In vitro biotransformation assays using fish liver cells: Comparing rainbow trout and carp hepatocytes.

Biotransformation assays using primary hepatocytes from rainbow trout, Oncorhynchus mykiss, were validated as a reliable in vitro tool to predict in vivo bioconcentration factors (BCF) of chemicals in fish. Given the pronounced interspecies differences of chemical biotransformation, the present study aimed to compare biotransformation rate values and BCF predictions obtained with hepatocytes from the cold-water species, rainbow trout, to data obtained with hepatocytes of the warm-water species, common carp (Cyprinus carpio). In a first step, we adapted the protocol for the trout hepatocyte assay, including the cryopreservation method, to carp hepatocytes. The successful adaptation serves as proof of principle that the in vitro hepatocyte biotransformation assays can be technically transferred across fish species. In a second step, we compared the in vitro intrinsic clearance rates (CLin vitro, int) of two model xenobiotics, benzo[a]pyrene (BaP) and methoxychlor (MXC), in trout and carp hepatocytes. The in vitro data were used to predict in vivo biotransformation rate constants (kB) and BCFs, which were then compared to measured in vivo kB and BCF values. The CLin vitro, int values of BaP and MXC did not differ significantly between trout and carp hepatocytes, but the predicted BCF values were significantly higher in trout than in carp. In contrast, the measured in vivo BCF values did not differ significantly between the two species. A possible explanation of this discrepancy is that the existing in vitro-in vivo prediction models are parameterized only for trout but not for carp. Therefore, future research needs to develop species-specific extrapolation models

Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species.

In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107

Biokinetics and dosimetry of 111In-DOTA-NOC-ATE compared with 111In-DTPA-octreotide

Purpose: The biokinetics and dosimetry of 111In-DOTA-NOC-ATE (NOCATE), a high-affinity ligand of SSTR-2 and SSTR-5, and 111In-DTPA-octreotide (Octreoscan™, OCTREO) were compared in the same patients. Methods: Seventeen patients (10 men, 7 women; mean age 60years), referred for an OCTREO scan for imaging of a neuroendocrine tumour (15), thymoma (1) or medullary thyroid carcinoma (1), agreed to undergo a second study with NOCATE. Whole-body anterior-posterior scans were recorded 0.5 (100% reference scan), 4, 24 and 48h (17 patients) and 120h (5 patients) after injection. In 16 patients the OCTREO scan (178 ± 15MBq) was performed 16 ± 5days before the NOCATE scan (108 ± 14MBq) with identical timing; 1 patient had the NOCATE scan before the OCTREO scan. Blood samples were obtained from 14 patients 5min to 48h after injection. Activities expressed as percent of the initial (reference) activity in the whole body, lung, kidney, liver, spleen and blood were fitted to biexponential or single exponential functions. Dosimetry was performed using OLINDA/EXM. Results: Initial whole-body, lung and kidney activities were similar, but retention of NOCATE was higher than that of OCTREO. Liver and spleen uptakes of NOCATE were higher from the start (p < 0.001) and remained so over time. Whole-body activity showed similar α and β half-lives, but the β fraction of NOCATE was double that of OCTREO. Blood T 1/2β for NOCATE was longer (19 vs. 6h). As a result, the effective dose of NOCATE (105μSv/MBq) exceeded that of OCTREO (52μSv/MBq), and the latter result was similar to the ICRP 106 value of 54μSv/MBq. Differential activity measurement in blood cells and plasma showed an average of <5% of NOCATE and OCTREO attached to globular blood components. Conclusion: NOCATE showed a slower clearance from normal tissues and its effective dose was roughly double that of OCTRE

Biokinetics and dosimetry of (111)In-DOTA-NOC-ATE compared with (111)In-DTPA-octreotide.

PURPOSE: The biokinetics and dosimetry of (111)In-DOTA-NOC-ATE (NOCATE), a high-affinity ligand of SSTR-2 and SSTR-5, and (111)In-DTPA-octreotide (Octreoscan?, OCTREO) were compared in the same patients. METHODS: Seventeen patients (10 men, 7 women; mean age 60 years), referred for an OCTREO scan for imaging of a neuroendocrine tumour (15), thymoma (1) or medullary thyroid carcinoma (1), agreed to undergo a second study with NOCATE. Whole-body anterior-posterior scans were recorded 0.5 (100 % reference scan), 4, 24 and 48 h (17 patients) and 120 h (5 patients) after injection. In 16 patients the OCTREO scan (178 ± 15 MBq) was performed 16 ± 5 days before the NOCATE scan (108 ± 14 MBq) with identical timing; 1 patient had the NOCATE scan before the OCTREO scan. Blood samples were obtained from 14 patients 5 min to 48 h after injection. Activities expressed as percent of the initial (reference) activity in the whole body, lung, kidney, liver, spleen and blood were fitted to biexponential or single exponential functions. Dosimetry was performed using OLINDA/EXM. RESULTS: Initial whole-body, lung and kidney activities were similar, but retention of NOCATE was higher than that of OCTREO. Liver and spleen uptakes of NOCATE were higher from the start (p &lt; 0.001) and remained so over time. Whole-body activity showed similar α and β half-lives, but the β fraction of NOCATE was double that of OCTREO. Blood T (1/2)β for NOCATE was longer (19 vs. 6 h). As a result, the effective dose of NOCATE (105 μSv/MBq) exceeded that of OCTREO (52 μSv/MBq), and the latter result was similar to the ICRP 106 value of 54 μSv/MBq. Differential activity measurement in blood cells and plasma showed an average of &lt;5 % of NOCATE and OCTREO attached to globular blood components. CONCLUSION: NOCATE showed a slower clearance from normal tissues and its effective dose was roughly double that of OCTREO

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging