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The interaction between heparin, a polyanion, and a polycationic dendrimer with a glycine core and lysine branches Gly–Lys63(NH2)64 has been investigated. Complexation was assessed by transmission electron microscopy, size and zeta potential measurements, methylene blue spectroscopy, and measuring the anti-coagulant activity of heparin in vitro and in vivo. Complete association between the heparin and the dendrimer occurred a 1:1 mass ratio (2:1 molar ratio or +/−charge ratio) with formation of quasi-spherical complexes in the size range of 99–147nm with a negative zeta potential (−47mV). Heparin–dendrimer (dendriplex) formation led to a concentration-dependent neutralization of the anticoagulant activity of heparin in human plasma in vitro, with complete loss of activity at a 1:1 mass ratio. The anticoagulant activity of the dendriplexes in Sprague-Dawley rats was also evaluated after subcutaneous administration with uncomplexed heparin as a comparator. The in vivo anticoagulant activity of heparin in plasma, evaluated using an antifactor Xa assay, was abolished after complexation. Measurement of [3H]-heparin showed that both free heparin and dendriplexes were present in plasma and in organs. Such data confirmed stably the formation of dendriplexes, which could be essential in developing novel dendrimer-based anti-angiogenic therapeutics suitable in combinatory therapeutics and theranostics.

Hexachloro-1:3-butadiene (HCBD) causes damage specifically to the renal proximal tubule in rats. In the present study, injury to the nephron of male Hanover Wistar rats was characterized at 24 h following dosing with HCBD in the range 5–90 mg kg−1 to determine the most sensitive biomarkers of damage, that is, the biomarkers demonstrating significant changes at the lowest dose of HCBD, using a range of measurements in serum and urine, renal histopathology, and renal and hepatic gene expression. Histologically, kidney degeneration was noted at doses as low as 10 mg kg−1 HCBD. Significant changes in the hepatic and renal gene expression categories of xenobiotic metabolism and oxidative stress were observed at 5 mg kg−1 HCBD, and in the kidney alone, evidence of inflammation at 90 mg kg−1 HCBD. Increases in the urinary excretion of α-glutathione S-transferase (α-GST) and kidney injury molecule-1 (KIM-1) were seen at 10 mg kg−1 HCBD, and increases in urinary excretion of albumin and total protein were evident at 15 mg kg−1 HCBD. The most sensitive, noninvasive biomarkers of HCBD-induced renal toxicity in Hanover Wistar rats were urinary α-GST and KIM-1. Urinary albumin measurement is also recommended as, although it is not the most sensitive biomarker, together with α-GST, albumin showed the largest relative increase of all the biomarkers investigated, and the protein is easily measured. Copyright © 2011 John Wiley & Sons, Ltd.

Oxysterols are present in mammalian brain at ng/g–μg/g levels while cholesterol is present at the mg/g level. This makes oxysterol analysis of brain challenging. In an effort to meet this challenge we have developed, and validated, an isolation method based on solid phase extraction and an analytical protocol involving oxidation/derivatisation (i.e., charge-tagging) followed by nano-flow liquid chromatography (nano-LC) combined with tandem mass spectrometry utilising multi-stage fragmentation (MSn). The oxidation/derivatisation method employed improves detection limits by two orders of magnitude, while nano-LC–MSn provides separation of isomers and allows oxysterol quantification. Using this method 13 different oxysterols have been identified in rat brain including 24S-hydroxycholesterol, 24S,25-epoxycholesterol and 7α,26-dihydroxycholest-4-en-3-one. The level of 24S-hydroxycholesterol in rat brain was determined to be 20.3 ± 3.4 μg/g and quantitative estimates were made for the other oxysterols identified. The presence of a large excess of cholesterol over oxysterol in brain raises the problem of autoxidation during sterol isolation and sample preparation. Thus, in parallel to identification studies, the degree of cholesterol autoxidation occurring during sterol isolation and analysis has been evaluated with the aid of [2H7]-labelled cholesterol and cholesterol autoxidation products identified.

The gastrointestinal (GI) tract is an important target organ for the toxicity of xenobiotics. The toxic effects of xenobiotics on this complex, heterogeneous structure have been difficult to model in vitro and have traditionally been assessed in vivo. The explant culture of GI tissue offers an alternative approach. Historically, the organotypic culture of the GI tract proved far more challenging than the culture of other tissues, and it was not until the late 1960s that Browning and Trier described the means by which intestinal tissues could be successfully cultured. This breakthrough provided a tool researchers could utilise, and adapt, to investigate topics such as the pathogenesis of inflammatory intestinal diseases, the effect of growth factors and cytokines on intestinal proliferation and differentiation, and the testing of novel xenobiotics for efficacy and safety. This review considers that intestinal explant culture shows much potential for the application of a relatively under-used procedure in future biomedical research. Furthermore, there appear to be many instances where the technique may provide experimental solutions where both cell culture and in vivo models have been unable to deliver conclusive and convincing findings.

This study describes the previously unreported intrinsic capacity of poly-L-lysine (PLL) sixth generation (G 6 ) dendrimer molecules to exhibit systemic antiangiogenic activity that could lead to solid tumor growth arrest. The PLL-dendrimer-inhibited tubule formation of SVEC4-10 murine endothelial cells and neovascularization in the chick embryo chick chorioallantoic membrane (CAM) assay. Intravenous administration of the PLL-dendrimer molecules into C57BL/6 mice inhibited vascularisation in Matrigel plugs implanted subcutaneously. Antiangiogenic activity was further evidenced using intravital microscopy of tumors grown within dorsal skinfold window chambers. Reduced vascularization of P22 rat sarcoma implanted in the dorsal window chamber of SCID mice was observed following tail vein administration (i.v.) of the PLL dendrimers. Also, the in vivo toxicological profile of the PLL-dendrimer molecules was shown to be safe at the dose regime studied. The antiangiogenic activity of the PLL dendrimer was further shown to be associated with significant suppression of B16F10 solid tumor volume and delayed tumor growth. Enhanced apoptosis/necrosis within tumors of PLL-dendrimer-treated animals only and reduction in the number of CD31 positive cells were observed in comparison to protamine treatment. This study suggests that PLL-dendrimer molecules can exhibit a systemic antiangiogenic activity that may be used for therapy of solid tumors, and in combination with their capacity to carry other therapeutic or diagnostic agents may potentially offer capabilities for the design of theranostic systems.

Hexachloro-1:3-butadiene (HCBD) causes kidney injury specific to the pars recta of the proximal tubule. In the present studies, injury to the nephron was characterized at 24 h following a single dose of HCBD, using a range of quantitative urinary measurements, renal histopathology and gene expression. Multiplexed renal biomarker measurements were performed using both the Meso Scale Discovery (MSD) and Rules Based Medicine platforms. In a second study, rats were treated with a single nephrotoxic dose of HCBD and the time course release of a range of traditional and newer urinary biomarkers was followed over a 25 day period. Urinary albumin (a marker of both proximal tubular function and glomerular integrity) and α-glutathione S-transferase (α-GST, a proximal tubular cell marker of cytoplasmic leakage) showed the largest fold change at 24 h (day 1) after dosing. Most other markers measured on either the MSD or RBM platforms peaked on day 1 or 2 post-dosing, whereas levels of kidney injury molecule-1 (KIM-1), a marker of tubular regeneration, peaked on day 3/4. Therefore, in rat proximal tubular nephrotoxicity, the measurement of urinary albumin, α-GST and KIM-1 is recommended as they potentially provide useful information about the function, degree of damage and repair of the proximal tubule. Gene expression data provided useful confirmatory information regarding exposure of the kidney and liver to HCBD, and the response of these tissues to HCBD in terms of metabolism, oxidative stress, inflammation, and regeneration and repair.

Capillaria hepatica is a nematode parasite of wild rodents and other mammals. Adult worms inhabit the liver. Recently, during the necropsy examination of a group of 160 rabbits from a commercial supplier, firm pale or cystic areas (1-5 mm) were noted on the liver in thirteen animals. On further investigation, these animals were found to be infected with C. hepatica. The histopathological features of the infection in the rabbit are described for the first time and diagnostic features recorded. Lesions were identified predominantly in portal tracts consisting of dilated bile ducts with luminal debris, peribiliary inflammatory cell infiltrates, and fibrosis. Large granulomas (macrogranulomas) were evident in portal areas and involved the bile ducts. Macrogranulomas contained collections of characteristic C. hepatica eggs, macrophages, eosinophils, and lymphocytes. Small granulomas (microgranulomas), characterized by epithelioid macrophages surrounded by lymphocytes and eosinophils, were also identified. C. hepatica eggs were also observed in the lumina of the bile ducts and gall bladder. No adult C. hepatica worms were identified. Oocysts of Eimeria stiedae were also evident in the biliary epithelium in some animals. The unique characteristics of the C. hepatica life cycle are described, and the differential diagnosis of hepatic capillariasis is discussed.

In vitro techniques for the culture of hemopoietic stem cells and committed hemopoietic progenitor cells in rat bone marrow have not been adequately described in the literature. In the present investigations, and using commercially available hemopoietic cytokines and growth factors, the conditions required to perform long-term bone marrow culture (LTBMC) using rat femoral bone marrow were studied, in conjunction with the colony-forming unit cell assay (CFU-C), to quantify the number of progenitor cells. CFU-C production by LTBMCs, set up using Iscove’s modified Dulbecco’s medium supplemented with fetal calf serum and horse serum, ceased after week 2 of culture. However, the duration of CFU-C production was significantly increased by supplementing LTBMCs with the cytokine recombinant mouse stem cell factor or recombinant rat stem cell factor.

Azathioprine (AZA) is a cytotoxic immunosuppressive drug used in the prevention of rejection in organ transplants and the treatment of auto-immune diseases. However, AZA is haemotoxic causing significant bone marrow depression. The present studies were to characterize the haemotoxicity of AZA in the female CD-1 mouse. In Experiment 1, a dose-ranging study, with AZA gavaged daily for 10 days, clinical evidence of toxicity was evident at 125 mg/kg and above. Experiment 2 was a dose–response study with AZA gavaged daily for 10 days at 40–120 mg/kg. At day 1 after the final dose, AZA induced a dose-related pancytopaenia, reduced femoral marrow cellularity, increases in serum levels of the cytokine fms-like tyrosine kinase 3 ligand, reduction in granulocyte-monocyte colony-forming units and erythroid colonies, and increased bone marrow apoptosis. Histology demonstrated hepatocyte hypertrophy, thymic atrophy, reduced splenic extramedullary haemopoiesis, and reduced cellularity of sternal bone marrow. In Experiment 3, AZA was dosed for 10 days at 100 mg/kg with autopsies at 1, 3, 9, 22, 29, 43 and 57 days postdosing. At 1, 3 and 9 days, haematological parameters reflected changes in Experiment 2. At 22/29 days, many blood parameters were returning towards normal; at 43/57 days, most parameters compared with controls. However, there was some evidence of a persistent (i.e. residual/late-stage) mild reduction in RBC and erythroid progenitor cell counts at day 43/57. We conclude that the CD-1 mouse provides an acceptable model for the haemotoxicity of AZA in man.

The concentration of the cytokine fms-like tyrosine kinase-3 ligand (FL) is elevated in the plasma of patients treated with chemotherapy or radiotherapy for malignant conditions. In addition, plasma FL is increased in patients with bone marrow failure resulting from stem-cell defects (e.g. aplastic anaemia). Our goal in the present study was to measure the concentration of serum FL in mice treated with the chemotherapeutic agent busulphan (BU) to induce bone marrow depression and relate changes in FL to effects on haemopoiesis. Female CD-1 mice were treated with BU (9.0 mg/kg) or vehicle by intraperitoneal injection on 10 occasions over 21 days. Animals were autopsied on days 1, 23, 72, 119 and 177 postdosing. A full blood count was performed, and serum prepared for FL analysis. Femoral marrow cell suspensions were prepared to assess the total femoral nucleated cell count (FNCC) and the number of committed haemopoietic progenitor cells (CFU-C). On days 1 and 23 postdosing, significant decreases were evident in many peripheral blood parameters; the FNCC and CFU-C were also reduced in BU-treated mice, in conjunction with increases in serum FL levels. On days 72, 119 and 177 postdosing, several peripheral blood and bone marrow parameters remained reduced and the concentration of serum FL continued to be significantly increased. Linear regression analysis demonstrated significant correlations between the concentration of serum FL in BU-treated mice and peripheral blood and bone marrow parameters; this suggests the possible use of serum FL as a potential biomarker for drug-induced bone marrow injury.

Multi-walled carbon nanotubes functionalized with diethylentriaminepentaacetic dianhydride (DTPA-MWNT) and radiolabeled with Indium-111, having therapeutic and diagnostic applications, were tracked in the systemic blood circulation and the excretory system using a microSingle Photon Emission Tomography (microSPECT) scanner. Quantitative Kaiser test was used to determine the number of free amino groups on the DTPA-MWNT. A suspension of PBS and DTPA-MWNT was intravenously injected by tail vein in the Wistar rats and the urine production, water consumption and body weight were observed for 24 hours. The rat was necropised and tissues of various organs were fixed and observed with a Nikon Microphot-FXA microscope coupled with digital camera. The Length of DTPA-MWNT used in the study is larger than the dimensions of the glomerular capillary wall, so it was excreted through urine after 24 hours post-administration.

The investigations aimed to evaluate the usefulness of cardiac troponins as biomarkers of acute myocardial injury in the rat. Serum from female Hanover Wistar rats treated with a single intraperitoneal (IP) injection of isoproterenol (ISO) was assayed for cardiac troponin I (cTnI) (ACS: 180SE, Bayer), cTnI (Immulite 2000, Diagnostic Products Corporation) and cardiac troponin T (cTnT) (Elecsys 2010, Roche). In a time-course study (50.0 mg/kg ISO), serum cTnI (ACS:180SE) and cTnT increased above control levels at 1 hour postdosing, peaking at 2 hours (cTnI, 4.30 μg/L; cTnT, 1.79 μg/L), and declined to baseline by 48 hours, with histologic cardiac lesions first seen at 4 hours postdosing. The Immulite 2000 assay gave minimal cTnI signals, indicating poor immunoreactivity towards rat cTnI. In a dose-response study (0.25 to 20.0 mg/kg ISO), there was a trend for increasing cTnI (ACS:180SE) values with increasing ISO dose levels at 2 hours postdosing. By 24 hours, cTnI levels returned to baseline although chronic cardiac myodegeneration was present. We conclude that serum cTnI and cTnT levels are sensitive and specific biomarkers for detecting ISO induced myocardial injury in the rat. Serum troponin values reflect the development of histopathologic lesions; however peak troponin levels precede maximal lesion severity.

In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

Spontaneous follicular and C-cell tumors of the thyroid gland in the Han Wistar rat were examined using two morphologic procedures. Firstly, in situ hybridization (ISH) was used to localize thyroglobulin (TG) and calcitonin (CT) mRNAs. Secondly, the proteins for these markers were detected using immunohistochemistry (IHC). The aim was to study the morphology of the tumors and to examine the usefulness of TG and CT markers in the differential diagnosis of these lesions. Follicular tumors with cystic, papillary and follicular patterns showed relatively consistent expression of TG mRNA by ISH, thereby confirming the diagnostic value of this technique. However, no staining for TG markers was observed in solid lesions. In general, C-cell tumors comprised well-differentiated cells that continued to express CT mRNA and peptides even after embolic spread and metastasis. Therefore, the performance of either ISH or IHC for CT markers can be used for diagnostic confirmation. Additional features noted in C-cell tumors included the appearance of tumor emboli or metastases in association with small primary lesions (less than 5 average follicular diameters in size) and the presence of eosinophilic (amyloid-like) material showing immunopositivity for CT peptides. Finally, evidence is provided for the sequestration of TG protein by proliferating C-cells.

Chloramphenicol (CAP) is haemotoxic in man, inducing two types of toxicity. First, a dose-related, reversible anaemia with reticulocytopenia, sometimes seen in conjunction with leucopenia and thrombocytopenia; this form of toxicity develops during drug treatment. The second haemotoxicity is aplastic anaemia (AA) which is evident in the blood as severe pancytopenia. AA development is not dose-related and occurs weeks or months after treatment. We wish, in the longer term, to investigate CAP-induced AA in the busulphan-pretreated mouse. However, as a prelude to that study, we wanted to characterize in detail the reversible haemotoxicity of CAP succinate (CAPS), administered at high dose levels in the mouse, and follow the recovery of the bone marrow in the post-dosing period. Female B6C3F1 mice were gavaged with CAPS at 0, 2500 and 3500 mg/kg, daily, for 5 days and sampled (n = 5) at 1, 7, 14 and 21 days post-dosing. Blood, bone marrow and spleen samples were analysed and clonogenic assays carried out. At day 1 post-dosing, at both CAPS dose levels, decreases were seen in erythrocytes and erythrocyte precursors; marrow erythroid cells were reduced. Reductions were also evident in splenic nucleated cell counts, blood high fluorescence ratio (HFR) reticulocyte counts and total reticulocyte counts; burst-forming units-erythroid and colony-forming units-erythroid showed decreases. At day 7 post-dosing (2500 mg/kg CAPS), there was regeneration of erythrocyte production, with marked splenic erythropoietic activity, and raised blood HFR reticulocytes. At day 7, at 3500 mg/kg CAPS, erythrocyte and reticulocyte parameters remained depressed. At 14 days post-dosing (2500 mg/kg CAPS), many erythrocyte parameters had returned to normal; at 3500 mg/kg CAPS, there was erythroid regeneration. By 21 days post-dosing, at both CAPS dose levels, most erythrocytic parameters were equivalent to control values. For leucocyte parameters, there was some depression at day 1 post-dosing (at both CAPS dose levels) and signs of recovery at day 7. At days 14 and 21 post-dosing, most leucocyte parameters were close to control values. Marrow smears at day 1 post-dosing (at both CAPS dose levels) showed vacuolation of early normoblasts, of myeloid and of monocytic precursors. We conclude that the administration of CAPS at 2500 and 3500 mg/kg for 5 days induced significant myelotoxicity in female B6C3F1 mice, with cessation of erythropoiesis at day 1 post-dosing; recovery was seen over the following 7/14 days. The blood HFR reticulocyte count was a precise indicator of CAPS-induced depressive effects and subsequent recovery. It is concluded that the administration of five daily doses of CAPS at 2500 and 3500 mg/kg to the female B6C3F1 mouse induces an anaemia with reticulocytopenia, in conjunction with leucopenia, in the immediate post-dosing period; no evidence was seen at 21 days post-dosing of peripheral blood pancytopenia or a hypocellular/acellular bone marrow, which are both characteristic features of AA in man.

Aplastic anaemia (AA) in man is an often fatal disease characterized by pancytopenia of the peripheral blood and aplasia of the bone marrow. AA is a toxic effect of many drugs and chemicals (e.g. chloramphenicol, azathioprine, phenylbutazone, gold salts, penicillamine and benzene). However, there are no widely used or convenient animal models of drug-induced AA. Recently, we reported a new model of chronic bone marrow aplasia (CBMA = AA) in the busulphan (BU)-treated mouse: eight doses of BU (10.50 mg/kg) were administered to female BALB/c mice over a period of 23 days; CBMA was evident at day 91/112 post-dosing with significantly reduced erythrocytes, platelets, leucocytes and nucleated bone marrow cell counts. However, mortality was high (49.3%). We have now carried out a study to modify the BU-dosing regime to induce CBMA without high mortality, and investigated the patterns of cellular responses in the blood and marrow in the post-dosing period. Mice (n = 64/65) were dosed 10 times with BU at 0 (vehicle control), 8.25, 9.0 and 9.75 mg/kg over 21 days and autopsied at day 1, 23, 42, 71, 84, 106 and 127 post-dosing (n = 7-15); blood and marrow samples were examined. BU induced a predictable bone marrow depression at day 1 post-dosing; at day 23/42 post-dosing, parameters were returning towards normal during a period of recovery. At day 71, 84, 106 and 127 post-dosing, a stabilized, late-stage, nondose-related CBMA was evident in BU-treated mice, with decreased erythrocytes, platelets and marrow cell counts, and increased MCV. At day 127 post-dosing, five BU-treated mice showed evidence of lymphoma. In this study, mortality was low, ranging from 3.1% (8.25 mg/kg BU) to 12.3% (9.75 mg/kg BU). It is concluded that BU at 9.0 mg/kg (or 9.25 mg/kg) is an appropriate dose level to administer (10 times over 21 days) to induce CBMA at approximately day 50-120 post-dosing.

Mitomycin (MMC), like many antineoplastic drugs, induces a predictable, dose-related, bone marrow depression in man and laboratory animals; this change is generally reversible. However, there is evidence that MMC may also cause a late-stage or residual bone marrow injury. The present study in female CD-1 mice investigated the haematological and bone marrow changes induced by MMC in a repeat dose study lasting 50 days. Control and MMC-treated mice were dosed intraperitoneally on eight occasions over 18 days with vehicle, or MMC at 2.5 mg/kg, autopsied (n = 6-12) at 1, 7, 14, 28, 42 and 50 days after the final dose and haematological changes investigated. Femoral nucleated bone marrow cell counts and levels of apoptosis were also evaluated and clonogenic assays carried out; serum levels of FLT3 ligand (FL) were assessed. At day 1 post-dosing, MMC induced significant reductions in RBC, Hb and haematocrit (HCT) values, and there were decreases in reticulocyte, platelet, and femoral nucleated cell counts (FNCC); neutrophil, lymphocyte and monocyte values were also significantly reduced. On days 7 and 14 post-dosing, all haematological parameters showed evidence of a return towards normal values, but at these times, and at day 28, values for RBC and FNCC remained significantly reduced in comparison with controls. At days 42 and 50 post-dosing, many haematological parameters in MMC-treated mice had returned to control levels; however, there remained evidence of late-stage effects on RBC, Hb and HCT values, and FNCC also continued to be significantly decreased. Results for granulocyte-macrophage colony-forming units and erythroid colonies showed a profound decrease immediately post-dosing, but a return to normal values was evident at day 50. Serum FL concentrations demonstrated very significant increases in the immediate post-dosing period, but a return to normal was seen at day 50 post-dosing; a relatively similar pattern was seen in the number of apoptotic femoral marrow nucleated cells. The histopathological examination of kidney tissues from MMC animals at day 42 and 50 post-dosing showed evidence of hydronephrosis with cortical glomerular/tubular atrophy and degeneration. It is therefore concluded that MMC administered on eight occasions over 18 days to female CD-1 mice at 2.5 mg/kg induced profound changes in haematological and bone marrow parameters in the immediate post-dosing period with a return to normal levels at day 50 post-dosing; however, there was evidence of mild but significant late-stage/residual effects on RBC and FNCC, and on cells of the erythroid lineage in the bone marrow.

We have recently developed a new model of drug-induced chronic bone marrow aplasia (CBMA) in the mouse, which shows features of aplastic anaemia (AA) in humans. Using a regimen of repeated doses of busulphan (BU) we induced late-stage (i.e. chronic) bone marrow depression. There are reports that indicate that other antineoplastic agents [e.g. chlorambucil (CHB), mitomycin, melphalan] may also cause CBMA in the mouse. Wishing to develop a model of CBMA in the rat, we investigated the potential of CHB to induce this change. Female Wistar Hanover (Wistar Han) rats were dosed with CHB (6.3 mg/kg intraperitoneally) on six occasions over 18 days. Animals (n=6–8) were killed and sampled on nine occasions (at 1, 3, 9, 16, 24, 38, 45, 59 and 65 days) after the final CHB dose. A full blood count was performed, and serum was prepared for FLT3 analysis; marrow smears were produced, and the spleen and sternum were placed in histological fixative; femoral marrow suspensions were prepared for assessment of the nucleated cell count [femoral nucleated cell count (FNCC)], levels of apoptosis, and the clonogenic potential of the marrow [colony forming unit cells (CFU-Cs)]. Our results showed that at days 1 and 3 post-dosing, in general, red blood cells (RBCs), lymphocytes and FNCC were significantly reduced in CHB-treated animals; reticulocytes were increased, and platelets and neutrophils were unaffected. At 9 days, parameters in CHB rats were returning to normal, but lymphocytes were still decreased. At 16 and 24 days, many blood parameters were normal, except for reduced lymphocyte counts; this pattern generally remained until the end of the study (day 65). Levels of apoptosis in marrow cells of CHB-treated rats were increased immediately post-dosing, and this elevation persisted until day 16; thereafter, levels were generally normal. Serum FLT3 ligand (FL) levels showed some evidence of increases in CHB rats after dosing. CFU-Cs/femur were significantly reduced in CHB animals after dosing, returning to normal values at day 24. In general, marrow smears showed reductions in the myeloid, erythroid and lymphoid lineages of CHB animals at days 1, 3 and 9, returning to normal at 16 and 24 days. Histology of the spleen showed severe depletion of the white pulp immediately post-dosing in CHB-treated animals. Therefore, it is concluded from these findings that CHB does not induce late-stage (i.e. chronic) bone marrow aplasia in the female Wistar Han rat.

A study has been carried out in the apolipoprotein (apo) E-deficient mouse to investigate the activity of lacidipine (a calcium antagonist with antioxidant properties) in inhibiting the development of atherosclerotic lesions; of particular interest were changes in the susceptibility of low-density lipoproteins (LDL) to oxidation. Mice receiving a Western-type diet to accelerate the development of atherosclerosis were treated orally with vehicle or lacidipine at 3 or 10 mg/kg/day for 8 weeks. Lacidipine treatment (at 3 or 10 mg/kg) had no effect on the plasma lipid profile. However, a significant (P < 0.01) dose-related reduction of 43 and 50% of the aortic lesion area in respect to vehicle-treated mice was observed. Moreover, the resistance of mouse plasma LDL to undergo lipid peroxidation was significantly (P < 0.01) increased in apo E-deficient mice treated with lacidipine. The native LDL-like particle, derived from apo E-deficient mice treated with lacidipine, contained significantly lower concentrations of malonyldialdehyde than the vehicle-treated control group (P < 0.01). After exposure to human umbilical vein endothelial cells, LDL-like particle vitamin E levels (expressed as area under the curve; AUC), were significantly higher (P < 0.01) in both the 3 and 10 mg/kg lacidipine-treated groups, in comparison with the vehicle-treated control animals. We conclude that lacidipine reduced the extent of the atherosclerotic area in hypercholesterolaemic apo E-deficient mice, and that this reduction may be associated with the capacity of the drug to decrease the susceptibility of LDL to oxidation.

C-cell tumours of the thyroid gland are among the most common spontaneous neoplasms of the laboratory rat. With the exception of calcitonin, little attention has been paid to the secretory peptides of C cells during the development of neoplasia. Of these peptides, somatostatin (SS) is of particular interest because it has been shown to have a direct anti-secretory effect on both thyroid follicular and C cells in vitro. In the present study, in situ hybridization and immunohistochemistry were used to investigate the expression of SS mRNA and SS peptides, in normal C cells and a range of spontaneous proliferative C-cell lesions in the Han Wistar rat. It was confirmed that a small minority of C cells in the normal rat thyroid gland produce and store SS peptides; however, approximately half of all C-cell adenomas and C-cell carcinomas stained positively for SS mRNA and peptides. SS expression was also observed in all metastatic deposits of carcinomas in drainage lymph nodes. From these observations, it appears that C-cell tumours are more likely to develop from SS-expressing stem cells, rather than from non-SS-expressing stem cells. In addition, a lack of differentiation of neoplastic C cells, or reversion to more primitive cell types, could account for increased number of cells expressing SS in C-cell tumours relative to the normal C-cell population. Finally, the mean percentage of cells that stained positively for SS mRNA and peptides appeared to be significantly higher in small C-cell tumours, suggesting that SS may have exerted a growth-controlling influence on these lesions.

Aplastic anaemia (AA) is characterized by hypocellular marrow, pancytopenia, and risk of severe anaemia, haemorrhage and infection. AA is often idiopathic, but frequently occurs after exposure to drugs/chemicals. However, the pathogenesis of AA is not clearly understood, and there are no convenient animal models of drug-induced AA. We have evaluated regimens of busulphan (BU) administration in the mouse to produce a model of chronic bone marrow aplasia showing features of human AA. Mice were given 8 doses of BU at 0, 5.25 and 10.50 mg/kg over 23 days; marrow and blood samples were examined at 1, 19, 49, 91 and 112 days after dosing. At day 1 post dosing, in mice treated at 10.50 mg/kg, nucleated marrow cells, CFU-GM and Erythroid-CFU were reduced. Similarly, peripheral blood erythrocytes, leucocytes, platelets and reticulocytes were reduced. At day 19 and 49 post dosing, there was a trend for parameters to return towards normal. However, at day 91 and 112 post dosing, values remained significantly depressed, with a stabilized chronic bone marrow aplasia. At day 91 and 112 post dosing, marrow cell counts, CFU-GM and Erythroid-CFU were decreased; marrow nucleated cell apoptosis and c-kit+ cell apoptosis were increased; peripheral blood erythrocyte, leucocyte, and platelet counts were reduced. We conclude that this is a model of chronic bone marrow aplasia which has many interesting features of AA. The model is convenient to use and has potential in several areas, particularly for investigations on mechanisms of AA pathogenesis in man.

In man, chloramphenicol (CAP), induces two major haemotoxic effects. First, a reversible, dose-related reticulocytopenia and anaemia developing during treatment. Second, a non-dose-related aplastic anaemia (AA), developing weeks after treatment, is often irreversible and fatal. In previous studies, we developed a mouse model of the reversible reticulocytopenia/anaemia using CAP succinate (CAPS); attempts to induce AA in the mouse with CAPS were unsuccessful; in the rat, CAPS induced only minimal haemotoxicity. We therefore wished to investigate haematological changes caused by CAPS in a third rodent, particularly in relation to the induction of significant 'late stage' bone marrow depression (AA). Female guinea pigs were gavaged with CAPS in three experiments. In a dose ranging study, CAPS (at 2500 and 3500 mg/kg) was administered daily for 9 days, and blood examined at 1 day post dosing. CAPS induced increased erythrocyte values (an apparent haemoconcentration effect), and reduced reticulocytes and femoral marrow nucleated cell counts (FNCC). In a second experiment, CAPS was given at 333, 666 and 1000 mg/kg (13 days); haematological changes were compared with results from the initial study, with evidence of dose-related effects. In a final experiment, CAPS was administered (825 mg/kg, 16 days) and blood studied at 1, 12, 28 and 63 days post dosing. At day 1, erythrocyte values were decreased (NS), and reticulocytes and FNCC were reduced; the marrow was hypocellular with erythroid depletion. At 12 and 28 days, values returned towards the normal range. At 63 days, parameters were normal. Thus, CAPS (825 mg/kg for 16 days) induced changes comparable to the reversible bone marrow depression seen in man; but there was no evidence of 'late stage' (i.e. at 63 days) marrow depression, as would be seen in a developing or overt marrow aplasia (AA). The guinea pig (like the mouse) is a model for the early events, but is not a good model for CAP-induced AA in man.

In toxicity studies, compound-induced changes are typically evaluated using a combination of endpoints and there are often a number of potential markers in biological fluids which can indicate toxic change in tissues and organs. However, some biomarkers are not specific to the organ of injury and therefore there is a continuing search for more sensitive and specific indicators of target organ toxicity. In experiments to assess the potential diagnostic usefulness of surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, skeletal muscle toxicity was induced in Wistar Han rats by administering 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD). The skeletal muscle toxicity was monitored using established endpoints such as increase in serum aldolase (Aldol), aspartate aminotransferase (AST) and histopathology, and also using SELDI retentate chromatography mass spectrometry of urine samples. Clear differences in urinary protein patterns between control and TMPD-treated animals were observed on the ProteinChip surfaces. Additionally a specific urine marker protein of 11.8 kDa was identified in TMPD-dosed rats, and the detection of the marker was related to the degree of skeletal muscle toxicity assessed by recognized clinical pathology endpoints. The 11.8 kDa protein was identified as parvalbumin-alpha. These experiments demonstrated the potential of urinary parvalbumin-alpha as a specific, noninvasive, and easily detectable biomarker for skeletal muscle toxicity in the rat and the potential of SELDI technology for biomarker detection and identification in toxicology studies.

Much toxicological research continues to be done using genetically undefined "outbred" stocks of mice and rats, although the case for using isogenic strains has been made repeatedly in the literature over a period of more than two decades. Also, very few studies are conducted using more than one strain, with the result that genetic variation in response is seldom apparent to the investigator. Here we report qualitative and quantitative strain differences in the haematological response to chloramphenicol succinate (CAPS) when administered by gavage at 500-2500 mg/kg for 7 days, to four inbred strains of mouse (C3H/He, CBA/Ca, BALB/c and C57BL/6) and one outbred stock (CD-1). CAPS caused anaemia and reticulocytopenia in all mouse strains, and leucopenia in the inbred strains but not in the outbred CD-1 stock. All four inbred strains showed significant (P0.01) responses to CAPS at lower dose levels than in CD-1 mice, which were phenotypically more variable than the inbred animals. A simulated experiment, using a sample of records from the present study, showed that the use of two mice at each dose level using CD-1, CBA, BALB/c and C57BL/6 (48 total mice), would have given a more sensitive experiment than the use of 47 CD-1 mice alone, and would also have shown that the response is partly strain dependent. These studies provide additional evidence that inbred strains, because of their greater sensitivity and other valuable properties, should be more widely used in toxicology.

Lacidipine is a widely used calcium-channel blocker, which has both long-lasting antihypertensive activity and also antioxidant properties. Previous studies have demonstrated the ability of lacidipine to reduce the development of atherosclerotic lesions in several animal models.The present study investigated the antiatherosclerotic potential of lacidipine in the apoE-deficient mouse, an experimental model of atherosclerosis showing progressively complex and widespread lesions which closely resemble the inflammatory-fibrous plaques seen in humans.Lacidipine was administered daily by gavage for 10 weeks at dose levels of 0 (control), 0.3, 1.0 and 3.0 mg/kg.Lacidipine administration reduces the extension of atherosclerotic lesions in the aorta of the apoE-deficient mouse without affecting plasma lipid levels. We also show that apoE-deficient mice have four-fold higher values of the proatherogenic peptide, endothelin, compared with the wild-type C57BL/6 mouse and that lacidipine administration reduced, in a dose-dependent manner, the concentrations of plasma endothelin.Lacidipine has anti-atherogenic effects in the apoE-deficient mouse, and reduces plasma endothelin concentrations.

The potential of the antibiotics chloramphenicol succinate (CAPS) and thiamphenicol (TAP) to induce aplastic anaemia in the female BALB/c mouse was investigated. CAPS was administered at 2000 mg/kg, and TAP at 850 mg/kg, daily by gavage, for 17 days. At 1, 13, 22, 41, 98 and 179 days after the final dose of each antibiotic, mice (n = 4 or 5) were sampled for haematological examination and haematopoietic stem cell assays. Both CAPS and TAP induced significant reductions in red blood cell count, haematocrit and haemoglobin values at day 1 post dosing; counts of colony-forming units-erythroid and colony-forming units-granulocyte-macrophage, were similarly significantly decreased at this time. All these reduced parameters returned towards normal at days 13 and 22. At days 41, 98 and 179, results for all haematological values and stem cell assays in both CAPS- and TAP-treated mice compared with the controls; there was no evidence of a reduction in peripheral blood values or bone marrow parameters at the later sampling points, as would be expected in a developing or overt bone marrow aplasia. We therefore consider that the administration of CAPS and TAP, which have been associated with the development of aplastic anaemia in man, induce a reversible anaemia, but not a chronic bone marrow aplasia, when given at haemotoxic dose levels for 17 days in the BALB/c mouse.

1 Chloramphenicol has been widely used in the treatment of serious infections including typhoid fever and meningitis. However, the drug is haemotoxic in man inducing firstly, a reversible, dose-dependent anaemia which develops during treatment, secondly, an often fatal aplastic anaemia with pancytopenia and acellular marrow, and thirdly, leukaemia. 2 We investigated the haemotoxicity of chloramphenicol succinate (CAPS) in female CD-1 mice in repeat dose studies, to compare the response with the reversible anaemia reported in man. Studies in male Wistar Hanover rats were also carried out. 3 CAPS was gavaged daily to mice at dose levels from 800–2000 mg/kg for seven days. Values were significantly reduced for reticulocytes at 1700 and 2000 mg/ kg, and for erythrocytes (RBC), haematocrit (HCT), and haemoglobin (Hb) at 2000 mg/kg. Platelet and white blood cell (WBC) counts were unaffected. 4 Mice were dosed with CAPS at 1400 mg/kg for 10 days and sampled at 1, 4 and 15 days after the last dose. At day 1 post dosing, RBC, HCT and Hb values were significantly reduced, but returned to normal (or above normal) by day 4 or 15. 5 CAPS from 2000–4000 mg/kg was gavaged to rats daily for 19 days. Hb values were significantly lower at 3600 and 4000 mg/kg; reticulocytes were not reduced. WBC and platelet counts, in general, were unaffected. 6 Levels of apoptosis in marrow mononuclear cells were increased in CAPS-treated mice, but not in CAPS-treated rats. Serum biochemistry parameters, in general, showed few changes of toxicological significance. 7 We conclude that the administration of CAPS to CD-1 mice induced haematological changes showing close parallels with the chloramphenicol-induced reversible anaemia seen in man.

The localization of surfactant protein (SP), A, B, C, and D mRNAs was examined in B6C3F1mice in the normal lung, and in a range of spontaneous proliferative lung lesions using nonisotopic in situ hybridization (ISH). The aim was to develop diagnostic markers, and if possible, throw further light on the histogenesis of these lesions. Tissues from 21 animals were examined, the lesions studied were: 4 alveolar epithelial hyperplasias, 12 alveolar/bronchiolar (A/B) adenomas, and 5 A/B carcinomas. Lung metastases of hepatocellular carcinoma (HCC) were used as controls. In the nonneoplastic lung, staining for SP A, B, and C mRNA was observed in normal and hyperplastic type II cells but not in the bronchiolar epithelium. SP mRN As were present in all lung tumors, with SPs A, B, and C being coexpressed in 10/12 (83%) of adenomas and 4/5 (80%) of carcinomas in both solid and tubulopapillary areas. No signals for SP D mRNA were noted in normal or neoplastic lung. Additionally, no staining for any SP transcript was observed in the HCC metastases examined. In summary, ISH for SP A, B, or C mRNA was a helpful aid in the diagnosis of proliferative lesions of the murine lung, enabling differentiation from hepatocellular metastases. Furthermore, this work provides strong support for the proposal that spontaneous lung tumors in B6C3F1mice are of alveolar, not bronchiolar origin, and consistently show type II cell differentiation. We suggest that such tumors should be referred to as alveolar adenomas and carcinomas.

Niosomes are vesicles formed by the self-assembly of nonionic surfactants in aqueous dispersions. They can entrap drugs and have been used experimentally as sustained drug delivery systems. Apart from conventional spherical niosomes, various types of vesicle ultrastructures can be formed by varying the composition of the vesicle membrane. Hexadecyl diglycerol ether (C16G2), cholesterol, and poly-24-oxyethylene cholesteryl ether (Solulan C24) in the ratio 91:0:9 gave polyhedral niosomes, whereas spherical and tubular niosomes are produced at a composition ratio of 49:49:2. The mean size of both polyhedral and spherical/tubular niosomes were within the range of 6 to 9 microm. Both types of vesicle were visualized by cryo-scanning electron microscopy. The properties of the two forms of niosomes were studied using luteinizing hormone releasing hormone (LHRH) as a model peptide. Analysis by high-performance liquid chromatography demonstrated high entrapment of LHRH acetate in polyhedral niosomes when prepared by remote loading methods using pH or (NH4)2SO4 gradients; in contrast, only low entrapment was achieved by passive loading methods (direct hydration at pH 7.4 or pH 3.0, dehydration-rehydration, and reversed-phase evaporation). In vitro studies demonstrated that both polyhedral and spherical/tubular niosomes were more stable in 5% rat skeletal muscle homogenate than in rat plasma. Also, polyhedral niosomes released more radiolabeled LHRH ([125I]LHRH) than spherical/tubular niosomes in both muscle homogenate and plasma. In clearance experiments in the rat, following intramuscular injection, both polyhedral and spherical/tubular niosomes gradually released [125I]LHRH into the blood, but some radioactivity remained at the injection site for 25 and 49 h, respectively. In contrast, [125I]LHRH in phosphate buffered saline was completely cleared from the injection site at 2 h. The release of drug is sustained by both niosome formulations, but spherical/tubular niosomes possess more stable membranes than polyhedral niosomes due to the presence of cholesterol.

Female B6C3F1 mice (12–14 weeks old) were dosed with busulphan (BU) by the intraperitoneal route at dose levels between 10 and 40 mg/kg on four occasions at 14 day intervals. Six weeks after the final dose, mice were given normal drinking water or drinking water containing chloramphenicol succinate (CAPS) at 4 mg/ml. Animals were killed at eight timepoints after the final BU dose, the last samples being taken at day 485 or 497 and a range of haematological parameters measured. During the experiment, no differences could be detected between mice receiving BU and CAPS and those receiving BU alone. Animals surviving to the end of the study displayed moderate leucopenia but no reduction in marrow cellularity nor was any effect on erythropoiesis apparent. Lymphocyte numbers were reduced in peripheral blood and bone marrow, and splenic cellularity was also reduced. Increased mortality was seen in animals which had received 40 mg BU/kg. In the first four months after BU dosing, animals killed due to ill health were pancytopenic with hypocellular marrows; deaths that occurred thereafter were due to lymphoma.

A non-experimental research design using questionnaires, was undertaken to find out what information out of that commonly given following myocardial infarction (MI), patients and their spouse/partners rate as being most and least important. These results were then compared with the results obtained from nurse subjects, who were given the same instrument to complete. Eighteen subjects were recruited for each of the three subject groups. Results indicated that some congruency existed between the three groups in terms of what they perceived as the most and least important categories of information. Yet, the scores for some informational categories included on the instrument, were significantly different between the nursing and two other groups (P < 0.01). However, in relation to the patient and spouse/partner groups, only a weak difference (P < 0.10) was found for the category 'dietary information'. These findings and others are discussed, and recommendations are made for improving the information giving process post-MI.

1 Chloramphenicol continues to be widely used in many parts of the world despite its known haematotoxicity. Until now, elucidation of the mechanisms involved and any attempt at amelioration of the toxic effects have been hampered by the lack of an animal model. 2 In this study neither acute nor chronic administration of chloramphenicol as its succinate ester in the drinking water produced anaemia in mice as assessed by changes in peripheral blood parameters. 3 Chloramphenicol could not be detected in the bone marrow when the antibiotic was administered either in the drinking water or by gavage, although it was detected in the serum. 4 In marrow taken from mice after chloramphenicol succinate administration and cultured in vitro, depression of the differentiation of immature committed erythroid progenitors occurred 15 min after administration of the antibiotic by gavage. However, recovery was beginning to occur at 48 h after administration of chloramphenicol succinate at 50 and 200 mg/kg and this was then followed by an `overshoot' response at the higher dose. A toxic effect was therefore achieved in the bone marrow but this was probably masked in the peripheral blood by enhanced proliferation. 5 Morphological evidence of apoptosis was seen in erythroid and myeloid precursors in mice treated with 200 mg/kg. 6 The data suggest that the effect of chloramphenicol was at the differentiation stage of the committed marrow progenitor cells rather than at the replication stage of the stem cells and therefore this response appears to mimic the reversible bone marrow depression seen in the treated patient.

Female B6C3F1 mice (12–14 weeks old) were dosed with busulphan (BU) by the intraperitoneal route at a range of dose levels between 10 and 40 mg/kg on four occasions at 14 day intervals. Six weeks after the final dose, mice were given normal drinking water or drinking water containing chloramphenicol succinate (CAP.S) at 4 mg/ml. Animals were killed at eight timepoints after the final BU dose, the final samples being taken at day 485 or 497. A range of haematological parameters were determined and lymphocyte subsets analysed. The inclusion of CAP.S in the drinking water did not affect the changes to haematological parameters or lymphocyte subsets induced by BU. Lymphocyte count was reduced to 51%–66% of control values in animals receiving 40 mg BU/kg on all occasions except day 85. However, the lymphocyte count was increased to 107%–143% of control values in animals receiving 10 mg BU/kg on all occasions except day 85. The large unstained cell (LUC) count was reduced to between 25%–47% of control values in animals receiving 40 mg BU/kg on all occasions, however, the LUC count was not increased at low BU dosage. At day 485/497, B lymphocyte counts were reduced to 48% (p

Demonstration of the improved doxorubicin pharmacokinetics and tumoricidal activity, after a single intravenous dose of 10mg kg-1 doxorubicin sorbitan monostearate (Span 60) based niosomes in the mouse adenocarcinoma (MAC) tumour model (Uchegbu et al., 1995) preceded the present study in which the activity of doxorubicin C16G2 (a hexadecyl diglycerol ether) based niosomes was evaluated against naive and established MAC tumour models. C16G2 niosomes were equiactive with doxorubicin solution. It is concluded that while in some tumour models, niosomal formulations demonstrate some advantages over the free drug, caution is advocated in the extrapolation of these results. The activity of doxorubicin C16G2 and Span 60 niosomes was also studied against a human ovarian cancer cell line and its doxorubicin resistant subline. There was a slight reduction in the IC50 against the resistant cell line when the drug was encapsulated in Span 60 niosomes in comparison to the drug in solution. Taking into account the in-vitro release characteristics of the various niosomal formulations, it is concluded that the use of niosomal formulations against multidrug resistance shows sufficiently encouraging results to warrant further study.

Purpose. Encapsulation of doxorubicin in niosomes was sought as a route to tumour targeting and improved tumoricidal through the alteration of doxorubicin pharmacokinetics and metabolism.

Hexachloro-1:3-butadiene (HCBD) causes segment-specific injury to the proximal renal tubule. A time course study of traditional and more recently proposed urinary biomarkers was performed in male Hanover Wistar rats receiving a single intraperitoneal (ip) injection of 45 mg/kg HCBD. Animals were killed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 28 postdosing and the temporal response of renal biomarkers was characterized using kidney histopathology, urinary and serum biochemistry, and gene expression. Histopathologic evidence of tubular degeneration was seen from day 1 until day 3 postdosing and correlated with increased urinary levels of α-glutathione S-transferase (α-GST), albumin, glucose, and kidney injury molecule-1 (KIM-1), and increased gene expression of KIM-1, NAD(P)H dehydrogenase, quinone 1, and heme oxygenase (decycling) 1. Histopathologic evidence of tubular regeneration was seen from day 2 postdosing and correlated with raised levels of urinary KIM-1 and osteopontin and increased gene expression of KIM-1 and annexin A7. Traditional renal biomarkers generally demonstrated low sensitivity. It is concluded that in rat proximal tubular injury, measurement of a range of renal biomarkers, in conjunction with gene expression analysis, provides an understanding of the extent of degenerative changes induced in the kidney and the process of regeneration.

Treatment of rats with beta-alanine increases the urinary taurine levels and markedly reduces the concentration of taurine in the liver. Dosing with carbon tetrachloride (CCl4) during treatment with beta-alanine results in a marked decrease in urinary taurine concomitant with a decrease in food intake. Treatment of animals with beta-alanine increases the hepatotoxicity of single doses of CCl4 as determined histologically and by measurement of serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. Urinary creatine is also raised significantly after the administration of CCl4 in beta-alanine-treated animals. However, the accumulation of triglycerides (TRIG) in the liver caused by dosing with CCl4 was not influenced by beta-alanine treatment. The data suggest that liver taurine levels may be an important factor in determining the degree of CCl4-induced cellular necrosis but not hepatic triglyceride accumulation.

We investigated the kinetics of circulating biomarker elevation, specifically correlated with morphology in acute myocardial injury. Male Hanover Wistar rats underwent biomarker and morphologic cardiac evaluation at 0.5 to seventy-two hours after a single subcutaneous isoproterenol administration (100 or 4000 µg/kg). Dose-dependent elevations of serum cardiac troponins I and T (cTnI, cTnT), and heart fatty acid–binding protein (H-FABP) occurred from 0.5 hour, peaked at two to three hours, and declined to baseline by twelve hours (H-FABP) or forty-eight to seventy-two hours (Serum cTns). They were more sensitive in detecting cardiomyocyte damage than other serum biomarkers. The Access 2 platform, an automated chemiluminescence analyzer (Beckman Coulter), showed the greatest cTnI fold-changes and low range sensitivity. Myocardial injury was detected morphologically from 0.5 hour, correlating well with loss of cTnI immunoreactivity and serum biomarker elevation at early time points. Ultrastructurally, there was no evidence of cardiomyocyte death at 0.5 hour. After three hours, a clear temporal disconnect occurred: lesion scores increased with declining cTnI, cTnT, and H-FABP values. Serum cTns are sensitive and specific markers for detecting acute/active cardiomyocyte injury in this rat model. Heart fatty acid–binding protein is a good early marker but is less sensitive and nonspecific. Release of these biomarkers begins early in myocardial injury, prior to necrosis. Assessment of cTn merits increased consideration for routine screening of acute/ongoing cardiomyocyte injury in rat toxicity studies.

The current studies demonstrate the effect of low-dose intraperitoneal (IP) administration of isoprotenerol (ISO) and subcutaneous (SC) versus IP routes of administration of ISO on serum cardiac troponin I (cTnI) levels in female Hanover Wistar rats, providing additional evidence to support acceptance of cTnI as a cardiac biomarker. At 2 hr postdosing with 0-500 microg/kg ISO, mean serum cTnI levels were increased in a dose-related fashion ator =10 microg/kg with no evidence of cardiac pathology. At 24 h, cTnI concentrations were generally at control levels, but histologic cardiomyocyte injury was evident in a proportion of the animals givenor =10 microg/kg. In a second experiment, rats given SC ISO at 5,000 microg/kg and necropsied at 0, 1, 2, and 4 hr postdosing had higher levels of serum cTnI than animals given the same dose IP.

Many anticancer drugs are myelotoxic and cause bone marrow depression; however, generally, the marrow/blood returns to normal after treatment. Nevertheless, after the administration of some anti-neoplastic agents (e.g. busulphan, BU) under conditions as yet undefined, the marrow may begin a return towards normal, but normality may not be achieved, and late-stage/residual marrow injury may be evident. The present studies were conducted to develop a short-term mouse model (a ‘screen’) to identify late-stage/residual marrow injury using a brief regimen of drug administration. Female BALB/c mice were treated with BU, doxorubicin (DOX), cisplatin (CISPLAT) or cyclophosphamide (CYCLOPHOS) on days 1, 3 and 5. In ‘preliminary studies’, a maximum tolerated dose (MTD) for each drug was determined for use in ‘main studies’. In main studies, mice were treated with vehicle (control), low and high (the MTD) dose levels of each agent. Necropsies were performed, and blood parameters and femoral/humeral nucleated marrow cell counts (FNCC/HNCC) were assessed on six occasions (from days 1 to 60/61 post-dosing). Late-stage/residual changes were apparent in BU-treated mice at day 61 post-dosing: RBC, Hb and haematocrit were reduced, mean cell volume/mean cell haemoglobin were increased and platelet and FNCC counts were decreased. Mice given DOX, CISPLAT and CYCLOPHOS, in general, showed no clear late-stage/residual effects (day 60/61). It was concluded that a brief regimen of drug administration, at an MTD, with assessment at day 60/61 post-dosing was a suitable short-term method/screen in the mouse for detecting late-stage/residual marrow injury for BU, a drug shown to exhibit these effects in man.

In 2005, the International conference on harmonization (ICH) recommended that all new human pharmaceuticals be tested for unintended immunomodulatory potential via a tiered approach. Included in this approach is a semiquantitative description of changes in the separate compartments of lymphoid tissue (also called enhanced histopathology). Chlorambucil was administered to Hanover Wistar rats at regular time points, followed by a treatment-free (recovery) period. Groups of treated and control animals were sacrificed regularly during both the treatment and recovery periods. Selected tissues were removed, weighed fresh and fixed in formalin, processed, and stained with hematoxylin and eosin. Blood samples and bone marrow smears were also obtained. With the use of enhanced histopathology, a description of the changes in lymphoid tissues and bone marrow was used as a means of assessing the susceptibility, and recovery, of the different lymphoid cell populations over time. A correlation with organ weights, flow cytometry data, and bone marrow cytology was achieved. The administration of chlorambucil in the Hanover Wistar rat provided a useful tool to examine the rate and sequence of changes in the lymphoid organs and bone marrow during treatment with, and the recovery from the effects of, a potent immunosuppressive agent.

Previous studies have shown that the enzyme copper/zinc superoxide dismutase (SOD-1) is increased in the urine of rats with carbon tetrachloride (CCl(4))-induced hepatotoxicity. The present experiments aimed to investigate further the usefulness of urinary SOD-1 as a non-invasive biomarker of liver injury. Two investigations were carried out, a dose response study and a time course study. In the dose response study, rats were given a single dose of CCl(4) at 0 (control), 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.80 ml/kg and urine samples collected from 12 to 36 h postdosing. In the time course study, rats were dosed at 0.80 ml/kg CCl(4) and urine sampled at 4, 12, 24 and 36 h postdosing. In both studies, the presence of SOD-1 in the urine was confirmed by Western blotting with an SOD-1 antibody. In the dose response study, serum SOD activity was elevated in all CCl(4)-treated animals and urinary SOD-1 activity was increased 2.2 times at the lowest dose (0.10 ml/kg) and 60.4 times at the highest CCl(4) dose level (0.80 ml/kg). In the time course study, urinary SOD-1 was first detected in samples collected from 4 to 12 h postdosing. We conclude that urinary SOD-1 has potential as a sensitive non-invasive biomarker of CCl(4)-induced hepatocellular injury.

Capillaria hepatica is a nematode parasite of wild rodents and other mammals. Adult worms inhabit the liver. Recently, during the necropsy examination of a group of 160 rabbits from a commercial supplier, firm pale or cystic areas (1-5 mm) were noted on the liver in thirteen animals. On further investigation, these animals were found to be infected with C. hepatica. The histopathological features of the infection in the rabbit are described for the first time and diagnostic features recorded. Lesions were identified predominantly in portal tracts consisting of dilated bile ducts with luminal debris, peribiliary inflammatory cell infiltrates, and fibrosis. Large granulomas (macrogranulomas) were evident in portal areas and involved the bile ducts. Macrogranulomas contained collections of characteristic C. hepatica eggs, macrophages, eosinophils, and lymphocytes. Small granulomas (microgranulomas), characterized by epithelioid macrophages surrounded by lymphocytes and eosinophils, were also identified. C. hepatica eggs were also observed in the lumina of the bile ducts and gall bladder. No adult C. hepatica worms were identified. Oocysts of Eimeria stiedae were also evident in the biliary epithelium in some animals. The unique characteristics of the C. hepatica life cycle are described, and the differential diagnosis of hepatic capillariasis is discussed.

Gaia, funded by ESA with EADS Astrium as the prime contractor, is an ambitious space observatory designed to measure the positions of around one billion stars with unprecedented accuracy and is currently planned for launch in 2011. The Gaia instrument will feature a focal plane containing 106 large area CCD91-72s manufactured by e2v technologies. This will be the largest CCD focal plane ever flown in space covering an area of 0.286m2. To ensure that the devices meet the required high specification, they undergo significant testing before being accepted by the end user. This involves geometrical, mechanical, environmental, endurance, electrical and electro-optical testing. With the flight phase contract for Gaia requiring the delivery of 130 flight grade devices (plus another 40 engineering devices of various grades), the volume of testing is an order of magnitude greater than and of similar timescale to, the typical space programmes e2v technologies are involved with. This paper will begin by providing an overview of the Gaia mission and the custom CCD91-72 that e2v technologies have designed for it. Next the various phases of the Gaia programme will be outlined and how e2v approached the test requirements for each stage. Problems encountered, lessons learned, and technical and logistical solutions implemented at each stage will be presented, to discuss how e2v technologies improved the quality of the test data whilst reducing the test times. There will be particular emphasis on the electro-optical testing and the test cameras on which this is performed.

Multiwalled carbon nanotubes (MWNTs) made highly water-dispersible by organic functionalization and radiolabeled with Indium-111 were shown to translocate through the kidney glomerular filter. On p. 225, multinational, multicenter research led by Kostas Kostarelos on dynamic imaging and tissue distribution of MWNTs in live animals is presented. The cover image presents a single plane single photon computed tomography whole body image of a rat following intravenous injection with the In-111 labeled MWNTs as seen through a 3D carbon NT projection. The radioactivity from carbon NTs was mainly localized in the kidneys, and accumulated in the bladder before clearance in the urine.

The aim of this study was the identification of a novel protein marker of hepatotoxicity in rat urine. Rats were dosed by gavage with carbon tetrachloride (CCl 4 ) to induce acute liver injury. Surface enhanced laser desorption/ionisation (SELDI) ProteinChip technology revealed the appearance of a 15.7 kDa protein in the CCl 4 -treated rat urine. One-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis (SDS–PAGE) identified an 18.4 kDa protein in the CCl 4 -treated rat urine. The appearance of either protein was coincident over a time course during which they first appeared at 12 h post-dosing, peaked at 36 h and had disappeared again within 3 days post-dosing. The protein was identified by in-gel digestion and nano-electrospray (nano-ES)-tandem mass spectrometry as Cu/Zn superoxide dismutase (SOD-1). SOD activity was found to be increased by 61.4-fold in CCl 4 -treated rat urine. Western blots of tissue homogenates from the rats revealed a time-dependent loss of SOD-1 from the livers of CCl 4 -treated rats matching the time course of SOD-1 appearance in urine. SOD-1 is not specifically located in liver; however, its appearance in urine in response to acute CCl 4 -induced hepatotoxicity is a novel finding; this coupled with loss from the liver following injury suggests urinary SOD-1 may be a potential marker of hepatotoxicity.

Presents a collection of slides covering the following topics: flight safety criticality; military system security; traceability; verifiability; through-life support capabilities; system complexity; sensitivity analysis; export licencing; and software source code access.