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PubMed1976

Localisation and tissue effects of tritiated dehydroretronecine in young rats.

Sixteen male Spraque-Dawley rats were injected with 3H-dehydroretronecine. The rats were subsequently evaluated for gross and microscopic changes. Scintillation counts and autoradiographic studies of the various organs demonstrated that the pyrrole accumulates in organs which were shown here and by others (Peterson et al; Allen and Hsu) to be affected most severely by dehydroretronecine and dehydroheliotridine. In the present experiment 3H-dehydroretronecine was shown to decrease the growth rate of the rats, decrease the percentage of circulating neutrophils and alter the hepatic mitotic index. Histologically, accumulations of label were seen in the glandular region of the stomach, in the liver, in Huxley's and Henle's layers of the hair follicles and in the epithelial cells lining the convoluted tubules of the kidney. Scintillation counts on the various tissues and autoradiographic evaluations of tissue sections indicate that there is a preferential localisation of radioactivity in the gastric mucosa which is postulated to be related to the pH in this area.

Animals↗
PubMed1987

Determination of erythrocyte life span in F-344, Wistar, and Sprague-Dawley rats using a modification of the [3H]diisopropylfluorophosphate ([3H]DFP) method.

The use of [3H]diisopropylfluorophosphate [( 3H]-DFP) to measure erythrocyte survival is well documented; however, the procedure has not been widely used in animal research primarily because it is more labor intensive than other methods. The [3H]DFP method was modified to simplify the preparation of samples for scintillation counting by using whole blood and to allow for small-volume serial blood sampling of individual animals. Eight-week-old F-344, Wistar, and Sprague-Dawley rats received 20 microCi [3H]DFP via the jugular vein. The latter two strains served to validate the procedure as significant data exist on the erythrocyte life spans of these rats. The red cell life span of the F-344 rat has not been previously investigated. Blood samples were collected from a tail vein at 8 days post-labeling and approximately three times per week until Day 33. Whole blood samples were individually prepared for scintillation counting. Blood radioactivity, corrected for sampling loss, was expressed as a percentage of the total activity at the time of initial sampling. Data were analyzed using the least-squares method of linear regression. Results, expressed as a mean erythrocyte life span in days (+/- 1 SE), for each strain were 66.3 +/- 1.6 for F-344, 59.8 +/- 2.2 for Wistar, and 61.0 +/- 1.3 for Sprague-Dawley rats. The results for the Wistar and Sprague-Dawley rats were in good agreement with published data for these strains, thus validating the modified [3H]DFP method for erythrocyte life span determination. In addition, the red cell life span of the F-344 rat was shown to be comparable with other rat strains.

Animals↗
PubMed1989

Fluoranthene-DNA adducts: identification and quantification by an HPLC-32P-postlabeling method.

A 32P-postlabeling procedure for identifying and quantifying fluoranthene (FA)-DNA adducts has been developed through modifications of the method of Randerath and collaborators. In this modified procedure, labeled adducts are separated chromatographically by high-pressure liquid chromatography (HPLC) and quantified by liquid scintillation counting. FA-modified DNA is digested to nucleotide 3'-monophosphates and nucleotide 3'-monophosphate adducts; unmodified nucleotides are then separated from adducts using a disposable C18 cartridge. Residual unmodified nucleotides, which reduce the efficiency of 32P-postlabeling of FA adducts, are removed by brief digestion with nuclease P1. This treatment selectively dephosphorylates unmodified nucleotide 3'-monophosphates, while FA adducts are minimally affected. FA adducts are then 5'-phosphorylated with polynucleotide kinase and [gamma-32P]ATP. Prolonged treatment with nuclease P1 then is employed to remove the unlabeled 3'-phosphate from adducted diphosphate nucleotides, following which adducts are separated by HPLC and quantified by liquid scintillation counting. Postlabeled microsomally-activated FA-modified DNA contained adducts derived from anti- and syn-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene. The identity of the major adduct in DNA-bound microsomally-activated FA was confirmed by this HPLC-32P-postlabeling method as an anti-2,3-dihydroxy-1,10b-epoxy-fluoranthene nucleotide adduct. Each step in the procedure was optimized with respect to experimental conditions, and the recovery of adducts was determined by analysis of DNA modified with [3H]FA. In repeated analyses of 2-50 micrograms DNA containing 1.8 adducts per 10(8) nucleotides, 10-15% of total DNA-bound [3H]FA was recovered as the major adduct; recovery was greater from DNA containing higher levels of adducts. The reproducibility of multiple analyses of the same sample was approximately 5%, and multiple analyses at different times were reproducible within experimental error. The limit of detection of the method was approximately 0.1 fmol adduct, representing a binding level of approximately 3 adducts per 10(8) nucleotides in 1 microgram DNA or approximately 1 adduct per 10(10) nucleotides in 500 micrograms DNA. Because the method is not limited with respect to the amount of DNA that can be subjected to analysis, the inherent sensitivity for adduct detection can be greatly enhanced by analysis of larger quantities of DNA.

Animals↗
PubMed1984

Distribution of [14C]aztreonam in rat tissue.

[14C]aztreonam was administered intramuscularly (50 mg/kg) to male and female rats. Groups of 10 rats (five male and five female) were sacrificed at 0.25, 2, 6, and 24 h after dosing. Blood and various tissues were removed from six rats (three male and three female) in each group for determination of total radioactivity and unchanged aztreonam by liquid scintillation counting and thin-layer radiochromatography. The remaining rats were prepared for whole-body autoradiography. Radioactivity was distributed rapidly to most tissues and was rapidly eliminated from blood into urine and bile. Concentrations of total radioactivity in kidney, liver, small and large intestines and their contents, and urinary bladder were higher than those in serum at all the times examined. Concentrations of unchanged aztreonam in serum, kidney, liver, and lung declined at about the same rate as did that of total radioactivity in the same tissues. The results of whole-body autoradiography essentially confirmed the results of the distribution of [14C]aztreonam as determined by liquid scintillation counting. No major differences between males and females were observed when concentrations in organs common to both were compared.

Animals↗
PubMed1993

The effect of aldosterone on phospholipid and phosphoinositide metabolism in rat submandibular gland.

It is well known that aldosterone stimulates Na+ transport in epithelia, including rat submandibular salivary gland. Aldosterone affects cells by causing nuclear-directed synthesis of aldosterone-induced protein. That protein then exerts its effect by: (1) phospholipid remodelling of the cell membrane (increasing permeability to Na+); (2) stimulating the Na+ pump; or (3) increasing ATP production by mitochondria. The purpose of this study was to determine if aldosterone stimulates membrane phospholipid or phosphoinositide turnover in the rat salivary gland and duct. Paired salivary glands were removed from 12 rats and main salivary-gland excretory ducts were removed from 16 rats. One-half of each group was treated with aldosterone (10(-6) M) while being incubated in Ringer's containing [32P]-orthophosphate for 2 h at 37 degrees C. The other half (controls) received no aldosterone during incubation. Phospholipids were extracted, separated and detected by thin-layer chromatography, autoradiography, and quantified by liquid scintillation counting. Results were expressed in dis/min (microM PO4)-1(h)-1. The turnover of membrane phospholipid fractions or percentage fractions was not changed significantly by aldosterone (p > 0.10). Paired submandibular salivary glands were also removed from 12 more rats and main submandibular excretory ducts from 18 rats. Tissue was incubated in 2 ml of Ringer's solution containing [3H]-myo-inositol for 2 h at 37 degrees C. Tissue was then placed in Ringer's containing LiCl and the experimental groups were challenged with aldosterone for 20 min at 37 degrees C. The tissues were then homogenized, and the inositol phosphate fractions quantified by column chromatography and liquid scintillation counting.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1979

The uptake in vitro of dyes, monosaccharides and amino acids by the filarial worm Brugia pahangi.

The uptake in vitro of various substances by Brugia pahangi was investigated using infective larvae obtained from Aedes aegypti and worms removed from Meriones unguiculatus at 2, 3, 10, 20 and 90 days post-infection. Worms incubated in growth medium 199 containing 1% Trypan blue possessed demonstrable dye in the oral orifice, the anterior oesophageal lumen and the external openings of the vulva and the cloaca or anus but the dye was not found in the gut lumen even after incubation for 24 h. No uptake of ferritin particles into the intestine of the worms was found and no fluorescence could be demonstrated in the gut lumen of worms incubated in medium containing 50% (v/v) fluorescein isothiocyanate-conjugated calf serum for up to 24 h. Trypan blue uptake by the gut of Aspiculuris tetraptera was clearly observed after incubation for several hours. The uptake of D-glucose and L-leucine by B. pahangi was demonstrated using autoradiographic and scintillation counting techniques and incorporation into worm tissues was detected. Glucose was found to be readily incorporated in the apical, glycogen-rich areas of the myocytes of worms of all ages studied and in the uterine epithelium of the adult female. In contrast, a lower incorporation of D-glucose was found in the eggs, embryos and vas deferens and especially in the gut. The incorporation of L-leucine occurred throughout the tissue of the worms during a 30 min incubation. Labelling was also located over the surface of the cuticle of the worms, when incubated for a period of 15 to 60 min in L-[H]leucine. Scintillation counting techniques demonstrated that there was no uptake of 14C-labelled L-glucose or sucrose by B. pahangi. The data presented on the uptake in vitro of nutrients or other compounds by infective larvae and adult stages of B. pahangi did not demonstrate an intestinal route of uptake but indicated that the transcuticular route of uptake may be employed.

Animals↗
PubMed1984

Quantitative autoradiography of muscarinic cholinergic receptors in the rat brain.

Using the in vitro autoradiographic technique with tritium-sensitive LKB sheet film and the liquid scintillation counting method, the distribution and the binding parameters of the muscarinic cholinergic receptors (MChR) were determined in various discrete regions of the rat brain. The results obtained in the present study were as follows: (1) Specific binding of [3H]QNB to the slide-mounted tissue sections increased slowly when incubated at room temperature; saturation occurred 2 h after incubation. Only 23% of [3H]QNB bound to the tissue section was dissociated 5 h after the addition of 20 microM atropine to the medium. These findings were very different from those obtained in the study using the tissue homogenates. (2) The regional distribution of MChR was determined using both autoradiographic and liquid scintillation counting methods. The distribution of MChR was heterogeneous, with highest densities in the striatum and nucleus accumbens and lowest in the globus pallidus, nucleus interpeduncularis and nucleus septi. Moreover, MChR were unevenly distributed within the subfields of each region. (3) In saturation binding studies using the slide-mounted tissue sections of 20 micron thickness the (Kd)app-values were similar but not exactly identical in 5 discrete regions, i.e. the striatum, somatosensory cortex, hippocampus (the subiculum + CA1 field), nucleus accumbens and gyrus dentatus, determined in the present study. The (Kd)app-value of each region was about 700 pM which was about 20 times higher than that obtained in the study using the tissue homogenates. However (Kd)app-values obtained with 5 and 10 micron tissue sections were approximately 3-fold lower.

Animals↗
PubMed1990

The comparative disposition of [14C]-fotemustine in non-tumourous and tumourous mice.

The distribution and excretion of radioactivity from [14C]-fotemustine was examined in mice with melanomas at different stages of development to determine whether the disease state substantially alters the disposition of the drug and its metabolites. Normal BDF1 mice and mice that had been subcutaneously grafted with B16 melanoma either 1, 3, 7, 14 or 21 days previously were used. The animals were killed at either 5 min, or at 3, 24 or 96 h after receiving an intravenous dose of [14C]-fotemustine (20 mg/kg) and were examined either by whole-body autoradiography or by liquid scintillation counting of excreta and tissues of interest. The majority of the [14C]-fotemustine dose was excreted in the urine, with similar amounts being measured in both non-tumourous animals (61.6% +/- 13.1%) and tumourous mice grafted 14 days previously (67.2% +/- 5.7%). Small amounts of radioactivity, again similar in both non-tumourous and tumourous mice, were recovered in the faeces (5.4% +/- 5.6% and 3.6% +/- 1.8%, respectively) and as carbon dioxide (7% +/- 3.5% and 6.4% +/- 1%, respectively), with minimal amounts being expired as chloroethanol (less than 1%). When mice were examined 5 min after dosing, there was extensive tissue distribution accounting for 75% +/- 10% of the dose. The highest concentrations determined by both whole-body autoradiography and liquid scintillation counting were measured in the excretory organs, with 33 and 28 micrograms Eq/g being found in the liver and kidney, respectively. High levels were also seen in the lung and plasma (19.8 and 19.5 micrograms Eq/g, respectively). Analysis of variance indicated that groups of tissues, such as the excretory organs, blood and plasma or the pigmented tissues, showed distinct but inconsistent patterns. Only tumours at 14 and 21 days of development were suitable for examination, and these showed levels of 12.1 micrograms Eq/g; however, the tumour-to-plasma ratio increased from between approx. 0.5 and 0.6 at 5 min to approx. 2 at 96 h after dosing, suggesting retention within the melanoma, whereas the ratio for the femur remained at approx. 1. Whole-body autoradiography showed that the distribution in the tumour was not uniform, but rather was concentrated in the peripheral area (presumably viable cells) as opposed to the central necrotic region. Thus, the high and sustained concentration of radioactivity found in the active cells of the melanoma may provide an explanation for the high efficacy of the drug.

Animals↗
PubMed1981

3H-fucose incorporation into bone following exposure to parathyroid hormone, dibutyryl cyclic-AMP and colchicine.

Radioautographic and scintillation counting procedures were used to examine the effect of parathyroid hormone (PTH), dibutyryl cyclic-AMP (DB-cAMP), and colchicine on the incorporation of 3H-fucose into macromolecular material in organ cultures of bone. Radioautography demonstrated 3H-fucose incorporation into bone cells, with the heaviest uptake occurring in osteoclasts. A minimal incorporation occurred in pre-osteoblasts and osteoblasts of the osteogenic periosteum, and in fibroblasts of the fibrous periosteum. PTH appeared to produce a heavier label in association with osteoclasts while decreasing the limited labeling associated with cells of the osteogenic and fibrous periosteum. DB-cAMP and colchicine both markedly reduced the labeling associated with osteoclasts, while the minimal labeling of other bone cells remained. By contrast, scintillation counting results indicated that PTH had little or no effect on 3H-fucose incorporation, while DB-cAMP and colchicine considerably reduced the amount of labeled macromolecular material. The incorporation of 3H-fucose into glycoproteins and the role of glycoproteins are discussed.

Animals↗
PubMedDate not supplied

[Significance of coagulation disorders and inflammatory immune reaction in an infectious model of rheumatoid arthritis. I. Systemic, shock-like coagulopathy and fibrin incorporation as indicators of rheumatoid manifestations in the erysipelas model].

A combined study employing plethysmographical, scintillation counting and coagulation methods indicates that a coagulation crisis in combination with fibrin overproduction may be an indicator of a beginning arthritis of rheumatoid character. Rats which received a single subcutaneous infection with erysipelas bacteria exhibit a shock resembling crisis two days post inoculation as substantiated by the consumption of coagulation factors II, V, VIII, XII and decrease of platelets. This consumption of coagulation factors is characterized by a rapid compensatory increase of platelets, antihaemophilic factor VIII and fibrin, 5 times more in the pig and 3 1/2 times more in the rat than in control animals. In adult rats the overproduction of fibrin is combined with an intense concealed consumption of fibrin in all organs of manifestation. Only in young rats an absolute consumption of fibrin is observed. The incorporation of fibrin into connective tissue is accompanied by fibrin consumption as demonstrated by immunofluorescence, by oedema of the paw, and by mesenchymal proliferation as substantiated by scintillation counting of incorporated 35SO4 and 3H-Proline, as markers for the beginning synthesis of ground substances and collagen. This model supports the importance of an initial vascular phase for the subsequent phase of manifestation in chronic rheumatoid diseases. It is discussed whether the organ specific permeability of the affected organs (joints, heart, arteries and eyes) may be a localizing factor of organ manifestation, parallel to the hormonal mesenchymal reaction.

Animals↗
PubMed1996

Cause and effect between concentration-dependent tissue damage and temporary cell proliferation in rat stomach mucosa by NaCl, a stomach tumor promoter.

This study was designed to test whether concentration or dose of NaCl was responsible for the initial tissue damage (after 1 min) and resulting temporary cell proliferation at 17 h in stomach mucosa of male F344 rats after gastric intubation of 0.65, 1.3, 2.6 and 3.7 M NaCl. Histological damage was studied by dual staining combining horseradish peroxidase-labeled Griffonia simplicifolia agglutinin-II staining (HRP-GSA-II) and periodic acid cold thionin-Schiff reaction (PATS). Cell proliferation was studied by measuring replicative DNA synthesis with liquid scintillation counting and by BrdU staining. NaCl at the same overall dose of 0.8 g/kg body weight induced different degrees of response depending on the concentration. For 4 ml of 0.65 M NaCl, there was no tissue damage after 1 min nor any increase in replicative DNA synthesis after 17 h in the pyloric mucosa. Administration of 1.3 M NaCl (2 ml), 2.6 M NaCl (1 ml) and 3.7 M NaCl (0.7 ml) induced concentration-dependent damage of the surface mucous cell layer after 1 min and increased replicative DNA synthesis after 17 h (P<0.05). Concentration-dependent increase in replicative DNA synthesis at 17 h was also induced with the same volume (1 ml) of 1.3, 2.6, and 3.7 M NaCl, while a volume-dependent increase in replicative DNA synthesis at 17 h was induced with 0.4, 0.7 and 1 ml of 3.7 M NaCl. However, a greater increase in replicative DNA synthesis was always observed when using higher NaCl concentrations at the same dose. Liquid scintillation counting was well-correlated with BrdU staining. These results suggest that a high concentration of NaCl is responsible for the initial tissue damage and resulting temporary cell proliferation during stomach tumor promotion.

Animals↗
PubMed1976

Uptake, distribution and metabolism of isoprenaline in the dog saphenous vein.

Dog saphenous vein strips were incubated with 2 muM 3H-isoprenaline. After 2 and 8 min of incubation small cross sections were cut and prepared for light and ultrastructural autoradiography. In addition, the accumulation of 3H-isoprenaline (3H-ISO) in the tissue as well as the formation of 3-O-methylisoprenaline (3H-OMI) were determined by liquid scintillation counting. The density of silver grains was 6.5 times higher on smooth muscle than on connective tissue. It decreased from the intima to adventitia when determined in the 2 min specimens, suggesting the existence of a selective transport of isoprenaline through the endothelium of the vein. The preferential distribution into the juxta-intimal part of the media which was observed after the 2 min incubation period was abolished by cortexone. Silver grain density, especially over the smooth muscle, was markedly increased by U-0521 and decreased by cortexone. Simultaneous treatment with cortexone and U-0521 led to a marked accumulation of silver grains over neuronal tissue; this accumulation was prevented by cocaine. Strips incubated at 0 degrees C and without oxygen showed almost no grains. In control conditions the saphenous vein strips removed 1968 +/- 19 pmoles-g-1 during 8 min of incubation. 86.3% of that value corresponded to 3H-OMI formation. O-methylation of 3H-ISO was strongly inhibited by U-0521 and, in a lesser degree, by cortexone. The influence of U-0521 on inhibition of O-methylation of 3H-ISO was not modified by cortexone. We found a linear correlation between silver grain densities per unit area of the vein strips and the 3H-isoprenaline accumulation determined by liquid scintillation counting, indicating a clear correspondence between silver grains and 3H-isoprenaline.

Animals↗
PubMed1981

Deposition and clearance of inhaled diesel exhaust particles in the respiratory tract of Fischer rats.

The fate of inhaled diesel particles was determined in male Fischer 344 rats using radioactive tracers of 131Ba and 14C. Test animals were exposed in a 'nose-only' inhalation chamber for 40-45 min to diluted diesel exhaust generated from diesel engines burning type 2D diesel fuel containing either 131Ba labelled barium dodecylbenzene sulfonate or 14C labelled n-hexadecane. Immediately after exposure, the deposition efficiency of inhaled diesel particles in the respiratory tract was determined to be 15 +/- 6% by external gamma counting of 131Ba and 17 +/- 2% by liquid scintillation counting of 14C in the lung tissue samples. Elimination of the particles was observed by measuring the 131Ba activity in which 40% of the initial deposition was excreted in the feces via the GI tract in 4 days. The long-term retention was determined with the 14C tag in the insoluble 'core' of the diesel particles. Two distinct phases of clearance were evident in the experimental data collected up to 105 days. Clearance half-times of 1 day and 62 days were found for mucociliary and alveolar clearance, respectively. A small fraction of the particles, about 6% of the initial deposition, was found in the mediastinal lymph nodes after 28 days. This demonstrated that the lymphatic system was also involved in the removal of diesel particles from the pulmonary airways.

Aerosols↗
PubMed1994

The radioanalytical laboratory at Pelindaba. Some applications for environmental and health monitoring.

This article deals with the radioanalytical work performed at Pelindaba, Atomic Energy Corporation of South Africa. It reports on the activities of a commercial laboratory, employing analytical techniques, such as total alpha/beta counting, alpha spectrometry, liquid scintillation counting, gamma spectrometry, and neutron activation analysis. Techniques discussed in more detail include semicyclic activation analysis for the determination of fluorine in vitamin mixes, as well as the determination of uranium and radium in urine samples for personnel monitoring.

Environmental Monitoring↗
PubMed1986

Use of 125I-labeled-histamine-cyclosporin C for monitoring serum cyclosporine concentrations in transplantation patients.

We synthesized an 125I-labeled-histamine-cyclosporin C tracer, to obviate the use of tritiated tracer in radioimmunoassay of cyclosporine. With this tracer, the assay results varied linearly with concentration up to at least 800 micrograms/L. The within-assay CV was 6.6% at 39 micrograms/L, 4.2% at 100 micrograms/L, and 7.0% at 300 micrograms/L (n = 15). The between-assay CV was 10.0, 6.4, and 7.8% for the same respective concentrations. Comparison with an assay involving tritiated tracer (x) showed good agreement of results: y = 3.81 + 0.927x (r = 0.975, n = 604). Analytical recovery ranged from 100 to 106%. We also compared another commercially available radioiodinated tracer ("125Iodocyclosporin"; Immunonuclear Corp.). Our tracer appeared to be more specific for cyclosporine, as determined by assaying chromatographic fractions of bile extract from a patient being treated with cyclosporine. Results with use of our tracer compared favorably with those obtained with the tritiated tracer, and our assay has the advantages of gamma counting vs liquid-scintillation counting.

Chromatography, High Pressure Liquid↗