Dosimetry of the PIM1 Pion Beam at the Paul Scherrer Institute for Radiobiological Studies of Mice.

Significant past work has identified unexpected risks of central nervous system (CNS) exposure to the space radiation environment, where long-lasting functional decrements have been associated with multiple ion species delivered at low doses and dose rates. As shielding is the only established intervention capable of limiting exposure to the dangerous radiation fields in space, the recent discovery that pions, emanating from regions of enhanced shielding, can contribute significantly to the total absorbed dose on a deep space mission poses additional concerns. As a prerequisite to biological studies evaluating pion dose equivalents for various CNS exposure scenarios of mice, a careful dosimetric validation study is required. Within our ultimate goal of evaluating the functional consequences of defined pion exposures to CNS functionality, we report in this article the detailed dosimetry of the PiMI pion beam line at the Paul Scherrer Institute, which was developed in support of radiobiological experiments. Beam profiles and contamination of the beam by protons, electrons, positrons and muons were characterized prior to the mice irradiations. The dose to the back and top of the mice was measured using thermoluminescent dosimeters (TLD) and optically simulated luminescence (OSL) to cross-validate the dosimetry results. Geant4 Monte Carlo simulations of radiation exposure of a mouse phantom in water by charged pions were also performed to quantify the difference between the absorbed dose from the OSL and TLD and the absorbed dose to water, using a simple model of the mouse brain. The absorbed dose measured by the OSL dosimeters and TLDs agreed within 5-10%. A 30% difference between the measured absorbed dose and the dose calculated by Geant4 in the dosimeters was obtained, probably due to the approximated Monte Carlo configuration compared to the experiment. A difference of 15-20% between the calculated absorbed dose to water at a 5 mm depth and in the passive dosimeters was obtained, suggesting the need for a correction factor of the measured dose to obtain the absorbed dose in the mouse brain. Finally, based on the comparison of the experimental data and the Monte Carlo calculations, we consider the dose measurement to be accurate to within 15-20%.

Eritrocitose primária em cão: relato de caso / Primary erythrocytosis in dog: case report

A eritrocitose absoluta primária, também denominada de policitemia vera, é um distúrbio mieloproliferativo crônico de causa desconhecida, caracterizado pela proliferação clonal de células-tronco eritróides neoplásicas. Acomete cães de meia-idade entre seis e sete anos. As manifestações clínicas mais comuns são letargia, fraqueza, poliúria, polidipsia, sangramentos como epistaxe, hematúria, hematoemese, hematoquezia, até mesmo convulsões e ataxia. O diagnóstico é baseado em valores altos de hematócrito, geralmente acima de 70%, excluindo-se as causas de eritrocitose secundária. As concentrações séricas de eritropoietina estão normais ou diminuídas. O tratamento consiste em flebotomia e administração de hidroxiuréia. Relata-se o caso de uma cadela, raça Bichon Frise, 11 anos, que, no início do quadro, apresentou hematócrito de 84%, letargia, ataxia, mucosas congestas, cianose de língua, poliúria e polidipsia. Realizou-se o tratamento com hidroxiuréia durante oito anos, na dose de 15 a 30 mg/kg, a cada 24 horas, sem ocorrência de efeitos colaterais ou recidiva das manifestações clínicas.(AU)

Primary absolute erythrocytosis, also termed polycythemia vera, is a chronic myeloproliferative disorder of unknown cause. It is characterized by clonal proliferation of neoplastic erythroid stem cells. It affects middle-aged dogs between 6-7 years. The most common clinical manifestations are lethargy, weakness, polyuria, polydipsia, and bleeding such as epistaxis, hematuria, hematoemese, and hematochezia. Seizures and ataxia are also common. Diagnosis is based on high hematocrit values, generally above 70% excluding the causes of secondary erythrocytosis. Serum concentrations of erythropoietin are at a normal level or decreased level. Treatments consists of hydroxyurea and phlebotomy management. It is reported that case of female Bichon Frise, 11 years old who onset of the disease had a hematocrit of 84%, lethargy, ataxia, congested mucous membranes, tongue cyanosis, polyuria and polydipsia. The treatment with hydroxyurea was performed for 8 years, at a dose of 15 to 20mg/kg, every 24 hours, without occurrence of side effects or recurrence of clinical manifestations.(AU)

Effect of heparin on cell proliferation: lack of correlation with heparin binding sites on cell membrane.

In this study an attempt was made to correlate the in-vitro anti-proliferative effect of heparin with the heparin binding on the cell surface. Cells with different sensitivities to the anti-proliferative effect of heparin (BHK-21, FAO, SMC, BAEC, A-431, V-79, and skin fibroblasts) were incubated with [3H]heparin either in the presence or in the absence of unlabelled heparin. A saturable binding was found only in BHK-21, FAO, SMC, BAEC and V-79. Scatchard analysis revealed the presence of a single class of binding sites. The binding of [3H] heparin was efficiently displaced by unlabelled heparin, pentosan polysulfate and low-molecular-weight heparin, but not by dermatan sulfate. Although the sensitivity to the anti-proliferative effect of heparin varied considerably among the cell types (BHK-21 > SMC, FAO > BAEC > V-79), there was no correlation between the reduction of proliferation of these cells and either their heparin binding capacity or the number of binding sites per cell.

Effect of heparin derived fractions on the proliferation and protein synthesis of cells in culture.

We investigated the effect of sulfated oligosaccharides derived from depolymerization of heparin on the proliferation and protein synthesis of smooth muscle cells (SMC), hamster kidney (BHK-21) and lung (V-79) fibroblasts, rat hepatoma cells (FAO) and human promyelocytes (HL-60). BHK-21 and FAO showed the highest sensitivity to heparin; V-79 and HL-60 cells were completely resistant. LMWH (Low Molecular Weight Heparin) (MW 4.5 kD) was as effective as unfractionated heparin in reducing cell proliferation. The oligo-derivative 381/1 (MW 2 kD) was effective only on FAO and BHK-21 cells; oligo-derivative 381/2 (MW 1KD) had a negligible effect. The anti-proliferative effect was associated with an increased secretion of some protein classes. This effect was not present in heparin-resistant cells. In conclusion when the molecular size of heparin derivative is reduced below 2 kD (i.e. the size of a hexasaccharide) the anti-proliferative activity decreases dramatically.

Choice and standardization of test protocols in cytotoxicology: A multicentre approach.

A major problem that interferes with the introduction of in vitro tests for toxicological risk assessment is that of defining reliable standardized protocols. This issue was approached in the present study with an interlaboratory comparison of three cytotoxicity assays detecting chemical toxicity as impairment of cell viability in confluent cultures, reduction of colony forming ability, and inhibition of cell proliferation over 3 days of treatment. The study was performed using V79 cells, which are unable to activate indirectly-acting xenobiotics, and six chemicals with different mechanisms of action: two antioxidants (butylated hydroxyanisole and butylated hydroxytoluene), an inhibitor of protein synthesis (cycloheximide), an alkylating agent requiring metabolic activation (cyclophosphamide), an uncoupler of oxidative phosphorylation (dinitrophenol), and a genotoxic metal salt (potassium dichromate). The three tests produced the same rank of relative toxic potency for the tested chemicals, based on LC(50) values. The cell viability test appeared to be the most suited for the screening of unknown chemicals, given its simplicity and better reproducibility.

Evaluation of metabolic endpoints of acute cytotoxicity in V79 fibroblasts.

A group of cytotoxicity tests that detect alterations in cell metabolism were applied to obtain a preliminary classification of test chemicals, based on their main mechanism of toxicity. V79 cells were exposed to toxic compounds in 'acute' treatments (up to 2 hr) and the specificity of different endpoints of cytotoxicity was compared. We tested five directly acting chemicals: butylated hydroxyanisole, butylated hydroxytoluene, cycloheximide, potassium dichromate [Cr(VI)] and dinitrophenol using four assays measuring; [(3)H]thymidine uptake and incorporation into DNA, [(3)H]leucine incorporation into proteins, ATP pool size and cellular energy charge, and oxygen consumption. In the thymidine-uptake test the radioactivity of the nucleotide pool was hardly affected by low concentrations of the toxic chemicals and was reduced by about 20-30% at the 10(-4)m concentration, except by butylated hydroxytoluene; the latter which caused a marked inhibition of [(3)H]thymidine uptake. Thymidine incorporation into DNA was more specifically altered, showing a net inhibition by potassium dichromate, in a concentration-dependent fashion, and by butylated hydroxytoluene. Protein synthesis was more inhibited by potassium dichromate and butylated hydroxytoluene than by the specific inhibitor, cycloheximide. All chemicals reduced ATP concentration, but the energy charge was scarcely affected, reflecting a parallel decrease of the other adenine nucleotides. The assay measuring early changes in oxygen consumption produced the expected results with dinitrophenol, the antioxidants and potassium dichromate, and showed that cycloheximide also inhibits mitochondrial respiration, in agreement with the observed fall of intracellular ATP. All the chemicals tested induced a range of effects on the metabolic parameters analysed but specific pathways of toxicity may be inferred by comparing the results of the individual tests.

Plasma lipoproteins, tissue cholesterol overload, and skeletal muscle apolipoprotein A-I synthesis in the developing chick.

In the present study we investigated the changes of plasma lipids, lipoproteins, and tissue lipids that occur during the late embryonic life (5 days before hatching) and the postnatal period (0, 2, 7, 14, and 30 days after hatching) of the chick. The chick emerges from the egg with extreme hypercholesterolemia associated with a high level of cholesterol-rich VLDL + IDL. The density gradient profile of plasma lipoproteins showed that the concentrations of VLDL + IDL and LDL decreased during the first week of postnatal life, whereas HDL concentration increased sharply around hatching and remained stable afterwards. All plasma lipoprotein classes of the newborn chick (2 days from hatching) were enriched in cholesterol and cholesteryl esters; 2 weeks after hatching, the relative amount of cholesterol and cholesteryl esters decreased. In the newborn chick, plasma VLDL + IDL consisted of two populations of cholesteryl ester-rich lipoproteins: the main one (designated apoB-VLDL) contained apoB and no apoA-I; the other (designated apoA-I-VLDL) contained predominantly apoA-I. In the newborn chick there was an accumulation of free and esterified cholesterol in the liver and, to a lesser extent, in the skeletal muscle. These cholesterol deposits were depleted 2 to 7 days after hatching. The depletion in skeletal muscle was preceded by and associated with a striking increase in the synthesis of apoA-I in this tissue, as demonstrated by immunological methods and apoA-I mRNA measurements. In addition, apoA-I-containing HDL were secreted in vitro by explants of skeletal muscle of the newborn chick. The synthesis of apoA-I in the skeletal muscle decreased to the level found in the adult animal 1 week after hatching. It is likely that the rise of HDL and apoA-I in plasma observed 1-2 days after hatching reflects the production of apoA-I-containing HDL by skeletal muscle. We suggest that the cholesterol overload in skeletal muscle might stimulate the production of apoA-I which, in turn, would promote the removal of cholesterol from this tissue. The hypothesis that metabolic stimuli play a role in inducing apoA-I synthesis in skeletal muscle is supported by the observation that feeding the newborn chick a diet rich in proteins and lipids and free of carbohydrates delays the fall of apoA-I mRNA which normally occurs 1 week after hatching.