Sieglinde Bahns

Comparative Localization of Cathepsin B Protein and Activity in Colorectal Cancer

Cathepsin B is a lysosomal cysteine proteinase that may participate in cancer progression. We compared localization of its protein and activity during progression of human colorectal cancer. In adenomas and carcinomas, protein expression and, particularly, activity were elevated compared with those in normal colorectal mucosa. In normal mucosa, cathepsin B protein expression was moderate in stroma and variable in epithelium, whereas activity was mainly present in distinct areas of stroma directly underneath the surface of the colon and in epithelium at the surface of the colon. Stroma in adenomas and carcinomas contained moderate to high protein levels but little activity except for areas of angiogenesis, inflammation, and necrosis, in which activity was high. In adenomas and the majority of well-differentiated carcinomas and moderately differentiated carcinomas, cathepsin B protein and activity were found in granular form in the epithelium, close to the basement membrane. Protein and activity levels were low and diffusely distributed in cancer cells in the remainder of the well-differentiated and moderately differentiated carcinomas and in all poorly differentiated carcinomas. Invasive fronts in most cancers contained moderate protein levels but high activity. We conclude that (a) activity localization is essential to understand the role of cathepsin B in cancer progression, and (b) cathepsin B activity in human colon is associated with invasion of cancer cells, endothelial cells, and inflammatory cells, and in cell death, both apoptotic and necrotic.

Effects of nanoparticles in Mytilus edulis gills and hepatopancreas : A new threat to marine life?

Every day new extraordinary properties of nanoparticles (a billionth of a meter) are discovered and worldwide millions are invested into nanotechnology and nanomaterials. Risks to marine organisms are still not fully understood and biomarkers to detect health effects are not implemented, yet. We used the filter feeding blue mussel as a model to analyse uptake and effects of nanoparticles from glass wool, a new absorbent material suggested for use in floating oil spill barriers. In both, gills and hepatopancreas we analysed uptake of nanomaterials by transmission electronmicroscopy (TEM) in unstained ultrathin sections over a period of up to 16 days. Lysosomal stability and lipofuscin content as general indicators of cellular pathology and oxidative stress were also measured. As portals of uptake, diffusion and endocytosis were identified resulting in nanoparticle accumulation in endocytotic vesicles, lysosomes, mitochondria and nuclei. Dramatic decrease of lysosomal membrane stability occurred after 12h of exposure. Lysosomal damage was followed by excessive lipofuscin accumulation indicative of severe oxidative stress. Increased phagocytosis by granulocytes, autophagy and finally apoptosis of epithelial cells of gills and primary and secondary digestive tubules epithelial cells indicated progressive cell death. These pathological responses are regarded as general indices of toxic cell injury and oxidative stress. By the combinational use of biomakers with the ultrastructural localisation of nanoparticle deposition, final evidence of cause-effect relationships is delivered.

Clonal adaptation of cancer cells in flatfish liver to environmental contamination by changes in expression of P-gp related MXR, CYP450, GST-A and G6PDH activity

Abstract Progression from eosinophilic foci to persistent basophilic foci and carcinomas was observed in pollution-induced hepatocellular carcinogenesis in European flounder (Platichthys flesus L.) in a similar sequence as in chemically induced liver cancer in mammals. Image analysis was used to quantify enzyme activity and protein expression as visualised by enzyme and immunohistochemistry in various stages of toxipathic lesions and carcinogenesis. In eosinophilic foci, growth advantage was achieved by increased production of NADPH and pentoses for biosynthesis and cell proliferation due to increased G6PDH capacity during the first steps of clonal adaptation. Simultaneously, oxyradical production by CYP450, phase I was reduced. Overexpression of P-gp mediating multi-xenobiotic resistance was noted in basophilic cell types which persisted during progression towards carcinomas. This was accompanied by increased protein levels of oxyradical scavenging GST-A. These changes are consistent with adaptation and phenotypic expression of the multidrug or xenobiotic resistance (MDR/MXR) type in hepatocarcinomas of European flounder.

Determination of kinetic properties of G6PDH and PGDH and the expression of PCNA during liver carcinogenesis in coastal flounder

Abstract Flounder sampled along a pollution gradient in the North Sea were investigated histochemically to determine in situ initial reaction velocities (Vini) and kinetic parameters (Vmax and Km values) of G6PDH and PGDH inside and outside hepatocellular foci and carcinomas. Reactions were monitored quantitatively in time using image analysis. PCNA was detected in serial sections as marker for cell proliferation by immuno-gold-silver labelling. Enzymic parameters were correlated with PCNA expression and histopathology. In all stages of carcinogenesis from early foci to well-organised adenomas and anaplastic carcinomas, higher Vini of G6PDH but not of PGDH and a higher PCNA labelling index (43–65%) than in surrounding liver tissue were found. PCNA expression in small satellites of carcinomas reflected their malignancy and were used to differentiate between early foci and invasively growing protrusions of carcinomas. Prolonged pollutant exposure partially inhibited or inactivated G6PDH (and not PGDH) in extrafocal liver tissue and this was compensated by a reduction in Km. In cancerous lesions, G6PDH levels were upregulated but the low Km values were kept to increase NADPH production in cancer cells that is required for biosynthesis during cell division and for xenobiotic biotransformation.

The liver of wrasse - morphology and function as a mirror of point source chemical impact.

Corkwing wrasse (Symphodus melops L.), a protogynous, non-migratory lipfish species, living close to rocky shores was chosen as an indicator species for the monitoring of biological effects of contaminants. Fish were caught by local fisherman at the Norwegian west coast in fjord sites within the framework of the EU BEEP project. The sites represented different point source impacts of (I) copper (a former copper mine), (II) polycyclic aromatic hydrocarbons (PAHs, aluminium smelter discharge), (III) formaldehyde plus PAHs (kelp-factory and influence of the aluminium smelter). Livers of wrasse were studied for histopathological alterations and compared to healthy livers of fish from a reference location. Besides liver morphology, different functional and metabolic parameters were measured to link pathological alterations to functional disorders. The integrity of the lysosomal compartment was tested by the assessment of lysosomal membrane stability (lys), and the accumulation of neutral lipids and lipofuscin. Activity and intracellular localisation of the NADPH-producing enzymes in the liver were assessed histochemically and measured by computer-assisted image analysis. Histopathological alterations were most severe at the site impacted by formaldehyde and PAHs. These findings were associated with highest tumor prevalence, lowest membrane stabilities in hepatocytes and highest accumulation rates of lipofuscin in the liver. The activities of the NADPH-producing enzymes phosphogluconate dehydrogenase (PGDH) and glucose-6-phosphate dehydrogenase (G6PDH) were significantly lower compared to unimpacted reference fish. Histopathological alterations showed clear differences dependent on the input source. Potential links between specific contaminant impact and functional and morphological disorders are discussed.

Vitellogenesis in flatfish liver with toxipathic lesions

Abstract Dab (Limanda limanda) displaying various stages of gonadal maturation were collected from two different sites (one was contaminated). Liver histopathology was complemented by detecting DNA strand breaks (TUNEL assay) and lysosomal latency. The yolk precursor protein vitellogenin and the multixenobiotic transporter (P-gp) were detected in liver by immunocytochemistry; NADPH-dependent EROD activity was measured biochemically. At the contaminated site liver pathologies were linked to significantly reduced lysosomal latency and there was a 40-fold increased DNA strand breaks in dab at all stages of maturity. EROD activity and P-gp expression, which were elevated 5–7-fold in immature dab at the contaminated site, decreased with the onset of gonadal development. Liver vitellogenin increased with different dynamics after the onset of gonadal development at both sites. At the contaminated site vitellogenin production in liver was reduced by 50% before spawning which might result in minimized serum levels and yolk internalization by oocytes with subsequent reduced internal food supply for embryos.

Localisation of a tumor-associated phenotype of Benz-aldehyde Dehydrogenase in liver carcinogenesis of flounder by quantitative histochemistry

Abstract Aldehyde dehydrogenases (ALDHs) oxidize endogeneous reactive aldehydes including lipid peroxidation products (propionaldehyde, malondialdehyde) and are also inducible by xenobiotics like DDT and TCDD. ALDHs are found in injured livers and cancers of contaminant-exposed flounder (Platichthys flesus L.) and showed identical characteristics as in hepatocellular carcinomas of rats by its cytosolic localisation, the preferential oxidation of aromatic compounds (benzaldehyde), dependency on NADP as coenzyme and sensitivity to the specific inhibitor disulfiram. Image analysis showed increased activity of NADP-dependent benzaldehyde dehydrogenase localised in a few cells of early eosinophilic foci and in basophilic adenomas, carcinomas and their invasively growing protrusions (satellites). Disulfiram sensitivity to differentiate between the xenobiotic-inducible isozyme and the tumor-specific isozyme in rats was highest in the early stages of carcinogenesis in the liver of fish. Moderate disulfiram inhibition was observed in non-neoplastic and extrafocal tissue. In neoplastic livers of flounder from contaminated areas (Elbe estuary), hepatocytes in early stages of carcinogenesis were best protected against accumulation of toxic aldehydes by increased activity of the specific tumor-associated isozyme.

Macrophage responses in xenobiotic-induced liver injury and carcinogenesis of a marine flatfish

Abstract In North Sea flounder ( Platichthys flesus L.) toxipathic liver lesions and progression to malignant hepatocellular carcinomas associated with metabolic changes (G6PDH, BzALDH, CYP450, GST, P-gp related MXR) were observed. Our interest is now directed towards the response of macrophages in toxipathic liver lesions and towards altered hepatocellular foci and carcinomas. The activity of macrophages was monitored by acid phosphatase activity. Simultaneously, lysosomal integrity and DNA strand breaks (TUNEL assay) were measured as biomarkers of cell injury and death in serial sections. Our results indicate that macrophage response and activity are primarily reduced in flounder from more contaminated habitats. During carcinogenesis, macrophage immigration is stimulated but activity in extrafocal tissue is downregulated in areas close to carcinomas. Macrophage centers are obviously ignored and circumgrown by invading cancer cells. We presently investigate whether cancer cell-specific signals, such as interleukin 10, transforming growth factor β and prostaglandin E 2 which induce macrophage inactivation in mammals, play a role in arrest of immune response against invading cancer cells in lower vertebrates.