation, we focussed on cAMP-dependent protein kinase A (PKA) and PKA-associated transcription factors. Transient transfection assays with rat hepatocyte cultures were performed, using a luciferase reporter gene, controlled by 1264 bp of the mdr1b gene 5'-flanking region (-1074 to +150bp). The high intrinsic mdrlb promoter activity in this system could be markedly decreased by overexpression (via transfection of expression vectors) of a PKA-inhibitor protein (pPKI) or of a dominant negative mutant of the cAMP responsive element (CRE)-binding protein (KCREB). KCREB co-transfected cells exhibited luciferase activities of ~45% of control cells. In conclusion, these data suggest that stimulation of the EP4-receptor, cAMP-dependent activation of PKA, and CREB or factors which are able to dimerize with CREB contribute to intrinsic mdr1b gene overexpression in primary rat hepatocyte cultures.