G. De Boeck

Effect of antivascular endothelial growth factor treatment on the intratumoral uptake of CPT-11

Promising preclinical activity with agents blocking the function of vascular endothelial growth factor (VEGF) has been observed in various cancer types, especially with combination therapy. However, these drugs decrease microvessel density, and it is not known whether this reduced vessel density (VD) results in decreased delivery of concomitantly administered classical anticancer drugs. We designed an in vivo study to investigate the relation between VEGF-blocking therapy, tumoral blood vessels, and intratumoral uptake of anticancer drugs. Nude NMRI mice bearing colon adenocarcinoma (HT29) were treated with the anti-VEGFmAb A4.6.1 or placebo. After 1 week, CPT-11 was administered 1 h prior to killing the animals. In A4.6.1 treated tumours, there was a significant decrease in VD, more pronounced with potentially functional large vessels than endothelial cords. Interestingly, a trend to increased intratumoral CPT-11 concentration was observed (P=0.09). In parallel, we measured an increase in tumour perfusion, as estimated by high-performance liquid chromatography determination of intratumoural Hoechst 33342 concentration. In the growth delay study, CPT-11 was at least equally effective with or without pretreatment with A4.6.1. These data suggest that tumour vascular function and tumour uptake of anticancer drugs improve with VEGF-blocking therapy, and indicate the relevance for further investigations.

Tumour necrosis factor alpha increases melphalan concentration in tumour tissue after isolated limb perfusion

Several possible mechanisms for the synergistic anti-tumour effects between tumour necrosis factor alpha (TNF-α) and melphalan after isolated limb perfusion (ILP) have been presented. We found a significant sixfold increase in melphalan tumour tissue concentration after ILP when TNF-α was added to the perfusate, which provides a straightforward explanation for the observed synergism between melphalan and TNF-α in ILP.

Phytate and phytase in fish nutrition.

Phytate formed during maturation of plant seeds and grains is a common constituent of plant-derived fish feed. Phytate-bound phosphorus (P) is not available to gastric or agastric fish. A major concern about the presence of phytate in the aquafeed is its negative effect on growth performance, nutrient and energy utilization, and mineral uptake. Bound phytate-P, can be effectively converted to available-P by phytase. During the last decade, phytase has been used by aqua feed industries to enhance the growth performance, nutrient utilization and bioavailability of macro and micro minerals in fish and also to reduce the P pollution into the aquatic environment. Phytase activity is highly dependent on the pH of the fish gut. Unlike mammals, fish are either gastric or agastric, and hence, the action of dietary phytase varies from species to species. In comparison to poultry and swine production, the use of phytase in fish feed is still in an unproven stage. This review discusses effects of phytate on fish, dephytinisation processes, phytase and pathway for phytate degradation, phytase production systems, mode of phytase application, bioefficacy of phytase, effects of phytase on growth performance, nutrient utilization and aquatic environment pollution, and optimum dosage of phytase in fish diets.

Nanotechnology in bio/clinical analysis

Nanotechnology is being exploited now in different fields of analytical chemistry: Single cell analysis; in chip/micro machined devices; hyphenated technology and sampling techniques. Secretory vesicles can be chemically and individually analyzed with a combination of optical trapping, capillary electrophoresis separation, and laser induced fluorescence detection. Attoliters (10(-18) l) can be introduced into the tapered inlets of separation capillaries. Chip technology has come of age in the field of genomics, allowing faster analyses, and will fulfil an important role in RNA and peptide/protein analysis. The introduction of nanotechnology in LC-MS and CE-MS has resulted in new findings in the study of DNA adduct formation caused by carcinogenic substances, including anticancer drugs. Sample handling and introduction also can benefit from nanotechnology: The downscaling of sample volumes to the picoliter level has resulted in zeptomole (10(-21)) detection limits in the single-shot mass spectrum of proteins.

The effects of silver on intestinal ion and acid-base regulation in the marine teleost fish, Parophrys vetulus

Exposure to elevated silver (as AgNO3) concentrations (6-9 microM) in seawater was associated with comparably high silver concentrations in the intestinal fluids of the lemon sole (Parophrys vetulus), and a tendency for reduced drinking rate. The effects of silver on intestinal ion and acid-base regulation were studied using in situ perfusion of the intestine. Intestinal net Cl- uptake was reduced from 0.4 to 0.1 and intestinal net Na+ uptake from 0.2 to 0 mmol kg(-1) x h(-1) during silver exposure (9 microM). At the same time, intestinal HCO3- net efflux was reduced from 0.2 to 0.1 mmol kg(-1) x h(-1). Both intestinal Na+ and Cl- uptake and Cl-/HCO3- exchange are thus sensitive to silver, but to different extents. None of the observed effects were reversible during 24 h of recovery. Intestinal water transport was highly variable in vivo in the perfused preparation, and no significant effect of silver exposure was observed. However, in vitro intestine preparations exhibited reduction of intestinal net water flux from 4 to 1 microl cm(-2) x h(-1) during silver exposure together with reduced unidirectional Cl- influx. Reduced water intake and transepithelial water transport in silver-exposed fish resulted in moderate hemoconcentration evident from higher hematocrit values, but not in increased plasma ion levels. The latter could reflect a compensatory response via increased branchial Na+/K+-ATPase levels, observed in silver-exposed fish, indicative of increased branchial ion transport capacity. Impairment of intestinal ion and water transport as a result of silver intake via drinking could be an important part of the fatal cascade of physiological effects observed in marine fish during acute silver exposure.

Isolated hypoxic hepatic perfusion with tumor necrosis factor-alpha, melphalan, and mitomycin C using balloon catheter techniques: a pharmacokinetic study in pigs

OBJECTIVE To validate the methodology of isolated hypoxic hepatic perfusion (IHHP) using balloon catheter techniques and to gain insight into the distribution of tumor necrosis factor-alpha (TNF), melphalan, and mitomycin C (MMC) through the regional and systemic blood compartments when applying these techniques. SUMMARY BACKGROUND DATA There is no standard treatment for unresectable liver tumors. Clinical results of isolated limb perfusion with high-dose TNF and melphalan for the treatment of melanoma and sarcoma have been promising, and attempts have been made to extrapolate this success to the isolated liver perfusion setting. The magnitude and toxicity of the surgical procedure, however, have limited clinical applicability. METHODS Pigs underwent IHHP with TNF, melphalan, and MMC using balloon catheters or served as controls, receiving equivalent dosages of these agents intravenously. After a 20-minute perfusion, a washout procedure was performed for 10 minutes, after which isolation was terminated. Throughout the procedure and afterward, blood samples were obtained from the hepatic and systemic blood compartments and concentrations of perfused agents were determined. RESULTS During perfusion, locoregional plasma drug concentrations were 20- to 40-fold higher than systemic concentrations. Compared with systemic concentrations after intravenous administration, regional concentrations during IHHP were up to 10-fold higher. Regional MMC and melphalan levels steadily declined during perfusion, indicating rapid uptake by the liver tissue; minimal systemic concentrations indicated virtually no leakage to the systemic blood compartment. During isolation, concentrations of TNF in the perfusate declined only slightly, indicating limited uptake by the liver tissue; no leakage of TNF to the systemic circulation was observed. After termination of isolation, systemic TNF levels showed only a minor transient elevation, indicating that the washout procedure at the end of the perfusions was fully effective. CONCLUSIONS Complete isolation of the hepatic vascular bed can be accomplished when performing IHHP using this balloon catheter technique. Thus, as in extremities, an ideal leakage-free perfusion of the liver can now be performed, and repeated, without major surgery. The effective washout allows the addition of TNF in this setting.

Metal accumulation and metallothionein induction in the spotted dogfish **Scyliorhinus canicula**

Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10microM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587microg/L), Cd (1124microg/L), Pb (2072microg/L), Cu (635microg/L), and Ag (1079microg/L and two additional exposures at 10microg/L and 1microg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.

Osmoregulation of the common carp (Cyprinus carpio) when exposed to an osmotic challenge assessed in-vivo and non-invasively by diffusion- and T2-weighted magnetic resonance imaging

Abstract The regulation of the total, extracellular and intracellular water content in the muscle and liver of the stenohaline common carp in response to an exposure of 1 wt.% NaCl was studied in an in-vivo and non-invasive way in conscious fish. The method of choice was in-vivo magnetic resonance imaging (MRI), which allowed study of dynamic changes in the free water content (T2) and the intra- versus extracellular water content of tissues (apparent diffusion coefficient, ADC). Also plasma osmolarity data were obtained which confirmed that the common carp is a hyperregulator and which were correlated in time with water content data. The demonstrated drop in T2-weighted signal intensity (T2w SI) values revealed water loss in both liver and muscle during the first days of salt exposure. As soon as the plasma osmolarity increased, recovery from the water loss was discerned in both tissues, as demonstrated by the increased T2w SI values, reaching after 21 days of exposure control values in muscle and twice the control value in the liver, reflecting the massive water gain of this organ. The observed ADC values increased linearly, revealing cell shrinkage the first day of exposure with subsequent continuous increase of the extracellular volume. No cell volume repair was observed during the investigated period, not even in the muscle tissue whose water content was restored after 21 days.

Social interactions, predation behaviour and fast start performance are affected by ammonia exposure in brown trout (Salmo trutta L.)

In fish, fast starts are brief, sudden accelerations during predator-prey encounters. They serve for escape and predation and are therefore ecologically important movements. Fast starts are generated by glycolytic muscle performance and are influenced by many internal and external factors. It is known that ammonia pollution has a major effect on the glycolytic muscle action, thus creating conditions in which fast start performance might be reduced and predation rates altered. Therefore, escape response and predation strikes were investigated in brown trout (Salmo trutta) of 10 and 20 cm body length exposed to an elevated (1 mg l(-1)) ammonia concentration for 24 and 96 h. Various locomotor and behavioural variables were measured. In C-starts, i.e. an escape start where the fish bends into a C-shaped position, ammonia exposure had no effect on response latency. After 96 h of exposure, cumulative distance, maximum swimming speed and turning radius of the prey were all significantly reduced and the escape went in no definite direction. The effect of ammonia exposure was more pronounced in large fish than in small fish. Predation strikes were also affected. Distance, speed and turning radius were significantly lower in exposed fish. Agonistic behaviour of dominant fish was significantly reduced and fish spent more time resting. Predator behaviour was also altered and the number of prey captured was reduced. This study shows that ammonia exposure affects brown trout escape response mainly through a reduction in fast start velocity and through an impairment of directionality. Thus, in addition to a reduced strength of the response, ammonia exposure could also reduce the fishs elusiveness facing a predator. Predation rate and social interactions are disrupted and predator-prey relationships could be altered.

Water household of the common carp,Cyprinus carpio, when submitted to an osmotic challenge, as determined by diffusion-weighted magnetic resonance imaging at 7 T

In vivo diffusion-weighted magnetic resonance imaging (MRI) was used to determine the effects of an osmotic challenge (1% NaCl) to a freshwater fish, the common carp (Cyprinus carpio). The imaged region covered organs such as the swimbladder, the liver, the kidney, the intestine, the spinal cord, and muscle tissue. A striking difference between salt-treated and control fish was found in the liver. The apparent diffusion coefficient value of livers from control fish was (0.39±0.16) 10−9 m2/s and of salt-treated fish was (1.23±0.14) 10−9 m2/s, which points to an increase in extracellular water content. These results were partially confirmed by a decrease in dry/wet weight ratio of the liver tissue. We also found increased levels of stress proteins in liver tissue. TheQ factor of the applied radiofrequency coil dropped dramatically when we performed experiments with salt-exposed fish, indicating an increased conductivity resulting from the increased ion concentration and osmolarity of the fish. The data on plasma osmolarity of salt-exposed fish confirm a significant osmolarity increase upon salt exposure (from 334 to 430 mOsm/kg) and exceeded the osmolarity of the salt water (324 mOsm/kg), indicating that carp tend to cope with an increased salinity by increasing the internal osmolarity (hyperosmotic regulation). These data demonstrate that diffusion-weighted MRI might be a useful and noninvasive tool in the study of osmotic challenges of aquatic organisms.