A multicentre study on spontaneous in-cage activity and micro-environmental conditions of IVC housed C57BL/6J mice during consecutive cycles of bi-weekly cage-change.

Mice respond to a cage change (CC) with altered activity, disrupted sleep and increased anxiety. A bi-weekly cage change is, therefore, preferred over a shorter CC interval and is currently the prevailing routine for Individually ventilated cages (IVCs). However, the build-up of ammonia (NH3) during this period is a potential threat to the animal health and the literature holds conflicting reports leaving this issue unresolved. We have therefor examined longitudinally in-cage activity, animal health and the build-up of ammonia across the cage floor with female and male C57BL/6 mice housed four per IVC changed every other week. We used a multicentre design with a standardised husbandry enabling us to tease-out features that replicated across sites from those that were site-specific. CC induce a marked increase in activity, especially during daytime (~50%) when the animals rest. A reduction in density from four to two mice did not alter this response. This burst was followed by a gradual decrease till the next cage change. Female but not male mice preferred to have the latrine in the front of the cage. Male mice allocate more of the activity to the latrine free part of the cage floor already the day after a CC. A behaviour that progressed through the CC cycle but was not impacted by the type of bedding used. Reducing housing density to two mice abolished this behaviour. Female mice used the entire cage floor the first week while during the second week activity in the latrine area decreased. Measurement of NH3 ppm across the cage floor revealed x3 higher values for the latrine area compared with the opposite area. NH3 ppm increases from 0-1 ppm to reach ≤25 ppm in the latrine free area and 50-100 ppm in the latrine area at the end of a cycle. As expected in-cage bacterial load covaried with in-cage NH3 ppm. Histopathological analysis revealed no changes to the upper airways covarying with recorded NH3 ppm or bacterial load. We conclude that housing of four (or equivalent biomass) C57BL/6J mice for 10 weeks under the described conditions does not cause any overt discomfort to the animals.

Treatment response assessment with (R)-[11CPAQ PET in the MMTV-PyMT mouse model of breast cancer.

BACKGROUND: The goal of the study was to assess the potential of the vascular endothelial growth factor receptor (VEGFR)-2-targeting carbon-11 labeled (R)-N-(4-bromo-2-fluorophenyl)-6-methoxy-7-((1-methyl-3-piperidinyl)methoxy)-4-quinazolineamine ((R)-[11C]PAQ) as a positron emission tomography (PET) imaging biomarker for evaluation of the efficacy of anticancer drugs in preclinical models. METHODS: MMTV-PyMT mice were treated with vehicle alone (VEH), murine anti-VEGFA antibody (B20-4.1.1), and paclitaxel (PTX) in combination or as single agents. The treatment response was measured with (R)-[11C]PAQ PET as standardized uptake value (SUV)mean, SUVmax relative changes at the baseline (day 0) and follow-up (day 4) time points, and magnetic resonance imaging (MRI)-derived PyMT mammary tumor volume (TV) changes. Expression of Ki67, VEGFR-2, and CD31 in tumor tissue was determined by immunohistochemistry (IHC). Non-parametric statistical tests were used to evaluate the relation between (R)-[11C]PAQ radiotracer uptake and therapy response biomarkers. RESULTS: The (R)-[11C]PAQ SUVmax in tumors was significantly reduced after 4 days in the B20-4.1.1/PTX combinational and B20-4.1.1 monotherapy groups (p < 0.0005 and p < 0.003, respectively). No significant change was observed in the PTX monotherapy group. There was a significant difference in the SUVmax change between the VEH group and B20-4.1.1/PTX combinational group, as well as between the VEH group and the B20-4.1.1 monotherapy group (p < 0.05). MRI revealed significant decreases in TV in the B20-4.1.1/PTX treatment group (p < 0.005) but not the other therapy groups. A positive trend was observed between the (R)-[11C]PAQ SUVmax change and TV reduction in the B20-4.1.1/PTX group. Statistical testing showed a significant difference in the blood vessel density between the B20-4.1.1/PTX combinational group and the VEH group (p < 0.05) but no significant difference in the Ki67 positive signal between treatment groups. CONCLUSIONS: The results of this study are promising. However, additional studies are necessary before (R)-[11C]PAQ can be approved as a predictive radiotracer for cancer therapy response.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Hepatic extraskeletal chondroblastic osteosarcoma with unusual angioinvasion of the caudal vena cava in a dog.

Extraskeletal osteosarcomas are rare malignant mesenchymal neoplasms that are able to directly produce osteoid, without requiring a cartilage template. The extraskeletal localization indicates that these neoplasms are not associated with pre-existing skeletal elements or periosteum. We describe the gross and histological findings of a 4-year-old male Rottweiler that presented with an extraskeletal chondroblastic osteosarcoma (also known as osteosarcoma of the chondroblastic subtype) originating from the liver and extending into the lumen of the caudal vena cava, passing through the right atrium and terminating in the right ventricle of the heart immediately below the pulmonary valve. In the liver, predominantly fusiform cells grew in loosely packed streams and whorls. In the vena cava, the neoplasm was multilobular with polygonal neoplastic cells scattered within lacunae in a chondroid matrix. In the cardiac lumen, neoplastic cells produced osteoid that showed multifocal mineralization. Immunohistochemical staining showed no cytokeratin and variable S-100 protein and vimentin immunoreactivity. To the best of our knowledge, this is the first report of a chondroblastic osteosarcoma arising in the liver and showing such extensive and unusual extension into the vasculature.

Case report: epitheliotropic lymphoma in a zebrafish (Danio rerio).

A proliferative disorder of lymphocytes was observed in a macroscopically normal young adult wild-type zebrafish (Danio rerio) during routine histological screening in a two-generation toxicity study. Proliferating lymphocytes were observed to invade the gill arches, infiltrate the cranial skeletal muscle and inner ear, and accumulate at distant sites in the frontal epidermis. The test compound, tetrabromobisphenol A (TBBPA, a flame retardant), is not known as a carcinogen and no tumours were detected in any of the 53 other fish in the study, including tank mates. Although neoplastic lymphoid proliferation in the thymus region is occasionally observed, we have never seen epitheliotropism in zebrafish during other similar exposure studies or brood stock. Our findings indicate that epitheliotropic lymphoma can occur spontaneously in zebrafish but at a low incidence.

Long-term exposure of European flounder (Platichthys flesus) to the flame-retardants tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD).

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are widely used flame retardants that have increasingly been found as contaminants in the aquatic environment. In the present study, European flounder (Platichthys flesus) were chronically exposed to TBBPA; (105 days) and HBCD (78 days), in a wide range including environmentally relevant concentrations. TBBPA was administered via the water, whereas HBCD was administered in food and sediment, or in sediment alone. Chemical analysis of muscle showed an average increase in internal concentrations of approximately two orders of magnitude for both compounds tested. Animals exposed to HBCD via sediment alone (8000 microg/g total organic carbon, TOC) showed a proportional increase of alpha-HBCD in muscle compared to animals exposed via food and sediment. In both studies, exposure to the test compounds did not affect general health and toxicity parameters (behavior, survival, growth rate, relative liver and gonad weight). Hepatic microsomal enzyme activities (TBBPA: EROD; HBCD: EROD, PROD, and BROD) were not induced by any of the tested chemicals. Aromatase activity in male gonads showed a mild increase with rising TBBPA levels. There were no morphological and immunohistochemical indications for increased production of the yolk precursor protein vitellogenin (VTG) in animals exposed to TBBPA and HBCD; immunochemical analysis of plasma VTG levels showed no dose response in animals exposed to TBBPA. In animals exposed to TBBPA, levels of the thyroid hormone thyroxin (T(4)) increased with internal concentrations of the test compound, possibly indicating competition of TBBPA for plasma protein binding. Triiodothyronin (T(3)) levels were not affected and histology showed no signs of altered thyroid gland activity. Other organs investigated (liver, gills, kidney, skin, and gonads) revealed no histological changes related to TBBPA or HBCD exposure. Overall, the present results indicate limited endocrine effects of these widely used flame retardants in a test species representative of European estuaries at environmentally relevant exposure levels and at internal levels up to 4300 ng TBBPA/g wet weight, and 446 microg HBCD/g lipid weight in flounder muscle.

Toxicity of tetrabromobisphenol A (TBBPA) in zebrafish (Danio rerio) in a partial life-cycle test.

Toxicological effects of the widely used flame retardant, tetrabromobisphenol A (TBBPA) were assessed in a partial life-cycle test with zebrafish (Danio rerio). Exposure of adult fish during 30 days to water-borne TBBPA in nominal concentrations ranging from 0 (control) to 1.5 microM was followed by exposure of the offspring in early life stages up to 47 days posthatching (dph) to the same concentrations. Adults exposed to 3 and 6 microM showed severe disorientation and lethargy shortly after beginning of exposure and were euthanized. Because semistatic exposure resulted in fluctuating water concentrations, pooled fish samples were chemically analyzed for internal dose assessment. Egg production was decreased in fish exposed to TBBPA concentrations of 0.047 microM and higher, and a critical effect level of 7.2 microg/g lipid with a lower 5% confidence limit of 3.9 microg/g lipid for 50% decreased egg production was calculated. Histology of adult ovaries indicated a relative increase of premature oocytes in two surviving females exposed to 1.5 microM. Hatching of TBBPA-exposed larvae was decreased except in animals exposed to 0.375 microM. In the highest exposure concentration, early posthatching mortality was high (81%) in larvae and the surviving juveniles showed a significant predominance of the female phenotype. Exposure of eggs from control parents up to 6 microM TBBPA resulted in increasing malformation and pericardial fluid accumulation from 1.5 microM; at higher concentrations, all embryos failed to hatch. The presented results indicate decreased reproductive success in zebrafish at environmentally relevant TBBPA concentrations.

In vivo and in vitro Ah-receptor activation by commercial and fractionated pentabromodiphenylether using zebrafish (Danio rerio) and the DR-CALUX assay.

The present study addresses the toxicity of a commercial pentabrominated diphenylether (PeBDE) flame retardant mixture, DE-71, in a model aquatic vertebrate. Four weeks' exposure of juvenile zebrafish (Danio rerio) to water-borne DE-71 resulted in dose-dependent induction of CYP1A immunoreactivity, predominantly in the endocardium and the endothelium of larger blood vessels, such as ventral aorta and branchial arteries, as well as the larger hepatic and pancreatic blood vessels. To investigate the impact of possible contaminating PBDD/Fs in the DE-71 product, the study was repeated after DE-71 had been fractionated into a non-planar (cleaned PBDEs) and a planar fraction (PBDD/Fs). Zebrafish were exposed under similar conditions to the planar and cleaned DE-71 fractions, and to uncleaned DE-71. In addition, the above fractions were chemically analyzed and tested in a reporter gene assay (DR-CALUX) for their aromatic hydrocarbon-receptor (AhR) stimulating potencies. A relatively strong CALUX response was detected from the planar DE-71 fraction (19.7ng TCDD equivalent (TEQ)/g DE-71), coinciding with a strong induction of CYP1A immunoreactivity in zebrafish. CYP1A immunoreactivity in zebrafish exposed to uncleaned DE-71 was intense, although the CALUX response was 10-fold less compared to the planar fraction. Only weak CYP1A immunoreactivity was found in fish exposed to cleaned DE-71, and none in control animals; no CALUX response was detected in cleaned DE-71. The present findings indicate that chemical impurities of the commercial PeBDE product account for AhR-mediated effects. Analytical isolation of a planar fraction from the commercial product increased the in vitro (DR-CALUX) signal 10 times. Immunohistochemistry showed a strong tissue specific reaction to DE-71 in vivo at these relatively low TEQ levels regardless of chemical pretreatment of the mix, reflecting the sensitivity of CYP1A induction in juvenile zebrafish to AhR agonists.

Some polybrominated diphenyl ether (PBDE) flame retardants with wide environmental distribution inhibit TCDD-induced EROD activity in primary cultured carp (Cyprinus carpio) hepatocytes.

Ethoxyresorufin-O-deethylase (EROD) activity, a catalytic function of the cytochrome P450 1A (CYP1A) microsomal oxygenase subfamily, is a popular biomarker for exposure to xenobiotics, polyhalogenated aromatic hydrocarbons (PHAHs) in particular. It has found wide use in aquatic pollution assessment both in vivo and in vitro. In such studies, subjects are often exposed to complex mixtures where various constituents can interfere with EROD-activity, possibly resulting in inadequate estimation of toxic hazard or biological response. The present study investigates the effects of polybrominated diphenyl ethers (PBDEs), a relatively new and increasingly detected group of environmental contaminants, on the validity of EROD activity as exposure marker in carp (Cyprinus carpio) hepatocytes. Freshly isolated hepatocytes of a genetically uniform strain of male carp were co-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at concentrations of 0, 1, 3, 10, 30, and 100 pM, and one of the highly purified PBDE/PCB congeners (at concentrations of 0, 0.25, and 2.5 microM) or cleaned-up and untreated DE-71 samples (0, 0.1, and 1 microM). PBDEs were selected from the 209 possible congeners based on their relative abundance in environmental samples: BDE-47, BDE-99, BDE-100, and BDE-153. A tentative metabolite of BDE-47, 6OH-BDE-47, was also included. In addition, a commercial pentabrominated dipenylether mixture (DE-71) was tested for interference with EROD activity both with and without clean-up by carbon fractionating which removed possible planar contaminants. Polychlorinated biphenyl (PCB)-153, a reported inhibitor of EROD activity in flounder, was included for comparison. Cells were cultured for a total period of 8 days; exposure started at day 3 after cell isolation. After 5 days of exposure, cell pellets were frozen before EROD activity was determined. Upon exposure to TCDD, the cells responded with increased EROD activity as expected. Significant reduction of TCDD-induced EROD activity was found in the presence of BDE-47, BDE-99, and BDE-153, but not with BDE-100 and 6-hydroxylated BDE-47. Of these PBDE congeners, the most abundant congener in environmental samples, BDE-47, exhibited the strongest inhibition (down to 6% of the TCDD control value). The cleaned-up fraction of commercial penta-BDE (DE-71) mixture proved an even more potent inhibitor, resulting in reduction of EROD activity to 4% of the control values observed at 1.0 microM. BDE-47 and BDE-153 did not reduce TCDD-induced EROD activity when added shortly prior to measurement, suggesting possible interaction with TCDD at the level of CYP1A biosynthesis. PCB-153 did not show significant effects on EROD activity in carp in this study. The present results indicate that environmentally relevant PBDEs can interfere with determination of EROD activity in vitro, at levels reported earlier for PCBs. The observation that detected PBDE levels are rising, stresses the need for caution when interpreting EROD data on environmental samples.

Toxicity of PCB-126 in European flounder (Platichthys flesus) with emphasis on histopathology and cytochrome P4501A induction in several organ systems.

A series of experiments was set up to elucidate the effects of pollution on marine and estuarine fish health, since the European flounder (Platichthys flesus) has shown a relatively high prevalence of (pre)neoplastic liver lesions and lymphocystis virus disease in Dutch coastal and estuarine waters. The hypothesis of a causal relationship between pollution and the above-mentioned diseases was supported by results from semi-field experiments. Therefore several laboratory experiments were carried out to substantiate causality further and to identify the xenobiotics that may play a major role in the field. The present study focuses on polychlorinated biphenyls (PCBs). European flounders (Platichthys flesus) were orally exposed to a single dose of 0, 0.5, 5 or 50 mg PCB-126/kg body weight under controlled laboratory conditions. The effects on liver, gills, gastrointestinal tract, gonads, spleen and mesonephros were examined histologically after 16 days. Induction and localization of cytochrome P4501A (CYP1A) immunoreactivity, and effects on hepatocyte proliferation were visualized immunohistochemically. Effects on thymus size were examined by morphometric analysis of serial sections. Three out of five animals of the highest dose group showed haemorrhages in the fins and tail after 16 days. All animals showed reduced activity in the later stages of the experiment, and some animals of the highest dose group discontinued feeding 14 days after exposure. Strong and exposure-related induction of CYP1A immunoreactivity was noted in hepatocytes, endothelium in all organs examined, and epithelium of the digestive tract and mesonephros at PCB-126 levels of 0.5, 5 and 50 mg/kg. In addition, the strong induction of CYP1A immunoreactivity in a distinct population of haematopoietic cells in the mesonephros and in circulating blood is remarkable, and has not been described previously in other fish species. Furthermore, a morphometrically determined significant reduction in relative thymus size was noted in animals exposed to 50 mg PCB-126/kg. Although the functional implications for the immune system of this reduction need to be further investigated, an impact on the specific resistance against infectious diseases as observed in the field, e.g. viral lymphocystis disease, is not implausible. In addition, a significant increase in absolute liver weight, in hepatosomatic index, and in number of proliferating hepatocytes [measured as immunoreactivity against proliferating cell nuclear antigen (PCNA)] was noted in animals of the highest dose group. From these findings we suppose that PCB-126 (and related chemicals) may play a role in the promotion of tumour development in the liver of European flounders as observed in the field. The results of the present experiment show relatively stronger effects than effects previously reported from experiments with TCDD, suggesting that the TEF of 0.005 assigned to PCB-126 from early life stage mortality experiments in rainbow trout (Oncorhynchus mykiss), underestimates the toxic potential of PCB-126.