Evaluation of four methods for scoring cytoplasmic grains in the in vitro unscheduled DNA synthesis (UDS) assay

The in vitro unscheduled DNA synthesis (UDS) assay measures DNA repair (incorporation of [³H]thymidine) following in vitro treatment of rat primary hepatocytes. The autoradiographic method was used to detect UDS by counting developed silver grains in the photographic emulsion overlaying nuclei and cytoplasmic areas of the hepatocytes. In this communication we report results using 4 scoring methods: (1) the most heavily labeled cytoplasmic areas adjacent to the nucleus (our standard method), (2) the cytoplasmic area left of the nucleus, (3) the cytoplasmic areas left and right of the nucleus, and (4) 2 cytoplasmic areas whose positions were selected at random. Rat primary hepatocyte cultures treated with a medium control, a solvent control (dimethyl sulfoxide) and 5 known genotoxic chemicals (2-acetylamino-fluorene, dimethylnitrosamine, diethylnitrosamine, methyl methanesulfonate and ethyl methanesulfonate) were scored using these 4 methods. The average or maximum cytoplasmic grain count was subtracted from the nuclear grain count to yield net grains/nucleus (NG). In general, NG counts for Methods 2,3 and 4 were similar, although shifted about 3–10 grains higher than Method 1 for controls and most treated groups. Methods 2, 3 and 4 showed more experiment-to-experiment variability in sensitivity for detecting statistically significant increases in treated groups than did our standard method. Thus, the alternative methods afforded no consistent improvements in sensitivity or reduction of variability for this assay. Subtraction of the average or the highest cytoplasmic count had virtually no effect on the sensitivity of the assay, but simply requires an appropriate adjustment of the criteria for a positive response.     

Statistical criteria for evaluating chemicals as positive or negative in the hepatocyte/DNA repair assay

The usefulness of the hepatocyte/DNA repair assay was enhanced when statistical techniques were applied to the evaluation of a chemical as positive or negative. Using our test procedure, we are 95% confident that the false positive rate is less than 1% if the net nuclear grain count for the test chemical is 3 counts higher than the solvent control for the same animal. Additionally, quantitation of the proportion of cells in repair should be used to corroborate the results of the net nuclear grain count and is recommended for inclusion in the criteria for evaluating a chemical.     

DNA repair synthesis induced by N-hydroxyurea, acetohydroxamic acid, and N-hydroxyurethane in primary rat hepatocyte cultures: comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increases over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genotoxicity may be a common property of aliphatic hydroxamic acids.     

不同因子驱动下通过不同途径发生的红树林斑块数量和面积变化量的计量方法

为深入阐明区域红树林空间演变机理,需对红树林斑块数量和面积在不同因子驱动下通过不同途径发生的变化量进行准确计量。提出了在GIS平台支持下的通过空间叠置分析方法进行斑块数量和面积变化量计量的两种方法——精确计量法和整体计量法。首先将前后两期通过遥感图像提取的红树林斑块分布图、遥感图像进行叠合,采用视觉信息叠合方法,将全部斑块划分为众多具有相同主要驱动因子和变化途径的分析单元;若前后期遥感图像能够精确配准,采用精确计量法计量:通过线与多边形叠置方法,用前期斑块的线状图对后斑块的面状图、后期斑块的线状图对前斑块的面状图分别进行切割,每个分析单元得到多个亚斑块,逐一确定每个亚斑块的驱动因子、变化途径,据此统计每个分析单元中斑块数量和面积在不同因子驱动下通过不同途径发生的变化量;若前后期遥感图像难以精确配准,采用整体计量法计量:对于每个分析单元,根据斑块恢复的难易程度、面积和斑块数量变化量的大小,确定其主要驱动因子和主要变化途径,该分析单元前、后期斑块数量和面积之差即为其在监测期间由该因子驱动通过该途径发生的斑块数量和面积变化量。尽管整体计量法对红树林空间演变机制分析的结果与精确计量法存在一定差异,但也属于定量分析范畴,都能深刻阐明红树林空间演变机制,能够全面、准确地反映了区域红树林斑块数量和面积在监测期内增加、减少的动态过程。     

Quantitative ultrastructural analysis of barley sperm

Summary Complete serial ultrathin sections of seven sperm pairs, computer-assisted measurements of cell, nuclear and organelle surface areas and volumes, and three-dimensional imagery were used to demonstrate that a process of cytoplasm and organelle elimination occurs during sperm maturation in barley. The number of mitochondria per sperm cell is reduced by 50%; sperm cell surface area and volume are reduced by 30% and 51% respectively. Mean volume and surface area per mitochondrion are significantly less in mature sperms. No examples of mitochondrial fusion or degeneration were observed within sperm cells. These data, along with observations of plasma membrane apposition and vesiculation within cytoplasmic extensions containing mitochondria, support the proposition that cytoplasm and organelle loss results primarily from the formation of cytoplasmic projections that are subsequently discarded from the sperm cell body. Comparisons of the quantitative data, including the number of mitochondria, indicate that differences between sperm cells of a pair are absent to very slight. Spatial organization within the pollen grain is such that the mature sperms, as well as the sperms and vegetative nucleus, are not in close proximity.     

Purkinje cell maturation in the light of RNA synthesis

Wistar rats 1- to 90-day-old received an injection of 3H-uridine and were killed 20 min to 44 h later. Autoradiographic examination revealed the highest grain count densities in Purkinje cell nuclei around postnatal day (PD) 6 while the incidence of labelled nuclei stayed at the peak values till PD 15. Silver staining of Purkinje cell nuclei showed that the expression of nucleolar r-RNA coding genes is maximal at PD 15; in some cells it even slightly exceeds adult values. After PD 15, the percentage of labelled Purkinje cell nuclei declined; this was more pronounced in the nucleolar region than outside the nucleolus. The percentage of cells with cytoplasmic labelling culminated on PD 15. The highest grain counts were found in Purkinje cell cytoplasm on PD 6 at 44 h p.i. interval. Reversal in nuclear grain counts at 2 and 6 h p.i. intervals observed between PD 15 and PD 25 suggests faster degradation, or processing and export, of a newly synthesized nuclear RNA in these age groups. Frequency distribution analysis of grain count densities revealed a small group of Purkinje cells with higher incorporation of 3H-uridine both in the nucleolar region and the whole nucleus at PD 15. In situ hybridization of 3H-r-RNA revealed a slight binding excess to DNA of some Purkinje cell nuclei but not in granule cells of 1-month-old rats. These data, together with those published recently by Brodsky et al. (1985), indicate an uneven structural organization and partial overexpression of the genom coding r-RNA synthesis in the population of Purkinje cells.     

Computerized morphometric discrimination between normal and tumoral cells in oral smears

The oral exfoliative cytology allows a quick and fairly accurate assessment of suspicious lesions of the oral cavity. Within this context, our paper proposes a quantitative approach, focusing on the construction of a classifier for detecting the presence of the tumoral cells on oral smears. The design of the classifier relies on a detailed computerized analysis of the individual morphometric features exhibited by two large known populations of normal and tumoral cells, respectively; the digital image processing was performed in the Zeiss KS400 environment. The classifier was implemented as a neural network with step activation function, whose parameters were obtained from an adequate training, based on the nuclear and cytoplasmic areas of the cells belonging to the two populations. Our procedure based on this classifier was meant to operate by identifying the tumoral or normal nature of any cell randomly selected from a smear. To identify the nature of an arbitrary cell, its nuclear and cytoplasmic areas are presented at the input of the classifier. The classification procedure was tested on several smears, and the results coincided with the pathological diagnosis in all the considered cases. The performances of our approach are discussed in comparison with other analytical methods previously reported in oral exfoliative cytology. These discussions emphasize the role of numerical information exploited for the classifier design, concluding that the individual morphometric features are more meaningful than the global characterization of smears by mean values.     

NUCLEAR INCORPORATION OF RADIOACTIVE DNA PRECURSORS AND PROGRESSION OF CELLS THROUGH S

Bone marrow blast cells of nine children with untreated acute leukaemia (five lymphoid, three myeloid, one monocytic), myeloid precursor cells of a haema-tologically normal child and thoracic duct lymph cells of a patient with sclerodermia were pulse-labelled in vitro with tritiated thymidine and/or tritiated cytidine. Combined radioautographic and cytophotometric techniques were used for the determination of the median nuclear size and the median grain count of labelled cells in different segments of S. It was found that the median grain count reached maximum values in the second or third quarter of S in all cell populations studied, and that the variation, during S, of the median grain count appeared to be independent of the median DNA synthesis times of the cell types investigated. In six cell populations a clear-cut inverse relation existed between the number and the median grain count of labelled cells in different segments of S. In three populations this relation was less apparent and in two it was not found at all.     

Quantitative and qualitative differences between benign and malignant mesothelial cells in pleural fluid

Quantitative as well as qualitative cellular parameters were investigated in 20 cases of reactive mesothelial proliferations and 40 cases of primary pleural mesotheliomas. The two groups showed statistically significant differences in the nuclear areas, cytoplasmic areas and standard deviations. In the mesotheliomas, the mean nuclear area and the mean cytoplasmic area were larger than in the reactive proliferations. Nine cases could not be properly classified with these quantitative parameters alone. Qualitative analysis revealed highly characteristic features in the mesotheliomas. Morula formation as well as irregular and coarse reticular chromatin patterns were strongly indicative of mesothelioma. The location and shape of the nucleus and the type and amount of cytoplasmic vacuolization gave additional information for distinguishing between the two groups.     

Planimetry of bronchoalveolar macrophages. Importance of preparation and staining techniques.

Bronchoalveolar lavage seems a well-established, valuable research tool in the study of alveolar macrophages. The influence of fixation, cytocentrifugation and staining procedures on the cellular and nuclear size has been investigated by planimetry. As a reference, mean profile areas of 109 and 39 microns 2 for cell and nucleus, respectively, were measured for alveolar macrophages suspended in the hemocytometer. For comparison, stained Cytospin preparations were measured. Unfixed cells were compressed during cytocentrifugation. The cellular profile areas for Cytospin preparations increased about 15% and 70% after May-Grünwald-Giemsa and Feulgen staining, respectively. The nuclear area was approximately 25% larger for both staining procedures as compared to the hemocytometer values. When the cells had been fixed prior to cytocentrifugation, these differences were less conspicuous. No significant differences were observed after May-Grünwald-Giemsa staining, showing a cellular area of 114 microns 2 and a nuclear area of 45 microns 2. Depending on the staining procedure, low nucleus:cell ratios (31%) were observed after Feulgen staining, while higher values (about 43%) were measured after May-Grünwald-Giemsa staining, regardless of which fixation or centrifugation procedure had been followed. In conclusion, these findings indicate that fixation should be carried out in order to prevent cell changes resulting from cytocentrifugation. Moreover, different staining procedures considerably influence the measurement of cellular and nuclear profile areas and the determination of nucleus:cell ratios.     

Combined blood cell counting and classification with fluorochrome stains and flow instrumentation.

A multiparameter flow cytophotometer was used to count and classify fixed human blood cells fluorochromed with a mixture of ethidium bromide (EB), brilliant sulfaflavine and a blue fluorescent stilbene disulfonic acid derivative (LN). The system measures light scattered by the cells and absorption at 420 nm for all cells. In addition, nuclear EB fluorescence (540 leads to 610 nm) and cytoplasmic fluorescence from LN (366 leads to 470 nm), brilliant sulfaflavine (420 leads to 520 nm) and EB exicted by energy transfer from LN (366 leads to 610 nm) are measured for all nucleated cells. This information is sufficient to perform red and white blood cell counts and to classify leukocytes as lymphocytes, monocytes, basophils, eosinophils or neutrophils. Light scattering and/or nuclear and cytoplasmic fluorescence values may be further analyzed to obtain the ratio of immature to mature neutrophils. Counts produced by the system are in reasonable agreement with those obtained by electronic cells counting and examination of Wright's-stained blood smears; some discrepancies appear to be due to systematic errors in the manual counting method.     

THE ROLE OF THE NUCLEOLUS : I. Tritiated Cytidine Activity in Liver Parenchymal Cells of Thioacetamide-Treated Rats

Male rats of the Sherman strain were fed for 2 weeks a diet of ground purina rat chow containing 0.04 per cent thioacetamide. Animals were injected intraperitoneally with tritiated cytidine, 200µc/100 gm body weight, and sacrificed in pairs, a control and a thioacetamide-treated rat, at prescribed intervals. Liver tissues were preserved with the freeze-substitution method and postfixed in anhydrous OsO₄. Other samples were fixed directly with an acetic acid-ethanol mixture (1:3). AR-10 stripping film was applied to 2- and 4-µ sections and exposed for appropriate lengths of time. Nuclear and nucleolar volumes were obtained by direct measurement. Cytoplasmic volumes were obtained with the aid of Chalkley ratios. Nucleolar and cytoplasmic RNA concentrations were calculated from cytophotometric extinction (E_{540 mµ}) measurements. Data were expressed as grains/unit area, grains/unit area/concentration (or specific activity) and grains/total structure. In the liver parenchymal cells of thioacetamide-treated rats, the nucleolus shows vast increases in volume, RNA content, and grain count/total structure, 14-fold, 25-fold, and over 30-fold, respectively. The nucleus increases 2-fold in volume and about 3-fold in total grain count. Cytoplasmic volume increases only 20 per cent and displays a total grain count about equal to that in the control. The time course of incorporation curves for nucleolus and non-nucleolar nucleus (NNN) contain 2 distinct turnover fractions, rapid and slow. Both fractions were increased after thioacetamide treatment but remained proportional to those of controls. The unique stimulated RNA turnover in the nucleus and nucleolus, coupled to a "normal" turnover in the cytoplasm, suggests that this nuclear-nucleolar loss of label does not represent an exclusive passage of formed nuclear RNA to the cytoplasm.     

Automated quantitative analysis of single and double label autoradiographs.

A method for the analysis of silver grain content in both single and double label autoradiographs is presented. The total grain area is calculated by counting the number of pixels at which the recorded light intensity in transmission dark field illumination exceeds a selected threshold. The calibration tests included autoradiographs with low (3H-thymidin) and high (3H-desoxyuridin) silver grain density. The results are proportional to the customary visual grain count. For the range of visibly countable grain densities in single labeled specimens, the correlation coefficient between the computed values and the visual grain counts is better than 0.96. In the first emulsion of the two emulsion layer autoradiographs of double labeled specimens (3H-14C-thymidin) the correlation coefficient is 0.919 and 0.906. The method provides a statistical correction for the background grains not due to the isotope. The possibility to record 14C tracks by shifting the focus through the second emulsion of the double labeled specimens is also demonstrated. The reported technique is essentially independent of size, shape and density of the grains.     

THE FINE STRUCTURE OF THE NUCLEUS AND NUCLEAR ASSOCIATIONS OF DEVELOPING CARPOSPORANGIA IN POLYSIPHONIA NOVAE-ANGLIAE (RHODOPHYTA)1

A high degree of activity of the nuclei in the developing carposporangia of the red alga Polysiphonia novae-angliae Taylor is described. Profiles of the nucleus are greatly convoluted, resulting in a much increased surface area. Regions where endoplasmic reticulum substitutes for the nuclear envelope are frequently observed. Various cytoplasmic reserves are associated with the nucleus during carposporangium maturation. Lipid bodies, fibrillar bodies, and striated vesicles (or cylindrical bodies) may occur within the nucleoplasm beneath areas of the nuclear envelope substituted by ER. Granules of Floridean starch are observed in proximity to the outer surface of these same areas as well as the nuclear membrane. The homologous nature of the nuclear envelope with the ER is stressed. The role of the nucleus as being actively involved in the synthesis of materials is suggested.     

Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy.

BACKGROUND: The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. METHODS: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. RESULTS: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error. CONCLUSIONS: The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.     

Dexamethasone and corticosterone receptor sites. Differential topographic distribution in rat hippocampus revealed by high resolution autoradiography

High resolution light microscopic autoradiography was used, together with regional surveys and combined acridine orange staining, to define in rat hippocampus cellular and subcellular sites of concentration and retention of 3H dexamethasone and to compare the topographic pattern of labeling with that of 3H corticosterone. Nuclear uptake of 3H dexamethasone in the hippocampus is demonstrated for the first time in vivo. With 3H dexamethasone, strongest nuclear radioactive labeling was observed in certain glial cells throughout the hippocampus, followed by strong nuclear labeling in most neurons in area CA1 and in the adjacent dorsolateral subiculum and weak nuclear labeling in granule cells of the dentate gyrus. Neurons in areas CA2, CA3, CA4, and in the dorsomedial subiculum and indusium griseum showed little or no nuclear labeling after 3H dexamethasone. With 3H corticosterone, strongest nuclear labeling was observed in neurons in area CA2 and in the dorsomedial subiculum and indusium griseum, followed by area CA1, then CA3 and CA4; the dentate gyrus contained scattered strongly labeled cells among cells with intermediate nuclear labeling. At the subcellular level, evidence for both nuclear and cytoplasmic accumulation of label was found. The results indicate that dexamethasone and corticosterone have both nuclear and cytoplasmic binding sites and that particular patterns of target cell distribution exist, characteristic for each agent. This suggests a differential regulation of cellular functions for the two compounds. Corticosterone nuclear binding appears to be more extensive and encompasses regions with dexamethasone binding. Whether in certain of these common regions corticosterone binds to the same receptor as dexamethasone, which seems possible, or to different receptors, remains to be clarified.     

A theoretical model for quantitatively inherited traits influenced by nuclear-cytoplasmic interactions

Summary Cytoplasmic genes of crop species exhibit non-Mendelian inheritance and affect quantitative traits such as biomass and grain yield. Photosynthesis and respiration are physiological processes responsible, in part, for the expression of such quantitative traits and are regulated by enzymes encoded in both the cytoplasm and nucleus. Cytoplasmic genes are located in the chloroplast and mitochondrial genomes. Unlike the nuclear genome, the cytoplasmic genomes consist of single, circular, double-stranded molecules of DNA, and in many crop species, the cytoplasmic genomes are inherited solely through the maternal parent. Maternal inheritance of cytoplasmic genomes and Mendelian inheritance of the nuclear genome were used to model the genotypic value of an individual. The model then was utilized to derive genetic variances and covariances for a random-mating population. Finally, the use of reciprocal mating designs to estimate variance components was investigated.Journal Paper No. J-12457 of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA 50011. Project No. 2447     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : IV. HeLa Tumor Strain Cells

Interferometric and photometric measurements have been made on HeLa cells, a strain of cells originally derived from a human carcinoma. From a study of the relations between successive physical measurements on individual cells, it was confirmed that, whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results on nuclear metabolism agree with others previously reported in cell strains derived from tumors; they contrast with results from freshly prepared normal cells, where the net syntheses of DNA, nuclear RNA, and protein are closely associated during interphase. Cytoplasmic measurements on HeLa cells showed that much of the net synthesis of cytoplasmic RNA is associated with DNA replication as in normal cells, and they failed to detect transfer from the nucleus of a stable RNA component synthesized independently from DNA replication. In auxiliary experiments, an inhibition of the onset of DNA synthesis was produced by a dose of X-rays; under these conditions it was shown that the major part of the accumulation of nuclear protein was independent of DNA replication and that the accumulation of nuclear RNA was equivalent to or slightly less than that of nuclear protein. About half the accumulation of cytoplasmic RNA was inhibited when DNA synthesis was blocked.     

Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.