Autoradiographic Grains Research Articles

Decision criteria for the in vitro rat hepatocyte UDS assay.

The induction of unscheduled DNA synthesis (UDS) in rat primary hepatocytes is used as an in vitro screen for genotoxicity. The purpose of this study was to develop criteria for statistical evaluation of autoradiographic grain counts from the UDS assay. Based on our historical solvent control population (N = 17), we first established a predictive two-sided symmetric tolerance interval providing 95% confidence for coverage of 99% of the distribution of the mean net nuclear grain count for a new solvent control observation. The tolerance interval was determined to run from -2.82 to 4.03 net nuclear grains. When the concurrent solvent control (C) proves admissible (-2.82 less than C less than 4.03), the mean net nuclear grain counts from treatment groups are compared to the concurrent solvent control value. If the difference exceeds 3.09 at two successive concentrations, the test compound is considered positive. We have tested eight carcinogens (MNNG, AFB1, 4DAB, 4NQO, DMBA, 2AAF, DEN and 2AA) utilizing these criteria. All eight were positive. Four non-carcinogens (2AB, pyrene, anthracene and alpha NA) were negative. Each of these chemicals was tested twice using our standard protocol, which employs a preliminary range-finding cytotoxicity assay. We conclude that these criteria provide adequate sensitivity and specificity to be applicable for the evaluation of the results of routine UDS assays with compounds of unknown genotoxic potential.

Effect of colchicine on iron transport during stages of maturation and enamel pigmentation in rat incisor enamel: an autoradiographic study using 55Fe.

An autoradiographic study on rats was carried out with 55Fe and colchicine in order to investigate the Arole of microtubules in the iron transport process from rat incisor ameloblasts to the enamel surface. In the control rats injected with 55Fe, autoradiographic silver grains were distributed over the ameloblasts during the whole stage of enamel maturation and over the enamel surface at the enamel pigmentation stage. After the colchicine injection, the area of ameloblasts with smooth distal cell membranes increased in the maturation stage. These ameloblasts were heavily injured, and the iron-containing pigment was released from the cell. The number of silver grains decreased in these ameloblasts. However, the silver grains increased in the area of ameloblasts with ruffled distal cell membranes. At the enamel pigmentation stage, the silver grains over the ameloblasts increased, and those over the enamel surface were reduced. The accumulation and dislocation of the iron-containing pigment was noted in the ameloblasts at this stage. Lumicolchicine injection did not affect the grain distribution. These findings suggest that microtubules regulate the transport and secretion process of iron in rat incisor ameloblasts.

Asialoorosomucoid hepatobiliary transport is unaltered by the loss of liver asialoglycoprotein receptors in aged rats.

The hepatobiliary transport of asialoorosomucoid (ASOR) was examined in aging male Fischer 344 rats. The time course of transport of 125I-ASOR from blood to bile was identical in both senescent and young adult rats. Peak secretion occurred at approximately 35 minutes after injection via the femoral vein. Total secretion of radiolabeled ASOR (3.6% of injected dose), bile secretion and rate of secretion of radiolabeled ligand (approximately 2% of administered dose/hr/gm bile/liver) were not significantly different for the two age groups. Determination of the binding capacity for 125I-ASOR with liver plasma membrane-enriched preparations showed the membranes from old animals capable of binding approximately 50% less radiolabeled ligand as the young adult animals. Analysis of the distribution of 125I-ASOR autoradiographic grains along the liver lobule indicated extensive uptake of ligand in Zone 2 and 3 cells in senescent animals, whereas uptake in young rats was essentially limited to Zone 1 parenchymal cells. These results indicate that, contrary to the age-related loss of hepatic receptors for dimeric IgA and the concomitant reduction in hepatobiliary secretion of IgA, loss of ASOR binding capacity on liver plasma membranes from old animals is not reflected in diminished hepatobiliary secretion of ASOR. The loss of ASOR binding capacity is offset by the recruitment of Zone 2 and 3 hepatocytes along the liver lobule. This result suggests that hepatic metabolism and hepatobiliary secretion of macromolecules which exhibit a lobular gradient of uptake (e.g. ASOR) will be relatively less affected by loss of receptors compared to ligands which do not display such a gradient (e.g. IgA).

Molecular and cytogenetic studies on nucleolar cistrons (rDNA) in mouse leukemia cells

The gene dosage change of nucleolar cistrons (rDNA) in tumor cells has not been extensively studied. The present studies showed that increased dosage, as well as abnormal distribution of rDNA, was frequently associated with leukemia cells of SL/Ni and AKR mice. In normal SL cells, 37%, 39%, and 25% of rDNA was located in nucleolar organizer regions (NOR) of chromosomes #12, #18, and #19, respectively. Increase of rDNA/DNA was shown by hybridization on filter membranes in SL1, SL2, SL3, and M1 leukemia cells. Direct measurement of rDNA/DNA in G 1 cells revealed an 11% increase in synchronized M1 cells. The increased rDNA dosage was explained by trisomy 12 in SL1 and SL2, the ectopic NOR of #9 in SL3, and the double t(X;19) marker chromosomes in M1. On the other hand, in normal AKR cells, 27%, 29%, and 45% of rDNA was assigned to NORs of chromosomes #15, #16, and #18, respectively. The relative rDNA distribution among NORs estimated by autoradiographic grain counting was suggested to be abnormal in AKR leukemia cells despite their normal karyotype; 36% rDNA was shown to be in chromosomes #15 and #16, respectively, by relative reduction in chromosome #18 in AKR1; the trisomy 15 explained the increased rDNA in AKR2; a relative increase was found in chromosome #15 in AKR3. These results were discussed with reference to the reported NOR involvement in chromosome translocation and amplification in tumor cells.

Observations on rRNA gene replication in Chara vulgaris evaluated by in situ [3H] rRNA-DNA hybridization

ABSTRACT In situ [3H]rRNA-DNA hybridization showed that: (1) during the cell cycle in antheridial filaments of Chara vulgaris the rDNA gene content doubles during a 2h period in mid-S-phase, which corresponds to about 13% of the duration of this phase; (2) doubling of the number of grains occurs during mid-S-phase irrespective of the developmental stage of the antheridial filaments; (3) the numbers of autoradiographic grains in comparable phases of successive cell cycles preceding spermatid formation are the same. Thus the gradual reduction of total nucleolar volume (estimated per nucleus) occurring in successive cell cycles preceding spermatid formation is not associated with a decrease in the number of rDNA genes.

Montage: a system for three-dimensional reconstruction by personal computer

This paper describes a simplified system for serial section three-dimensional (3-D) reconstruction. A set of 9 software programs runs on a standard personal computer and produces camera-ready illustrations suitable for publication. The user enters trace points on a digitizing tablet from sections that have been already aligned. A 3-D view of the reconstructed object is generated which can be displayed with hidden lines removed. Analysis of volume, surface area and autoradiographic grain density are performed automatically. A relational database query language allows display and analysis of a selected subset of the data. The system runs under the UNIX operating system which allows the programs to be easily transported to new hardware or modified for other purposes.

Autoradiographic localization of [ 3H]-guanidinoethylmercaptosuccinic acid binding sites in rat islets of Langerhans

Incubation of unfixed, frozen sections of rat pancreas with tritiated guanidinoethylmercaptosuccinic acid (GEMSA) resulted in a localization of [ 3H]-GEMSA autoradiographic grains over the islets of Langerhans, suggesting that a carboxypeptidase H-like enzyme occurs in the islet.

Pulmonary epithelial permeability in rats exposed to O3.

Measurements of pulmonary epithelial permeability were made in rats exposed for 2 h to either purified air or ozone (O3) at concentrations of 0.8 or 2 ppm. [99mTc]Diethylenetriaminepentaacetate ([99mTc]DTPA) (492 daltons) and 125I-labeled bovine serum albumin ([125I]BSA) (69,000 daltons) were injected intravenously, and the lungs were lavaged 6 min later. In rats exposed to air, transfer of the larger tracer molecule (BSA) from blood to the lavage fluid was less than that of the smaller molecule (DTPA) when the amount of tracer in the lavage fluid was calculated as percent of the counts in femoral artery blood 5 min after injection, i.e., 1 min prior to lavage. In rats exposed to O3, alveolar permeability increased in a dose-related fashion, the increase under all exposure conditions was greater for the smaller molecule than for the larger one, and the permeability was reversible with time. The increase in permeability from blood to air was comparable to the increase from air to blood reported earlier (Bhalla et al., 1986). The increased permeability provided an early and reliable indicator of short-term O3 exposure effect in rats. Autoradiography by electron microscopy identified multiple pathways for BSA transfer from blood to the alveolar space. Grains produced by [125I]BSA were localized over endothelial and epithelial cell surfaces, were associated with cytoplasmic vesicles, were over cell surface invaginations, and were found in the cytoplasm of apparently degenerating cells. Although defects in tight junctions of alveolar type I cells were observed in lungs of rats exposed to O3, autoradiographic grains also appeared in intercellular spaces, with the intercellular junctions remaining intact.

Autoradiographic location of beta-adrenoceptor subtypes in cat colon smooth muscle.

In order to localize beta-adrenoceptors 125I-(-)pindolol (IPIN) was used in binding to sections from cat colon. The binding characteristics for IPIN to beta-adrenoceptors on colon sections were estimated by demonstrating reversible binding in the presence of isoprenaline and by steroselective binding to the isomers of propranolol. The binding of IPIN to both beta 1- and beta 2-adrenoceptors was shown by biphasic displacement curves in the presence of the selective beta-adrenoceptor compounds betaxolol, ICI 118.551 and procaterol. The colon sections were found to contain proportions of beta 1-adrenoceptors (30-50%) and beta 2-adrenoceptors (50-70%). In the autoradiographic studies, 100% of the developed grains after exposure of IPIN to the photographic emulsion were displaced by 50 microM of isoprenaline. By microscopic counting at autoradiographic grains, 30-40% of the grains were found in the circular smooth muscle, while 60-70% of the grains were found in the longitudinal smooth muscle. A concentration of 2 nM ICI 118.551 completely displaced all grains in the circular smooth muscle and partly displaced those found in the longitudinal smooth muscle. A high concentration of ICI 118.551 (1 microM) displaced all grains above background from the smooth muscle. It is concluded that the circular smooth muscle only contains beta 2-adrenoceptors, while longitudinal smooth muscle may contain a proportion of beta 1-adrenoceptors. Whether such a location of beta adrenoceptors can be related to the beta 1-adrenoceptor-mediated inhibition of colon motility can not be clarified from these studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidal binding sites in the ampulla of the human fallopian tube — autoradiographic and biochemical study

Most of the morphological changes which occur during the different phases of the reproductive cycle are hormone-dependent. Thus, the aim of this work was to demonstrate the fluctuations in the availability of steroid binding sites in the ampullary segment of the tube during the menstrual cycle. Specimens were obtained during routine gynecological operations and were prepared for histological examination. In addition, determinations of specific binding of estradiol and progesterone (specific activity was about 100 Ci/mmol) were made in soluene-dissolved tissue of ampullary mucosa. Autoradiographic grain counts of tissues which were incubated with [ 3H]estradiol and [ 3H]progesterone were performed on histological sections. The epithelial cell binding capacity was higher than that of the cells in the underlying lamina propria and this was found throughout the cycle. Estrogen and progesterone binding capacity was higher during the proliferative phase and decreased markedly during the secretory phase. Tissue binding of steroids appears to be inversely related to serum levels, which are lower in the proliferative phase and rise during the secretory phase.

Autoradiographic localisation of muscarinic receptors on guinea pig intracardiac neurones and atrial myocytes in culture.

Muscarinic receptors were localised on cells cultured from the atria and interatrial septum of newborn guinea pig heart by autoradiography using [3H]propylbenzilylcholine mustard. All of the intracardiac neurones observed in these cultures were specifically labelled: both the neuronal cell body and processes were evenly labelled over their entire surface. Autoradiographic grains were also uniformly distributed over atrial myocytes in culture. This even pattern of labelling of both neurones and atrial myocytes was not changed by the substitution of serum-supplemented growth medium with serum-free, hormone-supplemented, defined medium. The demonstration of muscarinic receptors on intracardiac neurones has important implications for studies on the roles of these neurones in the mammalian heart.

Inhibition of intercellular communication between mouse hepatocytes by tumor promoters

Tumor promoters can inhibit gap junction-mediated intercellular communication in cultured cells. Since intercellular communication is thought to be important in normal cellular growth control, inhibition of intercellular communication by tumor promoters may be an important mechanism by which preneoplastic cells escape normal growth regulation and progress towards neoplasia. We have evaluated the effects of tumor promoters on intercellular communication between B 6C 3F 1 mouse hepatocytes in primary culture. “Donor” hepatocytes were labeled with 4 μCi [5- 3H]uridine/ml for 4 hr. Nonlabeled “recipient” hepatocytes were then plated onto and cocultured with the labeled donors. Hepatocyte cultures were then treated with either the tumor promoters or the solvent vehicle. After 2–24 hr, the cells were fixed and processed for autoradiography. Intercellular communication between donor and recipient hepatocytes was detected as an increase in autoradiographic grains over recipient cells in contact with donor cells, indicating the passage of labeled nucleotides from donor to recipient hepatocytes. Autoradiographic grains were not observed over recipient hepatocytes not in contact with donor cells thus indicating negligible transfer of labeled nucleotide through the medium. Intercellular communication in untreated and solvent vehicle treated hepatocytes was detected in approximately 80% of the donor-contacting recipients after 8–12 hr culture. Phenobarbital, DDT (1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane), Aroclor 1254, lindane (1,2,3,4,5,6-hexachlorocyclohexane, γ-isomer), and TPA (12- O-tetradecanoyl phorbol-13-acetate), at noncytotoxic concentrations, significantly decreased hepatocyte intercellular communication in a dose-dependent manner.

Visualization of O6-methylguanine in target cell nuclei of dimethylnitrosamine-treated human pancreas by a murine monoclonal antibody.

A murine monoclonal IgG2b with high affinity and specificity for O6-methylguanine was developed. This allowed visualization of this adduct in paraffin-embedded sections by indirect fluorescence or immunoperoxidase methods. The antibody was used to study and compare the formation and persistence of this adduct detected by immunohistochemistry and the presence of 14C methyl by autoradiography in sections from adult human pancreas explants exposed to [14C methyl]dimethylnitrosamine (DMN) and organ-cultured for up to 7 days. O6-methylguanine was detectable randomly and with varied intensity in parenchymal cell nuclei and cytoplasms, 6 h after exposure to DMN. Following RNase treatment, however, only 7.5% of nuclei revealed measurable staining. The positive nuclei decreased to 2.5% within 24 h and remained about the same throughout the 7 days of culture. This correlated well with the change in percentage of positive nuclei for 14C methyl autoradiographic grains which were selectively condensed over the O6-methylguanine-stained nuclei. The data demonstrate a subpopulation of repair-deficient parenchymal cells of human pancreas exposed to DMN.

Quantitativein VivoAutoradiographic Localization of [125I_Tyr11] Somatostatin-14-and [Leu8,d-Trp22-125ITyr-125] Somatostatin-28-Binding Sites in Rat Brain*

Quantitative in vivo autoradiography was used to identify and compare the regional distribution of specific binding sites for blood-borne [125I-Tyr11]somatostatin-14 (S-14 section) and [Leu8,D-Trp22-125I-Tyr25]somatostatin-28 (S-28 section) in the rat brain. Rats were given intracardiac injections of 17 pmol S-14 section (with or without unlabeled S-14) or 17 pmol S-28 section (with or without unlabeled S-28). After whole body perfusion and fixation, brains were processed for light microscopic autoradiography of S-14 section- and S-28 section-binding sites. Of the peripheral tissues, the adrenal glands showed the highest uptake of S-14 section and S-28 section (determined by counting) and were subsequently processed for autoradiography for comparison with brain. Specific autoradiographic grains (ARG) associated with both radioligands were identified only in the circumventricular organs (CVOs). The highest ARG density associated with S-14 section was found in the area postrema, followed in decreasing order by the subfornical organ and the organum vasculosum lamina terminalis region. Median eminence (ME) contained virtually no specific S-14 section ARG. As with S-14 section, the highest ARG density of S-28 section-binding sites was also found in the area postrema, which labeled approximately equally with the two radioligands. This was followed by the ME, subfornical organ, and organum vasculosum lamina terminalis. The overall patterns of labeling of the CVOs with S-14 section and S-28 section showed significant differences, especially in the ME. Within the ME, labeled S-28 section was concentrated in a broad band throughout the external zone in a location identical to that of immunoreactive S-14. Analysis of dose-response curves obtained with 0.3-30 nmol unlabeled S-14 or S-28 revealed IC50 values for S-14 3- to 6-fold lower than those for S-28 for all labeled CVOs. With both S-14 section and S-28 section, the labelling density of the adrenal glands was double that of the area postrema. Adrenal binding of the radioligands was confined to the cells of the zona glomerulosa. We conclude: specific high affinity binding sites for S-14 section and S-28 section exist in the CVOs and the adrenal glomerulosa; and the 3- to 6-fold higher affinity of binding of S-14 to CVOs compared to S-28 together with the dissimilar patterns of labelling of the different CVOs by the two radioligands suggest the existence of separate populations of S-14 and S-28 receptors.

Quantitation, with a new assay, of Theiler's virus capsid protein in the central nervous system of mice.

We developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with a primary antibody, a biotinylated secondary antibody, or 35S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. We showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, we studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. We found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells.

Autoradiographic studies of chromosome replication during the cell cycle of Streptococcus faecium.

Analysis of the distribution of autoradiographic grains around cells of Streptococcus faecium which had been either continuously or pulse-labeled with tritiated thymidine (mass doubling time, 90 min) showed a non-Poisson distribution even when the distribution of cell sizes in the populations studied was taken into account. These non-Poisson distributions of grains were assumed to reflect the discontinuous nature of chromosome replication. To study this discontinuous process further, we fitted an equation to the grain distribution observed for the pulse-labeled cells that assumed that in any population of cells there were subpopulations in which there were zero, one, or two replicating chromosomes. This analysis predicted an average time for chromosome replication and for the period between completion of rounds of chromosome replication and division of 55 and 43 min, respectively, which were in excellent agreement with estimates made by other techniques. The present investigation extended past studies in indicating that the initiation and completion of rounds of chromosome replication are poorly phased with increases in cell volume and that the amount of chromosome replication may be different in different cell halves.

Computerized pattern recognition used for grain counting in high resolution autoradiographs with low grain densities

Using a video-image system coupled to a minicomputer with commercial image handling software, autoradiographic grains displayed in dark-field arc counted with a fast (ca. 3.5 min for 120 000 μm 2) and reliable (false scores < 5%) grain-recognizing FORTRAN program executed in the users memory. The grain counts are printed in a raster of adjustable size overlying a bright-field image, so that the counts can be related directly to the underlying histological structures.

Age-related RNA polymerase I activity in isolated nuclei of PHA stimulated human lymphocytes

In order to extend to the immune system previous findings that there is an age-related loss of hybridizability of the genes for ribosomal RNA (rRNA) in several tissues of mice, dogs and humans, we have investigated the function of the genes for rRNA in human T lymphocytes. These cells were chosen because they show a substantial decline in function with age, greater than that of other components of the immune system. rRNA synthesis was determined by measuring tritiated-UTP incorporation into acid precipitable counts as a result of the action of RNA polymerase I in nuclei isolated from phytohemagglutinin (PHA) stimulated peripheral-blood lymphocytes from 24 young adult and old human donors. The number of PHA-responsive cells from each donor was determined by counting grains in autoradiographs after a pulse of tritiated-uridine had been administered to them. The aggregate PHA induced synthesis of rRNA in the cultures decraesed as a function of the age of the donor. However, the number of PHA-responsive cells also dropped with age. When the data are normalized for the number of PHA-responsive cells in each culture, it appears that rRNA synthesis per PHA-responding cell does not significantly decline with age, even though there is a suggestion of a decrease after corrections are made. On the average, differences between individuals of the same age group were as great or greater than age-related differences.

Quantitative in situ hybridization using initial velocity measurements

In situ hybridization can be used to quantitate viral RNA at the single cell level by measuring levels of hybridization after saturation hybridization with an excess of cDNA probe has been achieved (1,2). In this paper we describe an alternative approach which consists in measuring the initial hybridization rate using a low concentration of cDNA probe and a short hybridization time. Under these conditions, we obtained a linear relationship between the number of autoradiographic grains and the number of viral genomes per cell in the range of 600 to 60,000 copies per cell of a 7-kb RNA genome. This approach allows an accurate measurement of copy number in a range for which saturation in situ hybridization is very difficult to achieve.

Tracheal permeability in rats exposed to ozone. An electron microscopic and autoradiographic analysis of the transport pathway.

Exposure of rats to ozone (O3), 0.8 ppm increases the tracheal permeability to 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) about twofold but to 125I-bovine serum albumin (125I-BSA) to a lesser extent. It is generally believed that exposure to air pollutants causes perturbation of tight junctions and formation of intercellular channels for the passage of molecules from airway lumen to blood. We now report that a second mechanism, vesicular transport, is operative in the transepithelial movement of molecules, that this mechanism is speeded in tracheas of O3-exposed rats, and that there is a concurrent delay in movement of BSA from connective tissue to capillaries after O3 exposure. Horseradish peroxidase (HRP) instilled in trachea was taken up by endocytic vesicles, which could be localized in apical as well as basal regions of ciliated and nonciliated cells. A count of HRP-positive vesicles and measurement of their surface area revealed an approximate twofold increase in O3-exposed rats over that in control animals breathing clean air; this paralleled a twofold increase in transport of 99mTc-DTPA from tracheal lumen to blood. An autophagocytic process induced in tracheal epithelial cells by O3 is proposed. Despite the difference in the size of HRP and BSA, the 2 molecules migrated through common pathways and were colocalized in the luminal membranes as well as in endocytic vesicles and intercellular spaces in double labeling experiments involving simultaneous detection of HRP by cytochemistry and 125I-BSA by autoradiography. This procedure proved particularly useful in detecting a dramatic accumulation of 125I-BSA autoradiographic grains in subepithelial connective tissue and HRP accumulation in intercellular spaces and at the basal membrane-connective tissue junction in O3-exposed rats.(ABSTRACT TRUNCATED AT 250 WORDS)